3.43.4
© Springer-Verlag Berlin Heidelberg 2005
II.3.4 Benzodiazepines
by Hiroshi Seno and Hideki Hattori
Introduction
Benzodiazepines show antianxiety, hypnotic, anticonvulsant and muscle-relaxant e ects.  is
group of drugs has wide safety dose ranges; it means that the ratio of the LD
50
to the ED
50

(therapeutic index) is high. Because of its safety, benzodiazepines are being widely used in the
world. Some of benzodiazepines are also being abused or used for so-called “ drug facilitated
sexual assault”, and thus they are under the control of the Narcotics and Psychotropics Control
Law; in Japan, triazolam abuse has become one of the serious social problems. In this chapter, a
GC/MS method for simultaneous analysis of 22 kinds of benzodiazepines listed in
> Table 4.1
is described. In addition, the LC/MS analysis of triazolam, and its metabolites 4-hydroxy-
triazolam and α-hydroxytriazolam is also presented.
GC/MS analysis of benzodiazepines in blood and urine
Reagents and their preparation
•  e pure powder of the 22 kinds of benzodiazepines was donated by each pharmaceutical
manufacturers according to the authors’ request
a
(some of benzodiazepines now obtaina-
ble from Sigma, St. Louis, MO, USA).

• 1 M Sodium bicarbonate solution: a 8.4-g aliquot of sodium bicarbonate is dissolved in
distilled water to prepare 100 mL solution.

• 2 M Sodium acetate solution: a 27.5-g aliquot of sodium acetate is dissolved in distilled

5
(CH
2
)
2
N
C
2
H
5
F–
prazepam Cl CH
2
7
◃
H–
flutoprazepam Cl CH
2
7
◃
F–
dipotassium
clorazepate
Cl H H 3: CHCOOK
medazepam Cl CH
3
H 2: CH
2
clordiazepoxide Cl – H 2: CNHCH
3

3
; 8: –COCH
3
oxazolam Cl H H 4, 5: 2-methyloxazolo
mexazolam Cl H Cl 4, 5: 3-methyloxazolo
estazolam Cl – H 1, 2: triazolo
alprazolam Cl – H 1, 2: 1-methyltriazolo
triazolam Cl – Cl 1, 2: 1-methyltriazolo
midazolam Cl – F 1, 2: 1-methyltriazolo
etizolam – – Cl 1, 2: 1-methyltriazolo;
1, 5: 7-ethylthieno
brotizolam – – Cl 1, 2: 1-methyltriazolo;
1, 5: 7-bromothieno
285
Procedure
i. A 1-mL volume of whole blood or urine is mixed well with 8.5 mL distilled warter
b
in a
15-mL volume glass centrifuge tube with a ground-in stopper, followed by addition of
0.5 mL of 1 M sodium bicarbonate solution.
ii. A er it is vortex-mixed, it is centrifuged at 3,000 rpm for 10 min to obtain a supernatant
fraction.
iii. An Oasis HLB 3cc solid-phase extraction cartridge (Waters, Milford, MA, USA) is set on
a vacuum manifold, and 3 mL methanol and 3 mL water are passed through the cartridge
for conditioning
c
.
iv.  e supernatant fraction prepared at the step ii) is loaded on the Oasis HLB cartridge
c
.

 e quantitation
e
of the drugs was made by selected ion monitoring (SIM). Excellent quan-
titativeness could be con rmed in the range of 10–1,000 ng/mL of diazepam,  udiazepam,
 urazepam, prazepam,  utoprazepam, dipotassium clorazepate, medazepam, chlordiazepoxide,
 unitrazepam, alprazolam, midazolam, etizolam and brotizolam for both blood and urine.  e
detection limits of these 13 drugs were 1–5 ng/mL. For nitrazepam, mexazolam, nimetazepam,
clonazepam, bromazepam, to sopam, estazolam and triazolam, quantitativeness could be
observed in the range of 50–1,000 ng/mL with detection limits of 10–20 ng/mL, and for oxa-
zolam it could be observed in the range of 200–1,000 ng/mL with detection limits of 50 ng/mL
in urine and 100 ng/mL in blood.
⊡ Table 4.2
Retention times and principal mass spectral ions of benzodiazepines measured by GC/MS
Compound Retention
time (min)
Moleculer
weight
Principal ions m/z (% intensity)
medazepam 7.97 270 207 (100), 242 (91), 244 (30), 270 (20),165 (15)
fludiazepam 8.68 302 274 (100), 301 (96), 302 (92), 109 (43), 283 (37)
diazepam 8.94 284 283 (100), 256 (94), 284 (88), 221 (36), 110 (31)
dipotassium
clorazepate
9.28 409 242 (100), 270 (69), 103 (34), 89 (33), 76 (30)
chlordiazepoxide 9.31 299 282 (100), 124 (20), 247 (16), 220 (14), 89 (11)
oxazolam 9.72 328 251 (100), 253 (30), 70 (30), 105 (13), 77 (12)
midazolam 9.78 325 310 (100), 312 (30), 325 (20), 163 (12), 111 (12)
flunitrazepam 9.91 313 285 (100), 312 (99), 313 (95), 266 (58), 238 (37)
flutoprazepam 10.00 342 55 (100), 313 (67), 109 (61), 287 (42), 259 (37),
342 (29)

ated metabilites and their conjugates together with unchanged forms of benzodiazepines [3–5].
Using the benzophenone, detection and identi cation of a benzodiazepine or its metabolites
can be achieved by GC or GC/MS without any derivatization. However, it should be noted that
an unchanged benzodiazepine, its hydroxylated metabolite and its glucuronide metabolite all
give the same benzophenone; also there are many cases in which di erent benzodiazepines
give the same benzophenone.  erefore, by the benzophenone method, it is impossible to dis-
criminate among unchanged, hydroxylated and conjugated forms, and also among similar
types of benzodiazepines.
LC/MS analysis of triazolam and its metabolites
To analyze drugs in human specimens with high protein contents, such as blood and plasma,
the deproteinization procedure is generally essential. Recently, special column packing materi-
als are being developed from some manufacturers, which enable direct application of a crude
specimen without any prior deproteinization process. “Internal surface reversed phase col-
umn” is one of the columns of this type. In this section, a method of LC/MS analysis of triazolam
and its metabolites ( 4-hydroxytriazolam and α-hydroxytriazolam) using the above new column
without the need of deproteinization is presented.
Reagent and their preparation
• Triazolam can be purchased from Sigma (St. Louis, MO, USA). 4-Hydroxytriazolam and
and α-hydroxyltriazolam were purchased from Funakoshi, Tokyo, Japan
f
.
• 0.05 % Formic acid-containing 7.5 mM ammonium acetate solution (solution A): 578 mg
ammonium acetate and 0.5 mL formic acid are dissolved in distilled water to prepare
1,000 mL solution.
• 2 M Sodium acetate solution: 27.5 g of sodium acetate is dissolved in distilled water to
prepare 100 mL solution.
LC/MS analysis of triazolam and its metabolites