53

3.0 Review of Literature

The present chapter deals with the literature survey on pharmaceuticals and

biomedical analysis, determination and quantification of potential and degradation

impurities in anti cancerand antihypertensive active pharmaceutical ingredients using

the developed and validated, stability indicating, UPLC method. UPLC is a new

category of separation technique based upon well established principles of liquid

chromatography. Owing to its speed and sensitivity, this technique is gaining

considerable attention in recent years for pharmaceuticals and biomedical analysis.

The comprehensive literature survey revealed that the HPLC methods used for and

quantification of potential and degradation impurities in drug substances have low

sensitivity, highLOQ and long run time.

3.1 Impact of various impurities on quality safety and efficacy of drugs

Shokouh Taghipour Zahir et al studied cancer mortality worldwide. Study

indicating that lung carcinoma is the leading cause of Lung cancer and concluded that it

is reasonable to expect that early lung cancer detection, and appropriated treatment,

may improve surgical morbidity and mortality [1]. AhmedinJemal DVM etalstudied

cancer statistics each year and reported also examines cancer incidence, mortality, and

survival by site, sex, race/ethnicity, education,geographic area, and calendar year [2].

BrianJ. Druker et al have studied Imatinib as revolutionized drug therapy of chronic

myeloid leukemia (CML). In this study, authors reviewed the preclinical and clinical

development of imatinib for the therapy of CML, resistance and strategies that may help

to eliminate resistant or residual leukemia [6]. Alsante K M et al had studied a

multidisciplinary team approach to identify pharmaceutical impurities in this article. The

systematic strategy was successfully applied to the identification of an impurity in the

activepharmaceuticalingredient1-(1,2,3,5,6,7-hexahydro-s-indacen-4-yl)-3-[4-(1-

hydroxy -1-methyl-ethyl)-furan-2-sulphonylurea [8].

The ICH Guideline Q3A(R)addresses the chemistry and safety aspects of

impurities, including the listing of impurities in specifications and defines the thresholds

for reporting, identification and qualification [9]. ICH Guideline Q3B(R) specifically deals

with those impurities which might arise as degradation products of the drug substance

54

or arising from interactions between drug substance and excipientsor components of

primary packaging materials [10].

Ahuja S reviews the Impurities evaluationof pharmaceuticalsand some of the

applications of high-throughputliquid chromatography-massspectrometry (LC/MS)

techniques in the lead optimization phase of drug discovery.Several routinely used

techniques that are utilized in performing these ADME assays are described.The

preparation of biological samples is also presented in detail for the LC/MS quantitative

bioanalysis [13]. Sanjay B.Bari et al studied the identification of impurities by variety of

chromatographic and spectroscopic techniques like TLC, HPLC, HPTLC, AAS etc.

among all hyphenated techniques,themost exploited techniques,for impurity profiling of

drugs are LC-MS-MS, LC-NMR, LCNMR-MS, GC-MS, and LC-MS [14]. Goldman, J.M

et al studied Cytogenetic abnormalities Leading to Chronic Myeloid leukemia (cml)

which was probably the first form of leukemia to be recognized as a distinct entity. Until

the 1980s, CML was regarded as incurable and thus inexorably fatal [17]. Stegmeier F

et al studied the anticancer drug substances hold great promise for widening the

therapeutic window. In this article, the reviewes are discussed from the application of

imatinib revolutionized the treatment of chronic myeloid leukemia (CML)and other

targeted agents in clinical practice which leads into deep insights for development of

novel targeted therapies [19].

R. Kroes et al described the concept of the threshold of toxicological concern

(TTC) principle extends this concept by proposing that the TTC value can be identified

for many chemicals, in the absence of a full toxicity database, based on their chemical

structures and the known toxicity of chemicals which share similar structural

characteristics [21]. Colin F. Poole etal described the general concepts in column

chromatography are discussed. The column in gas chromatography, Instrumental

aspects of gas chromatography were discussed. J.C.Reepmeyeretal studied some

important aspects of the background of impurity related analytical studies

(toxicological, pharmacopoeial aspects, the characterisation of the sources of

impurities and the role of impurity profiling in various fields of drug research,

production and therapeutic use) are summarised. Identification and determination of

55

residual solvents and the determination of inorganic impurities is discussedin detailed

[33].

GVaidyanathan et al determined an isocratic reversed-phase liquid

chromatography method with UV detection has been developed for the purity

evaluation of imatinib mesylate in bulk drug. The method is selective and is capable of

detecting all process intermediates and other related compounds, which may be

present at trace levels in the drug substance [37]. G.Madhusudanreddy et al observed

seven impurities, in which four are new impurities in Rabeprazole sodium. All the

impurities were detected by a gradient high performance liquid chromatographic

(HPLC) method, whose area percentages were ranged from 0.05 to 1.0%.LCMS was

performed to identify the mass number of these impurities. A thorough study was

carriedout to characterize the impurities [38].

Somisetti Narender Rao et al have established the optimized conditions for the

bromination on compound and systematically studied the effect of base, catalyst,

solvent volume and temperature for the condensation of related compounds. They

developed and improved, high yielding, eco friendly and industrially feasible process

for Irbesartan intermediate with all the potential impurity levels in control [39].

K.Ramakrishna et al developed a gas chromatography–mass spectrometry (GC–MS)

method has been developed for the identification and determination of two

carcinogenic and genotoxic mesylate esters viz. methyl methane sulfonate (MMS) and

ethyl methane sulfonate (EMS) in imatinib mesylate.These mesylate esters were not

found in three different batches of pure and pharmaceutical formulations [40].

K.V.S.R.Krishna Reddy et al had studied the acid, base and oxygen stability of

risperidone, a novel anti-psychotic drug, has been evaluated storing the sample in

solution phase. One of the major degradation products has been identified and

characterized by using techniques namely IR, MS and NMR after isolation

bypreparative LC. The two major degradation products are identified by LC– MS/MS

and HPLC [41]. R. NageswaraRao et al have studied the reversed-phase high-

performance liquid chromatographic method was developed for determination of

process impurities and degradation products of Bicalutamide in bulk drug and

56

pharmaceutical formulations.The unknown process impurities and alkaline degradation

products were isolated and characterized by ESI-MS/MS, 1H NMR and FT-IR spectral

data [42]. D.Krishanamachary et al had determined one unknown impurity (degradation

product) present at a level below 0.1% in the initial samples increased to a level of

0.5% in 6M/40 degrees C/75% RH stability samples of risperidone tablets was detected

by gradient reverse-phase high-performance liquid chromatography (HPLC)[43].

P.Skett reviewed the analytical methods used to monitor genotoxic impurities.

Specifically, the impurities discussed here contain hydrazine,nitro,arylamine or

aldehyde functional groups as found in pharmaceuticals [46]. AndrewTeasdaleet al

examined the primary components of quality risk assessment for genotoxic impurities

(GTIs) a strategy, focusing specifically on the effective use of in silico assessment tools

to augment this process, in particular the calculation of theoretical purge factors based

on the physicochemical properties of a specific GTI and its interrelationship to the

process. Existing regulatory guidelines such as ICH Q3A (R2)/Q3B (R2)/Q3C (R4) do

not adequately address the requirements for controlling trace levels of GTIs [47].

A.G.Renwic ket.al. have studied the threshold of toxicological concern (TTC). TTC is a

level of intake predicted to be without adverse effects.The main advantage of the use

of TTCs is that the risk of low exposures can be evaluated without the need for

chemical-specific animal toxicity data. TTC shave been used for many years for

screening the safety of packaging migrants by certain regulatory bodies.

57

3.2 Theory of UPLC/LCMS techniques involved in the identification

quanitification of impurities in anticancer Active Pharmaceutical

Ingradients

The importance of drug impurity and stability related issues has also been

characterized by Smith R.et al and articles devoted to this subject. In the field of drug

analysis, the analytical investigation of bulk drug materials, the intermediates in their

synthesis, products of drug research, drug formulations, impurities and degradation

products, and biological samples containing the drugs and their metabolites is a very

important area of research [62]. The contributions of HPLC in drug analysis was

discussed by Georges.

For example, in 1983, a UV detector was applied almost exclusively, leaving a

little share to refractive index, fluorimetric, and electrochemical detection, but in 2005,

a mass spectrometer was applied as a detector inabout one-third of the analysis.

HPLC coupled with mass spectrometry, HPLC/MS (MS) or LC/MS(MS) due to high

sensitivity and selectivity have become the predominant method in bio assays and

pharmacokinetic and metabolic studies, as well as in the structure elucidation of drug

impurities and degradation products [63].

F.Erni et al studied and published the requirements for new pharmaceutical

products and their impact on applications of high-performance liquid

chromatography(HPLC) are discussed. The strengths and weaknesses of HPLC in this

context are evaluated and compared with current trends and expectation in separation

science [67]. R.A.W. Johnstone and M.E.Rose et al published a book describes the full

range of techniques and applications used in mass spectrometry. The authors

discussed powerful methods combining mass spectrometry with newer separation

techniques, the increased use of computers, and analysis of once difficult polar and

large-mass compounds such as proteins using new ionization methods [72].

58

The Van Deemter equation in chromatography relates the variance per unit

length of a separation column to the linear mobile phasevelocity by considering

physical, kinetic, and thermodynamic properties of a separation [76]. Tiller, P.R

reviewed on fast LC/MS analyses of small molecules that have been reported in peer

reviewed publications.

An examination of the current state of the literature, relating to "fast LC/MS",

should serve well to those new to LC/MS, and should help them in the development of

fast LC/MS methods that are effective in terms of both the chromatography and

utilization of the mass spectrometer [77].

Cheng, Y.-F et al described the use of conventional MS,GC-MSor LC-MS

methods to catalogue or screen for components of extracts including traditional

Chinese Medicines are discouraged, except where the authors can clearly demonstrate

that their contribution represents a significant extension of the capabilities

orapplications of mass spectrometry or new understanding of ion chemistry and related

disciplines [78]. U.D. Neue et al has outlined the reasoning for the development ofultra-

performance liquid chromatography(UPLC). Compared with HPLC,this new technique

makes it possible to achieve a higherseparation performance or a higher speed or a

combination of higher speed with higher separation performance [79].

GrittiF et al studied and suggested significant differences between the

structures of the porous silica in these different materials. Single-component adsorption

isotherm data were acquired by frontal analysis (FA) for phenol and caffeine on a new

C18-Chromolith column (Merck, Darmstadt, Germany),using a water-rich mobile phase

(methanol/water, 15/85, v/v) [86]. Mellors J.S et al described the use of 1.5 micron

porous ethyl bridged particles as a stationery phase support for reversed phase ultra-

high pressure liquid chromatography [89]. Alden B.A et al evaluated and the

characterization and evaluation of three novel 5- micron HPLC column packings,

prepared using ethyl-bridged hybrid organic/inorganic materials is described.

59

These highly spherical hybrid particles, which vary in specific surface area

(140, 187, and 270 m(2)/g) and average pore diameter (185,148, and 108 A), were

characterized by elemental analysis, SEM, and nitrogen sorption analysis and were

chemically modified in a two-stepprocess using octadecyl trichlorosilane and trimethyl

chlorosilane [90]. Wyndham KD et al described the characterization and evaluation of

C18 HPLC stationary phasesbased on ethyl-bridged hybrid organic/inorganic particles.

The characterization and evaluation of three novel 5-microm HPLC column packings,

prepared using ethyl-bridged hybrid organic/inorganic materials, is described.

Mechanical strength under conditions normally employed for traditional reversed

phase HPLC applications, using a high-pressure column flow test [91]. MacNair J.E et

al Ultra high pressure reversed phase capillary liquid chromatography isocratic and

gradient elutions using columns packed with 1 micron particles were discussed [95].

Kellog MD et al developed a rapid, accurate, and sensitive method for the

quantification of serum busulfan using direct inject tandem mass spectrometry.

Correlation with an established HPLC-UV method revealed a slope of 0.98, an

intercept of 0.1, and r = 0.95 (n = 48). No significant interfering substances or ion

suppression was identified [100]. M.Rauh et al [101] have developed a saliva method

as an alternative, less invasive way of quantifying busulfan in paediatric patients.

Cyclophosphamide and thiotepa are often co-prescribed for the treatment of advanced

breast, ovarian and testicular tumours.

Jonge ME et al developed simultaneous quantification of cyclophosphamide,4-

hydroxyCyclophosphamide ,N,N',N"triethylenethio phosphoramide(thiotepa) and

N,N',N"-triethylenephosphoramide(tepa) in human plasma was studied using high-

performance liquid chromatography coupled with electrospray ionization tandem mass

spectrometry. This robust and rapid LC/MS/MS assay is successfully applied for

routine therapeutic drug monitoring of CP, thiotepa and their metabolites in hospital

[102]. G. Remaud M et al developed asimple and reliable LC/MS-MS method allows

specific, sensitive and reproducible quantification of drugs in human plasma and can

be applied to further pharmacokinetic studies in patients treated with FT-based pro

drugs [103]. Swartz, M.E etaldescribed using sub‐2 µm particles and mobile phases at

highlinear velocities, and instrumentation that operates at higher pressures than those

60

used in HPLC,dramatic increases in resolution, sensitivity, and speed of analysis can

be obtained [104].

D. Goodalletal studied the use of elevated and high temperatures in high

performance liquid chromatography (HPLC) is reviewed, encompassing the range

ambient to 2500C and with examples covering separations of both small molecules

and biomolecules. Comparisons are made between temperature gradient and solvent

gradient elution, and requirements for successful elevated temperature HPLC are

Described[105].

Samples for thalidomide analysis should be buffered with Sorensen’s citrate

buffer to prevent spontaneous hydrolysis of the drugsamples are then stable at room

temperature for at least 24 h and at −200C for significantly longer [106]. C18 analytical

columns can be used for both these drugs. Although the structures of these drugs are

similar, it has been shown that APCI negative ionisation provides the most sensitivity

for thalidomide analysis [107], whereas APCI positiveionisationis preferred for

lenalidomide [108]. Tamoxifen is an oestrogen antagonist used to treat patients with

oestrogen-receptor positive breast cancer. It may be considered a pro drug as its

metabolites such as 4-hydroxy tamoxifen are more active than the drugitself, so it is

important to quantify of metabolites as well as the parentdrug. Both tamoxifen and

procarbazine can be measured in electrospray positive mode with a C18 analytical

column [111,112]. LC–MS/MS methods are available for the measurement of digoxin.

The majority use liquid–liquid extraction [113–115] with MTBE orchloroform, but SPE

extraction methods were also available [116,117], including a 96-well plate SPE

method [118] which may help to increase the throughput of the method. C18 is the

column of choice, and electrospray positive ionisation is used in the methods.

Lippert J.A et al studied the effect of column diameter on efficiency and

sensitivity. Columns packed with nonporous C6 particles produced higher efficiencies

than columns packed with a 1.5 micron porous octadecyl silane-modified material.

The new valve was more reproducible, convenient, and required much less sample

than previously used injection systems [120]. R.Nageswara Rao et al published an

extensive survey of the literature invarious analytical and pharmaceutical

61

chemistry related journals has been conducted and the high-performance liquid

chromatography (HPLC) methods which were developed and used for determination

of process-related impurities in drugs have been reviewed. This review covers the

time period from 1995 to 2001 during which around 450 analytical methods including

all types of chromatographic and hyphenated techniques were reported. A critical

analysis of the reported data has been carried out and the presentstate-of- art of

HPLC for determination of impurities of analgesic, antibiotic, anti-viral, anti-

hypertensive, anti-depressant, gastro-intestinaland anti neoplastic agents has been

discussed [124]. Kaila H.O et al have developed a simple and rapid ultra-performance

liquid chromatographic assay method for the simultaneous determination of aspirin,

clopidogrel bisulphate and atorvastatin calcium in capsule dosage form. Linearity

range for aspirin, clopidogrel bisulphate, atorvastatin calcium and Clopidogrel

bisulphate was found to be in 5-25 and 150-750 mcg/ml respectively. Accuracy,

Precision repeatability study were carried and found within range [135].

Saurabh K Sinhaet aldeveloped and validate d a simple and rapid isocratic

reversed-phasehigh-performance liquid chromatographic method (RP-HPLC)for the

simultaneous estimation of amlodipin and telmisartanin combined dosage form. The

method was validated in terms ofaccuracy, precision, linearity, range, and specificity,

limit of detection and limit of quantitation. Linearity for amlodipine besylate and

telmisartanwas established in the range of 5-30 and 10-60 g/mL, respectively.The

recoveries for the two compounds were above 96% [136].

Plumb R., et al discussed thetechnology of Ultra-performance liquid

chromatography (UPLC) utilizing sub-2 micron particles has been applied to the study

of in vivo drug metabolism, in particular the analysis of drug metabolites. The reduction

in peak width significantly increases analytical sensitivity by three-to five-fold.The

application of UPLC/MS resulted in the detection of additional drug metabolites,

superior separation and improved spectral quality [139]. Wilson I.D., et al described the

first functional genomic application of UPLC-MS technology is illustrated here with

respect to multivariate metabolic profiling of urines from males and females of two

groups of phenol typically normal mouse strains and a "nude mouse" strain. Also

compared this technology to conventional HPLC-MS under similar analytical conditions

62

and show improved phenotypic classification capability of UPLC-MS analysis together

with increased ability to probe differential pathway activities between strains as a result

of improved analytical sensitivity and resolution [141].

S.Gorog, described the general scheme set up for the estimation of the impurity

profile of bulk drug substances by the complex use of chromatographic,

spectroscopicand hyphenatedtechniques.Several examples are presented as

illustrations to the scheme from the authors' laboratory involving the use of

chromatographic methods such as thin-layer-(TLC), gas-(GC), (HPLC), spectroscopic

methods such as mass spectrometry (MS) and NMR spectroscopy as well as

hyphenated techniques (HPLC/diode-array UV, GC/MS and HPLC/MS) [146].

Robert E. Ardrey et al explained the basic principles of liquid chromatography

and mass spectrometry and then discussing the current applications and practical

benefits of LC-MS, along with descriptions of the basic instrumentation, this title will

prove to be the indispensable reference source for everyone wishing to use this

increasingly important tandem technique. Discusses current applications of LC-MS and

shows benefits of using this technique in practice [148].

Oliveira R V et al described a sensitive and specific liquid chromatography-

mass spectrometry (LC-MS) method has been developed and validated for the

enantioselective determination of ifosfamide. The enantioselective separations were

achieved using a mobile phase composed of 2-propanol:methanol (60:40, v/v) and a

flow rate of 0.5 ml/min. The method was applied to the analysis of plasma samples

obtained from a cancer patient who received 3.75 g/m(2)/day dose of (R,S)-ifosfamide

as a 96-h continuous infusion [152]. Quernin MH et al developed a new method

coupling liquid chromatography with mass spectrometry (LC-MS) and was validated for

the determination of busulfan concentrations in plasma. This method was used to

determine the pharmacokinetic parameters of busulfan after the first administration of 1

mg/kg orally, in 13 children receiving the drug as part of the preparative regimen for

bone marrow transplantation. Recovery was 86.7%, the limit of detection was 2.5 ng/ml

and linearity ranged from 5 to 2500 ng/ml [153].

63

D.R. Bunch et al studied a fast and simple LC-MS/MS method coupled with

turbulent flow online sample cleaning technology described here offers reliable busulfan

quantitation in serum or plasma with minimum manual sample preparation and was

fully validated for clinical use [154]. E.O. dos Reis et al described a sensitive and

specific assay for detection of busulfan in human plasma and the method was

successfully applied to analyze plasma samples obtained from six adults receiving

doses of 1 mg kg (-1) in a conditioning regimen prior to bone marrow transplantation

[155]. Kellogg MD et al developed a rapid, accurate, and sensitive method for the

quantification of serum busulfan using direct inject tandem mass spectrometry.

Correlation with an established HPLC-UV method revealed a slope of 0.98, an

intercept of 0.1, and r = 0.95 (n = 48).No significant interfering substances or ion

suppression was identified[156]. M.Rauh, et al described quantification of busulfan in

saliva and plasma in haematopoietic stem cell transplantation in children and

Validation of liquid chromatography tandem mass spectrometry method [157].

De Jonge ME et al established simultaneous quantification of cyclophosphamide,4-

hydroxycyclophosphamide,N,N',N"-triethylenethiophosphoramide(thiotepa) and

N,N',N"-triethylenephosphoramide (tepa) in human plasma was studied using high-

performance liquid chromatography coupled with electrospray ionization tandem mass

spectrometry [158].

G. Remaud, et al described simple and reliable liquid chromatography tandem

mass spectrometry (LC/MS-MS) method, chosen for its specificity of detection, for

simultaneously measuring in human plasma FT, 5-FU and 5-FUH2 [159]. Gu Y et al

developed a rapid, simple and sensitive LC/MS/MS method for the simultaneous

quantitation of tegafur (FT) and gimeracil (CDHP) in human plasma with a

concentration range of 20-5000 and 2-500 ng/mL, respectively. The analytes were

stable under all possible conditions of storing and handling for each compound [160].F.

Bai et al developed a sensitive method for the determination of lapatinib (GW572016) in

human plasma was developed using high-performance liquid chromatographic

separation with tandem mass spectrometric detection. Analytes were detected with a

good resolution. This method was validated over a linear range of 100-10,000

ng/mL[161].

64

Liquid chromatography-tandem mass spectrometric assays were developed for

the sensitive, rapid and high throughput bioanalysis of thalidomide in human plasma

and semen reported by S.K. Teo, et al. The methods successfully determined

concentrations of thalidomide from a clinical study to levels as low as 7 ng/ml plasma

and 8 ng/ml semen, respectively [162]. A. Tracquiet.al et al developed an original

method based upon high-performance liquid chromatography coupled to ion spray

mass spectrometry (HPLC-ISP-MS) has been developed for the identification and

quantification in plasma of several cardiac glycosides, namely digoxin, digitoxin,

lanatoside C and acetyl digitoxin [171].

Hashimoto Y, et al evaluated the pharmacokinetics of digoxin in humans, a

sensitive and specific LC/MS/MS method was developed and validated for the

determination of digoxin concentrations in human plasma. The method was shown to

be more sensitive, specific, accurate, and reproducible than common techniques such

as RIA. For detection, a LC/MS/MS system with electro spray ionization tandem mass

spectrometry in the positive ion-multiple reaction-monitoring (MRM) mode was used to

monitor precursor to product ions of m/z 798.5-51.5 for digoxin and m/z 782.5-35.5 for

the internal standard, digitoxin.Lindon JCet aldescribed HPLC-NMR and its application

to drug metabolism was studied. Evaluation of liquid chromatography coupled with

high-field 1H NMR spectroscopy for drug metabolite detection and characterization was

done. Evaluation of liquid chromatography coupled with high-field 1H NMR

spectroscopy for drug metabolite detection and characterization was discussed [175].

3.3 Literature survey on Gefitinib analytical methods

Dae Ho Lee etalretrospectively analysed and suggested gefitinib was of benefit

for chemotherapy-naïve patients who had good performance status and

adenocarcinoma histology. These findings are required to be validated in further

prospective clinical studies, which should include translational research characterizing

the molecular predictors[180]. Kataoka, et al has been reported, the accumulation of

clinical information and theunderlying mechanisms of gefitinib-induced interstitial

lungdisease (ILD) remain insufficient and unclea [181].

65

Asahina H, et al study showed that gefitinib is very active and well tolerated as

first-line therapy for advanced NSCLC with EGFR mutations. DNA was extracted from

paraffin-embedded tissues and EGFR mutations were analysed by direct sequence of

cancer products [185]. Murayama Y et al studied was prospectively designed to

evaluate a phase II study of gefitinib for non-small-cell lung cancer (NSCLC) patients

with epidermal growth factor receptor (EGFR) mutations [187]. This research showed

the need for screening for EGFR mutations in NSCLC patients. Sharyn D.Baker et al

developed a specific method for determination of Gefitinib in human plasma, mouse

plasma and tissues using high performance liquid chromatography coupled to tandem

mass spectrometry [195].

Ling-ZhiWang et al developed a novel, rapid and specific liquid

chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and

validated for the simultaneous quantification of gefitinib and its predominant metabolite,

O-gefitinib in human plasma. Chromatographic separation of analytes was achieved on

an Alltima C18 analytical HPLC column (150 mm × 2.1 mm, 5 μm) using an isocratic

elution mode with a mobile phase comprised acetonitrile and 0.1% formic acid in water

(30:70, v/v) [196].

Lionel Faivre et al developed asimple HPLC-UV method for the simultaneous

quantification of gefitinib and erlotinib in human plasma was reportedexclusively based

on mass spectrometry. This simple, sensitive, accurate cost- effective method can be

used in routine clinical practice to monitor gefitinib or erlotinib concentrations in plasma

from NSCLC patients [197]. XinZheng, et al developed a specific, sensitive, and rapid

method based on high-performance liquid chromatography coupled to tandem mass

spectrometry (LC–MS–MS). The analyte was detected by tandem mass spectrometry

operating in positive electrospray ionization modewith multiple reactions monitoring

(MRM) [198]. Feng Bai et al developed a highly sensitive liquid chromatography

electrospray tandem mass spectrometry (LC‐ESI‐MS/MS) method has been developed

for the measurement of gefitinib(ZD1839) in human plasma. The method was validated

over a linear range of 0.5–1000 ng/mL, using deuterated gefitinib (D8‐ZD1839) as the

internal standard (IS) [199].

66

Y. Hsieh et al developed the method for increasing the speed of HPLC-MS/MS-

based analyses, such as fast chromatography, direct sample injection, parallel

technologies and combined ionization interfaces are also presented in this review. In

addition, the special challenges when performing HPLC-MS/MS bio analysis, such as

the choice of ionization sources, matrixionization suppression and the potential for

endogenous interferences are addressed. The major high throughput strategies

including sample reduction and cassette dosingare discussed [200].

Mike S. Lee, et al described the utility of LC/MS techniques for accelerated

drug development and provides a perspective on the significant changes instrategies

for pharmaceutical analysis. Future applications of LC/MS technologies for accelerated

drug development and emerging industry trends are also discussed [202].

Signor Let al developed a qualitative and quantitative analysis of erlotinib

(RO0508231) and its metabolites was carried out on rat tissue sections from liver,

spleen and muscle which is in good agreement with the quantitative data obtained by

LC-MS/MS analysis [203].

Lankheet, N. et al developed a fast and accurate method for simultaneous

determination of anticancer tyrosine kinase (TKIs) dasatinib, erlotinib, gefitinib,

imatinib, lapatinib, nilotinib, sorafenib and sunitinib in humanplasma was developed

using high-performance liquid chromatography and detection with tandem mass

spectrometry (HPLC- MS/MS). This method was successfully applied for routine

therapeutic drug monitoring purposes in patients treated with the investigated TKIs

[204]. PVV.Satyanarayana et al developed a simple, specific, accurate and precise

reverse phase high performance liquid chromatographic method was developed and

validated for the estimation of Gefitinib in tablet dosage form. The proposed method

was successfully applied for the quantitative determination of Gefitinib in tablet

formulation [205].

67

V Kiran Kumar, et al developed a simple, precise, rapid and accurate reverse

phase HPLC method developed for the estimation of Gefitinib in tablet dosage form.

An Hypersil BDS RP C18,250x4.6 mm, 5 _m partical size, with mobile phase consisting

of 0.02 M Dipotassium Hydrogen ortho phosphate and Methanol in the ratio of 10:90

v/v was used. The flow rate was 1.0 ml/min and the effluents were monitored at 246 nm

[206].

3.4 Literature survey on Imatinib Mesylate analytical methods

Imatinib Drug Preparation was reported by Zimmermann,J. et al. The

condensation of 1-(3-pyridyl)ethanone (I) with dimethylformamidedimethylacetal (II)

gives 3-(dimethylamino)-1-(3-pyridyl)-2-propen-1-one (III), which is cyclized with 1-(2-

methyl-5-nitrophenyl)guanidine (IV) [obtained by reaction of 2-methyl-5-nitroaniline (V)

with cyanamide (VI)] in refluxing isopropanol to yield the pyrimidine derivative (VII).

Reduction of the nitro group of (VII) with H2 over Pd/C in THF affords the corresponding

amino compound (VIII), which is finally condensed with 4-(4-methylpiperazin-1-

ylmethyl)benzoyl chloride (IV) in pyridine [208].

Courtney M. Callis., et al proposes a risk assessment approach for developing

GTI limits based on shorter-term exposure durations by highlighting marketed

anticancer compounds with limited dosing schedules (e.g., 2 years). These limits are

generally higher than the defaulted threshold of toxicological concern (TTC of 1.5

μg/day) and can result in more easily developed and less complex analytical methods

[209]. Stefen Federal et al studied the Biology of Chronic Myeloid Leukemia and the

mechanisms of the disease Chromnic Myeloid Leukemia (CML). It was among the first

neoplastic diseases in which therapy with a biologic agent was found to suppress the

leukemic clone and proloneged survival [210].

D.Ivanovic, et al developed reversed-phase high-performance liquid

chromatographic (RP-HPLC) method has been developed and validated for the

simultaneous determination of imatinibmesylate and of the impurity productin Glivec

capsules (Novartis, Switzerland). The method was validated statistically for its

selectivity, linearity, precision, accuracy and robustness. Due to its speed and

accuracy, the method may be used for quality control analyses [211].

68

Muller, L., et al describeda procedure for testing, classification, qualification,

toxicological risk assessment, and control of impurities possessing genotoxic potential

in pharmaceutical products. Referencing accepted principles of cancer risk

assessment, this document proposes a staged threshold of toxicological concern (TTC)

approach for the intake of genotoxic impurities over various periods of exposure [213].

V.V.Vivekanand, et al described an isocratic reversed-phase liquid chromatography

method with UV detection has been developed for the purity evaluation of

imatinibmesylate in bulk drug. The method is selective and is capable of detecting all

process intermediates and other related compounds, which may be present at trace

levels in the drug substance [214]. Szczepek WJ, et al studiedthe decomposition

ofimatinibmesylate (ImM) under hydrolytic (neutral, acidic, alkaline), oxidative and

photolytic conditions was studied. The main degradation products under acidic and

alkaline conditions were isolated from the reaction mixtures and identified by the HPLC,

1H NMR and MS techniques. During stress study the suitability of the proposed HPLC

method to control purity of the samples was verified [215].

M. Medenica, et al determined an optimal RP-HPLC chromatographic

conditions for the separation of imatinibmesylate and its impurity STI 509-00

experimental design 2(4) was applied. All the factors that affect imatinibmesylate/STI

509-00 separation, as well as their mutual interactions were investigated. From the

experimentally determined capacity factor values, it was defined the factors that affect

to chromatographic system at the most [216].

Saranjit Singh et al subjected Ezetamib to different ICH prescribed stress

conditions. Degradation was found to occur in hydrolytic and to some extent in

photolytic conditions, while the drug was stable to oxidative and thermal stress. The

drug was particularly labile under neutral and alkaline hydrolytic conditions. A stability-

indicating HPLC method was developed for analysis of the drug in the presence of the

degradation products [218]. Sulfonic acid esters are considered as potentially

alkylating agents that may exert genotoxic effects in bacterial and mammalian cell

systems were discussed by Glowienke S, et al. In this study the iso-propyl esters of the

three sulfonic acids under study were found to be the strongest mutagens, either when

69

tested in the Ames test or the micronucleus assay, whereas p-toluenesulfonic acid iso-

butyl ester was the only compound shown to be devoid of a genotoxic potential in both

tests [225].

3.5 Literature survey on Bortezomib analytical methods

In 1936 diborane, B2H6, was a rare substance, prepared by Brown HC in less

than gram quantities in only two laboratories, that of Alfred Stock at Karlsruhe,

Germany, and of H. I. Schlesinger, at the University of Chicago. The existence of the

simplest hydrogen compound of boron, not as BH3, but as B2H6, was considered to

constitute a serious problem for the electronic theory of G. N. Lewis.The reactions of

diborane were under study at the University of Chicago by Professor H. I. Schlesinger

and his research assistant, Anton B. Burg, in the hope that a knowledge of the

chemistry would aid in resolving the structural problem [231]. Mikhael HJC et al

described Bortezomib is the first drug to act as a proteasome inhibitor, and has yielded

impressive anticancer effects in phase III trials in multiple myeloma. Its role in other

hematological malignancies is being evaluated.Proteasome inhibition, therefore,

represents a novel mechanism for anticancer therapy [232].

Richardson PG, et al studied combinations of Bortezomib plus established and

novel agents, such as melphalan-prednisone, dexamethasone, doxorubicin,

thalidomide-dexamethasone and, most recently, lenalidomide- dexamethasone, are

proving superior to or more promising than previous standards of care such as

bortezomib [233]. Wu S, et.al. described the degradation pathways of a peptide boronic

acid derivative, 2-Pyz-(CO)-Phe-Leu-B(OH)(2) was discussed. Degradation of 2-Pyz-

(CO)-Phe-Leu-B(OH)(2) under acidic and basic conditions seemed to be mediated by

an initial oxidative degradation pathway similar to that seen with the peroxide. In the

presence of hydrogen peroxide, the boronic acid group was cleaved from 2-Pyz-(CO)-

Phe-Leu-B(OH)(2) to give an alcohol with an apparent retention of the original stereo

chemistry. Subsequent isomerization and further hydrolysis were then seen [243].

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Ji Y Zhang, et al described a sensitive and specific liquid chromatography-

tandem mass spectrometry assay was developed. The chromatographic separation

was carried out on a narrow-bore reversed-phase Zorbax XDB-C(8) HPLC column with

a mobile phase of acetonitrile/water (40:60, v/v) containing 10 mm ammonium acetate

(pH 7.4). The analytes were ionized using negative-to-positive switch electrospray

mass spectrometry, then detected by multiple reaction monitoring with a tandem mass

spectrometer [244].

C. Rambabu, et al attempted to develop and validate a simple, economic,rapid,

and accurate method. In present investigation three simple and sensitive extractive UV-

Visible spectrophotometric methods have been developed and validated for the

determination of bortezomib in formulation dosage form [246]. J. S.Daniels, et al

described the Bortezomib [N-(2,3-pyrazine)carbonyl-L-phenylalanine-L-leucineboronic

acid] is a potent first-in-class dipeptidylboronic acid proteasome inhibitor that was

approved in May 2003 in the United States for the treatment of patients with relapsed

multiple myeloma where the disease is refractory to conventional lines of therapy.

Bortezomib was identified and characterized by liquid chromatography mass

spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry

(LC/MS/MS). The development of bortezomib has provided an opportunity to describe

themetabolism of a novel boronic acid pharmacophore [247].

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3.6 Literature survey on Eplerenone analytical methods

J.A. Spertus et al provides an overview of the considerations made regarding

quantification of a range of clinical and economic outcomes in the EPHESUS

(EplerenoneneuroHormonal efficacy and survival study) study, a 6200-patient,

randomized, controlled trial of aldosterone blockade in recent studies with Eplerenone,

a patients with heart failure as a complication of acute myocardial infarction [248].

J.Y.Zhang et al developed a sensitive and specific liquid chromatography-

tandemmass spectrometry assay was developed to quantifythe first selective

aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human plasma.

The chromatographic separation was carried out on a narrow-bore reversed-phase

Zorbax XDB-C(8) HPLC column with a mobile phase of acetonitrile/water (40:60, v/v)

containing 0.1 M ammonium acetate (pH 7.4) [262].

V.Rane, et al developed a novel stability-indicating reversed-phase (RP) HPLC

method has been developed and validated for quantitative analysis of eplerenone in the

bulk drug and in a pharmaceutical dosage form. UV detection was performed at 240

nm. The method was validated for linearity, accuracy (recovery), precision, specificity,

and robustness. Degradation was observed in acid, base, and 30% H2O2 .The

degradation products were well resolved from main peak [263].

Paraag Gide, et al developed arapid and simple high performance liquid

chromatography (HPLC) method with a UV detection (241 nm) was developed and

validated for estimation of Eplerenone from spiked human plasma. The analyte and

theinternal standard (valdecoxib) were extracted with a mixture of dichloromethane and

diethyl ether. The chromatographic separation was performed on a HiQSil C-18HS

column (250 mm×4.6 mm, 5 μm) with a mobile phase consisting of acetonitrile:water

(50:50, v/v) at flow rate of 1 mL/min [264]. Qian WJ, et al developed a sensitive high

performance liquid chromatography electrospray ionization-mass spectrometry (HPLC-

ESI-MS) method was established for the determination of eplerenone (EP) in human

plasma. EP was determined with electrospray ionization-mass spectrometry (ESI-MS)

in the selected ion monitoring (SIM) mode [265]. Brajesh Mahajan, et al developed a

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simple, selective and precise thin-layer chromatography/densitometry method has been

developed for determination of eplerenone, both as a bulk drug and in tablet

formulation. The study indicates that the drug is susceptible to acid, base hydrolysis,

oxidationand stable for photochemical and thermal conditions [266].

M. Naresh Chandra Reddy et al developed a simple reverse phase gradient

HPLC method used for simultaneous identification of Eplerenone impurities in market

tablet dosage form. Flow rate 1ml/min and injection volume 2μl chromatograms

observed at 240nm. validated its use full for stability studies and other pharmaceutical

analysis [267]. E. Lesellier, et aldescribes a new test designed in subcritical fluid

chromatography (SFC) to compare the commercial C18 stationary phase properties.

One hundred and twenty-nine columns were analysed by this simple and rapid test,

which allows a comparison of columns with the aim of helping along their choice in

HPLC [269].

3.7 Literature survey on NilotinibHCl analytical methods

Davies, A.E et al described a high performance liquid chromatography (HPLC)

method that separates two of the currently licenced tyrosine kinase inhibitors (TKIs);

nilotinib (AMN107, Tasigna) and imatinib (STI571, Glivec), together with its main

metabolite, CGP-74588, from human plasma. After solid phase extraction the drug mix

was separated through a Gemini C6-phenylcolumn (150 mm x 4.6mm, i.d.; 5 microm)

(Phenomenex). This simple and novel method may be used to quantify levels of TKIs

when used alone or in combination with drug treatments for clinical samples [277].

Parise RA, et al developed a LC-MS assay method for Nilotinib (AMN-107,

Tasigna) is a small-molecule inhibitor of BCR/ABL, approved for chronic myelogenous

leukemia. An LC-MS assay for sensitive, accurate and precise quantitation of nilotinib

in 0.2 mL human plasma or serum was developed and validated. Demonstrated the

suitability of this assay by quantitating plasma concentrations of nilotinib in a healthy

volunteer after oral administration of 400 mg Nilotinib [278]. Satyanarayana L et al

developed a simple, precise, rapid and accurate reverse phase HPLC method was

developed for the estimation of Nilotinib in capsule dosage form. A RP Inertsil ODS-3V

C-18, 250x4.6 mm, 5m partical size, with mobile phaseconsisting of 0.2 M Dipotassium

73

hydrogen orthophosphate in water pH-2.5 with orthophosphoric acid and Acetonitrile in

the ratio of 60:40 v/v was used. The flow rate was 1.0 ml/min and the effluents were

monitored at 266 nm. The retention time was 8.508 min. The method was validated by

determining its accuracy, precision and system suitability [279]. Yuki M, et alstudied

aprecise and convenient high-performance liquid chromatography (HPLC) method has

been established to assay nilotinib in human plasma. Chromatographic separation of

nilotinib was performed on a Li Chrosphere100 RP-18(e) column (250 mm×4.0 mm, 5

µm) using a mixture of acetonitrile and 0.01 M phosphate buffer (pH 3.0) (42:58, v/v)

under isocratic conditions at a flow rate of 1.0 ml/min with ultraviolet (UV) detection at

266 nm. The calibration curve showed linearity at concentrations between 250 ng/ml

and 5000 ng/ml (r(2)>0.999) [280]. A new method using high performance liquid

chromatography coupled with electrospray mass spectrometry is described for the

quantification of plasma concentration of tyrosine kinase inhibitors imatinib, dasatinib

and nilotinib. Chromatographic separation of drugs and Internal Standard (quinoxaline)

was achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18

reverse phase analytical column with 20 min of analytical run, at flow rate of 1 ml/min

[281]. Gotze L et al developed a simultaneous test for six TKIs (erlotinib, imatinib,

lapatinib, nilotinib, sorafenib, sunitinib) was developed using liquid chromatography

tandem mass spectrometry in a multiple reaction monitoring mode and separated using

a gradient of acetonitrile containing 1% formic acid with 10 mM ammonium ormiate on

an analytic RP-C18 column [282].

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3.8 Scope of the present study

Perusal of literature revealed that research investigation on Pharmaceuticals

and Biomedical analysis gained significant importance. In addition, determination and

quantification of impurities in anti cancer and Antihypertensive Active Pharmaceutical

Ingredients (APIs) using the developed and validated stability indicating UPLC

methods have been reported by several authors.

In development of new drugs, the determination of purity is important in order

to establish the acceptability of batches for safety assessment and clinical trials. These

activities are considered to be the primary objectives of drug manufactures. There is an

ever increasing interest in impurities present in the API’s.

In general, process impurities include trace substances resulting from the drug

substance synthesis, and traceside-reaction products or contaminants not removed by

the isolation and purification schemes. Regulatory authorities set various levels for

individual and total impurities in both drug substances and drug products that must be

reported and investigated. Ideally, the total amount of impurity, aswell as the quantity

ofeach individual impurity, should be monitored in the bulk drugs. The separation,

identification and determination of each impurity in a variety of sample matrices

complicate the development of analytical procedures.

Advanced analytical techniques, particularly the Ultra Performance

chromatographic techniques permit analysis to be carried out more rapidly in complex

matrices than conventional methods. Further, micro quantities of substances are

sufficient for carrying out the investigations. Today, the analytical methodology is in a

period of transition due to technological advances and modified policies of the

governmental regulatory agencies.

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The process of profiling involves, identification isolationby preperativeHPLC

and structural elucidation by LC/MS. LC–MS/MS is an increasingly important tool in

therapeutic drug monitoring as it offers increased sensitivity and specificity compared

to other methods, and may be the only viable method for quantifying drugs without

natural chromophores or fluorophores. For large molecules capillary electrophoresis is

useful and coupling to MS helps to elucidate the structure of impurities. Process

development has to make sure that during the scale up the product is within the set

specifications. Impurity profile employes degradation studies to identify potential

impurity candidates. It also involves development of a robust method as well as

validation of the method. In many countries the regulations and the practices of drug

manufacturing have changed because of the recently developed ability of analysis

equipped with modern instrumentation to determine low concentration of impurities.

HPLC and other chromatographic techniques are increasingly popular in the field of

pharmaceutical analysis. The anticancer active pharmaceutical ingredients are very

important therapeutic agents in treating patients with various types of cancer. There

are several kinds of anticancers and antihypertensive depending upon mode of action.

Moreover, reported analytical methods in literature lack the suitable procedure

for quantification and estimation of impurities in anticancer and antihypertensive drug

substances. Perusal of literature survey also indicates that, no UPLC methods with

identification of degradation impurities have yet been reported for the determination

and quantification of these drug substances. Therefore, it is thought to undertake the

development and validation of reversed phase UPLC method with lower LOQ for the

quantification of impurities and degradation products in the subjected drug substances.

The anticancer active pharmaceutical ingredients like ‘Gefitinib,

Imatinibmesylate, Bortezomib and NilotinibHydrochloride’ were selected for the

present study. The atypical antihypertenisive drug like ‘Eplerenone’ was selected for

development of analytical methods for impurity profiles in the present research study.

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In the present research investigation, author has been proposed to quantify

some of the life saving anticancer drugs with high therapeutic use employing

advanced analytical studies. Hence, efforts were made to identify and elucidate the

structure of those impurities using advanced techniques primarily such as

RRLC/UPLC, LC/MS. The other techniques FTIR and 1H /13c NMR spectral analysis

were employed wherever required. The present work has been proposed to develope

and validate new Ultra Performance (rapid) analytical methodologies for separation

and determination of trace level impurities of some of the popular i) Anticancer ii)

Antihypertensive drugs. This research investigation will add new dimensions to the

existing literature in the filed of chromatographic and spectroscopic studies on

pharmaceutical products.