Tài liệu Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 32) ppt

GC analysis [3, 6] Reagents and their preparation • Barbital, phenobarbital sodium, amobarbital sodium, pentobarbital sodium, secobarbital sodium, hexobarbital, thiopental sodium and thi

© Springer-Verlag Berlin Heidelberg 2005

by Masaru Terada and Ritsuko Watanabe

Introduction

Barbiturates are being widely used as antiepileptics, hypnotics and anaesthetics (> Figure 6.1 and > Table 6.1) Th 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

and Psychotropics Control Law in Japan; thus they are also important drugs in view of forensic toxicology Th 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

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

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 columnc ( DB-1, 15 m × 0.53 mm

i d., fi lm thickness 1 µm, J & W Scientifi c, Folsom, CA, USA)

Column temperature: 60 °C → 8 °C/min → 250 °C; injection temperature: 250 °C d; carrier gas (fl ow rate): He (15 mL/min); make-up gas (fl 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

i A 0.2-mL volume of serume, 1.3 mL distilled water, 1.0 mL of 0.2 M sodium acetate/acetic acid buff 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 Th 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 Th 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 curveg is constructed by spiking various concentrations of a barbiturate into serum Th 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 identifi 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

(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):

Assessment and some comments on the method

Th 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 Th 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) Th e on-column methylation is very rapid and simple Th 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

between mephobarbital and phenobarbital becomes impossible aft 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

• 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 columnh ( 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; fl 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 buff 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

(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

ii Th 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 Th e organic extract is evaporated to dryness under a stream of nitrogen with warming at

40 °C Th e residue is dissolved in 100 µL of the mobile phase

v A 10-µL aliquot of the above solution is injected into HPLC Th e peak area ratio obtained from a test specimen is applied to the calibration curvej 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 fl ow of mobile phase Th 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

was about 0.05 µg/mL except for thiopental (0.5 µg/mL) (> Table 6.4) Th e peak of pentobar-bital overlaps that of hexobarpentobar-bital; the peak of amobarpentobar-bital appears very close to these peaks

(> Figure 6.6).

Toxic and fatal concentrations

Th 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 aft er 3 h [6]; amobarbital 120 mg (oral), 1.8 µg/mL aft er 2 h [20]; pentobarbital 100 mg (oral), 1.2–3.1 µg/mL aft er 0.5–2 h [21]; thiopental 400 mg (intravenous), 28 µg/mL aft er 2 min,

7 µg/mL aft er 15 min, and 3 µg/mL aft er 90 min [22] Th 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

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)

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

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 107µg/mL As emergency treatments, 30 g

of activated charcoal and 30 g of sodium sulfate were administered orally Th e concentration of blood phenobarbital decreased to 72 µg/mL aft er 24 h; he thereaft er recovered without any complication

Fatal thiopental poisoning cases

Case 1 [24]: a 21-year-old female received intravenous administration of 17 mL of Th iobal (thiopental sodium solution) for artifi cial abortion at a women’s clinic, and fell into a shock state 20 min aft er Although various emergency treatments were made, she died 2 h aft er Th 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 Th 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) Th 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 Aft er injection of the 15 mL solution, he fell into

a shock state In spite of eff orts with various emergency treatments, he died 22 h later Th 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

chlorpromazine) and one capsule of Insumin (fl urazepam) were found From the stomach con-tents, 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 female was found dead by a passer-by at an open-air parking

lot close to her home almost in the nude with her clothes being scattered around her body In the previous night, the victim had drunk and eaten with her friends and left them Twenty four empty packages of Isomytal (amobarbital) were discovered at her home From her blood, amo-barbital at 10.6 µg/mL, phenobarbital at 28.6 µg/mL, promethazine at 2.8 µg/mL and chlor-promazine at 0.9 µg/mL were detected

Case 3 [29]: A 44-year-old male was found dead by his wife in his bedroom with smoke

from the right side of his mattress She called an ambulance, but he had been dead COHb concentration in his blood was 1 %; burning wounds found in his whole body were negative for vital reaction From his stomach contents, amobarbital, bromisovalum and levomepromazine were detected Th e drug concentrations in blood, the liver, brain and urine were: amobarbital,

25, 47, 30 and 7 µg/mL (g); bromisovalum, 37, 70, 57 and 6 µg/mL (g); levomepromazine, 1.5,

19, 2.0 and not detectable µg/mL (g), respectively

Notes

a) By the on-column methylation with TMAH, many compounds are methyl-derivatized

immediately (> Figure 6.3) Except for barbiturate drugs, this method is applicable to

glutethimide, primidone and oxazolo-benzodiazepines For thiopental, the derivatization reaction causes some decomposition products together with the methylated product; the analysis of the underivatized form of thiopental gives better results Th e 4 mM and 0.4 mM concentrations of TMAH are used for the FID and NPD, respectively

b) It should be cautioned that thiopental is desulfurized and decomposed by a peroxide in-cluded in diethyl ether

c) Similar types of columns from other manufacturers, such as SPB-1, Ultra#1, CBP-1 and CP-Sil5, can be used

d) When the injection temperature is not high enough (not higher than 200 °C), the effi ciency

of the on-column methylation is low

e) Blood plasma and whole blood can be also used

f) As extraction solvents, diethyl ether, ethyl acetate, chloroform and dichloromethane can be used; however, the present ethyl acetate/diethyl ether (1:1, v/v) gave the cleanest back-grounds with few impurity peaks for human specimens For metharbital, diethyl ether, ethyl acetate and dichloromethane give low extraction effi ciencies (46–63 %)

g) Each calibration curve showed good linearity in the range of 0.2–10 ng on-column (0.2–

10 µg/mL) Th e detection limits of this method were 60–90 µg/mL in serum Th e recovery rates of 0.1–5.0 µg of barbiturates spiked into 0.2 mL serum were 83–111 % Mass spectra, retention times, detection limits and each GC chromatograms for underivatized and meth-ylated barbiturates are shown in > Tables 6.2 and 6.3, and > Figure 6.5.

h) Similar types of ODS (octadecylsilane type silica gel) columns from other manufacturers, such as Zorbax ODS and Hypersil, can be also used However, the retention times may be longer, because of 5 µm of particle diameter

Survived and fatal poisoning cases