II.3.4 Benzodiazepines by Hiroshi Seno and Hideki Hattori

Home , Fludiazepam, Flutazolam, Flutoprazepam, Mexazolam, Nimetazepam, Oxazolam

3.4 II.3.4 Benzodiazepines by Hiroshi Seno and Hideki Hattori

Introduction

Benzodiazepines show antianxiety, hypnotic, anticonvulsant and muscle-relaxant eff ects. Th is group of drugs has wide safety dose ranges; it means that the ratio of the LD50 to the ED50 (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-hydroxytriazolam and α-hydroxytriazolam is also presented.

GC/MS analysis of benzodiazepines in blood and urine

Reagents and their preparation

• Th 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 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 × 0.25 mm i.d., fi lm thickness 0.25 µm, J & W Scientifi 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 temperature: 250 °C; carrier gas: He; its fl 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.

⊡ Table 4.1 Chemical structures of benzodiazepines

Compound R1 R2 R3 R4

diazepam Cl CH3 H–

fludiazepam Cl CH3 F–

flurazepam Cl C2H5 F–

(CH2)2N

C2H5

prazepam Cl CH2 7◃ H–

flutoprazepam Cl CH2 7◃ F– dipotassium Cl H H 3: CHCOOK clorazepate

medazepam Cl CH3 H 2: CH2

clordiazepoxide Cl – H 2: CNHCH3; 4: N→O

nitrazepam NO2 HH–

nimetazepam NO2 CH3 H–

clonazepam NO2 HCl–

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: –COCH3 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 GC/MS analysis of benzodiazepines in blood and urine 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. Aft 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 conditioningc. iv. Th e supernatant fraction prepared at the step ii) is loaded on the Oasis HLB cartridge c. v. Th 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. Th e chloroform layer (lower phase) is carefully transferred to a 4-mL volume glass vial using a Pasteur pipette. viii. Th e organic layer is evaporated to dryness under a stream of nitrogen. ix. Th e residue is dissolved in 50 µL methanol and a 2-µL aliquot is injected into GC/MSe.

Assessment and some comments on the method

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

⊡ Figure 4.1

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. 286 Benzodiazepines

chlordizepoxide, between fl utoprazepam and bromazepam and between fl urazepam and nitrazepam could not be achieved; the peak of tofi sopam showed tailing. Th e separation of other drugs was relatively good. Th e retention times, molecular weights and principal mass spectral ions of benzodiazepines are shown in > Table 4.2. Th e quantitatione of the drugs was made by selected ion monitoring (SIM). Excellent quantitativeness could be confi rmed in the range of 10–1,000 ng/mL of diazepam, fl udiazepam, fl urazepam, prazepam, fl utoprazepam, dipotassium clorazepate, medazepam, chlordiazepoxide, fl unitrazepam, alprazolam, midazolam, etizolam and brotizolam for both blood and urine. Th e detection limits of these 13 drugs were 1–5 ng/mL. For nitrazepam, mexazolam, nimetazepam, clonazepam, bromazepam, tofi 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 oxazolam 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 Moleculer Principal ions m/z (% intensity) time (min) weight 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 9.28 409 242 (100), 270 (69), 103 (34), 89 (33), 76 (30) clorazepate 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. LC/MS analysis of triazolam and its metabolites 287

In the analysis of benzodiazepines by GC and GC/MS, the decomposition of drugs due to heat frequently takes place. Th e decomposition is marked especially for oxazolam, cloxazolam, mexazolam, fl 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 fl unitrazepam, the nitro group is rapidly metabolized into an amino group (in the 7-position) aft er being absorbed into human body [2]. Th e above GC/MS method deals with detection and identifi cation of unchanged benzodiazepines. 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 hydroxylated metabilites and their conjugates together with unchanged forms of benzodiazepines [3–5]. Using the benzophenone, detection and identifi 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 diff erent benzodiazepines give the same benzophenone. Th 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 column” 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. 288 Benzodiazepines

LC/MS conditions

Column: Wakopak WS GP-N6 (150 × 4.6 mm i. d., Wako Pure Chemical Industries, Ltd., Osaka, Japan) g, h LC conditions: an LC 1100 Series high-performance liquid chromatograph (Agilent Tech- nologies, Palo Alto, PA, USA); mobile phases (isocratic mode): 100 % solution A for the initial 5 min (the eluate not introduced into MS)i, followed by solution A/acetonitrile (75:25, v/v) to be introduced into MS; fl ow rate: 0.3 mL/min. MS conditions: an LCQ ion trap mass spectrometer (Th ermoFinnigan Corp., San Jose, CA, USA); ionization: electrospray ionization (ESI); capillary temperature: 230 °C; sheath gas (fl ow

rate): N2 (80 units).

Procedures

i. Blood plasma specimen A 1-mL volume of plasma is mixed with 1 mL of solution A in a 4.5-mL volume glass vial; 20 µL of it is injected into LC/MSj.

ii. Urine specimen i. A 1-mL volume of urine is mixed with 50 µL of 2 M sodium acetate solution and 40 µL of β-glucuronidase solution k in a 4.5-mL volume glass vial, and incubated at 56 °C for 2 h. ii. Aft er cooling to room temperature, 1 mL of solution A is mixed with the above mixture, and a 20-µL aliquot of the solution is injected into LC/MS j.

Assessment of the method

> Figure 4.2 shows ESI mass spectra of triazolam, 4-hydroxytriazolam and α-hydroxytriazolam. Th e recoveries of triazolam and 4-hydroxytriazolam from plasma and urine were not less than 80 %; those of α-hydroxytriazolam 60–70 %. > Figure 4.3 shows mass chromatograms for triazolam, 4-hydroxytriazolam, α-hydroxytriazolam and alprazolam (IS). Under the present conditions, the separation of peaks of triazolam, 4-hydroxytriazolam and α-hydroxytriazolam was good. Th e detection limits obtained by mass chromatography were 20 ng/mL for triazolam and 40 ng/mL for both 4-hydroxytriazolam and α-hydroxytriazolam in plasma and urine. Th e quantitativeness could be observed in the range of 50–400 ng/mL for all compounds in both plasma and urine.

Toxic and fatal concentrations

Benzodiazepines generally show low toxicities and the fatal doses are not clear for many drugs. However, toxic blood levels were reported to be 5–20 µg/mL for diazepam, dipotassium clorazepate and chlordiazepoxide; not lower than 2 µg/mL for oxazepam, and not lower than 0.2–0.3 µg/mL for fl urazepam, lorazepam and nitrazepam. Th e fatal blood concentrations were reported to be not lower than 20 µg/mL for diazepam and chlordiazepoxide, and Toxic and fatal concentrations 289

⊡ Figure 4.2

ESI mass spectra of triazolam, 4-hydroxytriazolam and α-hydroxytriazolam. 290 Benzodiazepines

⊡ Figure 4.3

Mass chromatograms of triazolam, 4-hydroxytriazolam, α-hydroxytriazolam and alprazolam (IS). A 100-ng aliquot each of triazolam, 4-hydroxytriazolam and α-hydroxytriazolam and 50 ng of alprazolam were added to 1 mL blood plasma. Toxic and fatal concentrations 291

0.5–17 µg/mL for fl urazepam [6–8]. Deaths by benzodiazepine poisoning only are rare; simultaneous ingestion of other hypnotics or alcohol enhance the sedative and respiration-suppress- ing eff ects of benzodiazepines, resulting in deaths occasionally.

Notes a) Th e conventional Narcotics Control Law was changed into the Narcotics and Psychotropics Control Law in 1990 in Japan, and benzodiazepines have become included in the drug list to be controlled. Th erefore, at the present time, it is not easy to obtain them from each pharmaceutical manufacturer. b) By adding distilled water to whole blood, the erythrocytes are hemolyzed completely. c) Th e fl ow rate should not be higher than 1 mL/min especially under reduced pressure. It is usually not necessary to use a pump; without aspiration, the solution fl ows inside the cartridge only by natural gravity slowly. d) Since the polymer used for the Oasis HLB cartridge has high water retentativity, the drying of the cartridge during its manipulation does not aff ect recovery rates and reproducibility. Aft er a cartridge is washed with distilled water and its droplets by the action of natural gravity stop coming out, the cartridge is aspirated using a pump to remove water as much as possible; this results in the least contamination of the organic eluate by water. e) For quantitative analysis, one of other benzodiazepines can be chosen as IS. f) 4-Hydroxytriazolam and α-hydroxytriazolam had been able to be purchased from Funakoshi, Tokyo, Japan; however, they are not included in the list of their catalogue book 2001–2002. In the catalogue book 2002–2003 of Sigma, α-hydroxytriazolam (H2529) is listed as an uncontrolled substance and thus easily obtainable. g) Th e Wakopak WS GP-N6 packing material (Wakosil GP-N6) is made of silica gel support, into which a single modifi cation group with hydrophilic and hydrophobic properties is introduced. Large molecules, such as protein, pass through the column; while drugs are retained in the column because of the hydrophobic interaction. Th is column enables direct analysis of a crude specimen without any complicated pretreatment. For triazolam, 4-hydroxytriazolam and α-hydroxytriazolam, the separation of each peak was satisfactory with this column. With Wakopak WS GP-N6, analysis can be achieved without a column switch- ing device. h) A pre-column line fi lter is preferably connected with the column to prevent it from clog- ging. It is possible to analyze without the fi lter, but the column pressure is increased aft er 10–20 time use. i) Since proteins are usually eluted within 5 min, the eluate is not introduced into MS, but drained outside during this period. Aft er this time, the valve is switched to introduce clean eluate into MS not to contaminate MS with impurities. j) When the sample solution is turbid, the clear supernatant fraction aft er centrifugation is injected into LC/MS. k) As β-glucuronidase solution, type HP-2 (from Helix promatia), 132,500 units/mL (Sigma, St. Louis, MO, USA) was used. 292 Benzodiazepines

References

1) Hattori H, Suzuki O, Sato K et al. (1987) Positive and negative ion mass spectrometry of 24 benzodiazepines. Forensic Sci Int 35:165–179 2) Yamamoto I (1995) Yamamoto’s Dictionary of Drug Metabolism. Hirokawa Publishing Co., Tokyo, pp 301–302 (in Japanese) 3) Suzuki O, Hattori H, Asano M et al. (1987) Positive and negative ion mass spectrometry of benzophenones, the acid-hydrolysis products of benzodiazepines. Z Rechtsmed 98:1–10

4) Seno H, Suzuki O, Kumazawa T et al. (1991) Rapid isolation with Sep-Pak C18 cartridges and wide-bore capillary gas chromatography of benzophenones, the acid-hydrolysis products of benzodiazepines. J Anal Toxicol 15:21–24 5) Guan F, Seno H, Ishii A et al. (1999) Solid-phase microextraction and GC-ECD of benzophenones for detection of benzodiazepines in urine. J Anal Toxicol 23:54–61 6) Schonwald S (2001) Medical Toxicology. A Synopsis and Study Guide. Lippincot Williams & Wilkins, Phila delphia, USA, pp 345–347 7) Moffat AC, Jackson JV, Moss MS et al. (eds) (1986) Clarke’s Isolation and Identification of Drugs, 2nd edn. The Pharmaceutical Press, London 8) Winek CL (1989) Drug and Chemical Blood-Level Data 1988. Fisher Scientific, Pittsburgh, PA