GCMS analysis of benzodiazepines in blood and urine

Reagents and their preparation

• The pure powder of the 22 kinds of benzodiazepines was donated by each pharmaceutical manufacturers according to the authors' request a (some of benzodiazepines now obtainable 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 x 0.25 mm i.d., film thickness 0.25 |im, J & W Scientific, 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 temperature: 250 °C; carrier gas: He; its flow 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.

O Table 4.1

Chemical structures of benzodiazepines

Compound

Ri

R2

R3

R4

diazepam

Cl

CH3

H

-

fludiazepam

Cl

CH3

F

-

flurazepam

Cl

(CH2>2N(

/C2H5 F V2H5

p raze pa m

Cl

CH2-■

4 H

-

flutoprazepam

Cl

CH2-'

4 F

-

dipotassium clorazepate

Cl

H

H

3: CHCOOK

medazepam

Cl

CH3

H

2: CH2

clordiazepoxide

Cl

-

H

2: CNHCH3; 4: N^O

nitrazepam

no2

H

H

-

nimetazepam

no2

CH3

H

-

clonazepam

no2

H

Cl

-

flunitrazepam

no2

CH3

F

-

bromazepam

Br

H

-

5: C-pyridine

tofisopam

CH3CO

-

H

1: CHC2H5; 2: C-CH3; 3: N; 3' -OCH3; 4': -OCH3; 8: -COCH

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: 7-methyltriazolo

triazolam

Cl

-

Cl

1, 2: 7-methyltriazolo

midazolam

Cl

-

F

1, 2: 7-methyltriazolo

etizolam

-

-

Cl

1, 2: 7-methyltriazolo; 1, 5: 7-ethylthieno

brotizolam

-

-

Cl

1, 2: 7-methyltriazolo; 1, 5: 7-bromothieno

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. After 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 conditioningc.

iv. The supernatant fraction prepared at the step ii) is loaded on the Oasis HLB cartridge c.

v. The 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. The chloroform layer (lower phase) is carefully transferred to a 4-mL volume glass vial using a Pasteur pipette.

viii. The organic layer is evaporated to dryness under a stream of nitrogen.

ix. The residue is dissolved in 50 ^L methanol and a 2-^L aliquot is injected into GC/MSe.

Assessment and some comments on the method

The 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 dissolved in methanol. In this chromatogram, separation between dipotassium clorazepate and

O Figure 4.1

O Figure 4.1

Rohypnol Pippette Image

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.

chlordizepoxide, between flutoprazepam and bromazepam and between flurazepam and nitrazepam could not be achieved; the peak of tofisopam showed tailing. The separation of other drugs was relatively good. The retention times, molecular weights and principal mass spectral ions of benzodiazepines are shown in > Table 4.2.

The quantitatione of the drugs was made by selected ion monitoring (SIM). Excellent quan-titativeness could be confirmed in the range of 10-1,000 ng/mL of diazepam, fludiazepam, flurazepam, prazepam, flutoprazepam, dipotassium clorazepate, medazepam, chlordiazep oxide, flunitrazepam, alprazolam, midazolam, etizolam and brotizolam for both blood and urine. The detection limits of these 13 drugs were 1-5 ng/mL. For nitrazepam, mexazolam, nimetazepam, clonazepam, bromazepam, tofisopam, 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.

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.

* The ions used for SIM are shown in boldfaces.

In the analysis of benzodiazepines by GC and GC/MS, the decomposition of drugs due to heat frequently takes place. The decomposition is marked especially for oxazolam, cloxazolam, mexazolam, flutazolam 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 flunitrazepam, the nitro group is rapidly metabolized into an amino group (in the 7-position) after being absorbed into human body [2].

The above GC/MS method deals with detection and identification of unchanged benzodiazepines. To detect benzodiazepine metabolites from urine specimens, it is necessary to hydro-lyze the glucuronate conjugates of the drugs using p-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 identification 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 different benzodiazepines give the same benzophenone. Therefore, by the benzophenone method, it is impossible to discriminate among unchanged, hydroxylated and conjugated forms, and also among similar types of benzodiazepines.

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