Persona Et Al. - 2015 - Analytical Methodologies for the Determination of Benzodiazepines in Biological Samples

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Journal of Pharmaceutical and Biomedical Analysis 113 (2015) 239–264

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Journal of Pharmaceutical and Biomedical Analysis

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eview

nalytical methodologies for the determination of benzodiazepines iniological samples

arolina Personaa, Katarzyna Madeja,∗, Paweł Knihnickia, Wojciech Piekoszewskia,b

Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University, 30-060 Krakow, PolandLaboratory of High Resolution Mass Spectrometry, Regional Laboratory of Physicochemical Analysis and Structural Research, Faculty of Chemistry,

agiellonian University, 30-060 Krakow, Poland

r t i c l e i n f o

rticle history:eceived 22 October 2014eceived in revised form 5 February 2015ccepted 9 February 2015vailable online 17 February 2015

eywords:enzodiazepinesody fluids

a b s t r a c t

Benzodiazepine drugs belong to important and most widely used medicaments. They demonstrate suchtherapeutic properties as anxiolytic, sedative, somnifacient, anticonvulsant, diastolic and muscle relaxanteffects. However, despite the fact that benzodiazepines possess high therapeutic index and are con-sidered to be relatively safe, their use can be dangerous when: (1) co-administered with alcohol, (2)co-administered with other medicaments like sedatives, antidepressants, neuroleptics or morphine likesubstances, (3) driving under their influence, (4) using benzodiazepines non-therapeutically as drugs ofabuse or in drug-facilitated crimes. For these reasons benzodiazepines are still studied and determined ina variety of biological materials. In this article, sample preparation techniques which have been applied

lternative biological materialsample preparation techniquesnalytical methods

in analysis of benzodiazepine drugs in biological samples have been reviewed and presented. The nextpart of the article is focused on a review of analytical methods which have been employed for pharma-cological, toxicological or forensic study of this group of drugs in the biological matrices. The review waspreceded by a description of the physicochemical properties of the selected benzodiazepines and two,
very often coexisting in the same analyzed samples, sedative-hypnotic drugs.
© 2015 Published by Elsevier B.V.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
2. Sample preparation techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Sample preparation of conventional body fluids . . . . . . . . . . . . . . . . . . .2.2. Sample preparation of alternative biological materials . . . . . . . . . . . .

Abbreviations: 2D UTLC, two-dimensional ultra-thin-layer chromatography; ACN, azation; AP-MALDI-MS(MS), atmospheric pressure matrix-assisted laser desorption/ionizSTFA, N,O-bis-(trimethylsilyl)trifluoroacetamide; BZD, benzodiazepines; C16MIMBr, 1-ceide; CE, capillary electrophoresis; CEC, capillary electrochromatography; CEC-MS(TOF

onization; CPE, cloud-point extraction; CS, column-switching; CV, coefficient of variationive liquid–liquid microextraction; DPCAdSV, differential pulse cathodic adsorptive stripnfluence of drugs; ECD, electron capture detector; EDDP, 2-ethylidene-1,5-dimethyl-3,3-ay technique; ESI, electrospray ionization; GC, gas chromatography; HPLC, high performC, liquid chromatography; LC–HRMS, liquid chromatography–high resolution mass spuantification; MAE, microwave-assisted extraction; MALDI-MS, matrix-assisted laser deDEA, 3,4-methylenedioxy-N-ethylamphetamine; MDMA, 3,4-methylenedioxy-N-meth

arly imprinted solid-phase extraction; MRM, multiple reaction monitoring; MS, mass

-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide; NICI, negative-ion chemical ioDA, photodiode array detector; PFSPE, packed-fibre solid-phase extraction; PLS, partiheap Effective Rugged Safe; RAM, restricted access material; SRMM–MECC, stacking andodecyl sulfate; SERS, surface enhanced Raman spectroscopy; SPE, solid-phase extractioert-butyldimethylsilyl; THF, tetrahydrofuran; TMCS, trimethylchlorosilane; TOF, time-oPLC, ultra performance liquid chromatography; UV, ultraviolet detection.∗ Corresponding author. Tel.: +48 12 6635602; fax: +48 12 6635601.

E-mail address: [email protected] (K. Madej).

ttp://dx.doi.org/10.1016/j.jpba.2015.02.017731-7085/© 2015 Published by Elsevier B.V.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

cetonitrile; AP, atmospheric pressure; APCI, atmospheric pressure chemical ion-ation mass spectrometry; AuNPs, gold nanoparticles; BSA, bovine serum albumin;tyl-3-methylimidazolium bromide; C16MPYB, N-cetyl-N-methylpyrrolidinium bro-), capillary electrochromatography-time of flight mass spectrometry; CI, chemical

; DAD, diode array detector; DFSA, drug facilitated sexual assault; DLLME, disper-ping voltammetry; DPV, differential pulse voltammetry; DUID, driving under thediphenylpyrrolidine; EI, electron ionization; EMIT, enzyme-multiplied immunoas-

ance liquid chromatography; HS-SPME, headspace solid-phase microextraction;ectrometry; LLE, liquid–liquid extraction; LOD, limit of detection; LOQ, limit ofsorption/ionization-mass spectrometry; MDA, 3,4-methylenedioxyamphetamine;ylamphetamine; MECC, micellar electrokinetic chromatography; MISPE, molecu-spectrometry; MSTFA, N-methyl-N-(trimethylsilyl)trifluoroacetamide; MTBSTFA,

nization; NPD, nitrogen–phosphorus detector; PARAFAC, parallel factor analysis;al least squares; PSA, primary–secondary amine sorbent; QuEChERS, Quick Easy

reverse migration micelles–micellar electrokinetic chromatography; SDS, sodiumn; SPME, solid-phase microextraction; RSD, relative standard deviation; TBDMS,

f-flight; UA-DLLME, ultrasound-assisted dispersive liquid–liquid microextraction;

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240 K. Persona et al. / Journal of Pharmaceutical and Biomedical Analysis 113 (2015) 239–264

3. Analytical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2433.1. Chromatographic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

3.1.1. Liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2463.1.2. Gas chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

3.2. Chromatography-related techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2603.3. Electrochemical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2603.4. Miscellaneous methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

. . . . . .

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tetramethylammonium hydroxide and propyliodide (first step) andtriethylamine/propionic anhydride, 1:1, v/v (second step) [13],MTBSTFA/ACN/ethyl acetate (20:40:40, v/v/v) [14]. The extracts of

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Benzodiazepines (BZD) are a large group of drugs that werentroduced to medical practice in the 1960s. Owing to theirherapeutic effect (namely: anxiolytic, sedative, somnifacient, anti-onvulsant, diastolic and muscle relaxant), BZD belong to the mostften prescribed and applied pharmaceuticals [1]. Nowadays, therere over 50 BZD derivatives available worldwide, with the vastajority being under the international control of the Convention

n Psychotropic Substances. According to the International Nar-otics Control Board, in the last ten years the most commonly usedZD were: alprazolam, chlordiazepoxide, diazepam, flunitrazepam,

orazepam, lormetazepam, nitrazepam, temazepam, and triazolam1]. It is estimated that every year about 10–20% of adults livingn the developing countries take these drugs [2]. Furthermore, theffectiveness and safety in their use, as well as the low costs of theseypes of pharmaceuticals affect their spread.

Due to their properties, BZD belong to the most commonly pre-cribed medications worldwide and also, as a result, to the mostommonly abused pharmaceuticals. Despite the fact that overdosesappen more often than in the case of any other drugs, BZD areelieved to be relatively safe, mainly because of the ability of theuman organism to adapt quite quickly to an increased level ofhe drug in the blood stream. However, the safety of using BZDecreases when co-administered with alcohol or other sedatives,ntidepressants, neuroleptics and morphine-like substances, as itan cause a synergistic effect [1]. Taking BZD together with alcohols especially dangerous, as this combination may cause significantethargy, thus increasing the probability of traffic or home acci-ents [3,4]. Abuse of BZD action based on losing consciousnessnd memory, and sex crimes connected with them, stands for aarticular problem. A victim under the influence of the drug haso recollection of the events and, therefore, BZD determination iniological materials becomes essential evidence in drug-facilitatedrimes [5]. Another problem is connected with a number of casesf driving under the influence of drugs (DUID) [6,7]. The problems now so significant that in England and Wales, in the period fromebruary 2010 to March 2011, more than 100 cases (out of 376 con-rolled people) were reported as driving under the influence of BZD7].

The structures of BZD are diversed, however a heterocycliciazepine ring is most often condensed with a benzene ring. Deriva-ives of 1,4-benzodiazepine belong to the most common sub-group,ith their prototype of chlordiazepoxide, which is a derivative of

he amidine type. Introduction of a lactam structure in the place ofhe amidine group gave the most representative drugs in this group,hich may be considered as derivatives of diazepam (Table 1).

rom the physico-chemical point of view BZD are lipophilic com-ounds with a relatively high octanol-water partition coefficiente.g. for diazepam log P = 2.8). They are quickly absorbed upon
ral intake, and therefore the bioavailability depends mainlyn their form and the way of administration (e.g. bioavailabil-ty of diazepam from a tablet is 100% but from suppository
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

50–60%). Some physicochemical and pharmacological propertiesof individual drugs from the BZD group are presented in Table 1.

The metabolism of BZD covers 3 pathways: hydroxylation,demethylation and glucuronidation. Biotransformation reaction,where 1,4-benzodiazepines like diazepam, nordazepam (alsoknown as nordiazepam) and temazepam are mainly metabolizedand excreted as oxazepam or oxazepam glucuronide, belongs to thebest-known metabolic pathways. Similarly, clorazepate is metab-olized to oxazepam through nordazepam. Oxazepam is the mostoften found metabolite of the majority of 1,4-benzodiazepines inurine [1,2,8]. Most of the products of metabolism can also be phar-macologically active and may be used in treatment.

The hazardous interactions of BZD with alcohol and other phar-maceuticals exhibiting similar pharmacodynamic action, and theiruse in drug-facilitated crimes, as well as driving under the influ-ence of the drugs explains the legitimacy of BZD determinationand the search for more effective methods for isolation of themfrom biological material.

Our review covers development of analytical methodologies,including sample preparation techniques and subsequently usedanalytical methods for the determination of BZD drugs, which werereported in majority of the important publications over the last tenyears.

2. Sample preparation techniques

Generally, the choice of sample preparation technique dependson the following main factors: sample matrix, analyte, the aim ofanalysis, and the analytical technique used in connection with thelast issue.

Liquid–liquid extraction (LLE) and solid-phase extraction (SPE)procedures are predominately used for the isolation of BZD frombiomatrices. The use of these extraction techniques usually requiresan appropriate pretreatment of a sample, like adjustment of thesample pH or protein precipitation. Some BZD and/or their phaseI metabolites can be conjugated with glucuronic or sulfuric acid.Therefore, cleavage of conjugates is often performed; especiallyin urine. In the case of keratin matrices’ (hair, nails) analysis,additional initial procedures of washing, cutting and pulverizationbefore an extraction process are necessary.

In some cases of BZD determinations by the GC–MS method, theobtained extracts, especially from whole blood, as well as from oralfluid, were submitted to the derivatization step with such reagentsas N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTB-STFA) [9,10], acetonitrile (ACN)/MTBSTFA (80:20, v/v) [11,12],

urine samples, analyzed for oxazepam by the fluorescence method,were derivatized with cerium(IV), which is a catalyst, in concen-trated ortho-phosphoric acid (85%) [15].

K. Persona et al. / Journal of Pharmaceutical and Biomedical Analysis 113 (2015) 239–264 241

Table 1Physicochemical and pharmacological data for benzodiazepines and other sedative-hypnotic drugs [115].

Name Structure Dissociationconstant pKa

Partitioncoefficientlog Pa

Dailydose(mg)

Therapeuticconcentration(mg L−1)

Toxicity(mg L−1)

Biologicalhalf-life (h)

Volume ofdistribution(L kg−1)

Renal clearance(mL min−1 kg−1)

1,4-Benzodiazepine (R1–R7 = substituents dependent on a kind of benzodiazepine drug [116]).

Alprazolam 2.4 2.12 1–3(max. 10)

0.2–0.4b 0.1–0.4b 11–15c ∼0.7 ∼0.7

Clonazepam1.5

2.41 4–8 0.02–0.07c >0.1c 20–40c 3 1.5

10.5

Diazepam 3.3 2.8 5–30 0.1–1.0c >1.5c 20–100b 0.5–2.5 0.3–0.5

Flunitrazepam 1.8 2.1 0.5–2 0.0015–0.002c,d –e 16–35c 3.5–5.5 2

Oxazepam1.7

2.24 30–120 0.5–2c >2c 4–15c 0.5–2 1–2

11.6

Nitrazepam3.2

2.25 5–10 0.03–0.07c >0.2f 18–386 2–3 110.8

Nordazepam3.5

2.93 15 0.17c,g >0.587c 25–200c 0.5–2.5 0.1–0.312.0


Table 1 (Continued)

Name Structure Dissociationconstant pKa

Partitioncoefficientlog Pa

Dailydose(mg)

Therapeuticconcentration(mg L−1)

Toxicity(mg L−1)

Biologicalhalf-life (h)

Volume ofdistribution(L kg−1)

Renal clearance(mL min−1 kg−1)

Zolpidem 6.2 3.85 ca. 10 0.08–015b 0.5 1.7–2.5 0.5–0.7 0.26

Zopiclone –e –e 3.75–15 0.01–0.05b 0.15 3.5–6.5 1.3–1.6 13.9

a Octanol/water.b In serum.c In plasma.d After administration of 1–2 mg.

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e Undetermined.f In blood.g After administration of 10 mg.

Other non-classical extraction techniques for isolation of BZDrom biological samples, such as solid-phase microextractionSPME) [16,17], microwave-assisted extraction (MAE) [18–20] orloud-point extraction (CPE) [21] were also demonstrated. Inome cases of BZD determination in body fluids, by for examplehromatographic [22,23] or electrochemical [24,25] methods, noxtraction was performed and only protein precipitation and/orilution in an appropriate buffer, centrifugation and filtration wereecessary.

.1. Sample preparation of conventional body fluids

The sample preparation of whole blood, plasma/serum andrine is similar and, therefore, the sample preparation methodol-gy for these specimens may be described jointly.

For isolation of BZD from body fluids by the LLE technique, aariety of solvents or their mixtures have been used at alkaline,nd much rarely at slightly alkaline or acidic sample solu-ion. They include butyl acetate [11], n-butyl chloride [24,26],hlorobutane [27–29], chloroform [13,30], diethyl ether [31–34],thyl acetate [15,35–37], dichloromethane [38,39], toluene40], methyl tert-buthyl ether [41], n-hexane/dichloromethane70:30, v/v) [42,43], chloroform/isopropanol (9:1, v/v) [44] andichloromethane/isopropan (85:15, v/v) [45].

The SPE was the second technique as there is concern overts usability in the isolation of BZD drugs from body fluids.

any types of SPE sorbents were used, including ChemElut9,46], Bond-Elut Certify [27,47] Oasis HLB [33,48–51], Oasis

CX [14,52,53], C18 type [35,54–56], Abselut Nexus [57],solute C1 [58], Empore 3M [59] and SPEC DAU [60]. For elu-ion of BZD from column sorbents, the following solventsere applied: n-butyl acetate [9], methyl tert-buthyl ether

46], chloroform/isopropanol/concentrated ammonia (69:29:2,/v/v) [27], chloroform/isopropanol/concentrated ammonia78:20:2, v/v/v) [47], 5% ammonia in methanol [14], methanol48,50,51,54,56,58,59], ACN [49], dichloromethane/isopropanol75:25, v/v) [33], dichloromethane/isopropanol/ammoniumydroxide (80:20:2, v/v/v) [52], dichloromethane/isopropyl
lcohol/saturated ammonium (78:20:2, v/v/v) [53],ichloromethane/isopropanol/concentrated ammonia (80:20:2,/v/v) [61], methanol/ACN (1:1, v/v) [57], methanol/ACN1:5, v/v) [55], tetrahydrofuran (THF) (1:1, v/v) [59], diethyl
ether/hexane/methanol (50:30:20, v/v/v) [35], and ethylacetate/ammonium hydroxide (98:2) [60]. In one case, theuse of the dispersive-SPE technique for the isolation of 6 BZDand other medicaments from whole blood samples prior to theirdetermination by the GC–MS or LC–MS methods with an extractionrecovery from 70.3 to 85.3% was reported by Matsuta et al. [62].

The GC–MS or LC–MS/MS determinations of diazepam by itselfin plasma [16] or together with other medicaments in plasma andurine samples [17], preceded by SPME, were also performed. Afew coatings immobilized on the metal fibre core and consisting ofvarious types of silica particles (octadecyl, polar and cyano) immo-bilized on the metal fibre core were tested. Desorption of diazepamwas made using ACN/water (1:1, v/v) assisted with vortex agitation.In the first case [16] the diazepam recoveries were not given, andin the latter case [17] they were low; i.e. with a mean from 0.46 to0.60%, although the described LC–MS/MS method was suitable fordrug screening purposes.

Diazepam with its metabolite N-desmethyldiazepam, was alsoisolated from plasma samples using the packed-fibre solid-phaseextraction (PFSPE) [63]. A plasma sample (0.5 mL) was initially pre-cipitated with 40% perchloric acid, adjusted to pH 7.0 with 2 MNaOH, and then submitted to extraction on mini-columns packedwith electrospun polystyrene nanofibers (about 200–400 nm indiameter) with methanol as the eluent.

Two on line extractions, using column-switching (CS) [64] andan ion-exchange/reversed phase turbulent flow column [65], incombination with the LC–MS/MS method, for eight BZD in wholeblood and nine BZD together with other drugs in urine, correspond-ingly, were presented. In the study described by Bugey and Staub[64], restricted access material (RAM) sorbent monolithic silicasupport as the extraction precolumn was employed. This approachreduced the analysis time while maintaining the high efficiencyof the extraction process. Before extraction, whole blood sampleswere precipitated with ACN, but urine samples were only dilutedwith acetate buffer (pH 4).

Applications of the MAE technique for HPLC-DAD analysis ofthe selected BZD in plasma and urine were also reported [18]. 1 mLsamples taken from both of the biological materials were analyzed
using the same extraction conditions; with the solvent being chlo-roform/isopropanol (4:1, v/v), the temperature at 89 ◦C, and theextraction time at 13 min. The obtained extraction recoveries werehigh and ranged from 89.8 to 102.1%.

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In another case, a non-conventional CPE technique was appliedo a sequentional HPLC-DAD drug screening analysis, including twoZD drugs as the selected representatives for the acidic/neutralroup of medicaments [21]. The CPE conditions were as follows:ample pH 6, 1 mL of 7.5% Triton X-114 (surfactant used as thextraction medium), temperature and time of incubation with theurfactant: 40 ◦C and 30 min. After incubation, the sample was cen-rifuged and the surfactant-rich phase with the isolated BZD waseparated and dried under a stream of nitrogen. The dried micellarhase was dissolved in 150 �L of ACN. The obtained recoveries ofhe BZD were 52.7% (for lorazepam) and 67.0% (for alprazolam).

More details of the selected sample preparation proceduresor conventional body fluids combined with appropriate analytical

ethods are given in Tables 2–6.

.2. Sample preparation of alternative biological materials

Considering sample preparation, alternative biological speci-ens may be divided into unconventional body fluids (vitreous

umour and oral fluid/saliva), keratin samples (hair and nails) andther non-typical specimens including, for example, exhaled breathr fingerprint deposits.

Pretreatment (before extraction) of vitreous humour and salivas simple and it is usually limited to dilution with an appropriateuffer (adjusting the sample pH to acidic, neutral or alkalic) and/orrotein precipitation. In one case, when the appropriately sensitivePLC–MS/MS method was used, no extraction was needed [66]. Inther cases of these specimens’ preparation, SPE or LLE were mostften applied. However, the successful employment of the MAEechnique for vitreous humour samples after protein precipitationith ACN was also described [19]. The mean extraction recovery

f the commonly prescribed BZD from this material analyzed byhe HPLC-DAD method ranged from 87.6 to 101.2%. For SPE of BZDrom both unconventional body fluids, the following sorbents werepplied: Oasis HLB [67,68], Abselut Nexus [10,69], Isolute HCX [12],ond Elut Certify II [70] or mixed-mode columns CSDAU020 [71]. Asxtraction solvents/eluents were used: 10% acetic acid in methanol67], ethyl acetate [19,72] toluene/ethyl acetate (80:20, v/v),[12]nd ACN/ammonia (100:4, v/v),[12] diethyl ether/methylene chlo-ide (1:1, v/v) [73] acetone/chloroform (1:1, v/v) [70], diethylther [33], butyl chloride [74], chloromethane/isopropanol (75:25,/v) [68], 1-chlorobutane [75], 2% ammonia in ethyl acetate [71],ethanol/ACN (1:1, v/v) [69] and methanol [72]. In one case ofulti-drug GC–MS analysis of 30 drugs including BZD, in 0.25 mL

ral fluid, the derivatization step with an ACN/MTBSTFA reagentas also performed [12].

Analyses of keratin samples require more complex pretreat-ent procedures. First of all, the samples of these specimens

before or after cutting) should be carefully decontaminated fromxternal impurities using an appropriate solvent or sequence of sol-ents. Nails were decontaminated as limb samples (i.e. right hand,eft foot, etc.), then dried and cut into small pieces (1–2 mm) [76].he following sequences of washing reagents for the decontam-nation of hair samples were reported: distilled water, deionized

ater and ethanol [76], 0.1% sodium dodecyl sulphate, distilledater and dichloromethane [77], 0.1% sodium dodecyl sulphate,ater, methanol and water (three times) [20], isopropanol, phos-hate buffer and isopropanol; dichloromethane (twice rinsing)78], isooctane and acetone [79], water, acetone and hexane [80]ichloromethane and methanol (twice rinsed with each solvent)81], phosphate buffer pH 7.4, isopropanol and methylene chlo-ide [82], isopropanol (once washed) and water (twice washed)
83]. In the most often used procedures, after the cleaning step,he hair samples were dried, cut into small pieces (e.g. less than
mm) or appropriate segments (in the case of segmental analy-is, usually 1–3 cm) and then pulverized in a suitable device (ball

Biomedical Analysis 113 (2015) 239–264 243

mill, freeze mill, bead mill homogenizer or in an Eppendorff tubecontaining a tungsten carbide ball using a bench top shaker). Theappropriate amount of powder was weighed and incubated with orwithout sonication at room or elevated temperature (37–50 ◦C) forsome time (from 1 to overnight) in methanol [29,81], methanol/25%ammonium hydroxide (20:1, v/v) [77], phosphate buffers (pH 8.4or 7.4) [78,79,81,82] and borate buffer (pH 9.5) [20]. In most casesof nails and hair analysis, LLE was employed with small exceptionslike molecularly imprinted solid-phase extraction (MISPE) [77] orMAE [20]. In the first case of analyte separation, the extractionrecovery of nine BZD from hair ranged from 39 to 103%, and inthe latter case the extraction recovery for six BZD from this matrixwas high and equivalent to 86.9–93.4%. In one case, 32 BZD presentin a hair matrix were analyzed directly by the LC–MS/MS methodafter appropriate pretreatment, although no extraction data weregiven [81].

More details concerning the selected sample preparation proce-dures for alternative biological samples combined with appropriatedetection methods, are given in Tables 5 and 6, correspondingly.

The specificity of such biological samples as exhaled breathor fingerprints requires a specific approach for the preparation ofthem for analysis. The sampling of exhaled breath using appropriatedevices and preparation of such collected samples for LC–MS/MSanalysis was described in detail by Becket et al. [84]. Fingerprintsdeposited on glass cover slips were collected by dissolving them ina solution of dichloromethane/methanol (1:1, v/v), containing anappropriate internal standard. Then, the samples were evaporated,reconstituted with a mobile phase, and analyzed by LC–MS/MS [85].

3. Analytical methods

Over the last 10 years, a large number of methods for thedetermination of BZD in biological materials have been reported,among which the main role of chromatographic techniquesstill plays: liquid chromatography (LC) coupled with ultravioletdetection (UV) [35,38,40,42,54,57,58,63], diode array detection(DAD) [18,32,55,67,86–89], tandem mass spectrometry (MS/MS)[22,26,33,46,49,50,52,65,71,73,77,79,90–96] or time-of-flight massspectrometry (TOF-MS) [20,28,97] and, less frequently, gas chro-matography (GC) with nitrogen–phosphorus detection (NPD)[47,98], electron capture detection (ECD) [43,47] and mass spec-trometry (MS) [16,56,62,70] with chemical ionization (CI) [9,14,99]or electron ionization (EI) [11–13,69,100] mode. Despite theundeniable dominance of chromatographic techniques, it can beobserved increasing descriptions of use of alternative methods forthe detection and determination of BZD in biological matrices, suchas: immunoassay [74,101–104], fluorimetric [15], colorimetric[105] and chemiluminescence methods [30], as well as matrix-assisted laser desorption/ionization mass spectrometry (MALDI)[51] or Raman spectroscopy [106]. Chromatography-related meth-ods like sweeping capillary electrophoresis (CE) [39], sweepingmicellar electrokinetic chromatography (MECC) [53,107] or cap-illary electrochromatography (CEC) [36], besides electrochemicalmethods including, especially, some kind of voltammetry tech-niques such as DPCAdSV (differential pulse cathodic adsorptivestripping) [24], hydrodynamic [108], differential pulse voltam-metry (DPV) [59] or a microfluidic device with laser-inducedfluorescence detection [61] have been also introduced in the lastdecade.

In this chapter, the chief trends in the development of newanalytical methodology for BZD detection and determination in
biological samples, described in literature over the last 10 years,are demonstrated and discussed. Tables 7–10 summarize informa-tion on the equipment and validation parameters of the presentedanalytical methods and techniques.


Table 2Preparation of whole blood samples for the determination of benzodiazepines.

Analytes (n)a Sample volume(mL)

Initial samplepretreatment

Extraction conditions: column/eluentor extraction solventDerivatization reagent

Recovery (%) Analytical method Ref.

Solid phase extraction (SPE)Clonazepam, 7-

aminoclonazepam1.0 Sample pH 6.0

(phosphate buffer)Phenyl column/ethyl acetate MTBSTFA Mean 92 (for

clonazepam) andmean 90 (for 7-aminoclonazepam)

HPLC-PDAGC–(EI)-MS

[10]

Benzodiazepinesb

(4)–c Sample pH 6.0

(phosphate buffer)Bond Elut Certify/dichloromethane-isopropanol-ammonia (78:20:2,v/v/v)

88.8–97.9 GC-NPD/micro-ECD

[47]

Benzodiazepinesd

(11)0.5 sample pH 11.0

(borate buffer)ChemElut/methyl tert-butyl ether 71–96 UPLC–MS/MS [46]

Benzodiazepinese

(15)0.2 Sample pH 1.0

(1.5 M HCl)Oasis MCX/5% ammonia in methanolMTBSTFA-ACN-ethyl acetate(20:40:40, v/v/v)

80.6–91.4, exceptfor: oxazepam(70.8), lorazepam(62.2), clonazepam(61.8), and�-OH-alprazolam(61.1)

GC–(NICI)-MS [14]

Benzodiazepinesf

(16)1.0 Sample pH 6.0

(phosphatesolution) and thenalkalization (25%ammonia solution)

Bond Elut Certify/chloroform-isopropanol-concentrated ammonia(69:29:2, v/v/v)

47–110 HPLC–PDA-MS [27]

Benzodiazepines(6) and otherpsychotropicdrugsg

0.1 Sample pH ca. 5(acetic acid)

Sorbent: anhydrous MgSO4 andNaCl/ACN containing 0.2% acetic acid

74–82 GC–(EI)-MS, LC–MS [62]

Benzodiazepinesand otherpsychotropicdrugsh (10)

0.5 Dilution of a samplewith water andcentrifugation

Oasis HLB/methanol 70.3–85.3 UPLC–MS/MS [48]

Benzodiazepinesi

(16) andzaleplon,zopiclone

0.1 Sample pH 9.0(hydrophosphatebuffer)

ChemElut/n-butyl acetate MTBSTFA 88.8–97.9 GC–(NICI)-MS [9]

Liquid–liquid extraction (LLE)Benzodiazepinesj

(8)1.0 Sample pH 9.0

(sodiumhydrophosphate)

n-Butylchloride 74.3–105.7 LC–MS [26]

Benzodiazepinesk

(23)1.0 Sample pH 9.0

(phosphate buffer)ChloroformDerivatization (two stages):- propylation- propionylation

>74 GC–(EI)-MS [13]

Benzodiazepinesl

(14), zaleplon,zolpidem

0.5 Addition of 0.5 mL of0.5 M Na2HPO4

buffer

Butyl acetateACN/MTBSTFA (80:20, v/v)

88.2–107 GC–(EI)-MS [11]

Column switching (CS)Benzodiazepinesm

(8)0.05 Protein precipitation

(ACN)Extraction precolumns: RAM sorbentand monolithic silica support/5 mMammonium formate with formic acid(pH 3) – ACN (98:2, v/v)

68–113 LC–MS [64]

a Number of the analytes.b Flunitrazepam, nitrazepam, clonazepam, alprazolam.c No data was given.d Alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, lorazepam, midazolam, nitrazepam, nordazepam, oxazepam, phenazepam.e 7-Aminoclonazepam, �-OH-alprazolam, �-OH-midazolam, alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, lorazepam, midazolam, nitrazepam, nor-

dazepam, oxazepam, temazepam, triazolam.f 7-Aminoflunitrazepam, alprazolam, desalkylflurazepam, desmethylflunitrazepam, diazepam, flunitrazepam, flurazepam, �-OH-midazolam, lorazepam, midazolam,

nitrazepam, nordazepam, oxazepam, lormetazepam, bromazepam, clobazam.g Flunitrazepam, 7-aminoflunitrazepam, triazolam, �-OH-triazolam, brotizolam, �-OH-brotizolam, non-benzodiazepine psychotropic drugs: phenobarbital, chlorpro-

mazine, promethazine.h Bromazepam, diazepam, lorazepam, nordazepam, fluoxetine, haloperidol, olanzapine, paroxetine, quetiapine, risperidone.i Chlordiazepoxide, diazepam, nitrazepam, oxazepam, temazepam, bromazepam, clonazepam, flunitrazepam, midazolam, nordazepam, lorazepam, OH-alprazolam, tria-

zolam, alprazolam, phenazepam, OH-midazolam.j Clonazepam, diazepam, flunitrazepam, lorazepam, midazolam, N-desalkylflurazepam, nordazepam, oxazepam.k Diazepam, nordazepam, oxazepam, bromazepam, alprazolam, lorazepam, medazepam, flurazepam, fludiazepam, tetrazepam, chlordiazepoxide, clobazam, midazolam,

flunitrazepam, 7-aminoflunitrazepam, triazolam, prazepam, nimetazepam, temazepam, lormetazepam, clonazepam, camazepam.l Chlordiazepoxide, diazepam, nitrazepam, oxazepam, temazepam, bromazepam, medazepam, midazolam, nordazepam, lorazepam, �-OH-alprazolam, alprazolam,

phenazepam, �-OH-midazolam.m Clonazepam, diazepam, flunitrazepam, lorazepam, midazolam, N-desalkylflurazepam, nordazepam, oxazepam.


Table 3Preparation of plasma/serum samples for the determination of benzodiazepines.


Initial sample pretreatment Extraction conditions:column/eluent orextraction solvent

Recovery (%) Analyticalmethod

Ref.

Solid phase extraction (SPE)Clotiazepam 1.0 –b C-18/methanol Mean 93.1 GC–(EI)-MS [56]Diazepam, N-

desmethyldiazepam0.5 Protein precipitation (40%

perchloric acid), sample pH 7.0(2M NaOH)

Mini-columns packedwith electrospunpolystyrene nanofibers(about 200–400 nm indiameter)/methanol

51.7–83.2 HPLC-UV [63]

Diazepam,oxazepam

1.0 Hydrolysis with proteinase K at56 ◦C for 2–3 h

Extraction disccartridges (Empore3M)/methanol

–c Voltammetry [59]

Alprazolam and itstwo metabolites

ca. 1.0 Addition of 0.5 M NaOH Oasis HLB/ACN >85 LC–MS [49]

Benzodiazepinesd

(4)–c Sample pH 6 (phosphate

buffer)Bond ElutCertify/dichloromethane-isopropanol-ammonia(78:20:2, v/v/v)

90.7–97.5 GC-NPD/micro-ECD

[47]

Benzodiazepinese

(4)0.05 Protein precipitation (ACN) DSC-18/methanol 81.2–114 HPLC-UV [54]

Benzodiazepinesf

(6)0.5 Sample pH 7.4 (phosphate

buffer)Bond ElutCertify/chloroform-isopropanol (4:1,v/v)

ca. 90 HPLC-DAD [18]

Benzodiazepinesand tricyclicantidepressantsg

(10)

0.05 Protein precipitation (ACN) AbselutNexus/methanol-ACN(1:1, v/v)

90.7–97.5 HPLC-DAD [57]

Benzodiazepinesh

(15)0.5 Protein precipitation (ACN) Isolute C1/methanol Mean 93–100 HPLC-UV [58]

Benzodiazepinesi

(21)0.1 Addition of 10 mM ammonium

formateHLBcartridges/methanol

>58.1 (quazepam –45.8)

LC–MS/MS [50]

Solid phase microextraction (SPME)Diazepam and

other drugsj (4)1.5 –b Coatings of fibres

consisted of a mixtureof various types ofcoated silica (octadecyl,polar embedded andcyano) particlesDesorption: vortexagitation withACN/water (1:1, v/v)for 5 min

0.12–0.20 LC–MS/MS [17]

Liquid–liquid extraction (LLE)Lorazepam LLE Protein precipitation

(acetonitrile), supernatant pH9.5 (sodium carbonate–sodiumhydrogen carbonate buffer)

n-Hexane-dichloromethane(70:30; v/v)

Mean 75.3 HPLC-UV [42]

Diazepam LLE Sample pH 9.0 (borate buffer) n-Hexane-dichloromethane(70:30, v/v)

Mean 80.6 GC-ECD [43]

Lorazepam and itsglucuronidemetabolite

0.5 (withhydrolysis)1.0 (withouthydrolysis)

Hydrolysis with glucuronidaseat pH 5.0 (acetate buffer),hydrolyzate pH 10 (NaOH orcarbonate buffer)

Methyl tert-buthylether

83–87 LC–MS/MS [41]

Benzodiazepinesk

(4)0.5 –b Toluene Mean 59.8–98.1 HPLC-UV [40]

Benzodiazepinesl

(9)0.5 Sample pH 9 (borate buffer) Diethyl ether 70.3–86.9 LC–MS [33]

Benzodiazepines(41)

0.5 Sample pH 9.0 (borate buffer) 1-Chlorobutane –c LC–TOF-MS [28]

Benzodiazepinesand other drugsm

(5)

0.2 Addition of 0.1 M NaOH Diethyl ether >90 LC–MS/MS [31]

Dispensive liquid–liquid microextraction (DLLME)Benzodiazepinesn

(7)0.5 Addition of 0.1 M NaOH Chloroform

Dispenser: methanolWith addition ofsodium chloride

71–102 HPLC-PDAUPLC-PDA

[19]

Microwave-assisted extraction (MAE)Benzodiazepineso

(6)1.0 Sample pH 9.0 (borate buffer) Chloroform/isopropanol

(4:1, v/v); at 89 ◦C for13 min

89.8–102.1 HPLC-DAD [18]


Table 3 (Continued)


Initial sample pretreatment Extraction conditions:column/eluent orextraction solvent


Ref.

Cloud-point extraction (CPE)Alprazolam,

lorazepam1.0 Sample pH 7 Micellar medium: 7.5%

Triton X-114Incubation at 40 ◦C for25 minSolvent for micelarphase: ACN (150 �L)

64.2 (alprazolam)54.5 (lorazepam)

HPLC-DAD [21]

Directly (without extraction)Triazolam 1.0 Addition of acetate and 0.13%

formic acid, centrifugation andthen filtration

–b 86.4–92.7 LC–MS/MS [22]

Lorazepam 1.0 Protein precipitation (ethanol),filtration and dilution withBritton–Robinson buffer

–b Mean 82.6 Adsorptivecathodic pulsestrippingvoltammetry

[24]

Lorazepam 0.5 Protein precipitation(methanol) and dilution of thesupernatant with phosphatebuffer (pH 7.5)

–b ca. 100 Square wavevoltammetry

[25]

Temazepam,lormetazepam

–c Protein precipitation(methanol)

–b 98.7–99.2 (forenantiomers oftemazepam) ca. 97(for enantiomers oflormetazepam)

HPLC–UV [23]

a Number of the analytes.b No initial sample pretreatment or extraction was performed.c No data was given.d Flunitrazepam, nitrazepam, clonazepam, alprazolam.e Alprazolam, bromazepam, diazepam, flunitrazepam.f Alprazolam, bromazepam, diazepam, lorazepam, lormetazepam, tetrazepam.g Bromazepam, clonazepam, diazepam, flunitrazepam, alprazolam, lorazepam, amitriptyline, clomipramine, imipramine, doxepine.h Diazepam, oxazepam, alprazolam, bromazepam, brotizolam, clobazam, clonazepam, chlordiazepoxide, clotiazepam, delorazepam, flunitrazepam, flurazepam, lorazepam,

lormetazepam, triazolam.i Flurazepam, bromazepam, chlordiazepoxide, nitrazepam, clonazepam, flunitrazepam, estazolam, clobazam, lorazepam, alprazolam, triazolam, brotizolam, fludiazepam,

diazepam, quazepam, prazepam, 7-aminonitrazepam, 7-aminoclonazepam, 7-acetamidonitrazepam, N-desmethylclobazam, N-desmethyldiazepam.j Carbamazepine, propranolol, pseudoephedrine, ranitidine.k Diazepam, desmethyldiazepam, oxazepam, temazepam.l Midazolam, bromazepam, tetrazepam, alprazolam, lorazepam, triazolam, flunitrazepam, diazepam, lormetazepam.

m Alprazolam, clonazepam, estazolam, carbamazepine, phenytoin.etraze

3

33niocwi

tcsttps

(pabls

n Alprazolam, bromazepam, clonazepam, diazepam, lorazepam, lormetazepam, to Alprazolam, bromazepam, diazepam, lorazepam, lormetazepam, tetrazepam.

.1. Chromatographic techniques

.1.1. Liquid chromatography

.1.1.1. Plasma, serum, whole blood and urine samples. LC tech-iques are still the most widely employed in BZD determination

n blood, plasma or serum samples. In most cases, the combinationf LC with MS detection is utilized particularly in pharmacologi-al studies and the detection of metabolites but, despite this, HPLCith a UV or DAD detector still plays an important role, especially

n screening drug analysis.For screening and the determination of BZD, a combination of

he LC technique with TOF-MS, preceded by LLE, was employed toreate an exact mass database of 41 BZD and their metabolites inerum (as well as in urine and gastric samples) [28]. According tohe authors, the proposed LC-TOF–MS method could be a powerfulool in screening analyses of many analytes present in one sam-le due to fast mass spectral acquisition obtained with the highensitivity and mass accuracy.

A UPLC-TOF/MS technique with a C18 column100 mm × 2.1 mm × 1.7 �m) and gradient elution of the mobilehase (0.1% formic acid and ACN), preceded SPE of 46 drugs of
buse, including BZD from whole blood samples, was developedy Birkler et al. [97]. The values of the LOQ were below therapeutic
evels for each analyte. The application of the method for 167 bloodamples from potential victims of alleged sexual assaults indicated

pam.

the usefulness of the proposed chromatographic method in drugscreening analysis for forensic purposes.

A HPLC-DAD method with an environmentally friendly CPEtechnique was developed for the sequential isolation of alprazolamand other basic and acidic/neutral drugs from human plasma andserum samples [87]. The main advantage of the proposed proce-dure is its ability to determine compounds with different chemicalproperties (log P and pKa) in one chromatographic analysis. The lim-its of detection (LODs) for the studied medicaments correspondingto therapeutic or low toxic plasma concentration levels indicatethe possibility of utilizing the proposed procedure for toxicologicaldrug screening, which was confirmed by the analysis of real plasmasamples from patients suspected of drug overdosing.

LC with UV was also applied to a pharmacokinetic study oflorazepam in plasma from children with severe malaria and convul-sions after administration of a single dose of this drug [42]. The mainadvantage of the proposed method is its simplicity, sensitivity andrelatively inexpensive equipment and reagents, which is especiallyimportant in resource-poor countries for tracking the appropria-tion of the conducted therapy and concentration-dependent sideeffects of a drug.

A pharmacokinetic study of lorazepam in human plasma andurine was performed by the enantioselective LC–MS/MS method[41]. The proposed procedure made it possible to determine thepharmacokinetic parameters as an isometric mixture; e.g. the


Table 4Preparation of urine samples for the determination of benzodiazepines.

Analytes (n)a Samplevolume (mL)

Initial sample pretreatment Extraction conditions:column/eluent or extractionsolventDerivatization reagent


Solid phase extraction (SPE)Oxazepam,

diazepam3.0 Hydrolysis with �-glucuronidase at pH

4.6(Britton–Robinsonbuffer solution)

Extraction disc cartridges(Empore 3 M)/methanol-THF (1:1v/v)

–b Voltammetry [59]

Nimetazepam, 7-aminonimetazepam

1.0 Hydrolysis with �-glucuronidase at pH3.8 (sodium acetate buffer) at 37 ◦C for3 h

SPEC DAU/ethylacetate-ammonium hydroxide(98:2, v/v)

Mean 95 LC–MS/MS [60]

Benzodiazepinesc

(4)0.5 –d DSC-18 column

(before elution sample was washedwith a mixture of H2O-methanol;95:5 v/v)/methanol

81.0–115 HPLC-UV [54]

Benzodiazepinese

(6)0.1 Protein precipitation (ACN) LC-18 column/methanol-ACN

(50:50, v/v)88.7–99.3 HPLC-UV [55]

Benzodiazepinesf

(7)1.0 Hydrolysis with Helix pomatiaat pH

4.5 at 56 ◦C for 1.5 h, then alkalizationwith ammonium carbonate buffer (pH9.3) and purification with SPE

Oasis HLB/methanol –b AP-MALDI-MS/MSAP-MALDI-MS

[51]

Benzodiazepinesg

(8)0.5 Hydrolysis with �-glucuronidase at pH

4.0 (acetate buffer) at 60 ◦C for 2 h,adjusted to pH 7.5 (phosphate buffer)

Oasis MCX/dichloromethane-isopropanol-ammonium hydroxide(80:20:2, v/v/v)

54–86 LC–MS/MS [52]

Benzodiazepinesand analogsh

(21)

2.0 Sample pH 7.4 (Sorensen buffer) OasisHLB/dichloromethane-isopropanol(75:25, v/v)

Mean 77–110 LC–MS/MS [33]

Solid phase microextraction (SPME)Diazepam and

other drugsi (4)1.5 –d Coatings of fibres consisted of a

mixture of various types of coatedsilica (octadecyl, polar embeddedand cyano) particlesDesorption: vortex agitation withacetonitrile/water (1:1, v/v) for5 min

Mean 0.46–0.60 LC–MS/MS [17]

On-line extractionBenzodiazepines

(7) and otherdrugsj

0.5 Dilution with acetate buffer (pH 4),hydrolysis with�-glucuronidase/arylsulfatase at 40 ◦Cfor 90 min, centrifugation

Ion-exchange/reversed phaseturbulent flow column/10 mMammonium bicarbonate (pH 9)

78–116 (for alltested drugs)

LC–MS/MS [65]

Liquid–liquid extraction (LLE)Oxazepam 0.5 Protein precipitation, centrifugation

and adjustment to pH 9 (borate buffer)Ethyl acetateDerivatization: cerium (IV) (acatalyst) in concentratedortho-phosphoricacid, 85%)

–b Fluorimetricscreening method

[15]

Lorazepam 1.0 Addition of 0.2 MSodium carbonate buffer

Butyl chloride Mean 89.8 Adsorptivecathodic pulsestrippingvoltammetry

[24]

Clonazepam,diazepam

5.0 –d Chloroform Mean 98.5(diazepam)ca. 100(clonazepam)

Chemiluminescence [30]

Lorazepam andits glucuronidemetabolite

0.1 Hydrolysis with �-glucuronidase at pH4.2 (acetate buffer) at 56 ◦C for 1 h,then addition of 1 MNaOH (pH 10)

Tert-Buthyl ether Mean 88.7(lorazepamwithouthydrolysis)Mean 84.8 (totallorazepam, afterhydrolysis)

LC–MS/MS [41]

Benzodiazepines(5)k

1.0 Sample pH 9.5 (carbonate buffer) Ethyl acetate 70.5–96.7 LC–MS/MS [37]

Benzodiazepinesl

(10)1.0 Sample pH 6.0 (phosphate buffer) Ethyl acetate 81–98 CEC-TOF-MS [36]

Benzodiazepinesm

(11)1.0 Sample pH 6 (phosphate buffer), then

addition of 1 M NaOH (after first step ofLLE)

Ethyl acetate (the extract wasdissolved with 10% methanol andmixed with gold nanoparticles andMgCl2 as aggregating agent)

–b SERS [106]

Benzodiazepinesn

(21)3.0 Hydrolysis with �-glucuronidase at pH

6.0 and adjustment of sample to pH 7.5(phosphate buffer)

Dichloromethane-isopropanol(85:15, v/v)

Mean 56.2–98.8 LC–MS/MS [45]


Table 4 (Continued)


Initial sample pretreatment Extraction conditions:column/eluent or extractionsolventDerivatization reagent


Benzodiazepineso

(35)1.0 Hydrolysis with �-glusulase at pH 5.2

(sodium acetate buffer) at 37 ◦C for 4 h,then basification of the hydrolyzate(with 1.5 M sodium carbonate)

Chloroform-isopropanol (9:1, v/v) –b LC–TOF-MS [44]

Benzodiazepinesand theirmetabolites(41)

0.5 Hydrolysis with �-glucuronidase at pH5 (potassium acetate buffer) at 55 ◦Cfor 4 h, then after cooling, addition ofborate buffer (pH 9.0)

1-Chlorobutane –b LC–TOF-MS [28]

Abused drugsand hypnoticsp

0.5 Hydrolysis with �-glucuronidase at pH4.5 (sodium acetate), then adjustmentof the hydrolyzate to pH 9.6(ammonium hydroxide)

Dichloromethane –b Sweeping CE [39]

Directly (without of extraction)Benzodiazepinesq

(4)1.0 Addition of 10 mM ammonium acetate

and filtration–d 50.1–82.0 LC–MS/MS [110]

Benzodiazepinesr

(13)0.5 Protein precipitation with methanol

containing 0.2% formic acid, andhydrolysis with �-glucuronidase at60 ◦C for 90 min

–d Mean92.44–113.90

UPLC–MS/MS [96]

a Number of the analytes.b No data was given.c Alprazolam, bromazepam, diazepam, flunitrazepam.d No initial sample pretreatment or extraction was performed.e Alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, lorazepam.f Diazepam, lorazepam, midazolam, triazolam, nitrazepam, N-desalykylflurazepam, oxazepam.g 7-Aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, alprazolam, �-OH-alprazolam, oxazepam, 3-OH-diazepam, N-desmethyldiazepam.h 1-OH-midazolam, 4-OH-midazolam, midazolam, 7-aminoclonazepam, desmethylflunitrazepam, flunitrazepam, 7-aminoflunitrazepam, alprazolam, bromazepam, clon-

azepam, triazolam, lorazepam, 3-OH-flunitrazepam, zolpidem, zopiclone, flurazepam, desalkylflurazepam, �-OH-alprazolam, �-OH-triazolam, lormetazepam, loprazolam.i Carbamazepine, propranolol, pseudoephedrine, ranitidine.j Amphetamine, methamphetamine, MDA, MDMA, MDEA, EDDP, diazepam, nordazepam, oxazepam, bromazepam, lorazepam, OH-alprazolam, 7-aminoflunitrazepam,

zolpidem, zaleplon, zopiclone, tilidine, nortilidine, tramadol, and o-demethyltramadol, benzoylecgonine, morphine, codeine, dihydrocodeine, 6-monoacetylmorphine,buprenorphine, norbuprenorphine.

k Flunitrazepam, nimetazepam, nitrazepam, 7-aminonimetazepam, 7-aminoflunitrazepam.l Alprazolam, triazolam, chlordiazepoxide, lorazepam, nitrazepam, clonazepam, flunitrazepam, clorazepate, diazepam, prazepam.

m Alprazolam, midazolam, triazolam, 7-aminoflunitrazepam, chlordiazepoxide, clonazepam, diazepam, flunitrazepam, lorazepam, nordazepam, oxazepam.n Flunitrazepam, diazepam, demoxepam, medazepam, clonazepam, desalkylflurazepam, oxazepam, lormetazepam, alprazolam, lorazepam, nitrazepam, midazolam,

prazepam, temazepam, triazolam, nordazepam, flurazepam, N-�-OH-ethylflurazepam, 7-aminonitrazepam, 7-aminoflunitrazepam, bromazepam.o 7-Aminonorflunitrazepam, 7-acetamidonorflunitrazepam, bromazepam, 3-OH-flunitrazepam, 2-OH-flurazepam, diazepam, 7-aminoclonazepam, desmethylfluni-

trazepam, lorazepam, norfludiazepam, flunitrazepam, 7-aminoflunitrazepam, temazepam, lormetazepam, triazolam, nitrazepam, �-OH-triazolam, nordazepam, midazolam,prazepam, chlordiazepoxide, clobazam, flurazepam, clonazepam, desalkylflurazepam, 7-aminonitrazepam, alprazolam, oxazepam, �-OH-alprazolam, halazepam,medazepam, 3-OH-flunitrazepam, N-ethylnordazepam, demoxepam, chlorazepate.

p Morphine, ketamine, methamphetamine, codeine, alprazolam, clonazepam, diazepam, flunitrazepam, oxazeapm, �-OH-alprazolam, 7-aminoclonazepam, nordazepam,7-aminoflunitrazepam, N-demethylflunitrazepam.

m, nor

hmru

ozdofu

omseo

uoa

q Diazepam, nordazepam, temazepam, oxazepam.r Bromazepam, clobazam, �-OH-alprazolam, �-OH-triazolam, 7-aminoclonazepa

azepam, flurazepam.

alf-life distribution and elimination, volume of distribution,aximum plasma concentration and time of its reach, total and

enal clearance, plus the fraction of a lorazepam dose excreted intorine in an unchanged form.

A complex procedure for the determination of a numerous groupf 26 BZD, their metabolites, and non-BZD drugs: zolpidem andopiclone in plasma samples (as well as in urine and hair) waseveloped by Laloup et al. [29] and applied to authentic samplesriginated from forensic cases. The low LOQ value for three dif-erent types of biological matrices simultaneously indicates thesefulness of the method in cases of forensic investigations.

Dussy et al. [27] proposed a combination of LC with two typesf detector, photodiode array detector (PDA) and MS, for the deter-ination of BZD and its metabolites in 128 real plasma and serum

amples. According to the authors, the developed procedure isasily applicable and could be successfully utilized in routine lab-ratory practice.

Félix and Campèse [23] proposed a modification of a RAM col-mn for the separation of the enantiomers of BZD by introductionf a �-cyclodextrin carbamate on 70 A pore size amino silica gelnd coating the surface with bovine serum albumin (BSA). This

rdazepam, diazepam, nitrazepam, oxazepam, lorazepam, temazepam, desalkylflu-

RAM-Chiral column seems to be a good alternative for other tradi-tional equipment used for the direct analysis of drug enantiomersin plasma.

Cudjoe and Pawliszyn [109] investigated the possibility of appli-cation of the LC–MS/MS method integrated with a 96-well discSPE technique for the determination of nordazepam, diazepam,lorazepam and oxazepam, present in plasma and urine samples.The key advantages of this proposed method are its short time forone sample analysis (0.94 min), no use of vacuum pumps, low costsof chemical reagents (lower than those in traditional SPE), besidesthe low limit of quantification (LOQ) (0.2–2.0 ng mL−1), thus indi-cating the usefulness of this method in bioanalysis.

Umezawa et al. [110] proposed the LC–MS/MS method for thedetermination of diazepam and its three metabolites in urine. Theused hydrophilic polymer column (MSpak GF-310 4B) enableddirect injection of crude biological samples. The described methodis characterized by good validation parameters, such as: LOD and
LOQ 0.1 and 0.5 ng mL−1, respectively, and the precision being lessthan 9.6%. The usefulness of the proposed method for pharmaco-logical purposes was presented by analysis of human urine afteroral administration of diazepam.


Table 5Preparation of vitreous humour and oral fluid/saliva samples for the determination of benzodiazepines.


Initial sample pretreatment Extraction conditions: column/eluent orextraction solventDerivatization reagent


Ref.

Vitreous humourSolid phase extraction (SPE)Benzodiazepinesb (6) 0.5 Sample pH 6 (phosphate buffer) Oasis HLB/10% acetic acid-methanol >68.5 HPLC-DAD [67]

Microwave-assisted extraction (MAE)Benzodiazepinesb (6) 0.5 Protein precipitation (ACN), then

adjustment of sample to pH 9 (boratebuffer)

Ethyl acetate (at 98 ◦C for 10 min) Mean87.6–101.2

HPLC-DAD [19]

Oral fluid/salivaSolid phase extraction (SPE)Benzodiazepinesc (8) 0.5 Protein precipitation (ACN) Abselut Nexus/methanol-ACN (1:1, v/v) 96.0–108.6

(between days)HPLC-DAD [55]

Benzodiazepinesd (14) 1.0 Sample pH 6.0 (phosphate buffer and theQuantisal buffer)

Mixed-mode columns (CSDAU020)/ethylacetate – 2% ammonium hydroxide

81.4–90.2 LC–MS/MS [71]

Benzodiazepines (6)e andtricyclic antidepressants (4)f

0.5 Addition of methanol Abselut Nexus/methanol Mean ca. 100 HPLC-DAD [57]

Drugs of abuseg (30) 0.25 Sample pH 4.1 (phosphate buffer) Isolute HCX column/1 step: toluene-ethylacetate (80:20, v/v)2 step: ACN-aqueousAmmonia (100:4, v/v)Derivatization: ACN/MTBSTFA (4:2, v/v)and ACN/MTBSTFA (6:2, v/v)

62.3–79.9 GC–EI-MS [12]

Illicit and medicinal drugs andtheir metabolitesh (23)

0.5 Sample pH 9.0 (borate buffer) OASIS HLB/chloromethane-isopropanol(75:25, v/v)

76.2–80.7 LC–MS/MS [68]

Licit and illicit drugsi (49) 0.5 Sample pH 6.0 (phosphate buffer) Bond Elut Certify/acetone-chloroform(1:1, v/v) (for acidic and neutral drugs)

–j GC–MSLC–MS/MS

[70]

Liquid–liquid extraction (LLE)Benzodiazepinesk (9) 0.5 Sample pH 9.3 (ammonic carbonate

buffer)Diethyl ether 63.9–77.2 LC–MS [33]

Benzodiazepinesl (11) 0.45 Elution with methanol from collectingdevice, ultrasonication, centrifugationand alkalization

1-Chlorobutane and then borate-buffer(pH 9)

90–107 LC–MS/MS [75]

Benzodiazepinesm (25) 1.0 Sample pH 9.5 (sodium carbonate buffer) Ethyl acetate 82–99 LC–MS/MS [72]Benzodiazepines (14) and

hypnotics (3)n0.5 Sample pH 8.4 (phosphate buffer) Diethyl ether-methylene chloride

(1:1, v/v)>90 LC–MS/MS [73]

Benzodiazepines (4) and otherdrugso

0.2 Dilution or without dilution of a sampleand then basification

Butyl chloride –j Immunoassay(forscreening)LC–MS/MS

[74]

Directly (without extraction)Drugs (44) including

benzodiazepines (16)p0.5 Protein precipitation (ACN) –q 76–92 UPLC–MS/MS [66]

a Number of analytes.b Bromazepam, alprazolam, lorazepam, lormetazepam, diazepam, tetrazepam.c Bromazepam, clonazepam, diazepam, flunitrazepam, lorazepam, alprazolam, �-OH-alprazolam, �-OH-triazolam.d Bromazepam, alprazolam, clonazepam, lorazepam, oxazepam, diazepam, midazolam, flurazepam, flunitrazepam, nordazepam, triazolam, temazepam, nitrazepam,

chlordiazepoxide.e Bromazepam, clonazepam, flunitrazepam, lorazepam, alprazolam, diazepam.f Alprazolam, doxepine, amitryptyline, clomipramine.g Diazepam, ethylmorphine, nitrazepam, oxazepam, temazepam, clonazepam, midazolam, nordazepam, lorazepam, zolpidem, alprazolam, benzoylecgonine, metham-

phetamine, fentanyl hydrochlorides, 6-monoacetylmorphine, phenazepam, metadone, codeine, pholcodine, cocaine, amphetamine, 3,4-methylenedioxyamphetamine,3,4-methylenedioxyethylamphetamine, 3,4-methylenedioxymethamphetamine, 1-(10,30-benzodioxol-50-yl)-2-butanamine, N-methyl-1-(10,30-benzodioxol-50-yl)-2-butanamine, morphine, buprenorphine, norbuprenorphine, triazolam.

h Morphine, codeine, 6-monoacetylmorphine, methadone, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine,3,4-methylenedioxy-N-ethylamphetamine, benzoylecgonine, cocaine, delta-9-tetrahydrocannabinol, zolpidem, zopiclone, alprazolam, clonazepam, oxazepam, nordazepam,lorazepam, flunitrazepam, diazepam, diphenhydramine, amitriptyline.

i Morphine, codeine, dihydrocodeine, 6-acetylmorphine, methadone, EDDP, buprenorphine, norbuprenorphine, cocaine, benzoylecgonine, ecgonine methylester, cocaethylene, amphetamine, methamphetamine, 3,4-methylenedioxy-N-amphetamine, 3,4-methylenedioxy-N-methylamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, oxazepam, temazepam, nordazepam, diazepam, amitriptyline, chlorpromazine, clomipramine, cyclizine, diphenhydramine, doxepin, fluoxetine,imipramine, procyclidine, tramadol, chlorpheniramine.

j No data was given.k Midazolam, bromazepam, tetrazepam, alprazolam, lorazepam, triazolam, flunitrazepam, diazepam, lormetazepam.l Bromazepam, chlordiazepoxide, clonazepam, diazepam, flurazepam, flunitrazepam, lorazepam, midazolam, nordazepam, oxazepam, temazepam.

m 7-Aminoclonazepam, 7-aminoflunitrazepam, 7-aminonitrazepam, alprazolam, bromazepam, chlordiazepoxide, clobazam, clonazepam, clotiazepam, desalkylflurazepam,diazepam, estazolam, flunitrazepam, flurazepam, OH-alprazolam, OH-midazolam, OH-triazolam, lorazepam, lormetazepam, midazolam, nitrazepam, nordazepam, oxazepam,prazepam, temazepam, triazolam.

n 14-Benzodiazepines: alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordazepam,oxazepam, temazepam, tetrazepam, triazolam and 3 hypnotics: zaleplon, zopiclone, zolpidem.

o Clonazepam, diazepam, oxazepam, triazolam.p Alprazolam, bromazepam, clonazepam, delorazepam, desalkylflurazepam, diazepam, flunitrazepam, flurazepam, lorazepam, midazolam, nitrazepam, nordazepam, tria-

zolam, 4-OH-alprazolam, 7-aminoclonazepam, 7-aminonitrazepam.q No extraction was performed.


Table 6Preparation of keratin samples for the determination of benzodiazepines.

Analytes (n)a Sampleweight(mg)

Initial sample pretreatment Extraction conditions:column/eluent or extractionsolvent

Recovery(%)

Analyticalmethod

Ref.

NailsLiquid–liquid extraction (LLE)Benzodiazepinesb,

zopiclone andmetabolites (12)

ca. 50 Nail samples were decontaminated as limb samples(i.e. right hand, left foot, etc.) dried, and cut into smallpieces (1–2 mm)

Trifluoroacetic acid–methanol(1:50, v/v)

–c LC–MS/MS [76]

HairSolid phase extraction (SPE)Benzodiazepinesd (9) –c Hairs were decontaminated (with 0.1% sodium dodecyl

sulfate, distilled water and dichloromethane),sonicated for 1 h and incubated at room temperatureovernight in methanol–25% ammonium hydroxide(20:1, v/v)

Clean Screen (ZSDAU 020)/1 step: 2% ammoniated ethylacetate2 step: dichloromethane-isopropanol-ammoniumhydroxide (78:20:2 v/v/v)OrMISPE Diazepam MIPcartridges/15% (v/v) acetic acidin ACN

SPE: 55–94MISPE:39–103

LC–MS/MS [77]

Liquid–liquid extraction (LLE)Benzodiazepinese (3) 10 Hairs were subsequent washing (with phosphate

buffer pH 7.4, isopropanol and methylene chloride)and dried in 40 ◦C.Mother’s hair strands were cut into segments andnewborns’ hair samples were not segmented. Then,the hair was powdered in a ball mill and incubatedovernight with phosphate buffer (pH 7.4) at 50 ◦C

Diisopropylether 87–111.6 LC–MS/MS [82]

Benzodiazepinesf (18) 20 Hairs were segmented and rinsed twice with 5 mLdichloromethane, the segments were cut into smallpieces of less than 3 mm, and pulverized in a freezemill sonicated in phosphate buffer (pH 8.4) at roomtemperature for 1 h

Dichloromethane Mean39.4–102.6

LC–MS/MS [78]

Benzodiazepinesg (26) 50 Hairs were washed in isooctane followed by acetone.After drying, they were cut in segments of 1–3 cm andpulverized in a ball mill. Then, 50 mg aliquot wassonicated for 1 h and overnight incubated inphosphate buffer (pH 8.4) at 37 ◦C

Dichloromethane-diethyl ether(90:10, v/v)

55.2–120.1 LC–HRMS [79]

Benzodiazepinesh,zolpidem andzopiclone (28)

ca. 20 Hairs were decontaminated (with dichloromethane,water and methanol) under ultrasonication, dried andcut into segments in (1–3 cm). ∼20 mg sample waspulverized in ball meal and incubated in methanol at45 ◦C for 2 h with orbital shaking. After centrifugation,saturated ammonium chloride buffer (pH 9.2) wasadded

1-Chlorobutane 53.0–103.8 LC–MS/MS [29]

Benzodiazepinesi andzolpiden (32)

ca. 20 Hairs were washed twice with organic solvent(dichloromethane, methanol), taken to dryness undernitrogen stream and cut into small pieces, thenultrasonicated with a phosphate buffer (pH 8.4) up to1 h

Dichloromethane-diethyl ether(90:10, v/v)

39.4–115.7 LC–MS/MS [81]

Benzodiazepinesj andzopiclone (12)

ca. 50 Hairs were segmented axially (1–3 cm) from thescalp-end. The samples were soaked in distilled andthen deionised water, and decontaminated withethanol

Trifluoroacetic acid–methanol(1:50, v/v)

–c LC–MS/MS [76]

Benzodiazepines andhypnoticsk (24)

30 Hairs were segmented into 3–6 cm, the segments werewashed (with water, acetone and hexane). ∼30 mgsnippets were pulverized in an Eppendorff containinga tungsten carbide ball using a bench top shaker (for10 min, at frequency 30 Hz)

1 step: methanol2 step: 5 mM ammoniumformate buffer adjusted to pH3.5 with formic acid andmethanol (1:1, v/v)

16–108.5 LC–MS/MS [80]

Benzodiazepinesl (21)among 96 drugs

10 10 mg of segmented hair (1 cm) were rinsed once withisopropanol and twice with water, and then dried. Hairwas then pulverized in a bead mill homogenizer

Methanol–ACN–ammoniumformate, pH 5.3 (25:25:50,v/v/v)

57–90 UPLC–MS/MS [83]

Benzodiazepines andother drugsm (22)

20 Hairs were cut into 1 cm segments, washed withisopropanol, phosphate buffer and isopropanol in awater bath at 37 ◦C

ACN–25 mM formic acid (5:95,v/v)

25–58 LC–MS/MS [91]

Microwave-assisted extraction (MAE)Benzodiazepinesn (6) 45 Hairs were washed with 0.1% sodium dodecyl sulfate,

water, methanol and water (3x), dried and cut into2 mm pieces and then grounded, addition of boratebuffer (pH 9.5)

Ethyl acetate (at 75 ◦C for10 min at 1600 W)

86.9–93.4 UHPLC–MS–TOF [20]


Table 6 (Continued)

Analytes (n)a Sampleweight(mg)

Initial sample pretreatment Extraction conditions:column/eluent or extractionsolvent

Recovery(%)

Analyticalmethod

Ref.

Directly (without of extraction)Benzodiazepineso and

zolpiden (32)ca. 20 Hairs were washed twice with dichloromethane and

methanol, taken to dryness and cut into small pieces.Next, 20 ml Halazepam (I.S.) were added to the sample,together with 700 ml methanol and ultrasonicated for1 h

–p –c LC–MS/MS [81]

a Number of analytes.b Alprazolam, clobazam, N-desmethylclobazam, clonazepam, 7-aminoclonazepam, diazepam, nordazepam, lorazepam, midazolam, oxazepam, temazepam, triazolam,

N-desmethylzopiclone.c No data was given.d 7-Aminoflunitrazepam, chlordiazepoxide, diazepam, flunitrazepam, lorazepam, nitrazepam, nordazepam, oxazepam, temazepam.e Diazepam, nordazepam, oxazepam.f Alprazolam, �-OH-alprazolam, midazolam, �-OH-midazolam, triazolam, �-OH-triazolam, estazolam, diazepam, nordazepam, temazepam, oxazepam, clonazepam, 7-

aminoclonazepam, flunitrazepam,7-aminoflunitrazepam, nitrazepam, 7-amnionitrazepam, flurazepam.g 7-Aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, clordiazepoxide, midazolam, flurazepam, bromazepam, �-OH-alprazolam, nitrazepam, �-OH-

triazolam, oxazepam, estazolam, nordazepam, lorazepam, clonazepam, 2-OH-ethylflurazepam, alprazolam, triazolam, flunitrazepam, temazepam, etizolam, clotiazepam,delorazepam, clobazam, lormetazepam, diazepam, pinazepam, prazepam.

h 7-Aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clonazepam, flunitrazepam, clobazam, desmethylflunitrazepam, estazolam, nitrazepam, alprazolam,chlornordiazepam, temazepam, desalkylflurazepam, oxazepam, nordazepam, brotizolam, triazolam, lormetazepam, lorazepam, prazepam, clotiazepam, tetrazepam,diazepam, loprazolam, flurazepam, cloxazolam.

i Lorazepam, prazepam, brotyzolam, triazolam, clotiazepam, �-OH-triazolam, bromazepam, flurazepam, camazepam, �-OH-ethylflurazepam, nitrazepam, desal-cylflurazepam, temazepam, alprazolam, medazepam, diazepam, oxazepam, nordazepam, clobazam, chlordesmethyldiazepam, estazolam, clonazepam, demoxepam,7-amino-clonazepam, pinazepam, flunitrazepam, lormetazepam, 7-aminoflunitrazepam, etizolam, ketazolam, chlordiazepoxide, midazolam.

j Alprazolam, clobazam, N-desmethylclobazam, clonazepam, 7-aminoclonazepam, diazepam, nordazepam, lorazepam, midazolam, oxazepam, temazepam, triazolam,N-desmethylzopiclone.

k Alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, chlordiazepoxide, clonazepam, N-desalkylflurazepam, diazepam, flunitrazepam, flurazepam, �-OH-midazolam, lorazepam, lormetazepam, midazolam, nitrazepam, nordazepam, oxazepam, phenazepam, prazepam, temazepam, triazolam, zaleplon, zolpidem, zopiclone.

l 7-Aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, chlordiazepoxide, clozapine, bromazepam, midazolam, flurazepam, demoxepam, nitrazepam,oxazepam, clonazepam, lorazepam, alprazolam, triazolam, flunitrazepam, demethyldiazepam, temazepam, lormetazepam, phenazepam, diazepam.

m 7-Aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, oxazepam, diazepam, alprazolam, nicotine, cotinine, morphine, 6-monoacetylmorphine, codeine,amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, cocaine, benzoylecgonine, zopiclone, zolpidem, carisoprodol, meprobamate, buprenorphine,methadone.

n Alprazolam, estazolam, lorazepam, clonazepam, diazepam and tetrazepam.o Lorazepam, prazepam, brotyzolam, triazolam, clotiazepam, �-OH-triazolam, bromazepam, flurazepam, camazepam, �-OH-ethylflurazepam, nitrazepam, desal-

cylflurazepam, temazepam, alprazolam, medazepam, diazepam, oxazepam, nordazepam, clobazam, chlordesmethyldiazepam, estazolam, clonazepam, demoxepam,7 pam, e

tonumdmtn

diaemwetCt1Tc

flaae

-aminoclonazepam, pinazepam, flunitrazepam, lormetazepam, 7-aminoflunitrazep No extraction was performed.

The LC–MS/MS technique was applied by Lee et al. [37] forhe determination of nimetazepam (an N-methylated analoguef nitrazepam), a popular “date-rape drug” – flunitrazepam anditrazepam in urine samples originated from alleged sexual assaultrine specimens. It was stressed that the developed LC–MS/MSethod establishes better validation parameters than the other

escribed procedures that were applied to the same type ofatrix. Moreover, the performed study made it possible to confirm

hat nitrazepam and 7-aminonimetazepam are the metabolites ofimetazepam.

Owing to the higher concentrations and longer persistence ofrugs in urine than, for example, in blood, this biological matrix

s widely utilized in forensic analysis in driving re-licensing, carccidents, burglaries or alleged sexual violence cases. Salomonet al. [45] confirmed the usefulness of the fully validated LC–MS/MSethod for the determination of 17 BZD and their metabolites, asell as zolpidem and zopiclone, in urine in 329 forensic cases, after

nzymatic hydrolysis and LLE. Fast separation of the drugs (the totalime of one sample’s analyses was 8 min) was performed on the18 column (50 mm × 4.6 mm × 1.8 �m) with gradient elution ofhe mobile phase. The limits of quantification ranged from 1.7 to00.0 ng mL−1 with the inter- and intra-day precision below 11.8%.he proposed method could find an application in both toxicologi-al and forensic analyses.

Two fast, simple and sensitive methods based on turbulent
ow LC coupled with MS/MS, for the determination of BZD,mphetamines, designer amphetamines, benzoylecgonine, opi-tes and opioids in a urine sample were also applied in forensicxaminations for driving ability diagnostics in Germany [65]. The
tizolam, ketazolam, chlordiazepoxide, midazolam.

validation parameters for BZD, LOD and LOQ, were less than10 and 25 ng mL−1, respectively, and the linearity range was:25–250 ng mL−1, whilst the intra- and inter-day precision was lessthan 15%. The main advantage of the proposed methods is the lackof a laborious sample preparation step, which results in a shorten-ing of the analysis time.

3.1.1.2. Hair and nail samples. Because BZD are used as a “date-rape drug”, analysis of hair and nail samples is of great importancein drug-facilitated crime investigations, as well as in postmortemtoxicology. LC methods play an increasingly important role for thedetermination of BZD in these alternative matrices.

A LC–MS/MS drug-screening method was developed for thedetection of BZD implicated in DFSA concerning zopiclone andselected metabolites in hair and nail samples collected frompatients taking BZD or zopiclone in New Zealand [76]. The pro-posed procedure was also applied to hair analysis of a DFSA victim17 months after the crime, thus confirming the usefulness of alter-native biological samples in forensic toxicological investigations.

Xiang et al. [78] proposed a LC–MS/MS method for the quan-tification of 18 BZD in hair, which was successfully applied for theanalysis of hair segment samples 1 month after a single admin-istration of estazolam and for two samples that originated fromauthentic criminal cases. It was demonstrated that this method,with LOD 0.2 pg mg−1 and LOQ 0.5 pg mg−1, makes it possible to
detect estazolam in hair segments up to 2 cm length with a goodcorrelation between the dosage and segment concentration, whichindicates that segmental hair analysis seems to be a useful tool indrug-facilitated crimes.

252

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al. /

Journal of

Pharmaceutical

and Biom

edical A

nalysis 113

(2015) 239–264

Table 7Experimental conditions and validation parameters of liquid chromatography methods for the determination of benzodiazepines.

Analytes (n)a Sample Chromatographic column Type and speedof mobilephase flow(mL min−1)

Mobile phase LOD (ng mL−1) LOQ (ng mL−1) Accuracyb (%) Precision (%) (RSD) Ref.

Intraday Interday Intraday Interday

HPLC-DAD(UV)Lorazepam Children

plasmaSynergi Max RP150 mm × 4.6 mm × 4 �m

Isocratic2.5

A: 10 mM potassiumdihydrogenortophosphate buffer(pH 2.4)B: ACN (65:35, v/v)

2.5 10 –c 6.6–9.8 7.7–15.9 [42]

Model drugsd (8) Plasma Nucleosil C8250 mm × 4.6 mm × 5 �m

Gradient1

A: 0.002 M aqueousorthophosphoric acid(pH 3)B: ACN

250 –c –c 0.88–10.87 5.32–17.96 [87]

Benzodiazepines(6)e and tricyclicantidepressants(4)f

Plasma Kromasil C8150 mm × 4.6 mm × 5 �m

Gradient1.0

A: methanolB: ACNC: 0.05 M ammoniumacetate

110–1710 360–3910 –c <10 [57]

Urine 90–540 290–1750Benzodiazepinesg

(6)Vitreoushumour

XBrigdeTM Shiled RP18250 mm × 4.6 mm × 5 �m

Gradient0.8

A: ACNB: 0.02 M phosphatebuffer (pH 6)

10–13 30–35 87.6–101.2 <6.2 <7.9 [19]

LC–MSBenzodiazepinesh

(8)Whole blood Chromolith Performance

RP-18e100 mm × 4.6 mm

Isocratic1.5

A: 5 mM aq.ammonium formatewith formic acid (pH 3)B: ACN (65:35, v/v)

–c 2.5–5i 0–14j,k

0.6–19l<15 [64]

Benzodiazepinesm

(16)Whole bloodand serum

Restek Allure C18150 mm × 3.2 mm × 5 �mn

Gradient0.45

A: 5 mM ammoniumacetate (pH 4.75)B: ACNC: methanol

<1 10 –c 15–50 <3000 [27]

VWR superspher 60 RP select B125 mm × 3 mm × 4 �m

Gradient0.8

A: 50 mM potassiumdihydrogen phosphateB: ACN

<1 2–6

Benzodiazepineso

(3) among 12drugs

Plasma, urine,breath

Ethylene Bridged Hybridphenyl column100 mm × 2.1 mm × 1.7 �m

Gradient0.650

A: 2 mM ammoniumformate with 25%ammonia (pH 10)B: methanol withammonium formateand ammonia (as insolvent A)

1 pg/filer 2 pg/filer 85.2–99.3 3.7–11.8 [84]

Benzodiazepines(5)p

Urine ACE C18250 mm × 4.6 mm × 5 �m

Gradient0.8

A: 5% ACN with 0.1%formic acidB: 95% ACN with 0.1%formic acid

0.125–1 0.25–5 80.8–108.7 80.5–118.0 <4.6 <9.4 [37]

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Benzodiazepinesq

(25)Oral fluid Zorbax Eclipse XDB-C18

150 mm × 4.6 mm × 5 �mGradient1.0

A: 2 mM ammoniumformate/0.2% formicacid in waterB: 2 mM ammoniumformate/0.2% formicacid in ACN

0.01–0.5 0.1–0.5 10 <10 [72]

Benzodiazepinesr

and metabolites(28)

Hair Luna C18150 mm × 1 mm × 5 �m

Gradient0.1

A: water with 0.1%formic acidB: ACN with 0.1%formic acid

0.5–5 pg mg−1 1–10 pg mg−1 <20 2.4–20.1 2.8–20.1 [79]

LC–TOF-MSBenzodiazepiness

(15) among 46drugs

Whole blood ACQUITY UPLC BEH C18100 mm × 2.1 mm × 1.7 �m

Gradient0.6

A: 0.1% formic acidB: ACN

–c 0.06–27 ng g−1 –c <30 [97]

Benzodiazepinesand theirmetabolites (41)

Serum, urineand gastricsamples

Zorbax C18150 mm × 2.1 mm × 1.8 �m

Gradient0.25

A: 0.1% formic acidB: ACN (80:20, v/v)

0.16–32.1 –c –c –c [28]

Benzodiazepinest

(6)Hair Hypersil Gold Phenyl

50 mm × 2.1 mm × 1.9 �mGradient0.4

A: ACN with 0.1%formic acidB: ammonium formatebuffer

0.003–0.025ng mg−1

0.010–0.082ng mg−1

89.8–105.7 1.5–4.3 2.3–8.3 [20]

LC–MS/MSBenzodiazepinesu

(11)Ante-mortemandpost-mortemwhole blood

Acquity UPLC BEH C18100 mm × 2.1 mm × 1.7 �m

Gradient0.6

A: ACNB: and 5 mMammonium acetatebuffer (pH 5.0)

0.005–3.0 nM 0.6–75 nM –c 1–19 2–19 [46]

Benzodiazepines,metabolitesv

(26), zolpidemand zopiclone

Whole blood X-terra MS C18150 mm × 2.1 mm ×3.5 �m

Gradient0.2

A: 0.1% formic acidB: methanol

–c 1–2 pg mg−1 −18.0 to8.9

−20.0 to14.5

0.4–14.2 1.2–19.9 [29]

Urine 10–25 pg mg−1 −6.5 to19.4

−20.6 to14.1

0.8–10.2 1.0–20.8

Hair 0.5–10 pg mg−1 −15.2 to18.2

−3.8 to17.9

1.2–16.6 LOQ–1000pg mg−1

Benzodiazepinesand metabolitesw

(21)

Serum Unison C18150 mm × 2.0 mm × 3 �mCadenza C18150 mm × 2.0 mm × 3 �m

Gradient0.25

A: 10 mM aqueousammonium formatewith 0.1% formic acidB: methanol with 0.1%formic acid

0.3–11.4 0.8–38.1 75.8–117.1 87.8–116.8 1.9–19.8 3.3–16.1 [50]

Benzodiazepinesx

(4)Plasma –c Gradient

0.5A: ACNB: water

0.02–0.15 0.2–2.0 –c 3–13 6–9 [109]

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Table 7 (Continued)

Analytes (n)a Sample Chromatographic column Type and speedof mobilephase flow(mL min−1)

Mobile phase LOD (ng mL−1) LOQ (ng mL−1) Accuracyb (%) Precision (%) (RSD) Ref.


Urine 2–11 6–8Lorazepam and its

metaboliteglucuronide

Plasma Chiralpak OD-R250 mm × 4.6 mm × 10 �m

Isocratic1.0

A: ACNB: waterC: acid acetic(80:20:0.1, v/v/v)

–c 1.0 3.0 <15 [41]

Urine 10.0 13Benzodiazepinesy

(4)Urine Shodex MSpak GF–310 4B

50 mm × 3.9 mm × 6 �mGradientvariable

A: 10 mM ammoniumacetateB:ACN

0.1 0.5 89.2–112 <9.2 9.6 [110]

Benzodiazepinesand metabolitesz

(21), zolpidem,zopiclone

Urine Eclipse XDB C18 column50 mm × 4.6 mm × 1.8 �m

Gradient1.0

A: waterB: methanol

0.5–30 1.7–100 −21.9 to20.5

−15.2 to7.5

<12 <10 [45]

Illicit andmedicinal drugsand theirmetabolitesA

(23)

Oral fluid andpreserved oralfluid

Atlantis dC1850 mm × 2.1 mm × 3 �m

Gradient0.3

A: ACNB: 0.1% formic acid inwater

0.5 1 <15 <15 [68]

BenzodiazepinesB

(3)Mothers’ andnewborns’ hair

Zorbax Eclipse XDB-C18 RapisResolution HT50 mm × 4.6

Gradient0.5

A: 0.1% formic acid inwaterB: ACN

5 pg mg−1 10 pg mg−1 –c 0.5–21.4 –c [82]

BenzodiazepinesC

(18)Hair Resteck Allure PFP Propyl

100 mm × 2.1 mm × 5 �mGradient0.2

A: ACNB: 20 mM ammoniumacetate buffer with0.1% formic acid (pH4.0)

0.5–2 pg mg−1 –c <16.5 <14.8 <18.4 [78]

BenzodiazepinesD

(32)Hair Two Hypersil Gold

100 mm × 2.1 mm × 3 �mGradient0.2

A: 0.1% formic acid(v/v)B: ACN

0.1–1.0 pg mg−1 0.1–5.0 pg mg−1 0.6–21.0 0.4–18.2 [81] E

0.1–5.0 pg mg−1 0.5–20.0 pg mg−1 1.4–22.3 0.5–17.6 [81] F

BenzodiazepinesG,zopiclone andmetabolites (12)

Hair PhenomenexLuna50 mm × 2.0 mm × 3 �m

Gradient0.2

A: ACNB: 2 mM ammoniumformate

0.01–6.0 pg mg−1 –c 98–210 5–59 [76]

Nails 102–105 15–18BenzodiazepinesH

(21) among 96drugs

Hair Acquity UPLC HSS C18150 mm × 2.1 mm × 1.8 �m

Gradient0.4

A: 5 mM ammoniumformate (pH 3.0)B: 0.1% formic acid inACN

<0.001–<0.100 0.002–0.10 −20 to 22 3–20 4–27 [83]

Benzodiazepinesand hypnoticsI

(24)

Hair PhenomenexKinetex C1850 mm × 2.1 mm × 2.6 �m

Gradient0.75

A: 5 mM ammoniumformate buffer withformic acid (pH 3.5)B: methanol withammonium formate

–c 0.6–16 pg mg−1 −14.5 to14.2

≤15 [80]

Lorazepam Fingerprintdeposits

Waters C18150 mm × 2.1 mm × 1.7 �m

Gradient0.250

A: 10 mM ammoniumformate with 0.01%formic acid (v/v)B: ACN with 0.01%formic acid (v/v)

1.5 pg 5 pg –c <4.5 [85]

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Its 3-O-glucuronide 8 pg 30 pg

a Number of the analytes.b Accuracy was determined by authors in various manners.c No data was given.d Alprazolam, paracetamol, promazine, chlorpromazine, amitriptyline, salicyclic acid, opipramol, carbamazepine.e Bromazepam, clonazepam, flunitrazepam, lorazepam, alprazolam, diazepam.f Alprazolam, doxepine, amitryptyline, clomipramine.g Alprazolam, bromazepam, diazepam, lorazepam, lormetazepam, tetrazepam.h Clonazepam, diazepam, flunitrazepam, lorazepam, midazolam, N-desalkylflurazepam, nordazepam, oxazepam.i 80 ng mL−1 for desipramine.j 19.8 for desipramine.k LC–MS – monolith support.l LC–MS – RAM.

m Mordazepam 7-aminoflunitrazepam, alprazolam, desalkylflurazepam, desmethylflunitrazepam, diazepam, flunitrazepam, flurazepam, �-OH-midazolam, lorazepam, midazolam, nitrazepam, nordazepam, oxazepam,lormetazepam, bromazepam, clobazam.

n HPLC–PDA-MS.o Diazepam, oxazepam, alprazolam.p Flunitrazepam, nimetazepam, nitrazepam, 7-aminonimetazepam, 7-aminoflunitrazepam.q 7-Aminoclonazepam, 7-aminoflunitrazepam, 7-aminonitrazepam, alprazolam, bromazepam, chlordiazepoxide, clobazam, clonazepam, clotiazepam, desalkylflurazepam, diazepam, estazolam, flunitrazepam, flurazepam,

OH-alprazolam, hydrozymidazolam, OH-triazolam, lorazepam, lormetazepam, midazolam, nitrazepam, nordazepam, oxazepam, prazepam, temazepam, triazolam.r 7-Aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, clordiazepoxide, midazolam, flurazepam, bromazepam, �-OH-alprazolam, nitrazepam, �-OH-triazolam, oxazepam, estazolam, nordazepam, lorazepam,

clonazepam, 2-OH-ethylflurazepam, alprazolam, triazolam, flunitrazepam, temazepam, etizolam, clotiazepam, delorazepam, clobazam, lormetazepam, diazepam, pinazepam, prazepam.s 7-Aminoflunitrazepam, alprazolam, bromazepam, clobazam, lonazepam, desmethyldiazepam, diazepam, estazolam, flunitrazepam, lorazepam, lormetazepam, midazolam, nitrazepam, oxazepam, temazepam.t Alprazolam, estazolam, lorazepam, clonazepam, diazepam and tetrazepam.u Alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, lorazepam, midazolam, nitrazepam, nordazepam, oxazepam, phenazepam.v 7-Aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clonazepam, flunitrazepam, clobazam, desmethylflunitrazepam, estazolam, nitrazepam, alprazolam, chlornordazepam, temazepam, desalkylflurazepam, oxazepam,

nordazepam, brotizolam, triazolam, lormetazepam, lorazepam, prazepam, clotiazepam, tetrazepam, diazepam, loprazolam, flurazepam, cloxazolam.w Flurazepam, bromazepam, chlordiazepoxide, nitrazepam, clonazepam, flunitrazepam, estazolam, clobazam, lorazepam, alprazolam, triazolam, brotizolam, fludiazepam, diazepam, quazepam, prazepam, 7-aminonitrazepam,

7-aminoclonazepam, 7-acetamidonitrazepam, N-desmethylclobazam, N-desmethyldiazepam.x Nordiazepam, diazepam, lorazepam, oxazepam.y Diazepam, nordazepam, temazepam, oxazepam.z Flunitrazepam, diazepam, demoxepam, medazepam, clonazepam, desalkylflurazepam, oxazepam, lormetazepam, alprazolam, lorazepam, nitrazepam, midazolam, prazepam, temazepam, triazolam, zolpidem, nordazepam,

flurazepam, N-�-OH-ethylflurazepam, 7-aminonitrazepam, 7-aminoflunitrazepam, bromazepam.A Morphine, codeine, 6-monoacetylmorphine, methadone, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, benzoylec-

gonine, cocaine, delta-9-tetrahydrocannabinol, zolpidem, zopiclone, alprazolam, clonazepam, oxazepam, nordazepam, lorazepam, flunitrazepam, diazepam, diphenhydramine, amitriptyline.B Diazepam, nordazepam, oxazepam.C Alprazolam, �-OH-alprazolam, midazolam, �-OH-midazolam, triazolam, �-OH-triazolam, estazolam, diazepam, nordazepam, temazepam, oxazepam, clonazepam, 7-aminoclonazepam, flunitrazepam,7-aminoflunitrazepam,

nitrazepam, 7-amnionitrazepam, flurazepam.D Lorazepam, alprazolam, bromazepam, flurazepam, �-OH-ethylflurazepam, desalkyflurazepam, flunitrazepam, 7-aminoflunitrazepam, clonazepam, 7-aminoclonazepam, lormetazepam, clobazam, demoxepam, prazepam,

chlordiazepoxide, midazolam, nitrazepam, zolpidem, medazepam, oxazepam, diazepam, nordazepam, �-OH–triazolam, triazolam, temazepam, clotiazepam, estazolam, etizolam, chlordesmethyldiazepam, brotyzolam, ketazolam,pinazepam, halazepam.

E Data for LC–MS/MS with LLE.F Data for LC–MS/MS with direct injection.G Alprazolam, clobazam, N-desmethylclobazam, clonazepam, 7-aminoclonazepam, diazepam, nordazepam, lorazepam, midazolam, oxazepam, temazepam, triazolam, N-desmethylzopiclone.H 7-Aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, chlordiazepoxide, clozapine, bromazepam, midazolam, flurazepam, demoxepam, nitrazepam, oxazepam, clonazepam, lorazepam, alprazolam, triazolam,

flunitrazepam, demethyl diazepam, temazepam, lormetazepam, phenazepam, diazepam.I Alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, chlordiazepoxide, clonazepam, N-desalkylflurazepam, diazepam, flunitrazepam, flurazepam, �-OH-midazolam, lorazepam, lormetazepam, midazolam,

nitrazepam, nordazepam, oxazepam, phenazepam, prazepam, temazepam, triazolam, zaleplon, zolpidem, zopiclone.

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Table 8Experimental conditions and validation parameters of gas chromatography methods for the determination of benzodiazepines.

Analytes (n)a Sample Chromatographic column Temperature programme LOD(ng mL−1)

LOQ(ng mL−1)

Accuracyb (%) Precision (%) (RSD) Ref.


GC-(EI)-MSClonazepam, 7-

aminoclonazepamWhole blood HP-5MS

12 m × 0.2 mm × 0.33 �m150 ◦C (for 0.5 min)–300 ◦C(30 ◦C min−1; hold for 1 min)

1 5 –c <6 [10]

Benzodiazepinesd

(23)Whole blood HP-5MS

30 m × 0.25 mm × 0.25 �m120 ◦C (for 1 min)–295 ◦C(10 ◦C min−1; hold for 5 min)

0.52–58.47 1.58–177.2 <8.5 <11.1 [13]

Benzodiazepinesand metabolitese,zaleplon,zolpidem (14)

Whole blood DB-35 ms30 m × 0.32 mm × 0.25 �m

120 ◦C (for 1 min)–330 ◦C(15 ◦C min−1; hold for 2.80 min)

0.4–10 5–200 −16.3 to 17.8 2.4–10.7 3.8–13.8 [11]

Benzodiazepinesand otherpsychotropicdrugsf (6)

Whole blood DB-5MS(30 m × 0.25 mm × 5 �m)

80 ◦C (for 1 min)–320 ◦C(10 ◦C min−1; hold for 1 min)

10–1000 –c −10.4 to 9.0 −12.4 to 8.4 0.6–9.3 1.9–13.2 [62]

Pharmaceuticalsg

(40)Whole blood VF-5-MS and VF-X-MS

30 m × 0.25 mm × 0.25 �m50 ◦C (for 1 min)–200 ◦C(30 ◦C min−1)–280 ◦C(5 ◦C min−1)–300 ◦C(40 ◦C min−1; hold for 5 min)

<20 <39.0 –c <8 <20 [99]

Diazepam Plasma CROMA 530 m × 0.25 mm × 0.30 �m

160 ◦C–280 ◦C(15 ◦C min−1; hold for 10 min)

0.3 1.0 –c <14.0 [16]

Clotiazepam Plasma HP-5MS(30 m × 0.25 mm × 0.25 �m)

150 ◦C–210 ◦C (30 ◦C min−1)210 ◦C–270 ◦C (8 ◦C min−1)270 ◦C (hold for 0.5 min)

–c 5 99.0–107.9 92.4–101.3 3.3–13.1 1.0–13.5 [56]

Benzodiazepinesh

(3)Urine VF-5-MS

30 m × 0.25 mm × 0.25 �m60 ◦C (for 1 min)–280 ◦C(15 ◦C min−1; hold for 2.33 min)

–c 0.15–1.5 –c 2.0–4.9 4.1–12.4 [100]

Drugs of abusei

(30)Oral fluid Fraction Ij and IIIk: DB-35 ms

30 m × 0.32 mm × 0.25 �mFraction IIl: DB-5 ms30 m × 0.32 mm × 1.0 �m

Fraction If: 90 ◦C (for1 min)–330 ◦C(60 ◦C min−1; hold for4.50 min) Fraction IIh:130 ◦C (for 3 min)–300 ◦C(30 ◦C min−1; hold for 2 min)Fraction IIIg: 90 ◦C (for1 min)–330 ◦C(45 ◦C min−1; hold for 4.30 min)

–c <5 −15.4 to 15.0 <10 <13 [12] m

Psychoactivedrugsn (32)

Oral fluid Ultra 116.5 m × 0.2 mm × 0.11 �m

70 ◦C (for 2 min)–160 ◦C(30 ◦C min−1)–170 ◦C(5 ◦C min−1)–200 ◦C(20 ◦C min−1)–220 ◦C(10 ◦C min−1)–300 ◦C(30 ◦C min−1)

0.3–6.9 0.9–44.2 1.5–22.2 2.9–24.9 2.0–25.6 1.2–26.5 [69]

Licit and illicitdrugso (49)

Oral fluid HP-130 m × 0.25 mm × 0.25 �m

80 ◦C (for 2 min)–280 ◦C(10 ◦C min−1; hold for 5 min)

0.2–3.4 0.8–11.4 –c 1.8–17 0.4–6.5 [70]

GC-(NICI)-MSBenzodiazepinesp

(16) andzaleplon,zopiclone (16)

Whole blood DB-5HT30 m × 0.32 mm × 0.10 �m

180 ◦C (for 1 min)–350 ◦C(50 ◦C min−1)

–c 1–100 −12.5 to 16.0 1.99–29.6 [9]

Benzodiazepinesq

(15)Whole blood DB-5HT

(30 m × 0.32 mm × 0.1 �m)180 ◦C–325 ◦C(50 ◦C min−1; hold for 1 min)

0.24–0.62 0.72–1.89 89.5–110.5 <7 [14]

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GC-ECDBenzodiazepinesr

(4)Whole blood DB-5MS

(50 m × 0.2 mm × 0.33 �m)50 ◦C (for 4 min)–200 ◦C(7 ◦C min−1; hold for4 min)–300 ◦C (7 ◦C min−1;hold for 5 min).

0.04–0.290.14–1.54–c 1.05–2.08

[47]

Plasma 0.05–0.520.14–1.58 1.87–3.07Diazepam Plasma Glass column OV 17–3% on

80–100 mesh ChromosorbW/HP1.2 m × 4.5 mm

Column temperature: 260 ◦C;detector temperature: 310 ◦C

5 10 –c 4.6 7.9 [43]

GC-NPDBenzodiazepinesr

(4)Whole blood DB-5MS

(50 m × 0.2 mm × 0.33 �m)50 ◦C (for 4 min)–200 ◦C(7 ◦C min−1; hold for4 min)–300 ◦C (7 ◦C min−1;hold for 5 min).

0.08–0.950.25–2.88–c 2.38–4.68

[47]

Plasma 0.08–0.930.23–2.80 2.34–5.04Anaestheticss and

analgesicstUrine Alltech EG

30 m × 0.32 mm × 0.25 �m110 ◦C (for 1 min)–260 ◦C(32 ◦C min−1, hold for 5 min)

0.01–1.5 –c –c 7.7–12.6 [98]

a Number of analytes.b Accuracy was determined by authors in various manners.c No data was given.d Diazepam, nordazepam, oxazepam, bromazepam, alprazolam, lorazepam, medazepam, flurazepam, fludiazepam, tetrazepam, chlordiazepoxide, clobazam, midazolam, flunitrazepam, 7-aminoflunitrazepam, triazolam,

prazepam, nimetazepam, temazepam, lormetazepam, clonazepam, camazepam.e Chlordiazepoxide, diazepam, nitrazepam, oxazepam, temazepam, bromazepam, medazepam, midazolam, nordazepam, lorazepam, �-OH-alprazolam, alprazolam, flurazepam, phenazepam, �-OH-midazolam.f Flunitrazepam, 7-aminoflunitrazepam, triazolam, �-OH-triazolam, brotizolam, �-OH-brotizolam, non-benzodiazepine psychotropic drugs: phenobarbital, chlorpromazine, promethazine.g Acetaminophen, amitriptyline, articaine, atropine, benzocaine, biperidene, caffeine, cholesterol, codeine, diazepam, dienestrol, diethylstilbestrol, diphenhydramine, doxepin, escitalopram, estradiol, imipramine, lidocaine,

memantine, methylphenobarbital, nicotine, papaverine, paroxetine, phenobarbital, phenytoine, pridinol, prilocaine, procaine, scopolamine, sparteine, strychnine, testosterone propionate, theobromine, theophylline, thiopental,tiamulin, tiapride, tramadole, riphenylmethane tropine, xylazine.

h Diazepam, nordazepam, oxazepam.i Diazepam, ethylmorphine, nitrazepam, oxazepam, temazepam, clonazepam, midazolam, nordazepam, lorazepam, zolpidem, alprazolam, benzoylecgonine, methamphetamine, fentanyl hydrochlorides, 6-monoacetylmorphine,

phenazepam, metadone, codeine, pholcodine, cocaine, amphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, 3,4-methylenedioxymethamphetamine, 1-(10,30-benzodioxol-50-yl)-2-butanamine, N-methyl-1-(10,30-benzodioxol-50-yl)-2-butana-mine, morphine, buprenorphine, norbuprenorphine, triazolam.

j Most of the benzodiazepines, delta-9-tetrahydrocannabinol (�9-THC).k Opiates, cocaine, benzoylecgonine, midazolam, alprazolam and zolpidem.l Amphetamine-type stimulant drugs.

m Data for BZD.n Ethylamphetamine, phentermine, cocaine and metabolites (benzoylecgonine, cocaethylene, and ecgonine methyl esther), cannabinoids (delta-9-tetrahydrocannabinol, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol, 11-

OH-delta-9-tetrahydrocannabinol, cannabinol and cannabidiol), opiates (6-monoacetylmorphine, morphine and codeine), hypnotics (flurazepam, flunitrazepam, dipotassium chlorazepate, alprazolam, diazepam and oxazepam),antidepressant drugs (amitriptyline, paroxetine and sertraline), antipsychotic drugs (haloperidol, chlorpromazine and fluphenazine) chlormethiazole, loratidine, hydroxyzine, diphenhydramine, valproic acid and gabapentin.

o Morphine, codeine, dihydrocodeine, 6-acetylmorphine, methadone, EDDP, buprenorphine, norbuprenorphine, cocaine, benzoylecgonine, ecgonine methyl ester, cocaethylene, amphetamine, methamphetamine, 3,4-methylenedioxy-N-amphetamine, 3,4-methylenedioxy-N-methylamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, oxazepam, temazepam, nordazepam, diazepam, amitriptyline, chlorpromazine, clomipramine,cyclizine, diphenhydramine, doxepin, fluoxetine, imipramine, procyclidine, tramadol, chlorpheniramine.

p Chlordiazepoxide, diazepam, nitrazepam, oxazepam, temazepam, bromazepam, clonazepam, flunitrazepam, midazolam, nordazepam, lorazepam, OH-alprazolam, triazolam, alprazolam, phenazepam, OH-midazolam.q 7-Aminoclonazepam, �-OH-alprazolam, �-OH-midazolam, alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, lorazepam, midazolam, nitrazepam, nordazepam, oxazepam, temazepam, triazolam.r Flunitrazepam, nitrazepam, clonazepam, alprazolam.s Lidocaine, midazolam, diazepam, ketamine.t Fentanyl, remifentanyl, codeine.


Table 9Experimental conditions and validation parameters of chromatography-related methods for the determination of benzodiazepines.

Analytes (n)a Sample Chromatographiccolumn

Separation voltage[kV]

Mobile phase LOD(ng mL−1)

LOQ(ng mL−1)

Accuracyb

(%)Precision (%)(RSD)

Ref.

CEC-TOF-MSBenzodiazepinesc

(10)Urine Monolithic

fused-silica column500 mm

3.8 Methanol/water(50:50, v/v)with formicacid (0.1%, v/v)

0.6–1.8 1 –d 1.1–9.2 [36]

Sweeping CEAbused drugs and

hypnoticseUrine Uncoated

fused-silica504 mm × 5 �m

−15 75 mMphosphatebuffer (pH2.5)/methanol(70:30, v/v),SDS

20–50 –d <15.5 <15.5 [39]

Sweeping-MECCBenzodiazepinesf

(7)Urine

Pheonix400 mm × 5 �m −25

With C16MIMBr 9.39–38.25 31.3–127.5–d <8.26g [53]

With C16MPYB 4.68–9.75h 15.6–32.5Benzodiazepinesi

(3)Urine Fused silica

60 × 50 �m−25 3 mL

dichloromethane/isopropanol/ammoniumhydroxide(78:20:2, v/v/v)

5.6–13.4 –d –d <4.10 [107]

a Number of the analytes.b Accuracy was determined by authors in various manners.c Alprazolam, triazolam, chlordiazepoxide, lorazepam, nitrazepam, clonazepam, flunitrazepam, clorazepate, diazepam, prazepam.d No data was given.e Morphine, ketamine, methamphetamine, codeine, alprazolam, clonazepam, diazepam, flunitrazepam, oxazepam, �-OH-alprazolam, 7-aminoclonazepam, nordazepam,

7-aminoflunitrazepam, N-demethylflunitrazepam.f Diazepam, clorazepate, chlordiazepoxide, bromazepam, nitrazepam, alprazolam, flunitrazepam.

fmmacfad

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g For peak area.h For non-biological samples.i Flunitrazepam, 7-aminoflunitrazepam, N-desmethylflunitrazepam.

Segmental hair analysis could be also successfully employedor the evaluation of prenatal exposure to BZD. A LC–MS/MS

ethod was applied for the determination of diazepam and itsetabolites (nordazepam and oxazepam) in the hair of mothers

nd newborns at the level of 10 pg mg−1 [82]. A higher con-entration level of diazepam in newborns’ hair confirmed the
act that a foetus has a lower ability to metabolize drugs thandults. Application of the developed method in such cases asescribed in the paper may assess the health risk of a child and,
able 10alidation parameters of other methods for the determination of benzodiazepines in biol

Analytes (n)a Sample Analytical method LOD(ng mL−

Diazepam Plasma Voltammetry 21

Oxazepam Urine 12Lorazepam Urine, plasma Adsorptive cathodic

pulse strippingvoltammetry

19

Oxazepam Urine Fluorimetric screeningmethod

4.15

Clonazepam Bone, bonemarrow, blood

Colorimetric 8.9 × 10

ClonazepamUrine Chemiluminescence

28.0

Diazepam 19.0

7-aminoclonazepam

Human urine Ultrasensitiveimmunoassay

0.021

Benzodiazepinesd

(7)Urine AP-MALDI-MS 7.9–97

AP-MALDI-MS/MS <10 pmBenzodiazepinese

(11)Urine SERSf 2–10

a Number of the analytes.b Accuracy was determined by authors in various manners.c No data was given.d Diazepam, lorazepam, midazolam, triazolam, nitrazepam, N-desalykylflurazepam, oxe Alprazolam, midazolam, triazolam, 7-aminoflunitrazepam, chlordiazepoxide, clonazef Data for 7-aminoflunitrazepam, diazepam and nordazepam spiked into urine.

in turn, the implementation of an appropriate therapeutic inter-vention.

According to the authors [79], liquid chromatography-highresolution mass spectrometry (LC–HRMS) method for the simul-taneous determination of 28 BZD and their metabolites (whichinclude about 90% of the BZD registered on the Italian market)
in hair samples might be a suitable alternative for the conven-tional methods applied in clinical and forensic toxicology. In thereported procedure, separation of all the analytes was performed
ogical samples.

1)LOQ(ng mL−1)

Accuracyb (%) Precision (%) (RSD) Ref.

–c –c <2 [59]

–c –c 2.41 [24]

13.85 –c 7.14 [15]

−1 M –c 92.4–99.7 0.94–1.63 [105]

96.00.12–3.58 0.98–2.99 [30]

83.9–c –c <5 [61]

.0 pmol –c –c 1.2–11.7 [51]

ol–c –c –c [106]

azepam.pam, diazepam, flunitrazepam, lorazepam, nordazepam, oxazepam.

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n the C18 column (150 mm × 1 mm × 5 �m), and gradient elutionf water/ACN with an addition of 0.1% formic acid mobile phaseas used. The HRMS technique with a full scan mode seems to be aowerful tool for the exact determination of the molecular massesf analyzed compounds eluted from a HPLC column.

The UPLC–MS-TOF method, in combination with MAE, has beenroposed for the determination of 6 BZD in hair [20]. The valida-ion data, including the LOD and LOQ values, intra- and inter-dayrecision, and accuracy of the assay were provided. Applications ofhe optimized and validated method for the analyses of clinical hairamples indicate its usefulness in clinical, toxicological and forensicaboratories.

The UPLC–MS/MS method was also developed for the deter-ination of a wide group of 96 drugs, such as BZD, opiates,

mphetamines, hallucinogens, antihistamines, antidepressants,ntipsychotics, barbiturates and other sedatives, and muscle relax-nts in hair [83]. The low range of the LOD and LOQ, as well as aecovery value higher than 70%, and intra- and inter-day repeata-ility, indicate that the proposed procedure could be successfullytilized in toxicological, clinical, forensic and DFSA investigation.

.1.1.3. Oral fluid and vitreous humour samples. Oral fluid seems toe one of the alternative biological materials gaining much atten-ion in analysis where DUID is concerned. LC techniques are the

ost popular ones used for the determination of BZD in these kindsf biological samples.

Uddin et al. developed a simple, fast and reliable HPLC-DADethod for the determination of BZD mixtures with SPE from body

uids, such as plasma, urine [55,57] and saliva [86,111], whichould be applied in pharmacokinetic studies, therapeutic drugonitoring, and forensic toxicology. The separation of analytesas performed on a Kromasil C8 (250 mm × 5 mm × 5 �m) columnith a gradient flow of the mobile phase containing methanol, ACN

nd 0.05 M ammonium acetate. Due to the low LOD and LOQ val-es (0.08–0.34 ng mL−1 and 0.28–1.13 ng mL−1, respectively), it isossible to analyze spiked BZD and antidepressants at toxic andherapeutic levels in oral fluid samples, although the usefulness ofhis method in forensic and toxicological analysis should be con-rmed by application of the developed method for real oral fluidamples.

A fully validated LC–MS/MS method was also found to be a usefulool for the simultaneous determination of BZD among other drugsf abuse, such as morphine, codeine, amphetamine and its metabo-ites, cocaine, delta-9-tertrahydrocannabinol, etc. in oral fluid forurposes of DUID analysis [68]. Oral fluid samples were collectedith Stature Saliva SamplerTM, subjected to SPE, plus analyzed

n terms of their freeze-and-thaw stability and matrix effect. Thebtained validation parameters indicate that the proposed methodould be applied in DUID cases in the future.

Developed by Jang et al. [72], a LLE-LC–MS/MS method forhe determination of 25 BZD and zolpidem in oral fluid sam-les was successfully employed to authentic saliva specimensbtained from psychiatric patients who took these drugs reg-larly. It has been proven that some of the examined drugs,

ncluding alprazolam, clonazepam, diazepam, flunitrazepam, flu-azepam, lorazepam, zolpidem and/or their metabolites, coulde detected 1–18 h after administration, which indicates thesefulness of oral fluid as a material for forensic toxicological anal-sis.

LC techniques could be also applied to post-mortem species’nalysis, such as vitreous humour [19,67,93]. The main advantages
f this biological matrix are the low content of proteins, stabilitynd lack of pollutions. Determination of the analytes in this type ofatrix is particularly important in forensic cases where the body
ndergoes bleeding, decomposition or burning.


3.1.1.4. Miscellaneous samples. One of the most interesting applica-tions of the LC–MS/MS technique was an investigation of lorazepamand its 3-O-glucuronide in fingerprint deposits, which could beapplicable in DFSA analyses [85]. The samples were collected fromvolunteers after the oral administration of a 2 mg single dose oflorazepam. A sensitive procedure made it possible to determine thedrug and its metabolite at pictogram levels. The chromatogram wasachieved by the use of a C18 column (150 mm × 2.1 mm × 1.7 �m)and gradient elution of a mobile phase.

An interesting application of the LC–MS technique to non-conventional biological samples for the determination of BZD mayalso be afforded by analysis of exhaled breath, whose results werecompared with the plasma and urine levels of the tested drugs[84]. Using this method, it can be stated that analytical sensitiv-ity for BZD should be improved for an acceptable detection rate, inspite of the fact that the detection rate for most of the other inves-tigated substances appeared to be sufficiently high. Nevertheless,exhaled breath seems to be a convenient material for clinical andtoxicological analysis.

3.1.2. Gas chromatography3.1.2.1. Plasma, whole blood and urine samples. Gunnar et al. [9,11]presented a reliable, sensitive and quantitative procedure basedon tert-butyldimethylsilyl (TBDMS) derivatives for the determi-nation of 14 BZD, two �-hydroxy metabolites, and zolpidem andzaleplon in whole blood. In order to select the optimal derivatizingagents, three of the most common commercially available silylat-ing reagents (MSTFA, BSTFA + 1% TMCS and MTBSTFA) were tested[11]. Utilizing MTBSTFA made it possible to obtain the most stablederivatives, besides sensitive and repeatable GC–EI-MS results. Thesame derivatizing reagent was next employed by the same authorsfor the determination of BZD in whole blood by GC–NICI-MS [9]. Themain advantages of utilizing this chromatography technique werethe fast chromatographic separation (4.40 min), as well as a highsensitivity, even when a very small volume of samples (100 �L),was being analyzed. The repeatable, fully validated, method with alow LOQ range of 1–100 ng mL−1 could be a useful tool in clinicaland forensic toxicological laboratories.

Lower values of LOD (0.24–0.62 ng mL−1) and LOQ(0.72–1.89 ng mL−1) for 15 selected BZD determined in wholeblood utilizing the same chromatographic technique, GC withthe NICI-MS mode, were also obtained by Karlonas et al. [14].The usefulness of the proposed procedure was confirmed by itsapplication for the determination of trace concentrations of BZDdrugs in real blood samples.

The Quick Easy Cheap Effective Rugged Safe (QuEChERS)approach to determination of diazepam together with another 40pharmaceuticals present in whole blood samples, was describedby Plössl et al. [99] The proposed GC–MS with EI and CI ionizationmethods used for quantitative analysis of different types of medica-ments and toxins preceded by dispersive SPE with PSA (a sorbentfunctionalized with primary and secondary amines) seems to bean easy, cheap and fast screening procedure for therapeutic drugmonitoring and forensic and toxicological analysis.

The usability of a fully validated GC–EI-MS method in clinicaland forensic analysis for 23 of the most commonly administratedBZD present in whole blood was proved by Papoutsis et al. [13] byapplication of this analytical procedure in investigations of foren-sic and/or clinical samples that originated from cases concerningaccidental and suicidal poisoning. After LLE, the isolated drugswere derivatizated in two steps by propylation and propionyla-tion, and again subjected to an extraction step using a mixture of
hexane:dichloromethane (3:1, v/v).
Although the connection between GC and MS is currentlymost often applied for the determination of BZD in biologicalmaterial, Bravo et al. [47] developed a new dual column gas GC

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ethod using NPD and a micro-ECD for the determination of fluni-razepam, nitrazepam, clonazepam and alprazolam in whole bloodnd plasma samples. The combination of these two detectors withinhe same instrument represents a novel approach to drug analysisnd made it possible to determine the selected drugs at the level of.14–0.95 ng mL−1 in whole blood and 0.13–0.93 ng mL−1 in plasmaamples.

A GC–MS method associated with ECD and multiple reac-ion monitoring (MRM) was developed for the determination ofiazepam and its main metabolites, nordazepam and oxazepam

n urine samples without a need for the derivatization step [100].enon was used as a collision gas instead of “classic” gas (e.g. argon).he LLE performed in TOXI-TUBES® with the use of deionized wateras given an extraction recovery between 68 and 95% with the

oefficient of variation (CV) below 6%. The LOQs for diazepam,ordazepam and oxazepam were 0.15, 1.0 and 1.5 ng mL−1, respec-ively.

Raikos et al. [98] presented a sensitive GC-NPD method forhe simultaneous determination of widely used anaesthetics (lido-aine, midazolam, diazepam and ketamine) and analgesic drugsfentanyl, remifentanyl and codeine) in urine. In the proposed pro-edure, the optimization of headspace solid-phase microextractionHS-SPME) parameters, such as: SPME fibre, type and amount ofalt added, preheating and extraction time, extraction temperature,ample volume and desorption time, was made. The obtained LODsor all of the tested drugs were in the range of 0.01–1.5 ng mL−1,ith the RSD% less than 12.6. The usefulness of this method was

onfirmed by the determination of the anaesthetics and analgesicsn human urine from patients that had undergone coronary by-passurgery operations.

.1.2.2. Oral fluid samples. Gunnar et al. [12] developed a sin-le procedure for the simultaneous toxicological analysis of BZD,olpidem and other common illicit drugs in a 250 �L oral fluidample using a sequentional mixed-mode SPE and long-column30 m × 0.32 mm) fast GC–EI-MS. The derivatization of BZD wasarried out with a mixture of ACN and MTBSTFA (4:2, v/v). Theully validated and tested interlaboratory quality assurance methodeems to be a comprehensive tool for the screening and quantitativenalysis in toxicological, clinical and forensic laboratories.

A simple and reliable GC–EI-MS method was also applied forhe determination of BZD among a wide group of substancesrom other classes, such as: amphetamine derivatives, cocaine and

etabolites, cannabinoids, opiates, antidepressants and antipsy-hotic drugs in oral fluid samples [69]. All of the analytes weresolated from the samples by SPE and derivatized by MSTFA, andhe LOQs for all of them were in the range of 0.9–44.2 ng mL−1. Theeveloped procedure was applied for analysis of drugs in oral fluidamples taken from injured individuals attending the emergencyoom. The ability to determine a wide range of drugs via one stepnalysis, minimum handling and simplicity indicates the useful-ess of the proposed method in routine drug analysis; e.g. in DUIDases.

.2. Chromatography-related techniques

From 1990s chromatography-related methods like CEC or MECCave come to play increasing role in the drug analysis field, includ-

ng determination of BZD in biological matrices.Blas and McCord [36] developed a CEC method combined with

OF-MS for the determination of 10 selected BZD in urine samples.
o improve the sensitivity, the preconcentration of the analytes athe head of the column with a hexyl acrylate-based porous mono-ith was conducted. Although the proposed method is characterizedy satisfactory reproducibility (RSD% < 9.2%) and a low LOQ (less
Biomedical Analysis 113 (2015) 239–264

than 1.8 ng mL−1 for all analytes), a full validation procedure is stillrequired for its use in routine drug analysis.

Two types of cationic surfactants, N-cetyl-N-methylpyrrolidinium bromide (C16MPYB) and1-cetyl-3-methylimidazolium bromide (C16MIMBr), were testedas agents that enhanced the separation efficiency and detectionsensitivity during the sweeping-MECC analysis of seven BZD inspiked urine samples [53]. The optimization of separation andsweeping conditions were conducted by evaluating parameterssuch as the pH, concentration of the organic modifier and surfac-tants, as well as an injection volume. By applying C16MPYB, therecoveries of all of the analytes, after off-line SPE, were in therange of 77.0–88.3%, and the LOQ was less than 127.5 ng mL−1,thus confirming the usefulness of the proposed sweeping-MECCprocedure in the analysis of BZD in urine samples.

Chiang et al. [39] proposed a versatile, sensitive and selectiveon-line SRMM–MECC method for the simultaneous determinationof BZD among other drugs, such as morphine, codeine, ketamineand methamphetamine and their main metabolites in addicts’ urinesamples. The optimization process, taking into account the time ofanalysis and complete separation, was developed by the use of achemometric experimental design. Phosphate buffer (75 mM, pH2.5) and methanol (90:10, v/v) containing 65 mM sodium dodecylsulfate (SDS) was utilized as a sweeping buffer in the separationat −15 kV voltages. Under the optimized conditions, validationparameters such as the linearity range, LOD, accuracy, and intra-and inter-day precision were evaluated. The described procedurecould have an application in routine analysis of urine samples.

3.3. Electrochemical methods

The investigations on applications of electrochemical methodsto determinations of BZD primarily concern conventional materi-als, such as urine and plasma. As has been already studied [112]the 1,4-benzodiazepines are characterized by a relatively easilyreducible azomethine group. The second mechanism of redox pro-cesses is related to other electrochemically active groups, such asthe nitro, N-oxide and carbonyl groups. The development of newelectrochemical techniques is associated with many advantages,including the short time of analysis, good sensitivity and selectivity,and the low costs of instrumentation compared to other techniques.Despite the advantages mentioned above, papers emerging fromthis area concerned with BZD determination should be regarded asscientific reports. In this context, a very interesting review by Hon-eychurch and Hart [113] describing the pre-2006 years applicationsof LC coupled with electrochemical detection should be considered.

Two electrochemical methods for the determination of:lorazepam with the use of a hanging mercury drop electrode [24]and diazepam (in plasma) or oxazepam (in urine) using a glassycarbon electrode modified with bentonite [59] have been pre-sented. In both procedures, DPV was used, and comparable LODswere achieved (19 and 12 ng mL−1, correspondingly) with the pre-cision around 2% defined as relative standard deviation (RSD). InGhasemi’s paper [24], the decrease of LOD was obtained due toaccumulation of lorazepam at the working electrode with a poten-tial of 200 mV and the time of depolarization at 40 s, followed byreduction of this drug by a voltammetric scan using differentialpulse modulation. The credibility of the method was evaluated bythe determination of lorazepam in biological fluids (urine, plasma),with satisfactory results. For the determination of diazepam andoxazepam [59], the following experimental conditions were opti-
mized: modification of working electrode surface with 5% ofbentonite, buffer solution, ionic strength, pulse amplitude, andrepetition time (−125 mV; 0.7 s). Under optimal experimental con-ditions, a sensible, versatile and low cost method was developed to

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etermine the BZD in both biological fluids. Due to the complexityf the analyzed matrices, a SPE procedure was needed to separatehe medicaments before their determination.

The electrochemical detection was coupled with LC as describedy Honeychurch et al. [108]. Application of a cyclic and hydro-ynamic voltammetry helped to explain the electrochemicalechanism, which was exploited for BZD analysis and to find the

ptimal experimental conditions for this analysis. The proposedethod was based on dual-electrode detection with a glassy car-

on electrode in two cells (“generator” one with a potential 2.4 V,nd “detector” one with a potential 0.5 V) allowed to determineitrazepam with the LOD and precision equal to 5 ng mL−1 and 4.2%,espectively. According to the authors, this method is character-zed by the high selectivity of the dual-electrode detection system,nabling the elimination if interferences.

.4. Miscellaneous methods

There are several reports of attempts to develop the existingnalytical techniques or introduce new methods for the determi-ation of BZD in biological material.

The immunoassay techniques are further developed and appliedn the quick drug screening of biological material (usually urine orlasma), especially prior to chromatographic analysis, which servess a confirmation of the obtained immunoassay results.

The determination of 7-aminoclonazepam in human urine with very low LOD (0.021 ng mL−1) was presented by Chen at al. [61].he method uses an immunoassay microfluidic chip with laser-nduced fluorescence detection, and is based on water solubleenatured bovine serum albumin (BSA)-coated CdTe quantum dots.he total analysis time was 5 min, with the linear range between.1 and 60.1 ng mL−1.

The cross-reactivities and structure-reactivity relationships ofix BZD (3-hydroxyflunitrazepam, 7-aminonitrazepam, brotizo-am, delorazepam, pinazepam, �-hydroxymidazolam) not includedn the manufacturer’s instructions for EMIT® were investigated102]. LC–MS/MS was employed to confirm the concentration ofach of the analytes in spiked urine samples for EMIT® and toompare the quantitative results. Limited cross-reactivity towards-hydroxyflunitrazepam and 7-aminonitrazepam, as well as theossibility to obtain false positive and/or negative results, indi-ate that the described screening method is not always suitableor proper identification of these drugs, and the usefulness ofmmunoassay tests without additional confirmation for forensicurposes should be considered.

From the non-routinely used analytical methods for the deter-ination of BZD in biological matrices the following may beentioned: spectrophotometry, fluorometry, colorimetry, chemi-

uminescence and, especially, MALDI-MS or Raman spectroscopy.The chemometric approach to determine lorazepam by spec-

rophotometric technique was presented by Ghasemi and Niazi114]. Two methods were used: parallel factor analysis (PARAFAC)nd partial least squares (PLS) with optimized pH. Comparison ofhe results obtained by these methods for two biological matricesurine and plasma) shows the superiority of the PARAFAC model.

A spot colorimetric test sensor for clonazepam based on goldanoparticles (AuNPs) in the presence of melamine was proposedy Lodha et al. [105] Hydrogen bonding interactions between annalyte and melamine induced the aggregation of AuNPs and cor-elated with the colour change from red to blue. The most importantdvantages of the proposed method seem to be a high sensitivitynd selectivity, as well as minimal reagent consumption.

Searching for new analytical methods suited for BZD deter-ination in urine resulted in the usage of fluorimetric [15] and

hemiluminescence [30] detection of these medicaments. The for-er method consisted in oxazepam hydrolysis and cyclization,


a reaction catalyzed by Ce(IV) in phosphoric acid with propermedium to give 2-chloro-9(10H)-acronidine as a strong fluores-cent molecule. The latter was based on the catalytic effect of1-ehyl-3-methylimidazolium ethyl sulfate/copper on the chemilu-minescence reaction of clonazepam and diazepam; the oxidationof the drugs with hydrogen peroxide in a natural medium. Both ofthe methods were characterized by a LOD value at nanogram permillilitre.

The two-dimensional ultra-thin-layer chromatography (2DUTLC) and atmospheric pressure matrix-assisted laser desorp-tion/ionization mass spectrometry [AP-MALDI-MS(MS)] methodswere studied for the analysis of seven BZD in human urine sam-ples [51]. The repeatability of the 2D UTLC separation of analytesexpressed as RSD% was evaluated for the SPE-purified urine sam-ples in comparison to standard reference samples. The LOD for BZDmeasured by AP-MALDI-MS/MS was in the range of 0.4–84 pmol.The suitability of the proposed procedure was evaluated by appli-cation for quantitative screening analysis of an authentic urinesample collected 53 h after intake of a single dose of diazepam.According to the authors, the developed methodology could be analternative for routine LC–MS analysis in toxicological, pharmaceu-tical, forensic and metabolomics applications.

The applicability of surface enhanced Raman spectroscopy(SERS) for the screening analysis of the selected BZD spiked intourine was investigated by Doctor and McCord [106]. Urine samplesextracted by LLE with ethyl acetate were mixed with gold nanopar-ticles and 1.67 M MgCl2 as an aggregating agent and underwentthe SERS measurement. The proposed method appeared to be sta-ble, producing reproducible SERS intensities and frequencies thatallowed the determination of the selected BZD in urine samples atthe concentrations below 10 ng mL−1.

4. Conclusions

Medical and forensic importance of BZD imposes the necessityof searching for new biological matrices suitable for use in detectionand quantification of these medicaments. Generally, the materialsexamined for BZD may be divided into two classes: conventional(whole blood, plasma/serum, urine) and alternative (e.g. hair, oralfluid, vitreous humour, nails). In the last years, two non-typical(for this kind of analysis) samples: fingerprint deposits and exhaledbreath have been introduced and successfully analyzed.

Different chemical compositions of the above-mentioned matri-ces require specific and well selected methods for isolation of aparent drug and its biotransformation products, which in the nextstep of analysis (quantification) will allow to obtain credible results.

Among the main new trends in the presented analytical area,the following ones should be stressed:

- introducing new types of biological samples (e.g. fingerprintsdeposits, exhaled breath),

- application of new isolation techniques for BZD present in givenmatrices, e.g. dispersive liquid–liquid microextraction (DLLME),MAE or CPE,

- reducing sample amount taken for analysis (down to 50 nL in thecase of serum/plasma analysis and to10 mg in the case of hairanalysis).

A variety of analytical methods and techniques have beenemployed for detection and quantification of BZD drugs in biolog-ical materials.

Although such analytical techniques as: DPCAdSV, DPV orMALDI-MS have been successfully applied to BZD determination inbiological samples, LC, especially with MS(MS/MS) and DAD(UV)detection, and to a lesser degree GC with such detection methods

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peed, without the loss of sensitivity and repeatability of results.Among the main new trends in the presented analytical area,

he following ones should be mentioned:

increasing use of LC–MS(MS) techniques, improvement of analytical parameters of the commonly usedmethods for BZD analysis, e.g. HPLC-DAD(UV),

employment of new techniques for the determination of BZD inbiological matrices, e.g. 2D UTLC in combination with AP-MALDI-MS(MS) or SERS.

Generally, it may be stated that development of new method-logies for analysis of xenobiotics (including BZD) in biologicalatrices is focused on reduction of: the number of sample prepara-

ion steps, sample amount taken for analysis, consumption of timend reagents, and in general the costs of the whole analytical pro-ess, along with increase of specificity, accuracy and sensitivity ofhe method.

cknowledgements

K. Persona acknowledges financial support from the Interdis-iplinary PhD Studies project “Molecular Sciences for Medicine”co-financed by the European Social Fund within the Human Capitalperational Programme).

K. Persona received funds to prepare her doctoral dissertationrom the National Science Centre in the framework of a doctoralcholarship funding (ETIUDA) based on the decision no. DEC-014/12/T/NZ7/00262.

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Documents

Persona Et Al. - 2015 - Analytical Methodologies for the Determination of Benzodiazepines in Biological Samples