3.63.6 © Springer-Verlag Berlin Heidelberg 2005 II.3.6 Barbiturates by Masaru Terada and Ritsuko Watanabe Introduction Barbiturates are being widely used as antiepileptics, hypnotics and anaesthetics (> Figure 6.1 and > Table 6.1). e incidence of barbiturate poisoning cases tends to increase in Japan ( > Figure 6.2) [1]. A majority of the barbiturate drugs is being controlled by the Narcotics Structures of barbiturates. ⊡ Figure 6.1 302 Barbiturates and Psychotropics Control Law in Japan; thus they are also important drugs in view of forensic toxicology. e analysis of barbiturates in human specimens is being made by GC [2–6], GC/ MS [3, 6, 7–9], HPLC [10–13], LC/MS/MS [14], capillary electrophoresis [15] and immu- noassays [16, 17]. In this chapter, usual methods for analysis of barbiturates by GC and HPLC are presented. Incidence of fatal barbiturate poisoning cases. Since fatal cases due to Vegetamin® tablets containing phenobarbital are many, its incidence rate is also shown in this figure. ⊡ Figure 6.2 ⊡ Table 6.1 Properties and doses of barbiturates Action type Compound Route Therapeutic dose (g) Maximum dose (g) long-acting barbital phenobarbital phenobarbital sodium metharbital oral oral suppository oral 0.3 0.03–0.2 0.015–0.03 0.1 0.5 0.25 0.2 Intermediate acting amobarbital amobarbital sodium oral i.v./i. m. 0.1–0.3 0.25–0.5 0.5 short-acting pentobarbital calcium pentobarbital sodium secobarbital sodium hexobarbital oral i.v. i.v. oral 0.05–0.1 0.1 0.1–0.2 0.1–0.4 0.5 0.5 0.5 ultrashort-acting (anaesthetic) thiopental sodium thiamylal sodium i.v. i.v. 0.3–0.5 0.3–0.5 1.0 303 GC analysis [3, 6] Reagents and their preparation • Barbital, phenobarbital sodium, amobarbital sodium, pentobarbital sodium, secobarbital sodium, hexobarbital, thiopental sodium and thiamylal can be purchased from Sigma (St. Louis, MO, USA); pure powder of metharbital was donated by Dainippon Pharmaceu- tical Co., Ltd., Osaka, Japan. • Each barbiturate is dissolved in methanol to prepare 1 mg/mL stock standard solution and stored at –20 °C. • An on-column methylation reagent, 0.2 M trimethylanilium hydroxide (TMAH) (Pierce, Rockford, IL, USA), is diluted with methanol to prepare 4 mM TMAH or 0.4 mM TMAH solution to be used as an on-column methylation reagent ( > Figure 6.3) a . • Diethyl ether b used is of a special grade as pure as that used for analysis of the autoxidation value (AV) and peroxide value (POV) (Dojin Laboratories, Kumamoto, Japan). Other organic solvents are of the highest purity commercially available. • For calibration curves, various concentrations (4–200 ng/mL) of each barbiturate are pre- pared by diluting each 1 mg/mL solution with methanol, and a 10-µL each is evaporated to dryness under a stream of nitrogen, followed by addition of 0.2 mL serum. GC conditions Instrument: a Shimadzu GC-14A gas chromatograph (Shimadzu Corp., Kyoto, Japan). Column: a methylsilicone fused silica wide-bore capillary column c ( DB-1, 15 m × 0.53 mm i. d., lm thickness 1 µm, J & W Scienti c, Folsom, CA, USA). Column temperature: 60 °C → 8 °C/min → 250 °C; injection temperature: 250 °C d ; carrier gas ( ow rate): He (15 mL/min); make-up gas ( ow rate): He (30 mL/min); detectors: an FID and a nitrogen-phosphorus detector ( NPD). Methylation reaction of phenobarbital with TMAH. ⊡ Figure 6.3 GC analysis 304 Barbiturates Procedure i. A 0.2-mL volume of serum e , 1.3 mL distilled water, 1.0 mL of 0.2 M sodium acetate/acetic acid bu er solution (pH 6.0) and 6.0 mL of ethyl acetate/diethyl ether (1:1, v/v) f are placed in a glass centrifuge tube with a ground-in stopper. ii. e tube is shaken vigorously for 5 min. iii. It is centrifuged at 800 g for 5 min, and the organic layer is transferred to a glass vial with a conical bottom. iv. e organic extract is evaporated to dryness under a stream of nitrogen with warming at 40 °C; the residue is dissolved in 0.4 mL of 0.4 mM TMAH methanolic solution. v. A 1–2 µL aliquot of it is injected into GC; in this method, external calibration method is used. An external calibration curve g is constructed by spiking various concentrations of a barbiturate into serum. e peak area of a peak obtained from a test specimen is applied to the calibration curve to obtain its concentration in a specimen. For identi cation by GC/ MS, mass spectra are presented in > Table 6.2. ⊡ Table 6.2 Mass spectra of free forms and methyl derivatives of barbiturates Compound M. W. CI mode (isobutane) MH + EI mode base peak Other fragment ions metharbital 198 155 170, 112, 169 methylmetharbital 212 213 169 184, 126, 112 allobarbital 208 167 124, 80, 141, 106, 53 methylallobarbital 236 237 195 138, 194, 110, 221 amobarbital 226 156 141, 157, 55, 98 methylamobarbital 254 255 169 184, 112, 126 pentobarbital 226 141 156, 157, 55, 98 methylpentobarbital 254 255 169 184, 112, 126 secobarbital 238 167 168, 97, 124, 55 methylsecobarbital 266 267 196 195, 181, 138, 223 hexobarbital 236 221 81, 157, 80, 79, 155, 108, 53 methylhexobarbital 250 251 235 81, 79, 169 mephobarbital 246 218 117,118,146,103 methylmephobarbital 260 261 232 117, 146, 175, 77 phenobarbital 232 204 117, 146, 161, 77, 115 methylphenobarbital 260 261 232 117, 146, 175, 77 cyclobarbital 236 207 141, 81, 79, 67 methylcyclobarbital 264 265 235 169, 79 thiamylal 254 184 168, 167 thiopental 242 172 157, 173, 97, 69 Instrument: a Shimadzu QP-1000EX GC/MS instrument: column: DB-1 (15 m × 0.25 mm i.d., film thickness 0.25 µm); column temperature: 60° C (3 min) → 8° C/min → 290° C; injection temperature: 250° C; carrier gas (flow rate): He (1 mL/min); electron energy: 70 eV (EI), 200 eV (CI); reagent gas: isobutane. 305 Assessment and some comments on the method e GC analysis of barbiturates without any derivatization gives very low sensitivities, even if a non-polar fused silica wide-bore capillary column (DB-1) is used, except for metharbital and hexobarbital. e sensitivities of most barbiturates are enhanced several-fold to several ten-fold by the methyl-derivatization ( > Figs. 6.4 and 6.5, and > Table 6.3). e on-column methylation is very rapid and simple. e detection limits of the methyl-derivatives of barbiturates were 60–90 pg on-column with an NPD and 14–19 ng on-column with an FID. Methylmephobarbital is identical with methylphenobarbital; this means that discrimination Gas chromatograms of free forms (A) and methyl-derivatives (B) of barbiturates using an NPD. Amounts of barbiturates used for the free and derivatized forms were 2 and 1 ng on-column, respectively. 1: metharbital; 2: allobarbital; 3: amobarbital; 4: pentobarbital; 5: secobarbital; 6: hexobarbital; 7: phenobarbital. ⊡ Figure 6.4 GC analysis 306 Barbiturates between mephobarbital and phenobarbital becomes impossible a er methylation of these compounds. It should be cautioned that the peak of thiopental overlaps that of methylcyclo- barbital. HPLC analysis [10] Reagents and their preparation • Each barbiturate is dissolved in methanol to prepare 10 µg/mL standard solution and stored at –20 °C. • As internal standard (IS) solution, 5-(4-methylphenyl)-5-phenylhydantoin (Sigma) is dis- solved in methanol to prepare 10 µg/mL solution. Gas chromatograms for methyl derivatives of barbiturates spiked into distilled water (A) and human serum (B), and for blank human serum (C) using an NPD. The amount of each barbiturate spiked into distilled water or 0.2 mL plasma was 0.5 µg. The peak numbers are the same as specified in > Figure 6.4. ⊡ Figure 6.5 307 • Calibration curves: 10, 25, 50, 100, 500 and 1,000 µL of the standard solution of each bar- biturate were separately placed in glass centrifuge tubes together with 50 µL of IS solution, and evaporated to dryness under a stream of nitrogen, followed by addition of 0.5 mL se- rum each. HPLC conditions Column: a reversed phase column h ( ODS-80 Ts, 10 cm × 4.6 mm i.d., particle diameter 2 µm, Toso, Tokyo, Japan). Mobile phase: 8 mM phosphoric acid solution/acetonitrile (3:7, v/v). Detection wavelength: 215 nm; ow rate: 0.4 mL/min; temperature: room temperature. Procedure i. A 0.5-mL volume of blood (urine) i , 50 µL IS solution, 0.5 mL distilled water, 0.1 mL of 0.2 acetic acid/sodium acetate bu er solution (pH 6.0) and 3 mL of ethyl acetate/diethyl ether (1:1, v/v) are placed in a 10-mL volume glass centrifuge tube with a ground-in stopper. ⊡ Table 6.3 Retention times and detection limits for main barbiturates and their methyl derivatives measured by GC-NPD Compound Retention time (min) Detection limit (pg/on-column) metharbital 6.86 139 methylmetharbital 6.30 52 allobarbital 9.13 667 methylallobarbital 7.61 46 amobarbital 10.7 667 methylamobarbital 9.27 61 pentobarbital 11.0 667 methylpentobarbital 9.61 60 secobarbital 11.6 435 methylsecobarbital 10.2 48 hexobarbital 12.3 169 methylhexobarbital 11.9 52 mephobarbital 12.8 306 methylmephobarbital 12.2 57 phenobarbital 13.5 1,670 methylphenobarbital 12.2 57 cyclobarbital 13.7 1,360 methylcyclobarbital 12.4 89 thiopental 12.4 80 HPLC analysis 308 Barbiturates ii. e tube is well voltex-mixed or shaken for 2 min. iii. It is centrifuged at 800 g for 5 min, and the organic layer is transferred to a glass vial with a conical bottom. iv. e organic extract is evaporated to dryness under a stream of nitrogen with warming at 40 °C. e residue is dissolved in 100 µL of the mobile phase. v. A 10-µL aliquot of the above solution is injected into HPLC. e peak area ratio obtained from a test specimen is applied to the calibration curve j to calculate its concentration, which had been constructed with the spiked serum according to the above procedure. Assessment and some comments on the method In this method, the authors used an HPLC column with a particle diameter of 2 µm in place of 5 µm; the smaller diameter gives various advantages, such as higher sensitivity, rapid analysis and a smaller amount of the ow of mobile phase. e detection limit for most barbiturates HPLC chromatograms for blood extracts in the presence (b) and absence (a) of barbiturates. The concentration of each barbiturate spiked into blood was 0.1 µg/mL. 1: barbital; 2: allobarbital; 3: metharbital; 4: phenobarbital; 5: cyclobarbital; 6: pentobarbital; 7: hexobarbital; 8: amobarbital; 9: secobarbital; 10: thiopental; 11: IS [ 5-(4-methylphenyl)-5-phenylhydantoin]. ⊡ Figure 6.6 309 was about 0.05 µg/mL except for thiopental (0.5 µg/mL) (> Table 6.4). e peak of pentobar- bital overlaps that of hexobarbital; the peak of amobarbital appears very close to these peaks ( > Figure 6.6). Toxic and fatal concentrations erapeutic, toxic and fatal blood concentrations of some barbiturates are shown in > Table 6.5 [18, 19]. Every concentration shows a wide variation according to individuals. By single admin- istration, the dose (route), peak blood concentration and its time are: phenobarbital 50 mg (oral), 1.9 µg/mL a er 3 h [6]; amobarbital 120 mg (oral), 1.8 µg/mL a er 2 h [20]; pentobarbital 100 mg (oral), 1.2–3.1 µg/mL a er 0.5–2 h [21]; thiopental 400 mg (intravenous), 28 µg/mL a er 2 min, 7 µg/mL a er 15 min, and 3 µg/mL a er 90 min [22]. e minimum fatal doses are: amobarbital, about 1.5 g; pentobarbital, about 1 g; phenobarbital, about 1.5 g; and thiopental, about 1 g [19]. ⊡ Table 6.4 Retention times and detection limits for main barbiturates measured by HPLC Compound Retention time (min) Detection limit (µg/mL) barbital 3.8 0.05 allobarbital 5.1 0.05 metharbital 5.9 0.05 phenobarbital 6.1 0.05 cyclobarbital 7.1 0.05 pentobarbital 10.6 0.05 hexobarbital 10.6 0.05 amobarbital 10.9 0.05 secobarbital 14.2 0.05 thiopental 21.8 0.5 ⊡ Table 6.5 Blood concentrations of main barbiturates (µg/mL) Compound Therapeutic conc. Toxic conc. Fatal conc. Reference amobarbital 1–5 2–12 10–30 >9 13–96 9–72 [18] [19] pentobarbital 1–3 1–10 >5 >8 10–169 8–73 [18] [19] phenobarbital 10–40 2–30 40 – 60 4–90 >80 4–120 [18] [19] thiopental 1–42 4.2–134 >7 10–400 6–392 [18] [19] Toxic and fatal concentrations 310 Barbiturates Survived and fatal poisoning cases Survived phenobarbital poisoning case [23] A 26-year-old male ingested 2–3 g of phenobarbital plus phenytoin. Upon arrival at a hospital, blood phenobarbital concentration was as high as 107 µg/mL. As emergency treatments, 30 g of activated charcoal and 30 g of sodium sulfate were administered orally. e concentration of blood phenobarbital decreased to 72 µg/mL a er 24 h; he therea er recovered without any complication. Fatal thiopental poisoning cases Case 1 [24]: a 21-year-old female received intravenous administration of 17 mL of iobal (thiopental sodium solution) for arti cial abortion at a women’s clinic, and fell into a shock state 20 min a er. Although various emergency treatments were made, she died 2 h a er. e blood concentration of thiopental of this victim was 8.2 µg/mL. Case 2 [25]: a 26-year-old male (son) and a 54-year-old female (mother). At their wit’s end due to domestic violence by the son, his father intended to commit triple-suicide. He injected about 12 mL (about 1.75 g thiopental) and about 15 mL (about 1.5 g thiopental) of thiopental solution into his son and wife intravenously, respectively; both of them were killed. He intro- duced car exhaust into his car room and also tried to inject the same solution intravenously by himself to commit suicide, but his trials were abortive. e blood thiopental concentrations of his son and wife were 6.9 and 4.4 µg/mL, respectively. Case 3 [25]: a 25-year-old female was found dead in her room; she had committed suicide by injecting an alate needle into a vein of the right dorsum pedis for drop infusion of thiopen- tal (its amount infused not clear). e concentration of thiopental in her heart blood was 6.3 µg/mL; the amount administered was estimated to be about 1.5 g. Fatal pentobarbital poisoning case [26] A 3.2-year-old male. A doctor directed a nurse to inject 50 mg pentobarbital into the child intramuscularly; but the nurse misunderstood the direction. She began intravenous injection of 50 mL pentobarbital solution to the child. A er injection of the 15 mL solution, he fell into a shock state. In spite of e orts with various emergency treatments, he died 22 h later. e pentobarbital concentration in his blood was 17.5 µg/mL (total amount injected estimated about 500 mg). Fatal poisoning cases due to a combined drug or multiple drugs including a barbiturate Case 1 [27]: a 40–45 year-old (estimated) female was found dead on a bed of a hotel. In her room, 2 tablets of Vegetamin (a combined drug containing, phenobarbital, promethazine and [...]... (2000) Evaluation of Emit Tox benzodiazepine and barbiturate assays on the Vitalab Viva analyser and FPIA on the Abbott ADx analyser Clin Chem Lab Med 38:615–618 18) Winek CL (1994) Drug & chemical blood-level data 19) Moffat AC, Jackson JV, Moss MS et al (eds) (1986) Clarke’s Isolation and Identification of Drugs The Pharmaceutical Press, London Survived and fatal poisoning cases 20) Inaba T, Tang BK,... Treatment of phenobarbital overdose with activated charcoal JAMA 247:2400–2401 24) Furuno J, Sugawara N (1972) On a fatal case of thiopental shock and the metabolism Jpn J Legal Med 26:71–77 (in Japanese with an English abstract) 25) Tadano Y, Yamakawa M, Miura K et al (1989) Cases of homicide and suicide using thiopental sodium Res Pract Forensic Med 32:117–121 (in Japanese with an English abstract)... National Research Institute of Police Science, Chiba (in Japanese) 2) Terada M, Yamamoto T, Kuroiwa Y et al (1992) Gel permeation chromatographic clean-up prior to determination of barbiturates in blood extract by gas chromatography Jpn J Forensic Toxicol 10:31–37 3) Terada M, Shinozuka T, Yasuda M et al (1992) Simultaneous determination of acidic and neutral drugs by wide bore column gas chromatography... 37:193–200 10) Tanaka E, Terada M, Tanno K et al (1997) Forensic analysis of 10 barbiturates in human biological samples using a new reversed-phase chromatographic column packed with 2-micrometre porous microspherical silica-gel Forensic Sci Int 85:73–82 11) Hart AP, Mazarr Proo S, Blackwell W et al (1997) A rapid cost-effective high-performance liquid chromatographic (HPLC) assay of serum lamotrigine after... (1970) A case of fatal pentobarbital poisoning – Estimation of pentobarbital dosage from the blood concentration – Jpn J Legal Med 24:68–72 (in Japanese with an English abstract) 27) Kageura M, Hara K, Hieda Y et al (1987) Forensic toxicological study on a combined drug (phenobarbital, promethazine and chlorpromazine) Res Pract Forensic Med 30:83–87 (in Japanese with an English abstract) 28) Terada M, Yasuda... liquid-liquid extraction and using HPLC conditions routinely used for analysis of barbiturates Ther Drug Monit 19:431–435 12) Biosca YM, Sagrado S, Camanas RMV et al (1999) Determination of barbiturates in urine by micellar liquid chromatography and direct injection of sample J Pharm Biomed Anal 21:331–338 13) Borregon PFG, Lores M, Cela R (2000) Analysis of barbiturates by micro-high-performance liquid...Survived and fatal poisoning cases chlorpromazine) and one capsule of Insumin (flurazepam) were found From the stomach contents, phenobarbital, promethazine and chlorpromazine were detected Drug concentrations in blood, the liver and kidney were: phenobarbital, 98.0, 106 and 105 µg/g; promethazine, 5.05, 38.2 and 6.92 µg/g; chlorpromazine, 1.68, 22.1 and 3.03 µg/g, respectively Case 2 [28]: A 30-year-old... liquid chromatography with post-column photo-chemical derivatization J Chromatogr A 870:39–44 14) Spell JC, Srinivasan K, Stewart JT et al (1998) Supercritical fluid extraction and negative ion electrospray liquid chromatography tandem mass spectrometry analysis of phenobarbital, butalbital, pentobarbital and thiopental in human serum Rapid Commun Mass Spectrom 12:890–894 15) Ferslew KE, Hagardorn AN, McCormick... barbiturates in blood and urine J Forensic Sci 42:1160–1170 8) Hall BJ, Brodbelt JS (1997) Determination of barbiturates by solid-phase microextraction (SPME) and ion trap gas chromatography-mass spectrometry J Chromatogr A 777:275–282 9) Kudo K, Nagata T, Kimura K et al (1988) Toxicological analysis of thiamylal in biological materials by gas chromatography/mass spectrometry Forensic Sci Int 37:193–200... McCormick WF (1995) Application of micellar electrokinetic capillary chromatography to forensic analysis of barbiturates in biological fluids J Forensic Sci 40:245–249 16) Schwenzer KS, Pearlman R, Tsilimidos M et al (2000) New fluorescence polarization immunoassays for analysis of barbiturates and benzodiazepines in serum and urine: performance characteristics J Anal Toxicol 24:726–732 17) Charlier CJ, Plomteux . of fatal barbiturate poisoning cases. Since fatal cases due to Vegetamin® tablets containing phenobarbital are many, its incidence rate is also shown in. used is of a special grade as pure as that used for analysis of the autoxidation value (AV) and peroxide value (POV) (Dojin Laboratories, Kumamoto, Japan).