Bio-Sample Preparation and Gas Chromatographic Determination of Benzodiazepines—A Review

12 Pages·2013·0.28 MB·English

JournalofChromatographicScience2013;51:587–598
doi:10.1093/chromsci/bms263 AdvanceAccesspublicationJanuary25,2013 ReviewArticle
Bio-Sample Preparation and Gas Chromatographic Determination
of Benzodiazepines—A Review
MohammadNasirUddin1*,VictoriaF.Samanidou2andIoannisN.Papadoyannis2
1DepartmentofChemistry,UniversityofChittagong,Chittagong-4331,Bangladesh,and2LaboratoryofAnalyticalChemistry,
AristotleUniversityofThessaloniki,Greece
*Authortowhomcorrespondenceshouldbeaddressed.Email:[email protected]
Received26September2012;revised22December2012 D
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Benzodiazepines have become commonly prescribed medicines increased anxiety. Adverse effects are more likely when high ad
e
worldwideinthetherapyofanxiety,sleepdisordersandconvulsive potency, short half-life benzodiazepines are prescribed in large d
attacksbecausetheyarerelativelysafe,withmildsideeffects.The and prolonged doses. There is some inconsistent evidence that fro
m
availability of rapid, sensitive and selective analytical methods is patients with schizophrenia may be at a lower risk of depend- h
essentialforthedeterminationofthesedrugsinclinicalandforensic ence. Consequently, benzodiazepines are frequently encoun- ttp
s
cases. Benzodiazepines are usually present at trace levels (mg/mL tered in both clinical and forensic toxicological analyses. For ://a
or ng/mL) in acomplex biological matrix, and the potentially inter- identification purposes, urine or plasma are the preferred ca
d
feringcompoundsneedtoberemovedbeforeanalysis.Therefore,a matrices because the concentrations of benzodiazepines are e
m
samplepreparationtechniqueisoftenmandatory,bothtoextractthe higher(1,2).Biologicalmatricesalsoincludeblood,urine,bile, ic
.o
drugsofinterestfromthematricesandtoincreasetheirconcentra- vitreous humor, liver, kidney, skeletal muscle, brain, adipose u
p
tion. An extended and comprehensive review is presented herein, tissue,bonemarrowandlung. .c
o
focusing on bio-sample preparation (pretreatment, extraction and Recent quantitative methodologies for the analysis of benzo- m
/c
derivatization) and gas chromatographic methods applied for the diazepines in serum, plasma or whole blood include micellar h
ro
quantificationof1,4-benzodiazepines. liquidchromatography(MLC)(3),micellarelectrokineticchro- m
s
matography(MEKC)(4),potentiometry(5),spectrophotometry c
m(6u),nflouasosraiyme(t9r)y, (l7iq)u,icdapcilhlarorymealteocgtrraopphhyor(eLsCis)(C(1E0),(181))a,nldiqiumid- i/article
chromatography–mass spectrometry (LC–MS) (12) and liquid -a
b
Introduction chromatography–tandemmassspectrometry(LC–MS-MS)(13), stra
Benzodiazepines have hypnotic, anxiolytic, anticonvulsant, ultra-performance liquid chromatography (UPLC) (14), gas c
myorelaxant and amnestic propertiesthat are useful forawide chromatography(GC)(16),dual-columnGC(17),GC–MS(18) t/51
range of conditions. Their primary clinical indications are for and GC–MS-MS (19). However, GC–MS based techniques /7/5
anxiety and sleep disorders, although they are also commonly remain the method of choice for many routine laboratories 87
used for rapid tranquillization in psychotic or manic agitation due to the separation efficiency, versatility, ease of operation /47
1
and for some movement disorders. However, benzodiazepines and maintenance, and lower costs of the analyses and invest- 0
6
lack convincing evidence for exerting substantial long-term mentexpensesofananalyticalsystemcomparedtoLC–MS-MS. 6 b
antipsychotic benefits in patients with schizophrenia. Benzo- Moreover, the unexpected consequences of matrix-dependent y g
diazepinesareofteninvolvedinintoxications. ion suppression complicate the optimization of LC–MS-MS ue
s
The structural elucidation of 1,4-benzodiazepine can be techniques, especially when using electrospray ionization t o
described as the following. A seven-membered ring of carbon (ESI) or atmospheric pressure chemical ionization (APCI). n 2
atomswithonenitrogeniscalledanazepinering,whereaswith Furthermore, GC can be used with various detection systems 6 N
two nitrogen atoms, it is called a diazepine ring in which the like flame-ionization detection (FID), nitrogen-phosphorus de- o
v
e
topmostnitrogenisdesignatedpositionone.Therefore,usinga tection (NPD) or electron-capture detection (ECD) and MS m
b
standard numbering, the complete name of this molecule is with the different ionization modes [electron impact (EI) and e
1,4-diazepine.Abenzeneringfusedonthe10and11-positions chemical ionization (CI)] or scanning modes [full scan, r 2
0
of the 1,4-diazepine ring (Figure 1) forms 1,4-benzodiazepine. selected-ionmonitoring(SIM),multiple-iondetection(MID)or 18
TheadditionofanN-methylgrouptothenitrogenatposition1 atmosphericpressurephotoionization(APPI)](20).
leads to the prototype benzodiazepine–diazepam, which as a Procedures for quantification of drugs reviewed by different
representative drugexhibits allthree ofthebasicbenzodiazep- authors (21, 22) included benzodiazepines in the bio-samples
ineeffects;e.g.,skeletalmusclerelaxation,anticonvulsantactiv- blood, plasma, serum or oral fluid (saliva) using LC coupled
ity and anti-anxiety effects, at doses far lower than those that with single-stage or tandem mass spectrometry. Literature
causeataxia(lossofbalance).Dependinguponthesubstituents, devoted to drug testing in hair by high-performance liquid
over50derivativeshavecurrentlybeenidentified(1,2). chromatography (HPLC)/GC (23) has been reviewed. A review
Benzodiazepine treatment is also associated with adverse addressed the use of HPLC and CE as affinity separation
effects, including excessive sedation, cognitive impairment, de- methods to characterize drugs or potential drug–biopolymer
pendence, discontinuation or withdrawal symptoms such as interactions (24). Only one elaborate report covered the
#TheAuthor[2013].PublishedbyOxfordUniversityPress.Allrightsreserved.ForPermissions,pleaseemail:[email protected]
Choiceofsamples
Themostcommonsamplesusedfortheanalysisofbenzodiaze-
pines are human serum, plasma, blood, rat liver, urine and hair
(human or rat). Most recently, attention has been given to the
analyses of breast milk and oral fluid. In recent years, studies
have included plasma of rat, sheep, bovine, dog, monkey and
rabbit, rat brain, beverage, larvae, human muscle and brain
membrane. Blood, plasma and serum can often be deprotei-
nized and hair needs incubation in most cases, whereas urine
mayrequirehydrolysispriortotheisolationprocedure.
D
o
Hydrolysisofurine:Enzymatichydrolysis w
n
The chemical hydrolysis of conjugates with hydrochloric acid lo
FCi.g1u,r4e-ben1z.odCihaezempiicna-2l-osnteruDct.uDreiaszepoafmA. 1,4-diazepine, B. 1,4-benzodiazepine, or sodium hydroxide is not recommended for 1,4-benzodiaze- aded
pines because they can be hydrolyzed to the corresponding fro
benzophenones in strongly acidic or basic media (26). m
Enzymatic digestion generally causes hydrolysis without deg- h
ttp
radation of the parent molecule to the corresponding benzo- s
phenone. Most investigators prefer enzymatic digestion ://a
c
(hydrolysis) of plasma, urine, hair and tissue samples of benzo- ad
e
diazepinesbeforeextractiontoliberatetheconjugatedfraction m
of the drug, especially forold stains strongly bound to the ma- ic.o
terial. Benzodiazepines and their metabolites in urine have up
been determined by GC–MS after special sample preparation .co
m
including enzymatic hydrolysis, special solid-phase extraction /c
(SPE)andsilylation(27). hro
Borreyetal.(27,28)incubatedurineat568Cfor1h,buffered m
s
with 0.2M sodium acetate (pH 4.5) and 5,500 U of Helix ci/a
pomatia b-glucuronidase for hydrolysis. After centrifugation rtic
prior to SPE, 1M sodium hydroxide and phosphate buffer (pH le
Figure2. Numberofpublishedpapersperyearby1996-2012forthedetermination 6.8)wereaddedtothesupernatant.Otherauthorsappliedsimilar -ab
ofbenzodiazepines. procedures for hydrolysis, with slight variations including tem- stra
c
peratures(50–608C),theamountandsourceofenzymeused,pH t/5
ofbuffer(4–5)andtimeofincubation(2–4h).b-Glucuronidase 1/7
chromatographicmethodsHPLC,LC–MS,GCandGC–MSpub- as an appropriate enzyme has been used to release benzodiaze- /58
lished over the years 1992 to 1997 for the measurement pines from their conjugates with glucuronic acid (30–33, 34). 7/4
of benzodiazepines in biological samples (25). This review Gluculase(b-glucuronidaseþsulphatase)hasalsobeenpreferred 71
0
includes published GC methods since 1996 for the determin- toreleasebenzodiazepinesfromanytypeofconjugate(35,36). 66
ation of benzodiazepines in biological samples, which includes by
elaborate sample preparation procedures. The number of pub- gu
Proteinremoval:Blood,plasmatreatment e
lishedpapersperyear(1996–2012)ispresentedinFigure2. s
PubMed, Science Direct, Scopus, Google, SpringerLink and Proteinremovalfrombloodsamplescanbeperformedbyvarious t on
Interscience websites were searched to collect references methods.Ultramicro-filtrationandequilibriumdialysis(37)ofthe 26
severaltimesduringtheperiodof2009–2012. sampleremoveproteins,butthe ultramicro-filtrateorthe dialys- N
o
ate contains only the free fraction of the drug. The method v
e
usually used for precipitation of plasma or serum proteins con- m
b
SamplePreparation sists of mixing one volume of plasma or serum with three er 2
volumes of acid, 6% (m/v) HClO (38, 39), 10% (m/v) 0
4 1
1,4-Benzodiazepines are usually present at trace levels (mg/mL trichloroacetic acid (40) or organic solvent (41–43), methanol, 8
or ng/mL) in a complex biological matrix and the potentially isopropanol, acetonitrile, chloroform or acetone followed by
interfering compounds need to be removed before analysis. vortex-mixing and centrifugation, which releases the 1,4-
Sample pretreatment and protein removal followed by benzodiazepinesfromprotein-bindingsites,removing99%ofthe
extraction should be capable of concentrating the sample and proteins.Recoveriesoftheboundportionofdrugaredependent
reducingtheamountofinterferingsubstances.Nitrobenzodiaz- uponthenatureofthe1,4-benzodiazepinesandtheprecipitation
epines (clonazepam, flunitrazepam) are converted rapidly to agents.Anotherwaytorelease1,4-benzodiazepinesfromproteins
the corresponding 7-amino metabolite, almost quantitatively without precipitation isthe addition of fattyacidsthat compete
(23). Consequently, these 7-amino forms should be specifically with 1,4-benzodiazepines for binding sites of proteins (44, 33,
targetedinpostmortemcases. 45),whichdisruptsthestructureofproteins.
588 Uddinetal.
Treatmentofhair:Bufferincubation four benzodiazepines (diazepam, nordazepam, oxazepam and
Aswithurinesamples,acidoralkalinehydrolysiswerefoundto temazepam) in 11 biological matrices [blood, urine, bile, vitre-
be unsuitable to extract the target drugs from the hair matrix ous humor, liver, kidney, skeletal muscle, brain, adipose tissue,
becausetheseleadtodecompositionintocorrespondingbenzo- bonemarrow(BM)andlungofrabbitspecimens](19).
phenones. Therefore, mild alkaline incubation conditions are Theorganicsolventsusuallychosenarediethylether,chloro-
usuallyappliedwithgoodextractionefficiency.Methanoloram- form, ethyl acetate and butyl acetate. Additionally, neutral
moniacal methanol (35) incubation can be applied. Reports are toluene,benzene,heptaneandhexaneareused,towhichasmall
available in which incubation was conducted using Sorensen amount of a more polar solvent (methylene chloride, isoamyl
buffer (pH 7.545–48), 0.5M Na HPO (pH 8.531,49), 1N alcohol or isopropanol) is added (34, 46–48, 50, 51). However,
2 4
NaOH proteinaseK50,amixtureofb-glucuronidase/arylsulfa- therearenoparticularreferencesforanysolventorcombination
15
taseatpH4.035,bothmethanoland0.1Mhydrochloricacid(52) ofsolventstobeemployedfortheirextraction.Oneadvantageof
oran 8M urea–0.2M thioglycolate solution (pH 3) (53). Before solventswithlowboilingpointsisthattheycanbereadilyevapo- D
o
incubation,hairsamplesarewashedwithhotwater(53),isopro- ratedfortherecoveryofdrugs.Diethylether,however,isdisad- w
n
panol (54), acetone and methanol, alone or consecutively with vantaged by its volatility and inherently high danger from fire. lo
a
one or more solvents. In most cases, dichloromethane (47–49) Usually, a solvent evaporation step is required after extraction. de
w(va/sv)usseoddiufomrwdoasdheicnyglspuulfrapteos(e5s5,)w.Ritahtohnaiereinxccoerpptoioranteudsinwgith0.1d%i- Tprheevepnotsesdiblbeyadsislaonrpiztaiotinonofotfhegladsrsuwgaroentoorthbey gthlaesswinacrleuscioannboef d from
azepam,flurazepamandmedazepamwasincubatedinsevendif- l–2% of alcohol (ethanol or 3-methylbutan-l-ol) in a nonpolar h
ferent incubation media (proteinase K, methanol–ammonia, extractantsuchashexaneorheptane. ttps
methanol–trifluoroacetic acid, Soerensen buffer, 1M NaOH, ://a
c
b-glucuronidase/arylsulfataseandbiopurase),andthehighestre- Solid-phaseextraction ad
e
coveries for all three drugs were found in acidic methanol ex- m
traction (51). Benzodiazepine concentrations are generally low, TrehteainSePdE oanpprsooalicdh-p,hiansewahdicsohrbceonmtspofoulnlodwseodf binytesreelsetctaivree ic.o
sstoatGe-Cof–-tMheS--naretgamtievtehocdhefmoricatelsitoinngizabteionnzo(dNiaCzIe)pirneepsreisnenhtusmthane eseluvteiroanl,ahdavsanbteaegnes,initnecnlsuedlyingdelveeslsopoerdg.anIticissoplrveefnertreudsadgue,entoo up.com
hair,duetothehighelectrophiliccharacteroftheanalytes(16). foaming problems, shorter sample preparation time or minimal /c
h
handling time, high recoveryeven at low concentrations, clean ro
m
sample extracts and little or no need for concentration of the s
c
ExtractionTechniques extract (28, 29, 31, 33, 36, 52, 53, 59). In SPE, the sample is i/a
Chromatographic techniques, with few exceptions, require poured directly onto a cartridge packed with solid adsorbent rtic
le
some form of isolation procedures to separate the benzodiaze- (alumina, silica, chemically bonded silica, florisil or non-ionic -a
b
pcainteegsorfirzoemd inbtioololigqiuciadl–mliqautridiceesx.trTachteiosen (pLrLoEc)e,dSuPrEesorcmanicrboe- ovarriioounsicsizeexschanandgweitrhesainws)i.deHorawnegveero,fdadisspoorsbaebnletscaarertrciodmgemseorf- strac
extraction(SPME,LPME)anddialysis.Theconditionsofsample cially available. These cartridges selectively adsorb benzodiaze- t/5
1
preparation and extraction procedures adopted by some pines and their metabolites from biofluids at a pH of 9.0–11.0. /7
/5
authors for the determination of benzodiazepines and their Undesirable compounds that are also adsorbed may be 8
7
metabolitesbydifferentmethodsaresummarizedinTableI. removed by washing with an appropriate solvent or buffer. /4
7
Drugs and related compounds are then eluted by passing an 1
0
6
appropriate elution organic solvent through the cartridge. This 6
b
makes sample cleanup simpler, quicker and less laboriousthan y
Liquid–liquidextraction g
thetraditionalsolventextractionprocedure.Theefficiencyand u
LLE is the most widely used method for the pretreatment of e
biological samples. Benzodiazepines and their metabolites are reproducibilityareasgoodasthoseofLLE(18). st o
Sample preparation included mixed-mode SPE for the isola- n
usuallyextractedastheneutralmoleculesfrombiofluidswitha 2
tion of midazolam and its biologically active metabolite, 6
range of organic solvents under weakly alkaline conditions, N
1-hydroxymidazolam,inrabbitplasma(60).Thetargetanalytes, o
with recoveries in excess of 90%. Some workers find it un- v
35 benzodiazepines, were isolated from urine by SPE using e
necessary to alkalize samples, because the pK values of benzo- m
Oasis MCX extraction columns followed by a supported LLE b
diazepines are considerably below the physiological pH (56). e
Some others recommend the use of back-extraction with method (61). The extraction and cleanup of benzodiazepines r 20
(clobazam, medazepam, midazolam and diazepam) in plasma 1
aqueous acid solutions and basifying followed by extraction 8
was optimized using an automated ASPEC system with
withorganicsolvent (25),butnoparticularadvantageoverthe
Supelclean LC-18 (100mg; 40mm), disposable extraction car-
use of solvents alone has been achieved. Solvent polarity and
tridges (DEC). Analytes were eluted from the DEC with ethyl
pH of the aqueous phases are the major factors to be consid-
acetateandmonitoredonlinewithGC–NPD(62).
ered. The pH should be adjusted to a value at which the drug
isintheneutralformbutnothydrolyzed.
ThesamplepreparationconsistedofLLE,followedbyderiva- Microextraction
tizationinthepostmorteminvestigationsbyGC–MSforphena-
zepam (57). The 23 most commonly used benzodiazepines in Solid-phasemicroextraction
blood were extracted by LLE with chloroform at pH 9 (58). SPME has two processes, which are equilibrium between ana-
Three-step LLE was developed r to quantify citalopram and lytesandthefibercoating anddesorption tothemobilephase.
Bio-SamplePreparationandGasChromatographicDeterminationofBenzodiazepines—AReview 589
5
9
0 TableI
U BiologicalSamplePreparationfortheDeterminationofBenzodiazepinesbyGasChromatography*
d
d
in
e Analytes Matrix Samplepretreatmentandextraction Derivatization Recovery Reference
t
al. (%) D
LLE o
w
FNZ,7-AFNZ Plasma, LLE:acidhydrolysisandextracted Heptafluorobutyrate — 69 nlo
blood a
d
FANLPZ,,TALFN(dZ4,)d(iIaSz)-d5(IS) RHaatirhair LL(t8oLLl0EEu:::e2din0nie,cg–uevbs/mtaveet)dethdwyilneitnhSeo1crMehnloNsroaidnOe’sH(b7au:t3ff4,e0vr8/(Cvp)Hat7p.6H),9e.0x,trsaocdteiudminbdoireattheybleutfhfeerr;–ecxhtlroarcotfioornm: BHSepTtFaAfluaonrdob1u%tyrTiCcManShydride–ethylacetate(2:1,v/v) 9425,–9909 1456 ed from
NDZ,OZ,BRZ,DZ,LZ,FNZ,ALP,TL,PRZ-d5(IS) Hair LeLthEe:rin–ccuhbloartoefdorimn1(8m0L:2o0f,Sv/ovre)nson’sbuffer(pH7.6),extractedwith5mLofdiethyl Derivatizedusing35mLBSTFA–TMCSfor20minat708C 48–90 47 http
s
FNZ Hair L(8L0E::2i0n,cuvb/avt)ioninSorenson’sbuffer(pH7.6),extractedwithdiethylether–chloroform Derivatizationwithheptafluorobutyricanhydride — 59 ://ac
LZ Hair LLE:incubationinSorenson’sbuffer(pH7.6),extractedwithdiethylether–chloroform Derivatizationbysilylation — 72 a
d
(80:20,v/v)atpH8.4 e
m
FNZ,DFNZ,7-AFNZ Blood LLE:extractedwithdiethylether,diisopropyletherandtoluene–isoamylalcohol 80 80 ic
mixture(95:5,v/v) .o
DZ,DDZ,OZ,TZ,LZ,DXP,NCBZ,NZ, Blood,urine LLE/SPE:hydrolyzedbyadding10mLofb-glucuronidaseþ1MacetatebufferpH4.8, SilylatedwithMTBSTFAwith1%TBDMSCl — 33 u
p
1-HMDL,ALP,1-HAL,1-HTL,CLZ,FNZ, extractedwithethylacetateatpH7.4.SPE-HCXcolumns:elutedwithethylacetate .c
DFNZ,PNZ,LRZ om
NDZ,OZ Hair LLE:incubatedinSorenson’sphosphatebuffer,pH7.6,extractedwith5mLdiethyl Derivatizedbysilylationusing35mLBSTFAþ1%TMCS 68–77 45 /c
ether–chloroform(80:20,v/v) h
FNZ,7-AFNP,DFNZ,7-ADFNZ,7-AFNZ-d3(IS) Serum,urine LLE:40%phosphatebuffer(pH9.0)and4mLofethylacetate 4-pyrolidinopyridine,and100mLofheptafluorobutyric 96 40 rom
anhydride s
DTMLZLZZ,,,,ATNNZLD,PDZAZ(,,LISBPD)R,ZZM,,ODMZL,Z,B1(-IRSHZ)A,LCPD,O1,-HPMNZD,LN,FZZ, (IS) WSWehhruoomlleebblloooodd LLLLLLEEE:::0M0.5.5eMtmhsaLondooilfuic0m.s5ohMlyudtNirooanx2iþdHePO2þ4mtbLoulfuofefenrn,e-ebxuttryalcatecedtawteith5mLofbutylacetate Derivatizedwithdinacetonitrile–MTBSTFA(80:20,v/v) 8—985––110092 456956 ci/article
CLZ Plasma LLE:extractedwith1.0mLofboratebufferandtoluene–dichloromethane Derivatizedwith30mLofBSTFAwith1%TMCS 92–107 71 -ab
7-AFNZ Urine L(7L0E::3p0H,v9/.0v)(Na2CO3/Na2HCO3buffer),extractedwithethylacetate. Derivatizingagent:TMS-derivativeusingMSTFA — 77 strac
CDO,ESZ,FNZ,TL,PRZ(IS) Rathair, LLE:incubationwithSorenson’sbuffer,n-hexane–ethylacetate(7:3,v/v), Derivatizedethylacetate–BSTFA(2:1v/v)orderivatized 98 50 t/5
plasma 7extractionprocedures with50mLofBSTFA 1
SPE /7
OZ,LZ,NDZ,DZ,FNZ,LRZ Hair SPE:hydrolyzedwith70mLofb-glucuronidase/arylsulfatasefor2hat408C; Elutedwith2mLofacetone–dichloromethane(3:l,v/v). 50–95 35 /58
SPR:ChromabondC 7
20benzodiazepines,FDZ(IS) Wholeblood SPE:OasisHLBcartrl8idge,elutedwith5mLofCH2Cl2 44–88 41 /47
ALP,FZ,OZ,LZ,DZ,TZ,MDL,NDZ,PRZ, Urine SPE:incubatedwith100mLphosphatebuffer/b-glucuronidase, DerivatizedBSTFAcontaining1%TMCSand50mLofethyl — 30 1
0
DA-FZ,a-HALP,a-HMDL,a-HTL, ZSDAU020columns,rinsedwith2mLofhexane acetate 6
6
2-HEFZ,7-AFNZ,7-ACLZ,7-ANZ b
CLZ,7-ACLZ,DZD5(IS) Urine SPE:incubatedwithb-glucuronidaseenzyme.HCXisolute(10mL,200mg) Elutedwith3mLdichloromethane–isopropanol– — 32 y
column ammoniumhydroxide(78:20:2,v/v/v) gu
CTZ Plasma SPE:StrataC18-Ecartridge Elutedwith1mLofmethanol 93–101 67 es
OZ,LZ,DZ,TZ,NDZ,a-HALP,a-HTL Bloodand Mixed-modeSPE DerivatizedwithMTBSFTA 87 t o
urine n
ELanditsprimarymetabolites Wholeblood SPE TMSderivatization 84 26
ALP,a-HALP,4-HALP,FNZ,7-AFNZ,DFNZ,FZ,HEFZ, Urine SPE:incubatedH.pomatiab-glucuronidaseelutedwith1mLofmethanol Derivatization:pyridine–aceticanhydride(1:1,v/v)was .79 27,28 N
N-DAFZ,KTZ,OZ,LRZ,LZ,TL,a-HTL,N-MCLZ,(IS) added o
v
DZ,NDZ,7-AFNZ,deuteratedanalytes(IS) Hair SPE:incubatedin8Murea–0.2Mthioglycolatesolution(pH3); Derivatization:heptafluorobutyricanhydride(HFBA) — 52 e
m
ChromabondC18columns b
22benzodiazepines Plasma, SPE:Toxi-tubeA.extractedacetonitrile Silylatedwith40mLofBSTFA 67.8–93.2 65 e
urine r 2
FNZ,DFNZ,7-AFNZ Urine SPE:extractedbyBondElutCertify 90 81 01
FNZ,7-AFNZ,NZ(IS) Whole SPE:solidphase(CECN)butylendcapped,elutedwith2(cid:2)3mLethyl Derivatizingreagent:ethylacetate(25mL)and25mLof 83–77 70 8
blood,urine acetate–methanol(80:20,v/v) PFPAwasadded
CLZand7-ACLZ Hair SPE:Mixed-modeisoluteHCX,elutedusingmethylenechloride– Derivatizedusing50mLofHFBAat608Cfor30min — 51
isopropanol–ammoniumhydroxide(78:20:2,v/v)
OralfluidSPE:phosphatebuffer(0.02N,pH6.0),BondElutCertify130mg,elutedwithmixtureDerivatization:HFBA83–9068ofdichloromethane–isopropanol–ammonia PlasmaSPME:proteinswerethereafterprecipitationbyadditionof100Lof1MTCA5Macetatebuffer,pH7,wasaddedtothesupernatant—39m,1.4gofNaCO,Derivatizingagents:aceticanhydride–pyridine—38Urine,serumHT-SPME:hexane–ethylacetate,1:1(vv)1.4gofNaHCO/þ32315.1gofNaClatpH9or70%perchloricacidand2mLof-hexanenPlasmaSPME:deproteinizedwith1.6Mperchloricacid—37Urine,serumSPME-ACAXDVBcoatedfiber,sodiumchloride(1.2g)andbuffer(pH7)solution68–7764/Urine,LPME:proteinremoval,0.1Mphosphatebuffer(pH7.5)andmethanolAcceptorsolution:butylacetate–1-octanol(1:1,vv)was64–10062/plasmaused;amixtureofhexylether–1-octanol(1:3,vv)/ m(BRZ),brotizolam(BRL),camazepam(CMZ),chlordiazepoxide(CDO),clinazolam(CNZ),clobazam(CBZ),cloxazolam(CXL),clonazepam(CLZ),clorazepate(CLP),clotiazepam(CTZ),delorazepammoxepam(DXP),diazepam(DZP),ethylloflazepate(ELP),ethylflurazepam(EFZ),estazolam(ESZ),etizolam(EL),fludiazepam(FDZ),flunitrazepam(FNZ),flurazepam(FZ),flutazolam(FTZ),flumazenilDZ),ketazepam(KTZ),loprazolam(LRL),lorazepam(LRZ),lormetazepam(LRZ),medazepam(MZ),mexazolam(MXL),midazolam(MDL),nordazepam(NDZ),nimetazepam(NTZ),nitrazepam(NZ),phenazepam(PNZ),pinazepam(PZ),prazepam(PRZ),quazepam(QZ),temazepam(TZ),tetrazepam(TTZ),-tofisopam(TTZ),triazolam(TL),andamino(A),acitamido(Ac),hydroxyl(H),methyl(M), iItwctoti(ttamSiLTNfiUwDIplsmaTcascsutiduatydntntniehihhiyFeazPrpniahlbtimsrhhooyo-ntieiieeqaseamrhLemieeepidwMaptteaerannneectntnlczataunhhNprpolteeiiehlrsnphrkly)neiarkglahsmEn(idram-aiswyaemle,oZestaPnplloupeadiedsmmoertpoeefsLsdiamomr)Vwaeidiatadpoein-sfpislnrsnml6PymhpfasettaisHsirierDiipoafanoiolge0aMnssndeatmethpeoioucgimutsmPlpl,tahmoplnnFsgn0c,eeeerohaynnoalEtanpLber)ryeerstSoafsercopsl1oennsriaoaweadCiolyoPcddaorrufepaodmhrst.tmronShcbcwtpa2uplasMreoeeruatlmleyidPuehelosepmuaorlaPavoossrctapefamilEesswmnEoordrlotztfisyanygLnrrhctlaayteoavciifetpeu–oreaobci-mRhoaimmrmcaonlrniisdcaimcdttadpeawLysyinGcalaraneneParehinedyauorenlppoesaClqltetmGa.e-dCallstnibeeht5nvmyhmeeSiolluteean,to,0nofineeFpstydCdio0xupteowctecia.afdoembbatcil2nhagotmetlclpn(ampeeistmipeierrelreoacpnorynnPfhaesoiairaramapfinrarnanntooptmlpplnetiPnpirtnewnieauhycnprlcnnmreabhspnaiopalll)atoattes(cibaeieoelh.dtgticoeidllcrlleayip2luGaeohhyaydetsdonsyAeehrpfiritdchr0ers(sdommmeasweCnqaoaesdtna(mbimeom6hhor0oilfstattrnwp6surnsiyffeaeteeir3mifimabitladaoe.ateeo4auhmsvasleyfsterdrsni)mbtuolwtoeirofyrtnna,)neupaai.amsirtesIehswcmeqsst.gr(tpclstntieirnAaia(erlitir3nocntiffeausairtzHzsttmetrtitoinrtoe7eienetldenhmoaeawnehcwmamnme,rsfiFaexsca)idtptrisegd)etaee:spend,i)ia.obeaniotot.bfitlnoows-odln.rweorsjdev.edtdalLa(lltiIrtetnnbapauhefoeaorteTirn3hfeoePaccwcuptpetomtbsctmreadtdq8dtthiesMheottie(hsccohoousenreoiietuaeeri–-nfatasooiacplner(hnldoaEencdchadiydsiep0u4,m2mnolmrub,eefincaekncvyi.se.ape0s0imbac2inlltordoinsiodneexlaialdeSei)n0nsyleioyGiidafnimtyefrcpomtstoaflr,ydsaelonatirsfipyaw,gsChnyeeblailtttbtlhmnbaizivtstvsflciyohhitrra3scnmueciihdyo,eoeieeoaebutionzrenit0cvpisteeiahnlmcnngodelnfee1nonuicuai0eapydnsnoehtteretnupj.mfihuoocde-ho2ouioretwtsenxmatmlsosaecsumifrsfucer(nsdsantmmaedrimmeeed2esosaprtasstohmptmibizbteoonalsifaanhi5mfromdsoevleoeSoflfcrscsmmrleeapdsuioiaPcivnfplnvomulalwpttsbicnfilauleynsttefitzMiwpponzamerfolLindorohhneetiallaoneeeponomwlldvaaalar)ninodddlElgeeeeyaas)srtf----------ll.. Downloaded from https://academic.oup.com/chromsci/article-abstract/51/7/587/471066 by guest on 26 Novem
FNZ,7-AFNZ,(IS)d7 MicroextractionDZ,PRZ(IS)FNZ MDL,PNZ(IS)NDZ,OZDZ,N-DDZ,PRZ(IS) *Note:alprazolam(ALP),adinazolam(ADZ),bromazepa(DLZ),desmethyldiazepam(DDZ),deltiazepam(DTZ),de(FMZ),helazepam(HZ),haloxazolam(HLZ),imidazenil(Inorfludiazepam(NFDZ),oxazepam(OZ),oxazolam(OXL),nor(N),desmethyl(D),desalkyl(DA)assubstituent. DDtdmastrrbsiiaehileevzlleetalioanceiiacrrttorwtodoizyaipveovmgsaeadoiacdrntsrpfaoiaitzacpsiomGctzatzoshshahteCiaipmitgetrpoit.oiotnoopiinoDunmopinnofinieeneausacdodsarrtnaoeyikfsovndht.nstgpaeths,aerIthc.vteiarazotaepeBnatssfdhhetdiematgiuitesholcntsocrteen(oimhrt3firp,eifuo3caoesbsscni,aalealea3ntyknetpobi6ltshnanaalir,ylletsryet3iaaphotf7itcbdoeeanni,heoesrnac4.nitonktree2hzecrsMvc,otaeoea5taoosdrcidii6eorrflmiyiaees,nnluzm6sopagtepst3vrtha,iipooeh,nonepirvn6rgnny,eer5dseomsa,vidiushnvgv6aeenioniogr6drnndiiali,oiddvtuelnt6-usahrontgt7tsioehfdiv)oz-mesi.efncfateurrihotumznistivriohceessoaahinddee---l ber 2018
Bio-SamplePreparationandGasChromatographicDeterminationofBenzodiazepines—AReview 591
The sample pretreatment allows the derivatization of the 35
benzodiazepines with N, O-bis(trimethylsilyl)trifluoroacetamide
plus 1% trimethylchlorosilane (61). By means of acylation
(41, 49, 52, 53, 68) or silylation (19, 60) of benzodiazepines,
derivatization achieved better thermal stability, and generally
produce derivativesd with well defined mass spectra. The
reagents used for derivatization (acylation) in different
methods include heptafluorobutyric anhydride (41, 49, 52, 53,
68), heptafluorobutyrate (69), heptafluorobutyric anhydride–
ethylacetate(2:1,v/v)(47),aceticanhydride–pyridine(28,29,
39), ethyl acetate and pentafluoropropionic anhydride (PFPA)
(70). The most frequently used derivitizing (silylation) agents D
o
include conventional trimethysilyl (TMS) derivatives like w
n
N-methyl-N-(trimethysilyl)-trifluoroacetamide (MSTFA) (71), N, lo
a
O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% d
e
t4r6im, 4e8th,y1l6c,h7lo1r)osainladneter(tT-bMuCtySl)dwimitehtheytlh-dyelriavcaetitvaetes,(N5-9m,e3th1,yl4-N2-, d from
Figure 3. Comparison on different methods applied for benzodiazepines
(tert-butyldimethylsilyl)-trifluoroacetamide(MTBSTFA)with1% h
tert-butyldimethylsilyl chloride (TBDMSCl) (34) or acetonitrile determination. ttps
(80:20,v/v)(50).TheEI–GC–MSmethodforthedetermination ://a
c
of benzodiazepine and their metabolites in blood includes two transforminfrared(FTIR)],andelectro-analytical(potentiomet- a
d
stages of derivatization using tetramethylammonium hydroxide ric, voltammetric and polarographic) methods have all been em
and propyliodide (propylation), in addition to a mixture of used for the analysis of benzodiazepines. Different methods ic
.o
triethylamine–propionicanhydride(propionylation)(58). applied for the analysis of benzodiazepines are graphically pre- u
p
TMCS acts as a catalyst to increase the silylating power of sented in Figure 3, which implies that GC curved the second .co
BSTFA.Silylderivativesareformedbythedisplacementreaction positionfortheirdetermination. m
/c
of active protons as a nucleophilic attack of the more electro- hro
negative hetero-atom upon the silicon atom of the silylating m
s
reagent.Variousbenzodiazepineswithpolarfunctionalhydroxyl Chromatographicmethods ci/a
and/oraminegroupspossessing active protonscanbe silylated Inthecontextofthisreview,therearefourprincipalchromato- rtic
bytheTMSorTBDMSalkylsilylgroup,whichaddsthemolecular graphic procedures used to separate and detect benzodiaze- le
weight, andcorrespondingly, themass-to-charge ratio (m/z) of pinesfromeachotherandothercomponentsinextracts.These -ab
s
the analyte by 72 or 114, respectively. The use of t-butyl- areHPLC,GC,TLCandmicellar(electrokinetic)capillarychro- tra
dimethylsilyl derivatives as a replacement for the conventional matography (MECC). Chromatographic methods can either be ct/5
trimethylsilylderivativesoffersexcellentspectralcharacteristics usedtoscreenbio-samplessuchasblood,plasma,serum,urine 1
/7
for all common benzodiazepines. TBDMS derivatives are less orhairforthepresenceofoneormorebenzodiazepines,orto /5
8
sensitivetomoisturebecausetheirhydrolyticstabilityisgreater. confirmthepresenceofoneormorebenzodiazepinesfollowing 7
/4
The sensitive GC–MS evidence of benzodiazepines as tri- aninitialimmunoassayorotherscreeningtest.Duetothecom- 7
1
methylsilyl derivatives was foundin EI modein a urineanalysis plexityofbiologicalsamples,achromatographicseparationstep 06
6
by Balikova et al. (26). The silylation procedure using is required for the analysis of drugs in such samples. b
y
MTBSTFA–TBDMSCl is generally suitable for benzodiazepines. Chromatography can be avoided by the use of enzyme multi- g
u
However, the amino groups of the 7-amino metabolites of clo- plied immunoassay techniques, but these are not specific for e
s
nazepam, flunitrazepam and nitrazepam did not silylate in the each drug. TLC is a valuable technique as an initial screening t o
n
silylation procedure (MTBSTFAwith 1% TBDMSCl), and conse- method to narrow the possible identities of unknown drugs in 2
6
quently,theLODsofthesecompoundswerehigh(34).Gunnar biological samples. However, it is relatively nonspecific, time N
et al. (49) evaluated many the most common commercially consuming and provides only semi-quantitative data. GC ov
e
available silylating reagents, MSTFA, BSTFAþ1% TMCS, methodsofferexcellentsensitivityinthepicogramtonanogram m
b
MTBSTFAandTBDMS.DerivativesformedbyTBDMSprovedto rangeandlinearityoverawideconcentrationrange.However,it e
be more stable, reproducible and sensitive than corresponding possessessomedrawbacksover HPLC,suchaslengthycleanup r 20
1
TMS derivatives in the GC–EI–MS analysis of benzodiazepines. procedures, and in some cases, the formation of more volatile 8
Furthermore, they are reported to have more specific mass derivativesorhydrolysispriortoanalysis.Furthermore,thehigh
fragmentationandhigherm/zvaluesinEImassspectra. temperaturesrequiredforelutioncanleadtotheon-columnde-
compositionofcertainbenzodiazepines.
ClassificationofAnalyticalMethods Gaschromatography:Columnanddetection
Chromatography[LC(HPLC,UPLC,MLC,chiralseparation),GC
and thin-layer chromatography (TLC)], capillary electrosepara- Column
tion (MEKC, capillary electrochromatography, CE), immuno- GC methods generally use a fused-silica capillary column, al-
assay, photometry [ultraviolet (UV) spectrometry, Fourier though one has used a deactivated metal capillary column
592 Uddinetal.
TableII
GasChromatographicConditionsfortheDeterminationofBenzodiazepines
Analytes Matrices GCconditions Results Reference
D
Column Detection/ Range,recovery(%),LOD o
interfaces w
n
lo
NDZ,OZ Hair HP-5MScapillarycolumn,5%phenyl–95%methylsiloxane,30m(cid:2)0.25mm, MS-NCI 0.1–20.0ng/mg,68–77,NDZ:0.01,OZ:0.005ng/mg 45 a
0.25mm de
MFNZZ,,N7D-AZF,NDZZ,P,NOZZ(,ISB)RZ,CDO,PNZ,NZ,LRZ, BWlohoodle,ubrlionoed 3H0P-m5MDSB-c3a5pimllasry(0c.o3l2ummnm(,102.2m5(cid:2)mm0.2filmm)ms,il0ic.3a3campmillafirylmc)olumn MMSS--ESIIM —0–,18080–n1g0/9m,L0,.483––1087n,g5/mngL/mL,1ng/mL 7409 d fro
TZ,ALP, m
MDL,1-HAL,1-HMDL,FZ(IS) h
ALP,FZ,OZ,LRZ,DZP,TZ,MDL,NDZ,PRZ, Urine RTX-200capillarycolumn(15m(cid:2)0.25mm(cid:2)0.25mm) MS Upto1.5mg/mL,—,2.5ng/mL 30 ttp
s
DaA-H-FAZL,P,a-HMDL,a-HTL,2-HEFZ,7-AFNZ, ://a
c
7-ACLZ,7-ANZ a
d
CDLZZP,,7F-ZA,CMLZZ,,DOZZPD5(IS) RUaritnehair, THCP--15McaSpicllaapryil,la2ry0cmol(cid:2)um0n.2(530mmm,(cid:2)0.22550mmmmfi(cid:2)lm0.25mm) MMSS--ENICI PClLaZs:m5a0:–04.1,0–0200pmg/gm/mL,L,—ha,i5r:00p.1g–/m10Lng/mg,98, 5302 em
plasma 0.01–0.5pg/mg ic.o
CLZ,7-ACLZ,DZP-d5(IS) Tablet HP-5MScapillarycolumn(30m(cid:2)250mm;0.25mm) MS-NCI 1.2–23pg/mg,—,0.4pg/mg 51 up
FNZ,7-AFNZ,d7(IS) Oralfluid HP-1capillarycolumn(30m(cid:2)250mm(cid:2)0.25mm) MS-NCI-SIM 0.1–5.0mg/L,83.3–89.6,0.05–0.1mg/L 68 .c
7-AFNZ Urine HP-5MScapillarycolumn(30m(cid:2)0.25mm,0.25mm) MS-SIM 0–25ng/mL,—,2.5ng/mL 77 o
DZP,NDZ,BRZ,MZ(IS) Wholeblood DB5-MS,20m(cid:2)0:18mm,0.18mm MS-TOF 50–300ng/mL,r2¼0.99,—,— 55 m/c
FNZ,7-AFNZ,deuterated(IS) Hair — FNZ:2.5–200pg/mg,7-AFNZ:0.5–100pg/mg 82 h
DZP,DAFZ,NDZ,MDL,OZ,TZ,LRZ,ALP,CLZ Wholeblood DB-5MSfused-silicacapillary(30m(cid:2)0.25mmi.d.,0.25mm) EI-MS-SIM 50–1,000ng/mL,5–50ng/mL 85 ro
m
Bio-Sample NFPEMlLhuDD,enZLnai,t,ar-OazPHezZNEep,LZpaT,am(Z8mIS-H)EL BP1WPll1laaohssomommdleaaat,rbicluoeroisnde FFFDHuuuBesssw-eee5ldddMe---tssstSiiilllPiiicccfauaaacskecccaooodrlllduuusmmmilHicnnnPa-(((5333cM000apSmmmillac(cid:2)(cid:2)(cid:2)aryp000i(ll3...a2220r555ymmmmco(cid:2)mmmlu,,,m0000.n2...2225(5553m0mmmmmmmmi(cid:2).fififidlllmmm.,0)))0.2.255mmmm,)0.25mmfilm) EGGGMICCC-SM–––-SSMMMI-MMSSS--SMMSS 5001...–10505––4013–,0n0g300/,60mm0gn0L/g/nL,gm/rL2m,¼L0.002.959n9g,/—m,L1ng/mL 1865384367 sci/article
Pre ALP,a-HALP,4-HALP,FNZ,7-AFNZ,DFNZ,FZ, Urine SGEBP1capillarycolumn(30m(cid:2)0.25mm,0.25mmfilm),Restekhydro-guard MS-EI/SIM 5–100ng/mL,r2¼0.995,(cid:3)79,LOQ: 27,28 -ab
parationan F(HNINSE-DFZ)ZA,,F7Z-A,FKNTZZ,OZ,LRZ,LZ,TL,a-HTL,N-MCLZ, Hair column(5m(cid:2)0.32mmi.d.) MS-NCI —1.0,–—1.,7LnOgQ/:m0L.5–2.3pg/mg 74 stract/51
d DZP,NDZ,7-AFNZ,deuteratedanalyte(IS) Hair HP-Ultra2capillarycolumn(12m(cid:2)0.2mm(cid:2)0.33mmfilm) MS-SIM —,—,0.02–0.15ng/mg 52 /7
Gas FNZ Urine,serum HP5capillarycolumn(30m(cid:2)0.25mm) MS-SIM — 38 /58
C DZP,DDZ,OZ,TZ,LRZ,DXP,NCBZ,NZ, Blood,urine HP-1capillarycolumn(12m(cid:2)0.20mmwith0.33mmfilm) MS —,—,5–40ng/mL 33 7
hro 1-HMDL,ALP, /47
m 1-HAL,1-HTL,CLZ,FNZ,DFNZ,PNZ, 1
ato MZ,FTZ,OXL,MDL,BRZ,HLZ,FZ,CLZ,ALP, Wholeblood BPX5capillarycolumn(15m(cid:2)0.32mm,film0.25mm),SGE MS-SIM 5–500ng/mL,r2.0.995,44–138,0.2–20ng/mL 41 06
graph TCLD,OD,ZC,XL,FNZ,NTZ,MXL,NZ,ESZ,EL,BRL, 6 by
ic FDZ(IS) g
D u
e CLZ,7-ACLZ Hair — MS-NCI 7-ACZ:1–1,000pg/mg,CZ:10–400pg/mg,—,— 75 e
term FNZ,7-AFNZ Urine — MS-NCI —,—,LOQ:FNZ,100,AFNZ:10pg/mL 73 st o
ination 3AFN5LPZB,,eT7nL-zAo(FddN4ia)Zz,e(IpdSii)naze-sd5(IS) UHRaaritinrehair FCRueasspetiledlak-rs-y2ili0cc0aoluccmaoplunilm,la5nry%(c3po0hluemmny(cid:2)nl–(019.552%5mmm(cid:2)me0t,h.02y.s52il5moxmmanm,e0fi.(23lm50)mmm(cid:2)) 0.25mm) MGMCSS–--NNTCCOIIF--SMIMS U1100p—,t2o0320550p0nggp//gm/mgmL,g9,09,21–59p9g,/LmOgQ,:325pgp/gm/mgg 164506 n 26 N
ofBe OMZid,aLzRoZla,mN,D1Z-,hDydZPro,xFyNmZid,azolam HRaabirbitplasma FHuPs-eUdlt-rsaili2caccaopliullamrync(3o0lummn(cid:2)(102.2m5(cid:2)mm0,.20m.2m5m(cid:2)m0fi.3lm3)mmfilm) MGCS–-SMIMS-NCI —2–,25000–n9g5/,m0L.01–2.0ng/mg 3559 ove
nzo CLZ,CLZ-d5(IS) Plasma HP-5column(12m(cid:2)0.25mm,0.33mmfilm) MS-NCI 1–25ng/mL,—,0.1ng/mL 71 mb
diaze A7-LAPF,NFZN,ZD,FFNZ,Z,KT7Z-A,DLRFNZ,ZT,LH,EaFZ-H,ANL-,D4A-FHZA,L, Drugs BphPa1sec)apillarycolumn(30m(cid:2)0.25mm,0.25mmfilm;nonpolarpolydimethylsiloxane MS-EI-SIM —,—,0.1–0.5ng/mL 28 er 2
pin OZ,a-HTL 01
es— CTZ Plasma Fused-silicacapillarycolumn(30m(cid:2)0.25mm,0.25mmfilm) MS 5–200ng/mL,r2¼0.9985,92–101,LOQ:ng/mL 67 8
22Benzodiazepines Drugs,Blood, DB5-MScapillarycolumn(30m(cid:2)0.25mmi.d.)filmthickness:0.25mm Iontrap-MS-MS Detectionthresholds:10–500pg/mL,blood:67–75, 65
A
R urine urine:85–93,—
eview N(ISD)Z,OZ,BRZ,DZP,LRZ,FNZ,ALP,TL,PRZ-d5 Hair H30Pm5-M(cid:2)S0.c2a5pimllamry(cid:2)co0lu.m25n,m5m%fiplmhenyl–95%methylsiloxane, MS-NCI r2¼0.930–1.0,47.6–90,1–20pg/mg 47
59 (continued)
3
ContinuedTableII AnalytesMatricesGCconditionsResultsReference ColumnDetectionRange,recovery(%),LOD/interfaces 50DrugsofabuseOralfluidFused-silicacolumn(30m0.25mm,0.25mfilm)MS-MSD-EI-NICI17m(cid:2)13BenzodiazepinesUrineRTX-200capillarycolumn(30m0.25mm,0.25m)MS-ESI—83m(cid:2)(IS)Plasma,urineDB-5MScolumn(25m0.2mm,0.33mfilm)MSUpto200ngmL,96.1ngmL40FNZ,7-AFNP,DFNZ,7-ADFNZ,7-AFNZ-dm//(cid:2)3128Date-rapedrugsUrineFused-silicacolumn(30m0.25mm,0.25mfilm)EI-MS58m(cid:2)ClobazamandnorclobazamFused-silicacolumn(30m0.25mm,0.25mfilm)MS0.72and36ngmL76m/(cid:2)23BenzodiazepinesBloodFused-silicacolumn(30m0.25mm,0.25mfilm)EI-MS57m(cid:2)DZP,NDZ,OZ,TZ,LRZUrine,serum30m0.25mmPTE-5poly(5%diphenyl–95%dimethyl-siloxane)columnFID-MS0.1–2gmL,—,0.02ng–0.1gmL64mm//(cid:2)(filmof0.25m)mDZP,OZ,TZ,NDZ,LRZUrine,serumSPB-5(30m0.25mm),PTE-5poly(5%diphenyl–95%dimethyl-siloxane),FID-MS0.1–2gmL,—,0.02–0.10gmL64mm//(cid:2)filmof0.25mm2¼0.9994,—,0.10nmolmL39DZP,PRZ(IS)PlasmaDB-I(methylsilicon)column,30m0.2mm,0.25mfilmFID-NPD0.25–7.5nmolmL,rm//(cid:2)MDL,DZP,MZ,NZ,N-DCBZPlasmaProcolumn;3m0.32mm15m0.32mmcontainedSE-54FID-NPD0.1–2.0ngmL,65–80,LOQ:5–25ngmL36//(cid:2)(cid:2)with0.25mfilm;fusedsilicam2¼0.996,64–104,0.020–0.11562DZP,N-DDZ,PRZ(IS)Urine,plasmaSPB-1(30m0.25mm,0.25mfilm)NPD0.5–8.0nmolmL,rm/(cid:2)nmolmL/2¼0.992–7,70,0.5–2ngmL61MZ,DZP,CBZ,MDLPlasmaSPB-5(15m0.32mm)withafilmof0.25mNPD5–50ngmL,rm//(cid:2)DZP,MDL,PRZ,FZ,HEFZ,LRZ,NDZ,FNZ,Blood,plasmaFusedsilicacapillarycolumnwas10m0.53mmi.d.,2mfilmthickness63NiECD—,plasma:59–92,blood:53–99,2–14ngmL82m/(cid:2)NFNZ,DAFZ,CLP,CBZ,BRZ2¼0.9996,95–102,0.5ngmL66TL,ALP(IS)SerumNC-17capillarycolumn(OV-17;15m0.25mm,0.25m)63NiECD0–10ngmL,rm//(cid:2) *Note:alprazolam(ALP),adinazolam(ADZ),bromazepam(BRZ),brotizolam(BRL),camazepam(CMZ),chlordiazepoxide(CDO),clinazolam(CNZ),clobazam(CBZ),cloxazolam(CXL),clonazepam(CLZ),clorazepate(CLP),clotiazepam(CTZ),delorazepam(DLZ),desmethyldiazepam(DDZ),deltiazepam(DTZ),demoxepam(DXP),diazepam(DZP),ethylloflazepate(ELP),ethylflurazepam(EFZ),estazolam(ESZ),etizolam(EL),fludiazepam(FDZ),flunitrazepam(FNZ),flurazepam(FZ),flutazolam(FTZ),flumazenil(FMZ),helazepam(HZ),haloxazolam(HLZ),imidazenil(IDZ),ketazepam(KTZ),loprazolam(LRL),lorazepam(LRZ),lormetazepam(LRZ),medazepam(MZ),mexazolam(MXL),midazolam(MDL),nordazepam(NDZ),nimetazepam(NTZ),nitrazepam(NZ),norfludiazepam(NFDZ),oxazepam(OZ),oxazolam(OXL),phenazepam(PNZ),pinazepam(PZ),prazepam(PRZ),quazepam(QZ),temazepam(TZ),tetrazepam(TTZ),-tofisopam(TTZ),triazolam(TL),andamino(A),acitamido(Ac),hydroxyl(H),methyl(M),nor(N),desmethyl(D),desalkyl(DA)assubstituent. tbLpticTwgbft(du0scflbtczieb((hsmTidocoaM17DSf((0Ma10mirponsnroeeoyeIia2654tlpeOoroomevoe.uee5...eixeraagh–olpTe1000ihnpaSSaLmccuesti8626ipeTCrieronetnatnnm)/toaectmnDa2pzurjr08MNpttoia–ewietnh,),)tnecmaonzimnhzeeewazrlselfaiieae0tue5lnapslrlocoodmo1oetCoonztracSleeoe27igapaiwnmoddrrttygiien–ebdunnpo.utr(opm)MndegonefttinNI/9y6e7aepaimdirie,4atbroaoheas0nnsalirs/oninlfimndmu)wg)iwwergaaecntaC1Saezn0esolondscnmrnt.eem,danHetoodms0r/cnnzhhaaidcEa0–d)g0LlhppifemgiIautastacve1rirerotem5t1omr,t,yhPehgraIn,hml.Nt/n–ouyemarhhhawiy05das2pieptod%daleba-(oapretmmlnublnrnlduiggh1erat5oCoe2snm6ro–otni–feicounioianteooaflOrmsnzgrrooam0nhhaetflMfrfhy6yLoitdpmtIdn5eac5lonalpuh(lenezx/uiebur0iiauoebeVy)lterecv(rae40ito.nmsbdSohmdrtN(msltdoesr0arhlzrr.ctleoitm(e2naamst-dhe8oi07hhnepdeditelraoeda2f1GoetentaCgnmc9aaIp,(gopnniou)yof3nsaospg(rte7inTe87raznnm5repzom)e.zChhiso(;LIlgsigotnormdfi)iw/ztifire-ayzoetap)roMran6ad%hirsgiOah.raaso/Tohrrmnmolhlv(nhenupnrytthdaewo8(mpy/cectso(smitomy–v-RTa(dSiieaehaDlntesdnesa3amh5epdcleiowmLdo)lamr5rlinwfnl9saciiaibAshioeinmcsdwo3,aLs0snmeaht,nnswelnip3(t2zdehli5theLubyheebntSiceod.)zcceie)l(0y7,eeaflnoe,ic)aoh5qri%agCai,asaemeeTvdSeoldlacl,.n8mfcriupiyfb-0geaniostma0vwqsuuzo,InoodpnO(oseflmerIomS)nphha,ayn0otnyMniq.aadno5Cu6anlp1nrizimnIurrintnaeiogl7rviTima.(dnisuMnhetipbfaps2saohae)2e–le)ietle–ntn/o1ir0tzbhaasaziszee-tinnrtuamno)ahnddehrhad0slSs–vm0meiilubh60gpNot)e(iaaesyst,sartltldmoclgwfiPioyecaaei.,4p.thy2,zhl.),toaeiamrreipalt1a16iC5egeti1climslctthBaidmoaue2lLnurna0bnzyemsdodoaezalawtaea0ht.uta–5aiz-eLinrOi-pimp)ernhlsnerrnuemnwhtietnte1zcda1lilt8-oneanOe.pto0eeaiigbyiiplcdaygpeitettzCtyDt7othuocsy,lmpngondIhdn.or/mho(loiohai/oeDadal2(ao)ongpsar1naod,ond/sEapngemdabztla6snmmotprwaie5nnnaeinomhvnipwvmhefeSeirnoosEe(eLnitn3fdfinorsaoLleewdianreI-tLseo6frondwandglltrOIeev,)ghtpud)Lraeersbalyolfd,eeEnanh5gfreiTui,gocnaaeii/zaimirsocpcnDemam0vfamn(cnawtptIidryctta)ssfl/ttwaeseerimds(nlstit2hisperissag.th.ledvlcouhohbvm4d4uezesnopeuoaoeoaiweiwm9neieefdveomLiAormleal2demdstnntnpcsimdoaxrpxndtte)ngawpaapth3eohsnhntdisiss)mieidebfusaeoissefezlnqesentetgprhaxoeymbbd–iuotasiIfbb1nrfemiretnmlemscesaeuttsau,g/ooIollma.ramafysyuhaerN1ee0eeas(i.pa.gsnmdxahonmesdzirrfvr(zewTpsic7uybn5flns(n0oittaiauteNCdoiplr6deoGMiAesnyntm7ehettlo1lmhh(ctoursztzgm%mnuteslpphlope7fyeupIho4ylaldc4ttCeeoaohox)reo,lSnailtyrL0pacoeoayda)aait,sa.eelr6ezrne)chadldia5mtummiO.b–ne.nmthceedsldnmid,atdeflie3d4t(sudhnba–oyiie0nNnuonofLdrotreaetyMai3aeaseeshoD.see0neosleea–duaari4iOwdssiv,ooezlzcip,t8oiopttdAaoti00tdbzzirianfheiSo,bg18leieeerhhstltenuiwnoi)vadDonnedeegenm0850Puyadeeeuhrnt.e0,rfpp,cyyrcasCrgr((exspp/.oubld..Tmtthds–nma00llitilte57(7Tohhawiendmgaaaass.nuonstttttrGiEddioz4t2M22flt117timoaaiihhhchheneeabmmbgaiisroteeernoa-illeen7.atLCii)))s––hhnniiinndod53eeeeeee/yySaasssssrrr---------...,,,,,, 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594 Uddinetal.
The determination of some benzodiazepines in plasma with The rapid detection and quantification of 35 benzodiaze-
thermionic detection, FID or NPD was presented (37) after pines in urine was performed by GC–time-of-flight (TOF)-MS
online coupling of dialysis via a precolumn, or rather, a trap- (61). TOF-MS offers new perspectives for forensic toxicology.
ping column as a preparation technique. Diazepam in human Qualitative and quantitative analyses of a mixture of three
plasmawas detected byeither FID or NPD, with an LOD value selected benzodiazepines (diazepam, nordazepam and broma-
of 0.10 nmol/mL (40). FID detected many acid or basic drugs, zepam) were performed to compare gas chromatography
including benzodiazepines in human plasma, with an LOD of (GC–TOF-MS, quadrupole GC–MS and GC–ECD). The experi-
0.5–2.0mg/mL (79, 80). The LOD ranged from 0.020–0.115 ment included the analysis of real human blood for bromaze-
nmol/mL in the determinationof diazepam and themetabolite pamanddemonstrated highsensitivityandhighselectivity due
N-desmethyldiazepam in plasma and urine after LPME by NPD to the high quality of mass spectra obtained by MS-TOF (56).
(63). The ASPEC system was optimized for the determination Atypicaltotalionchromatogramofninebenzodiazepinesfrom
of four benzodiazepines in plasma; the analytes were detected a50ng/mLcalibratorispresentedinFigure4. D
o
by the same NPD, providing an LOD of 0.5–2ng/mL (62). Allgaschromatographicmethodsusedultra-purityheliumas w
n
Amethodforthedeterminationoftraceamountsoftriazolamin carrier gas with few exceptions: one used hydrogen (16) and lo
a
serum by deactivated metal capillary GC with electron-capture another used a mixture of argon–methane (9:1, v/v) (87), in a d
e
tdreiatezoctlaiomni(nNiseErCuDm)(w65as).eCstoamblpisahreatdivteolyd,eEtCerDmwinaes0s.u5pnpgo/smedLtoof sDtaebuitleitryatsetdudiyntoefrnsaolmsetabnednazrdosdia(z8e7p)inweesraendcotmhemironmlyetaubsoelditeisn. d from
provide the best LOD ((cid:4)1ng/mL) for 1.0mL (40, 65), al- most of the cited references, reflecting their wide commercial h
though NPD provided LODs down to 5–25ng/mL (63, 62). availability and their general acceptance as ideal internal stan- ttps
ECD (81) or benchtop ion MS-MS (82) have been applied for dards. Table II summarizes the conditions and experimental ://a
c
the identification and determination of flunitrazepam and its results of some gas chromatographic methods for the deter- a
d
metabolites in blood and urine, respectively. GC–MS with the minationof1,4-benzodiazepines. em
mass selective detector (MSD) operating in either EI or ic
.o
negative-ion chemical ionization (NICI) mode was employed u
p
fortheanalysisof50drugsofabuse,includingbenzodiazepines .co
(18). A highly sensitive micro-plate enzyme immunoassay Conclusion m
/c
screening of benzodiazepines and their major metabolites in GC–MS methods were, as expected, well represented and still hro
biological fluids was confirmed by GC–NICI (83, 84). Etizolam offerthebestmeanstoconfirmbenzodiazepines.Mostofthese m
s
and its primary metabolites in whole blood were determined methods derivatized the benzodiazepines, both to improve ci/a
by ion trap GC–MS-MS (85). A rapid and efficient EI-GC–MS spectral definition and to reduce on-column thermal degrad- rtic
method in SIM mode was optimized for the screening and ation. The most favoured derivative was the conventional TMS, le
quantitation of benzodiazepines in blood, with LODs from 5 to although the TBDM derivative offered improved spectral prop- -ab
s
50ng/mL(86). erties. Conventional LLE or SPE are still widely used by most tra
c
t/5
1
/7
/5
8
7
/4
7
1
0
6
6
b
y
g
u
e
s
t o
n
2
6
N
o
v
e
m
b
e
r 2
0
1
8
Figure 4. Total ion chromatogram of a 50ng mL21 calibrator. Peak identification: 1, diazepam-d5 and diazepam; 2, desalkylflurazepam-d4-TBDMS and
desalkylflurazepam-TBDMS; 3, nordiazepamd5-TBDMS and nordiazepam-TBDMS; 4, midazolam-d4 and midazolam; 5, oxazepam-d5-diTBDMS and oxazepam-diTBDMS; 6,
temazepam-d5-TBDMSand temazepam-TBDMS; 7,lorazepam-d4-diTBDMSand lorazepam-diTBDMS;8, clonazepam-d4-TBDMS and clonazepam-TBDMS;and 9, alprazolam-d5
andalprazolam. ReproducedfromNicholasB.Tiscione,XiaoqinShan,IleneAlfordandDustinTateYeatman.QuantitationofBenzodiazepinesinWholeBloodbyElectronImpact-
GasChromatography-MassSpectrometry,JournalofAnalyticalToxicology,Vol.32,October2008,page644-652,Figure1,bypermissionofOxfordUniversityPress.
Bio-SamplePreparationandGasChromatographicDeterminationofBenzodiazepines—AReview 595
researchers as isolation steps with no obvious relative advan- benzodiazepines and methadone; Journal of Chromatography B,
tages. SPMEorLPMEofferrelativelynewapproachesto sample (2011);879(25):2642–2652.
preparation.Theserequirefewerorganicsolvents,whichisim- 14. Sauve, E.N., Langødega˚rd, M., Ekeberg, D., Øiestad, A˚.M.L.;
Determinationofbenzodiazepinesinante-mortemandpost-mortem
portant from an ecological and analytical viewpoint. They are
wholebloodbysolid-supportedliquid–liquidextractionandUPLC–
also fast, solvent-free and provide excellent performances.
MS/MS;JournalofChromatographyB,(2012);883–884:177–188.
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most commonly used with ECD, although some papers found Detectionofalprazolaminhairbynegativeionchemicalionization
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the ionization method with SIM, although the use of NCI 201–209.
16. Rasanen, I., Neuvonen, M., Ojanpera¨, I., Vuori, E.; Benzodiazepine
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Dimethylpolysiloxane (100%) and 5% diphenyldimethylpolysi-
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o
separationcapabilitiesformostderivatizedbenzodiazepines. dated method for the detection and quantitation of 50 drugs of w
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a
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596 Uddinetal.