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

Journal of Chromatographic Science 2013;51:587– 598
doi:10.1093/chromsci/bms263 Advance Access publication January 25, 2013
Review Article
Bio-Sample Preparation and Gas Chromatographic Determination
of Benzodiazepines—A Review
Mohammad Nasir Uddin1*, Victoria F. Samanidou2 and Ioannis N. Papadoyannis2
1
Department of Chemistry, University of Chittagong, Chittagong-4331, Bangladesh, and 2Laboratory of Analytical Chemistry,
Aristotle University of Thessaloniki, Greece
*Author to whom correspondence should be addressed. Email: [email protected]
Received 26 September 2012; revised 22 December 2012
Benzodiazepines have become commonly prescribed medicines
worldwide in the therapy of anxiety, sleep disorders and convulsive
attacks because they are relatively safe, with mild side effects. The
availability of rapid, sensitive and selective analytical methods is
essential for the determination of these drugs in clinical and forensic
cases. Benzodiazepines are usually present at trace levels (mg/mL
or ng/mL) in a complex biological matrix, and the potentially interfering compounds need to be removed before analysis. Therefore, a
sample preparation technique is often mandatory, both to extract the
drugs of interest from the matrices and to increase their concentration. An extended and comprehensive review is presented herein,
focusing on bio-sample preparation ( pretreatment, extraction and
derivatization) and gas chromatographic methods applied for the
quantification of 1,4-benzodiazepines.
Introduction
Benzodiazepines have hypnotic, anxiolytic, anticonvulsant,
myorelaxant and amnestic properties that are useful for a wide
range of conditions. Their primary clinical indications are for
anxiety and sleep disorders, although they are also commonly
used for rapid tranquillization in psychotic or manic agitation
and for some movement disorders. However, benzodiazepines
lack convincing evidence for exerting substantial long-term
antipsychotic benefits in patients with schizophrenia. Benzodiazepines are often involved in intoxications.
The structural elucidation of 1,4-benzodiazepine can be
described as the following. A seven-membered ring of carbon
atoms with one nitrogen is called an azepine ring, whereas with
two nitrogen atoms, it is called a diazepine ring in which the
topmost nitrogen is designated position one. Therefore, using a
standard numbering, the complete name of this molecule is
1,4-diazepine. A benzene ring fused on the 10 and 11-positions
of the 1,4-diazepine ring (Figure 1) forms 1,4-benzodiazepine.
The addition of an N-methyl group to the nitrogen at position 1
leads to the prototype benzodiazepine –diazepam, which as a
representative drug exhibits all three of the basic benzodiazepine effects; e.g., skeletal muscle relaxation, anticonvulsant activity and anti-anxiety effects, at doses far lower than those that
cause ataxia (loss of balance). Depending upon the substituents,
over 50 derivatives have currently been identified (1, 2).
Benzodiazepine treatment is also associated with adverse
effects, including excessive sedation, cognitive impairment, dependence, discontinuation or withdrawal symptoms such as
increased anxiety. Adverse effects are more likely when high
potency, short half-life benzodiazepines are prescribed in large
and prolonged doses. There is some inconsistent evidence that
patients with schizophrenia may be at a lower risk of dependence. Consequently, benzodiazepines are frequently encountered in both clinical and forensic toxicological analyses. For
identification purposes, urine or plasma are the preferred
matrices because the concentrations of benzodiazepines are
higher (1, 2). Biological matrices also include blood, urine, bile,
vitreous humor, liver, kidney, skeletal muscle, brain, adipose
tissue, bone marrow and lung.
Recent quantitative methodologies for the analysis of benzodiazepines in serum, plasma or whole blood include micellar
liquid chromatography (MLC) (3), micellar electrokinetic chromatography (MEKC) (4), potentiometry (5), spectrophotometry
(6), fluorimetry (7), capillary electrophoresis (CE) (8) and immunoassay (9), liquid chromatography (LC) (10, 11), liquid
chromatography– mass spectrometry (LC – MS) (12) and liquid
chromatography– tandem mass spectrometry (LC –MS-MS) (13),
ultra-performance liquid chromatography (UPLC) (14), gas
chromatography (GC) (16), dual-column GC (17), GC– MS (18)
and GC–MS-MS (19). However, GC –MS based techniques
remain the method of choice for many routine laboratories
due to the separation efficiency, versatility, ease of operation
and maintenance, and lower costs of the analyses and investment expenses of an analytical system compared to LC– MS-MS.
Moreover, the unexpected consequences of matrix-dependent
ion suppression complicate the optimization of LC–MS-MS
techniques, especially when using electrospray ionization
(ESI) or atmospheric pressure chemical ionization (APCI).
Furthermore, GC can be used with various detection systems
like flame-ionization detection (FID), nitrogen-phosphorus detection (NPD) or electron-capture detection (ECD) and MS
with the different ionization modes [electron impact (EI) and
chemical ionization (CI)] or scanning modes [full scan,
selected-ion monitoring (SIM), multiple-ion detection (MID) or
atmospheric pressure photoionization (APPI)] (20).
Procedures for quantification of drugs reviewed by different
authors (21, 22) included benzodiazepines in the bio-samples
blood, plasma, serum or oral fluid (saliva) using LC coupled
with single-stage or tandem mass spectrometry. Literature
devoted to drug testing in hair by high-performance liquid
chromatography (HPLC)/GC (23) has been reviewed. A review
addressed the use of HPLC and CE as affinity separation
methods to characterize drugs or potential drug –biopolymer
interactions (24). Only one elaborate report covered the
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Choice of samples
The most common samples used for the analysis of benzodiazepines 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 deproteinized and hair needs incubation in most cases, whereas urine
may require hydrolysis prior to the isolation procedure.
Figure 1. Chemical structures of A. 1,4-diazepine, B. 1,4-benzodiazepine,
C. 1,4-benzodiazepin-2-one D. Diazepam
Figure 2. Number of published papers per year by 1996-2012 for the determination
of benzodiazepines.
chromatographic methods HPLC, LC –MS, GC and GC –MS published over the years 1992 to 1997 for the measurement
of benzodiazepines in biological samples (25). This review
includes published GC methods since 1996 for the determination of benzodiazepines in biological samples, which includes
elaborate sample preparation procedures. The number of published papers per year (1996 –2012) is presented in Figure 2.
PubMed, Science Direct, Scopus, Google, SpringerLink and
Interscience websites were searched to collect references
several times during the period of 2009 –2012.
Sample Preparation
1,4-Benzodiazepines are usually present at trace levels (mg/mL
or ng/mL) in a complex biological matrix and the potentially
interfering compounds need to be removed before analysis.
Sample pretreatment and protein removal followed by
extraction should be capable of concentrating the sample and
reducing the amount of interfering substances. Nitrobenzodiazepines (clonazepam, flunitrazepam) are converted rapidly to
the corresponding 7-amino metabolite, almost quantitatively
(23). Consequently, these 7-amino forms should be specifically
targeted in postmortem cases.
588 Uddin et al.
Hydrolysis of urine: Enzymatic hydrolysis
The chemical hydrolysis of conjugates with hydrochloric acid
or sodium hydroxide is not recommended for 1,4-benzodiazepines because they can be hydrolyzed to the corresponding
benzophenones in strongly acidic or basic media (26).
Enzymatic digestion generally causes hydrolysis without degradation of the parent molecule to the corresponding benzophenone. Most investigators prefer enzymatic digestion
(hydrolysis) of plasma, urine, hair and tissue samples of benzodiazepines before extraction to liberate the conjugated fraction
of the drug, especially for old stains strongly bound to the material. Benzodiazepines and their metabolites in urine have
been determined by GC –MS after special sample preparation
including enzymatic hydrolysis, special solid-phase extraction
(SPE) and silylation (27).
Borrey et al. (27, 28) incubated urine at 568C for 1 h, buffered
with 0.2M sodium acetate (pH 4.5) and 5,500 U of Helix
pomatia b-glucuronidase for hydrolysis. After centrifugation
prior to SPE, 1M sodium hydroxide and phosphate buffer (pH
6.8) were added to the supernatant. Other authors applied similar
procedures for hydrolysis, with slight variations including temperatures (50–608C), the amount and source of enzyme used, pH
of buffer (4–5) and time of incubation (2–4 h). b-Glucuronidase
as an appropriate enzyme has been used to release benzodiazepines from their conjugates with glucuronic acid (30–33, 34).
Gluculase (b-glucuronidase þ sulphatase) has also been preferred
to release benzodiazepines from any type of conjugate (35, 36).
Protein removal: Blood, plasma treatment
Protein removal from blood samples can be performed by various
methods. Ultramicro-filtration and equilibrium dialysis (37) of the
sample remove proteins, but the ultramicro-filtrate or the dialysate contains only the free fraction of the drug. The method
usually used for precipitation of plasma or serum proteins consists of mixing one volume of plasma or serum with three
volumes of acid, 6% (m/v) HClO4 (38, 39), 10% (m/v)
trichloroacetic acid (40) or organic solvent (41–43), methanol,
isopropanol, acetonitrile, chloroform or acetone followed by
vortex-mixing and centrifugation, which releases the 1,4benzodiazepines from protein-binding sites, removing 99% of the
proteins. Recoveries of the bound portion of drug are dependent
upon the nature of the 1, 4-benzodiazepines and the precipitation
agents. Another way to release 1,4-benzodiazepines from proteins
without precipitation is the addition of fatty acids that compete
with 1,4-benzodiazepines for binding sites of proteins (44, 33,
45), which disrupts the structure of proteins.
Treatment of hair: Buffer incubation
As with urine samples, acid or alkaline hydrolysis were found to
be unsuitable to extract the target drugs from the hair matrix
because these lead to decomposition into corresponding benzophenones. Therefore, mild alkaline incubation conditions are
usually applied with good extraction efficiency. Methanol or ammoniacal methanol (35) incubation can be applied. Reports are
available in which incubation was conducted using Sorensen
buffer ( pH 7.545–48), 0.5M Na2HPO4 ( pH 8.531,49), 1N
NaOH15 proteinase K50, a mixture of b-glucuronidase/arylsulfatase at pH 4.035, both methanol and 0.1M hydrochloric acid (52)
or an 8M urea–0.2M thioglycolate solution (pH 3) (53). Before
incubation, hair samples are washed with hot water (53), isopropanol (54), acetone and methanol, alone or consecutively with
one or more solvents. In most cases, dichloromethane (47–49)
was used for washing purposes, with one exception using 0.1%
(v/v) sodium dodecylsulfate (55). Rat hair incorporated with diazepam, flurazepam and medazepam was incubated in seven different incubation media (proteinase K, methanol–ammonia,
methanol–trifluoroacetic acid, Soerensen buffer, 1M NaOH,
b-glucuronidase/arylsulfatase and biopurase), and the highest recoveries for all three drugs were found in acidic methanol extraction (51). Benzodiazepine concentrations are generally low,
so GC–MS-negative chemical ionization (NCI) represents the
state-of-the-art method for testing benzodiazepines in human
hair, due to the high electrophilic character of the analytes (16).
Extraction Techniques
Chromatographic techniques, with few exceptions, require
some form of isolation procedures to separate the benzodiazepines from biological matrices. These procedures can be
categorized into liquid –liquid extraction (LLE), SPE or microextraction (SPME, LPME) and dialysis. The conditions of sample
preparation and extraction procedures adopted by some
authors for the determination of benzodiazepines and their
metabolites by different methods are summarized in Table I.
Liquid –liquid extraction
LLE is the most widely used method for the pretreatment of
biological samples. Benzodiazepines and their metabolites are
usually extracted as the neutral molecules from biofluids with a
range of organic solvents under weakly alkaline conditions,
with recoveries in excess of 90%. Some workers find it unnecessary to alkalize samples, because the pK values of benzodiazepines are considerably below the physiological pH (56).
Some others recommend the use of back-extraction with
aqueous acid solutions and basifying followed by extraction
with organic solvent (25), but no particular advantage over the
use of solvents alone has been achieved. Solvent polarity and
pH of the aqueous phases are the major factors to be considered. The pH should be adjusted to a value at which the drug
is in the neutral form but not hydrolyzed.
The sample preparation consisted of LLE, followed by derivatization in the postmortem investigations by GC– MS for phenazepam (57). The 23 most commonly used benzodiazepines in
blood were extracted by LLE with chloroform at pH 9 (58).
Three-step LLE was developed r to quantify citalopram and
four benzodiazepines (diazepam, nordazepam, oxazepam and
temazepam) in 11 biological matrices [blood, urine, bile, vitreous humor, liver, kidney, skeletal muscle, brain, adipose tissue,
bone marrow (BM) and lung of rabbit specimens] (19).
The organic solvents usually chosen are diethyl ether, chloroform, ethyl acetate and butyl acetate. Additionally, neutral
toluene, benzene, heptane and hexane are used, to which a small
amount of a more polar solvent (methylene chloride, isoamyl
alcohol or isopropanol) is added (34, 46–48, 50, 51). However,
there are no particular references for any solvent or combination
of solvents to be employed for their extraction. One advantage of
solvents with low boiling points is that they can be readily evaporated for the recovery of drugs. Diethyl ether, however, is disadvantaged by its volatility and inherently high danger from fire.
Usually, a solvent evaporation step is required after extraction.
The possible adsorption of the drug onto the glassware can be
prevented by silanization of glassware or by the inclusion of
l–2% of alcohol (ethanol or 3-methylbutan-l-ol) in a nonpolar
extractant such as hexane or heptane.
Solid-phase extraction
The SPE approach, in which compounds of interest are
retained on solid-phase adsorbents followed by selective
elution, has been intensely developed. It is preferred due to
several advantages, including less organic solvent usage, no
foaming problems, shorter sample preparation time or minimal
handling time, high recovery even at low concentrations, clean
sample extracts and little or no need for concentration of the
extract (28, 29, 31, 33, 36, 52, 53, 59). In SPE, the sample is
poured directly onto a cartridge packed with solid adsorbent
(alumina, silica, chemically bonded silica, florisil or non-ionic
or ionic exchange resins). However, disposable cartridges of
various sizes and with a wide range of adsorbents are commercially available. These cartridges selectively adsorb benzodiazepines and their metabolites from biofluids at a pH of 9.0 –11.0.
Undesirable compounds that are also adsorbed may be
removed by washing with an appropriate solvent or buffer.
Drugs and related compounds are then eluted by passing an
appropriate elution organic solvent through the cartridge. This
makes sample cleanup simpler, quicker and less laborious than
the traditional solvent extraction procedure. The efficiency and
reproducibility are as good as those of LLE (18).
Sample preparation included mixed-mode SPE for the isolation of midazolam and its biologically active metabolite,
1-hydroxymidazolam, in rabbit plasma (60). The target analytes,
35 benzodiazepines, were isolated from urine by SPE using
Oasis MCX extraction columns followed by a supported LLE
method (61). The extraction and cleanup of benzodiazepines
(clobazam, medazepam, midazolam and diazepam) in plasma
was optimized using an automated ASPEC system with
Supelclean LC-18 (100 mg; 40 mm), disposable extraction cartridges (DEC). Analytes were eluted from the DEC with ethyl
acetate and monitored online with GC –NPD (62).
Microextraction
Solid-phase microextraction
SPME has two processes, which are equilibrium between analytes and the fiber coating and desorption to the mobile phase.
Bio-Sample Preparation and Gas Chromatographic Determination of Benzodiazepines—A Review 589
590 Uddin et al.
Table I
Biological Sample Preparation for the Determination of Benzodiazepines by Gas Chromatography*
Analytes
Matrix
Sample pretreatment and extraction
Derivatization
Recovery
(%)
Reference
LLE: acid hydrolysis and extracted
Heptafluorobutyrate
—
69
FNZ, AFNZ, diaz-d5 (IS)
Plasma,
blood
Hair
Heptafluorobutyric anhydride –ethyl acetate (2:1,v/v)
45, 90
46
ALP, TL (d4) (IS)
Rat hair
BSTFA and 1% TCMS
92– 99
15
NDZ, OZ, BRZ, DZ, LZ, FNZ, ALP, TL, PRZ-d5 (IS)
Hair
Derivatized using 35 mL BSTFA –TMCS for 20 min at 708C
48– 90
47
FNZ
Hair
Derivatization with heptafluorobutyric anhydride
—
59
LZ
Hair
Derivatization by silylation
—
72
FNZ, DFNZ, 7-AFNZ
Blood
80
80
DZ, DDZ, OZ, TZ, LZ, DXP, NCBZ, NZ,
1-HMDL, ALP, 1-HAL, 1-HTL, CLZ, FNZ,
DFNZ, PNZ, LRZ
NDZ, OZ
Blood, urine
LLE: incubated in Sorenson’s buffer (pH 7.6), extracted in diethylether –chloroform
(80:20, v/v)
LLE: digested with 1M NaOH at 408C at pH 9.0, sodium borate buffer; extraction:
toluene – methylene chloride (7:3, v/v)
LLE: incubated in 1 mL of Sorenson’s buffer (pH 7.6), extracted with 5 mL of diethyl
ether –chloroform (80:20, v/v)
LLE: incubation in Sorenson’s buffer (pH 7.6), extracted with diethylether –chloroform
(80:20, v/v)
LLE: incubation in Sorenson’s buffer (pH 7.6), extracted with diethylether –chloroform
(80:20, v/v) at pH 8.4
LLE: extracted with diethyl ether, diisopropyl ether and toluene –isoamyl alcohol
mixture (95:5, v/v)
LLE/SPE: hydrolyzed by adding 10 mL of b-glucuronidase þ 1M acetate buffer pH 4.8,
extracted with ethyl acetate at pH 7.4.SPE-HCX columns: eluted with ethyl acetate
Silylated with MTBSTFA with 1% TBDMSCl
—
33
Hair
Derivatized by silylation using 35 mL BSTFA þ 1% TMCS
68– 77
45
FNZ, 7-AFNP, DFNZ, 7-ADFNZ, 7-AFNZ-d3 (IS)
Serum, urine
LLE: incubated in Sorenson’s phosphate buffer, pH 7.6, extracted with 5 mL diethyl
ether –chloroform (80:20, v/v)
LLE: 40% phosphate buffer (pH 9.0) and 4 mL of ethyl acetate
4-pyrolidinopyridine, and 100 mL of heptafluorobutyric
anhydride
96
40
DZ, NDZ, BRZ, MZ (IS)
TL, ALP (IS)
MZ, NDZ, DZ, OZ, BRZ, CDO, PNZ, NZ,
LZ, TZ, ALP, MDL, 1-HALP, 1-HMDL, FZ (IS)
CLZ
Whole blood
Serum
Whole blood
LLE: Methanolic solution þ 2 mL of n-butyl acetate
LLE:0.5M sodium hydroxide þ toluene
LLE: 0.5 mL of 0.5M Na2HPO4 buffer, extracted with 5 mL of butyl acetate
Derivatized with dinacetonitrile –MTBSTFA (80:20, v/v)
—
95– 102
88– 109
55
66
49
Plasma
Derivatized with 30 mL of BSTFA with 1% TMCS
92– 107
71
7-AFNZ
CDO, ESZ, FNZ, TL, PRZ (IS)
Urine
Rat hair,
plasma
LLE: extracted with 1.0 mL of borate buffer and toluene –dichloromethane
(70:30, v/v)
LLE: pH 9.0 (Na2CO3/Na2HCO3 buffer), extracted with ethyl acetate.
LLE: incubation with Sorenson’s buffer, n-hexane –ethyl acetate (7:3, v/v),
7 extraction procedures
Derivatizing agent: TMS-derivative using MSTFA
Derivatized ethyl acetate– BSTFA (2:1 v/v) or derivatized
with 50 mL of BSTFA
—
98
77
50
Eluted with 2 mL of acetone –dichloromethane (3:l, v/v).
50– 95
35
44– 88
—
41
30
—
32
93– 101
67
87
LLE
FNZ, 7-AFNZ
SPE
OZ, LZ, NDZ, DZ, FNZ, LRZ
20 benzodiazepines, FDZ (IS)
ALP, FZ, OZ, LZ, DZ, TZ, MDL, NDZ, PRZ,
DA-FZ, a-HALP, a-HMDL, a-HTL,
2-HEFZ, 7-AFNZ, 7-ACLZ, 7-ANZ
CLZ, 7-ACLZ, DZ D5 (IS)
CTZ
OZ, LZ, DZ, TZ, NDZ, a-HALP, a-HTL
EL and its primary metabolites
ALP, a-HALP, 4-HALP, FNZ, 7-AFNZ, DFNZ, FZ, HEFZ,
N-DAFZ, KTZ, OZ, LRZ, LZ, TL, a-HTL, N-MCLZ, (IS)
DZ, NDZ, 7-AFNZ, deuteratedanalytes (IS)
22 benzodiazepines
FNZ, DFNZ, 7-AFNZ
FNZ, 7-AFNZ, NZ (IS)
CLZ and 7-ACLZ
Hair
Whole blood
Urine
Urine
Plasma
Blood and
urine
Whole blood
Urine
Hair
Plasma,
urine
Urine
Whole
blood, urine
Hair
SPE: hydrolyzed with 70 mL of b-glucuronidase/arylsulfatase for 2 h at 408C;
SPR: Chromabond Cl8
SPE: Oasis HLB cartridge, eluted with 5 mL of CH2Cl2
SPE: incubated with 100 mL phosphate buffer/b-glucuronidase,
ZSDAU020 columns, rinsed with 2 mL of hexane
Derivatized BSTFA containing 1% TMCS and 50 mL of ethyl
acetate
SPE: incubated with b-glucuronidase enzyme. HCX isolute (10 mL, 200 mg)
column
SPE: Strata C18-E cartridge
Mixed-mode SPE
Eluted with 3 mL dichloromethane – isopropanol –
ammonium hydroxide (78:20:2, v/v/v)
Eluted with 1mL of methanol
Derivatized with MTBSFTA
SPE
SPE: incubated H. pomatia b-glucuronidase eluted with 1 mL of methanol
TMS derivatization
Derivatization: pyridine –acetic anhydride (1:1, v/v) was
added
Derivatization: heptafluorobutyric anhydride (HFBA)
.79
84
27,28
—
52
Silylated with 40 mL of BSTFA
67.8 –93.2
65
90
83– 77
81
70
—
51
SPE: incubated in 8M urea– 0.2M thioglycolate solution (pH 3);
Chroma bond C18 columns
SPE: Toxi-tube A. extracted acetonitrile
SPE: extracted by Bond Elut Certify
SPE: solid phase (CECN) butyl endcapped, eluted with 2 3 mL ethyl
acetate –methanol (80:20, v/v)
SPE: Mixed-mode isolute HCX, eluted using methylene chloride –
isopropanol –ammonium hydroxide (78:20:2, v/v)
Derivatizing reagent: ethyl acetate (25 mL) and 25 mL of
PFPA was added
Derivatized using 50 mL of HFBA at 608C for 30 min
*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), and amino (A), acitamido (Ac), hydroxyl (H), methyl (M),
nor (N), desmethyl (D), desalkyl (DA) as substituent.
37
64
62
—
68 –77
64 –100
MDL, PNZ (IS)
NDZ, OZ
DZ, N-DDZ, PRZ (IS)
Plasma
Urine, serum
Urine,
plasma
Acceptor solution: butyl acetate –1-octanol (1:1, v/v) was
used; a mixture of hexyl ether –1-octanol (1:3, v/v)
39
38
—
—
5M acetate buffer, pH 7, was added to the supernatant
Derivatizing agents: acetic anhydride –pyridine
SPME: proteins were there after precipitation by addition of 100 mL of 1M TCA
HT-SPME: hexane –ethyl acetate, 1:1 (v/v) þ 1.4 g of NaHCO3, 1.4 g of Na2CO3,
15.1 g of NaCl at pH 9 or 70% perchloric acid and 2 mL of n-hexane
SPME: deproteinized with 1.6 M perchloric acid
SPME-A CAX/DVB coated fiber, sodium chloride (1.2 g) and buffer (pH 7) solution
LPME: protein removal, 0.1M phosphate buffer (pH 7.5) and methanol
Plasma
Urine, serum
Microextraction
DZ, PRZ (IS)
FNZ
68
83 –90
Derivatization: HFBA
SPE: phosphate buffer (0.02N, pH 6.0), BondElut Certify 130 mg, eluted with mixture
of dichloromethane –isopropanol –ammonia
Oral fluid
FNZ, 7-AFNZ, d7 (IS)
Unlike HPLC or LC, in a GC system after analyte extraction
with simultaneous in situ derivatization (acetylation or silylation), the SPME device is transferred to the GC injector for
thermal desorption. For the quantitation of midazolam and diazepam in human plasma or urine (38 –40) by the SPME
method a polyacrylate coated fiber was used. Solvent-modified
SPME offers sufficient enrichment for bioanalysis, high selectivity and a short sample preparation time.
Liquid-phase microextraction
The LPME device applied to determine diazepam and
N-desmethyldiazepam in plasma consisted of a porous hollow
fiber of polypropylene filled with extraction solvent (25 mL).
It was immersed in human urine sample with continuous vibration at 600 rpm for 50 min. An aliquot of the extraction solvent
with preconcentrated analytes was injected directly into the
capillary gas chromatograph (63). A new polyvinylidene difluoride (PVDF) hollow fiber (200 mm wall thickness, 1.2 mm internal diameter and 0.2 mm pore size) was compared with two
other polypropylene (PP) hollow fibers (200, 300 mm wall
thickness, 1.2 mm internal diameter and 0.2 mm pore size)
in the automated hollow fiber (HF)-LPME of flunitrazepam
(FLNZ) in biological samples (64).
Dialysis
In the last few years, dialysis has gained popularity as a sample
preparation technique in the determination of traces of analytes in protein-containing matrices, because the use of a
semi-permeable membrane offers the possibility of removing
macromolecular sample constituents: the dialysis membranes
are designed to allow only small molecules to be sampled.
Therefore, no sample pretreatment is required because clean
chromatograms can often be obtained. It has been successfully
applied to a variety of biomedical, food and environmental
samples prior to GC analysis. In addition, efficient sample
cleanup and analyte enrichment can be combined in the
system in a fully automated way, if a trace-enrichment precolumn is incorporated in the setup to overcome the dilution of
the sample caused by the dialysis step. The cleanup of plasma
in an online SPE –GC approach for the determination of benzodiazepines was based on performing the dialysis of samples
using water as the acceptor phase and trapping the diffused
analytes on a PLRP-S copolymer precolumn, in which desorption was made with ethyl acetate (37).
Derivatization
Derivatization is one of the factors for the improvement of sensitivity in GC. Derivatization, silylation or acylation avoid thermal
decomposition of the thermolabile benzodiazepines in the chromatograph to detect more analytes. Moreover, derivatization
allows a significant reduction in peak tailing, providing much
sharper chromatographic peaks than corresponding underivatized compounds. It significantly increases the signal-to-noise
ratio of the peaks, and thus the detection thresholds of the
related compounds. Better separations of many underivitized
benzodiazepines have also been achieved using various fused
silica as stationary phases (33, 36, 37, 42, 56, 63, 65, 66, 67).
Bio-Sample Preparation and Gas Chromatographic Determination of Benzodiazepines—A Review 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–
ethyl acetate (2:1, v/v) (47), acetic anhydride–pyridine (28, 29,
39), ethyl acetate and pentafluoropropionic anhydride (PFPA)
(70). The most frequently used derivitizing (silylation) agents
include conventional trimethysilyl (TMS) derivatives like
N-methyl-N-(trimethysilyl)-trifluoroacetamide (MSTFA) (71), N,
O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1%
trimethylchlorosilane (TMCS) with ethyl acetate (59, 31, 42,
46, 48, 16, 71) and tert-butyl dimethyl-derivatives, N-methyl-N(tert-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) with 1%
tert-butyldimethylsilyl chloride (TBDMSCl) (34) or acetonitrile
(80:20, v/v) (50). The EI –GC –MS method for the determination
of benzodiazepine and their metabolites in blood includes two
stages of derivatization using tetramethylammonium hydroxide
and propyliodide ( propylation), in addition to a mixture of
triethylamine– propionic anhydride ( propionylation) (58).
TMCS acts as a catalyst to increase the silylating power of
BSTFA. Silyl derivatives are formed by the displacement reaction
of active protons as a nucleophilic attack of the more electronegative hetero-atom upon the silicon atom of the silylating
reagent. Various benzodiazepines with polar functional hydroxyl
and/or amine groups possessing active protons can be silylated
by the TMS or TBDMS alkylsilyl group, which adds the molecular
weight, and correspondingly, the mass-to-charge ratio (m/z) of
the analyte by 72 or 114, respectively. The use of t-butyldimethylsilyl derivatives as a replacement for the conventional
trimethylsilyl derivatives offers excellent spectral characteristics
for all common benzodiazepines. TBDMS derivatives are less
sensitive to moisture because their hydrolytic stability is greater.
The sensitive GC –MS evidence of benzodiazepines as trimethylsilyl derivatives was found in EI mode in a urine analysis
by Balikova et al. (26). The silylation procedure using
MTBSTFA– TBDMSCl is generally suitable for benzodiazepines.
However, the amino groups of the 7-amino metabolites of clonazepam, flunitrazepam and nitrazepam did not silylate in the
silylation procedure (MTBSTFA with 1% TBDMSCl), and consequently, the LODs of these compounds were high (34). Gunnar
et al. (49) evaluated many the most common commercially
available silylating reagents, MSTFA, BSTFA þ 1% TMCS,
MTBSTFA and TBDMS. Derivatives formed by TBDMS proved to
be more stable, reproducible and sensitive than corresponding
TMS derivatives in the GC –EI –MS analysis of benzodiazepines.
Furthermore, they are reported to have more specific mass
fragmentation and higher m/z values in EI mass spectra.
Figure 3. Comparison on different methods applied for benzodiazepines
determination.
transform infrared (FTIR)], and electro-analytical ( potentiometric, voltammetric and polarographic) methods have all been
used for the analysis of benzodiazepines. Different methods
applied for the analysis of benzodiazepines are graphically presented in Figure 3, which implies that GC curved the second
position for their determination.
Chromatographic methods
In the context of this review, there are four principal chromatographic procedures used to separate and detect benzodiazepines from each other and other components in extracts. These
are HPLC, GC, TLC and micellar (electrokinetic) capillary chromatography (MECC). Chromatographic methods can either be
used to screen bio-samples such as blood, plasma, serum, urine
or hair for the presence of one or more benzodiazepines, or to
confirm the presence of one or more benzodiazepines following
an initial immunoassay or other screening test. Due to the complexity of biological samples, a chromatographic separation step
is required for the analysis of drugs in such samples.
Chromatography can be avoided by the use of enzyme multiplied immunoassay techniques, but these are not specific for
each drug. TLC is a valuable technique as an initial screening
method to narrow the possible identities of unknown drugs in
biological samples. However, it is relatively nonspecific, time
consuming and provides only semi-quantitative data. GC
methods offer excellent sensitivity in the picogram to nanogram
range and linearity over a wide concentration range. However, it
possesses some drawbacks over HPLC, such as lengthy cleanup
procedures, and in some cases, the formation of more volatile
derivatives or hydrolysis prior to analysis. Furthermore, the high
temperatures required for elution can lead to the on-column decomposition of certain benzodiazepines.
Classification of Analytical Methods
Gas chromatography: Column and detection
Chromatography [LC (HPLC, UPLC, MLC, chiral separation), GC
and thin-layer chromatography (TLC)], capillary electroseparation (MEKC, capillary electrochromatography, CE), immunoassay, photometry [ultraviolet (UV) spectrometry, Fourier
Column
GC methods generally use a fused-silica capillary column, although one has used a deactivated metal capillary column
592 Uddin et al.
Table II
Gas Chromatographic Conditions for the Determination of Benzodiazepines
Analytes
Matrices
GC conditions
Results
Reference
Column
Detection/
interfaces
Range, recovery (%), LOD
MS-NCI
0.1 –20.0 ng/mg, 68– 77, NDZ: 0.01, OZ: 0.005 ng/mg
45
MS-SIM
MS-EI
0 –100 ng/mL, 83 –87, 5 ng/mL, 1 ng/mL
—, 88– 109, 0.4 –10 ng/mL
70
49
Bio-Sample Preparation and Gas Chromatographic Determination of Benzodiazepines—A Review 593
NDZ, OZ
Hair
FNZ, 7-AFNZ, NZ (IS)
MZ, NDZ, DZP, OZ, BRZ, CDO, PNZ, NZ, LRZ,
TZ, ALP,
MDL, 1-HAL, 1-HMDL, FZ (IS)
ALP, FZ, OZ, LRZ, DZP, TZ, MDL, NDZ, PRZ,
DA-FZ,
a-HALP, a-HMDL, a-HTL, 2-HEFZ, 7-AFNZ,
7-ACLZ, 7-ANZ
CLZ,7-ACLZ, DZP D5 (IS)
DZP, FZ, MZ, OZ
Blood, urine
Whole blood
HP-5 MS capillary column, 5% phenyl –95% methylsiloxane, 30 m 0.25 mm,
0.25 mm
HP-5MS capillary column (12 m 0.2 mm, 0.33 mm film)
30 m DB-35 ms (0.32 mm, 0.25 mm film) silica capillary column
Urine
RTX-200 capillary column (15 m 0.25 mm 0.25 mm)
MS
Up to 1.5 mg/mL, —, 2.5 ng/mL
30
Urine
Rat hair,
plasma
Tablet
Oral fluid
Urine
Whole blood
Hair
Whole blood
Whole blood
11 matrices
Plasma, urine
Blood
Plasma
Urine
HP-5MS capillary column (30 m 250 mm 0.25 mm)
TC-1 capillary, 20 m 0.25 mm, 0.25 mm film
MS-NCI
MS-EI
32
50
HP-5 MS capillary column (30 m 250 mm; 0.25 mm)
HP-1capillary column (30 m 250 mm 0.25 mm)
HP-5MS capillary column (30 m 0.25 mm, 0.25 mm)
DB5-MS, 20 m 0:18 mm, 0.18 mm
—
DB-5MS fused-silica capillary (30 m 0.25 mm i.d., 0.25 mm)
DB-5MS fused silica capillary (30 m 0.25 mm i.d., 0.25 mm)
Fused-silica column (30 m 0.25 mm, 0.25 mm film)
Fused-silica column (30 m 0.25 mm, 0.25 mm film)
Fused-silica column (30 m 0.25 mm, 0.25 mm film)
Hewlett Packard HP-5MS capillary column (30 m 0.25 mm, 0.25 mm film)
SGE BP1 capillary column (30 m 0.25 mm, 0.25 mm film), Restek hydro-guard
column (5 m 0.32 mm i.d.)
MS-NCI
MS-NCI-SIM
MS-SIM
MS-TOF
CLZ: 50 –4,000 pg/mL, —, 50 pg/mL
Plasma: 0.1 –20 mg/mL, hair: 0.1 – 10 ng/mg, 98,
0.01– 0.5 pg/mg
1.2 –23 pg/mg, —, 0.4 pg/mg
0.1 –5.0 mg/L, 83.3 –89.6, 0.05 –0.1 mg/L
0 –25 ng/mL, —, 2.5 ng/mL
50– 300 ng/mL, r 2 ¼ 0.99, —, —
FNZ: 2.5 –200 pg/mg, 7-AFNZ: 0.5 –100 pg/mg
50– 1,000 ng/mL, 5 –50 ng/mL
5 –50 ng/mL
CLZ, 7-ACLZ, DZP-d5 (IS)
FNZ, 7-AFNZ, d7 (IS)
7-AFNZ
DZP, NDZ, BRZ, MZ (IS)
FNZ, 7-AFNZ, deuterated (IS)
DZP, DAFZ, NDZ, MDL, OZ, TZ, LRZ, ALP, CLZ
EL, a-HEL, 8-HEL
NDZ, OZ, TZ
Flunitrazepam
Phenazepam
MDL, PNZ (IS)
ALP, a-HALP, 4-HALP, FNZ, 7-AFNZ, DFNZ, FZ,
HEFZ,
N-DAFZ, KTZ, OZ, LRZ, LZ, TL, a-HTL, N-MCLZ,
(IS)
FNZ, 7-AFNZ
DZP, NDZ, 7-AFNZ, deuterated analyte (IS)
FNZ
DZP, DDZ, OZ, TZ, LRZ, DXP, NCBZ, NZ,
1-HMDL, ALP,
1-HAL, 1-HTL, CLZ, FNZ, DFNZ, PNZ,
MZ, FTZ, OXL, MDL, BRZ, HLZ, FZ, CLZ, ALP,
TL, DZ,
CDO, CXL, FNZ, NTZ, MXL, NZ, ESZ, EL, BRL,
FDZ (IS)
CLZ, 7-ACLZ
FNZ, 7-AFNZ
FNZ, 7-AFNZ, diaz-d5 (IS)
ALP, TL (d4) (IS)
35 Benzodiazepines
Midazolam, 1-hydroxymidazolam
OZ, LRZ, NDZ, DZP, FNZ,
CLZ, CLZ-d5 (IS)
ALP, FNZ, FZ, KTZ, LRZ, TL, a-HAL, 4-HAL,
7-AFNZ, DFNZ, 7-ADFNZ, HEFZ, N-DAFZ,
OZ, a-HTL
CTZ
22 Benzodiazepines
NDZ, OZ, BRZ, DZP, LRZ, FNZ, ALP, TL, PRZ-d5
(IS)
EI-MS-SIM
EI-MS-MS
GC – MS-MS
GC – MS-MS
GC – MS
MS-SIM
MS-EI/SIM
0.1 –1,000 ng/mL, 0.025 ng/mL
0.004 –3,600 ng/mL
1.5 –300 mg/L, r 2 ¼ 0.999, —, 1 ng/mL
5 –100 ng/mL, r 2 ¼ 0.995, 79, LOQ:
1.0 –1.7 ng/mL
51
68
77
55
82
85
84
18
63
56
37
27, 28
Hair
Hair
Urine, serum
Blood, urine
HP-Ultra 2 capillary column (12 m 0.2 mm 0.33 mm film)
HP 5 capillary column (30 m 0.25 mm)
HP-1 capillary column (12 m 0.20 mm with 0.33 mm film)
MS-NCI
MS-SIM
MS-SIM
MS
—, —, LOQ: 0.5 –2.3 pg/mg
—, —, 0.02 –0.15 ng/mg
—
—, —, 5 –40 ng/mL
74
52
38
33
Whole blood
BPX 5 capillary column (15 m 0.32 mm, film 0.25 mm), SGE
MS-SIM
5 –500 ng/mL, r 2 . 0.995, 44 –138, 0.2 – 20 ng/mL
41
Hair
Urine
Hair
Rat hair
Urine
Rabbit plasma
Hair
Plasma
Drugs
—
—
Capillary column, 5% phenyl – 95% methysiloxane (30 m 0.25 mm)
Restek-200 capillary column (15 m 0.25 mm, 0.25 mm)
Fused-silica column (30 m 0.25 mm, 0.25 mm film)
Fused-silica column (30 m 0.25 mm, 0.25 mm film)
HP-Ultra 2 capillary column (12 m 0.2 mm 0.33 mm film)
HP-5 column (12 m 0.25 mm, 0.33 mm film )
BP 1 capillary column (30 m 0.25 mm, 0.25 mm film; nonpolar poly dimethylsiloxane
phase)
MS-NCI
MS-NCI
MS-NCI-SIM
MS-NCI
GC – TOF-MS
GC – MS-NCI
MS-SIM
MS-NCI
MS-EI-SIM
7-ACZ: 1 –1,000 pg/mg, CZ: 10– 400 pg/mg, —, —
—, —, LOQ: FNZ, 100, AFNZ: 10 pg/mL
10, 200 pg/mg, 90, 15 pg/mg, 3 pg/mg
Up to 250 pg/mg, 92 –99, LOQ: 25 pg/mg
10—350 ng/mL
2 –200 ng/mL
—, 50– 95, 0.01 –2.0 ng/mg
1 –25 ng/mL, —, 0.1 ng/mL
—, —, 0.1 –0.5 ng/mL
75
73
46
15
60
59
35
71
28
Plasma
Drugs, Blood,
urine
Hair
Fused-silica capillary column (30 m 0.25 mm, 0.25 mm film)
DB5-MS capillary column (30 m 0.25 mm i.d.) film thickness: 0.25 mm
MS
Ion trap-MS-MS
67
65
HP 5-MS capillary column, 5% phenyl –95% methyl siloxane,
30 m 0.25 mm 0.25 mm film
MS-NCI
5 –200 ng/mL, r 2 ¼ 0.9985, 92 –101, LOQ: ng/mL
Detection thresholds: 10 –500 pg/mL, blood: 67 –75,
urine: 85 –93, —
r 2 ¼ 0.930 – 1.0, 47.6– 90, 1 – 20 pg/mg
47
(continued)
Plasma
Plasma
Urine, plasma
DZP, PRZ (IS)
MDL, DZP, MZ, NZ, N-DCBZ
DZP, N-DDZ, PRZ (IS)
MZ, DZP, CBZ, MDL
DZP, MDL, PRZ, FZ, HEFZ, LRZ, NDZ, FNZ,
NFNZ, DAFZ, CLP, CBZ, BRZ
TL, ALP (IS)
*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), and amino (A), acitamido (Ac), hydroxyl (H), methyl (M),
nor (N), desmethyl (D), desalkyl (DA) as substituent.
66
0 –10 ng/mL, r 2 ¼ 0.9996, 95 –102, 0.5 ng/mL
63 Ni ECD
NC-17 capillary column (OV-17; 15 m 0.25 mm, 0.25 mm )
Serum
61
82
NPD
63Ni ECD
SPB-5 (15 m 0.32 mm) with a film of 0.25 mm
Fused silica capillary column was 10 m 0.53 mm i.d., 2 mm film thickness
Plasma
Blood, plasma
62
Urine, serum
DZP, OZ, TZ, NDZ, LRZ
FID-NPD
FID-NPD
2
Blood
Urine, serum
FID-MS
Oral fluid
Urine
Plasma, urine
Urine
50 Drugs of abuse
13 Benzodiazepines
FNZ, 7-AFNP, DFNZ, 7-ADFNZ, 7-AFNZ-d3 (IS)
128 Date-rape drugs
Clobazam and norclobazam
23 Benzodiazepines
DZP, NDZ, OZ, TZ, LRZ
0.72 and 36 ng/mL
NPD
0.5 –8.0 nmol/mL, r 2 ¼ 0.996, 64– 104, 0.020 – 0.115
nmol/mL
5 –50 ng/mL, r 2 ¼ 0.992 –7, 70, 0.5 –2 ng/mL
—, plasma: 59 –92, blood: 53 –99, 2 –14 ng/mL
0.1 –2 mg/mL, —, 0.02 –0.10 mg/mL
39
36
64
0.1 –2 mg/mL, —, 0.02 ng –0.1 mg/mL
0.25 –7.5 nmol/mL, r ¼ 0.9994, —, 0.10 nmol/mL
0.1 –2.0 ng/mL, 65– 80, LOQ: 5 –25 ng/mL
17
83
40
58
76
57
64
MS-MSD-EI-NICI
MS-ESI
MS
EI-MS
MS
EI-MS
FID-MS
Fused-silica column (30 m 0.25 mm, 0.25 mm film)
RTX-200 capillary column (30 m 0.25 mm, 0.25 mm)
DB-5 MS column (25 m 0.2 mm, 0.33 mm film)
Fused-silica column (30 m 0.25 mm, 0.25 mm film)
Fused-silica column (30 m 0.25 mm, 0.25 mm film)
Fused-silica column (30 m 0.25 mm, 0.25 mm film)
30 m 0.25 mm PTE-5 poly (5% diphenyl –95% dimethyl-siloxane) column
(film of 0.25 mm)
SPB-5 (30 m 0.25 mm), PTE-5 poly (5% diphenyl –95% dimethyl-siloxane),
film of 0.25 mm
DB-I (methylsilicon) column, 30 m 0.2 mm, 0.25 mm film
Procolumn; 3 m 0.32 mm 15 m 0.32 mm contained SE-54
with 0.25 mm film; fused silica
SPB-1 (30 m 0.25 mm, 0.25 mm film)
—
Up to 200 ng/mL, 96.1 ng/mL
Range, recovery (%), LOD
Column
Reference
Results
Detection/
interfaces
GC conditions
Matrices
Analytes
Table II Continued
594 Uddin et al.
coated with OV-17 (RASCOT NC-17) (65). As expected, fused
silica used in all GC columns has ranged from 100% methylsilicone to a mixed phenylmethylsilicone phases. Although trifluoropropylmethyl or cyanopropylphenyl phases have also
been used, no report is available during the study period. the
types of column phases are basically of low polarity methylsilicone (40), polymethylsilicon SPB-1 (63), dimethylpolysiloxane
(28, 29), 5% phenyl –95% methyl- or dimethylsiloxane PTE-5
(66) or HP-5MS (5%-phenyl)-methylpolysiloxane (46 –48, 71).
Temperature programming is utilized in most methods, which
is necessary for assays involving the detection of many benzodiazepines due to the wide polarity differences. The initial GC
oven temperature has begun at 608C, which was increased at a
certain rate to the final temperature at approximately 3008C.
Detection
Spectral characteristics for some selected benzodiazepines
found in many references are shown in Table II. Nordiazepam,
oxazepam, bromazepam, diazepam, lorazepam, flunitrazepam,
alprazolam and triazolam in human hair have been detected by
MS in NCI with methane as reactant gas, in which the LOD was
1 –20 pg/mg (48). The quantification of lorazepam in human
hair after derivatization by silylation was performed by the
same MS –NCI (73). The quantitation of flunitrazepam and its
major metabolite 7-amino-flunitrazepam in urine (74), hair (47,
75) and oral fluid (68), clonazepam and its metabolites in hair
(52, 76) and urine (33), nordiazepam and oxazepam in hair
(46) and alprazolam in hair (16) has been conducted using MS
in NCI detection mode, with methane as the ionization gas,
resulting in the corresponding LOD values 3 –15 and 05 –2.3
pg/mg, 10 –100 pg/mL, 0.05 –0.1 ng/mL, 0.4 pg/mg, 50 pg/mL,
0.005– 0.01 ng/mg and 25 pg/mg.
Clobazam and norclobazam were detected by GC–MS (77).
MS with EI ionization in SIM mode has been for the determination many of benzodiazepines, with LOD values found to be
1.0 –1.7 ng/mL (28) and 0.1– 0.5 ng/mL (29) for urine, 0.02 –
0.08 ng/mg for hair (53), 0.2 –20 ng/mL (42) and 0.4 –10 ng/
mL for whole blood (50) and 0.1 –0.25 ng/mg for rat hair (51).
The determination of flunitrazepam and its metabolites in
whole blood and urine (78, 70), midazolam in plasma (38), triazepam in human muscle (62), 15 low-dosed benzodiazepines
in pure drugs (29) has also been conducted by MS detection
employing the same mode of detection resulting in LOD values
between 0.5 –5.0 ng/mL.
The use of NCI with methane as reagent gas afforded a
greatly enhanced LOD compared to EI mode (71). Nineteen
benzodiazepines and two thienodiazepines were well separated
from each other on their SIM chromatograms and also on the
total ion chromatogram (TIC) (42). Ion trap mass spectrometry
(ITMS) with electrospray ionization (ESI) was employed for the
determination of five benzodiazepines in aqueous solution,
urine and serum, in which the LOD was found to be 0.02 –
0.1 mg/mL (66). ITMS was also used for the simultaneous detection of 22 benzodiazepines in both EI and NCI modes for
both derivatized (silylated) and underivatized molecules. The
LOD was 10– 500 pg/mL for all, but 1 ng/mL for three. A comparison clearly showed that silylation provides much lower detection thresholds; hence, the improved sensitivity, and both
ionization modes provide equivalent sensitivities (67).
The determination of some benzodiazepines in plasma with
thermionic detection, FID or NPD was presented (37) after
online coupling of dialysis via a precolumn, or rather, a trapping column as a preparation technique. Diazepam in human
plasma was detected by either FID or NPD, with an LOD value
of 0.10 nmol/mL (40). FID detected many acid or basic drugs,
including benzodiazepines in human plasma, with an LOD of
0.5 –2.0 mg/mL (79, 80). The LOD ranged from 0.020–0.115
nmol/mL in the determination of diazepam and the metabolite
N-desmethyldiazepam in plasma and urine after LPME by NPD
(63). The ASPEC system was optimized for the determination
of four benzodiazepines in plasma; the analytes were detected
by the same NPD, providing an LOD of 0.5 –2 ng/mL (62).
A method for the determination of trace amounts of triazolam in
serum by deactivated metal capillary GC with electron-capture
detection (Ni ECD) was established to determine 0.5 ng/mL of
triazolam in serum (65). Comparatively, ECD was supposed to
provide the best LOD (1 ng/mL) for 1.0 mL (40, 65), although NPD provided LODs down to 5 –25 ng/mL (63, 62).
ECD (81) or benchtop ion MS-MS (82) have been applied for
the identification and determination of flunitrazepam and its
metabolites in blood and urine, respectively. GC– MS with the
mass selective detector (MSD) operating in either EI or
negative-ion chemical ionization (NICI) mode was employed
for the analysis of 50 drugs of abuse, including benzodiazepines
(18). A highly sensitive micro-plate enzyme immunoassay
screening of benzodiazepines and their major metabolites in
biological fluids was confirmed by GC –NICI (83, 84). Etizolam
and its primary metabolites in whole blood were determined
by ion trap GC –MS-MS (85). A rapid and efficient EI-GC –MS
method in SIM mode was optimized for the screening and
quantitation of benzodiazepines in blood, with LODs from 5 to
50 ng/mL (86).
The rapid detection and quantification of 35 benzodiazepines in urine was performed by GC –time-of-flight (TOF)-MS
(61). TOF-MS offers new perspectives for forensic toxicology.
Qualitative and quantitative analyses of a mixture of three
selected benzodiazepines (diazepam, nordazepam and bromazepam) were performed to compare gas chromatography
(GC–TOF-MS, quadrupole GC –MS and GC–ECD). The experiment included the analysis of real human blood for bromazepam and demonstrated high sensitivity and high selectivity due
to the high quality of mass spectra obtained by MS-TOF (56).
A typical total ion chromatogram of nine benzodiazepines from
a 50 ng/mL calibrator is presented in Figure 4.
All gas chromatographic methods used ultra-purity helium as
carrier gas with few exceptions: one used hydrogen (16) and
another used a mixture of argon –methane (9:1, v/v) (87), in a
stability study of some benzodiazepines and their metabolites.
Deuterated internal standards (87) were commonly used in
most of the cited references, reflecting their wide commercial
availability and their general acceptance as ideal internal standards. Table II summarizes the conditions and experimental
results of some gas chromatographic methods for the determination of 1,4-benzodiazepines.
Conclusion
GC– MS methods were, as expected, well represented and still
offer the best means to confirm benzodiazepines. Most of these
methods derivatized the benzodiazepines, both to improve
spectral definition and to reduce on-column thermal degradation. The most favoured derivative was the conventional TMS,
although the TBDM derivative offered improved spectral properties. Conventional LLE or SPE are still widely used by most
Figure 4. Total ion chromatogram of a 50 ng 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-TBDMS and temazepam-TBDMS; 7,lorazepam-d4-diTBDMS and lorazepam-diTBDMS; 8, clonazepam-d4-TBDMS and clonazepam-TBDMS; and 9, alprazolam-d5
and alprazolam. Reproduced from Nicholas B. Tiscione, Xiaoqin Shan, Ilene Alford and Dustin Tate Yeatman. Quantitation of Benzodiazepines in Whole Blood by Electron ImpactGas Chromatography-Mass Spectrometry, Journal of Analytical Toxicology, Vol. 32, October 2008, page 644-652, Figure 1, by permission of Oxford University Press.
Bio-Sample Preparation and Gas Chromatographic Determination of Benzodiazepines—A Review 595
researchers as isolation steps with no obvious relative advantages. SPME or LPME offer relatively new approaches to sample
preparation. These require fewer organic solvents, which is important from an ecological and analytical viewpoint. They are
also fast, solvent-free and provide excellent performances.
Conventional GC methods using fused capillary columns were
most commonly used with ECD, although some papers found
NPD a useful alternative detector. EI was generally used as
the ionization method with SIM, although the use of NCI
provided significant sensitivity, with LODs to 0.1 ng/mL.
Dimethylpolysiloxane (100%) and 5% diphenyldimethylpolysiloxane phases were most commonly used and provided good
separation capabilities for most derivatized benzodiazepines.
References
1. Uddin, M.N.; Estimation of 1,4-benzodiazepines in biological
samples: HPLC, the Versalite Method; LAP LAMBERT Academic
Publishing, Germany, (2012), pp. 18–45.
2. Uddin, M.N., Samanidou, V.F., Papadoyannis, I.N.; Sample preparation
overview
for
the
chromatographic
determination
of
1,4-benzodiazepines in biological matrices, Chapter 7. Benthem
Science Publishers, (2010), pp. 84 –107.
3. Esteve-Romero, J., Carda-Broch, S., Gil-Agust, M., Capella-Peiro, M.,
Bose, D.; Micellar liquid chromatography for the determination of
drug materials in pharmaceutical preparations and biological
samples; Trends in Analytical Chemistry, (2005); 24(2): 75 –91.
4. Berzas, J.J., Castaneda, G., Pinilla, M.J.; Determination of clobazam,
clorazepate, flurazepam and flunitrazepam in pharmaceutical preparations; Talanta, (2002); 57: 333– 341.
5. Salem, A.A., Barsoum, B.N., Izake, E.L.; Potentiometric determination
of diazepam, bromazepam and clonazepam using solid contact ionselective electrodes; Analytical Chimca Acta, (2003); 498: 79– 91.
6. Salem, A.A., Barsoum, B.N., Izake, E.L.; Determination of bromazepam and clonazepam in pure and pharmaceutical dosage forms
using chloranil as a charge transfer complexing agent; Analytical
Letters, (2002); 35(10): 1631– 1648.
7. Dolejsova, J., Solich, P., Polydorou, Ch.K., Koupparis, M.A.,
Efstathiou, C.E.; Flow-injection fluorimetric determination of
1,4-benzodiazepines in pharmaceutical formulations after acid hydrolysis; Journal of Pharmaceutical Biomedical Analysis, (1999);
20: 357–362.
8. Tomita, M., Okuyama, T.; Application of capillary electrophoresis to
the simultaneous screening and quantitation of benzodiazepines;
Journal of Chromatography B, (1996); 678: 331–337.
9. Moore, K.A., Werner, C., Zannelli, R.M., Levine, B., Smith, M. L.;
Screening postmortem blood and tissues for nine cases of drugs of
abuse using automated microplate immunoassay; Forensic Science
International, (1999); 106: 93–102.
10. Uddin, M.N., Samanidou, V.F., Papadoyannis, I.N.; Development and
validation of an HPLC method for the determination of six
1,4-benzodiazepines in pharmaceuticals and human biological
fluids; Journal of Liquid Chromatography & Related Technology,
(2008); 31(9): 1258– 1282.
11. Uddin, M.N., Samanidou, V.F., Papadoyannis, I.N.; Validation of
SPE-HPLC determination of 1,4-benzodiazepines and metabolites in
blood plasma, urine, and saliva; Journal of Separation Science,
(2008); 31: 3704–3717.
12. Ishida, T., Kudoa, K., Hayashidac, M., Ikeda, N.; Rapid and quantitative screening method for 43 benzodiazepines and their metabolites, zolpidem and zopiclone in human plasma by liquid
chromatography/mass spectrometry with a small particle column;
Journal of Chromatography B, (2009); 877(25): 2652–2657.
13. de Jager, A. D., Bailey, N. L.; Online extraction LC– MS/MS method
for the simultaneous quantitative confirmation of urine drugs of
abuse and metabolites: Amphetamines, opiates, cocaine, cannabis,
596 Uddin et al.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
benzodiazepines and methadone; Journal of Chromatography B,
(2011); 879(25): 2642– 2652.
Sauve, E.N., Langødega˚rd, M., Ekeberg, D., Øiestad, A˚.M.L.;
Determination of benzodiazepines in ante-mortem and post-mortem
whole blood by solid-supported liquid–liquid extraction and UPLC–
MS/MS; Journal of Chromatography B, (2012); 883–884: 177–188.
Hiild, K.M., Crouch, D.J., Wilkins, D.G., Rollins, D.E., Maes, R.A.;
Detection of alprazolam in hair by negative ion chemical ionization
mass spectrometry; Forensic Science International, (1997); 84:
201– 209.
Rasanen, I., Neuvonen, M., Ojanpera¨, I., Vuori, E.; Benzodiazepine
findings in blood and urine by gas chromatography and immunoassay; Forensic Science International, (2000); 112(2): 191– 200.
Langel, K., Gunnar, T., Ariniemi, K., Rajama¨ki, O., Lillsunde, P.; A validated method for the detection and quantitation of 50 drugs of
abuse and medicinal drugs in oral fluid by gas chromatography–
mass spectrometry; Journal of Chromatography B, (2011);
879(13-14): 859–870.
Cartiser, G.N., Be´valot, F., Le Meur, C., Gaillard, M.Y.D., Hubert, N.,
Guitton, J.; Gas chromatography–tandem mass spectrometry assay
for the quantification of four benzodiazepines and citalopram in
eleven postmortem rabbit fluids and tissues, with application to
animal and human samples; Journal of Chromatography B, (2011);
879(27): 2909–2918.
Marchi, I., Schappler, J., Veuthey, J., Rudaz, S.; Development and validation of a liquid chromatography– atmospheric pressure photoionization –mass spectrometry method for the quantification of
alprazolam, flunitrazepam, and their main metabolites in haemolysed blood; Journal of Chromatography B, (2009); 877(23):
2275–2283.
Odou, P., Robert, H., Luyckx, M., Brunet, C., Dine, T., Gressier, B.,
et al.; A routine HPLC method for monitoring midazolam in serum;
Biomedical Chromatography, (1997); 11: 19– 21.
Bugey, A, Rudaz, S., Staub, C.; A fast LC-APCI/MS method for analyzing benzodiazepines in whole blood using monolithic support;
Journal of Chromatography B, (2006); 832: 249–255.
Sachs, H., Kintz, P.; Testing for drugs in hair: Critical review of chromatographic procedures since 1992; Journal of Chromatography
B, (1998); 713:147–161.
Bertucci, C., Bartolini, M., Gotti, R., Andrisano, V.; Drug affinity to
immobilized target bio-polymers by high-performance liquid chromatography
and
capillary
electrophoresis;
Journal
of
Chromatography B, (2003); 797: 111– 129.
Drummer, O.H.; Methods for the measurement of benzodiazepines
in biological samples; Journal of Chromatography B, (1998);
713: 201–225.
Panderi, I., Archontaki, H., Gikas, E., Parissi-Poulou, M.; Acidic hydrolysis of bromazepam studied by high performance liquid chromatography: Isolation and identification of its degradation products;
Journal of Pharmaceutical and Biomedical Analysis, (1998);
17: 327–335.
Balikova, M., Maresova, V., Vecerkova, J.; The sensitive GC-MS evidence of benzodiazepines in urine as trimethylsilylderivatives;
Soudnı´ le´karstvı´, (1999); 44(3): 34 –42.
Borrey, D., Meyer, E., Lambert, W., Van Peteghem, C., De Leenheer,
A.P.; Simultaneous determination of fifteen low-dosed benzodiazepines in human urine by solid-phase extraction and gas chromatography–mass spectrometry; Journal of Chromatography B, (2001);
765: 187–197.
Borrey, D., Meyer, E., Lambert, W., Van Calenbergh, S., Van
Peteghem, C., De Leenheer, A.P.; Sensitive gas chromatographic –
mass spectrometric screening of acetylated benzodiazepines;
Journal of Chromatography A, (2002); 910: 105–118.
Lozano-Chaves, M.E., Palacios-Santander, J.M., Cubillana-Aguilera, L.M.,
Naranjo-Rodriguez, I., Hidalgo-Hidalgo-de-Cisneros, J.L.; Modified
carbon-paste electrodes as sensors for the determination of
1,4-benzodiazepines: Application to the determination of diazepam
and oxazepam in biological fluids; Sensors and Actuators B, (2006);
115: 575–583.
47. Cirimele, V., Kintz, P., Ludes, B.; Screening for forensically relevant
benzodiazepines in human hair by gas chromatography-negative
ion chemical ionization-mass spectrometry; Journal of
Chromatography B, (1997); 700: 119– 129.
48. Cirimele, V., Kintz, P., Mangin, P.; Determination of chronic flunitrazepam abuse by hair analysis using GC–MS– NCI; Journal of
Analytical Toxicology, (1996); 20: 596–598.
49. Gunnar, T., Ariniemi, K., Lillsunde, P.; Determination of 14 benzodiazepines and hydroxy metabolites, zaleplon and zolpidem as
tert-butyldimethylsilyl derivatives compared with other common
silylating reagents in whole blood by gas chromatography–mass
spectrometry; Journal of Chromatography B, (2005); 818:
175– 189.
50. Scott, K.S., Nakahara, Y.; A study into the rate of incorporation of
eight benzodiazepines into rat hair (HPLC, GC-MS); Forensic
Science International, (2003); 133: 47 –56.
51. Negrusz, A., Bowen, A.M., Moore, C.M., Dowd, S.M., Strong, M.J.,
Janicak, P.G.; Deposition of 7-aminoclonazepam and clonazepam in
hair following a single dose of klonopin; Journal of Analytical
Toxicology, (2002); 26(7): 471–478.
52. Yegles, M., Marson, Y., Wennig, R.; Influence of bleaching on stability of benzodiazepines in hair; Forensic Science International,
(2000); 107: 87– 92.
53. Negrusz, A., Bowen, A.M., Moore, C.M., Dowd, S.M., Strong, M.J.,
Janicak, P.G.; Deposition of 7-aminoclonazepam and clonazepam in
hair following a single dose of klonopin; Journal of Analytical
Toxicology, (2002); 26(7): 471–478.
54. Ariffin, M.M., Miller, E.I., Cormack, P.A.G., Anderson, R.A.;
Molecularly imprinted solid-phase extraction of diazepam and its
metabolites from hair samples; Analytical Chemistry, (2007); 79:
256– 262.
55. Aebi, B., Sturny-Jungo, R., Bernhard, W., Blanke, R., Hirsch, R.;
Quantitation using GC–TOF-MS: Example of bromazepam; Forensic
Science International, (2002); 128: 84 –89.
56. Kriikku, P., Wilhelm, L., Rintatalo, J., Hurme, J., Kramer, J., Janpera¨, I.;
Phenazepam abuse in Finland: Findings from apprehended drivers,
post-mortem cases and police confiscations; Forensic Science
International, (2012); 220(1– 3): 111– 117.
57. Papoutsis, I.I., Athanaselis, S.A., Nikolaou, P.D., Pistos, C.M.,
Spiliopoulou, C.A., Maravelias, C.P.; Development and validation of
an EI –GC– MS method for the determination of benzodiazepine
drugs and their metabolites in blood: Applications in clinical and
forensic toxicology; Journal of Pharmaceutical and Biomedical
Analysis, (2010); 52(4): 609– 614.
58. Adamowicz, P., Kal a, M.; Simultaneous screening for and determination of 128 date-rape drugs in urine by gas chromatography– electron ionization-mass spectrometry; Forensic Science International,
2010; 198(1-3): 39 –45.
59. Kaartama, R., Jarho, P., Savolainen, J., Kokki, H., Lehtonen, M.;
Determination of midazolam and 1-hydroxymidazolam from plasma
by gas chromatography coupled to methane negative chemical ionization mass spectrometry after sublingual administration of midazolam; Journal of Chromatography B, (2011); 879(19):
1668–1676.
60. Arnhard, K., Schmid, R., Kobold, U., Thiele, R.; Rapid detection and
quantification of 35 benzodiazepines in urine by GC-TOF-MS;
Analytical Bioanalytical Chemistry, (2012); 403(3): 755– 68.
61. Louter, A.J.H., Bosma, E., Schipperen, J.C.A., Vreuls, J.J., Brinkman,
U.A.Th.; Automated on-line solid-phase extraction-gas chromatography with nitrogen-phosphorus detection: determination of benzodiazepines in human plasma; Journal of Chromatography B,
(1997); 689: 35– 43.
62. Ugland, H.G., Krogh, M., Rasmussen, K.E.; Liquid-phase microextraction as a sample preparation technique prior to capillary gas
chromatographic-determination of benzodiazepines in biological
matrices; Journal of Chromatography B, (2000); 749: 85– 92.
63. Cui, S., Tan, S., Ouyang, G., Pawliszyn, J.; Automated polyvinylidene
difluoride hollow fiber liquid-phase microextraction of flunitrazepam in plasma and urine samples for gas chromatography/tandem
=
30. Klette, K.L., Wiegand, R.F., Horn, C.K., Stout, P.R., Magluilo, J., Jr.;
Urine benzodiazepine screening using Roche online KIMS immunoassay with b-glucuronidase hydrolysis and confirmation by gas
chromatography– mass spectrometry; Journal of Analytical
Toxicology, (2005); 29: 193–200.
31. Kintz, P., Villain, M., Cirimele, V., Pepin, G., Ludes, B.; Windows of
detection of lorazepam in urine, oral fluid and hair, with a special
focus on drug-facilitated crimes; Forensic Science International,
(2004); 145: 131–135.
32. Negrusz, A., Bowen, A.M., Moore, C.M., Dowd, S.M., Strong, M.J.,
Janicak, P.G.; Elimination of 7-aminoclonazepam in urine after a
single dose of clonazepam; Analytical Bioanalytical Chemistry,
(2003); 376: 1198–1204.
33. Rasanen, I., Neuvonen, M., Ojanpera, I., Vuori, E.; Benzodiazepine
findings in blood and urine by gas chromatography and immunoassay; Forensic Science International, (2000); 112: 191– 200.
34. Laloup, M., Fernandezm, M.M.R., Wood, M., Maes, V., De Boeck, G.,
Vanbeckevoort, Y., et al.; Detection of diazepam in urine, hair and
preserved oral fluid samples with LC-MS-MS after single and
repeated administration of myolastan and valium; Analytical
Bioanalytical Chemistry, (2007); 388: 1545–1556.
35. Yegles, M., Mersch, F., Wennig, R.; Detection of benzodiazepines
and other psychotropic drugs in human hair by GC/MS; Forensic
Science International, (1997); 84: 211–218.
36. Herraez-Hernandez, R., Louter, A.J.H., de Merbel, V., Brinkman,
U.A.Th.; Automated on-line dialysis for sample pregaration for gas
chromatography: Determination of benzodiazepines in human
plasma; Journal of Pharmaceutical and Biomedical Analysis,
(1996); 14: 1077–1087.
37. Frison, G., Tedeschi, L., Maietti, S., Ferrara, S.D.; Determination of
midazolam in human plasma by solid phase microextraction and
gas chromatography/mass spectrometry; Rapid Communication
Mass Spectroscopy (2001); 15: 2497– 2501.
38. Staerk, U., Kulpmann, W.R.; High-temperature solid-phase microextraction procedure for the detection of drugs by gas chromatography–mass spectrometry; Journal of Chromatography B, (2000);
745: 399–411.
39. Krogh, M., Grefslie, H., Rasmussen, K.E.; Solvent-modified solidphase microextraction for the determination of diazepam in human
plasma samples by capillary gas chromatography; Journal of
Chromatography B, (1997); 689: 357–364.
40. Snyder, H., Schwenzer, K.S., Pearlman, R., McNally, A.J., Tsilimidos,
M., Salamone, S.J., et al.; Serum and urine concentrations of flunitrazepam and metabolites, after a single oral dose, by immunoassay and
GC-MS; Journal of Analytical Toxicology, (2001); 25(8): 699– 704.
41. Inoue, H., Maeno, Y., Iwasa, M., Matoba, R., Nagao, M.; Screening and
determination of benzodiazepines in whole blood using solid-phase
extraction and gas chromatography/mass spectrometry; Forensic
Science International, (2000); 113: 367– 373.
42. Honeychurch, K.C., Smith, G.C., Hart, J.P.; Voltammetric behavior of
nitrazepam and its determination in serum using liquid chromatography with redox mode dual-electrode detection; Analytical
Chemistry, (2006); 78: 416– 423.
43. Yuan, H., Mester, Z., Lord, H., Pawliszyn, J.; Automated in-tube solidphase microextraction coupled with liquid chromatography–electrospray ionization mass spectrometry for the determination of
selected benzodiazepines; Journal of Analytical Toxicology,
(2000); 24(8): 718– 725.
44. Abu-Qare, A.W., Abou-Donia, M.B.; Chromatographic method for
the determination of diazepam, pyridostigmine bromide, and their
metabolites in rat plasma and urine; Journal of Chromatography
B, (2001); 754: 503– 509.
45. Kintz, P., Cirimele, V., Vayssette, F., Mangin, P.; Hair analysis for nordiazepam and oxazepam by gas chromatography-negative-ion
chemical
ionization
mass
spectrometry;
Journal
of
Chromatography B, (1996); 677: 241–244.
46. Cirimele, V., Kintz, P., Staub, C., Mangin, P.; Testing human hair for
flunitrazepam and 7-amino-flunitrazepam by GC/MS-NCI; Forensic
Science International, (1997); 84: 189–200.
Bio-Sample Preparation and Gas Chromatographic Determination of Benzodiazepines—A Review 597
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
598 Uddin et al.
76. Pok, P.P., Mauras, M., De Saint Le´ger, M., Kuhlmann, E.,
Charpenel-Durat, C., Navarette, C., et al.; Blood concentrations of
clobazam and norclobazam in a lethal case involving clobazam,
meprobamate and clorazepate; Legal Medicine, (2010); 12(6):
300– 304.
77. Wang, P., Liu, C., Tsay, W., Li, J., Liu, R.H., Wu, T., et al.; Improved
screen and confirmation test of 7-aminoflunitrazepam in urine
samples for monitoring rlunitrazepam (rohypnol) exposure;
Journal of Analytical Toxicology, (2002); 26(7): 411– 418.
78. Qiu, F.H., Liu, L., Luo, Y., Liu, F, Lu, Y.Q.; Systematic analysis of basic
drugs in plasma using X-5 solid-phase extraction GC-FID and
GC-MS; Yao Xue Xue Bao, (1996); 31(4): 296–299.
79. Qiu, F.H., Liu, L., Luo, Y., Liu, F., Lu, Y.Q.; A double column double
pH solid phase extraction and capillary GC/FID method for rapid
simultaneous determination of acidic and basic drugs in human
plasma; Yao Xue Xue Bao, (1996); 31(3): 205– 208.
80. Chl obowska, Z., S’wiegoda, C., Kos’cielniak, P., Piekoszewski, W.;
Identification and determination of flunitrazepam and its metabolites in blood by gas chromatography; Chemia Analityczna,
(2004); 49(1): 71 –77.
81. Nguyen, H., Nau, D.R.; Rapid method for the solid-phase extradion and GC-MS analysis of flunitrazepam and its major metabolites in urine; Journal of Analytical Toxicology, (2000); 24(1):
37– 45.
82. Negrusz, A., Moore, C., Deitermann, D., Lewis, D., Kaleciak, K.,
Kronstrand, R., et al.; Highly sensitive micro-plate enzyme immunoassay screening and NCI-GC-MS confirmation of flunitrazepam and
its major metabolite 7-aminoflunitrazepam in hair; Journal of
Analytical Toxicology, (1999); 23(6): 429– 435.
83. DeRienz, R.T., Holler, J.M., Manos, M.E., Jemionek, J., Past, M.R.;
Evaluation of four immunoassay screening kits for the detection of
benzodiazepines in urine; Journal of Analytical Toxicology,
(2008); 32: 433– 437.
84. Nakamae,
T., Shinozuka, T., Sasaki, C., Ogamo, A.,
Murakami-Hashimoto, C., Irie, W., et al.; Case report: Etizolam and
its major metabolites in two unnatural death cases; Forensic
Science International, (2008); 182: 1– 6.
85. Tiscione, N.B., Shan, X., Alford, I., Yeatman, D.T.; Quantitation of
benzodiazepines in whole blood by electron impact-gas
chromatography-mass spectrometry; Journal of Analytical
Toxicology, (2008); 32: 644– 652.
86. Goldberger, B.A., Chronister, C.W., Merves, M.L.; Quantitation of
benzodiazepines in blood and urine using gas chromatographymass spectrometry (GC-MS); Methods in Molecular Biology,
(2010); 603: 75– 87.
87. Skopp, G., Po¨tsch, L., Ko¨nig, I., Mattern, R.; A preliminary study on
the stability of benzodiazepines in blood and plasma stored at 4oC;
International Journal of Legal Medicine, (1998); 111: 1–5.
=
64.
mass spectrometry; Journal of Chromatography A, (2009);
1216(12): 2241– 2247.
Nishioka, R.; Determination of triazolam in serum by deactivated
metal capillary gas chromatography with electron-capture detection; Journal of Chromatography B, (1996); 681: 401–404.
Luo, Y., Pan, L., Pawliszyn, J.; Determination of five benzodiazepines
in aqueous solution and biological fluids using solid-phase microextraction with carbowax/DVB fiber coating; Journal of
Microcolumn Separations, (1998); 10(2): 193–201.
Ahn, S., Maeng, H., Koo, T., Kim, D., Shim, C., Chung, S.; Quantification
of clotiazepam in human plasma by gas chromatography–mass spectrometry; Journal of Chromatography B, (2006); 834: 128–133.
Samyn, N., De Boeck, G., Cirimele, V., Verstraete, A., Kintz, P.;
Detection of flunitrazepam and 7-aminoflunitrazepam in oral fluid
after controlled administration of rohypnol; Journal of Analytical
Toxicology, (2002); 26(4): 211– 215.
El-Sohly, M.A., Feng, S., Salamone, S.J., Brenneisen, R.; GC– MS determination of flunitrazepam and its major metabolite in whole blood
and plasma; Journal of Analytical Toxicology, (1999); 23(6):
486–489.
Hackett, J., Elian, A.A.; Extraction and analysis of flunitrazepam/
7-aminoflunitrazepam in blood and urine by LC–PDA and GC–MS
using butyl SPE columns; Forensic Science International, (2006);
157: 156–162.
de Ming, S., Zhang, S., Kohlhof, K.; Quantitative determination of
clonazepam in plasma by gas chromatography-negative ion chemical ionization mass spectrometry; Journal of Chromatography B,
(1996); 686: 199–204.
Pirnay, S., Ricordel, I., Libong, D., Bouchonnet, S.; Sensitive method
for the detection of 22 benzodiazepines by gas chromatography-ion
trap tandem mass spectrometry; Journal of Chromatography A,
(2002); 954: 235–245.
Cirimele, V., Kintz, P., Mangin, P.; Detection and quantification of
lorazepam in human hair by GC-MS/NCI in a case of traffic accident; International Journal of Legal Medicine, (1996); 108(5):
265–267.
Negrusz, A., Moore, C.M., Stockham, T.L., Poiser, K.R., Kern, J.L.,
Palaparthy, R., et al.; Elimination of 7-aminoflunitrazepam and flunitrazepam in urine after a single dose of rohypnol; Journal of
Forensic Sciences, (2000); 45(5): 1133–1141.
Negrusz, A., Moore, C.M., Hinkel, K.B., Stockhamm, T.L., Verma, M.,
Strong, M.J., et al.; Deposition of 7-aminoflunitrazepam and flunitrazepam in hair after a single dose of rohypnol; Journal of Forensic
Science, (2001); 46(5): 1143–1151.
Negrusz, A., Moore, C. M., Kern, J. L., Janicak, P. G., Strong, M. J.,
Levy, N.A.; Quantitation of clonazepam and its major metabolite 7aminoclonazepam in hair; Journal of Analytical Toxicology,
(2000); 24(7): 614– 620.