Review

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Benzodiazepines: sample preparation and

HPLC methods for their determination in
biological samples

Benzodiazepines (BDZs) belong to a group of substances known for their sedative, antidepressive, muscle relaxant,
tranquilizer, hypnotic and anticonvulsant properties. Their determination in biological fluids is essential in clinical
assays as well as in forensics and toxicological studies. Researchers focus on the development of rapid, accurate,
precise and sensitive methods for the determination of BDZs and their metabolites. A large number of analytical
methods using different techniques have been reported, but none can be considered as the method of choice.
BDZs are usually present at trace levels (microgram or nanogram per milliliter) in a complex biological matrix and
the potentially interfering compounds must be isolated by various extraction techniques before ana­lysis. An
extended and comprehensive review is presented herein, focusing on sample preparation (pretreatment and
extraction) and HPLC conditions applied by different authors. These methods enable bioanalysts to achieve detection
limits down to 1–2 ng/ml using UV/diode array detection, readily available in most laboratories, and better than
1 ng/ml using electron capture detection, which is lower than that obtained using a nitrogen phosphorus detector.
MS interfaced with electrospray ionization offered a similar sensitivity, while negative chemical ionization MS or
sonic spray ionization MS provided sensitivity down to 0.1 ng/ml.

Benzodiazepines (BDZs) represent a large and vulnerable to BDZs and are often dependent on Victoria F Samanidou†,
important class of psychotherapeutic agents, act- their effects [10,12] . Since BDZs are widely seen in Mohammad N Uddin &
ing on the CNS by enhancing the actions of a clinical and forensic cases, their measurements in Ioannis N Papadoyannis
natural brain chemical, g-aminobutyric acid [1] . samples of biological origin are widely exercized. †
Author for correspondence
Over 50 of these compounds have been inves- Benzodiazepines are clinically effective and Laboratory of Analytical
tigated worldwide for biological activity since represent a large range of potencies at low doses Chemistry, Department of
the introduction of chlordiazepoxide in 1960 [2] . ranging from less than 1–30 mg to over 100 mg, Chemistry, Aristotle University
Due to their tranquilizer, antidepressive and resulting in blood concentrations ranging from of Thessaloniki, Thessaloniki,
sedative properties, they have become the most sub-nanogram per milliliter (10–500 ng/ml) to GR-541 24, Greece
frequently prescribed drugs for the treatment of near-microgram per milliliter levels [8] . They Tel.: +30 231 099 7698
anxiety, sleep disturbance and status epileptics undergo extensive metabolism by CYP mono- Fax: +30 231 099 7719
[3–5] . As the worldwide demand for BDZ anxio- oxygenases and many of their metabolites are E‑mail: [email protected]
lytics and hypnotics is extremely large, they con- pharmacologically active. Therefore, it is essen-
tinue to be developed, evaluated and introduced tial that the assay methods are selective, sen-
for clinical use. They are also commonly used as sitive and specific (i.e., capable of separating
‘date-rape’ drugs. Often, young illicit drug users and determining the parent drug as well as its
are being abused, and in large doses BDZs cause major metabolites). Benzodiazepines
profound behavioral effects [6,7] . They are also In recent years, a large number of analyti- A class of psychotherapeutic
agents
used in the treatment of alcohol withdrawal, to cal and pharmacological studies on BDZs and
relieve tension in the pre-operative period and to their metabolites in biological samples have
induce amnesia in surgical procedures [8] . Owing been described. The dominant assay meth- Biological samples
to their potential for abuse, probably due to the ods include HPLC [13] , GC [14] , micellar LC Include blood, serum, plasma,
fact that they are efficacious and relatively safe (MLC) [15] , micellar electrokinetic chromatog- urine and hair, for example
drugs, BDZs are frequently present in the blood raphy (MEKC) [16] , potentiometry [17] , spec-
of drivers involved in traffic accidents [9] . BDZs trophotometry [18] , fluorimetry [19] , CE [20] and
may also cause or contribute to sudden death if immunoassay [21] . Chromatographic methods,
misused [10] ; they are often subject to overdose particularly HPLC and GC, are most com-
in suicide attempts [11] . Their continued abuse monly used to identify specific BDZs present
leads to dependence [12] . Older populations are in a sample, sometimes initially screened by one

10.4155/BIO.09.43 © 2009 Future Science Ltd Bioanalysis (2009) 1(4), 755–784 ISSN 1757-6180 755
Review | Samanidou, Uddin & Papadoyannis
Chromatographic methods of the immuno­assay-based kit methods. HPLC, detail the procedures for the detection of opi-
Methods for separation of a technique of increasing importance for these ates, cocaine, amphetamines and cannabis and
compounds of a mixture, based purposes, is being used with UV, fluorescence focused on GC–negative chemical ionization–
on the positioning between the or electrochemical detection and even with MS, MS and HPLC/HPLC as increasing the number
mobile and stationary phases
applying the recently developed soft ionization of procedures for BDZ ana­lysis in hair. One elab-
interfaces, such as thermospray ionization, elec- orate report covered chromatographic methods
trospray ionization (ESI), sonic spray ionization (HPLC, LC–MS, GC and GC–MS) published
(SSI) or atmospheric pressure chemical ioniza- from 1992 to 1997 for the measurement of BDZs
tion (APCI) with high sensitivity and selectivity. in biological samples, and included some sample
ESI is the most commonly used ionization tech- preparation procedures [12] .
nique for qualitative and quantitative ana­lysis of The application of CE to the detection and
BDZs [12,22,23] . The application of HPLC–ESI– determination of 1,4-BDZ tranquilizers in for-
MS/MS for qualitative and quantitative ana­lysis mulations and biological samples has been criti-
of BDZs was first described in 1996 [24] . cally evaluated [34] . The use of HPLC and CE
The complexity of biological samples such as as affinity separation methods to characterize
urine, blood and plasma demands a powerful drugs or potential drug–biopolymer interac-
sample preparation technique. This is usually tions has been addressed elsewhere [35] . A fur-
Sample preparation
the most critical and time-consuming step for ther review deals with the different methods of
Sample pretreatment prior
drug determination in biological matrices [25] . ana­lysis of certain tranquilizers (pheno­thiazines,
to ana­lysis by various
extraction techniques Moreover, due to this reason, the analytical thioxanthenes and BDZ derivatives) in biologi-
methods are frequently hampered by impuri- cal fluids of pharmaceutical interest [36] .
ties, which can cause severe inconvenience in This article reviews published methods from
the quantification process of the drugs [26] . 1996 up to 2008, covering sample prepara-
Therefore, an extensive sample preparation tion and chromatographic conditions (column,
technique is always mandatory to extract and mobile phase and detection method) for the
concentrate compounds of interest in order to determination of BDZs in biological samples
obtain reliable analytical results removing impu- and pharmaceutical formulations.
rities contained in human body fluids. Several
sample preparation procedures have been applied Method
for the extraction and isolation of drugs with Pubmed, ScienceDirect, Scopus and Springerlink
Extraction
different degrees of purity [24] . Usually, this websites were searched for the collection of
Isolation of analytes of interest
involves the most common liquid–liquid extrac- references several times between 1996 and 2008.
from matrix
tion and SPE [27] . SPE offers the potential for
specific and accurate sample preparation and, BDZs: structure, properties
with the use of new and advanced technology, & metabolism
SPE can easily be automated [28,29] . Several „„Structure
methods based on SPE (online extraction, col- A seven-membered ring of carbon atoms with
umn-switching techniques, solid-phase microex- one nitrogen is known as an azepine ring while,
traction [SPME] or direct injection of samples with two nitrogen atoms, it is known as a diaz-
to an HPLC column with back flushing) have epine ring, where the topmost nitrogen is desig-
also been successfully described. In addition, a nated position one. Using standard numbering,
recently developed HPLC column consisting of the complete name of this molecule is 1,4-diaz-
a highly cross-linked hard gel polymer station- epine. A benzene ring fused on the 10 and
ary phase of polyvinyl alcohol has been applied 11 positions of the 1,4-diazepine ring (Table 1)
to the direct injection of human plasma and forms 1,4-BDZ [2] .
urine samples with no extraction procedure or Almost all active BDZs are based on the
column-switching technique [29] . 5-aryl-1,4-BDZ structure with a carbonyl group
Benzodiazepines are included in a number (ketone) at position two, except those possess-
of reviews by different authors [22,23,31–32] for ing a fused heterocyclic ring or a thionyl group
quantification of drugs in blood, plasma, serum and a halogen/nitro group at position seven. The
or oral fluid (e.g., saliva) using LC–MS or aryl substituent at the fifth position is usually
LC–MS/MS. Papers that have been published phenyl (e.g., oxazepam) or 2-halophenyl (loraz-
between 1992 and 2007 devoted to drug ana­lysis epam or flurazepam) [37,38] . The addition of an
in hair by HPLC or GC, have been compre- N-methyl group to the nitrogen at position 1,
hensively reviewed [32] . The review described in leads to the prototype BDZ: diazepam, which,

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 Benzodiazepines: sample preparation & HPLC methods for their determination | Review

Table 1. Selected 1,4-benzodiazepines.

R1
R2
N

R3

N
R7

R5

1,4-benzodiazepine
Benzodiazepines R1 R2 R3 R5 R7
Bromazepam H =O H 2’-pyridyl Br
Camazepam CH3 =O (CH3)2-N-COO- Phenyl Cl
Clonazepam H =O H 2-Cl-phenyl NO2
Clorazepate H =O COOH Phenyl Cl
Chlordiazepoxide* - CH3NH H Phenyl Cl
Delorazepam H =O H 2-Cl-phenyl Cl
Demoxipam* H =O H Phenyl Cl
Diazepam CH3 =O H Phenyl Cl
Ethyl loflazepate H =O CH3CH2COO 2-F-phenyl Cl
Fludiazepam CH3 =O H 2-F-phenyl Cl
Flunitrazepam CH3 =O H 2-F-phenyl NO2
Flurazepam (C2H5)2-N-CH=CH- =O H 2-F-phenyl Cl
Halazepam CF3CH2 =O H Phenyl Cl
Lorazepam H =O OH Phenyl Cl
Lormetazepam CH3 =O OH Phenyl Cl
Medazepam CH3 H H Phenyl Cl
Nordazepam H =O H Phenyl Cl
Norfludiazepam H =O H F-phenyl Cl
Nimetazepam CH3 =O H Phenyl NO2
Nitrazepam H =O H Phenyl NO2
Oxazepam H =O OH Phenyl Cl
Phenazepam H =O H Cl-phenyl Br
Pinazepam CH=C-CH2 =O H Phenyl Cl
Prazepam Cyclopropyl methylene =O H Phenyl Cl
Quazepam CF3CH2 =S H 2-F-phenyl Cl
Temazepam CH3 =O OH Phenyl Cl
Tetrazepam CH3 =O H 1,2-dihydro cyclohexyl Cl
*
R4 = N-oxide and double bond at C1-C2.

as a representative drug, exhibits all three of the and chloroform, but only slightly soluble in
basic BDZ effects (i.e., skeletal muscle relaxation, n-hexane or n-heptane and practically insoluble
anticonvulsant activity and anti-anxiety) at doses in water [2] . On the other hand, the salt forms
far lower than those that cause ataxia (loss of bal- (e.g., chlordiazepoxide and flurazepam hydro-
ance). Depending upon the substituents, more chlorides, loprazolam methanesulphonate and
than 50 derivatives have been identified. dipotassium clorazepate) are water-soluble.
Stock solutions of BDZs in methanol, etha-
„„Properties: solubility & stability nol or acetonitrile are stable stored at 4°C for
Benzodiazepines are weak basic drugs, and as at least 6 months [39] . Concentration of BDZs
free bases, are lipid-soluble and water-insolu- in blood and plasma samples stored at 4°C for
ble [38] . 1,4-BDZs are soluble in organic solvents some time prior to ana­lysis should be inter-
such as methanol, ethanol, dimethyl formamide preted cautiously [40] . For longer storage periods,

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Review | Samanidou, Uddin & Papadoyannis
biological samples should be frozen at -20°C, drug-facilitated crime cases when the crimes are
under such conditions BDZs are stable for at reported after a long period of consumption,
least 3–6 months [41] . human hair is considered as the best specimen.
For abuse drug testing in employees in work
„„Metabolism places, saliva can provide a quick and non-
1,4-BDZs can undergo different metabolic reac- invasive specimen. Blood, plasma and serum
tions. Most BDZs are extensively metabolized are often deproteinized and hair needs incu-
by two phases: phase I is predominantly deal- bation in most cases, while urine may require
kylation, aliphatic and aromatic hydroxylation, hydrolysis prior to the isolation procedure.
reduction and acetylation; phase II conjugation 1,4-BDZs are usually present at trace levels
reactions consist largely of glucuronides [2] . In (µg/ml or ng/ml) in a complex biological matrix
most cases, phase I metabolites retain some bio- and the potentially interfering compounds need
logical activity but phase II conjugates, highly to be removed before ana­lysis. Sample pretreat-
polar glucuronides, are usually pharmacologi- ment includes protein removal followed by
cally inactive and can be rapidly removed from extraction and it should be capable of concen-
circulation by the kidneys [12] . trating the sample and reducing the amount of
interfering substances.
„„Metabolism phase I
Oxidation „„Hydrolysis of urine: enzymatic hydrolysis
Many 1,4-BDZs are biotransformed by oxida- Chemical hydrolysis of conjugates with hydro-
tive reactions in the liver, primarily by demeth- chloric acid or sodium hydroxide is not rec-
ylation or dealkylation of the nitrogen in posi- ommended for 1,4-BDZs because they can be
tion one and hydroxylation of the carbon in hydrolyzed to the corresponding benzophe-
position three. nones in strongly acidic or basic media [46] .
Enzymatic digestion [47] generally causes
Reduction hydrolysis without degradation of the parent
1,4-BDZs with 7-nitro substituents, such as molecule to the corresponding benzophenone.
nitrazepam, flunitrazepam, nimetazepam and Most investigators prefer enzymatic hydrolysis
clonazepam represent a subgroup of drugs that of plasma, urine, hair and tissue samples before
are metabolized by reduction of the nitro group extraction to liberate the conjugated fraction
to form the corresponding biologically inac- of the drug, especially for old stains strongly
tive 7-amino and 7-acetamido derivatives or by bound to the material [38] . The procedure is the
demethylation to form N-desmethyl metabo- same with the variation of hydrolysis conditions
lites. The N-desmethyl metabolites are further including temperature (50–60°C), the amount
hydroxylated and subsequently converted to and source of enzyme used, pH of buffer (4–5)
N-glucuronides, which are target metabolites and time of incubation (2–4 h). b-glucuroni-
in urine and post-mortem blood [42–45] . In dase has been used to release BDZs from their
urine, BDZs are predominantly excreted as conjugates with glucuronic acid [43,48–51] . Urine
glucuronideconjugates. was incubated at 37°C for 1–4 h buffered with
sodium acetate 0.2 M (pH 4.5–5.2) and Helix
„„Metabolism phase II pomatia b-glucuronidase for hydrolysis [47,52] .
Conjugation
The 3-hydroxy substitution of some 1,4- „„Protein removal: blood & plasma treatment
BDZs, such as oxazepam, temazepam or loraz- Protein removal from blood samples can be
epam allows direct conjugation to glucuronic performed by various methods, including
acid, yielding pharmacologically inactive, ultramicrofiltration [11] and equilibrium dialy-
water‑soluble glucuronide conjugates that are sis [53] . Usually, the method used for precipita-
excreted in urine [2,12] . Usually, an enzymatic tion of plasma or serum proteins consists of
hydrolysis is required in order to liberate 1,4- mixing one volume of plasma or serum with
BDZs from their conjugates. three volumes of acid (6% m/v HClO4 [54,55] or
concentrated H3PO4 [56,57]) or organic solvent
Sample preparation (methanol [58,59] , isopropanol [60,61] , acetoni-
The most common samples used for the ana­lysis trile [62–66] , chloroform [67] or acetone [61]) or a
of BDZs are whole blood, urine, serum/plasma mixture of both followed by vortex-mixing and
(human/rat), rat liver and hair (human/rat). In centrifugation, which releases the 1,4-BDZs

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 Benzodiazepines: sample preparation & HPLC methods for their determination | Review
from protein-binding sites, thus removing 99% hydrolyzed. A single‑step extraction involving
of the proteins [68] . Recoveries of the bound 1 ml of sample and 5 ml of organic solvent
portion of drug are dependent upon the nature is sufficient for all BDZs except midazolam,
of the 1,4-BDZs and the precipitation agents oxazepam and lorazepam [2] . However, some
[2] . Another way to release 1,4-BDZs from pro- 1,4-BDZs were subjected to a double extraction
teins without precipitation is the addition of owing to their lower lipid solubility, and the
acids (acetic acid 1 M, formic acid 0.2% or combined extracts are evaporated to dryness
ammonium acetate 26.7 mM plus formic acid before chromatography [86] .
0.97%) that compete with 1,4-BDZs for bind- Although there is a plethora of extraction
ing sites of proteins [11,29,63,69,70] or the addi- solvents used for the extraction of BDZs from
tion of alkyl sulphates such as sodium octylsul- biological fluids, there are no particular refer-
phate, or sodium dodecylsulfate , that disrupt ences employing a specific solvent or combina-
the structure of proteins [13] . tion of solvents. One advantage of low-boiling
solvents is that they can be readily evaporated
„„Treatment of hair: buffer incubation for the recovery of drugs. Diethylether, how-
Acid or alkaline hydrolysis was found to be ever, has the disadvantage of its volatility
unsuitable for extracting the target drugs from and inherently high risk of fire, although it is
the hair matrix, leading to decomposition widely used [42,81,82,87–89] . Usually, a solvent-
into the corresponding benzophenones [33,71] . evaporation step is required after extraction.
Methanol [51,72–75] or ammoniacal metha- Solid-phase extraction was carried out by
nol [76,77] incubation can be used, but poor classical sorbents: octyl or octadecylsilane-
chromatograms with endogenous peaks were bonded cartridges (C8 or C18 ). These are com-
obtained. A series of reports describe methods to monly used for the rapid preparation of blood,
avoid this problem by incubation in buffer, such plasma, serum or urine samples to determine
as Soerensen buffer (pH 7.5) [78–80] , NH4OH
25% [79] , Na 2HPO4 0.5 M (pH 8.5) [49,81,82] ,
NaOH 1 M [79] , proteinase K [79,83] , a mixture CS Others
4% 2%
of b-glucuronidase/arylsulfatase (pH 4.0),
DI
thioglycolate and glycerine [79] . Before incu- 8%
bation, hair samples are washed with hot SPE
water [72–74] , methanol [74,76] or consecutively 34%
by one or more solvents [76] . In most cases,
SPME
dichloromethane [73,74,78,81,82] or sodium 6%
dodecylsulfate 0.1% [13,72,73,77] was used for
washing [72–74,76–78,81,82] .

Extraction techniques
Liquid–liquid extraction is the most widely
used method for the pretreatment of common
biological samples, as shown in Figure 1. BDZs
and their metabolites are usually extracted as
the neutral molecules from biofluids with a
range of organic solvents under weakly alkaline
conditions, with recoveries exceeding 90% [38] .
Some analysts find it unnecessary to alkalize
samples, since the pK values of BDZs are con-
siderably below physiological pH [45,84,85] . The
use of back extraction with aqueous acid solu-
LLE
tions and basifying followed by extraction with
46%
organic solvent [12,26] is also recommended, but
no particular advantage over the use of solvents
alone is achieved. Solvent polarity and pH of
the aqueous phases are the major factors to be Figure 1. Sample-preparation techniques for the determination of
considered. pH should be adjusted to a value at benzodiazepines in biological samples.
LLE: Liquid–liquid extraction; SPME: Solid-phase microextraction.
which the drug is in the neutral form, but not

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Review | Samanidou, Uddin & Papadoyannis
BDZs [12,24,28,90–92] . A sequential SPE protocol divinylbenzene (PDMS/DVB) was proved to
has been developed for the simultaneous deter- be more suitable than carbowax/templated
mination of six BDZs and four tricyclic anti- resin (Carbowax/TPR-100) for the determi-
depressants in biological fluids by HPLC–UV nation of delorazepam in urine by HPLC–UV
using Nexus™ cartridges [93] . Polymeric car- because PDMS/DVB was capable of the most
tridges, such as Oasis™ HLB [94] for 21 BDZs efficient extraction with the same equilibrium
in urine and Abselut Nexus [95] for diazepam, time of approximately 30 min [3] .
flunitrazepam, nitrazepam, oxazepam in serum Automated and direct in-tube extraction of
and urine, offered some advantages: they can several BDZs (clonazepam, diazepam, oxaz-
be used at pH 1–14 and with many different epam, temazepam, nordazepam, 7-aminofl-
polar and apolar organic solvents (e.g., metha- unitrazepam and N-desmethylflunitrazepam)
nol, chloroform and diethylether). Contrary from human serum and urine was applied;
to classical reversed phase (RP) silica extrac- where analytes were extracted from the sample
tion columns, it is easier to find an appropriate directly into an open tubular capillary column
extraction condition for a specific compound equipped with highly biocompatible SPME
and especially for a mixture of analytes with capillary of RAM, ADS [11,24,97] .
different chemical properties, such as polarity, Molecularly imprinted (MI) SPE proto-
pH and affinity [95] . cols for diazepam have been developed by
Solid phase micro extraction was applied Ariffin [77] and Anderson [98] , which could be
using various alkyldiol-silica (ADS) restricted- used as complementary methods to classical
access materials (RAM), as the SPME coating, SPE for the ana­lysis of BDZ in hair samples. As
which was able to simultaneously fractionate the molecularly imprinted polymers (MIP) possess
protein component from a biological sample [96] . a group-selective binding nature they have been
Among two silica fibers, polydimethylsiloxane/ successfully applied to some other BDZ drugs
(diazepam, nordiazepam, nitrazepam, chlordi-
azepoxide, temazepam, oxazepam, lorazepam,
Electro-analytical TLC f lunitrazepam and 7-aminof lunitrazepam).
Photometry 3% 1% The methods detected BDZs at higher con-
3% centrations than classical SPE as the MIP pro-
IMA
5% cedure produced extracts with fewer matrix
interferences than the classical SPE method.
Electro-separation Column switching was employed to elute the
7%
extracted analytes from the precolumn into a
HPLC analytical column. Column switching
was applied to the simultaneous determina-
tion of five frequently prescribed BDZs: clon-
azepam, diazepam, midazolam, oxazepam and
LC flunitrazepam and its main metabolites (nor-
9% f lunitrazepam, 7-amino- and 7-acetamido-
flunitrazepam). The use of a biocompatible
extraction column as the precolumn enabled
repeated direct injection of serum, plasma and
urine supernatant of cell culture or other com-
plex matrices into the HPLC system without
any clean-up procedure [76,99–103] . Application
of monolithic supports to online extraction
GC
11% and LC–MS ana­lysis of BDZs is the first pub-
HPLC lished work dealing with online extraction
61% by column switching on whole blood. Other
column-switching methods are generally
applied to plasma or serum samples [104] . An
automated method for bioequivalence studies
Figure 2. Analytical techniques used for the determination of has been developed using online SPE coupled
1,4-benzodiazepines in human biological fluids. with HPLC–MS/MS for quantification of
ICA: Ischemia modified albumin; TLC: Thin-layer chromatography.
bromazepam in human plasma [65,105] .

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 Benzodiazepines: sample preparation & HPLC methods for their determination | Review

Table 2. Chromatographic conditions for the determination of benzodiazepines by HPLC.

Sample Column Mobile phase LOD Ref.
Alprazolam
Serum, plasma Novapak C18 ACN/MeOH/buffer (30:2:200 v/v/v) 1.5 ng/ml [28]
Tablet Discovery™ C8 KH2PO4 /ACN (43:57 v/v) 1 ng/ml [150]
Tablet Hypersil™ C18 ACN/buffer (55:45 v/v) [171]
Tablet ODS-C18 ACN/buffer (55:45 v/v) 200 ng/ml [170]
Tablet Develosil C8 ACN/buffer (30:70 v/v) 0.5 ng/ml [148]
Blood, urine Inertsil™ C8 CH3COONH4 /CH3OH/ACN (3:57:10 v/v/v) 3 ng/ml [89]
Blood, urine XTerra C18 MeOH/buffer, gradient 3 ng/ml [74]
Plasma, saliva XTerra C18 ACN/0.1% HCOOH gradient 1 ng/ml [39]
Plasma Develosil C8 ACN/buffer (30:70 v/v) 0.5 ng/ml [148]
Plasma C18 column MeOH/buffer (40:60 v/v) 0.05 ng/ml [191]
Plasma RP-18 CN Heptane/EtOH/AA (78:17.6:4.4 v/v/v) 6 ng/ml [154]
Hair X Terra C18 ACN/0.1% HCOOH, gradient 1 pg/mg [81]
Hair X Terra C18 ACN/buffer, gradient 0.4 pg/mg [82]
Larvae Zorbax® SB-phenyl Ternary, gradient 2 pg/g [155]
Drug RP-18 RP-18 ACN/buffer (65:35 v/v) 3 ng/ml [160]
Urine Symmetry® C18 ACN/CH3COONH4, gradient 0.07–0.3 ng/ml [48]
Plasma ChromSep glass, KH2PO4 (pH 4.8)/MeOH/ACN (45:45:10 v/v/v) [168]
Inertsil ODS
Rat hair, plasma Mightysil RP-18 Water/ACN/1% AcOH, gradient 0.04–1.6 ng/ml [13]
Tablet RP-18 RP- 18 Water/ACN (60:40 v/v); acetate/ACN (65:35 v/v) [210]
Tablet Hypersil ODS ACN/phosphate buffer (55:45 v/v) 200 ng/ml [172]
Urine Zorbax SB-C18 0.01% HCOOH in ACN/water (33:67 v/v) [132]
Plasma, urine Kromasil™ C8 CH3OH/CH3COONH4 /CH3CN, gradient 20 ng/ml [202]
Plasma, urine Kromasil C8 CH3OH/CH3COONH4 /CH3CN, gradient 0.08–1.17 ng/µl [93]
Plasma, urine, saliva Kromasil C8 CH3OH/CH3COONH4 /CH3CN, gradient 0.02–0.47 ng/µl [203]

Bromazepam
Serum Hypersil ODS-C18 MeOH/NaH2PO4 (45:55 v/v) 50 ng/ml [124]
Blood, urine Inertsil C8 CH3COONH4 /CH3OH/CH3CN (3:57:10 v/v/v) 3 ng/ml [89]
Plasma, oral fluid XTerra RP-18 ACN/0.1% HCOOH, gradient 0.5 ng/ml [39]
Hair XTerra MS C18 ACN/0.1% HCOOH, gradient 2 pg/mg [81]
Plasma Chromsep 100–103 BDS CH3COONH4 /MeOH (2:8 v/v) 0.4 ng/ml [207]
Plasma Genesis C18 ACN/0.1% TFA (50:50 v/v) 2 ng/ml [187]
Pharmaceutical LiChrospher-100 RP-18 Water/MeOH/triethylamine (70:30:0.1 v/v/v) 100–600 ng/ml [17]
Urine, tablets LiChrospher-100 RP-18 Water/MeOH/triethylamine (70:30:0.1 v/v/v) 300–430 ng/ml [158]
Blood LiChrospher-100 RP-8 Phosphate (pH 6)/ACN (55:45 v/v) 6.5–123 ng/ml [163]
Plasma, urine SB-C18 CH3COONH4 /ACN, gradient [138]
Drugs ODS-C8 column MeOH/ACN/KH2PO4 (pH 6) 125–248 ng/ml [46]
(26.5:21.5:52 v/v/v)
Whole blood Novapak, C18 MeOH/CH3COONH4 (60:40 v/v) 0.05–0.5 ng/ml [18]
Urine Zorbax SB-C18 0.01% HCOOH in ACN/water (33:67 v/v) [109]
Plasma, urine Kromasil C8 CH3OH/CH3COONH4 /CH3CN, gradient 20 ng/ml [181]
Plasma, urine Kromasil C8 CH3OH/CH3COONH4 /CH3CN, gradient 80–117 ng/ml [75]
Plasma, urine, saliva Kromasil C8 CH3OH/CH3COONH4 /CH3CN, gradient 20–470 ng/ml [182]

Brotizolam
Whole blood Novapak C18 MeOH/CH3COONH4 (60:40 v/v) 0.05–0.5 ng/ml [18]
Plasma, urine Shodex MSpak GF-310 4B CH3COONH4 /ACN, gradient 2–5 ng/ml [22]
Urine CAPCELL PAK C18 AcONH4 /ACN, gradient 2–10 ng/ml [39]
Hisep: Hydrophobic shielded phase; LOD: Limit of detection; ODS: Octadecyl silica; TFA: Trifluoroacetic acid.

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Table 2. Chromatographic conditions for the determination of benzodiazepines by HPLC (cont.).

Sample Column Mobile phase LOD Ref.
Clonazepam
Serum, plasma Novapak C18 ACN/MeOH/KH2PO4 (30:2:100 v/v/v) 1.6 ng/ml [28]
Whole blood Novapak C18 MeOH/CH3COONH4 (60:40 v/v) 0.05–00.5 ng/ml [24]
Serum Hypersil ODS® C18 MeOH/NaH2PO4 (45:55 v/v) 0.05 ng/ml [124]
Plasma Jones Genesis® C8 ACN/water/HCOOH (90:9:1 v/v/v) 0.5 ng/ml [149]
Water/HCOOH (99.9:5:0.05 v/v/v)
Urine Symmetry C18 ACN/CH3COONH4, gradient 0.63–63.15 ng/ml [48]
Hair XTerra MS C18 ACN/0.1% HCOOH, gradient 2 pg/mg [81]
Plasma Novapak C18 ACN/acetate (40:60 v/v) 2 ng/ml [123]
Whole blood Chromolith RP-18e Phosphate buffer/ACN (70:30 v/v) 10 ng/ml [4]
Larvae, puparia Zorbax SB-phenyl ACN/MeOH/CH3COONH4, gradient 6.28 pg/mg [155]
Pharmaceutical LiChrospher-100 RP-18 Water/MeOH/triethylamine (70:30:0.1 v/v/v) 100–630 ng/ml [17]
Plasma, urine, saliva LiChrospher-100 RP-8 Acetate buffer/ACN (55:45 v/v) 2.0–14.1 ng/ml [67]
Urine Supelcosil C18 Water/MeOH (52:48 v/v) 46–750 ng/ml [134]
Urine, tablets LiChrospher-100 RP-18 Water/MeOH/triethylamine (70:30:0.1 v/v/v) 300–430 ng/ml [158]
Urine, plasma Supelcosil C18 Water/MeOH (54:46 v/v) 24–52 ng/ml [99]
Plasma Ultrasphere Octyl Acetate buffer/ACN/triethylamine 6 ng/ml [147]
(70:30:0.01 v/v/v)
Human hair LiChrospher Select B Phosphate buffer/ACN, gradient 0.15 ng/mg [76]
Blood LiChrospher-100 RP-8 Phosphate (pH 6)/ACN (55:45 v/v) 6.5–123 ng/ml [163]
Plant tissue Knauer Eurospher RP-18 Water/0.05% TFA/ACN, gradient [5]
Serum C8 LiChrospher Select B Phosphate buffer/ACN (65:35 v/v) 2–3.5 ng/ml [198]
Whole blood LiChrospher Select B KH2PO4 (pH 2.1)/ACN (65:35 v/v) 350 ng/ml [165]
Plasma, urine Kromasil C8 CH3OH/CH3COONH4 /CH3CN, gradient 20 ng/ml [202]
Plasma, urine Kromasil C8 CH3OH/CH3COONH4 /CH3CN, gradient 80–117 ng/ml [93]
Plasma, urine, saliva Kromasil C8 CH3OH/CH3COONH4 /CH3CN, gradient 20–470 ng/ml [203]
Tablet YMC Slimbore C18 Water/MeOH/ACN (40:30:30 v/v/v) 24 ng/ml [143]
Plasma Symmetry C18 ACN/20 mM KH2PO4, gradient 2 ng/ml [121]
Whole blood Chromolith RP-18e HCOONH4 /ACN (65:35 v/v) 1.5 ng/ml [181]
Urine, serum Supelco C18 MeOH/CH3COONH4 (60:40 v/v) 0.02–1.5 ng/ml [11]
Serum C8 LiChrospher Select B 30 mM phosphate/ACN (94:6 v/v) 18–24 ng/ml [102]
tablet HiQ SiL C18 ACN/tetrabutylammonium hydrogen sulfate [139]
(55:45 v/v)
Chlordiazepoxide
Serum Hypersil ODS-C18 MeOH/NaH2PO4 (45:55 v/v) 50 ng/ml [124]
Hair Gemini C18 HCOONH4 /ACN, gradient 0.09–0.14 ng/mg [73,77]
Plasma ChromSep glass, KH2PO4 (pH 4.8)/MeOH/ACN (45:45:10 v/v/v) [168]
Inertsil ODS
Rat hair, plasma Inertsil ODS-2 Phosphate/MeOH (50:50 v/v) 0.01–0.5 ng/mg [79]
Hair Luna C18 Water/ACN/MeOH (30:5:65 v/v/v) 0.12 ng/ml [75]
Plasma, urine SB-C18 CH3COONH4 /ACN, gradient [138]

Clobazam
Hair XTerra MS C18 ACN/0.1% HCOOH, gradient 2 pg/mg [81]
Plasma Chromolith™ Perform Phosphate buffer (pH 3.5)/ACN (70:30 v/v) 2 ng/ml [84]
RP-18e
Blood Symmetry C18 MeOH/water, gradient 1.0 ng/ml [127]
Plasma Hisep column ACN/ammonium acetate (15:85 v/v) 160–500 ng/ml [106]
Serum, urine Supelcosil LC-8-DB CH3CN/water/0.5 M KH2PO4 0.8 ng/ml [179]
Hisep: Hydrophobic shielded phase; LOD: Limit of detection; ODS: Octadecyl silica; TFA: Trifluoroacetic acid.

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 Benzodiazepines: sample preparation & HPLC methods for their determination | Review
Table 2. Chromatographic conditions for the determination of benzodiazepines by HPLC (cont.).
Sample Column Mobile phase LOD Ref.
Clotiazepam
Dietary supplement Wakosil® 5 C18 5 mM heptane sulfonic acid/ACN (13:7 v/v) 50–800 ng/ml [131]
(pH 2.4, HCLO4)
Urine Superiorex ODS, ACN/water (35:65 v/v), gradient 1–6 ng/ml [177]
semimicro
Demoxipam
Urine REMEDi HS system 15 ng/ml [184]

Hair Luna C18 Water/ACN/MeOH (30:5:65 v/v/v) 0.12 ng/ml [75]

Diazepam
Whole blood Novapak C18 MeOH/CH3COONH4 (60:40 v/v) 0.05–0.5 ng/ml [24]
Urine, serum LiChrospher RP-60 MeOH/water/ACN (1:1:1 v/v/v) [91]
Select B
Blood, urine Inertsil C8 CH3COONH4 /CH3OH/CH3CN (3:57:10 v/v/v) 3 ng/ml [89]
Hair Gemini C18 HCOONH4 /ACN, gradient 0.09–0.14 ng/mg [73,77]
Urine Symmetry C18 ACN/CH3COONH4, gradient 6–30 ng/ml [48]
Plasma, oral fluid Xterra RP-18 ACN/0.1% HCOOH, gradient 0.5 ng/ml [39]
Hair Xterra MS C18 ACN/0.1% HCOOH, gradient 2 pg/mg [81]
Dietary supplement Wakosil 5 C18 5 mM heptane sulfonic acid/ACN (13:7 v/v) (pH 500–800 ng/ml [131]
2.4, HClO4)
Plasma XTerra MS C18 74% MeOH/0.1% HCOOH 2 ng/ml [130]
Plasma ChromSep glass, KH2PO4 (pH 4.8) MeOH/ACN (45:45:10 v/v/v) [168]
Inertsil ODS
Whole blood Chromolith RP-18e Phosphate buffer/ACN (70:30 v/v) 10 ng/ml [4]
Blood Supelcosil C18 Water/MeOH (50:50 v/v) 20–35 ng/ml [96]
Larvae, puparia Zorbax SB-phenyl ACN/MeOH/20mM CH3COONH4, gradient 6.28 pg/mg [155]
Tablets Hibar Li-Chrosorb RP-18 MeOH/phosphate buffer (1:1 v/v) [159]
Pharmaceuticals LiChospher-100 RP-18 Water/MeOH/triethylamine (70:30:0.1 v/v/v) 100–630 ng/ml [17]
Plasma, urine, saliva LiChrospher-100 RP-8 Acetate buffer/ACN (55:45 v/v) 2.0–14.1 ng/ml [67]
Urine Supelcosil C18 Water/MeOH (52:48 v/v) 46–750 ng/ml [134]
Urine, tablets LiChrospher-100 RP-18 Water/MeOH/triethylamine (70:30:0.1 v/v/v) 300–430 ng/ml [158]
Serum LiChrospher-100 RP-18e Water/MeOH, gradient 22–29 ng/ml [25]
Tablets Supelcosil LC-18 ACN/water (30:70 v/v), gradient 110 ng/ml [178]
Rat plasma, urine µ-Bondapak C18 MeOH/ACN/water (10:40:50 v/v/v) 20–50 ng/ml [70]
Plasma, urine Kromasil C8 CH3OH/CH3COONH4 /CH3CN, gradient 20 ng/ml [202]
Plasma, urine Kromasil C8 CH3OH/CH3COONH4 /CH3CN, gradient 80–117 ng/ml [93]
Plasma, urine, saliva Kromasil C8 CH3OH/CH3COONH4 /CH3CN, gradient 20–470 ng/ml [203]
Urine, plasma Supelcosil C18 Water/MeOH (54:46 v/v) 24–52 ng/ml [99]
Plasma Ultrasphere C18 ODS 10 mM KH2PO4 /ACN (69:31 v/v) 5.0 ng/ml [64]
Dog plasma Discovery C18 ACN/0.01MCH3COONH4, gradient 0.28 ng/ml [135]
Human hair LiChrospher Select B 20 mM phosphate buffer/ACN, gradient 0.15 ng/mg [76]
Blood LiChrospher-100 RP-8 Phosphate (pH 6)/ACN (55:45 v/v) 6.5–123 ng/ml [163]
Plant tissue Knauer Eurospher RP-18 Water/0.05% TFA/ACN, gradient [5]
Serum C8 LiChrospher Select B Phosphate buffer/ACN (65:35 v/v) 2–3.5 ng/ml [198]
Hair Luna-C18 Water/ACN/MeOH (30:5:65 v/v/v) 0.12 ng/ml [75]
Plasma, urine SB-C18 CH3COONH4 /ACN, gradient [138]
Plasma, urine Hypersil BDS RP-18 MeOH/ACN/KH2PO4 (50:10:40 v/v/v) 4 ng/ml [45]
Urine Superiorex ODS, ACN/water (35:65 v/v), gradient 1–6 ng/ml [177]
semimicro
Hisep: Hydrophobic shielded phase; LOD: Limit of detection; ODS: Octadecyl silica; TFA: Trifluoroacetic acid.

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Table 2. Chromatographic conditions for the determination of benzodiazepines by HPLC (cont.).

Sample Column Mobile phase LOD Ref.
Delorazepam
Serum, urine Symmetry Shield RP-8 ACN/0.1M KH2PO4 (40:60 v/v) [95]
Rat cerebrospinal Aquasil C18 0.1% HCOOH/ACN, gradient 0.04–0.1 ng/ml [66]
fluid
Whole blood Chromolith RP-18e HCOONH4 /ACN (65:35 v/v) 1 ng/ml [181]
Rat plasma, urine µ-Bondapak C18 MeOH/ACN/water (10:40:50 v/v/v) 100 ng/ml [70]
Urine, serum Supelco C18 MeOH/CH3COONH4 (60:40 v/v) 0.02–1.5 ng/ml [11]
Urine Zorbax SB-C18 0.01% HCOOH in ACN/water (33:67 v/v) [132]
Plasma LiChrospher RP-18 ACN/water (45:55 v/v) 1 ng/ml [156]
Urine, hair, oral fluid Gemini C18 0.1% HCOOH/MeOH, gradient 10 ng/ml [51]
Serum LiChrospher-C8 Select B 30 mM phosphate/ACN (94:6 v/v) 18–24 ng/ml [102]
Hair Gemini, Synergi Hydro, Different 0.02–0.09 ng/ml [209]
Zorbax Stablebond-
Phenyl
Plasma Chromolith RP-18e Phosphate/MeOH/ACN (63:10:27 v/v/v) 2 ng/ml [182]
Urine Supelcosil LC-18-DB ACN/water (65:35 v/v) 1–5 ng/ml [3]
Plant tissue Knauer Eurosphere Water/0.05% TFA/ACN, gradient [5]
100 RP-18
Plasma, urine SB-C18 CH3COONH4 /ACN, gradient [138]

Tetrazepam
Plasma, oral fluid Xterra RP-18 ACN/0.1% HCOOH, gradient 0.5 ng/ml [39]
Hair Xterra MS C18 ACN/0.1% HCOOH, gradient 2 pg/mg [81]

Tofisopam
Dietary supplement Wakosil 5 C18 5 mM heptane sulfonic acid/ACN (13:7 v/v) 500–800 ng/ml [131]
(pH 2.4, HClO4)
Serum CAPCELL PAK C18 UG 120 43% ACN/0.1% phosphoric acid in 5 mM sodium 2 ng/ml [103]
octanesulfonate, gradient
Diltiazepam
Drugs RP-8,18 LiChrospher-100 ACN/EtOH/KH2PO4 (25:5:70 v/v/v) 25–75 ng/ml [133]
monolithic
Nucleosil C18 MeOH/HCOONH4 (80:20 v/v)
Estazolam
Serum Hypersil ODS-C18 MeOH/NaH2PO4 (45:55 v/v) 50 ng/ml [124]
Rat hair, plasma Inertsil ODS-2 Phosphate/MeOH (50:50 v/v) 10–500 pg/mg [79]
Rat hair, plasma Mightysil RP-18 Water/ACN 1% AcOH, gradient 0.4–1.6 ng/ml [13]
Plasma Develosil C8-5 0.5% KH2PO4 /ACN (70:30 v/v) 0.5 ng/ml [148]
Serum CAPCELL PAK C18 0.1% acetic acid/40% ACN, gradient 20–80 ng/ml [136]
Plasma, urine SB-C18 CH3COONH4 /ACN, gradient [138]
Urine Zorbax SB-C18 0.01% HCOOH in ACN/water (33:67 v/v) [132]
Urine CAPCELL PAK C18 AcONH4 /ACN, gradient 2–10 ng/ml [50]

Etizolam
Serum Hypersil ODS-C18 MeOH/NaH2PO4 (45:55 v/v) 50 ng/ml [124]
Plasma, urine Shodex MSpak GF-310 4B CH3COONH4 /ACN, gradient 2–5 ng/ml [29]
Urine CAPCELL PAK C18 AcONH4 /ACN, gradient 2–10 ng/ml [50]

Flumazenil
Plasma Spherisorb (ODS) 10 mM ammonium acetate/ACN (75:25 v/v) < 1 ng/ml [169]
Hisep: Hydrophobic shielded phase; LOD: Limit of detection; ODS: Octadecyl silica; TFA: Trifluoroacetic acid.

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 Benzodiazepines: sample preparation & HPLC methods for their determination | Review

Table 2. Chromatographic conditions for the determination of benzodiazepines by HPLC (cont.).

Sample Column Mobile phase LOD Ref.
Flunitrazepam
Plasma R.Sil CN MeOH/KH2PO4 (17:83 v/v) 2.5–10 ng/ml [42]
Whole blood Novapak C18 MeOH/CH3COONH4 (60:40 v/v) 0.05–0.5 ng/ml [24]
Urine, serum LiChrospher RP-60 MeOH/water/ACN (1:1:1 v/v/v) [91]
Select B
Blood, urine Inertsil C8 CH3COONH4 /CH3OH/ACN (3:57:10 v/v/v) 3 ng/ml [89]
Hair Gemini C18 HCOONH4 /ACN, gradient 0.09–0.14 ng/mg [73,77]
Urine Symmetry C18 ACN/CH3COONH4, gradient 0.6–31.0 ng/ml [48]
Blood, urine Synergi RP C18 MeOH/0.1% TFA, gradient 1–5 ng/ml [135]
Plasma, oral fluid Xterra RP-18 ACN/0.1% HCOOH, gradient 0.5 ng/ml [39]
Whole blood Chromolith RP-18e Phosphate buffer -ACN (70:30) 10 ng/ml [4]
Larvae, puparia Zorbax SB-phenyl ACN/MeOH/CH3COONH4, gradient 6.28 pg/mg [155]
Rat hair, plasma Inertsil ODS-2 Phosphate/MeOH (50:50 v/v) 0.01–0.5 ng/mg [79]
Tablets Hibar Li-Chrosorb RP-18 MeOH/phosphate buffer (1:1 v/v) [159]
Urine, plasma Novapak C18 Water/ACN/MeOH (49.5:5.5:45 v/v/v) 0.025–20.20 ng/ml [43]
Plasma, urine, saliva LiChrospher-100 RP-8 Acetate buffer/ACN (55:45 v/v) 2.0–14.1 ng/ml [67]
Plasma Symmetry C18 ACN/0.1% formic acid, gradient 0.25 ng/ml [57]
Human hair LiChrospher Select B Phosphate buffer/ACN, gradient 0.15 ng/mg [76]
Blood LiChrospher-100 RP-8 Phosphate (pH 6)/ACN (55:45 v/v) 6.5–12.3 ng/ml [163]
Serum LiChrospher-C8 Select B Phosphate buffer/ACN (65:35 v/v) 2–3.5 ng/ml [198]
Serum Superspher-100 RP-18 ACN/water (40:60 v/v) 0.19 ng/ml [161]
Whole blood LiChrospher Select B KH2PO4 (pH 2.1)/ACN (65:35 v/v) 350 ng/ml [165]
Hair Luna C18 Water/ACN/MeOH (30:5:65 v/v/v) 125 ng/ml [75]
Serum, urine Superspher RP-18 ACN/50 mM HCOONH4 (45:55 v/v) 0.2–1.0 ng/ml [44]
Urine Superiorex ODS, ACN/water (35:65 v/v), gradient 1–6 ng/ml [177]
semimicro
Urine, plasma ChromSpher C8 MeOH/isopropylamine, gradient 10–25 ng/ml [186]
Serum, urine Symmetry Shield RP-8 ACN/0.1M KH2PO4 (40:60 v/v) [95]
Whole blood Chromolith RP-18e HCOONH4 /ACN (65:35 v/v) 1 ng/ml [181]
Urine, serum Supelco C18 MeOH/CH3COONH4 (60:40 v/v) 0.02–1.5 ng/ml [11]
Urine Shield RP-18 1–3 ng/ml [65]
Urine CAPCELL PAK C18 AcONH4 /ACN, gradient 2–10 ng/ml [50]
Urine LiChrospher 60 Select B ACN/0.05M phosphate (36:64 v/v) 2 ng/ml [101]
Plasma LiChrospher Select B RP-8 ACN/phosphate buffer (35:65 v/v) 12–20 ng/ml [100]
Serum C8 LiChrospher Select B 30 mM phosphate/ACN (94:6 v/v) 18–24 ng/ml [102]
Plasma, urine Kromasill C8 CH3OH/CH3COONH4 /CH3CN, gradient 20 ng/ml [202]
Plasma, urine Kromasill C8 CH3OH/CH3COONH4 /CH3CN, gradient 80–117 ng/ml [93]
Plasma, urine, saliva Kromasill C8 CH3OH/CH3COONH4 /CH3CN, gradient 200–470 ng/ml [203]

Flutazolam
Serum Hypersil ODS-C18 MeOH/NaH2PO4 (45:55 v/v) 50 ng/ml [124]

Flurazepam
Plasma ChromSep, Inertsil ODS KH2PO4 /MeOH/ACN (45:45:10 v/v/v) [168]
Whole blood Chromolith RP-18e Phosphate buffer/ACN (70:30 v/v) 10 ng/ml [4]
Plasma, urine, saliva LiChrospher-100 RP-8 Acetate buffer/ACN (55:45 v/v) 2.0–14.1 ng/ml [67]
Hair Luna C18 Water/ACN/MeOH (30:5:65 v/v/v) 120 ng/ml [75]
Whole blood Chromolith RP-18e HCOONH4 /ACN (65:35 v/v) 1 ng/ml [181]
Urine CAPCELL PAK C18 AcONH4 /ACN, gradient 2–10 ng/ml [50]
Hisep: Hydrophobic shielded phase; LOD: Limit of detection; ODS: Octadecyl silica; TFA: Trifluoroacetic acid.

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Table 2. Chromatographic conditions for the determination of benzodiazepines by HPLC (cont.).

Sample Column Mobile phase LOD Ref.
Haloxazolam
Serum Hypersil ODS-C18 MeOH/NaH2PO4 (45:55 v/v) 50 ng/ml [124]

Imidazenil
Rat plasma RP-18-CN, normal phase N-hexane/ethanol/acetic acid (78:17.6:4.4 v/v/v) 2 ng/ml [154]

Ketazepam
Whole blood Novapak C18 MeOH/CH3COONH4 (60:40 v/v) 0.05–0.5 ng/ml [24]

Loprazolam
Whole blood Novapak C18 MeOH/CH3COONH4 (60:40 v/v) 0.05–0.5 ng/ml [24]

Lorazepam
Serum Hypersil ODS-C18 MeOH/NaH2PO4 (45:55 v/v) 50 ng/ml [124]
Hair Gemini C18 HCOONH4 /ACN, gradient 0.09–0.14 ng/mg [73,77]
Serum Perfectsil ODS-3 NH4H2PO4 (pH 5.8)/MeOH (1:1 v/v) 1 ng/ml [85]
Plasma, oral fluid XTerra RP-18 ACN/0.1% HCOOH, gradient 0.5 ng/ml [39]
Hair XTerra MS C18 ACN/0.1% HCOOH, gradient 2 pg/mg [81]
Plasma Synergi Max phenyl-RP KH2PO4 (pH 2.4)/ACN (65:35 v/v) 2.5–10 ng/ml [62]
Plasma Zorbax Eclipse XDB C18ACN/10mM HCOOH (65:35 v/v) 0.10 ng/ml [107]
Plasma ChromSep, Inertsil ODSKH2PO4 (pH 4.8)/MeOH/ACN (45:45:10 v/v/v) [168]
Whole blood Chromolith RP-18e Phosphate buffer/ACN (70:30 v/v) 10 ng/ml [4]
Larvae, puparia Zorbax SB-phenyl ACN/MeOH/CH3COONH4, gradient 6.28 pg/mg [155]
Plasma Hisep column ACN/ammonium acetate (15:85 v/v) 160–500 ng/ml [106]
Plasma, urine Shodex MSpak GF‑310 4BCH3COONH4 /ACN, gradient 2–5 ng/ml [29]
Plasma, urine, saliva LiChrospher-100 RP-8 Acetate buffer/ACN (55:45 v/v) 2.0–14.1 ng/ml [67]
Blood LiChrospher-100 RP-8 Phosphate (pH 6)/ACN (55:45 v/v) 6.5–123 ng/ml [163]
Hair Luna C18 Water/ACN/MeOH (30:5:65 v/v/v) 120 ng/ml [75]
Urine, oral fluid, hair XTerra C18 ACN/HCOONH4, gradient 0.02–0.5 ng/ml [49]
Whole blood Chromolith RP-18e HCOONH4 /ACN (65:35 v/v) 1 ng/ml [181]
Urine CAPCELL PAK C18 AcONH4 /ACN, gradient 2–10 ng/ml [50]
Rat plasma Cyclobond-I-2000 RSP ACN/1% triethylamineacetate/water 10 ng/ml [111]
(19:8:73 v/v/v)
Plasma Shodex ORpak CDBS-453 ACN/NaCl (13:87 v/v) 1 ng/ml [112]
Plasma, urine Kromasill C8 CH3OH/CH3COONH4 /CH3CN, gradient 20 ng/ml [202]
Plasma, urine Kromasill C8 CH3OH/CH3COONH4 /CH3CN, gradient 80–117 ng/ml [93]
Plasma, urine, saliva Kromasill C8 CH3OH/CH3COONH4 /CH3CN, gradient 0.20–24.7 ng/ml [203]

Lormetazepam
Whole blood Novapak C18 MeOH/CH3COONH4 (60:40 v/v) 0.05–0.5 ng/ml [24]
Plasma, oral fluid Xterra RP-18 ACN/0.1% HCOOH, gradient 0.5 ng/ml [39]
Hair Xterra MS C18 ACN/0.1% HCOOH, gradient 2 pg/mg [81]
Plasma ChromSep, Inertsil ODS KH2PO4 (pH 4.8)/MeOH/ACN (45:45:10 v/v/v) [168]
Urine CAPCELL PAK C18 AcONH4 /ACN, gradient 2–10 ng/ml [50]

Medazepam
Urine, serum LiChrospher RP-60 MeOH/water/ACN (1:1:1 v/v/v) [91]
Select B
Plasma, urine SB-C18 CH3COONH4 /ACN, gradient [138]
Urine Superiorex ODS, ACN/water (35:65 v/v), gradient 1–6 ppb [177]
semimicro
Midazolam
Rat brain Rainin C8 Microsorb MeOH/ACN/0.025M KH2PO4 (33:37:30 v/v/v) 30 ng/mg [145]
Plasma Symmetry C8 ACN/THF/phosphate (35:5:60 v/v/v) 4 ng/ml [26]
Hisep: Hydrophobic shielded phase; LOD: Limit of detection; ODS: Octadecyl silica; TFA: Trifluoroacetic acid.

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Table 2. Chromatographic conditions for the determination of benzodiazepines by HPLC (cont.).

Sample Column Mobile phase LOD Ref.
Midazolam (cont.)
Rat serum Symmetry C18 MeOH/ACN/CH3COONa (10:23:67 v/v/v) 4 ng/ml [87]
Plasma, oral fluid Xterra RP-18 ACN/0.1% HCOOH, gradient 0.5 ng/ml [39]
Hair Xterra MS C18 ACN/0.1% HCOOH, gradient 2 pg/mg [81]
Plasma HyPURITY Elite C18 Water/ACN (75:25 v/v), 0.1% HCOOH 2 ng/ml [142]
Plasma Symmetry Shield RP-8 NH4OH/MeOH (35:65 v/v) 0.002–0.15 ng/ml [58]
Plasma, saliva Xterra1 RP-18 ACN/HCOOH, gradient 0.1–0.5 ng/ml [162]
Dog plasma Hypersil 100 C18 MeOH/water/acetate buffer 0.1 ng/ml [56]
Plasma STR ODS-2 HClO4 /ACN/phosphate (57.9:0.1:42 v/v/v) 0.2 ng/ml [189]
Plasma STR ODS-C18 KH2PO4 buffer (pH 4.6)/HClO4 (60%)/acetonitrile 0.2 ng/ml [172]
(58.9:0.1:41 v/v/v)
Whole blood Chromolith RP-18e Phosphate buffer/ACN (70:30 v/v) 10 ng/ml [4]
Plasma Symmetry C8 0.1 M KH2PO4 /ACN (70:30 v/v) 900 ng/ml [146]
Rat hair, plasma Mightysil RP-18 Water/ACN 1% AcOH, gradient 1.6 ng/ml [13]
Plasma C18 Novapak ACN/phosphate buffer, gradient [100]
Human hair LiChrospher Select B 20 mM phosphate buffer/ACN, gradient 0.15 ng/mg [76]
Blood LiChrospher-100 RP-8 Phosphate (pH 6)/ACN (55:45 v/v) 6.5–123 ng/ml [163]
Serum LiChrospher-C8 Select B Phosphate buffer (pH 2.1)/ACN (65:35 v/v) 2–3.5 ng/ml [198]
Whole blood LiChrospher Select B KH2PO4 (pH 2.1)/ACN (65:35 v/v) 350 ng/ml [165]
Plasma Spherisorb silica MeOH/0.02% perchloric acid (30:70 v/v) 3 ng/ml [188]
Plasma Ultrasphere ODS ACN/MeOH/acetate buffer (35:5:60 v/v/v) 2.4 ng/ml [173]
Plasma Hypersil C18 BDS 10 mM Na2HPO4 /ACN, gradient 0.4 ng/ml [144]
Plasma Fused-silica, Kromasil ACN/MeOH/CH3COONH4 /CH3COOH (37.5:37.5 0.5–2 ng/ml [54]
RP-18 :25:1 v/v/v)
Serum Cyanopropyl ACN/CH3COONa, 0.02 M (80:20 v/v) 3 ng/ml [153]

Plasma YMC-Pak Pro C18 CH3COONH4 /MeOH (1:1 v/v) 26.3 pg/mg [205]
Plasma Luna C18 ACN/CH3COONH4 (52:48 v/v) 0.65–60.68 ng/ml [86]
Whole blood Chromolith RP-18e HCOONH4 /ACN (65:35 v/v) 1 ng/ml [181]
Plasma, oral fluid Luna C18 0.1% CH3COOH/ACN, gradient 0.025 ng/ml [137]
Plasma YMC-Pack Pro C18 CH3COONH4 /MeOH (50:50 v/v) 0.1 ng/ml [59]
Urine CAPCELL PAK C18 AcONH4 /ACN, gradient 2–10 ng/ml [50]
Serum C8 LiChrospher Select B 30 mM phosphate/ACN (94:6 v/v) 18–24 ng/ml [102]

Nitrazepam
Serum, plasma Novapak C18 ACN/MeOH/KH2PO4 (30:2:100 v/v/v) 1.5 ng/ml [28]
Whole blood Novapak C18 MeOH/CH3COONH4 (60:40 v/v) 0.05–00.5 ng/ml [24]
Urine, serum LiChrospher RP-60 MeOH/water/ACN (1:1:1 v/v/v) [91]
Select B
Serum Hypersil ODS-C18 MeOH/NaH2PO4 (45:55 v/v) 50 ng/ml [124]
Hair Gemini C18 HCOONH4 /ACN, gradient 0.09–0.14 ng/mg [73,77]
Urine Symmetry C18 ACN/CH3COONH4, gradient 0.60–30 ng/ml [48]
Serum Hypersil C18 MeOH/acetate buffer (60:40 v/v) 5 ng/ml [1]
Dietary supplement Wakosil 5 C18 5 mM heptane sulfonic acid/ACN (13:7 v/v) 500–800 ng/ml [131]
(pH 2.4, HClO4)
Tablets Hibar LiChrospher RP-18 MeOH/phosphate buffer (1:1 v/v) [159]
Plasma Hisep column ACN/ammonium acetate (15:85 v/v) 160–500 ng/ml [106]
Plasma, urine, saliva LiChrospher-100 RP-8 Acetate buffer/ACN (55:45 v/v) 2.0–14.1 ng/ml [67]
Blood LiChrospher-100 RP-8 Phosphate (pH 6)/ACN (55:45 v/v) 6.5–123 ng/ml [163]
Hisep: Hydrophobic shielded phase; LOD: Limit of detection; ODS: Octadecyl silica; TFA: Trifluoroacetic acid.

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Review | Samanidou, Uddin & Papadoyannis

Table 2. Chromatographic conditions for the determination of benzodiazepines by HPLC (cont.).

Sample Column Mobile phase LOD Ref.
Nitrazepam (cont.)
Hair Luna C18 Water/ACN/MeOH (30:5:65 v/v/v) 0.12 ng/ml [75]
Urine Superiorex ODS, ACN/water (35:65 v/v), gradient 1–6 ng/ml [177]
semimicro
Serum, urine Symmetry Shield RP-8 ACN/0.1M KH2PO4 (40:60 v/v/v) [95]
Urine CAPCELL PAK C18 AcONH4 /ACN, gradient 2–10 ng/ml [50]
Rat plasma, brain Develosil ODS-5 Water/ACN/MeOH (60:38:2 v/v/v) 2 ng/ml [53]
microdialyate
Oxazepam
Hair Xterra MS C18 ACN/0.1% HCOOH, gradient 2 pg/mg [81]
Urine Symmetry C18 ACN/CH3COONH4, gradient 6–30 ng/ml [48]
Dietary supplement Wakosil 5 C18 5 mM heptane sulfonic acid:ACN (13:7 v/v) 500–800 ng/ml [131]
(pH 2.4, HClO4)
Plasma XTerra MS C18 74% MeOH/0.1% HCOOH 2 ng/ml [130]
Whole blood Chromolith RP-18e Phosphate buffer/ACN (70:30 v/v) 10 ng/ml [4]
Blood Supelcosil C18 Water/MeOH (50:50 v/v) 20–35 ng/ml [96]
Larvae, puparia Zorbax SB-phenyl ACN/MeOH/CH3COONH4, gradient 6.28 pg/mg [155]
Plasma Hisep column ACN/ammonium acetate (15:85 v/v) 160–500 ng/ml [106]
Plasma, urine, saliva LiChrospher-100 RP-8 Acetate buffer/ACN (55:45 v/v) 2.0–14.1 ng/ml [67]
Urine Supelcosil C18 Water/MeOH (52:48 v/v) 46–750 ng/ml [134]
Serum LiChrospher-100 RP-18e Water/MeOH, gradient 22–29 ng/ml [25]
Urine, plasma Supelcosil C18 Water/MeOH (54:46 v/v) 24–52 ng/ml [99]
Dog plasma Discovery C18 ACN/0.01MCH3COONH4, gradient 0.12 ng/ml [135]
Human hair LiChrospher Select B Phosphate buffer/ACN, gradient 0.15 ng/mg [76]
Plasma m-Bondapack C18 MeOH/phosphate buffer (50:50 v/v) 10 ng/ml [55]
Blood LiChrospher-100 RP-8 Phosphate (pH 6)/ACN (55:45 v/v) 6.5–123 ng/ml [163]
Serum C8 LiChrospher Select B Phosphate buffer (pH 2.1)/ACN (65:35 v/v) 2–3.5 ng/ml [198]
Whole blood LiChrospher Select B KH2PO4 (pH 2.1)/ACN (65:35 v/v) 350 ng/ml [165]
Hair Luna C18 Water/ACN/MeOH (30:5:65 v/v/v) 120 ng/ml [75]
Plasma, urine SB-C18 CH3COONH4 /ACN, gradient [138]
Plasma, urine Hypersil BDS RP-18 MeOH/ACN/KH2PO4, 0.05 M (50:10:40 v/v/v) 4 ng/ml [45]
Serum, urine Symmetry Shield RP-8 ACN/0.1 M KH2PO4 (40:60 v/v) [95]
Rat cerebrospinal Aquasil C18 0.1% HCOOH/ACN, gradient 0.04–0.1 ng/ml [66]
fluid
Whole blood Chromolith RP-18e HCOONH4 /ACN (65:35 v/v) 1 ng/ml [181]
Urine, serum Supelco C18 MeOH/CH3COONH4 (60:40 v/v) 0.02–1.5 ng/ml [11]
Serum LiChrospher-C8 Select B 30 mM phosphate/ACN (94:6 v/v) 18–24 ng/ml [102]
Rat plasma Cyclobond-I-2000 RSP ACN/1% triethylamineacetate/water 10 ng/ml [111]
(19:8:73 v/v/v)
Oxazolam
Serum Hypersil ODS-C18 MeOH/NaH2PO4 (45:55 v/v) 50 ng/ml [124]
Dietary supplement Wakosil 5 C18 5 mM heptane sulfonic acid/ACN (13:7 v/v) 500–800 ng/ml [131]
(pH 2.4, HClO4)
Prazepam
Plasma XTerra MS C18 74% MeOH/0.1% HCOOH 2 ng/ml [130]
Larvae, puparia Zorbax SB-phenyl ACN/MeOH/CH3COONH4, gradient, 6.28 pg/mg [155]

Triazolam
Whole blood Novapak C18 MeOH/CH3COONH4 (60:40 v/v) 0.05–0.5 ng/ml [24]
Serum Hypersil ODS-C18 MeOH/NaH2PO4 (45:55 v/v) 50 ng/ml [124]
Hisep: Hydrophobic shielded phase; LOD: Limit of detection; ODS: Octadecyl silica; TFA: Trifluoroacetic acid.

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 Benzodiazepines: sample preparation & HPLC methods for their determination | Review

Table 2. Chromatographic conditions for the determination of benzodiazepines by HPLC (cont.).

Sample Column Mobile phase LOD Ref.
Triazolam (cont.)
Plasma MS pakGF polymer CH3COONH4 /ACN, gradient 0.1 ng/ml [69]
Plasma, oral fluid Xterra RP-18 ACN/0.1% HCOOH, gradient 0.5 ng/ml [39]
Hair Xterra MS C18 ACN/0.1% HCOOH, gradient 2 pg/mg [81]
Dietary supplement Wakosil 5 C18 5 mM heptane sulfonic acid/ACN (13:7 v/v) (pH 500–800 ng/ml [131]
2.4, HCLO4)
Larvae, puparia Zorbax SB-phenyl ACN/MeOH/CH3COONH4, gradient, 6.28 pg/mg [155]
Rat hair, plasma Inertsil ODS-2 Phosphate/MeOH (50:50 v/v) 0.01–0.5 ng/mg [79]
Rat plasma, brain Develosil ODS-5 Water/ACN/MeOH (60:38:2 v/v/v) 2.1–0.7 ng/ml [60]
Rat hair, Mightysil RP-18 Water/ACN/1% AcOH, gradient [72]
human hair
Serum CAPCELL PAK C18 0.1% acetic acid/40% ACN, gradient 20–80 ng/ml [136]
Plasma, urine SB-C18 CH3COONH4 /ACN, gradient [138]
Hair-shafts, root Mightysil RP-18 Water/ACN/AcOH, gradient [72]
Rat hair 3‑µm micro ACN/1% CH3COOH, gradient [190]
Urine Zorbax SB-C18 0.01% HCOOH in ACN/water (33:67 v/v) [132]
Rat plasma, brain Develosil ODS-5 Water/ACN/MeOH (60:38:2 v/v/v) 2 ng/ml [53]
microdialyate
Plasma Thermo C18 ACN/water/HCOOH (35:65:0.2 v/v/v) 0.02 ng/ml [145]

Temazepam
Hair Gemini C18 HCOONH4 /ACN, gradient 0.09–0.14 ng/mg [73,77]
Hair XTerra MS C18 ACN/0.1% HCOOH, gradient 2 pg/mg [81]
Plasma ChromSep glass, KH2PO4 /MeOH/ACN (45:45:10 v/v/v) [168]
Inertsil ODS
Urine Supelcosil C18 Water/MeOH (52:48 v/v) 46–750 ng/ml [134]
Blood Supelcosil C18 Water/MeOH (50:50 v/v) 20–35 ng/ml [96]
Larvae, puparia Zorbax SB-phenyl ACN/MeOH/CH3COONH4; gradient, 6.28 pg/mg [155]
Serum LiChrospher-100 RP-18e Water/MeOH, gradient 22–29 ng/ml [25]
Rat plasma, urine µ-Bondapak C18 MeOH/ACN/water (10:40:50 v/v/v) 20–50 ng/ml [70]
Urine, plasma Supelcosil C18 Water/MeOH (54:46 v/v) 24–52 ng/ml [99]
Dog plasma Discovery C18 ACN/0.01 M CH3COONH4, gradient 0.12 ng/ml [135]
Plant tissue Knauer Eurospher RP-18 Water/0.05% TFA/ACN, gradient [5]
Hair Luna C18 Water/ACN/MeOH (30:5:65 v/v/v) 120 ng/ml [75]
Plasma, urine Hypersil BDS RP-18 MeOH/ACN/KH2PO4 (50:10:40 v/v/v) 4 ng/ml [45]
Rat cerebrospinal Aquasil C18 0.1% HCOOH/ACN, gradient 0.04–0.1 ng/ml [66]
fluid
Rat plasma, urine µ-Bondapak C18 MeOH/ACN/water (10:40:50 v/v/v) 100 ng/ml [70]
Urine, serum Supelco C18 MeOH/CH3COONH4 (60:40 v/v) 0.02–1.5 ng/ml [11]
Rat plasma Cyclobond-I-2000 RSP ACN/1% triethylamineacetate/water 10 ng/ml [111]
(19:8:73 v/v/v)
Hisep: Hydrophobic shielded phase; LOD: Limit of detection; ODS: Octadecyl silica; TFA: Trifluoroacetic acid.

Nonextraction techniques used for BDZ Analytical techniques

involve dialysis [53] , where no sample pretreatment „„General techniques
is required as clean chromatograms can often be Broad classification of techniques employed for
obtained. Direct injection after protein precipita- the determination of BDZs include:
tion and filtration has also been applied [94,106– n LC ([including HPLC, UPLC, MLC and
108] . However, direct injection of complex samples
chiral separation], GC and TLC);
leads to contamination of chromatographic col-
umns, impairing their performance. To avoid n Capillary electro-separation (MEKC, CEC
these problems, sample clean-up is required. and CE);

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Review | Samanidou, Uddin & Papadoyannis
n Immunoassay; oxazepam, lorazepam, temazpeam, lormetaz-
n Photometry (UV spectrometry and Fourier epam [90,110–120] . HPLC columns and mobile
transform infrared spectroscopy); phases used are summarized in Table 2 .
n E le c t ro - a n a ly t ic a l (p otent iome t r ic , HPLC conditions: column, mobile phase &
voltammetric and polarographic) methods. detection
Distribution of their applications is illustrated Column
in Figure 2. Although columns containing silica, anion-
exchanger, cation-exchanger, Durapak OPN and
„„Chromatography Carbowax 400-coated support have been widely
Following an initial immunoassay or other used for BDZ separation from biological samples
screening test, chromatographic methods can with UV detection since the early days of HPLC
either be used for confirming the presence of one in the 1970–1980s, these methods are not sensitive
or more BDZs or their quantitation in biologi- and selective enough to analyze nanogram levels
cal samples such as blood, plasma/serum, urine of BDZs and their metabolites [2] . RP columns
and hair. Due to the complexity of biological have become most popular because they require
samples, a chromatographic separation step is relatively little maintenance, perform equally well
required for the ana­lysis of drugs in such sam- with eluent gradients, facilitate injection of aque-
ples. TLC is a valuable technique as an initial ous samples and a wide variety of drugs of differ-
screening method to narrow the possible iden- ent polarity can be separated [38] . The packing
tities of unknown drugs in biological samples. materials used for RP columns for BDZ ana­lysis
However, it is relatively nonspecific, time-con- are chemically bonded silica containing octyl
suming and provides only semiquantitative data. (C8), octadecyl (C18), cyano (CN) and phenyl
GC methods offer excellent sensitivity, but pos- groups. The high-purity synthetic silica-based
sess some drawbacks over HPLC, such as lengthy columns resolve the problems of peak broaden-
cleanup procedures and, in some cases,formation ing encountered from the silanol interactions with
of more volatile derivatives or hydrolysis prior basic compounds. It has been shown that the use
to ana­lysis [42] . Furthermore, the high tempera- of a synthetic silica-based stationary phase mark-
tures required to elute can lead to on-column edly improved the resolution of clonazepam in
decomposition of certain BDZs [109] . For these plasma compared with classic RP columns [121] .
reasons; attention has shifted to the develop- A large number of HPLC methods for the
ment of HPLC methods for the determination separation and determination of BDZs from bio-
of these drugs in body fluids [22] . HPLC offers logical samples (plasma, serum, oral fluid, urine,
several advantages such as the relatively simple hair and cerebrospinal fluid) have been reported
extraction procedures used and the fact that using RP C18 columns, such as:
derivatization is not usually necessary, if UV/
n Novapak for flunitrazepam and its metabolites
HPLC or single-wavelength detectors are used
in urine [43] , alprazolani, clonazepam and
for detection. Operation at ambient temperature
nitrazepam in human serum or plasma [28] ,
allows the determination of nonvolatile, polar,
brotizolam, clonazepam, desmethyl fluni-
high mass molecules or thermally labile BDZs
trazepam, diazepam, flunitrazepam, keta-
for which GC is not applicable. The strong
zolam, loprazolam, lormetazepam, nitrazepam
absorption in the 230–260-nm region gives
and triazolam in whole blood [24] , midazolam
sensitivity in the nanogram range and linearity
in plasma [122] , and clonazepam in human
over a wide concentration range. Moreover, if the
plasma [123] ;
detection system is not destructive, the eluted
drugs can be recovered for further examination n Hypersil for frequently-used BDZs in human
[22,30] . Therefore, HPLC offers an attractive serum [124] , and nitrazepam in serum [61] ;
analytical alternative for the routine determi-
nation of 1,4-BDZs in biological samples. It is n Gemini molecularly imprinted solid phase
recognized that biological activity of many drugs extraction (MISPE) for 7-aminoflunitrazepam,
is strongly related to chirality, hence the study flunitrazepam, oxazepam, lorazepam, chlor­
of their configurational stability is important diazepoxide, temazepam, diazepam, nor-
[110] . Chiral HPLC is a well-established area of diazepam, nitrazepam in hair [73] , diazepam
bioanalytical chemistry and is often used for in urine, hair, oral fluid [51] , and diazepam and
enantioseparations of selected BDZs: diazepam, its metabolites in hair [77] ;

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 Benzodiazepines: sample preparation & HPLC methods for their determination | Review
n Atlantis d for diazepam in plasma of rat and n CAPCELL PAK for estazolam and triazolam
rabbits [125], and alprazolam, bromazepam, clon- in serum [136] ;
azepam, 7-aminoclonazepam, diazepam, N-des- n Aquasil for diazepam and its major metabolites
methyldiazepam, 3-OH-diazepam, fenazepam,
in rat cerebrospinal fluid [66] ;
f lunitrazepam, 7-aminof lunitrazepam,
lorazepam, nitrazepam, 7-aminonitrazepam, n Luna for diazepam, desmethyldiazepam,
oxazepam in oral fluid [126]; oxazepam and temazepam in human hair [75] ,
midazolam and its hydroxyl metabolites in
n Symmetry for 7-aminonitrazepam, 7-amino- human plasma and oral fluid [137] ;
clonazepam, 7-aminoflunitrazepam, alpra-
zolam, a-hydroxy alprazolam, oxazepam, n SB-(stable bond) for oxazolam, chlordiazep­
3-OH-diazepam and N-desmethyldiazepam oxide, carbamazepine, medazepam, esta-
in human urine [48] , flunitrazepam and its z ola m, nor f lud ia z epa m, t ria z ola m,
metabolites in human plasma [57] , clobazam delorazepam and bromzepam in plasma,
in whole blood [127] , midazolam in rat bile [128] ; urine [138] ;
n RP-Synergi for flunitrazepam and its metabo- n Nucleosil for diltiazem in drugs [133] ;
lite (7-aminoflunitrazepam) in blood and n HiQ SiL for escitalopram, clonazepam in
urine [129] ; combined tablet dosage form [139] ;
n XTerra for diazepam, nordiazepam, temazepam, n Thermo for triazolam and its metabolites in
oxazepam, 7-aminoflunitrazepam, N-desmeth- human plasma [140] .
ylflunitrazepam and clonazepam in urine and
XTerra MS C18 column has been used in LC–
serum samples [11] , lorazepam in urine, hair,
MS/MS methods for a large number of BDZs in
oral fluid [49] , alprazolam, 7-aminoclonazepam,
human blood, plasma, urine [74,94,141] . C18 analyti-
7-aminoflunitrazepam, bromazepam, cloba-
cal columns, Symmetry for rat serum [87], Luna for
zam, diazepam, lorazepam, lormetazepam,
rat plasma [88] , Aquasil [86] , HyPURITY Elite [142]
midazolam, nordiazepam, oxazepam,
and YMC-Pak Pro [59] for human plasma and
temazepam, tetrazepam, triazolam in hair [81] ,
Hypersil 100 for dog plasma [56] have been used to
alprazolam in hair [82] , and prazepam and its
separate midazolam and its metabolites. Separation
metabolites in human plasma [130] ;
was also performed on C18 3-µm capillary column
n Wakosil for oxazolam, nitrazepam, oxazepam, Hypersil BDS (0.8 ×150 mm) for midazolam and
tofisopam, triazolam, clotiazepam and its metabolite, 1-hydroxy midazolam in human
diazepam in dietary supplements [131] ; plasma by HPLC–UV [129] and on Atlantis (2.1
× 50 mm) for 32 drugs, including some BDZs, in
n Zorbax for lorazepam in human plasma [107] , oral fluid by LC–MS/MS [126] methods. Knauer
bromazepam, carbamazepine, estazolam, nor- Eurospher 100 C18 column (100 × 2.0 mm,
fludiazepam, alprazolam and triazolam in 5 µm) was used to investigate radioligand recep-
human urine [132] , and diltiazem in drugs [133] ; tor binding to central benzodiazepin receptor for
n Uptisphere ODB for alprazolam, bromazepam, clonazepam, delorazepam, diazepam and temaze-
clonazepam, diazepam, f lunitrazepam, pam in brain membrane by HPLC–ESI–MS/
lorazepam, midazolam, nordiazepam in MS [5] . Among the four C18 columns studied,
hair [78] , bromazepam, clonazepam and Phenomenex Bondex 10 (300 × 3.9 mm; 10 µm),
metabolites in urine, hair [80] ; Micra Scientific NPS octadecyl silica (ODS) (33 ×
4.6 mm; 1.5 µm), YMC Slimbore (150 × 3.0 mm;
n Supelcosil for clonazepam, oxazepam, 3.0 µm) and Alltech Solvent Miser (150 × 2.1 mm;
temazepam, nordazepam and diazepam in 5.0 µm), the reproducibility of assay results on the
urine [99,134] ; smallbore column was excellent for the determina-
tion of clonazepam in a commercial tablet dosage
n µ-Bondapak for oxazepam in human
form [143] . A RP chromatographic C18 column,
plasma [55] , and diazepam and its metabolites
TSK gel Super-ODS (100 × 4.6 mm, 2 µm), based
N-desmethyldiazepam and temazepam in rat
on silica gel has been reported for the simultane-
plasma and urine [70] ;
ous determination of 12 BDZs in human serum,
n Discovery for diazepam and its metabolites achieving better resolution and faster separation
N-desmethyldiazepam and temazepam in dog than that of conventional Hypersil ODS (100 ×
plasma [135] ; 4.6 mm, 5 µm) column [124] . Midazolam and its

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Review | Samanidou, Uddin & Papadoyannis
metabolite, 1-hydroxymidazolam, were deter- Lee et al. separated triazolam, etizolam, bro-
mined in human plasma by capillary HPLC tizolam and lorazepam from human plasma and
using Hypersil C BDS 3-µm capillary column urine samples with an MSpak GF polymer col-
(150 × 30.8 mm) [144] . umn (50 × 4.6 mm, 6 µm) [29,69] . Column was
C8 analytical columns have been used in a suited to eliminating proteins, nucleic acids and
number of HPLC methods for separation and polysaccharides from biological samples, because
quantitation of BDZs in biofluids. The separa- their molecular size is too large to enter the pores
tion of midazolam and its major metabolites was of the stationary phase, whereas drugs with small
carried out using a RP system, with a C8 Rainin molecular size can enter and be retained at the
Microsorb column in rat brain ana­lysis [145] and polyvinyl alcohol phase.
with Symmetry in plasma ana­lysis [26,146] . The A series of HPLC–UV methods have been
HPLC system was equipped with a C8 analytical reported for quantification of BDZs: bro-
column, Ultrasphere for clobazam [147] , Develosil mazepam, clonazepam nitrazepam, f luni-
for estazolam [148] , ChromSpher for flunitraz- trazepam, nordiazepam, diazepam and its
epam and its metabolites [24,42] and Inertsil for major metab­olites as well as photodegradation
four other BDZs’ [89] determination in biological products of alprazolam, in pure forms, tab-
fluids (urine and plasma). X-Terra MS C8 column let formulations and biological fluids; plasma
(100 × 2.1 mm, intradermal 3.5 µm) was used for and urine, using RP-18 columns, such as
the simultaneous determination of diazepam with LiChrospher [17,18,156–158] , RP-18 [159,160] and
avizafone, atropine and pralidoxime in human Hypersil BDS [45] . Other RP-18 columns such
plasma [108] . as Mightysil for alprazolam, estazolam and
Clonazepam in human plasma was deter- midazolam and their metabolites in rat hair
mined using Jones Genesis [149] , while degrada- and plasma [13] and triazolam and its hydroxy
tion product of alprazolam, 7-chloro-1-methyl-5- metabolites in hair shaft and hair root [72] ,
phenyl-[1,2,4]triazolo[4,3-a]quinolin-4-amine or Superspher for flunitrazepam and its metabo-
triazolaminoquinoleine, in tablets was identified lites in blood [44] and flunitrazepam in human
and quantified using the ODS Hypersil [150] col- serum [161] , Kromasil for midazolam and its
umn. LC–MS-(time of flight [TOF]) procedure three metabolites in human plasma [54] , X-terra
was reported for the screening of 22 BDZs in for midazolam, bromazepam, tetrazepam,
human urine, where separation was achieved on a alprazolam, lorazepam, triazolam, flunitraz-
Luna column [151] . Alprazolam, chlordiazepoxide, epam, diazepam and lormetazepam in plasma
diazepam, flurazepam, lorazepam, nitrazepam and oral fluid [39] and midazolam in human
and nordiazepam as bulk powders and pharma- plasma and saliva [162] , Shield for midazolam
ceutical formulations were investigated using and hydroxy-metabolites in plasma [58] and
Perfectsil Target ODS-3(125 × 4 mm, internal flunitrazepam and 7-aminoflunitrazepam in
diameter 5 µm) column [152] . human urine [65] and LiChrospher RP select B
Two HPLC–UV reports for serum are avail- for diazepam, N-desmethyldiazepam, nitraz-
able, one for the monitoring of midazolam using epam, flunitrazepam and medazepam in human
cyanopropyl precolumn [153] , Nucleosil CN serum and urine [91] were used for their iden-
connected to Intersphere CN column, and the tification and determination by LC–MS or
other for quantitative ana­lysis of flunitrazepam LC–MS/MS techniques.
and metabolites using RSil CN column [42] . A A LiChrospher RP-8 column was used [67,72,163]
normal-phase RP-18-CN column (250 × 4 mm, for the determination of common BDZs
5 µm) was used to separate a BDZ against imida- (i.e., triazolam and their hydroxy metabolites)
zenil extracted from a plasma sample after parti- in forensic samples like hair. Determination
tioning with diethyl ether [154] . of diazepam, flunitrazepam, nitrazepam and
Reversed phase phenyl-bonded columns used oxazepam in serum and urine, using Symmetry
include Zorbax SB [155] for the simultaneous Shield [95] or identification and quantification of
quantitation of ten BDZs in Calliphora vicina lar- 23 BDZs in plasma, using Superspher RP Select
vae and puparia by LC–MS/MS and Synergi Max B column have been reported [164] . Semi-micro
RP [62] , for the determination of lorazepam in LiChrospher Select B (125 × 3 mm, 5-µm) col-
human plasma by HPLC–UV method. Genesis umn was used for the determination of clonaz-
Phenyl-120 (150 × 2.1 mm, 4-mm) column was epam, diazepam, flunitrazepam, midazolam and
used for the determination of diazepam in urine oxazepam in human hair samples [76] . The deter-
by LC–MS–ESI [47] . mination of BDZs in urine, plasma or serum was

772 Bioanalysis (2009) 1(4) future science group

 Benzodiazepines: sample preparation & HPLC methods for their determination | Review
performed using column-switching technique 2,3-BDZs and their metabolites in rat plasma.
and separating them by LiChrospher RP-18 HPLC–UV methods to analyze BDZs in human
ADS (25 × 4 mm) [101] , LiChrospher Select B urine using semimicro column, Superiorex
(125 × 3 mm, 5 µm) [100,102] and C18 (a silicone (250 × 1.5 mm, 5 µm) [177] , triazolam in rat
polymer coated silica-gel-based) CAPCELL PAK plasma [60] and brain microdialysate [53] using
UG 120 (150 × 1.5 mm, 5-µm) [76] or CAPCELL Develosil ODS‑5, have been described.
PAK UG 120 (250 × 1.5 mm, 5-µm) [103] col- Delorazepam in spiked human urine samples [3],
umn. RP LiChrospher-100 RP-18e (150 × 4 mm, diazepam in multicomponent tablet formulations
5 µm) was equipped to LiChrospher-100 RP-18e [178] and clobazam and N-desmethylclobazam in
(4 cm × 4 mm, 5-µm) guard column for the human serum and urine [179] were determined by
determination of several BDZs in human serum HPLC–UV using Supelcosil LC-18-DB column
by in-tube extraction through ADS-SPME (250 × 4.6 mm, 5 µm). LC–ESI–MS was used for
capillary [25] . the simultaneous determination of seven BDZs;
Photostability of alprazolam in tablets was diazepam, nordiazepam, temazepam, oxazepam,
studied after isolation and structural elucida- 7-aminoflunitrazepam, N-desmethyl flunitraz-
tion of degradation products using an RP-8 epam and clonazepam in urine and serum sam-
column, RP-18 (5 µm, 200 × 4.6 mm) [160] . ples by automated in-tube SPME connected to a
LiChrospher Select B (125 × 3 mm, 5 µm) was Supelco LC-18 column [97] .
used for monitoring the stability of BDZs in Most of conventional HPLC methods are
whole blood stored at varying temperatures [165] . time consuming because of the low flow-rates
The effect of formaldehyde on stability on ten employed due to high back pressure. The use of a
BDZs alprazolam, chlordiazepoxide, diazepam, monolithic column may be an alternative to this
flunitrazepam, flurazepam, lorazepam, mid- problem to achieve faster separation at elevated
azolam, oxazepam, prazepam and triazolam was flow rates without generating a high back pres-
evaluated using the C18 colum, YMC-Pack Pro sure and without any loss of chromatographic
(2 × 100 mm) [166] . The degradation of hydroly­ properties in comparison to conventional col-
tically labile drugs; flunitrazepam, temazepam, umns [180] . The ChromolithTM is a monolithic
oxazepam, lorazepam, nitrazepam, diazepam HPLC column comprised of a silica rod with
and cocaine in blood spots on filter paper during a biporous structure made up of macropores
a storage period of 1 month was studied using (2 µm) and mesopores (13 nm), which offers
the Gemini C18 column (150 × 2 mm, internal a high porosity (80%) compared with usual
diameter 5 µm) [167] . columns. Macropores form a dense network
The use of a RP ODS-column such as through which the mobile phase can rapidly flow
Ultrasphere for diazepam in plasma [64] , Inertsil-2 and mesopores form a fine internal structure and
for chlordiazepoxide, diazepam, estazolam, create a large specific surface area [84,181] .
flunitrazepam, flurazepam, medazepam, oxaz- An HPLC–UV method using a Hisep col-
epam and triazolam in rat hair [79] , Inertsil-3 umn has been developed for the determination
for diazepam, clorazepinic acid, alprazolam, of selected BDZs (nitrazepam, clobazam, oxaz-
flurazepam, desalkylflurazepam, nordazepam, epam and lorazepam) in plasma. The hydro-
oxazepam, lormetazepam, temazepam and loraz- phobic shielded phase (Hisep) column is silica
epam in human plasma [168] , Perfectsil Target-3 based and covered with a polymer consisting of
for lorazepam in human serum [85] , Spherisorb hydrophobic regions in a hydrophilic network.
for flumazenil in plasma [169] and Hypersil for Small analytes, such as drugs, penetrate the
alprazolam in tablets [170,171] have been used in hydrophilic network and are retained by the
HPLC–UV methods. A kinetic study on the hydrophobic moieties. The hydrophilic network
acidic hydrolysis of bromazepam was carried shields protein molecules from contact with the
out HPLC–UV using ODS-C8 column (250 × surface and the hydrophobic groups and, thus,
4.6 mm intradermal, 5 µm) and 2-amino-5-bro- these molecules are not retained. The column
mophenyl-2-pyridylmethanone was identified as has the ability to exclude proteins and avoid the
the final product [46] . Midazolam and its metab- column-packing deterioration over a wide pH
olite 1,4-hydroxymidazolam were determined by range (2.0–7.5) [106] .
automated column switching techniques using REMEDi HS drug-profiling system is an auto-
C18 STR ODS-2 [172] and Ultrasphere 3 [173] in mated HPLC system that utilizes online sample
plasma. Partisil 10 [174,175] and Ultrasphere [176] clean-up using a four-column separation, scan-
columns were used for the determination of ning UV detection and a software algorithm.

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Review | Samanidou, Uddin & Papadoyannis
Essien et al. [182] confirmed the presence of butylamine, ethanol, tetrahydrofuran [26,172] and
demoxepam in human urine following separa- ion-pairing reagents such as 1-haptanesulfonic
tion on a REMEDi system as to establish the acid [131] to prevent peak tailing, the major prob-
administration of chlordiazepoxide, a common lem in RP HPLC ana­lysis of BDZs [121] . Peak
BDZ [182] . Urinary BDZs and their metabolites tailing was completely omitted using a mobile
(lorazepam, 7-aminoflunitrazepam, demoxepam, phase consisting of a mixture of methanol and
7-aminoclonazepam and a-hydroxyalprazolam) ammonium dihydrogen phosphate 0.05 M
after b-glucuronidase hydrolysis were assessed (50:50 v/v) adjusted to pH 5.8 with ammo-
using the automated REMEDi HS system [183] . nia [152] . In a microHPLC–MS system (Kromasil
Results demonstrated that it had distinct advan- RP-18, 250 × 300 µm, 5 µm), midazolam and its
tages over GC–MS because of the simplicity of three metabolites in human plasma were eluted
the assay (capability to identify), less time was in isocratic mode with a quarternary solvent mix-
required for analyses and the additional informa- ture consisting of ACN, methanol, ammoniu-
tion (detection of metabolite [demoxepam]) con- macetate and acetic acid 5 mM (37.5:37.5:25:1
cerning the parent BDZ (e.g., chlordiazepoxide) v/v/v/v) at a flow rate of 10 µl/min [54] . The polar
that GC–MS could not detect because of ther- organic mobile phase, a quarternary mixture of
mal decomposition (nordiazepam). However, the acetonitrile, absolute ethanol and triethylamine
bonded silica sorbent product in the analytical glacial acetic acid (100:1:0.001:0.001 v/v/v) was
column is stable within a pH range of approxi- used for the determination of oxazepam enan-
mately 2–8. Siloxane linkages are cleaved below tiomers in plasma extracts from rabbits [111] .
pH 2, while silica substrate is susceptible to dis- Configurational stability of these enantiomers
solution in aqueous solutions at a pH above 8. in body fluids is important to study as the S[+]
In practice, bonded silica may be used at a pH enantiomer is pharmacologically active with
above 8, since degradation of the sorbent is a higher apparent affinity (100- to 200-fold) for
finite process and a pre- or guard-column packed the receptor binding site than its R[-] antipode.
with microparticulate silica (guard columns may
contain preferably the same or sometimes differ- Detection
ent packing material that is used in the analytical UV detection with a variable-wavelength detector
column) is included to saturate the eluent before Among the widely used detectors (UV, MS, flu-
it enters the analytical column. It can consider- orimetric and electrochemical), UV is still the
ably prolong the lifetime of the analytical col- most popular for HPLC BDZ assays due to high
umn [184] . These assays were fully validated with absorption in the range of 200–240 nm.
respect to accuracy, precision, reproducibility and Flunitrazepam and its major metabolites
detection limits in respective cases. (norflunitrazepam, 7-aminoflunitrazepam and
7-acetamidoflunitrazepam) in serum, plasma and
Mobile phases urine after a mixed-mode SPE [190] or an online
Chromatographic separation is generally per- immunoaffinity [191] extraction procedure were
formed at room temperature under isocratic detected by UV HPLC separation, which yields
conditions. However, elevated temperatures (40– a limit of detection (LOD) of at least 1 ng/ml
50°C), gradient elution conditions or a consider- in serum or plasma for all analytes. The use of
ation of both can be also used. The mobile phases UV detection for the determination of clobazam
usually consist of a mixture of organic solvents and N-desmethylclobazam in human serum and
and water or an aqueous buffer. The organic sol- urine [179], clobazam in human blood [192] , diaz-
vents used are acetonitrile or methanol or com- epam and nordiazepam in plasma samples [156] ,
binations of both, whereas buffers (e.g., acetate, or triazolam in human muscle [193] provided
formate, phosphate, sulphate or NH4OH) are LOD values within the range 0.05–01 ng/ml.
used at weakly acidic pH, adjusted by the cor- The simultaneous determination of fentanyl and
responding acid to achieve optimum conditions midazolam in plasma using UV yielded a LOD
for the separation. Trifluoroacetic acid [5,129,185] , of 10 ng/ml and appeared to be suitable for phar-
perchloric acid [186,187] , acetic acid 0.1% [137,188] macokinetic studies [186] . HPLC methods have
or formic acid 0.1% [189] were also used in com- been developed for the simultaneous determina-
bination with one or two organic solvents. To the tion of 15 BDZs in human plasma [194] or seven
mobile phase some authors have added an organic frequently prescribed 1,4-BDZs in bulk powder
modifier such as N,N-dimethyloctylamine [133] , or formulated in tablets or capsules using UV
triethylamine [17,18,147] , isopropylamine [184] , detection [195] .

774 Bioanalysis (2009) 1(4) future science group

 Benzodiazepines: sample preparation & HPLC methods for their determination | Review
UV detection with diode-array detector midazolam and their metabolites in rat hair [13] ,
UV detection with diode-array detection offers and etizolam, brotizolam and lorazepam in
real advantages to ana­lysis, both for the identi- plasma and urine [29] . Better LOD values within
fication of peaks and in order to establish their 0.02–2.0 ng/ml for some BDZs (diazepam,
purity, although the sensitivity of this type of nordiazepam, temazepam, oxazepam, 7-amino-
HPLC detector is still relatively low. Detection flunitrazepam, N-desmethyl flunitazepam and
limits hovered around 10–20 ng/µl [4,89,146,184] clonazepam) in urine and serum samples were
depending on the extinction coefficient of mea- found [11] . LOD values of 20–35 ng/ml were
sured BDZs, the volume of sample extracted, found for the determination of diazepam and its
the detection wavelength and the method used. metabolites N-desmethyldiazepam, oxazepam
A LOD value of approximately 1–2 ng/µl was and temazepam in whole blood by automated
reported for a number of BDZs (clobazam, in-tube SPME coupled with LC–ESI–MS [96] .
clonazepam, delorazepam, diazepam, flunitraz- The LOD was approximately 0.2–1.0 ng ml
epam, 7-aminoflunitrazepam, midazolam and when MS was interfaced with APCI for the
oxazepam) in whole blood and urine [3,127,129,196] . quantification of 23 BDZs in plasma [164] ,
Direct ana­lysis of 1-OH midazolam glucuronide flunitrazepam and its metabolites in serum [44]
in human serum detected at 254 nm gave a LOD and midazolam and 1-hydroxymidazolam in
of 0.04 ng/µl [197] . Analysis of clobazam and its plasma [86] . LC–TOF– MS for the quantitation
active metabolite norclobazam in plasma and of 22 BDZs in human urine gave a LOD value
serum [198] and determination of several BDZs of 0.5–3.0 ng/ml [151] .
in forensic samples (blood, plasma or urine) [199] The combination of MS/MS provides a good
resulted in LOD values of 4.5 ng/ml and 10–30 example of the separation power of HPLC with
ng/ml, respectively. Authors successfully deter- the sensitivity and specificity of MS detection.
mined six BDZs [200] , as well as two metabo- LOD was found within the range 0.05–0.5 ng/
lites [201] , by different HPLC methods in plasma, ml for the determination of BDZs along with
urine and saliva samples resulting in LOD values other drugs in whole blood interfacing MS/MS
between 0.02 and 0.47 ng/µl. with thermo­spray ionization [24] . Interfacing with
APCI yielded a LOD of 0.025–22.5 ng/ml for
MS detection flunitrazepam and its major metabolites 7-ami-
A direct-probe MS technique, which is particu- noflunitrazepam and N-desmethylflunitrazepam
larly useful for the identification of thermally in plasma [57] and for lorazepam in urine, oral
labile compounds, was applied to confirm the fluid and hair [49] . Again, for the determina-
presence of demoxepam during urine screen- tion of midazolam and 1-hydroxymidazolam
ing by multicolumn HPLC with 2D data ana­ in monkey plasma, the LOD was 0.026–20.113
lysis [182] . A sensitive LC–ESI–MS method for ng/ml when MS/MS was interfaced with SSI
simultaneous determination of triazolam and [203] . A segmental ion-spray LC–MS/MS ana­
its hydroxy metabolites in hair has been devel- lysis of 11 BDZs in human hair of psychiatric
oped [188] . In HPLC determination of mid- patients was reported by Kronstrand et al. [83].
azolam and its major metabolite 1-hydroxymi- Interfacing with ESI mode, bromazepam in
dazolam, MS detection interfaced with SSI human plasma was determined with a LOQ of
provided a LOD of 0.08 ng/ml in plasma [59] 0.2 ng/ml [105] and alprazolam and a-hydroxyal-
and MS detection interfaced with ESI a LOD prazolam in plasma was determined with a LOD
of 0.5–2.0 ng/ml in plasma [54,142,162] and 0.1 of 0.05 ng/ml [189] . Midazolam and its hydroxyl
ng/ml in saliva [162] . SSI provided better LOD metabolites were determined in human plasma
values, probably due to much higher signal (sen- and oral fluid by LC–ESI ion trap MS/MS with
sitivity) for analytes in SSI than for ESI [202] . a LOD of 0.025 ng/ml [137] . Two HPLC–MS/
ESI–MS resulted in LOD values between 0.025 MS methods, one interfacing with APCI for the
and 1.0 ng/ml for the determination of cloba- simultaneous detection of 18 BDZs and metabo-
zam and flunitrazepam in whole blood, plasma, lites in human blood [204] and the other inter-
urine or oral fluid by other chromatographic facing with ESI for the determination of bro-
methods [39,43,127] . Using the same ESI interface, mazepam in human plasma [205], were described.
LOD values within 2–5 ng/ml were obtained An HPLC method was developed and validated
for the quantitation of prazepam and its major for the simultaneous identification and quanti-
metabolites (oxazepam and nordiazepam) in fication of some BDZs in urine, serum, plasma
human plasma [48] , alprazolam, estazolam, and meconium [206] and in hair with a LOD of

future science group www.future-science.com 775

Review | Samanidou, Uddin & Papadoyannis
0.02–0.09 ng/mg, when MS/MS was interfaced temperature, they can be converted into highly
with ESI [207] . LC– TOF–MS procedure is pre- fluorescent derivatives in strong acid–alcoholic
sented for the simultaneous ana­lysis of 35 BDZs mixtures. Another way to get a fluorescence
in urine in a single run with excellent LOD val- enhancement is the use of low temperatures
ues of 0.03 –3.0 ng/ml [52] . (77 K) or the use of a laser as an excitation
source. During the period covered by this review,
Electrochemical detection no reports are available based on these types of
Most BDZs have a 4,5-azomethine group includ- fluorescence. Rather, labeled ligand (coumarin-
ing other electroactive, groups such as nitro, labeled desethyl­flumazenil) has been coupled to
N-oxide or hydroxyl. The presence of easily- a metabolite of the BDZ antagonist flumazenil
reducible nitro and/or azomethine (>C=N-) (desethylflumazenil) giving a coumarin fluo-
groups in BDZs allows their electrochemical rophor, which was then quantitated with a RP
detection and, hence, development of a sensitive HPLC system with a fluorescence detector [290] .
electrochemical detector operation in the reduc- However, it can reduce the need for excessive
tion mode would extend the potential of HPLC. clean-up due to its greater selectivity. The nature
This is a relatively new area and very few reports and concentration of the acid and the addition of
are available. Reductions or oxidations are usually an alcohol, such as methanol or ethanol, affect
performed on carbon and mercury electrodes. the fluorescence intensity. In addition, online
Electrochemical detection has been used to a derivative formation or low temperature lumi-
lesser extent in HPLC, due to the possible con- nescence is not as easy as UV to incorporate in
tamination of electrodes and the need for a small an HPLC apparatus detector.
cell volume. Advances in electrode design should
encourage wider applications in the future. The „„Internal standard
ideal working electrode material should have A compound from the same family of BDZs
large anodic and cathodic potential ranges with at a certain concentration is usually used as an
low background currents and the properties of internal standard to minimize errors arising
the electrode surface should not change with from losses during sample treatment and due
time. Glassy-carbon and carbon-paste electrodes to variation in column separation or detector
are particularly well suited for detection in the response [28,42,86,88,127,148] . More recently, deu-
anodic range. Electrodes made from glassy car- terated (tetra, penta or hepta) molecules of the
bon have a relatively large cathodic range, but analytes have been widely used as an internal
need a cumbersome polishing procedure, while standard [47,108,125].
carbon-paste electrodes exhibit a particularly low
background in the anodic region and fresh elec- Future perspective
trode surface can be obtained by simply removing The necessity for rapid, sensitive and specific
the top layer of the carbon paste. assays for BDZs, which is obvious from the
Honeychurch et al. have successfully devel- publication of numerous papers, will continue
oped a HPLC method with dual-electrode elec- to increase. HPLC methods are mainly pre-
trochemical detection in the redox mode for the ferred for the determination of BDZs and their
determination of nitrazepam in serum, elucidat- metabolites in biological samples. Most meth-
ing the electrochemical mechanism occurred at ods have an acceptable sensitivity for routine and
a glassy-carbon electrode [61] . Wilhem et al. have research applications, except for labile BDZs (i.e.,
developed and optimized dual-mode detection ketazolam, temazepam or oxazepam) that are
systems for the determination of BDZs based on not well detected by many of the methods. RP
HPLC with UV detection in series with reduc- columns are the most popular HPLC columns
tive electro­chemical detection at the hanging because they require relatively little maintenance
mercury drop electrode [67,208] . Depending on and a wide variety of drugs of different polarity
the actual BDZ species, detection limits were in can be separated. Long ana­lysis time in many
the range of 2.0–14.1 ng/ml. cases can be reduced by using monolithic col-
umns, which can provide faster separation at
Fluorescence detection elevated flow rates without generating high back
The sensitivity and specificity of detection can pressure and with satisfactory resolution. UV is
be further improved by the use of a fluorescence still the most popular detection technique for
detector although it is less-widely used. Although HPLC BDZ assays due to the high absorption in
BDZs do not yield intense fluorescence at room the range of 200–240 nm. Diode-array detectors

776 Bioanalysis (2009) 1(4) future science group

 Benzodiazepines: sample preparation & HPLC methods for their determination | Review
offer real advantages to ana­lysis, making it pos- Financial & competing interests disclosure
sible to identify peaks and establish peak purity. The authors have no relevant affiliations or financial
MS and MS/MS using different interfaces have involvement with any organization or entity with a finan-
recently become the first choice for BDZ ana­lysis cial interest in or financial conflict with the subject matter
with respect to sensitivity and identification. This or materials discussed in the manuscript. This includes
trend will continue by taking into consideration employment, consultancies, honoraria, stock ownership or
that the use of new simplified instruments with options, expert t­estimony, grants or patents received or pend-
user-friendly software will become more wide- ing, or royalties.
spread. Simply, it will become a major success in No writing assistance was utilized in the production of
BDZ ana­lysis in the next few years. this manuscript.

Executive summary
„„ HPLC is the main choice for the determination of benzodiazepines (BDZs) and their metabolites in biological samples, since it offers
several advantages.
„„ The extraction procedures used are relatively simple and the formation of derivatives is usually not necessary if the detection system is

UV spectrophotometry or amperometry.
„„ LC–MS (MS/MS) is now dominating for BDZ ana­lysis and this detection technique gets relatively little treatment compared with other

detection methods and may not be superior to other methods.

„„ Sonic spray ionization interface is a new approach and has proved to be very similar or, in some cases, superior compared with the

widely used electrospray ionization. Therefore, for pharmacokinetic studies and forensic toxicology cases, this ion source will become
more important in the future in LC–MS or LC–MS/MS assays.
„„ Reverse-phase columns prevail; however, other packing materials, with the addition of organic modifiers or other chromatographic

mechanisms, could be considered for reducing peak tailing.

„„ Solid-phase extraction and liquid–liquid extraction have been extensively successfully applied in extracting drugs from biological fluids.

It has become widely recognized that these methods are time consuming, tedious and often require complicated procedures and
preconcentration of the extract prior to instrumental ana­lysis.
„„ The ultimate choice of an assay method for BDZ determination will be considered by the aim of application (routine monitoring,

pharmacokinetics, overdose studies and forensic medicine), the chemical characteristics of the BDZ of interest, the expertise of the
analyst, the equipment available, the desired sensitivity and specificity, and the time involved in method development or adaptation
and validation.

5 Kavvadias D, Abou-Mandour AA, Czygan F 11 Yuan H, Mester Z, Lord H, Pawliszyn J.

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