Identification and differentiation of barbiturates, other sedative-hypnotics and their metabolites in urine integrated in a general screening procedure using computerized gas chromatography-mass spectrometry

Jorrrnol o/‘Cllromotog,trph?:. 530 (1YYO) 307-326 Biomrdid Appliwtiuns Elsevier Science Publishers B.V.. Amsterdam

CHROMRTO.

5373

Identification and differentiation of barbiturates, other sedative-hypnotics and their metabolites in urine integrated in a general screening procedure using computerized gas chromatography-mass spectrometry” HANS

II. MAURER

(First rcccivcd

March

12th. 1990; revised manuscript

received

April 28th.

1090)

ABSTRACT A gas chromatographic-mass cntiation

other hypnotics barbital.

acecarbromal,

(scc)butabarbital.

cyc]opentobarbita].

hcptabarbital.

phenobarbital.

thiopental,

meprobamatc.

pentobarbital.

for the identification

amobarbital.

butobarbital.

diethylallylacetamide.

vinharhital

is described

in urine. The following

allobarbital.

butalbital.

hexobarbttal.

methyprylone.

thiobutaharbital,

procedure

and their metabolites

could be detected:

bromisoval,

cycloharbital. namate.

spectrometric

of sedative-hypnotics

dipropylbarbital,

phenobarbital.

and vinylhital.

aprobarbital,

carhromal.

mcthaqualone.

24 barbiturates

glutethimide.

propallylonal.

The procedure

liquid extraction,

derivatization

by acetylation.

mass spectrometry.

141, 167. 169; 207. 221 and 235. the presence indicated.

The identity

v-isual or computerized preparation.

of positive comparison

mass chromatograms,

stgnals

separation

by capillary

lising mass chromatography of barbiturates.

is integrated

of the stored reference

secobarbital. in a general

and identih-

with the selected ions W: 83, I 17. and or their metabolites

mass chromatograms

full mass spectra

mass spectra

ethi-

methyl-

over 300 drugs and isolation by liquid-

gas chromatography.

other hypnotics

in the rcconstructcd

guaifenesin.

pyrithyldione.

presented

brallo-

crotylbarbital.

methohexital.

screening procedure (general unknown analysis) for several groups of drugs detecting over 1000 of their mctabolites. It includes cleavage of conjugates by acid hydrolysis. cation by computcri7ed

barbital,

clomethiazole.

metharbital,

and ditferand thirteen

with the reference

and gas chromatographic

was confirmed spectra.

retention

was by a

The sample

indices are

documented.

li’!TRODUCTlON

The sedative-hypnotic drugs are one of the largest groups of drugs, usually classified into barbiturates, bromureids, benzodiazepines, and antihistamines. They are widely used for the treatment of insomnia, anxiety states and convulsive disorders as well as for anaesthetic and preanaesthetic medication. Because of their central nervous and respiratory depressant effect, they may cause. alone or in combination with other drugs and/or ethanol, severe accidental or suicidal

0378-4347~90.‘s03.50

:c

I990 Elsevier Science Publishers

B.V

308

H. H. MAIJRER

intoxication for which treatment is necessary. Furthermore, they may impair the fitness to drive a car and to work at machines even after therapeutic doses. In particular, the barbiturates and benzodiazepines may lead to a drug dependence. The medical use of barbiturates has been declining in recent years, because their margin of therapeutic safety is relatively narrow [2]. However, they are still misused by heroin fixers to ease the withdrawal symptoms from heroin or to augment to effects of “weak heroin”. For all these reasons, sedative-hypnotics may be encountered in clinical or forensic toxicological analysis. Before quantification in plasma, the drugs, which are usually unknown, must be first identified. Identification methods for benzodiazepines [3] and antihistamines [4-71 have already been published. Detection of some of the barbiturates and other hypnotics using thin-layer chromatography (TLC) [8--l 11, high-performance liquid chromatography (HPLC) [8, 12-151, gas spectromctry chromatography (GC) [8,16-201 and gas chromatography-mass (GC-MS) [16,21] has been described. Furthermore, fluorescence polarization immunoassays [22], enzyme immunoassays [23] or radioimmunoassays [24] are commercially available for the detection and quantification of barbiturates. However, in clinical and forensic toxicology, immunological results must be confirmed by a non-immunological method. especially by GC-MS. However, none of these procedures allows the rapid and specific identification and differentiation of all barbiturates and other sedative-hypnotics. The aim of this study was to investigate whether and how barbiturates and other sedative-hypnotics can be detected within the “general-unknown” analysis procedure that has proved to be successful for several hundred psychotropic, antihistaminic, analgesic and cardiovascular drugs and their metabolitcs [3-7:25361. Urine was used for this general screening procedure. because the concentrations of drugs and their metabolites are higher in urine than in plasma. Because most of the toxicologically relevant drugs are excreted in urine in a completely metabolized and conjugated form, at least in the later phase of excretion, conjugates were cleaved by acid hydrolysis, which can be completed more quickly than enzymic hydrolysis. Polar metabolites were derivatized by acetylation. This paper describes a computerized GC-MS method for the identification and differentiation of barbiturates, other sedative-hypnotics and their mctabolites in urine, integrated in a general unknown analysis procedure. EXPERIMENTAL

Appara tm

A Hewlett-Packard (HP) Series 5890 gas chromatograph, combined with an HP MSD Series 5970 mass spectrometer and an HP Series 59970 C workstation, was used. The GC conditions were as follows: splitless injection mode; column, HP capillary (12 m x 0.2 mm I.D.), cross-linked methylsilicone, 0.33 pm film thickness; column temperature, programmed from 100 to 3 10°C in 30”Cmin,

CC-MS

OF BARBITURATES

309

initial time 3 min, final time 5 min; injection port temperature, 270°C; carrier gas, helium; flow-rate 1 ml/min. The MS conditions were as follows: scan mode; ionization energy, 70 eV; ion-source temperature, 220°C; capillary direct interface heated at 260°C. Exact measurement of retention indices was performed on a Varian Series 3700 gas chromatograph. The column effluent went to a flame-ionization’detector and a nitrogen-sensitive flame-ionization detector after a 1:1 split by a splitter made from nickel tubing. The column was a steel tube (60 cm x 2 mm I.D.) packed with Chromosorb G HP (100@120 mesh) coated with 5% OV-101. The column and injector temperatures were identical with those used for GCMS, and the temperature for the detectors was 270°C. Nitrogen was used as carrier gas at a flow-rate of 30 mlimin. Urine

samples

The investigations were performed apeutic doses of hypnotics.

on urine from in-patients

trcatcd with ther-

Hydrolysis and extraction procedure A lo-ml volume of urine was refluxed with 3 ml of 37% hydrochloric acid for 15 min. Following hydrolysis, ca. 3 g of potassium hydroxide pellets were added and the resulting solution was mixed with 10 ml of 30% aqueous ammonium sulphate to obtain a pH between 8 and 9. This solution was extracted with a 1O-ml portion of dichloromethanee2-propanol-ethyl acetate (1: 1:3). Phase separation was accomplished by centrifugation. The organic extract was transferred to a pear-shaped flask and carefully evaporated to dryness. The temperature should not be too high and the vacuum not too strong, because hypnotics with relatively low molecular masses can be partly evaporated. Acefylation The extracted residue was acetylated for 30 min at 60°C with 100 ~1 of acetic acid anhydride pyridine (3:2). The acetylation mixture was then carefully cvaporated to dryness and the resultant residue dissolved in 100 ~1 of methanol. A 0.552 ,IL~volume of this sample was injected into the gas chromatograph. Gas chromatographic-mass spectrometric analysis Full mass spectra were recorded at a speed of 1 scan/s and stored on a hard disk during the temperature-programmed GC analysis. Using mass chromatography with the ions m/z 83. 117, 141, 167, 169,207,221 and 235, the stored spectra were evaluated. The identity of positive signals in the reconstructed mass chromatograms was confirmed by a visual comparison of the full mass spectra with reference spectra (Fig. 1) or by a computer library search [37]. The reconstruction of the mass chromatograms, the recognition of the top of the peaks and the library search of the peak underlaying mass spectra can be automated by conaection of the corresponding computer programs using so-called macros [25].

2:

tWROBRHATE

, BROMISOVK

ETHItMRTE

RRTIFRC

13;

100

-AL

120

100

‘IO0

200

8

71

v,;/,

II,

(NOR- )

I,,

100

II

IS0

1, , , $

100

IO0

LOO RCECFIRBRCML-WRRTIFRCT lCRR5ROl4lDE) CRRBROf4tlL-WRRTIFRCT (CRRBROHIDEI

69

1I

CLOIKTHIRZOLE

ERRBITAL tlETHMBITRL-Ii

DIETliYLRLLYLRCETW4IllE

200

(

,

, ,

200

J, , , , , , ,

, Il., , , , ,

7

GC-MS

OF BARBITURATES

311

BUTOBRRBITAL

VINYLBITRL-II

F

(HO-1

100

ioo -HZ0

100

-MO

100

[HO-1

L

20

19

VINBRRBITRL

VINBRRBITAL-I4

+-%i--

156

200

/‘n4,197

200

24

22

I(ETHYPRYLONE-II

I

CRRBROIIRL RCECARBROMRL-tl

69

BRRLL~BRRBITRL

100

100

‘100

ICRRBROHRL)

100

(0X0-1

IPENTOBM?BITRL)

1,‘1\,:“;,.;.,,

I

PENTOBRRBITRL THIOPENTRL-M

,<;,

,

i

200

j;

168

200

, , , ,

ioo

300

, , , , , , , ,

200

I),, , , i”:

156

N

”

SECOBRR8ITRL

BUTRLBITAL

Fig. 1.

27

100

200

JL-CI-

168

100

L

200

II

7

100

100 CROTYLBRRBITRL-I!IHO- -HZ0

ALLOBRRBIlRL

31

30

4

120

100

q

111

CROTYLBRRBITRL

100

100 M'ROBRRBITRL PROPRLLYLONRL-R (DESBROWO-)

100 BRRLLOBRRBITRL-H (DIHYDRO-1

67

PROPRLLYLONAL

1 I

I

,

*,

200

,

,

,

,

,

,

300

,

314

H. H. MAURER

L

&

I..

6y/

Fig. 1

PROPRLLYCONRL-N

,L ,~,

-HZ0

100 (DESBROHO-HO-I

IHO-)

141

i

I’:,,

1.1. /

100 IDESBRONO-HO-)

a ;.

,“d,

E;;

431

RNOBfiRB17RLdl

ERALLOBRRBITRL-N

121

/

ZOO

L

IEl

/

221

n+

,“,:.

46

IHO-

(0X0-1

too

‘IO0

UIPROPYLBRRBITRL-M

DIPROPYLBAREITRL-N

[‘r;;ooi ,::::i ,

IS?

ISOKR

I RC

200

181

200

,k

so0

/ l.L-_ 169

CYCLOBIRBITRL-N

HEXOBFIRBITR-N

100 (HO-)

(HO-1

ioo

-HEE

-HZ0

100 PH~NOBRi?BITRL HEXRNILI-N (PtKNOERREI767Ll METHYLPHENOBRRBITRL-fl (NCR-1 PRIMIDONE-tl

200

200

loo

lPHENOBRRBIlALl

54

BUTALBITR-N

SECOBRRUITRL-N

IHO-)

100

100 (HO-1 -HP0

zoo

200

ilt

‘X-MS

OF BARBITURATES

317

67

-HZ0

IHO-PHENYL-)

!lIISOOCTYLFHTHRLRTE

CLUTETHIHIDE-M

GLUTETHIf4lDE-N

ANDROSTERONE

RC

RTHYLPNENOBRRBITRL-M

PHENOBRRBITRL-Ii IHO-) ,KTHYLFHENOBRRBITRL-M

IHO-)

RC

AC PRIlllUONE-H LNOR-HO-) RC

IHO-PHENOBRRBITRLI

RC

/

290

CC-MS

OF BARBITURATES

31Y

320

RESULTS

H. H. MAURER

AND

DISCUSSION

The results of the studies are summarized in Table I. ITsing mass chromatography with the eight proposed ions, the possible presence of barbiturates, other sedative-hypnotics and!or their metabolites in urine could be indicated selectively. In contrast to mass fragmentography, mass chromatography is based on the full scan mode and. therefore, the specific identification is carried out by comparison of the peak underlying full mass spectrum with reference spectra. Some of the compounds are derivatized by acetylation (see Derivative column in Table 1). The mass spectra numbers in Fig. 1, the molecular masses and the GC retention indices are given. These indices were determined using temperatureprogrammed GC combined with flame ionization detection (FID) and nitrogensensitive FID. In our experience, retention indices provide preliminary indications of the possible presence of the compounds and may be useful to workers without a GCMS facility. Data are given for only those metabolitcs that were frequently found. All of the listed metabolites are not detected in every sample owing to inter-individual differences in metabolism. or variable times which elapsed after administration. The mass spectra and retention indices of the less abundant metabolites will be included in a forthcoming handbook and computer library [37,38]. The data for androsterone, the dehydration products of androsterone and cholesterol, two unidentified endogenous biomolecules and diisooctylphthalate a widespread softener of plastics ~ arc included. because these compounds can be indicated by the mass chromatograms. Some artifacts were formed during sample preparation and/or GC. Acecarbromal and carbromal were partly altered to the corresponding amide carbromide. Bromisoval was also altcrcd in part to an artifact, probably also to the amide. Alcoholic hydroxy groups were dehydrated to the corresponding alkenes (- HZ0 in Table I and Fig. 1). Because the dehydration artifacts were formed during acid hydrolysis, the corresponding intact molecules were found after enzymic hydrolysis. The data for the intact molecules will be included in a forthcoming handbook and computer library [37,38]. The full mass spectra for the specific identification of the selectively indicated compounds are shown in Fig. 1. They are listed primarily in order of ascending mass of the ion with the largest mass detected in the spectrum. For the same nominal mass value, the spectra are arranged in order of ascending retention indicts. Formulae are proposed for probable metabolite structures. Interferences by other drugs are improbable because the identity of the peaks observed in the mass chromatograms can be positively confirmed by a visual comparison of the underlying mass spectrum with reference spectra (Fig. 1 and ref. 3X) or by a computer library search [37]. The following drugs lead to common metabolites and/or artifacts: acecarbromal and carbromal, aprobarbital and propallylonal, barbital and metharbital. butabarbital and thiobutabarbital, pento-

(M)

Amobarbital

M (HOP)

Aprobarbital

Barbital

208

226

224

21 I)

184

M (HO ) Butobarbital

M (0x0.)

M (IIO-)

Carbromal

M;Artifacl

Clomethiazole

M (Deschloro-di-HO-)

M (2-HO-)

240

226

270

236

193

161

1x3

219

(carbromide)

Butalbital

224

212

M (HO

210

) -H20

Butabarbital

- H20

A(.

AC

AC

5

2

I

s

7

2 0.1

0.1

0.6

3x

39

0.9

25

65

100

I8

24

<

48

2

83

4

3

35

Bromisoval

212

5 2

67

Bromisoval

222

4

63

I5

2

6

41

8X

6

4

3

49

45

6

M (Dihydro-)

5 100

100

100

I5

63

2

97

2

37

73

22

0.9

(%)

0.3

0.3

1

I6

IX

0.2

2

I7

1

0.1

0.2

25

0.2

0.2

2

0.2

I

5

0.3

I

1590

1420

1230

1215

1515

1940

1880

1665

1440

1690

1905

1655

1510

1540

2040

1970

1795

1850

1500

1610

1830

1710

IS15

1215

Retention

37

13

7

x

24

47

35

20

S6

21

33

IS

2

39

40

30

41

2:

31

42

34

25

24

8

No.

MS

METABOLITES

I

THEIR

I595

100

AND

I

0.2

SEDATIVE-HYPNOTICS

13

0.5

0. I

intensity

OTIIER

2

0.7

12

4

2

5

Rclativc

M (HOP)

artifact

(carbromide)

Derivative”

OF BARBITURATES.

302

224

DETECTION

AND ACETYLATION

FOR THE

288

Brallobarhital

M (Desbromo-HO-)

286

-H20

M (Carhromal)

Allobarbital

236

Acccarbromal-M/artifact

Drug!mctabolilc

I93

Mass

Mol.

ACID

IlYDROLYSIS

PROGRAMME

AFTER

URINE

I

MONITORING

TABLE IN

F

2

E

$-

* m _~

M (HOP) ISOMER

M (HO-)

Cilutcthimide

M (HO-ethyl-)

M (HO-phcnyl-)

Guaifcncsin

M (0-desmethyl-)

M (HO-)

M (HO-m&boxy-)

Ethinamate’

Heptabarbital

M (HO-)

Hexobarbital

270

270

217

275

275

2X2

310

340

370

167

250

24X

236

AC AC

M(H0

M (0X0-)

Meprobamate

Methaqualone

M (2-Formyl-)

M (2-HO-methyl-)

M (4’-HO-)

250

218

250

264

308

30x

4

88

6

3

2

I

I

IS

19 100

99

0. I

4

6

AC

1

0.3

4

1 9

4

2

AC

AC

AC

AC

6

5

2

AC AC

3

0.5

22

I 00

100

80

5

3

2530

2440

2240

2155

1785h

2055

1970

1855

2300

2070

I 395h

2265h

223Sh

1920”

I865”

2250

2060

1830

2000

1950

I870

1650

12x5

8

AC

234

) -H20

H20

ISOMER

2

M (0x0-)

226

1

Dipropylbarbilal

212

1865

2

2190

74

73

64

61

3

60

51

53

59

38

I

x0

78

75

69

67

66

36

48

46

45

11

5

I6

62

52

Diclhylallylacctamidc

2

Cyclopentobarbiial

5

100

155

5

55

1970

234

-H20

2 I70

M (0x0.)

6

250

7

M (HO-)

234

62

Cyclobarbital

236

100

32

-H20

26

MS

1600

Retention

1620

(“A)

M (HO-)

intensity

Crotylbarbilal

Rclativc

210

Dcrivativc”

208

(M) NO.

Drug/mctabolitc

Mass

Mol.

f c

3:

?

I 2

0.5

1

2 4

I IO0 5

M (HOP)

Phenobarbital

M (HOP)

Propallylonal

M (Desbromo-)

M (Dcsbromo-HO-)

224

232

290

288

210

226

AC

0.2

biomolecule

Androsterone

Androsterone

Cholesterol

Diisooctylphthalate

biomolecule

M (HO-)

Endogenous

Endogenous

224

222

332

212

368

390

These drugs can bc detected

c

of urine.

FID.

AC -__..

only in a direct extract

be detected

AC

These compounds

’

cannot

- H20

- H20

- H20

*

acetylated.

6

Vinylbital

222

AC

38

M (HO-)

224

2

23

13

28

Vinbarbital

224

-1120

M (Pentobarbital)

M (HO-pentobarbital)

226

M (Butabarbital)

Thiopental

242

44

0.9

19

IO

14

0.3

0.5

6

0.5

3

3

6

2

0.5

2

I6

29

84

83

0.6

212

15

I 3 2 2 I 1 1 0.9

9

10

43

5

M (HO-m) - H20

29

Thiobutabarbital

’

2

228

Secobarbital

AC

0.6

x

236

Pyrithyldione

167

238

~ H2O

by nitrogen

29

I

Pentobarbital

- H20

x4

2

M (0x0-)

197

0.1

226

~ H20

M (HO-)

181

AC

100 100 100

Methyprylone

183

290

2

s 4

I

2

41

1

8

0.2

6

0.4

2

4

2 2 4 100

0.5 0.5

2575”

1950

2540”

3030h

2240h

2S80h

1995

1145

I165 2020

I 4

16

12

68

79

65

71

19

4

17

18

43

21

IS

I655

54 49 1790

1970

28

Y 17’)s

1520

31 44

I 740 I X90

I

6

70 29 1770

1610

1875

0.4

4

II

1965 2360

57

6

2

0.4

70

17

98

25

2

0.3

18.55

4

0.2

77

78

3

100

99

2

43 50

21

1740 1x90

1870

0.4

0.9

70

71

SO

63 58

6

IS40

1525

2360

1965 2330

I

1895

17x0

I 0.3 4

62

5

I

I0

4 12

0.3

I

1500

145.5

14 12 22

0.2

M (H@) M (Nor-HO-)

304

5

29

M (Nor-)

232 AC

2

38

Methylphcnobarbital

14

246

4

Methohexital

0.2

262

22

12

M (Nor-)

184

97

20

Metharbiial

198

G

;;1 VI

F

z =i c

;il

%

$

2

324

H. H. MAURER

Fig. 2. Mass chromatograms pentobarbital

from

(I) and its dehydrated

lite (4). and diisooctylphthalate

the urine hydroxy

of a patient metabolite

(5). a widespread

softener

suspected

of misusing

(2), mcthaqualone of plastics

hypnotics.

(3) and its 2-hydroxy

(mass spectra

indicating metabo-

Nos. 21, 43. 61~ 73 and

68 in Fig. I).

barbital and thiopental, and phenobarbital. methylphenobarbital and the anticonvulsant primidone [36]. Guaifenesin has a common metabolite with the muscle relaxant methocarbamol and a common metabolite with the P-blocker oxprenolol[33]. However, the detection of the parent drugs and/or further unique metabolites allowed a differentiation. Acecarbromal and carbromal can only be differentiated in a direct extract of native urine [37739] The detection limits of the parent compounds were between 10 and 20 ng/ml of urine. The sensitivity of the method was sufficient to detect therapeutic concentrations of the studied drugs. Because urine concentrations of sedative-hypnotics are relatively high, the relatively poor analytical recovery of only 40&80% caused by hydrolysis and weakly alkaline pH is of minor relevance. However, diethylallylacetamide, ethinamate and pyrithyldione could only be detected in a direct extract of native urine [39]. To illustrate the method, mass chromatograms from the urine of a patient suspected of misusing hypnotics is shown in Fig. 2. Peaks 1 and 2 indicate pentobarbital and its dehydrated hydroxy metabolite, peaks 3 and 4 methaqualone and its 2-hydroxy metabolite, and peak 5 indicates diisooctylphthalate (mass spectra Nos. 21, 43, 61, 73 and 68 in Fig. 1). CONCLUSIONS

The procedure presented allows the identification and differentiation of therapeutic concentrations of barbiturates> other sedative-hypnotics and their metabolites in urine, integrated in a general unknown analysis procedure for the detec-

GC-MS

OF BARBITURATES

325

tion of several categories of drugs like benzodiazepines [3,26], antihistamines [447], butyrophenone neuroleptics [27], analgesics [28], opioids [29], antidepressants [30], neuroleptics [31], antiparkinsonians [32], P-blockers [33], antiarrhythmics [34], laxatives [35] and anticonvulsants [36]. The identified sedative-hypnotics can then be quantified in plasma using methods described by Mangin ez al. [14], Rop et al. [15], Svinarov and Dotchev [40] or reviewed by Gupta [41]. ACKNOWLEDGEMENTS

The author thanks Prof. Dr. K. Pfleger for his support, A. Weber for plotting the structures, and the Bundesminister fur Jugend, Familic, Frauen und Gesundheit for sponsorship with instruments.

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