Tài liệu Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 30) doc

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

water to prepare 100 mL solution.
GC/MS conditions
Column: a DB-5 fused silica capillary column (30 m × 0.25 mm i.d.,  lm thickness 0.25 µm,
J & W Scienti c, Folsom, CA, USA).
GC conditions; instrument: a GC-17A gas chromatograph (Shimadzu Corp., Kyoto, Japan);
column (oven) temperature: 150 °C (1 min) → 20 °C/min → 300 °C (6.5 min); injection tempera-
ture: 250 °C; carrier gas: He; its  ow rate: 0.9 mL/min; sample injection: splitless mode for
1 min, followed by the split mode.
MS conditions: a QP-5050A mass spectrometer (Shimadzu Corp.); ionization: EI; electron
energy 70 eV; interface temperature: 250 °C.
284 Benzodiazepines
⊡ Table 4.1
Chemical structures of benzodiazepines
Compound R
1
R
2
R
3
R
4
diazepam Cl CH
3
H–
fludiazepam Cl CH
3
F–
flurazepam Cl C
2
H

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

; 4: N→O
nitrazepam NO
2
HH–
nimetazepam NO
2
CH
3
H–
clonazepam NO
2
HCl–
flunitrazepam NO
2
CH
3
F–
bromazepam Br H – 5: C-pyridine
tofisopam CH
3
CO – H 1: CHC
2
H
5
; 2: C–CH
3
; 3: N; 3’:
–OCH
3
; 4’: –OCH

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
.

v.  e cartridge is washed with 3 mL distilled water
d
.
vi. A target drug is eluted with 3 mL chloroform
c
into a 5-mL volume glass tube with a
conical bottom.
vii.  e chloroform layer (lower phase) is carefully transferred to a 4-mL volume glass vial
using a Pasteur pipette.
viii.  e organic layer is evaporated to dryness under a stream of nitrogen.
ix.  e residue is dissolved in 50 µL methanol and a 2-µL aliquot is injected into GC/MS
e
.
Assessment and some comments on the method
 e recovery rates of the drugs from blood and urine were not less than 60 %. > Figure 4.1
shows a total ion chromatogram (TIC) for the authentic standards of benzodiazepines dis-
solved in methanol. In this chromatogram, separation between dipotassium clorazepate and
TIC for the authentic standards of 22 benzodiazepines.
1: medazepam, 2: fludiazepam, 3: diazepam, 4: dipotassium clorazepate, 5: chlordiazepoxide,
6: oxazolam, 7: midazolam, 8: flunitrazepam, 9: flutoprazepam, 10: bromazepam, 11: prazepam,
12: nimetazepam, 13: mexazolam, 14: flurazepam, 15: nitrazepam, 16: clonazepam, 17: estazolam,
18: alprazolam, 19: tofisopam, 20: etizolam, 21: triazolam, 22: brotizolam.
⊡ Figure 4.1
GC/MS analysis of benzodiazepines in blood and urine
286 Benzodiazepines
chlordizepoxide, between  utoprazepam and bromazepam and between  urazepam and
nitrazepam could not be achieved; the peak of to sopam showed tailing.  e separation of
other drugs was relatively good.  e retention times, molecular weights and principal mass
spectral ions of benzodiazepines are shown in
> Table 4.2.

 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)

bromazepam 10.01 315 90 (100), 326 (92), 315 (91), 77 (91), 317 (86)
prazepam 10.10 324 55 (100), 91 (91), 269 (75), 295 (68), 324 (46)
241 (36)
nimetazepam 10.30 295 267 (100), 294 (77), 248 (63), 295 (62), 220 (34)
mexazolam 10.75 363 251 (100), 253 (30), 70 (22), 139 (11), 236 (9)
flurazepam 10.83 387 86 (100), 99 (7), 58 (6), 387 (2)
nitrazepam 10.91 281 280 (100), 253 (95), 234 (80), 264 (61), 206 (58)
clonazepam 11.56 315 280 (100), 314 (87), 315 (68), 288 (54), 89 (52)
estazolam 12.03 294 259 (100), 293 (65), 294 (64), 205 (59), 89 (50)
alprazolam 12.42 308 279 (100), 273 (90), 308 (88), 204 (84), 102 (82)
tofisopam 12.97 382 382 (100), 326 (87), 341 (63), 353 (33), 156 (31)
etizolam 13.32 342 342 (100), 344 (47), 313 (37), 266 (31), 125 (24)
triazolam 13.50 342 313 (100), 315 (76), 238 (71), 75 (62), 342 (52)
brotizolam 13.82 392 394 (100), 392 (77), 245 (43), 118 (38), 123 (27)
* The ions used for SIM are shown in boldfaces.
287
In the analysis of benzodiazepines by GC and GC/MS, the decomposition of drugs due to
heat frequently takes place.  e decomposition is marked especially for oxazolam, cloxazolam,
mexazolam,  utazolam and haloxazolam having oxazolo rings in their structures [1]. In such
cases, the relatively low injection temperature and the use of a wide-bore capillary column with
short length (15 m) can protect the drugs from their heat decomposition to some extent.
For nitro-group containing drugs, such as nitrazepam, nimetazepam, clonazepam and
 unitrazepam, the nitro group is rapidly metabolized into an amino group (in the 7-position)
a er being absorbed into human body [2].
 e above GC/MS method deals with detection and identi cation of unchanged benzodi-
azepines. To detect benzodiazepine metabolites from urine specimens, it is necessary to hydro-
lyze the glucuronate conjugates of the drugs using β-glucuronidase; the resulting free forms
with hydroxyl groups should be derivatized before GC (/MS) analysis.
When benzodiazepines are treated in strong acid, they are hydrolyzed into benzophenones,
which are very stable against heat; the benzophenones can be also obtained from the hydroxyl-

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