Mass spectrometry in the pharmacokinetic studies of anticancer natural products

MASS SPECTROMETRY IN THE PHARMACOKINETIC STUDIESOF ANTICANCER NATURAL PRODUCTS

Sara Crotti,1,2*y Bianca Posocco,1y ElenaMarangon,1yDonato Nitti,3

Giuseppe Toffoli,1 andMarco Agostini2,31Experimental and Clinical Pharmacology Unit, Centro di RiferimentoOncologico, IRCCS National Cancer Institute, Via Franco Gallini 2, 33081Aviano (PN), Italy2Istituto di Ricerca Pediatrica - Citt!a della Speranza, Corso Stati Uniti 4,35127, Padova, Italy3 Surgical Clinic, Department of Surgical, Oncological and Gastroenterologi-cal Sciences, University of Padova, Via Nicolo Giustiniani 2, 35128, Padova,Italy

Received 27 April 2015; accepted 29 June 2015

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.21478

In the history of medicine, nature has represented the mainsource of medical products. Indeed, the therapeutic use ofplants certainly goes back to the Sumerian and Hippocratesand nowadays nature still represents the major source fornew drugs discovery. Moreover, in the cancer treatment,drugs are either natural compounds or have been developedfrom naturally occurring parent compounds firstly isolatedfrom plants and microbes from terrestrial and marineenvironment. A critical element of an anticancer drug isrepresented by its severe toxicities and, after administration,the drug concentrations have to remain in an appropriaterange to be effective. Anyway, the drug dosage definedduring the clinical studies could be inappropriate for anindividual patient due to differences in drug absorption,metabolism and excretion. For this reason, personalizedmedicine, based on therapeutic drug monitoring (TDM),represents one of most important challenges in cancertherapy. Mass spectrometry sensitivity, specificity and fast-ness lead to elect this technique as the Golden Standardfor pharmacokinetics and drug metabolism studies thereforefor TDM. This review focuses on the mass spectrometry-based methods developed for pharmacokinetic quantificationin human plasma of anticancer drugs derived from naturalsources and already used in clinical practice. Particularemphasis was placed both on the pre-analytical and analyti-cal steps, such as: sample preparation procedures, samplesize required by the analysis and the limit of quantificationof drugs and metabolites to give some insights on theclinical practice applicability. # 2015 Wiley Periodicals,Inc. Mass Spectrom Rev

Keywords: natural products; anticancer drug; pharmacoki-netics; liquid chromatography-mass spectrometry

I. INTRODUCTION

Anticancer drugs possess a narrow therapeutic window: there-fore, to be effective, the drug concentrations have to remain inan appropriate range: too high levels will increase toxicity andtoo low levels may not produce benefits. The therapeuticwindow, defined during the development of a drug, could beinappropriate for an individual patient due to differences in drugabsorption, metabolism and excretion. Moreover, the coexis-tence of genetic polymorphisms in transporters, enzymes,targets and receptors could result in appearance of toxicities.

For these reasons, personalised therapy is the major goal incancer management. About 30–60% of drugs are used withoutany benefit for patients. This implies not only ethical issues, butalso economical considerations due to the high costs ofanticancer therapies. Besides the genetic background, even otherfactors can affect plasma drug concentrations: liver or renalfunction, sex and age of patients, interaction between co-medications. Therapy personalization protocols aim to optimizethe treatment according to patient’s characteristics and therapeu-tic drug monitoring (TDM) is therefore crucial for a realpersonalized medicine. Indeed, TDM represents a turning pointon the correct dosing of chemotherapeutic drugs.

Mass spectrometry can play an important role in thisfield, having all the instrumental capabilities in terms ofsensitivity, specificity and fastness. In this review the authorswould provide an exhaustive view of clinical application ofmass spectrometry techniques to pharmacokinetic studies ofanticancer natural products administered in humans. Thenatural compounds and their semisynthetic derivatives aredivided into nine main classes and discussed in terms ofactivity, toxicity, dosage and their peculiar pharmacokineticcharacteristics. Mass spectrometry-based methods applied topharmacokinetic studies in human samples are presented anddiscussed in terms of analytical performances, validationassay and results reliability. Publications describing methoddevelopment and validation of assays used to quantifynatural antitumor products in human samples were searchedtrough public accessible database (PubMed). The keyword“assay” was combined with keywords related to the name ofdrugs of interest. The results were limited to publications inEnglish and those published until February 2015. Cited

†Sara Crotti, Bianca Posocco, and Elena Marangon contributed equallyto this work.!Correspondence to: Sara Crotti, Fondazione Istituto di RicercaPediatrica Citt!a della Speranza, Corso stati Uniti, 4 - 35127 Padova,Italy. E-mail: s.crotti@irpcds.org

Mass Spectrometry Reviews# 2015 by Wiley Periodicals, Inc.

references were considered and included when inclusioncriteria were met.

II. NATURAL PRODUCTS AS CYTOTOXIC AGENTS

Many anticancer drugs are either natural compounds or havebeen developed from naturally occurring parent compounds.The use of natural products, especially plants, in medicine isprobably as ancient and as the medicine itself and naturerepresented the basis of several medicine traditions. Indeed,throughout the ages and across the world humans have reliedon nature as a source of medical products. From Mesopotamiawith the earliest records, dating from around 2600 BC, toChina, with the Chinese Materia Medica (Huang, 1999), andIndia, with documentation of the Indian Ayurvedic system datesfrom before 1000 BC (Dev, 1999), the use of plant-derivedsubstances has been extensively documented. Afterwards,Greeks and Romans contributed substantially to the rationaliza-tion of plant-derived drugs use in the ancient Western world(https://www.nlm.nih.gov/; Cragg &Newman, 2013).

Traditional Medicines and plant products continue to play akey role in human healthcare. On the one side, plant-derivedtraditional medicines are still used by approximately 65% of theworld population for their primary healthcare, as estimated bythe World Health Organization (WHO) in 1985 (Farnsworthet al., 1985). On the other side, Traditional Medicine and plantproducts play an important, even though more indirect, role indrug discovery. For example, the antimalarial drug quinine wasat first isolated from the bark of Chinchona species that wasused by indigenous groups in the Amazon region for thetreatment of fever, in 1820. Furthermore, the antihypertensiveagent reserpine isolated from Rauwolfia serpentine, was used inAyurvedic medicine for the treatment of snakebite and otherailments (Dev, 1999). Moreover, despite pharmaceutical indus-try has recently explored other ways to discover new com-pounds, such as de novo chemical synthesis, high-throughputscreening, in silico modelling or biotechnology, nature stillrepresents the major source of new and effective drugs. Indeed,it has been estimated that 38 new drugs, originated from plants,

microorganisms, and living marine or terrestrial organisms,have been launched on the market between 2000 and 2010(Brahmachari, 2012). Furthermore, among the 1073 smallmolecules, New Chemical Entities (NCEs) classified over theperiod 01/1981–12/2010 by Cragg and Newman (2013), only36% can be considered as truly synthetic compounds. Consider-ing cancer treatment area, 75% of drugs belongs to the naturallyderived or inspired category.

The reasons why natural products are of primary interest indrug development are mainly two: the complexity and moleculardiversity coupled with the highly selective and specific biologi-cal activities possessed (Cragg & Newman, 2013), defined as“purposeful design” by Mishra and Tiwari (2011). In addition,during the years, an increasing interest arose on the productionof semisynthetic derivatives of the natural active substance (thelead compound) to improve the activity or to reduce side effects.This was the case of the well known acetylsalicylic acid(aspirin1) derived from salicylic acid isolated from Salix alba,and marketed in the 1899.

Figure 1 reports some examples of natural sources of activecompound from plants and terrestrial or marine microbes.

In the cancer treatment, plants have a long history: some ofthe best-known drugs are the so called vinca alkaloids [isolatedfrom the Madagascar periwinkle Chataranthus roseus (vanDer Heijden et al., 2004)], paclitaxel [discovered in the leavesof various Taxus species] (Wani et al., 1971), and irinotecan[a camptothecin semi-synthetically derivative isolated fromCamptotheca acuminata] (Wall & Wani, 1995). Even, micro-organisms are a prolific source of bioactive metabolites and haveprovided some of the most important pharmaceutical products,such as the antitumor antibiotics: doxorubicin [isolated fromStreptomyces peucetius (Arcamone et al., 1969)], bleomycin[produced by Streptomyces verticillus (Umezawa et al., 1966)],and mitomycin [produced by Streptococcus caespitosus (Wa-kaki et al., 1958)]. Contrary to plants, marine organisms do nothave a significant history of use in Traditional Medicine.Nevertheless, the development of diving techniques (1970s)allows the marine environment to be deeply explored as a sourceof bioactive compounds (Martins et al., 2014). For example,

FIGURE 1. Some examples of natural source of anticancer drugs.

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spongiuridine and spongothymidine, extracted from the spongeCryptotheca crypta, provided the basis for the chemical synthe-sis of cytarabine, whereas trabectedin was isolated fromEcteinascidia turbinate, a Caribbean sea squirt.

Despite the essential role in medicine history played byterrestrial sources, it has been estimated that only 6% of theapproximately 300000 species of higher plants have beensystematically investigated for drug development. Furthermore,it has been estimated that much less that 1% of microorganismsseen microscopically have been cultivated (Cragg & Newman,2013). These two evidences, in addition to the unexploredpotential of the marine environmental, clearly indicate thatnature remains an “untapped potential” that could continue to bea major source for new drugs discovery (Cragg & Newman,2013).

The present review focuses on the anticancer drugs fromnatural source already used in clinical practice. The Table 1reports the list of drugs here described. At the best of ourknowledge, no reports on mass spectrometry-based pharmacoki-netics (PK) quantification the anticancer enzymes (L-Asparagi-nase) are published; for this reason this class is not considered inthis review.

III. PHARMACOKINETIC PARAMETERS

Pharmacokinetics describes the temporal patterns of response todrug administration following acute or chronic dosing. PKdetermination is necessary to provide a rational basis for drugdesign, drug formulation and dosage regimen design. Poor

pharmacokinetic properties of a drug may limit its clinicalapplication. In particular, PK studies are essential to determinehow the body handles drug, that is, how drug is absorbed,distributed, metabolized and eliminated. All these processes areinfluenced by patient’s characteristics (i.e., genetics, body size,age, and co-morbidity) and by dosage, drug formulation, routeof administration and by the possible co-administration of otherdrugs. By definition, when a drug is administered intravenously,its bioavailability is 100% and, on the contrary, when a drug isadministered via other routes, its bioavailability decreasesbecause of several losses during the absorption phase.

Drug circulating in the bloodstream is responsible ofpharmacological activity and its level is regulated by severalprocesses: the organs uptake; the drug binding with plasmaproteins, red cells or platelets; the permeability of tissuemembranes and the drug metabolism and elimination.

Drug elimination occurs by two processes, excretion andmetabolism; excretion is the irreversible loss of chemicallyunchanged drug in urine and in faeces, and metabolism is theconversion of one chemical species to another.

Drug metabolism is obtained by two types of enzymaticreactions: phase I (biotransformation) characterized by reactionsof oxidation, hydroxylation, reduction and hydrolysis and phaseII (conjugation) characterized by reactions of addition of a newfunctional group such as glucuronide, sulphate, methyl andacetyl groups, glutathione and amino acids. Even if drugmetabolism is the physiological way to detoxification, somemetabolites can retain (or increase) the pharmacologicalactivity.

TABLE 1. List of natural compounds and semisynthetic derivates marketed for anticancer therapy.

Class Lead compound semisynthetic derivatives Source

A. Vinca AlkaloidsVinblastine Vindesine PVincristine Vinorelbine; Vinflunine P

B. Taxanes Paclitaxel Docetaxel; Cabazitel; Nab-paclitaxel P

C. Epothilones Epothilones A-F Ixabepilones BD. Camptothecin Analogs Camptothecin Topotecan; Irinotecan P

E. Antibiotics

Dactinomycin BDoxorubicin Idarubicin

BDaunobicin Epirubicin

Mitoxantrone BBleomycins BMitomycin B

F. EpipodophyllotoxinsEtoposide PTeniposide P

G. Marine ProductsTrabectedin MCytarabine M

H. Retinoids Retinol Tretinoin PI. Histone Deacetylase

Inhibitors Vorinostat B

Source: B, bacterial; P, plant; M, marine.

Mass Spectrometry Reviews DOI 10.1002/mas 3

PHARMACOKINETIC STUDIES OF ANTICANCER NATURAL PRODUCTS &

Measuring drug plasma concentration in samples collectedat specific time points, it is possible to obtain the plasmaconcentration-time profile and the shape and the mathematicalelaboration of this profile provide the main pharmacokineticparameters reported in Table 2.

IV. MASS SPECTROMETRY FORPHARMACOKINETIC ANALYSIS

In order to define the pharmacokinetic profile of a compound,the method and the analytical technique used are fundamental.The higher the method sensitivity, the better the description ofthe drug kinetics, in terms of a much longer monitoring of drugconcentration, which also means a better measurement of thearea under the concentration-time curve (AUC) and a descrip-tion of the half-life in the terminal phase (t1/2). In the last35 years, there have been significant improvements in analyticaltechnologies applied in cancer pharmacology to measure drugconcentration and to study drug metabolism. At the beginning,the concentration data from plasma or other biological matrixwere usually obtained by LC-UV/VIS methods. The next stepwas to prefer, when possible, the use of a fluorescence detector(FLD), but the real change in bio-analysis began with the

development of bench-top mass spectrometry instruments,combined with liquid chromatography (LC-MS). Within a fewyears, LC-MS has become the method of choice for quantitativedrug analysis to support pharmacokinetics and drug metabolismstudies (Hopfgartner & Bourgogne, 2003). Coupling the massspectrometer with LC provided significant improvements inassay sensitivity, specificity and capability to analyze sampleswith very different concentration ranges. The increase insensitivity and specificity caused three important effects:

i) the possibility to detect drugs and metabolites at very lowconcentration;

ii) the possibility to use very small amount of sample (that isparticularly important in preclinical studies conducted insmall animals or in paediatric studies);

iii) a selective analytes detection in presence of complexmatrices such as tissues or whole blood.

Mass spectrometry owes its success to its performances indrug quantitative (PK) and qualitative (metabolites identifica-tion) analysis. Accurate and sensible quantitation is obtained byoperating in tandem mass (MS/MS) mode (Saint-Marcoux et al.,2007). MS/MS is necessary because of possible interfering

TABLE 2. Main pharmacokinetic parameters and their clinical significance

Parameter Name Significance Key features

Maximumplasma

concentration

The highest drug concentration observed in plasma following

administration

Cmax and Tmax arecorrelated and both

depend on how quicklythe drug enters into

and is eliminated fromthe bodyTmax

Time untilCmax is

reachedThe time at which the highest drug

concentration occurs

Area under theconcentration -

timecurve

The measure of the total systemicexposure to the drug

It represents theamount of unchangeddrug that has reachedthe general circulation

and it is useful to define

the bioavailability of adrug

VdVolume of

distributionThe apparent volume into which the

drug is dissolved

It depends on bindingto plasma proteins andtissues and it is usefulto correlate the drug

concentration in plasma with its

amount in the body

t1/2Half-life in theterminal phase

The time taken for the plasmaconcentration to fall by one half once

distribution equilibrium has beenachieved

It is independent of theamount of drug in the

body and it is useful forthe det ermination ofthe frequency of drug

administration

Cl ClearanceThe rate of drug elimination by all routesnormalized to the concentration of the

It is the sum of all organs clearance,

especially hepatic and renal clearance

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compounds present in the biological sample exhibiting the sameinteger mass, while the fragmentation pattern is compoundspecific. Tandem mass experiments performed by means oftriple quadrupole instrumentations, are obtained through colli-sion-induced dissociation (CID). Detection is based on selectivereaction monitoring (SRM); sometimes also called multiplereaction monitoring (MRM), based on monitoring of fragmenta-tion reaction(s) from the analyte molecular ion to analytespecific fragment ion(s) (de Hoffmann, 1996). The combinationof parent mass and its fragment ions is used to monitorselectively the compound that has to be quantified.

However, even if the need for chromatographic separationis often low with MRM detection mode thanks to its specificity,co-eluting matrix components may cause problems in theionization process by the so called matrix effect. Moreover,other factors such as the drug-proteins binding ant the analyteinstability during the untreated sample storage could affect thequantitative drug analysis. Sample preparation is one of the mosttime-consuming steps in the bioanalysis aiming to isolate,clean-up and pre-concentrate analytes of interest from biologicalmatrices (Nov"akov"a, 2013). Sample preparation proceduresmainly employed in PK studies are schematized in Figure 2.Solid phase extraction (SPE), liquid-liquid extraction (LLE) andprotein precipitation (PP) are considered the conventionalsample preparation techniques still highly employed in contrastto modern approaches such as on-line techniques or micro-extractions (Nov"akov"a, 2013). Basically, LLE and SPE provideselective analytes recovery (depending on the solvent and/orstationary phase choice) and result in cleaner extracts withrespect to PP. SPE has advantages in terms of less sampleamount required, a minor solvent consumption, and the possibil-ity to be used in on-line systems.

In LC-MS analysis the analytes are introduced into the ionsource of mass spectrometer after their separation in a LC column.There are different types of MS ion sources, but the mostcommonly employed in pharmacokinetic studies is Electrospray

Ionization (ESI), an atmospheric pressure ionization. ESI is a softionization technique—as very little internal energy is retained bythe analyte after ionization—and does not cause decomposition oflabile compounds. It is characterized by an efficient ion produc-tion, mainly by protonation or cationization reactions, and it canoperate in either positive or negative ionmode.

In a typical ESI source, schematized in Figure 3, thesolution is injected in a stainless steel capillary. Betweenthis capillary and their counter electrode, a voltage in the orderof 3–5 kV is applied. Under these conditions, the formation of asolution cone just outside the capillary occurs. The coneformation is due to the presence of charged species inside thesolution, which experiment the electrostatic field existingbetween the capillary and the counter electrode (Taylor, 1964).After the cone production, the droplets formation from the coneapex is observed, charged droplets further migrate through theatmosphere to the counter electrode (Taylor, 1964). Dropletsformation is strongly influenced by solvent chemical–physicalcharacteristics, ionic analytes concentration, inorganic saltsconcentration, and the applied voltage. The so generatedcharged droplets, decrease their radius after solvent evaporationstill conserving their total charge amount. The energy requiredfor the solvent evaporation is due to the environment thermalenergy, further enhanced through by the use of a heated capillaryor by collisions with heated gas flow. As the droplet radiusdecreases, the surface charge density increases; when the radiusreaches the Rayleigh stability limit, the electrostatic repulsionequals the surface tension. For lower radii, the chargeddroplets are unstable and decompose through a process defined“Columbic Fission” (Rayleigh, 1882). This produces smallerdroplets that ultimately liberate unsolvated charged analytemolecules. Alternatively, the “ion evaporation” mechanism hasbeen proposed, based on the direct emission of ions from thecharged droplets occurring when the surface charge densityshows a large increase. Far to be fully understood, themechanism(s) of gas phase ions production from the small/

FIGURE 2. Schematic representation of the conventional sample preparation procedures used in PK.

highly charged droplets has been investigated by several authors,as discussed in some recent reviews (Kebarle & Verkerk, 2009;Crotti et al., 2011).

Typically, in ESI the production of smaller droplets isenhanced by lower mobile phase flow rate and by the use ofvolatile mobile phases; the pH and volatility of the LC eluenthas a very high role in ionization efficiency and thus detectionsensitivity.

Another, commonly used, ionization type is atmosphericpressure chemical ionization (APCI), which is based on theinteraction of solution vapours with a corona discharge, leadingto gas phase ionization reactions. APCI has been used typicallyfor less polar (neutral) analyte compounds, such as steroid-likecompounds.

Physicochemical properties (i.e., hydrophilicity and ioniza-tion behavior) of the analyte have a major role in affecting themethod performance in both chromatographic separation stepand mass spectrometric detection. The detection response ofcompounds varies due to the ionization efficacy and co-presenceof interfering molecules, thus quantitative analysis by LC-MSshould require that pure standards are available for each analyte.The use of isotope labelled compounds as internal standards isrecommend to reduce those problems caused by matrix effect.

V. CLASSES OF ANTICANCER NATURALPRODUCTS

The natural and semisynthetic compounds discussed in thepresent review have peculiar mechanisms of action aimed todestroy tumoral cells by blocking important targets of cellsurvival and replication. A schematic representation of thesemechanisms of action, together with the sites of action and thecellular toxicities, is reported in Figure 4 for each drug discussedin the present review. All the pharmacological and the PK dataof discussed drug are reported in according to FDA database(http://www.fda.gov/default.htm) unless otherwise stated.

A. Vinca Alkaloids

Vinblastine and vincristine (Fig. 5) are two alkaloidsderived from tryptophan decarboxylation and dimerization.

They have been isolated from the Catharanthus roseusamong the 130 alkaloids identified after purification (van DerHeijden et al., 2004). Vinca alkaloids are microtubule-interfer-ing agents. Microtubules are part of cytoskeleton—within thecell’s cytoplasm—and are involved in numerous cellular func-tions, including the maintenance of cell shape, intracellulartransport, secretion and neurotransmission. Microtubulesare made up of polymers of tubulin. Tubulin molecules, intheir turn, are made up of a heterodimer consisting of a- andb-tubulin subunits. Microtubules are highly dynamic andunstable structures that are constantly incorporating free dimersand releasing dimers into the soluble tubulin pool. Vincaalkaloids bind to beta-tubulin causing microtubules polymeriza-tion inhibition and stopping cell division in a cycle-specificmanner. Vinca alkaloids are used for treatment of leukemias,lymphomas, and testicular cancer. Vincristine is also used fortreating paediatric leukemias and solid tumors such as Wilms’tumor, neuroblastoma, and rhabdomyosarcoma. Vinblastine isgiven by intravenous (i.v.) infusion with doses of 0.3mg/kgevery 3-weeks in a typical regiment for testicular cancer, whilevincristine is administered at standard doses of 1.4–2mg/m2

weekly (Brunton et al., 2011). Vinca alkaloids possess longterminal half-life time (range 19–155 hr), are extensivelymetabolized by the liver and the metabolites are mainly excretedin the bile (Robieux et al., 1996), while less than 15% is foundunchanged in urines. Vinorelbine is a semi-synthetic derivativeshowing less neurotoxicity than vincristine precursor; its deriva-tive vinflunine is a bi-fluorinated molecule with superiorantitumor activity both in vitro and in vivo. Vindesine (VDS) isthe only semi-synthetic derivative of vinblastine.

Vincristine and vinorelbine largely bind to platelets (Sethiet al., 1981; Urien et al., 1993; Gauvin et al., 2002), as aconsequence the blood to plasma ratio (B:P) is >1 (Gauvinet al., 2002). Higher blood concentrations can introduce bias inthe evaluation of pharmacokinetic parameters (e.g., AUC) sincedrug can be released from platelets during plasma preparationprocedure. Due to this aspect, some authors suggest to determinedrug dosage in blood instead of plasma (Puozzo et al., 2007).Vinca alkaloids are able to bind plasma proteins also; a fullyFDA-validated method has been developed by Damen et al. inorder to obtain a simultaneous quantification of vincristine in

FIGURE 3. Ions formation in positive electrospray ionization. Under these conditions the capillary is placed at apositive voltage, while the counter electrode is placed to a negative voltage. Reproduced with permission from(Crotti et al., 2011).

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whole plasma and in plasma ultrafiltrate with LC-MS/MS(Damen et al., 2009c). By means of 30 kDa (MWCO) centrifu-gal filters the unbound fraction of vincristine was determined in150mL of sample and the total amount of drug in plasma hasbeen quantified in 30mL of sample, after a simple proteinprecipitation (PP) treatment using methanol/acetonitrile (50:50,v/v). By this validated method drug concentration was quantifiedup to 72 hr in plasma samples of a child treated with 1.4mg ofvincristine and free vincristine has been detectable up to 3 hr inplasma ultrafiltrate (Damen et al., 2009c).

Early attempt for vinca alkaloids quantification werebased on radio immunoassay (RIA) (Teale et al., 1977; Nelson,1982), LC-UV (Embree et al., 1997) or LC-ECD methods(Vendrig et al., 1988). Quantification was limited to the lowconcentration reached and the lacks of specificity in distinguishconcomitant metabolites. MS introduction increases specificity(Ram"rez et al., 1997), clarifies metabolites structure (Puozzo

et al., 2007; de Graeve et al., 2008), and makes possible thesimultaneous quantification of metabolites (Zorza et al., 2007;Dennison et al., 2008). Most of published LC-MS/MS methodsare based on positive Electrospray ionization of vincristine(Skolnik et al., 2006; Lee et al., 2007b; Dennison et al., 2008;Damen et al., 2009d; Guilhaumou et al., 2010), while only tworeported the use of APCI method (Schmidt et al., 2006; Coronaet al., 2008). The presence of basic amines in its structureensures good ionization efficiency, with quantitation limitsdown to 0.05 ng/mL samples when operating in high tempera-ture ESI conditions (Lee et al., 2007b; Dennison et al., 2008).The MRM transition monitored was m/z 825>765 for almost allpapers, with the exception of the method proposed by Dennisonet al. (2008), where doubly charged precursor ions and doublycharged product ions are used for PK studies.

Early LC-MS methods developed for vincristine required2/1.5mL of plasma samples (Ram"rez et al., 1997), while

FIGURE 4. Levels of cellular toxicity and mechanisms of action of natural anticancer drugs.

FIGURE 5. Structures of vinca alkaloids of natural and semi-synthetic origin. The tryptophan-derived moietiesare shown in bold.

recently published methods lowered plasma samples volumesdown to 0.1mL (Corona et al., 2008; Damen et al., 2009c). Theproposed extraction procedures were based on traditional off-line SPE (Skolnik et al., 2006; Lee et al., 2007b; Guilhaumouet al., 2010) or LLE (Dennison et al., 2008); the bestperformance in term of recovery % (90–95%) were ensured by aPP followed by an on-line SPE purification (Corona et al.,2008). In all the above mentioned methods, the internal standardchosen was the semi-synthetic drug vinorelbine and chro-matographic separation were performed in presence of ammoni-um acetate both in acidic or basic conditions. A step forwardrealistic application of TDM for vincristine was the set-up ofdried blood spot (DBS) quantification analysis developed byDamen et al. (2009b). Authors justified the necessity of asimpler method for sampling, storing and shipping bloodsamples of patients located far away from their laboratory. Bythis method, 40mL of blood samples have been analyzed after a0.25-in.-diameter punch sampling and 15min extraction in asonic bath. This method has been applied only to healthyvolunteers but compared to other methods, it seems morefeasible to the application in the paediatric field.

Vinorelbine metabolic pathway has been characterized (VanHeugen et al., 2001; de Graeve et al., 2008) by ESI-MS/MS andthe quantification of active metabolite is essential in the defining ofactivity/toxicity levels. Multi drug therapy is commonly performedin cancer treatment and pharmacokinetic alteration can occur, as inthe case of patients treated with vinorelbine in combination withlapatinib. UHPLC-MS/MS analysis revealed that vinorelbineclearance decreased statistically (Rezai et al., 2011). Recently,methods have been developed leading to fast and sensitive (subppb) analysis (Damen et al., 2009a; Qian et al., 2011).

PK in paediatric patients has been extensively evaluated inthe recent years (Groninger et al., 2002; Schmidt et al., 2006;Damen et al., 2009c; Guilhaumou et al., 2010) due to the largeinterest of vinca alkaloids in the paediatric leukemias treatment.In some papers, the very low amount of plasma (30mL)necessary to vincristine analysis was due to an increasedionization efficiency obtained with ESI sources (Damen et al.,2009c; Guilhaumou et al., 2010). In other papers, the minimalsample size necessary to perform the analysis by LC-MS wasaround 200mL of plasma (Guilhaumou et al., 2010). The APCI-MS method developed by Schmidt et al. and applied to study thevincristine levels in 29 infant plasma samples still uses arelatively large amount of plasma (0.5–1mL) and requires atime-consuming SPE procedure, but ensure enough sensitivitywith an LLOQ of 0.18 ng/mL (Schmidt et al., 2006).

B. Taxanes

Taxanes represent a very important class of anticancer agentsavailable for clinical use since 1990s. Currently, two taxanes,paclitaxel (Taxol1) and docetaxel (Taxotere1) (Fig. 6), areincluded in multidrug regimens for the therapy of several solidtumours, such as ovary, breast, head and neck, prostate and non-small cell lung cancers. Paclitaxel and docetaxel share the samespectrum of clinical activity but they differ in their toxicity.Paclitaxel is a natural product isolated in 1971 (Wani et al.,1971) from the bark of the Pacific Yew (Taxus brevifolia) whiledocetaxel, a semi-synthetic taxane analogue derived from theEuropean Yew (Taxus baccata) (Vaishampayan et al., 1999),was identified in the 1980s (Gligorov& Lotz, 2004).

FDA approved Taxol for the treatment of ovarian cancer in 1992and docetaxel approval was obtained in 1996 (http://dtp.nci.nih.gov/timeline/flash/FDA.htm). A 130 nm albumin-bound particleform of paclitaxel (Abraxane1) was approved in 2005 for breastcancer treatment (Gradishar et al., 2005) and in 2010 cabazitaxel(Jevtana1) (Fig. 6), the fourth taxane to be approved as a cancertherapy, was licensed for the treatment of hormone-refractoryprostate cancer.

Taxanes, like vinca alkaloids, are microtubule-interferingagents (see Fig. 4). Taxanes bind to the b-tubulin subunit of thetubulin heterodimer, accelerate the polymerization of tubulinand stabilize the resultant microtubules inhibiting their depo-lymerization. This inhibition results in the arrest of the celldivision cycle which triggers the cell signalling cascade, leadingto apoptosis of cancer cells (Schiff & Horwitz, 1980; Schiff &Horwitz, 1981).

Paclitaxel is insoluble in aqueous solution and it is thereforeformulated in 50% ethanol and 50% cremophor-EL (a polyox-yethylated castor oil derivative) to improve its solubility(Gelderblom et al., 2001). This vehicle is responsible for a highrate of hypersensitivity reactions. Patients receiving this formu-lation are protected by pre-treatment with a histamine H1-and H2-receptor antagonist and a glucocorticoid. Paclitaxel isgiven only intravenously because of its poor oral bioavailability(5–8%). Paclitaxel is administered as a 3-hr i.v. infusion of 135–175mg/m2 every 3 weeks or as a weekly 1-hr i.v. infusion of80–100mg/m2. Prolonged infusions of 96 hr have been evaluat-ed in different tumor histologies and are active (Brunton et al.,2011). Elimination of paclitaxel has been found to have a three-phase elimination curve (Fig. 7) (Huizing et al., 1997).

FIGURE 6. Chemical structures of taxanes.

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Paclitaxel showed a disproportionate increase in plasmaCmax and AUC as the dose increased, suggesting saturation ofelimination at higher concentrations of paclitaxel (Henningssonet al., 2001; Mross et al., 2000). Several studies with paclitaxelgiven as a 6-hr i.v. infusion documented non-linear pharmacoki-netics with doses higher than 250mg/m2 (Brown et al., 1991;Grem et al., 1987; Wiernik et al., 1987), while others reportedthat a lower dose of 135mg/m2 is the critical threshold for non-linear kinetics (Ohtsu et al., 1995; Panday et al., 1998). Similarfindings were noted with 3-hr i.v. infusion schedules in patientswho received doses of 135 and 175mg/m2, (Gianni et al., 1995;Ohtsu et al., 1995; Ye et al., 2000) and in pharmacokineticstudies in children showing non-linear disappearance of pacli-taxel with saturation of elimination pathways and tissuedistribution (Sonnichsen et al., 1994). Paclitaxel is bound toproteins in plasma, tissues, and tubulin. In plasma, proteinbinding is 95–98% ((Longnecker et al., 1987; Wiernik et al.,1987). Paclitaxel is bounded mainly to a1- acid glycoproteinand, to a minor extent, to albumin and lipoproteins (Kumaret al., 1993).

Supporting extensive drug binding in vivo, total volumes ofdistribution have been reported as significantly variable, depen-dent from the dose/schedule and larger than that of total bodywater, ranging from 50L/m2 to over 650L/m2 (Wiernik et al.,1987; Brown et al., 1991; Tamura et al., 1994). In addition,paclitaxel shows high distribution in specific tissue of organs askidney, lung, spleen, and in third space fluids, including asciticand pleural fluid (Wiernik et al., 1987; Glantz et al., 1995).Paclitaxel does not penetrate in tumour sanctuary tissues,including testicles and brain, in which it is undetectable, and it isnot present in cerebral spinal fluid (Glantz et al., 1995; Lesseret al., 1995). Despite the fact that paclitaxel is highly bound toplasma proteins and in tissues (tubulin), it is readily clearedfrom plasma. Paclitaxel clearance is nonlinear and decreaseswith increasing dose or dose rate. Paclitaxel disappearedfrom plasma with a half-life of 10–14 hr and a clearance of 15–18 L/hr/m2 (Brunton et al., 2011). Paclitaxel undergoes hepaticoxidative metabolism by the cytochrome P450 family and the

major route of elimination is biliary excretion (Monsarrat et al.,1993; Walle et al., 1995). In humans, renal excretion andother extrahepatic excretion mechanisms account for less than10% of elimination (Longnecker et al., 1987; Wiernik et al.,1987; Baker et al., 2002). The major human metaboliteidentified is 6a-hydroxypaclitaxel which is inactive. However,multiple hydroxylated products were found in plasma (Cresteilet al., 1994).

The best model to predict the relationship between thepaclitaxel plasma concentration and the effects is the thresholdmodel, in which the length of time that paclitaxel concentrationexceeds a threshold concentration is predictive of toxicity.According to some studies, the duration of paclitaxel concen-trations above a threshold value correlates with not only thetoxicity, but also the antitumor activity (Sonnichsen et al., 1994;Gianni et al., 1995; Huizing et al., 1998; Mielke et al., 2005)

The most concerning side effects of paclitaxel are neutrope-nia and neuropathy. Many patients experience myalgias forseveral days. Mucositis is prominent in 72- or 96-hr i.v. infusionsand in weekly schedule. Hypersensitivity reactions occurred inpatients receiving paclitaxel infusions of 1–6 hr but have largelybeen averted by premedication. Premedication is not necessarywith 96-hr i.v. infusion (Brunton et al., 2011).

Docetaxel, being slightly more soluble than paclitaxel, isadministered in polysorbate 80 and it is associated with alower incidence of hypersensitivity reactions than paclitaxeldissolved in Cremophor. However, pre-treatment with a gluco-corticoid prior to therapy is required to minimize the severity ofhypersensitivity reactions (Brunton et al., 2011). Docetaxel haslow (<10%) and highly variable oral bioavailability (Sparre-boom et al., 1996). In general, the pharmacokinetic profile of i.v.docetaxel is characterized by substantial inter-patient variability(Bruno et al., 1998, 2001; Rosing et al., 2000). In a populationpharmacokinetic study of more than 600 patients receivingdocetaxel 75–100mg/m2, the median clearance was 36L/hr,representing approximately a variation of 3.5-fold in thispopulation (Bruno et al., 2001). In a more recent study in whichpatients received docetaxel 75mg/m2 approximately 10-foldvariation in drug clearance was observed (Slaviero et al., 2004).The docetaxel dosage used for treating cancer patients rangesfrom 35 to 100mg/m2 as a 1-hr i.v. infusion administered onceevery 3 weeks or once a week for three consecutive weeks. Thecomparative pharmacokinetic parameters of docetaxel adminis-tered weekly at a dose of 35mg/m2 and 3-weekly scheduleswere summarized by Baker et al. (2006). Docetaxel pharmaco-kinetics is linear for doses lower or equal to 115mg/m2.

Docetaxel is extensively bound to albumin and a1-acidglycoprotein and the latter is the main determinant of variabilityin docetaxel serum binding (Urien et al., 1996). Sequentialmetabolism (CYP-mediated hydroxylation) and biliary excre-tion are the principal pathways of excretion of docetaxel (Shouet al., 1998; Baker et al., 2009). The metabolites demonstratesubstantially reduced cytotoxic activity against cancer cells andbone marrow compared with the parent drug, making biotrans-formation a major route of inactivation (Sparreboom et al.,1996; Monsarrat et al., 1997). The principal site of metabolismis the tert-butylpropionate side chain which undergoes a seriesof oxidation reactions (Rosing et al., 2000). The observed largepharmacokinetic variability for docetaxel has important clinicalconsequences: for a 50% decrease of Cl, neutropenia increase of450% (Bruno et al., 1998) and even a 25% decrease of Cl

FIGURE 7. Plasma concentration-vs-time profiles of paclitaxel in threepatients treated at different dose levels: 100 (*), 200 (&), 250 ( ) mg/m2.Reproduced with the permission of (Huizing et al., 1997).

comported a 150% increase of cases with febrile neutropenia.Another study, with docetaxel administered as 3-weekly regi-mens, demonstrated that AUC was the only significant predictorof toxicity, as grade 3/4 mucositis, grade 3/4 diarrhoea, severeasthenia and febrile neutropenia (Bruno et al., 2001). Docetaxelexposure has also been related to the efficacy of treatment, inparticular AUC was a significant predictor of time to tumourprogression in NSCLC (Bruno et al., 1998). Docetaxel causesgreater degrees of neutropenia than paclitaxel but less peripheralneuropathy and asthenia and less frequent hypersensitivity.Fluid retention is a progressive problem with multiple cycles ofdocetaxel therapy, leading to peripheral oedema, pleural andperitoneal fluid (Brunton et al., 2011).

Nab-paclitaxel is soluble in aqueous solutions and can besafely administered without prophylactic antihistamines orsteroids. This reformulation of paclitaxel has increased cellularuptake via an albumin-specific mechanism (Yardley, 2013).Nab-paclitaxel achieves a higher serum concentration of pacli-taxel compared to Cremophor-solubilized paclitaxel but even anincreased clearance therefore, the two formulations show asimilar drug exposure (Gardner et al., 2006).

Different dosing regimens have been evaluated but nab-paclitaxel is usually administered intravenously over 30min at260mg/m2 every 3 weeks. Nab-paclitaxel produces increasedrates of peripheral neuropathy compared to Cremophor-deliv-ered paclitaxel but rarely causes hypersensitivity reactions(Brunton et al., 2011).

Cabazitaxel is a semi-synthetic derivative of the naturaltaxoid 10-deacetylbaccatin III. It is lipophilic, practicallyinsoluble in water and soluble in alcohol. It was approved bythe U.S. Food and Drug Administration (FDA) for use incombination with prednisone for treatment of patients withhormone-refractory metastatic prostate cancer previouslytreated with a docetaxel-containing regimen. The recommendeddose is 25mg/m2 administered every three weeks as a 1-hr i.vinfusion. Since severe hypersensitivity reactions can occur,premedication with corticosteroids and H2 antagonists isindicated. Unlike other taxane compounds, this agent is a poorsubstrate for the membrane-associated, multidrug resistance,P-glycoprotein (P-gp) efflux pump and may be useful fortreating multidrug-resistant tumors (Abidi, 2013; Cheetham &Petrylak, 2013). In addition, cabazitaxel penetrates the blood-brain barrier (Semiond et al., 2013).

Based on the population pharmacokinetic analysis, after ani.v. dose of cabazitaxel 25mg/m2 every three weeks, the meanCmax, reached at the end of the 1-hr i.v. infusion, was 226 ng/mL (CV 107%) in patients with metastatic prostate cancer. Themean AUC in patients with metastatic prostate cancer was 991ng " hr/mL (CV 34%). No major deviation from the doseproportionality was observed from 10 to 30mg/m2 in patientswith advanced solid tumors. The volume of distribution was4,864 L at steady state and the plasma clearance of 48.5 L/hr inpatients with metastatic prostate cancer.

In vitro, the binding of cabazitaxel to human serum proteinswas 89 to 92%. Cabazitaxel is mainly bound to human serumalbumin (82%) and lipoproteins. In in vitro experiments,cabazitaxel resulted equally distributed between blood andplasma. Cabazitaxel is extensively metabolized in the liver(>95%), mainly by the CYP3A4/5 isoenzyme and to alesser extent by CYP2C8. Seven metabolites were detectedin plasma (including the 3 active metabolites issued from

O-demethylation), with the main one accounting for 5% ofcabazitaxel exposure. Even if this drug is minimally excretedvia the kidney, around 20 metabolites of cabazitaxel are excretedinto human urine and faeces. The most concerning side effectsof cabazitaxel are: neutropenia, febrile neutropenia, hypersensi-tivity reactions, gastrointestinal symptoms and renal failure.

Many publications have been published describing methodsbased on LC-UV assays developed to quantify taxanes inbiological matrices such as rat, mouse or rabbit serum, plasma,tissue, tumour, urine and faeces and human serum, plasma andurine (Kim et al., 2005; Suno et al., 2007; Kumar et al., 2012;Wei et al., 2014). These bioanalytical methods were developedto support taxanes development in preclinical and clinicalsettings to define the disposition of these drugs. As this reviewaims to summarize LC-MS assays developed to quantify taxanesin human samples, we have not mentioned LC-UVassays and allthe methods determining these drugs on matrices derived fromanimals. Besides the LC-UV assays, to quantify taxanes, eventubulin-based biochemical assays, immunoassays and micellarelectrokinetic chromatography (MEKC) have been developed.Tubulin-based biochemical assay is characterized by a minimalpre-treatment of sample but its sensitivity is limited comparedto LC-UV and LC-MS methods (Hamel et al., 1982; Moraiset al., 2003). Even if immunoassays (Grothaus et al., 1993;Svojanovsky et al., 1999; Sheikh et al., 2000) are characterizedby a good LOQ (below 0.5 ng/mL), this method could beinvalidated by cross reactivity of metabolites to antibodiesresulting in decreasing of concentration accuracy. MEKCcombines chromatographic and electrophoretic separation prin-ciples and the separation is based on the differential partitioningof an analyte between the two-phase system: the mobile aqueousphase and micellar pseudostationary phase. In the methodspublished (Hempel et al., 1996; Rodriguez et al., 2012a,b)MEKC was coupled to a UV detector and even if MEKC ischaracterized by a higher separation power and a less sensitivityto endogenous components compared to LC, LLOQ in plasmawas between 50 ng/mL (Hempel et al., 1996) and 220 ng/mL(Rodriguez et al., 2012b) and the run times were similar to thoseobtained with LC-UV (14–20min).

With the always stronger need of quantifying taxanes insamples derived from clinical studies or from treated patientsdue to the great inter-patient variability in order to expectresponse and toxic effects, and thanks to the identification ofliquid chromatography coupled with mass spectrometry as theGold Standard of drug testing, many LC-MS/MS assays havebeen developed and validated. A complete list of publishedmethods is reported in Table 3. There is also a quantificationmethod, of paclitaxel and docetaxel in human plasma, using MSwith a single quadrupole (Parise et al., 2003). The concentrationrange is therefore defined on the basis of the expected taxane/sconcentrations in the sample. Since the matrix effect could bedefined the Achille’s heel of quantitative LC–electrospray–tandem mass spectrometry and only few methods employedstable isotope labelled internal standard (IS) that minimizes thematrix effect, for the others the cleaning-up of the samplesrepresented a very important step. Three different extractionprocedures, PP, LLE and SPE are described to clean up thebiological samples. PP is the fastest extraction method and itwas employed by 6 authors: some of them (Yamaguchi et al.,2012; Marzinke et al., 2013) used PP alone as sample prepara-tion while some other associated PP to SPE (Sottani et al., 1998;

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tier K

1 0.4 5 0.24

7.5 10

7 7 3 7 12

2’,7

P P P P, O

P P P P P

p-3’

3.Listofpublicationsrelatedtothequantificationoftaxanes

inhuman

samples

Continued

0.1 1 5 8 2 0.

5 2.4 8 6 2.5 9 10

P S P S P P P,B P nr

500 nr

3’-O

3.Continued

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tier K

5 0.87

1 2 0.5

7 2 4 4 8 9 9

P P P P P P P P P P P

3’-O

tier K

IS,internal

standard;PCX,paclitaxel,6-O

H-PCX¼6-alpha-hydroxy-paclitaxel;p-3

0 -OH-PCX,p-3

0 -hydroxy-paclitaxel;DOC,docetaxel;M1-4,proposedmetabolites;

CAB,cabazitaxel;

P,plasm

a;S,serum;nr,notreported;SPE,solidphaseextraction;LLE,liquid–liquid

extraction;PP,protein

precipitation;TFC,turbulent-flow

chromatography;(o),on-line;

(a),automatic,

(sa),semi-automatic.! totalandunboundpaclitaxel;!!multidrugdosage;

!!! oralfluid.

Gardner et al., 2008; Corona et al., 2011; Navarrete et al., 2013).Navarrete et al., for PP, used a solution of zinc sulphate inmethanol (30:70 v/v) in order to obtain the lysis of erythrocytesand consequently a very clean supernatant (Navarrete et al.,2013). Gardner et al. and Yamaguchi et al. used only acetoni-trile, the former to quantify paclitaxel following Abraxanetreatment and the latter to determine docetaxel (Gardner et al.,2008; Yamaguchi et al., 2012). The extraction volume was oneto threefold the volume of the biological sample to treat.

Besides the methods already cited because employing aSPE extraction added to the PP, the are five methods based onSPE alone (Schellen et al., 2000; Parise et al., 2003; Grozavet al., 2004; Green et al., 2006; Yamaguchi et al., 2012). Threemethods, developed for the quantification of paclitaxel, de-scribed on line SPE (Schellen et al., 2000; Grozav et al., 2004;Yamaguchi et al., 2013) and this technique is characterized byless sample handling time than off-line SPE guaranteeing thesame level of cleaning-up. SPE procedure could be affected bycarry-over effect therefore, it should be defined during thevalidation process. To overcome this problem, Grozav et al.introduced a washing step of the extraction column before thesample injection.

The extraction procedure more frequently described inliterature to quantify taxanes is LLE (Alexander et al., 2003;Basileo et al., 2003; Wang et al., 2003; Baker et al., 2004;Mortier et al., 2004; Stokvis et al., 2004a; Guitton et al.,2005; Kuppens et al., 2005; Mortier et al., 2005a,b; Gao et al.,2006; Gardner et al., 2006; Mortier & Lambert, 2006a,b;Vainchtein et al., 2006; Zhang & Chen, 2008; Hendrikx et al.,2011; Zhang et al., 2011; Grobosch et al., 2012; Hendrikx et al.,2013; Kort et al., 2013b). LLE is more time-consuming thanPP and on-line SPE but, with this procedure, the best LLOQs intaxane quantification were obtained: 0.1 ng/mL for paclitaxel(Alexander et al., 2003; Grobosch et al., 2012) and docetaxel(Kort et al., 2013a). Currently, the two solvents giving the bestresults for taxane quantification in terms of extraction recovery,matrix effect and higher sensitivity are: diethyl-ether andtertbutylmethylether.

As regards the chromatographic analysis, both LCand UHPLC were used to quantify taxanes in human samples.UHPLC is a derivative of high-pressure liquid chromatography:UHPLC column packing particle size decreases compared toHPLC column, therefore efficiency and consequently resolutionincreases (Novakova & Vlckova, 2009). By making use of thesmaller particles, the speed of analysis and peak capacity i.e.,number of peaks resolved per unit time, can be prolonged to themaximum values and these values are much better than thevalues achieved earlier by HPLC.

Two UHPLC-MS/MS methods were developed for thedetermination of the first two taxanes approved, paclitaxel(Zhang et al., 2008) and docetaxel (Du et al., 2013) and, even ifthe chromatographic separation is obtained in less than 3min,the sensitivity seems not to increase because the LLOQ values5 ng/mL (Zhang & Chen, 2008) and 2 ng/mL (Du et al., 2013)resulted comparable to those obtained by the use of HPLC.

The most used chromatographic columns are C18 and C8

reversed-phase silica-based while Green et al. applied a C12

column (Green et al., 2006). The elution mobile phase in themajority of published methods is made up of at least 50% (v/v)of organic solvent (acetonitrile and/or methanol). The authorsset up the chromatographic analysis in order to increase

selectivity, reduce interferences and run time and to obtain agood peak shape. Mortier et al. (2004) described ion suppressionof paclitaxel and docetaxel by the formulation vehicle (polysor-bate 80 and Cremophor EL) due to carryover in subsequent runswith an isocratic LC elution therefore the same group developeda chromatographic method applying a gradient run with acolumn wash (Mortier et al., 2005b).

The most used ionization source was ESI operating inpositive ion mode. Other techniques described for paclitaxelquantification in human serum or plasma are APCI (Mortieret al., 2004 Schellen et al., 2000) and sonic spray ionization(SSI) (Green et al., 2006). Some authors reported that thecomposition of the mobile phase can modify the response ofall the approved taxanes (Alexander et al., 2003; Wang et al.,2003; Stokvis et al., 2004a; Guitton et al., 2005; Kuppenset al., 2005; Gao et al., 2006; Vainchtein et al., 2006; Hendrikxet al., 2011; Kort et al., 2013a). Guitton et al. noticed that theacid modifier in the mobile phase could enhance the responseand that acetic acid provided more efficient ionization thanformic acid (Guitton et al., 2005). Moreover, they tested arapid gradient of acetonitrile with 0.1% acetic acid from 50%to 95%, observing an important decrease in the docetaxelresponse (Guitton et al., 2005). Stokvis et al. tested severalmobile phase additives in flow injection analysis experiments todetermine their influence on the MS response of paclitaxel andthey determined that an alkaline mobile phase could increase theresponse. They obtained the best results in terms of signal-to-noise ratio and peak width using an alkaline mobile phasecontaining 10mM ammonium hydroxide in combination with acolumn stable at high pH values (Stokvis et al., 2004b).Vainchtein et al. (Vainchtein et al., 2006) proved that the samewas true for paclitaxel metabolites: they observed the highestsignal-to-noise ratio using 10mM ammonium hydroxide in theeluent. The authors proposed that positive ionization inthe electrospray ion source in the presence of ammoniumhydroxide most likely results from ion–molecule reactionsbetween the analyte molecule and ammonium ions or collision-induced dissociation of ammonium adducts of the analyte underinfluence of the electrospray voltage as previously described byZhou et al. (Zhou & Cook, 2000). Even Kuppens et al. observedthat an alkaline mobile phase favoured the high MS responseand they used a mobile phase of methanol containing 10mMammonium hydroxide (7:3 v/v) (Kuppens et al., 2005).

To detect cabazitaxel and docetaxel, Kort et al. (2013a)tested an acidic eluent (0.1% formic acid) against 10mMammonium hydroxide, as aqueous phase, and they found a twoto threefold decrease in peak intensity for cabazitaxel. There-fore, ESI in positive mode combined with an alkaline eluentincreased the sensitivity of a basic compound like cabazitaxel,compared to the use of an acidic eluent. Gao et al. (2006)demonstrated that another way to improve paclitaxel anddocetaxel detection sensitivity is the adding of alkylamineadditives due to a mechanism of suppression of multiplemolecular ions originating from different cationization phenom-ena through preferential formation of a predominant alkylamineadduct ion, as shown in Figure 8. The response can be increasedby the adding of additives that limit the formation of adducts andAlexander et al. reported that an acidic mobile phase wasrequired to suppress formation of sodium adducts of paclitaxelin favour of the protonated parent ion (Alexander et al., 2003).The authors chose acetic acid instead of trifluoroacetic acid

& CROTTI ET AL.

because the latter caused a significant suppression of absolutesignal. Even if additives are mainly added to the mobile phasewith the aim of limiting adduct formation, some authors usedthem to promote the formation of adducts during the ionizationprocess (Zhang & Chen, 2008). In some cases, the response ofthe sodium adduct of taxanes was higher than the protonatedparent ion response and the authors selected the sodium adductas quantification ion (Zhang & Chen, 2008; Zhang et al., 2011;Navarrete et al., 2013). The common denominator of thesemethods is the linearity in a wide calibration range, featureuseful to avoid the sample dilution before the treatment.

C. Epothilones

Epothilones have emerged as a promising new anticancercompounds belonging to the microtubule stabilizing antimitoticagents class, a series of anti-neoplastic molecules witha common mechanism of action involving tubulin binding(paclitaxel is the lead compound of this class). The interesttoward these compounds arose from the necessity to overcomesome problems related to taxane-based therapy, such as thedifficulty with formulation and administration and susceptibilityto resistance conferred by the drug efflux pump P-glycoprotein

FIGURE 8. Mass spectra of paclitaxel with (A) and without (B) octylamine showing the suppression of multiplemolecular ions through preferential formation of a predominant alkylamine adduct ion. Reproduced with thepermission of (Gao et al., 2006).

(P-gp) (Goodin et al., 2004). Indeed, epothilones bind to andstabilize microtubules in a manner similar but not identical tothat of paclitaxel and are effective in paclitaxel-resistant tumourmodels (Goodin et al., 2004; Heinz et al., 2005; Forli, 2014).Epothilones were discovered as cytotoxic agents from amyxobacterium, Sorangium cellulosum, isolated in 1985 fromsoil of the bank of the Zambesi River in southern Africa (Gerthet al., 1996). Six natural epothilones (A–F), and synthetic andsemisynthetic analogs are currently in various stages of clinicaldevelopment and only ixabepilone (IXEMPRA), a semisynthet-ic analogue of epothilone B, was approved in 2007 by FDA forbreast cancer (Brunton et al., 2011). Ixabepilone presents achemically modified lactam substitution of the naturally existinglactone. Indeed, the epothilones showed a lower activity in vivothan that obtained from in vitro test due to the instability of theirlactone ring: thus, the lactone oxygen in the epothilone B wassubstituted with a nitrogen (Fig. 9), yielding to a new compound,ixabepilone, not susceptible to esterases (Brunton et al., 2011).

Ixabepilone binds directly to b-tubulin subunits on micro-tubules, thus blocking cells in the mitotic phase of the celldivision cycle, leading to cell death (Goodin et al., 2004; Heinzet al., 2005). Moreover, ixabepilone possesses low in vitrosusceptibility to multiple tumour resistance mechanisms includ-ing efflux transporters, such P-glycoprotein (Brunton et al.,2011).

The recommended dosage of IXEMPRA is 40mg/m2

administered intravenously over 3-hr every 3 weeks. It isindicated in patients with metastatic breast cancer resistant orpre-treated with anthracyclines and resistant to taxanes. Ixabepi-lone is also indicated as monotherapy in metastatic breast cancerpatients who have previously progressed through treatment withanthracyclines, taxanes and capecitabine (Brunton et al., 2011).The PK of ixabepilone was linear at doses of 15–57mg/m2.Following administration of a single 40mg/m2 dose of IXEM-PRA in patients with cancer, the mean Cmax (typically occurredat the end of the 3-hr i.v. infusion) was 252 ng/mL (coefficient ofvariation, CV 56%) and the mean AUC was 2143 ng " hr/mL(CV 48%). In vitro, the binding of ixabepilone to human serumproteins ranged from 67 to 77%. Ixabepilone is extensivelymetabolized by hepatic cytochromes P450 and more than 30metabolites are excreted into human urine and faeces (Brunton

et al., 2011). No single metabolite accounted for more than 6%of the administered dose and the biotransformation productswere not active when tested for in vitro cytotoxicity against ahuman tumour cell line. Ixabepilone has a terminal eliminationhalf-life of approximately 52 hr.

At our knowledge, the first reported applications of massspectrometry to this anticancer drug was related to two humanmass balance studies (Beumer et al., 2007; Comezoglu et al.,2009). Human urine, faeces and plasma samples were analyzedby LC-AMS (Accelerator Mass Spectrometry) to determine thebiotransformation profiles of [14C]ixabepilone. AMS permitsthe measurement of elemental isotopes at the individual atomlevel (Garner, 2000). The principle behind AMS is the separa-tion of individual positively charged atoms through mass, chargeand momentum differences. In order to obtain the high-energycharge state required for separation, negative atoms are acceler-ated through a high voltage field (up to 10 million volts)generated by a tandem Van de Graaff accelerator. For 14C, AMScounts the number of individual atoms rather than measuringradioactive decays.

The mass balance is an elaborated pharmacokinetic investi-gation employing a radioactive tracer. The typical decaycounting method used for 14C detection, that is, liquid scintilla-tion counting, could not be conducted due to instability (auto-radiolysis) of [14C]ixabepilone at the specific activity requiredfor the conduct of such study (100mCi of [14C]). Since [14C]ixabepilone was sufficiently stable at low specific activity (1-2nCi/mg), the AMS was applied instead of the more commoncounting method. These studies showed how ixabepilone iscleared via urinary and fecal excretion through a variety ofmetabolic and degradative processes (Beumer et al., 2007;Comezoglu et al., 2009). Both studies presented as additionalaim the quantification of ixabepilone in plasma and urinesamples (Beumer et al., 2007) and the characterisation of thedrug and its metabolites in plasma, urine and faeces samples(Comezoglu et al., 2009) using conventional mass spectrometrymethods. In the first case (Beumer et al., 2007), the applicationof a validated method is reported but no reference for a completedescription of the analytical method is provided. However, abrief description of the method is presented: the samples wereprecipitated with acetone, the supernatant was further extractedwith 1-chlorobutane and the organic layer was evaporated todryness, reconstituted and then injected into the LC-MS.Chromatographic separation was achieved isocratically using asmobile phase acetonitrile-0.01M ammonium acetate (pH 5.0)(65:35). The standard curve ranged from 2 to 500 ng/mL andBMS-212188, a structural analogue of ixabepilone, was used asinternal standard. Eight patients receiving 70mg of [14C]ixabepilone containing 80 nCi of radioactivity as 3-hr i.v.infusion were enrolled in this single-dose study.

Afterwards, Comezoglu et al. (2009), parallel to thedetermination of the biotransformation profiles of ixabepiloneusing LC-AMS, tried to characterize the drug metabolites usingLC-MS/MS method. In this study, plasma samples collected at 4and 8 hr after the end of the infusion from eight patients werepulled across subjects and 1mL of this pooled plasma was addedwith 2mL of acetonitrile to precipitate proteins. After beingvortexed and centrifuged, the supernatant was removed and theresidue was washed with 1mL of acetonitrile:water (2:1, v/v).The washing and the original supernatant were combined,concentrate to near dryness, and reconstituted in 300mL of

FIGURE 9. Chemical structures of epothilone B and its analogueixabepilone.

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0.005% aqueous acetic acid:ethanol (8:2, v/v) for the analysis.Ixabepilone was separated from its degradants (degradation ofixabepilone involves an intramolecular cyclization to give anoxazine compound, that is subsequently hydrolyzed to a diolderivative of ixabepilone or isomerizes) with a chromatographicgradient of the mobile phases consisted of 0.05% acetic acid inwater and 0.05% acetic acid in acetonitrile. The mass spectrom-eter operated in ESI conditions (positive ion mode). Theyobserved in plasma samples (as well as in urine and fecalsamples) the three degradants of ixabepilone (above described)together with other oxidation products that could be derivedfrom the degradants or ixabepilone itself.

More recently, a full development and validation ofanalytical method for the determination of ixabepilone in humanplasma samples has been published (Xu et al., 2010). In thiscase, the sample preparation was based on simple PP with a120 ng/mL working internal standard (BMS-212188) solutionprepared in acetonitrile. The chromatography was conductedunder isocratic conditions and the mobile phase consisted ofacetonitrile:10mM ammonium acetate, pH 5.0 (70:30, v/v). Themethod is very fast with a total run time of 2.5min. The massspectrometer operated in negative ion mode, using multiplereaction monitoring (MRM). Indeed, contrary to what isexpected for these kind of molecules and what is reported in theprevious method (Comezoglu et al., 2009), both ixabepilone andBMS-212188 demonstrated good ionization efficiency in nega-tive ion mode. The precursor-to-product transitions followedwere: 505> 405 and 492> 392m/z for ixabepilone and theinternal standard, respectively. A nine-point calibration curveranging from 2 to 500 ng/mL of ixabepilone was used induplicate in each analytical run, one at the beginning and theother at the end of the run. However, they observed a curvesplitting, since the responses of the first curve did not match thatof the second curve. They hypothesized the source of thisdiscrepancy may involve an adsorption of ixabepilone tosurfaces of the LC-MS, systems due to the interaction of theunique closed ring structure of the drug with metal ions of theinstrument. To overcome this problem they proposed a pre-treatment of the source through the infusion of a solutioncontaining 1mg/mL of the two analytes for at least 10minimmediately prior to starting the analytical run, in order toreduce potential active sites on the surfaces. The validation ofthe method was conducted according to the FDA guidance.Moreover, a cross-validation was conducted using QC samplesprepared at two different laboratories and no significant differ-ence between the data generated by this procedure wasobserved. The method was applied to a later phase and life-cyclemanagement clinical studies and the concentration-versus-timeprofile of ixabepilone of a patient that received 40mg/m2 of thedrug as a continuous 3-hr i.v. infusion was reported. Finally,they proposed some modifications in the chromatographicconditions in order to separate ixabepilone from its degradants(Comezoglu et al., 2009) and, so, to determine ixabepilone afteroral administration. A representative chromatogram of a patientsample dosed with the oral formulation, reported in Figure 10,showed the presence of a degradant (BMS-590113).

D. Camptothecin Analogs

The camptothecins are potent, cytotoxic antineoplastic agentsthat target the nuclear enzyme topoisomerase I (TOP1).

Camptothecin (CPT), the lead compound of this class, was firstisolated from the bark of the Chinese tree, Camptothecaacuminata (Wall &Wani, 1995). Camptothecin carboxylate wastested clinically in the mid 1970s and showed anticanceractivity, but was discontinued because of its severe andunpredictable toxicity, principally myelosuppression and hem-orrhagic cystitis (Brunton et al., 2011). After the discovery thatTOP1 was the cellular target of this drug, more soluble and lesstoxic analogs of CPT, topotecan (TPT) and irinotecan (CPT-11),were successfully developed. They have activity in colorectal,ovarian, and small cell lung cancer (Brunton et al., 2011).Several aspects make the camptothecins pharmacologicallyunique. Indeed, TOP1 is their only target, as it has been shownusing yeast cells, which become totally resistant to CPT whenthe TOP1 gene is removed (Wall & Wani, 1995). Moreover, allcamptothecins have a fused five-ring backbone that includes alabile lactone ring (at physiological pH): the hydroxyl group andS-conformation of the chiral center in the lactone ring arerequired for biological activity (Brunton et al., 2011). Thecamptothecins bind to and stabilize the normally transient DNA-topoisomerase I cleavable complex, leading to the accumulationof single-strand breaks in DNA. The collision of a DNA

FIGURE 10. Chromatogram of a patient sample dosed with the oralformulation of ixabepilone. Reproduced with permission from Xu et al.(2010).

replication fork with this cleaved strand of DNA causes anirreversible double-strand DNA break, ultimately leading to celldeath (Brunton et al., 2011). Since the cytotoxic activity ofcamptothecins depends on cellular cycle and is more pro-nounced during S phase, a sufficient exposure of tumour cells todrug concentrations above a minimum threshold is necessary forthe implementation of the cytotoxic activity of this agents(Lorusso et al., 2010; Brunton et al., 2011).

1. Topotecan

TPT is a semisynthetic camptothecin analogue with a basicdimethylamino group that increases its water solubility(Fig. 11). It is approved for intravenous administration and it isindicated for previously treated patients with ovarian and smallcell lung cancer. The dose limiting toxicity with all dosingschedules is neutropenia, with or without thrombocytopenia,and this significant haematological toxicity has limited its use incombination with other active agents in the diseases abovereported (Brunton et al., 2011). The recommended topotecandosing regimen for the ovarian cancer and small cell lung canceris a 30min i.v. infusion of 1.5mg/m2/day for 5 consecutive daysevery 3 weeks. In combination with cisplatin, the topotecan dosefor cervical cancer is 0.75mg/m2 on days 1–3, repeated for21 days (Brunton et al., 2011). TPT exhibits a linear pharmaco-kinetics, and it is rapidly eliminated from systemic circulation(it primarily undergoes renal excretion) (Lorusso et al., 2010;Brunton et al., 2011). Only 20–30% of the total drug in plasmais found to be in the active lactone form. Indeed, at pH $4,the lactone is exclusively present, whereas the ring-openedhydroxyl-acid form predominates at physiologic pH. Thebiological t1/2 of total TPT, which ranges from 3.5–4.1 hr, isrelatively short compared to that of other camptothecins. Plasmaprotein binding of TPT is low (32–35%), but the volume ofdistribution is high (%20 L/m2) both in animal models and inhuman (mean 132 L/m2), indicating a good tissue penetration(Lorusso et al., 2010). The primary hepatic metabolite oftopotecan is N-desmethyl topotecan, and the mean ratio ofmetabolite to parent AUC was about 3% for total TPT and TPTlactone following intravenous administration.

The analytical methods for topotecan quantification inhuman plasma have involved liquid chromatography almostalways with fluorescence detection (Rosing et al., 1995; Rosinget al., 1999; Bai et al., 2003; de Vries et al., 2007; Vali et al.,2005) with only two procedures based on mass spectrometry(Baczek et al., 2012; Li et al., 2013). However, mass spectrome-try was successfully applied also for the determination of TPT inother matrices such as: rat serum (Shin et al., 2009), rat evertedgut sacs (Arellano et al., 2010), and beagle dog plasma (Ye et al.,

2013). To the best of our knowledge Baczeck et al. (2012) wasthe first to apply mass spectrometry detection for the determina-tion of topotecan in human plasma. This methods is based on thesimple deproteinization of 100mL of plasma with 100mL ofmethanol and 100mL of 0.2M ammonium formate buffer(pH¼ 3). The acidic condition allows keeping the drug in thelactone form, in order to quantify total topotecan (as lactoneform). The supernatant was directly injected into the LC systemand data were acquired in single ion monitoring mode to detectthe characteristic pseudomolecular [MþH]þ ion of the com-pound (monoisotopic mass 422m/z). The spectrometer operatedin ESI conditions (positive ion mode). The total time of a singledetermination procedure, including equilibration of the column,was 20min. The validation of this method was conducted inaccordance with the guidelines of the Food and Drug Adminis-tration. The standard curve resulted linear (r2¼ 0.999) over theconcentration range 1–150 ng/mL and the peak height was usedto calculate the topotecan concentration, so no internal standardwas used. The plasma stability of topotecan was solely assessedanalysing spiked plasma samples at three concentration levels(5, 25, and 100 ng/mL) immediately after the preparation andthen again after 6 hr in the refrigerator (4–8˚C). The reportedmethod was applied to a clinical study aimed at the determina-tion of topotecan exposure in paediatric acute myeloid leukae-mia (AML) patients. Indeed, in the case of combining topotecanwith cladribine for recurrent/refractory paediatric AML thereare recommendations to determine the appropriate dose oftopotecan in daily administration for 5 days in order to obtainthe targeted single day systemic exposure of 140' 20 ng/mL hrof topotecan in plasma and avoiding the dose-limiting toxicity(Inaba et al., 2010). The study shows how the pharmacokineticdata obtained over the 5-days course treatment with topotecanfrom one patient allowed the establishment of the appropriatedose of the drug and resulted in achieving the final AUC value of140' 20 ng/mL hr.

An UHPLC-tandem mass spectrometry method was devel-oped by Li et al. (2013) for the quantification of both totaltopotecan and topotecan in the lactone form. Also in this case, asimple PP step with cold methanol ((20˚C, 300mL) was usedfor sample pre-treatment (100mL of plasma). The supernatantwere directly injected onto the analytical column for the analysisof the lactone form while, prior to quantifying total topotecanconcentration, the supernatant were acidified with 5% formicacid (pH 1.73) to fully convert the carboxylate form to thelactone form. The spectrometer operated with ESI source inthe positive ion mode and multiple reaction monitoring. Theanalytes were detected by following the transitions 422> 377and 440> 331m/z corresponding to the loss of the amino-alkylside chain (-N(CH3)2). The mass spectrum of topotecan wasreported also in the previously mentioned method, showing thesame molecular ion fragmentation. The total time of a singledetermination procedure was 5min. The study analyzed top-otecan over different pH conditions: it was stable as the lactoneform at low pH (pH $4) while only the carboxylate peak wasobserved at pH 10. At pH 8, the two forms coexisted. Theauthors reported the attempt to use 4S-9-Morpholinomethyl-10-hydroxycamptothecin acetate salt, an analogue of topotecanlactone form, as IS. However, this analogue compound seemedto be not stable and, since there was no satisfactory alternativeIS available, topotecan concentration was calculated using peakintensity. The method was validated with respect to selectivity,FIGURE 11. Topotecan structure.

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extraction recovery, matrix effect, linearity, intra and inter-day precision, accuracy, and stability, according to FDA guide-lines. Acceptable linearity was achieved in the range of 0.5–100 ng/mL and 0.1–50 ng/mL for total topotecan and topotecanin the lactone form, respectively. The r2 value was greater than0.995 in all validation batches and the signal-to-noise ratios atthe LLOQ concentration level (0.5 and 0.1 ng/mL for totaltopotecan and topotecan in the lactone form, respectively) wasgreater than 20. Unlike the previous method, in this case a fullystability assessment of topotecan in plasma was performed,covering the expected handling conditions of clinical samplesunder a variety of storage and processing conditions: at roomtemperature for 4 hr, into the autosampler (4˚C) for 24 hr andstored at (80˚C for 3 months. Also, the stability of topotecan instock solution was assessed at(20˚C for 3 months. This methodwas developed in order to determine the absolute bioavailabilityand pharmacokinetics of a new oral topotecan formulation(Xinze1) in patients with small-cell lung cancer (SCLC). Themean plasma concentration-versus-time curves of total top-otecan and topotecan in the lactone form determined from fivepatients with SCLC, treated with topotecan orally and byintravenous administration, were also reported.

2. Irinotecan

CPT-11 differs from TPT in that it is a prodrug. Indeed, it isactivated by the enzyme carboxylesterase to SN-38 (Mathijssenet al., 2001) that, compared with the parent drug, is 100- to1000-times more cytotoxic (Newton et al., 2012). As reported inFigure 12, SN-38 is formed from irinotecan by carboxylester-ase-mediated cleavage of the carbamate bond between thecamptothecin moiety and the dipiperidino side chain. Bothirinotecan and SN-38 exist in an active lactone form and aninactive hydroxy acid anion form. A pH-dependent equilibriumexists between the two forms such that an acid pH promotes theformation of the lactone, while a more basic pH favours thehydroxy acid anion form. After formation, SN-38 can be further

inactivated by conjugation with glucuronic acid by forming theSN-38 glucuronide (SN-38G) through an enzymatic reactionmediated by the UDP-glucuronosyl transferase 1A1 isoform(UGT1A1) (Mathijssen et al., 2001). Moreover, oxidativemetabolites (for instance APC and NPC) have been identified inplasma, all of which result from CYP3A-mediated reactionsdirected at the bispiperidine side chain (Fig. 13). Biliaryexcretion appears to be the primary elimination route of CPT-11,SN-38 and its metabolites. CPT-11 exhibits a linear pharmacoki-netics. After intravenous infusion, CPT-11 plasma concentra-tions decline in a multiexponential manner, with a meanterminal elimination half-life of about 6–12 hr while the meanterminal elimination half-life of the active metabolite SN-38 isabout 10–20 hr. The AUC of SN-38 is only 4% of the AUC ofCPT-11, suggesting that only a relatively small fraction of thedose is ultimately converted to the active form of the drug(Brunton et al., 2011). Monitoring of total (lactone andcarboxylate forms) CPT-11 and total SN-38 has essentially thesame clinical significance as the monitoring of lactone forms ofthe two agents, because the pharmacokinetics of total CPT-11and total SN-38 are significantly correlated with those of lactoneirinotecan and lactone SN-38, respectively (Ragot et al., 1999).CPT-11 and SN-38 have differential binding affinity for severalplasma proteins: indeed, from 30% to 68% of CPT-11 is boundto plasma protein (predominantly albumin) while SN-38 ishighly bound (approximately 95% bound). Currently, CPT-11 is

FIGURE 13. Chemical structures of irinotecan and its main metabolites. CPT11: (7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin); SN-38: 7-ethyl-10-hydroxycamptothecin; SN-38G: SN-38 glucuro-nide; APC: 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin; NPC: 7-ethyl-10-(4-amino-1-piperidino) carbonyloxy-camptothecine.

FIGURE 12. Metabolic pathway of irinotecan (CPT-11).

mainly used in colorectal cancer (CRC) diagnosed patients withmetastases, with recorded relapse or progression after applica-tion of standard 5-fluorouracil (5-FU)-based therapy (Wu &Yeo, 2011). In patients with advanced CRC CPT-11 is used asfirst line therapy in combination with other chemotherapeutic(i.e., FOLFIRI: infusional 5-fluoruracil, leucovorin, and irinote-can) or biological (i.e., cetuximab and bevacizumab) agents(Brunton et al., 2011).

It has also been proposed the use of SN-38 as directanticancer drug, but its development has been largely hinderedby the poor solubility and stability of SN-38 in pharmaceuticallyacceptable solvents (Pal et al., 2005). For this reason, aliposome-based SN-38 formulation (LE-SN38) was developedin order to overcome the aforementioned problems, and toprovide a mechanism of direct delivery of SN-38 without in vivoenzymatic conversion. To support preclinical trials an LC-MS/MS method was developed to determine the total SN-38concentration in human plasma spiked with LE-SN38 (Khanet al., 2003). Anyway, several methods have been developed forthe determination of CPT-11 and its metabolites in humanplasma, most of them based on the use of fluorescence detectors,as reviewed by Mullangi et al. (2010) and, more recently, byChen et al. (2012). Among them, several methods have beendescribed for the simultaneous determination of the lactone andcarboxylate forms. In these methods extraction and chromatog-raphy were generally performed at the pH of equilibrium (pH5.5) between the two forms, condition that may not allow thecomplete cleavage and extraction of the albumin-bound carbox-ylate form. In other studies, the carboxylate form concentrationwas deduced from the difference between total and lactone formconcentration. In this case, the instability of CPT-11 and SN-38at room temperature requires rapid freezing of clinical samplesafter blood collection to prevent significant degradation oflactone into carboxylate form. The determination of total CPT-11 and SN-38 concentration (conducing sample preparation andanalysis at pH <5) overrides this requirement. Indeed, amongthe analytical methods based on mass spectrometry listed inTable 4, almost all were developed for the determination of thetotal concentration of CPT-11 and its metabolites as lactoneform. Noteworthy, CPTwas used in all of these analytical assaysas the natural IS.

For instance, Ragot et al. (1999) developed an LC-MS/MSmethod for the quantification of CPT-11 and SN-38 based onsimple PP treatment of 200mL of human serum in acidicconditions (100mL of the supernatant was acidified with 20mLof sodium citrate buffer- 2.5M, pH 2) for CPT-11, while SN-38was extracted by a liquid–liquid procedure and the dry extractswere redissolved in 50mL of a mixture of acetonitrile- 5mMammonium formate (pH 3). This extraction procedures, as wellas the mobile phases, were optimized in order to transform thecarboxylic into lactone form and maintain the total amount ofCPT-11 and SN-38 under this latter form through the chro-matographic separation. This was verified by the absence of anychromatographic peak corresponding to the carboxylate form.Anyway, this first example is quite complicate because theyassessed two different methods for the preparation and theanalysis of each compounds. Although FDA guidance was notjet published, this method was validated by examining recovery,inter- and intra-day precision and linearity with the same criteriarequired by the FDA guidelines, while it lacks in matrix effect,accuracy and stability assessment. Acceptable linearity was

achieved in the range of 0.5–100 ng/mL and 10–10000 ng/mLfor SN-38 and total CPT-11, respectively. The suitability forclinical samples was demonstrated by the determination of CPT-11 and its metabolite in a blood sample obtained from a 31-year-old male patient treated with 350mg/m2 of CPT-11 and thechromatograms of CPT-11 and SN-38 were showed (serumsample taken 14-hr after i.v. infusion).

Sai et al. (2002) developed an LC method coupled withfluorescence and mass spectrometric parallel detection of CPT-11, SN-38, SN-38G, and APC, as well as other minormetabolites including NPC, demethylated (M1), hydroxylated(M2) and dehydrogenated (M3) metabolites. This method ischaracterized by a protein extraction of the analytes from humanplasma with perchloric acid/methanol; quantification of majormetabolites (SN-38G, SN-38 and APC) with a fluorescencedetector (FLD), where the limits of quantitation by FLD were2.5 ng/mL for SN-38G and APC, 5 ng/mL for CPT-11, and 1 ng/mL for SN-38, respectively and parallel selective monitoring ofthe metabolites including minor metabolites with a massselected detector. In case of mass spectrometry detection, rangesof linearity were 10-800 for SN-38G and APC, 10-4000 forCPT-11 and 2–80 ng/mL for SN-38. Despite they obtained alower LLOQ in case of FLD, UV or fluorescent detection maysometimes suffer from interfering compounds when samplesfrom patients treated with multiple drugs are analyzed. Indeed,this method was not applied directly for the analysis of patientplasma samples but for the analysis of CPT-11 metabolitesproduced in human liver microsomes. This group reported theuse of commercially supplied reference standard for SN-38G.Anyway, SN-38G reference compound is not easy to findcommercially available and many alternatives have beenproposed: standard custom-synthesized by the analytical labora-tory (Chen et al., 2012), biosynthesis of SN-38G from SN-38precursor and the uridine-diphosphate (UDP) glucuronic acidsubstrate through the action of the UDP-glucuronosyl transfer-ase 1A1 isoform enzyme (Corona et al., 2010), and hydrolysis ofSN-38G in plasma by b-glucuronidase (Zhang et al., 2009). Inthis latter case, the quantification of SN-38G was obtainedthrough the increase of SN-38 after treatment. This LC-MS/MSmethod was developed for the quantification of CPT-11, SN-38and SN-38G (without the use of SN-38G reference standard) inhuman plasma samples. Sample preparation is based on simplePP treatment of 100mL of plasma with 400mL of acetonitrile:methanol (2:1), the supernatant was then evaporated to drynessand dissolved in 100mL of acetonitrile: water (12:88). Notewor-thy, no mention was done about the equilibrium between thecarboxylate and lactone form and the sample treatment wasconducted without acidic condition. The validation of themethod was conducted through the assessment of: specificity,linearity of calibration curves (CPT-11 and SN-38 curves werelinear over the range of 10–2000 and 0.5–200 ng/mL, respec-tively), LLOQ, precision and accuracy, matrix effect, recoveryand stability. Application to clinical samples was done by theanalysis of blood samples obtained from a metastatic CRCpatient after intravenous administration of 180mg/m2 of CPT-11. The plasma concentration of CPT-11, SN-38 and SN-38Gwere reported for samples obtained at 0.5, 2.5, and 4.5 hr afterdrug administration.

Corona et al. (2010) developed an LC-MS/MS method forthe determination of CPT-11, SN-38, SN-38G, APC and NPC inhuman plasma. SN-38G was obtained by biosynthesis as

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described above, and the concentration of the resulting SN-38Gstock solution was assessed by measuring the molar content ofthe SN-38 resulting from the complete enzymatic conversion ofSN-38G through the b-glucuronidase treatment. Sample treat-ment required simple PP of 100mL of human plasma with200mL of acetonitrile. Subsequently, 100mL of supernatantwere diluted with two volumes of an aqueous solution contain-ing 1% formic acid. CPT-11 and its main metabolites wereseparated under gradient chromatography with water containing0.01% formic acid and acetonitrile as mobile phases within6min. The analysis was performed in MRM using ESI source;quantification was performed with the following MRM transi-tions: m/z 587> 167 for CPT-11, m/z 393> 349 for SN-38, m/z569> 393 for SN-38G, m/z 619> 393 for APC, and m/z519> 393 for NPC. Noteworthy, these transitions, the samereported in the previous paper for CPT-11, SN-38, and SN-38G,represent the common fragmentation pathways. For a detaileddescription of the principal fragmentation patterns related tothese analytes see D’Esposito et al. (2008), Corona et al. (2010),Marangon et al. (2015). The validation process was conductedin accordance with the FDA guidelines and linearity wasachieved in the range of 0.2–200 ng/mL for NPC and SN-38,0.5–500 ng/mL for SN-38G and APC and 2-2000 for CPT-11.To test the applicability of this method, they monitored CPT-11and its main metabolites in 14 plasma samples obtained from anadult male CRC patient receiving 180mg/m2 of CPT-11 (2-hri.v. infusion) in FOLFIRI regimen. Plasma samples wereobtained immediately before and at 0.5, 1.0, 1.75, 2.0, 2.5, 3.0,4.0, 6.0, 8.0, 10.0, 14.0, 26.0, and 50.0 hr after the start of theinfusion. The PK profiles of CPT-11 and its metabolites showedthe typical biphasic decay pattern with a fast initial eliminationrate followed by a slow elimination process. Moreover, theplasma AUC of the SN-38 active metabolite was approximately45-fold lower than that of the parent drug.

The concentration ranges of calibration curves of CPT-11,SN-38, SN-38G, and APC were modified in a subsequentmethod, developed in the same laboratory, in order to make theanalytical assay more suitable for a dose escalation phase Iclinical study (Marangon et al., 2015). This LC-MS/MSmethod,validated according to FDA and EMA guidelines, showedlinearity in the range of 10-10000 for CPT-11, 1-500 for SN-38and SN-38G, and 1-5000 for APC. In addition, in order toquantify irinotecan and its main metabolites on pluri-treatedpatients’ plasma, more than one transition for each analyte werefollowed (one used as quantified and two as qualifier ions).Moreover, the reference standard of SN-38G was used, avoidingany interferences related to the enzymatic conversion of SN-38.Sample treatment (100mL of human plasma) was based on PPtreatment with methanol in acidic condition (0.1% of aceticacid). This method was applied to a pharmacokinetic study inpatients with metastatic CRC enrolled in a genotype-guidedphase I study of high-dose CPT-11 (260–370mg/m2) adminis-tered in FOLFIRI plus bevacizumab regimen. Patients receivedCPT-11 as a 120min i.v. infusion on days 1 and 15 of a 28-daytreatment cycle and blood samples were collected on days 1–3and 15–17 of the first cycle of treatment (before drug administra-tion, and at 1.0, 2.0, 2.25, 2.50, 3.0, 4.0, 6.0, 8.0, 10.0, 14.0,26.0, 50.0 hr following the start of the irinotecan infusion). Alsoin this case, the pharmacokinetic profiles appeared to decline ina bi-exponential manner, with a rapid initial phase and anextended terminal phase.

Even if most of the analytical methods reported consistedin sample treatment based on protein precipitation, moresuitable for clinical application of the analytical method, someprocedures required solid phase extraction. For instance,the LC-MS/MS method developed by D’Esposito et al. (2008)for the determination of CPT-11, SN-38 and APC involves asample treatment based on SPE conducted in acidic condition.The validated method was applied to investigate CPT-11biotransformation in microsomal fractions from healthy individ-uals and patients with liver disease. Indeed, the ULOQ of 25 ng/mL in plasma for both CPT-11 and APC is too low to describethe pharmacokinetic curve of these compounds in patientsreceiving 125mg/m2 or more (depending on the therapyschemes) of CPT-11. More recently, Chen at al. (2012)developed and validated, according to the FDA guidance, anUHPLC-MS/MS based on SPE, conducted in acidic condition,for the quantification of CPT-11, SN-38 and SN-38G (customsynthesized). This method involved the use of three differentinternal standards: irinotecan-d10 for CPT-11, tolbutamide forSN38 and CPT for SN-38G. The calculated detector response ofthe irinotecan/irinotecan-d10, SN38/tolbutamide and SN-38G/camptothecin ratio versus the nominal concentration displayed alinear relationship in the range of 0.5–100 ng/mL for SN-38 andSN-38G and 5–1000 ng/mL for CPT-11. The validated methodwas applied to study the clinical PK on patients in a phase Iclinical trial of irinotecan formulated into drug-eluting beads fortrans-arterial hepatic chemoembolization. Plasma concentrationversus time curves for each analyte up to 78 hr post dose inpatients were reported and CPT-11 demonstrated an approxi-mately 10-fold higher maximum plasma concentration com-pared to SN38 and its glucuronidated metabolite.

E. Cytotoxic antibiotics

Each antibiotic presented in this section has its unique mecha-nism of action based on indirect DNA damage. The currentclassification is based on their microbial origin.

1. Chromomycins

This class is constituted exclusively by dactinomycin, also knowas actinomycin-D, an antibiotic discovered in 1940. The termchromomycin come from the planar phenoxazone derivativewhich give the characteristic yellow-red color (Fig. 14). Thisplanar structure is responsible for the pharmacological effects,possessing an intercalating properties between adjacent gua-nine-cytosine base pairs. The strong dactinomycin-DNA com-plex causes the block of RNA polymerase activity: as a result theinhibition of rapidly proliferating cells occurs. This antibiotic isimportant for the treatment of most of paediatric tumors such as:Wilms’ and Edwings’ tumors, the gestational trophoblasticneoplasia, and rhabdomyosarcoma (Brunton et al., 2011).Dactinomycin is administered by i.v. injection at 10–15mg/kgfor five days. Up to now, no metabolites of actinomycin D havebeen detected in vitro or in vivo and it is excreted intact mainlythrough bile and urines. The drug half-life is 36 hr (Benjaminet al., 1976) and toxicity onset few hours after administration,with hematopoietic suppression and pancytopenia; concomitantdermatological manifestation include erythema, desquamationand alopecia. Actinomycin D is easily ionizable by ESI-MS,with the proton located preferentially on the primary amine of

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phenoxazone. The semisynthetic derivative 7-amino-actinomy-cin-D is commonly used as internal standard and the monitoredtransitions were: m/z 1256> 858 for actinomycin and m/z1271> 873 for the internal standard 7-amino-actinomycin-D(Skolnik et al., 2006). The product ions for both actinomycin-Dand its IS derived from the breakdown of cyclopeptide ring andthe loss of H-Val-Pro-Sar-Meval-OH moiety (Barber et al.,1988). The first LC-MS pharmacokinetic study in two paediatricpatients monitored the drug profile after a single bolusadministration (Veal et al., 2003). This method reached adiscrete sensitivity (LLOQ 1 ng/mL, linearity range 1–100 ng/mL) which ensured the actinomycin-D quantification in plasmaover 48 hr. By this method, a total of 117 paediatric patientshave been monitored in the first 24 h after drug administration(Veal et al., 2005; Hill et al., 2014). In two paediatric patients,actinomycin D has been monitored (Skolnik et al., 2006; Leeet al., 2007a) in a combination therapy with vincristine. The LC-MS/MS method proposed by Skolonik et al. has been furtherameliorated to obtain a higher sensitivity (LLOQ 0.05 ng/mL;linearity range 0.05–25 ng/mL) and fully validated following theFDA guidelines (Lee et al., 2007a) before performing a PKstudy in a treated patient over 168 hr after drug administration(Lee et al., 2007b). By this method both drugs were extracted bySPE cartridge and separated by a C18 column in presence of5mM of ammonium acetate (pH<3.5); ESI ionization wasperformed with the temperature of the heated nebulizer set at450˚C, in order to obtain the complete desolvatation of ions,which ensured the highest sensitivity. As multidrug therapyactinomycin-D and vincristine are used for the treatment ofsome childhood solid tumors such as Wilms’ tumor andrhabdomyosarcoma and their simultaneous quantification isessential for determining the pharmacokinetics parameters inpaediatric patients. For this reason, several methods faced thesimultaneous quantification of both drugs have been developed(Skolnik et al., 2006; Lee et al., 2007b; Damen et al., 2009c,d).More recently, an interesting application of dried blood spot(DBS) sampling to actinomycin-D and vincristine LC-MS/MSanalysis has been published by (Damen et al., 2009b) which leadto perform PK studies in 40mL of blood sample after a fastextraction (15min) in a sonic bath with a solution of acetoni-trile–methanol–water (1:1:1, v/v/v).

2. Anthracyclines

This class contains several active molecules characterized by thepresence of a tetracyclic ring structure linked to a unit of

daunosamine, an uncommon sugar. Anthracycline antibiotics(Fig. 15) were isolated from Streptomyces peucetius in 1958 andthe main molecules in this class are: doxorubicin (DOX),daunobicin, epirubicin, and idarubicin. Idarubicin, a semisyn-thetic analogue, is characterized by the absence of the methoxygroup in the anthracycline ring, which increases the compoundlipophilicity and ameliorates the cellular uptake.

Doxorubicin and epirubicin are used in human solidtumours therapy, whereas daunobicin and idarubicin displayhigher activity against acute leukemias (Brunton et al., 2011).Idarubicin and daunobicin are administered by i.v. injection for3 days at typical doses of 12mg/m2/day and 25–45mg/m2/dayrespectively; doxorubicin is administered as a single i.v. of 60–75mg/m3, repeated after 21 day. These agents are characterizedby several mechanisms of action, based both to direct DNAdamage (by DNA intercalation) both to indirect DNA damage(by free radical production and direct action against the enzymeTopoisomerase II). In all cases, DNA duplication and replicationis impeded, ultimately causing cell death. Acute and chronicadverse effects such as: myelosuppression, neutropenia, leuco-poenia, and cardiotoxicity have a strong dose-limiting conse-quence (Danesi et al., 2002). Anthracyclines exhibit fluorescentproperties, which make them suitable for methods based on LC-FLD (Fogli et al., 1999; Bellott et al., 2001). MS-based methodsreached comparable sensitivity, some of them managing theanalysis of several anthracyclines in a single run (Lachatre et al.,2000a). Anthracyclines are analyzed by ESI in the positive ionmode, which exhibit better sensitivity compared to APCI(Arnold et al., 2004).

The pharmacokinetic profile of traditional doxorubicin(DOX) formulation has been monitored in a treated patients upto 72 hr after i.v. infusion by operating with the in-sourcefragmentation mode (ISD) method developed by Lachatre et al.(2000b). This method has been validated for the quantificationof epirubicin, doxorubicin, daunorubicin and idarubicin in therange 2.5–2000 ng/mL and for the quantification of the three

FIGURE 14. Chemical structure of dactinomycin.

FIGURE 15. Chemical structure of the anthracycline antibiotics doxorubi-cin, daunobicin, epirubicin, and idarubicin.

metabolites doxorubicinol, daunorubicinol and idarubicinolin the range 2.5–200 ng/mL. Analytes were extracted bySPE from 0.5mL of serum and a simple isocratic separation on aC18 column was used to resolve four anthracyclines andthree reduced metabolites. Doxorubicin and its metabolitedoxorubicinol have been monitored in 23 patients by anotherLC-MS/MS method based on SPE extraction and separationonto C18 column (Di Francesco et al., 2007); the relatively highLLOQ of 7.2 ng/mL (linearity range 7.2–984 ng/mL and 3.04–104 ng/mL for doxorubicin and doxorubicinol, respectively)showed that approximately 50% of patients’ plasma levels werebelow the limit at 48 hr after drug administration. Even if othermethods report slighter lower LOD, no complete PK profile ofpatients treated has been reported (Lachatre et al., 2000a) or nohuman samples have been analyzed (Arnold et al., 2004).

Doxorubicin efficacy is limited when systemically adminis-tered and several formulations (e.g. liposomes, pegylated lip-osomes, microspheres loading, and peptides-conjugates) havebeen employed in order to improve its pharmacokinetic profile.The PK of pegylated liposomal doxorubicin shows a characteris-tic profile with extended half-life (20–30 hr) and a reducedvolume of distribution which give rise to an AUC at least 60-fold higher compared to traditional doxorubicin formulation(Gabizon et al., 2003). The release of doxorubicin in plasma byHepaSpere, a formulation used for transcatheter arterial chemo-embolization (TACE), has been investigated by MS (Sottaniet al., 2013b; Malagari et al., 2014). HepaSphere are drug-eluting beads used for local treatment of hepatocellular carcino-ma. Without HepaSpere, the drug release after administration ismassive, leading to higher plasma levels and increased systemictoxicity (Fig. 16). The drug-eluting beads avoid unwanted

doxorubicin loss in plasma during the chemoembolizationprocedure and ensure a slow release up to 48 hr (Malagari et al.,2014). In the paper of Sottani et al., both doxorubicin anddoxorubicinol can be detected up to seven days after treatmentfollowing the transitions m/z 544> 361, 397 and m/z 546> 363,397 respectively with an LLOQ of 1 ng/mL (Sottani et al.,2013a).

A peptide–doxorubicin conjugate has been proposed for thetreatment of prostate cancer (Di Paola et al., 2002). Desai et al.provided reliable data on the in vivo drug metabolism after thelocal cleavage of the peptide portion by prostate-specific antigen(PSA). Analyses were performed in 0.5mL of plasma treatedwith C8 SPE before chromatographic separation. Metaboliteshave been detected by a combined LC-MS/FLD methodworking with a 80:20 (FLD:MS) flow split (Desai et al., 2004)with simultaneous detection of two main active metabolites:doxorubicin and the partial clavered peptide, the leucine–doxorubicin. Further metabolites, such as: doxorubicinol, leu-cine–doxorubicinol, and the 7-deoxydoxorubicinol aglyconewere simultaneously detected also and quantified in MRM whita switched positive/negative ionization.

PK of liposomally encapsulated daunorubicin has beeninvestigated also (Guaglianone et al., 1994; Feldman et al.,2011). The method employed by Guaglianone et al. was an LC-FLD, which provides good separation of parent drug frommetabolites, but does not distinguish free daunorubicin fromthat still entrapped in the liposomal fraction. Conversely,Feldman et al. used a validated analytical methods based on LC-MS/MS for the quantification of total daunorubicin (bothencapsulated and non-encapsulated fraction) in plasma oftreated patients (Feldman et al., 2011). Even if the massspectrometry method was not clearly discussed, the assaysincluded an additional step for dissolving the liposome in orderto liberate the encapsulated daunorubicin.

Epirubicin has been monitored in plasma samples usingboth APCI (Wall et al., 2007) and ESI (Lachatre et al., 2000b)with equivalent sensitivity (LLOQ 1.0 ng/mL) whereas the firstone employed a fast liquid-liquid extraction procedure withisopropanol instead of SPE extraction proposed by Lachatreet al. The UHPLC-MS method developed by Li et al. showed aslight improved performances with an LLOQ of 0.5 ng/mL (Liet al., 2005), and this method was employed to quantify plasmalevels of epirubicin in three treated patients. The comparativePK of two different drug eluting beads (DC Bead1 andHepaSphere Microsphere1) pre-loaded with epirubicin for thetransarterial chemoembolization (TACE) has been performed(Sottani et al., 2012). Authors showed a persistent drug elutionfor both types of microparticles after a Cmax reached at 5minafter the injection. Authors faced the extensive binding ofepirubicin to the serum proteins treating the serum samples with2.0mL 0.1N hydrochloric acid before a fully validated LC-ESI-MS/MS assay procedure (Sottani et al., 2009).

3. Anthracenediones

Antracene-9,10 diones are molecules involved in severalbiological processes. Ametantrone is the lead compound of thisclass, while the most active compound is its 1,4 dihydroxyanalogue, the mitoxantrone (Cheng & Zee-Cheng, 1983).Mitoxantrone (MTX) structure is depicted in Figure 17. Itpossesses a similar mode of action of doxorubicin with less toxic

FIGURE 16. Transcatheter arterial chemoembolization (TACE) PK pro-files in patients receiving 50mg of conventional doxorubicin formulation(cTACE, light green line) and patients receiving 50mg of doxorubicin loadedonto drug-eluting beads (Hepasphere). Reproduced with permission fromMalagari et al. (2014).

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side effects and lower cardiotoxicity. It was shown to beeffective against numerous types of tumors: acute myeloidleukaemia, non-Hodgkin lymphoma, advanced breast andadvanced primary liver cancer. The typical administrationprotocol is 12–14mg/m2 once every 21 days in patients withlymphomas and solid tumors, while patients with leukaemiareceive 12mg/m2 per day for three days (Brunton et al., 2011).

The mechanism of action of antracenediones involvesinterstrand DNA cross-links through their aminoalkyl moietyintercalation (Skladanowski & Konopa, 2000; Rosu et al., 2006)or the formation of a covalent bind MTX–DNA after celloxidative metabolism (Panousis et al., 1994). A direct-infusionanalysis by negative ion ESI-MS of MTX in presence ofequimolar amount of duplex and tripled DNA strands highlight-ed its ability to form complex of 1:2 (duplex DNA:MTX) and1:1 (triplex DNA:MTX) binding stoichiometry (Wan et al.,2008). An increase of MTX concentration gives arise to abinding stoichiometry up to 1:4 (DNA:MTX) with duplex DNAstrand and up to 1:2 for triplex DNA strand. Beside the directDNA binding, mitoxantrone possesses strong activity as topo-isomerase II inhibitor also and its use is associated with thedevelopment of therapy related acute myeloid leukaemia (t-AML). MTX metabolism has been investigated only after i.v.

bolus because of its tissutal toxicity. Following the intravenousadministration, MTX is metabolized to the mono- and dicarbox-ylic acid derivatives, as well as glucuronide conjugates. Theposition of glucuronide conjugates has been investigated byMS/MS and its has been proposed to occurs both at the phenylenedi-amine moiety (Rossato et al., 2013) and at the hydroquinone(Blanz et al., 1991), but only this last has been unequivocallyrecognized by 13C-NMR (Blanz et al., 1991).

Mitoxantrone PK has been investigated by LC-ECD (Canalet al., 1993) and LC-UV/VIS (Ehninger et al., 1985; Richardet al., 1992). At the best of our knowledge, no reports on massspectrometry-based pharmacokinetic analysis of MTX in humanplasma samples have been presented. On the contrast, aninteresting method has been developed for MTX quantificationin rat plasma using palmatine as internal standard. The fast LC-MS/MS separation was achieved in a chromatographic run timeper sample of 2.5min using buffered methanol, instead ofacetonitrile, which ensured higher sensitivity. MTX was ionizedby ESI(þ) with the proton located preferably on the basicethylenediamine moiety and it was monitored using the transi-tion m/z 445> 88. Plasma samples were extracted by means of aLLE using 10mL of a saline/ascorbate solution and 50mL of a0.05M borax-sodium carbonate buffer (pH 10.8); by thismethod a linear range of 0.5–500 ng/mL and a lower limit ofquantification (LLOQ) of 0.5 ng/mL (Zhang et al., 2010) wereensured.

4. Bleomycins

Bleomycins are natural glycopeptide antibiotics comprising: thebleomycins A2 and B2 produced by Streptomyces verticillus(Fig. 18) and an antibiotic produced by Streptococcus caespito-sus, the mitomycin. They possess a wide applications range inthe treatment of lymphomas (Hodgkin and non-Hodgkin), thegerm cell tumors and the squamous cell cancers. The mechanismof action is iron-mediated and consists in free radicals produc-tion which is able to cause single or double-stranded DNA

FIGURE 17. Chemical structure of mitoxantrone.

FIGURE 18. Structures of bleomycin A2 and B2.

rupture. ESI-MS has been used to demonstrate the bleomycinsactivation (Sam et al., 1994) and its ability to cleave DNAstrands (Harsch et al., 2000). These glycopeptide antibioticsshow slight myelo- and immunosuppressive activity, whichmake them suitable in multidrug therapy with other drugs. Ifadminister by i.v. bolus at 15mg/m3, the observed Cmax rangebetween 1 and 5mg/mL, with an t1/2 of 3 hr. Bleomycins aremetabolized by a specific hydrolases present in tissues and liverbut not in skin, which represent the major site of toxicity(Brunton et al., 2011).

Up to now, plasma detection and quantification of bleomy-cin has been investigated exclusively by LC-UV (Joerger et al.,2005) and LC-FLD (Mahdadi et al., 1991) and the first massspectrometry-based method has been published early in 2015(Galba et al., 2015). The proposed method by Galba et al.showed the good performance in terms of hydrophilic interac-tion liquid chromatography (HILIC)-based separation withrespect to conventional C18 and C8 stationary phases tested. Themethod has been validated in term of LOD, LLOQ, linearityrange, precision, and recovery and accuracy. The identificationof bleomycin A2 and B2 in plasma spiked samples were alsodemonstrated.

5. Mitomycin

Mitomycin (Fig. 19) is used to treat cancer of breast, bladder,oesophagus, stomach, pancreas, lung, and liver. It is known tocause strong myelosuppression (both leucopoenia and thrombo-cytopenia) in treated patients and for this reason, loco-regionaldrug therapy is preferred. Mitomycin is metabolized byenzymatic reduction to give a number of active forms, whichexert antineoplastic activity through the formation of inter- andintrastrand DNA cross links, alkylation, and DNA strandbreakage with free radicals production. Currently used methodsfor mitomycin dosage in human plasma are based on LC-UV(Cerretani et al., 2002) and very few application of massspectrometry to mitomycin quantification are reported in theliterature. For instance, thirty human plasma samples of patientsreceiving a corneal topical application of mitomycin-C wereevaluated by a LC-MS method having a declared LLOQ of10 ng/mL. No drug was detectable in plasma and the analyticalprocedure is not discussed in details (Crawford et al., 2013). Inanother paper, a UHPLC-MS/MS method has been developedand validated in rabbit plasma (Tang et al., 2012). Even if PK inanimals is not in the line of this review, we aim to discuss it asmethod easily adaptable to human samples. Authors performedan LLE with ethyl acetate, using triamcinolone acetonide asinternal standard with declared recoveries from 85% to 115% atthree different spiked levels (1, 5, 100, and 800 ng/mL). A fast

chromatographic separation (less than 3min) was performedonto a C18 column in isocratic conditions. By monitoring thetransition m/z 335> 242 mitomycin was quantified reachinggood sensitivity levels (LLOQ 1 ng/mL).

F. Epipodophyllotoxins

Two semisynthetic derivatives of podophyllotoxin, extractedfrom mandrake plant, have significant anticancer activity:etoposide and teniposide, both reported in Figure 20 (Bruntonet al., 2011). These two drugs are similar in their actions and inthe spectrum of human tumours affected. Both etoposide andteniposide are thought to exert their antitumor activity throughthe stabilization of cleavable topoisomerase II/DNA complexesleading to cell death. Cells in the S and G2 phases of the cellcycle are most sensitive to etoposide and teniposide.

1. Etoposide

In addition to DNA topoisomerase II-mediated chromosomalbreakage reported above, cytotoxicity of etoposide also involvesa secondary mechanism related to the etoposide catechol, one ofetoposide major metabolite obtained through the O-demethyla-tion by cytochrome P450 3A4 (Zheng et al., 2006). Indeed, thiscatechol metabolite can undergo sequential one- and two-electron oxidations to form etoposide semi-quinone and etopo-side quinone, respectively, which have been recognized ascytotoxic metabolites. Following redox cycling results in thegeneration of reactive oxygen species, such as hydrogenperoxide and superoxide, which undergo Fenton chemistry formhydroxil radicals. These hydroxil radicals are able to eitherdamage DNA directly or induce the formation of lipid hydro-peroxides, which break down to DNA-reactive aldehydicbifunctional electrophiles (Zheng et al., 2006).

FIGURE 19. Structure of mitomycin.FIGURE 20. Chemical structures of etoposide, etoposide catechol andteniposide.

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Etoposide is primarily used, in combination with cisplatinand bleomycin, for treatment of testicular tumours and, incombination with cisplatin and ifosfamide, for small cell lungcarcinoma (Brunton et al., 2011). It is also active against non-Hodgkin’s lymphomas, acute non-lymphocytic leukaemia, andKaposi sarcoma associated with acquired immunodeficiencysyndrome (AIDS). In combination therapy for testicular cancerthe intravenous dose of etoposide is 50–100mg/m2 for 5 days, or100mg/m2 on alternate days for three doses (Brunton et al.,2011). For small cell lung carcinoma, the intravenous dosage incombination therapy is 35mg/m2/day for 4 days or 50mg/m2/day for 5 days. It can also be administered orally and for smallcell lung cancer the dose is twice the i.v. dose. Indeed, oraladministration of etoposide results in variable adsorption thataverages 50% (Brunton et al., 2011). In combination withifosfamide and carboplatin, it is frequently used for high-dosechemotherapy in total doses of 1500–2000mg/m2. Noteworthy,therapy with etoposide is associated with leukaemia withchromosomal translocations, especially translocations of theMLL gene at chromosome band 11q23, as a treatmentcomplication.

After intravenous administration, the disposition of etopo-side is best described as a biphasic process with a distributionhalf-life of about 1.5 hr and terminal elimination half-liferanging from 4 to 11 hr. It presents a linear PK over a range of100–600mg/m2, but Cmax and AUC values exhibit marked intra-and inter-subject variability. In vitro, etoposide is highly proteinbound (97%) to human plasma proteins.

Most of the methods commonly used for the determinationof this drug in human plasma include LC with UV detection(Pigatto et al., 2015), fluorescence detection (Manouilov et al.,1998) and electrochemical detection (Cai et al., 1999).

At the best of our knowledge, the first method for thequantification of etoposide in human plasma based on massspectrometry was developed by Chen and Uckun (2000). Thismethod employs LC-electrospray mass spectrometry (positiveion mode) using atmospheric pressure ionization (API) inter-face. Sample treatment was based on LLE of 100mL of plasma(or 200mL of serum) with 7mL of chloroform and, aftercentrifugation, the organic phase was dried and the residue wasreconstructed in 50mL of acetonitrile. The calibration curveswere linear over a range of 6–6000 ng/mL in plasma and 7–3000 ng/mL in serum. The validation study was conducted onlythrough the assessment of the extraction recovery and the intra-and inter-assay accuracy and precision. The applicability of theassay to clinical samples was demonstrated by measuringetoposide concentration in four cancer patients (disease unspeci-fied), who received etoposide intravenously via 1-hr i.v. infusionat dose of 50mg/m2.

One year later, Pang et al. (2001) proposed an LC-MS/MSmethod for the simultaneous determination of etoposide and itscatechol metabolite in human plasma. Indeed, etoposide proteinbinding appeared to be dependent upon serum albumin concen-trations and presents significant inter-patient variations. In orderto define whether there were inter-individual differences inetoposide catechol binding, they developed a method for thedetermination of etoposide and its catechol metabolite asprotein-free and total concentration in human plasma samples.To quantify total etoposide and its catechol, they conducted a PPtreatment to 50mL of plasma while for the quantification ofprotein-free etoposide and its catechol metabolite, 500mL of

plasma were filtered and centrifuged in order to remove proteinand protein-bound etoposide and its metabolite before the PPtreatment. Sample treatment was conducted in presence ofascorbic acid in order to avoid the oxidation of etoposidecatechol to form etoposide quinone. Therefore, any quinone thatwas eventually present in the plasma was previous reduced tothe catechol form. The separation between etoposide and itscatechol metabolite was achieved using a chromatographicgradient with the mobile phases consisting of 5mM HCOONH4

and 0.1% aqueous formic acid solution with 10% acetonitrile asA and 90% acetonitrile as B. Quantification was conducted inSRMmode using the product ions obtained from the ammoniumadducts [MþNH4]

þ. Acceptable precision and accuracywere obtained for concentrations in the calibration curveranges 0.2–100mg/mL and 10–5000 ng/mL for etoposide andcatechol metabolite, respectively, and 25–15000 ng/mL and2.5–1500 ng/mL for protein-free etoposide and etoposide cate-chol, respectively. This method was applied to pharmacokineticstudies in paediatric cancer patients receiving a 100mg/m2 doseof etoposide as 1-hr i.v. infusion daily for five days.

More recently, besides the development of an LC-MS/MSmethod for the simultaneous determination of seven commonlyused anticancer drugs in human plasma, among which there isthe etoposide (Zhou et al., 2012), just one method was publishedfor the quantification of etoposide and its catechol metabolite inhuman plasma (Liao et al., 2014). The group of Liao proposed astrategy for correcting matrix effect in various biofluids using apost column infused-internal standard (PCI-IS) method incombination with matrix normalization factors (MNFs). Theresponse ratios of the analyte and PCI-IS in different biofluidswere used to calculate the MNFs, which were then been used toquantify the target analyte in various biofluids by using the sameLC–ESI–MS data with the same calibration curve generatedfrom standard solutions. Regarding plasma samples, they chooseto use the PP treatment (4x volume of methanol). Similarly tothe previous method, etoposide and etoposide catechol wereseparated during the chromatographic run using a gradient withmobile phases consisted of 0.1% aqueous formic acid (solventA) and 0.1% formic acid in acetonitrile (solvent B). The massspectrometer was configured in the multiple reaction monitoringmode and followed the transitions: 589> 229 and 589> 185m/zfor etoposide, 575> 229 and 575> 185m/z for etoposidecatechol, and 657> 229m/z for teniposide (used as internal

FIGURE 21. Common fragmentation pathways for etoposide, etoposidecatechol and teniposide.

standard). Figure 21 reports the common fragmentation path-ways for these analytes proposed in the illustrated methods. Thisanalytical method was validated in terms of accuracy, linearity,precision and sensitivity. The LLOQ of etoposide and etoposidecatechol were 10 ng/mL for both analytes (10–500 ng/mL rangeof concentration for calibration curves). To demonstrate thefeasibility of MNFs and the PCI-IS method for clinical measure-ment, plasma and cerebrospinal fluid samples from one patientwith a brain malignancy receiving etoposide treatment (intrave-nous administration of 70mg/m2) were analyzed during twotreatment cycles. In order to ensure the correctness of theproposed method, the plasma samples were analyzed by theestablished internal standard correction method and the PCI-ISwith MNF correction method. The similar pharmacokineticprofiles seemed to indicate the proposed method is accurate.

2. Teniposide

Teniposide differs from etoposide by the substitution of athenylidene group on the glucopyranoside ring (Fig. 20). Thepharmacokinetic characteristics of teniposide differ from thoseof etoposide. Teniposide is more extensively bounded to plasmaproteins and its cellular uptake is greater. Indeed, in vitro plasmaprotein binding of teniposide is >99%. Moreover, the highaffinity of teniposide for plasma proteins may be an importantfactor in limiting distribution of drug within the body. Tenipo-side is intravenously administered in dosage that ranges from50mg/m2/day for five days to 165mg/m2/day twice weekly(Brunton et al., 2011). The drug is primarily given for acuteleukaemia in children and monocytic leukaemia in infants, aswell as glioblastoma, neuroblastoma and brain metastases fromsmall cell carcinomas of the lung.

To date, there have been little research on analyticalmethods to determine teniposide in pharmacokinetic applica-tions. Analytical methods have been developed for the quantifi-cation of teniposide based on electrochemical detection(Sinkule & Evans, 1984; Canal et al., 1986), UV detection(Nagai et al., 1998) and fluorescence detection.

To our knowledge, the first report of the development,validation and application of a UPLC-ESI-MS/MS method forthe determination of teniposide and its application to apharmacokinetic study was published by Wang et al. (2009).Anyway, this method was applied to an in vivo study in rats andfor this reason it will not be discussed in the present review.

G. Marine Products

The marine environment is recognized to be a huge sourceof metabolites of great interest for the pharmaceuticalindustry. The most interesting compounds are secondarymetabolite produced for defence purposes by sessile marineinvertebrates. Two marine-derived natural compounds areactually marketed for anticancer therapy: the cytarabine andthe trabectedin (Figs. 22 and 23, respectively).

1. Cytarabine

Spongouridine and spongothymidine are among the first bioac-tive compounds extracted in 1950s from a marine source, thesponge Cryptotheca crypta. This compound is characterized bythe presence of an ara-nucleosides (i.e., an arabinose unit instead

of ribose) that provided the basis for the chemical synthesis ofcytarabine in 1959. Cytarabine possesses short plasma t1/2 andlow bioavailability; typical therapeutic regimens consist incontinuous i.v. infusion for 7 days. To exert its anticanceractivity, cytarabine is intracellularly converted into the activeform cytarabine triphosphate that inhibits the DNA synthesis viacompetition with the natural nucleotide deoxycytidine triphos-phate. Conventional reverse phase chromatography is not usefulto adequately retains cytarabine, since this compound is highlypolar; to overcome this problem ion-pairing chromatography(Hsieh & Duncan, 2007) and the modified C18 high strengthsilica T3 (Hilhorst et al., 2011) have been employed. The naturalcompound cytidine, present in plasma, is the main interferingcompound to separate having both identical precursor andfragment ions (transition followed m/z 244> 112). Cytarabinehas been quantified in human plasma samples with goodsensitivity (linearity range: 0.5–500 ng/mL) after opportunesamples stabilization with tetrahydrouridine. This step isreported to prevent the ex vivo conversion of cytarabine by theplasmatic enzyme cytidine deaminase (Hilhorst et al., 2011).Sample preparation procedure performed by Hilhosrst et al.consisted in a cation-exchange SPE extraction, while Demerset al. used a PP extraction which not ensured comparablesensitivity having an LLOQ of 10 ng/mL (Demers et al., 2013).

FIGURE 22. Chemical structure of marine drug cytarabine.

FIGURE 23. Chemical structure of trabectedin.

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2. Trabectedin

Trabectedin is a marine antineoplastic alkaloid isolated from theCaribbean sea squirt (Ecteinascidia turbinate). Its mechanismof action differs from that of alkylating agents, as it binds theminor groove of DNA and the formed adduct interacts withproteins of the DNA repair complex causing the double-strandbreak. Actually it is approved for the treatment of advanced softtissue sarcoma and ovarian cancer in Europe. Trabectidin isadministered at 1.3mg/m2 by a 24-hr i.v. infusion every 3weeks: the observed t1/2 is in the range 24–40 hr.

Some analytical methods for the quantification of trabecte-din in human plasma by LC-MS have been proposed (Rosinget al., 1998 Stokvis et al., 2004b Zangarini et al., 2014). All ofabove cited methods operated in Electrospray positive ionmode, since the basic nature of alkaloids make them easilyionisable by protonation reactions.

In the first method, 0.5mL of plasma were extracted on aSPE cyano column in presence of acidic conditions and the LC-MS/MS analysis has been performed on a C18 column inisocratic conditions (methanol/water 75:25 v/v in presence ofammonium acetate 5mM and 0.4% formic acid). This methodprovided high sensitivity (LLOQ of 0.01 ng/mL) and the PKparameters of three low-doses treated patients was obtainedduring the 24-h i.v. infusion procedure (at 2, 6 and 23.5 hr) andafter the infusion (at 5, 10, 15, 30 and 60min and 2, 4, 6, 9, 12and 24 hr). Stokvis et al. published in 2004 a method withcomparable sensitivity (LLOQ 0.05 ng/mL) but with a less time-expensive procedure based on the on-line sample clean-up by aC18 trapping column. Plasma samples were firstly diluted twofold with acidified water before the injection onto the trappingcolumn, but residual proteins caused interference with thepurification procedure; the PP procedure before the on-linesample clean-up has been introduced to increase the methodrobustness. After a complete method validation procedure, theconcentration curve in a patient receiving 1.2mg/m2 by a 3-hri.v. infusion was monitored up to 70 hr (Stokvis et al., 2004b).

Zangarini et al. developed in 2014 an easier procedure toperform PK of trabectedin: they used a LLE extraction with0.1M HCL in methanol to extract 0.1mL of a treated patient’plasma. Quantitative data were obtained monitoring the transi-tion m/z 744> 495 for trabectedin and m/z 747> 253 for thedeuterated trabectedin (IS) in the linearity range 0.025–1 ng/mL(Zangarini et al., 2014).

H. Retinoids

Retinoids, derivatives of vitamin A (retinol), represent a class ofnatural and synthetic compounds that exhibit vitamin A-likebiological activity or bind to nuclear receptor for retinoids. Theyplay an important role in several physiological processes, suchas vision, regulation of cell proliferation, differentiation, andbone growth, immune defence, and tumour suppression (Brun-ton et al., 2011). Retinoids are mainly taken up in the human dietas proretinoid carotenoids, retinyl esters, and retinol. Retinol ismetabolized into various more polar metabolites, such asretinoic acid (RA) isomers and oxidized metabolites. Monitor-ing endogenous retinoids concentration and its biologicallyactive geometric isomers in various biological systems has beenimportant to understand vitamin A functions and mechanisms ofaction in health and disease. For this reason, many bioanalytical

methods have been published in the last 30 years to determineendogenous retinoids levels not only in serum or plasma, butalso in several tissues such as embryo-, kidney- and liver-homogenates, as reviewed by Gundersen (2006).

The biology and pharmacology of retinoids involve alsocancer treatment. Among all the compounds belonging to thisclass, the most important for chemotherapy is tretinoin, i.e., at-RA (Fig. 24), which induces a high grade of complete remissionin acute promyelocytic leukaemia (APL) administered as asingle agent, and, in combination therapy with anthracyclines,cures most patients with this disease (Brunton et al., 2011).

Under physiological conditions, the retinoic acid receptor-a dimerizes with the retinoid X receptor and forms a complexthat binds at-RA tightly. This binding displaces a repressor fromthe complex promoting differentiation of cells of multiple types.In APL cells, endogenous concentration of retinoid is notenough to displace the repressor. at-RA also binds and activatesretinoic acid receptor-g promoting a repopulation of the bonemarrow cells. Tretinoin is orally administered with a dosingregimen of 45mg/m2/day until 30 days after remission isachieved (Brunton et al., 2011). In human pharmacokineticsstudies, administered drug was well absorbed into the systemiccirculation and approximately two-thirds of the administeredradiolabel was recovered in the urine. The terminal eliminationhalf-life of tretinoin following initial dosing is 0.5–2 hr inpatients with APL. More than 95% of at-RA resulted bound inplasma, predominately to albumin.

Retinoids analysis in biological samples is a challengingtask, due to low endogenous concentrations that range from 1 to5 ng/mL in human plasma (Eckhoff & Nau, 1990; Gundersenet al., 2007), complexity of biological samples, and the labilenature of these compounds. Indeed, as retinoids are sensitive tolight, in order to avoid undesired isomerisation, in all of thisanalytical methods samples preparation and handling wereconducted under yellow light or, more recently, under red light(Gundersen et al., 2007; Arnold et al., 2012; Peng et al., 2014).Moreover, the use of stable isotopes as internal standard, besidesprovides the best performance for this purpose, allows tomonitor in real time the occurrence of artificial isomerisation, asreported by Gundersen et al. (2007), Arnorld et al. (2012) andNapoli et al. (1985). Alternatively, acitretin was used as internalstandard (Eckhoff & Nau, 1990; Lehman & Franz, 1996).

FIGURE 24. Chemical structures of all-trans-retinoic acid and acitretin.

The first quantification problem arises from the impossibili-ty to have blank human plasma/serum samples since retinoidsare endogenously present in these matrices. In order to overridethis problem, different strategies have been proposed: Napoliet al. (1985) obtained samples free of retinoids by irradiatingplasma or serum at 4˚C for 24–36 hr with a UV lamp whileEckhoff and Nau (1990) dissolved standard retinoids in reti-noids-free 5% solution of pure bovine serum albumin inphosphate-buffered saline (PBS). More recently, calibrationcurves were constructed spiking standard retinoids in fetal calfserum (Ruhl, 2006) as well as in charcoal-treated human serum,that presents a normal range of triglycerides and cholesterol tomimic the extraction environment of clinical samples (Arnoldet al., 2012).

Extraction procedure of the analytes from human plasma orserum, usually obtained from healthy volunteers, commonlyrequired SPE (Eckhoff & Nau, 1990; Lehman & Franz, 1996) aswell as LLE with hexane (Napoli et al., 1985; Arnold et al.,2012). In the method proposed by Ruhl (2006), serum sampleswere simply treated with PP using isopropanol, dried undervacuum and injected into the LC system after resuspension inthe LC solvent, while Gundersen et al. (2007) described a mono-phase extraction with acetonitrile performed in black 96-wellmicrotiterplates.

Separation of the analytes was achieved applying chro-matographic conditions quite similar over the different methodsdeveloped: the column commonly used was the C18 (Lehman &Franz, 1996; Gundersen et al., 2007; Peng et al., 2014) (C30 inthe case of the method proposed by Ruhl (2006) and an amidecolumn in the case of the assay developed by Arnold et al.(2012)) with gradients formed from mobile phases composed ofwater and solvents (generally acetonitrile or methanol, ormixture of both) acidified with 0.1% formic acid (Gundersenet al., 2007; Arnold et al., 2012) or 0.1% acetic acid (Lehman &Franz, 1996). Total run times were in the range of 7min(Gundersen et al., 2007) to 30min (Lehman & Franz, 1996).

Napoli et al. (1985) and, later, Eckhoff and Nau (1990)developed a GC-MS method able to distinguish all-trans-RA(at-RA) from total RAs, and able to determine at-RA, 13-cis-RA and 13-cis-4-oxoretinoic acid (13-cis-4-oxoRA), respective-ly. In these cases, an additional methylation procedure of RAswith diazomethane was necessary in order to obtain methylreti-noate derivatives that could be analyzed by gas chromatography.Both assays were based on selective monitoring of the negativeion (chemical ionization performed with methane), obtainingvalues of LOD of 75 pg (Napoli et al., 1985) and of LLOQ of0.5 ng/mL (Eckhoff & Nau, 1990) for all the analytes.

Lehman and Franz (1996) proposed a quantification assayfor at-RA and 13-cis-RA that involves a derivatization ofendogenous RAs to their pentafluorobenzyl esters followed byseparation and isolation by reverse phase LC (twoC18 columnsin series). The LC eluate was then directed to a mass spectrome-ter via particle beam interface and the analytes were quantifiedby SIM of the RAs carboxylate anion produced by negativechemical ionization using methane as reagent gas. The methodproposed is more sensitive than the previous assays as it has aLOD of 25 pg and a LLOQ of 0.25 ng/mL.

No derivatization of retinoids was required in the subse-quent reverse phase LC methods based on the use of massspectrometer operating in positive ion APCI mode for thequantification of: 1) at-RA, 9-cis-RA and 4-oxo-RA in human

serum (Ruhl, 2006); 2) 4-oxo-RA, 4-oxo-13-cis-RA, 13-cis-RA,at-RA and at-retinol in human plasma (Gundersen et al., 2007);3) different isomers of RA (at-RA, 9-cis-RA and 13-cis-RA)and oxidized metabolites (4-oxo-13-cis-RA, 4-oxo-at-RA, 4-hydroxy-at-RA and 4-hydroxy-9-cis-RA) in human serum(Arnold et al., 2012). In the first case no IS was required and aLOD of 7 pg and a LLOQ of 0.2 ng/mL were obtained, in thesecond one a LOD$ 2 pg and a LLOQ$ 2 ng/mL for RAs wereachieved and in the latter case LLOQ was 0.05 nM for theisomers of RA and 6 nM for the oxidized metabolites.

Besides the different analytical methods developed for thequantification of endogenous retinoids reported and describedabove, at the best of our knowledge, only one assay has beenpublished with a bioequivalence study of at-RA as anticancerdrug (Peng et al., 2014). The numerous difficulties involved inthe quantification of retinoids and discussed above are alsoreported in this study: samples preparation and collection werecarried out in dark under red light in order to avoid undesiredisomerisation, for the preparation of calibration standard samplesand quality control samples, blank plasma with sunshineexposure treatment for 6 h was used and analytes extraction from500mL of human plasma was conducted with LLE using 2mLof methyl thert-buthyl ether (acitretin was used as IS). After areverse phase chromatography (C18 column), quantification ofat-RA was done with mass spectrometer operating in MRMmode via the positive ESI interface. The validation procedureswere conducted in accordance with the FDA guidelines and thecalibration curve was linear over the range of 0.45–217 ng/mL,being the LLOQ equal to 0.45 ng/mL. This method was appliedto a bioequivalence study of two formulation of at-RA: 29healthy volunteers were enrolled and randomly oral administeredwith a single dose of 20mg of at-RA soft capsule or at-RAcapsule with 200mL of water. Blood samples were collected 24,20, 12, and 0 hr prior to drug administration and at 0.25, 0.5,0.75, 1, 1.5, 2.5, 3, 4, 6, 8, and 10 hr after drug administration.The formulations were considered bioequivalent because thedifference between two compared pharmacokinetic parameterswas found statistically insignificant.

I. Histone Deacetylase Inhibitors

Vorinostat (ZOLINZA), also known as suberoylanilide hydroxa-mic acid (SAHA), belongs to a class of anticancer agents that actin a new field of cancer research: the epigenetics. Indeed,epigenetic changes in gene expression may play an importantrole in cancer growth (Mann et al., 2007). Vorinostat is a potentinhibitor of histone deacetylases (HDACs), enzymes that cata-lyze the removal of acetyl groups from the lysine residues ofproteins, including histones and transcription factors. Vorinostatbinds to a zinc ion in the catalytic domain of the enzyme thusincreasing histone acetylation and gene transcription. Pharma-cological inhibition of HDACs causes re-expression of proteinsthat promote apoptosis and cell differentiation while inhibitscell cycling and cell division (Brunton et al., 2011). After oraladministration (once-daily dose of 400mg), vorinostat isinactivated by glucuronidation of the hydroxyl amine group toform vorinostat glucuronide. Moreover, it undergoes hydrolysisof the terminal carboxamide bond followed by oxidation of thealiphatic side chain to obtain 4-anilino-4-oxobutanoic acid(Fig. 25). These two metabolites are pharmacologically inactive(Brunton et al., 2011). Vorinostat has been approved in 2006 by

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FDA for the treatment of cutaneous manifestations of cutaneousT-cell lymphoma in patients with progressive, persistent, orrecurrent disease on or following two systemic therapies (Mannet al., 2007).

In the literature, few LC-MS/MS methods for the quantifica-tion of vorinostat in human biofluids are reported. At the best ofour knowledge, the first analytical method based on massspectrometry was developed by Du et al. (2005) for thequantification of vorinostat and its two main metabolites inhuman serum. This assay required an high turbulence liquidchromatography online extraction technology, which allowed theseparation of the analytes from the proteins and other hydrophilicmacromolecules through a size-exclusion mechanism. Theanalytes were then transferred directly to the analytical column.Other extraction procedures applied to vorinostat required simplePP with acetonitrile (Patel et al., 2008), also followed by solventevaporation and resuspension in mobile phase before the analysisof the drug and its main metabolites (Parise et al., 2006).Recently, Liu et al. (2014) proposed the use of SPE for theextraction of vorinostat and its main metabolites from plasmasamples and LLE for the extraction of these analytes fromperipheral blood mononuclear cells (PBMCs).

In two methods, the chromatographic separation of vorino-stat and its main metabolites was achieved using C18 columnswith gradient elution. The mobile phases used were: water andacetonitrile, both with 0.1% acetic acid (Du et al., 2005; Pariseet al., 2006). In the method developed by Liu et al. (2014), themobile phases used were 5mM ammonium acetate buffer with0.1% acetic acid (A), acetonitrile (B) and methanol (C). For theseparation of vorinostat and its metabolites in plasma samples,the isocratic mobile phase was 65% A/15% B/20% C, while, inorder to increase the sensitivity of PBMCs analysis, a gradientelution was developed.

In most of these analytical assays, the mass spectrometrydetection was conducted with ESI source operating in positiveion mode (turbo ion spray source was used by Du et al. (2005)).As internal standards were always used the analytes deuterium-

labelled (d5): d5-vorinostat, d5-vorinostat glucuronide and d5-4-anilino-4oxobutanoic acid. The lower LLOQ for vorinostatwas achieved by the method of Patel et al. (2008), with a valueof 1 ng/mL and an acceptable linearity observed in the range of0.5–1000 ng/mL. In the method proposed by Du et al. (2005),the standard curves resulted linear over the concentration rangesof 2-500, 5-2000 and 10-2000 ng/mL for vorinostat, vorinostatglucuronide and 4-anilino-4oxobutanoic acid, respectively, withthe corresponding LLOQ at 2, 5, and 10 ng/mL. The same valueof LLOQ (3 ng/mL) was obtained for all the three analytes in themethod developed by Parise et al. (2006), and acceptablelinearity was observed in the range of 3–1000 ng/mL. Theapplicability of this assays to clinical samples was demonstratedby measuring vorinostat and its main metabolites in serumsamples obtained from a 74-year-old male patient before and at0.5, 1, 1.5, 2, 3, 4, and 6 hr after the oral administration of200mg of vorinostat. The plasma concentration versus timeprofiles of vorinostat, vorinostat glucuronide and 4-anilino-4oxobutanoic acid were reported. The maximum serum concen-tration of vorinostat (384 ng/mL) observed from patient sampleswas below the ULOQ (1000 ng/mL).

The method developed by Liu et al. (2014) for thedetermination of vorinostat and its two main metabolites inhuman plasma and in PBMCs was validated according to FDAand EMA guidelines. The three analytes concentrations in studysamples were measured within the calibration range 11-11000 ng/mL (plasma samples) and 0.1-10 ng vorinostat/3) 106

cells (PBMCs samples). Study samples were obtained throughthe incubation of citrated fresh whole blood samples fromhealthy volunteers with different concentration of vorinostat at37˚C for 2 hr. Subsequent separation of plasma and PBCMsallowed the analyses of vorinostat distribution between thesetwo matrices and so to verify the effective drug concentrations atthe site of target cells.

VI. CONCLUSIONS AND FUTURE PERSPECTIVES

In the literature there is a wide diversity of assays developed fordrug quantification. The coupling of liquid chromatography andmass spectrometry has had such an important impact on drugquantification that this technique has become the Gold Standardof drug testing. The high selectivity and the high sensitivity ofliquid chromatography-tandem mass spectrometry make possi-ble the sample volume can be reduced up to performing theanalysis in dried blood spot and the quantification in micro-samples. This feature renders LC-MS/MS suitable to supportstudies employing micro-dosing or metronomic dosing, paediat-ric studies or when the quantification of metabolites is requiredbecause usually they are present in low concentrations.

Since high performance liquid chromatography in combi-nation with mass spectrometry has been extensively applied inthe last decades to quantify anticancer drugs of natural originand their metabolites, this review presented an overview ofpublications describing the LC-MS methods in human samples.A large number of papers here discussed has been validatedaccording to FDA and/or EMA guidelines, in order to assure theresults quality.

Further developments in LC coupled to MS for drugquantification has been obtained and will be still expected. Forexample, the introduction of pneumatically-assisted or heatedsources has lead to improve the ionization efficiency with the

FIGURE 25. Chemical structures of vorinostat, vorinostat glucuronide and4-anilino-4-oxobutanoic acid.

increase of instrumental sensitivity. The parallel improvementof mass spectrometer analyzer performances in terms ofaccurate mass measurements has increased the ability inmetabolites identification and in high-throughput analyses(Gomez-Canela et al., 2013). This is a focal point when facingthe multi-drug therapies, for which a comprehensive quantifica-tion in a single LC-MS analysis is very helpful.

Anyway, it is important to notice that besides the widelyemployed LC-MS/MS approach, new MS techniques or theapplication of other mass analyzer than triple quadrupole todrug quantification in human samples has led, in recent years, togood results. For instance, the possibility to perform quantitativeanalysis of pharmaceutical compounds by MALDI-TOF hasbeen demonstrated: the choice of matrices avoiding the interfer-ences in the low-molecular weight regions and the opportunedata processing made possible to quantify HIV proteasesinhibitors in peripheral blood cells (van Kampen et al., 2006).Another MALDI-TOF quantitative approach has been recentlyproposed by Calandra et al. (2014), who investigated thefeasibility of this technique to quantify some antineoplasticdrugs of natural origin, such as irinotecan (CPT-11) and themain metabolite of paclitaxel (6-a-hydroxy paclitaxel), inhuman plasma. Other recently-proposed approaches, based onambient mass spectrometry techniques, demonstrated the possi-bility to perform the PK analysis in a less time-expensive waythanks to the combination of sampling and ionization proceduresin one step. Even if the absence of sample preparationprocedures and chromatographic separations could in principlenegatively affect the quantitative performances, the straightfor-ward benefits in terms of velocity should also be considered.Both Direct Analysis in Real Time-Mass Spectrometry (DART-MS) (Zhao et al., 2008; Yu et al., 2009; Crawford et al., 2011)and Paper Spray Mass-Spectrometry (PS-MS) (Espy et al.,2012; Su et al., 2013) have been positively applied to drugquantification in biological samples. With the introduction ofambient mass spectrometry techniques, we expect that one ofthe most interesting application of mass spectrometry in theclinical practice will be the TDM performed in “real time”making the personalization of therapy feasible.

ABBREVIATIONS

APCI atmospheric pressure chemical ionizationAUC area under the curveC18 octadecyl carbon chainDART direct analysis in real timeECD electrochemical detectorESI electrospray ionizationFDA Food and Drug AdministrationFLD fluorescence detectori.v. intravenousLC liquid chromatographyLLE liquid-liquid extractionLOD limit of detectionLLOQ lower limit of quantitationMALDI matrix-assisted laser desorption/ionizationMRM multi reaction monitoringMS mass spectrometrySPE solid phase extractionPK pharmacokineticsPP protein precipitation

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