application of gas chromatography/ mass … · application of gas chromatography/ mass spectrometry...

APPLICATION OF GAS CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS) TO THE ANALYSISOF BENZODIAZEPINES

Dariusz B£ACHUT1, Marta BYKAS-STRÊKOWSKA1, Ewa TARACHA2,Bogdan SZUKALSKI2

1Department of Criminalistics, Internal Security Agency, Warsaw2Department of Neurochemistry, Institute of Psychiatry and Neurology,Warsaw

ABSTRACT: A gas-chromatographic-mass spectrometric method was developed forthe simultaneous analysis of 18 benzodiazepines in human urine. Separation of thiscomplex mixture of benzodiazepines was achieved on three capillary columns withphases: SPB-OCTYL, SPB-1, HP-5MS. The thermal instability of benzodiazepinesin the chromatographic process may lead to erroneous interpretation of obtained re-sults. This is especially significant in the case of examinations carried out within theframework of expert casework requested by the administration of justice, and thuspotentially influencing the outcome of legal proceedings.

The practical usefulness of the developed conditions was checked by means of uri-nalysis of two patients from the Neurosis Clinic and one patient undergoing metha-done treatment at the Department of Detoxification of Institute of Psychiatry andNeurology. Screening tests carried out on these patients to check abstinence compli-ance revealed compounds from the benzodiazepine group. The urine of the patientsfrom the Neurosis Clinic contained tetrazepam and nordiazepam, whereas that ofthe patient undergoing methadone therapy contained nordiazepam, amphetamine,promethazine and three of its metabolites, nicotine and three of its metabolites,a metabolite of carbamazepine and also methadone and its two metabolites.

KEY WORDS: Benzodiazepine; Decomposition; Gas chromatography/Mass spec-trometry.

Problems of Forensic Sciences, vol. LIX, 2004, 5–37Received 31 August 2004; accepted 29 September 2004

INTRODUCTION

Beside typical narcotics such as heroin, cocaine, derivatives of Indianhemp and derivatives of phenylisopropylamine (amphetamine), widely usedmedicines are also abused, which, in addition to their intended pharmaco-logical action, evoke euphoric effects and could lead to dependence. Thisgroup includes benzodiazepines, which evoke a good mood (euphoria, “mel-

lowing”, calming down), and increase the psychotropic effects of other psy-choactive compounds (cocaine, heroin, derivatives of phenylisopropylamine,alcohol). Some of them have an intoxicating effect and are used in criminalcases as “date rape” type drugs [36], serving to stupefy victims in order tocarry out a punishable offence, e.g. rape or underhand obtaining of PINnumber or bank account number.

Benzodiazepines are available on the pharmaceutical market and aretherefore relatively easy to obtain in contrast to typical narcotics, whichcan only be obtained on the illegal market. As a consequence of the non-med-ical use of benzodiazepines, legal regulations relating to their turnover anduse have been introduced. The Drug Counteraction Act [10] in Poland in-cluded 34 derivatives of benzodiazepine, listing them on the register ofpsychotropic compounds, in group VI-P. Flunitrazepam was treated morerestrictively by the Polish legislature, being classified into group III-P.

Because of the widespread use of benzodiazepines both in therapy and fornon-medical purposes, many methods of benzodiazepines detection andquantification have been developed. Whereas analysis of confiscated evi-dence material in the form of pills and capsules does not pose any major ana-lytical problems, detection and identification of particular benzodiazepinesin urine or blood is a difficult several-step analytical task.

Rapid detection of benzodiazepines in biological material has been madepossible by homogenous methods [2, 12, 39], such as the enzymatic-immuno-logical method (EMIT), the fluorescence-polarisation method (FPIA) andthe radioimmunological method (RIA). Their advantages are simplicity andspeed of test performance, without a troublesome extraction step, and insome cases it is also possible to determine the approximate concentration ofanalyte. A disadvantage is their low specificity, caused by the phenomenonof cross reactivity with compounds of similar structure. These methods onlyallow group identification (i.e. the class of compound) and a very approxi-mate estimation of concentration, which is the resultant of concentrationsand reactivity of all substances which react with the test. They do not, how-ever, enable identification of individual compounds. Another problem is thetoo high detection limit, which is also termed the “cut-off value” – it may be in-sufficiently sensitive for benzodiazepines monitoring as their therapeutic dosesare very low. This group includes among others: triazolam (dose 0.125–0.25 mg,concentration in plasma 2–20 ng/ml), flunitrazepam (dose 0.5–1 mg, concen-tration in plasma 5–15 ng/ml) and loprazolam (dose 0.5–1 mg, concentration inplasma 3–10 ng/ml) [4].

The following chromatographic methods, amongst others, are used rou-tinely for benzodiazepines identification in forensic laboratories: thin layerchromatography (TLC), high performance liquid chromatography (HPLC),gas chromatography (GC), and also spectroscopic methods – mass spectrom-

6 D. B³achut, M. Bykas-Strêkowska, E. Taracha et al.

etry (MS) and infrared spectrometry (FTIR). Chromatographic techniques(GC and HPLC) allow separation of complex mixtures of benzodiazepinesand when coupled with spectroscopic methods in a GC/MS system [11, 29,38], or an HPLC/MS system [21, 25, 37, 40], enable identification of individ-ual benzodiazepines in biological material (urine, blood, saliva, hairs) notonly from tablets but also from mixtures with other substances.

Increased sensitivity and selectivity in relation to classic MS detectioncan be obtained by application of tandem mass spectrometry (MS/MS) [19,22, 28] and chemical ionisation by negative ions (NICI). Certain structuralfeatures of benzodiazepines, i.e. the relatively high content of nitrogen at-oms and halogens (increasing the affinity of a molecule with low-energy elec-trons) and also the presence of condensed rings (stabilizsing the negativecharge of a molecular ion) make these compounds ideal subjects for analysisby GC-NICI. Song et al. [33] determined clonazepam in plasma with a detec-tion limit of 0.25 ng/ml on the basis of the above mentioned technique. Thesame authors [34] obtained a much lower detection limit of 0.1 ng/ml whenthey determined flurazepam and its metabolites in rat plasma [34]. The se-lectivity and sensitivity of the GC/NICI technique turned out to be especiallyuseful in the determination of trace amounts of various derivatives ofbenzodiazepines and their metabolites in hair [6, 7, 27].

Metabolic processes of benzodiazepines occur in the organism, consistingin hydroxylation of aliphatic fragments and aromatic rings, de-alkylation,reduction of the nitro group and acetylation [35]. Metabolites and also someprimary compounds are coupled with glucuronic acid in the final phase oftransformation. It is necessary to free them from these compounds before be-ginning to analyse benzodiazepines by the GC method. This is done by acidor enzymatic hydrolysis.

The first of these leads to the formation of benzophenones, which arecharacterised by relative volatility and good chromatographic properties[16]. It should also be mentioned that in the 1960s and 1970s, when onlypacked chromatographic columns were available with thermally unstablestationary phases, transformation of some high molecular-mass benzo-diazepines into more volatile benzophenones was the only way of detectingthem by the GC technique. Although it is simple to carry out, this methodhas one serious shortcoming: hydrolysis of various benzodiazepines resultsin only one benzophenone (Figure 1). This makes identification of the pri-mary compound impossible.

Enzymatic hydrolysis by means of �-glucuronidaze is used to break downglucuronate and release benzodiazepine in unchanged form. This enzymeonly acts on �-glucosidase bonds of glucuronates and releases a compoundcoupled with glucuronic acid.

Application of gas chromatography ... 7

An important factor in the analytical cycle when studying biological ma-terial is correct procedure of analyte separation from the sample matrix. Thefollowing are used for isolation of analytes from a matrix: conventional liq-uid-liquid extraction (LLE) with the use of various solvents [1], solid phaseextraction (SPE) on columns with octadecyl phase [14, 32], octyl phase [13],phenyl phase, cyclohexyl phase [5] and with mixed phases [15, 26], and alsosolid phase micro-extraction (SPME) [30]. Liquid-liquid extraction is betterfor broad screening of biological material. However, solid phase extractiongenerally gives better results (purity of extract, recoveries) in the case ofanalysis directed towards determination of a selected derivative of benzo-diazepine.

A positive result obtained by homogeneous methods has to be verified bya selected confirmatory test. One can choose from among chromatographic


Fig. 1. Benzodiazepines which create the same benzophenone.

methods (TLC, GC, HPLC, EC) and hyphenated techniques (GC/MS,HPLC/MS, EC/MS) depending on the character of analysis, access to suitableequipment and the initiative of the scientist carrying out the analysis.

Benzodiazepines and their metabolites differ in having various locationsof functional groups. Some of these are part of the primary structure ofa compound and some of them are the result of metabolic processes. This de-termines, among other things, the varying thermal stability of benzodia-zepines, which should be taken into account during interpretation of spectraobtained by the GC/MS method.

Chromatographic analysis of benzodiazepines is frequently preceded bya derivatization step, which consists in replacement of the labile hydrogen ofactive functional groups by trimethylsilane groups [33, 38], acetyl groups[3], heptafluorobutyric groups [6, 7] and alkyl groups [17, 24]. The obtainedderivatives are thermally stable but they show (e.g. trimethylsilane ethers)sensitivity to traces of water. Therefore, reagents with a modified, spheri-cally extended alkyl system, which create derivatives that are more resis-tant to the hydrolysis process, are used for silanisation. Tert-butyldi-methylsilane ethers of oxazepam, temazepam, lorazepam, �-hydroxyalpra-zolam, �-hydroxytriazolam and 2-hydroxyethylflurazepam have excellentspectral properties, their mass spectra being characterised by the presenceof an intensive ion M��-57 [9, 20].

Readers interested in the methodology of benzodiazepines analysis areadvised to refer to a monographic publication [31], review articles [8, 23] andnumerous original papers.

AIM OF THE RESEARCH

The aim of this study was to work out conditions of separation and identi-fication of 18 benzodiazepines by the use of chromatographic techniques(GC/MS and GC/FID) and to apply the elaborated method to identification ofbenzodiazepines in patients’ urine samples. An additional aim was to evalu-ate the thermal durability of analysed benzodiazepines, because it was ob-served that some of them did not give an analytical signal in the form of anindividual peak. This indicates that in the injector of the chromatograph andalso during elution they are partially or totally decomposed. The phenome-non of thermal decomposition of benzodiazepines has been described earlier[18]. However, it seems to be advisable to define the quantitative nature ofthis phenomenon in the GC/MS system that has been applied in the de-scribed research.


MATERIALS AND METHODS

Standards and reagents

Standards of 7-aminoflunitrazepam, alprazolam, bromazepam, cama-zepam, chlorodiazepoxide, clobazam, clonazepam, nordiazepam, diazepam,estazolam, flunitrazepam, flurazepam, dipotassium clorazepat, loprazolam,lorazepam, lormetazepam, medazepam, nitrazepam, oxazepam, temaze-pam, tetrazepam, triazolam are from Sigma Chemicals Co. (St. Louis, USA).Their structures are presented in Figure 2.

Solvents and other reagents were produced by Merck KgaA (Darmstadt,Germany), Lancaster (Morecambe, United Kingdom) and Sigma ChemicalsCo. (St. Louis, USA) and they did not require additional purification.


Fig. 2. The structure of analysed benzodiazepines.

Standards of benzodiazepines were prepared as 1 mg/ml solutions ina mixture of methanol and ethyl acetate (1:4).

Materials

Urine of patients from the Detoxification Department (sample no. 3025)and Institute of Psychiatry and Neurology (samples no. 3526 and 4278) – inwhom screening analysis had revealed the presence of substances from thebenzodiazepine group – was used to verify the applied methodology.

Solid phase extraction

The extraction was performed by means of columns (Bond Elut CertifyExtraction Columns – Varian) with siliceous sorbent, which were located inan appliance with a regulated vacuum (Analitychem Vac Elut SPS 24TM –Varian). Columns were prepared by bathing under low pressure with 2 ml ofmethanol and 2 ml of 0.1 M phosphoric buffer (pH 6), and next 5 ml of centri-fuged urine mixed with 2 ml of 0.1 M phosphoric buffer was put onto a col-umn. The column was not allowed to dry. Columns were washed with1 M acetic acid (1 ml), dried for 5 minutes with maximum air flow, washedonce again with methanol (6 ml) and dried for 2 minutes. Elution was carriedout with 2% (v/v) solution of ammonia (25%) in ethyl acetate (2 ml). Theeluate was dried in a stream of dry nitrogen and the dry remains were dis-solved in 50 �l of ethyl acetate. 1 �l of extract was injected onto the capillarycolumn, which was the equivalent of 0.1 ml urine.

Acid hydrolysis of loprazolam

2 ml of water and 2 ml of hydrochloric acid were added to 2 mg ofloprazolam and heated for half an hour in a reverse cooler. The solution wasneutralised by solid sodium hydrocarbonate and extracted by methylenechlorine. The extract was evaporated to dryness, dissolved in 30 �l of ethylacetate and analysed by the chromatographic method.

Equipment

Gas chromatograph HP 5890, series II, with a flame ionisation

detector (FID)

The temperature of the injector was 250oC, and the detector: 280oC. He-lium was used as the carrier gas. Analytical conditions, which were differentfor various columns used for analysis, are presented in Table I.



TA

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ine-

flu

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26.4

521

.35

18.6

314

.69

16.7

628

3,25

5,55

4,24

0C

16H

14F

N3O

283.

11

(16)

alpr

azol

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–22

.74

g)16

.78

19.4

730

8,27

9,27

3,20

4,23

9C

17H

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lN4

308.

08

(10)

brom

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26.5

521

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19.1

314

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16.6

823

6,31

5,28

6,20

8,17

9C

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rN3O

315.

00

(17)

cam

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17.8

9g)21

.89

16.8

419

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271,

255,

299,

371

C19

H18

ClN

3O3

371.

10

(6)

chlo

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f)19

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f)20

.93

f)15

.90

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f)28

2,29

9,77

,241

,124

283,

282,

220,

247,

124

C16

H14

ClN

3O

C16

H14

ClN

3

299.

08

283.

09

(7)

clob

azam

23.9

519

.35

18.2

8g)

14.2

516

.04

300,

255,

77,2

83,2

31C

16H

13C

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230

0.07

(14)

clon

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.80

24.6

820

.57

g)15

.98

g)18

.55

g)28

0,31

4,31

5,28

6,23

4C

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10C

lN3O

331

5.04

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ium

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f)18

.80

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f)13

.78

f)15

.59

f)24

2,24

1,26

9,27

0,77

C16

H11

ClN

2O4K

240

8.97

(Ml)

nor

diaz

epam

23.2

518

.80

18.0

413

.81

15.6

224

2,24

1,29

6,27

0,77

C15

H11

ClN

2O27

0.05

(4)

diaz

epam

21.6

217

.56

17.8

013

.40

15.0

225

6,28

3,28

4,22

1C

16H

13C

lN2O

284.

07

(15)

esta

zola

m16

.43

19.0

925

9,20

5,29

4,23

9,10

1C

16H

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lN4

294.

07

(9)

flu

nit

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pam

24.8

019

.83

18.7

014

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16.5

231

2,28

5,31

3,26

6,23

8C

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N3O

331

3.09

(12)

flu

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pam

25.7

720

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20.4

415

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17.7

786

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387,

245,

315

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387.

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(B)

lopr

azol

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241,

276,

139,

111

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327

6.03

(3)

lora

zepa

m21

.27

f)17

.12

f)17

.46

f)13

.20

f)14

.78

f)23

9,27

4,30

2,75

C15

H10

Cl 2

N2O

232

0.01

(11)

lorm

etaz

epam

26.2

820

.79

–15

.05

17.1

830

3,30

7,11

1,33

4,75

C16

H12

Cl 2

N2O

233

4.03

(1)

med

azep

am18

.87

14.9

516

.38

12.0

013

.35

242,

207,

270,

165

C16

H15

ClN

227

0.09

(13)

nit

raze

pam

29.4

022

.98

20.3

9g)

15.5

3g)

17.8

4g)

253,

206,

234,

281,

222

C15

H11

N3O

328

1.08

(2)

oxaz

epam

20.2

3f)

16.0

0f)

17.0

0f)

12.6

3f)

14.1

7f)

268,

239,

205,

233,

77C

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6.05

(8)

tem

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.5f)

19.5

8f)

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256,

300,

239

C16

H13

ClN

2O2

300.

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(18)

tria

zola

m–

––

17.9

520

.54

313,

238,

342,

203,

279

C17

H12

Cl 2

N4

342.

04

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Gas chromatograph HP 6890 coupled to mass detector HP 5973

An HP-5MS capillary column, 30 m � 0.25 mm � 0.25 �m was used foranalysis of urine samples. The temperature of the injector was 250oC. He-lium was applied as the carrier gas and the flow capacity was 0.6 ml/min.Registration of the total ion current was carried out in the mass range from40 to 400 amu. The following oven temperature programme was applied: aninitial temperature of 110oC, which was maintained for 2 minutes, and thenwas increased by 15oC/min up to 255oC and by 8oC/min up to 300oC. The finaltemperature was maintained for 5 minutes. The duration of analysis wasabout 23 minutes. Analysis of standards was performed in split injection op-tion 1:50, and urine extract – in splitless injection option. The sample injec-tion volume V was 0.4 �l.

Types of tested columns and chromatographic parameters are presentedin Table I.

RESULTS AND DISCUSSION

Decomposition of benzodiazepines during chromatographicanalysis

During analysis of nitrazepam and clonazepam, apart from the peaks ofthese substances, other peaks were also observed, caused by products of theirpartial thermal decomposition: 7-aminonitrazepam and 7-aminoclonazepam.The chromatogram of clonazepam together with MS spectra of the primarycompound and the product of its decomposition are presented in Figure 3.


Fig. 3. Chromatographic peak of clonazepam – the primary compound and product ofits decomposition – 7-aminoclonazepam with MS spectra.

Thus, aromatic 7-nitro compounds (nitrazepam and clonazepam) arepartially reduced to appropriate amines during chromatographic analysis.This process depends on the type of column and the temperature of elution(Figure 4). A higher temperature and longer duration of stay of analyte ina column cause an increase in quantity of degradation products.

A single peak was obtained during chromatographic analysis of chloro-diazepoxide, whose mass spectrum corresponded to a compound of molecu-lar mass 283 amu. The molecular mass of chlorodiazepoxide is 299 amu.Therefore, it is the spectrum of the compound without an oxygen atom (Fig-ure 5).

One could conclude that chlorodiazepoxide, containing in its structure anN-oxygen system, undergoes thermal decomposition – this is a quantitativeprocess (Figure 6) that takes place in the injector during sample injectiononto a chromatographic column.

Mass spectra of lorazepam and oxazepam are characterised by molecularions m/z [ ]Moxazepam 18�

�� and m/z [ ]Mlorazepam 18�

��. Mass spectra of products ofdecomposition of both compounds are presented in Figures 7 and 8.


Fig. 5. Mass spectrum of product of thermal decomposition of chlorodiazepoxide.

Fig. 4. Thermal decomposition of nitrazepam (R = H) and clonazepam (R = Cl).

One could conclude on the basis of obtained spectra that compounds with�-hydroxyketone structure (lorazepam and oxazepam) undergo a thermal


Fig. 8. The mass spectrum of product of lorazepam decomposition.

Fig. 7. The mass spectrum of product of oxazepam decomposition.

Fig. 6. Thermal decomposition of chlorodiazepoxide.

regrouping process, in the course of which dehydration occurs leading to con-traction of the ring and creation of a durable aromatic structure (Figure 9).

Two well-separated peaks are present on chromatograms of lormetaze-pam and temazepam. The more intensive one corresponds to the product ofregrouping of the primary particle. The second one, which corresponds toa product with a 2,3-diketone grouping, is caused by decomposition. Its massspectrum is characterised by the presence of a molecular ion lighter by2 amu than the primary benzodiazepine molecule (Figures 10 and 11).


Fig. 10. Chromatogram of temazepam and MS spectrum of: a) – product of regroup-ing of primary molecule, b) – product of decomposition.

Fig. 9. Thermal regrouping (a) and decomposition (b) of oxazepam (R = H) andlorazepam (R = Cl).

Lormetazepam and temazepam, which contain N-methyl-�-hydroxy-ketone groups, lose a hydrogen atom during partial decomposition to �,�-di-ketones. Temazepam and lormetazepam, in spite of a structural similarityto their N-demethylation analogues (oxazepam and lorazepam), undergo de-composition by another mechanism (Figure 12).

The amount of benzodiazepine introduced onto the chromatographic col-umn is also important [18]. The shape of the peak is asymmetrical and the


Fig. 12. Thermal decomposition of temazepam (R = H) and lormetazepam (R = Cl).

Fig. 11. Chromatogram of lormetazepam and MS spectrum of: a) – product of re-grouping of primary molecule, b) – product of decomposition.

mass spectrum of the product of decomposition is practically invisible whena very small sample is injected.

Analysis of the dipotassium salt of clorazepat is noteworthy, because itrequires isolation from an acid environment – in contrast to all analysedbenzodiazepines in the presented research. The dipotassium salt of 7-chloro-5-phenyl-2,2-dihydroxy-1H-1,4-benzodiazepin-3-carboxy acid, undergoesdecarboxylation and dehydration during chromatographic analysis, beingtransformed into nordiazepam (Figure 13).

Loprazolam, due to its too high molecular mass, does not give a signal inthe form of a chromatographic peak, when injected on tested columns. How-ever, the product of its acid hydrolysis, 2-amine-5-nitro-2-chlorobenzo-phenone, gives an analytical signal in the form of a peak (Figure 14). Thismakes identification of loprazolam possible. It should, however, be remem-bered that the product of acid hydrolysis is identical to the product ofclonazepam hydrolysis. This may constitute an analytical trap for the per-son performing the analysis for forensic purposes.

The thermal decomposition of benzodiazepines during chromatographicanalysis constitutes an important analytical problem, which may lead to in-correct interpretation of results of forensic expert studies carried out at thebehest of the administration of justice, and thus potentially having an influ-ence on legal proceedings. In the case of studies using the GC/MS method,the basis of identification is the results of a performed instrumental analy-


Fig. 13. MS spectrum of nordiazepam, product of thermal decomposition of di-potassium clorazepat.

sis. These results indicate the presence of a particular compound in analysedevidence material (a mass spectrum is the basis of identification in massspectrometry). It is always necessary to prove that the product of thermaldecomposition of the analysed benzodiazepine which has been identified bythe mass detector is specific for the benzodiazepine that was injected ontothe chromatographic column. An expert who issues an opinion has to re-member that the mass spectrum of a product of decomposition only consti-tutes indirect evidence of the presence of a particular benzodiazepine in theevidence material. This is especially significant in a situation where the re-sult of identification of a particular compound can result in legal penalties asindicated in legislation.

Summarising, it should be concluded that:1. Partial decomposition of temazepam, nitrazepam, clonazepam and

oxazepam leads to obtaining chromatograms containing peaks of pri-mary compounds and peaks of products of decomposition.

2. Decomposition or regrouping of chlorodiazepoxide, lorazepam andclorazepatic acid leads to a chromatographically stable product.

3. Active functional groups in the structure of an analyte could cause ir-reversible adsorption on the stationary phase of a column and hence anincorrect peak shape (e.g. “tailing”). This concerns derivatives thatcontain hydroxyl groups and – to a lesser degree – primary and sec-ondary amine groups.


Fig. 14. The MS spectrum of the product of acid hydrolysis of loprazolam.

Separation of a complex mixture of benzodiazepines and theiridentification in urine

The effectiveness of separation of benzodiazepines was analysed on5 capillary columns which had various chromatographic parameters: length,internal diameter and thickness of stationary phase. The polarity of phasesof applied columns increased according to the following series: SPB-Octyl <SPB-1< HP-5MS < HP-35 < HP-30+. The absolute retention times of individ-ual compounds or products of their decomposition are presented in Table I.

Chromatograms of all thermally stable derivatives were obtained in ap-plied analytical conditions thanks to the use of columns with non-polar sta-tionary phases (SPB-1, SPB-Octyl, HP-5MS); the following compounds andproducts of their decomposition have similar retention times: nordiaze-pam/chlorodiazepoxide and alprazolam/camazepam on a column with anHP-5MS phase, flunitrazepam/bromazepam, camazepam/alprazolam on a col-umn with an SPB-1 phase and flurazepam/nitrazepam on a column with anSPB-Octyl phase. An increase in polarity of phase (HP-35 and HP-50+) hada negative influence on chromatographic parameters of analysed com-pounds: an increase in the breadth of the half height of peaks, a significantreduction in their intensity and the occurrence of so-called tailing.

Unsatisfactory results were obtained when derivatives with a triazolegroup were analysed, i.e. alprazolam, triazolam and estazolam.

The chromatogram of a mixture of 18 benzodiazepines obtained by theGC/MS method is presented in Figure 15.

Optimal conditions of separation of the above mentioned standard sub-stances were applied to analysis of biological material.

A chromatogram of the urine extract of a patient undergoing methadonetreatment (sample no. 3025) is presented in Figure 16.

A peak of nordiazepam and a peak of amphetamine were identified, andalso the following peaks: methadone and its metabolites, nicotine and itsmetabolites, promethazine and its metabolites and a metabolite of carbama-zepine (Table II). The presence of nordiazepam and amphetamine peaks isevidence that a patient did not comply with abstinence rules for patients un-dergoing methadone treatment, and the presence of promethazine and itsmetabolites was evidence that the patient took medicine not prescribed bya doctor. The presence of nicotine and its metabolites proves that the patientis a smoker. Methadone was administered to the patient at a dose of80 mg/day as part of therapy for patients addicted to opiates, and carma-zepine, whose metabolite is 9-formyloacrydyne, at a dose of 15 mg/kg of bodyweight per day in order to prevent convulsions.

Figures 17 and 18 present two chromatograms of urine extracts of pa-tients of the Clinic of Neurosis in Warsaw.



Fig. 16. Chromatogram of total ion current (TIC) of a patient’s urine extract (sampleno. 3025). Identified compounds are presented in Table II.

Fig. 15. Chromatogram of a mixture of eighteen benzodiazepine medicines.1 – medazepam; 2 – oxazepam; 3 – lorazepam; 4 – diazepam; 5 – dipotassium clora-zepat; 6 – chlorodiazepoxide; 7 – clobazam; 8 – temazepam; 9 – flunitrazepam;10 – bromazepam; 11 – lormetazepam; 12 – flurazepam; 13 – nitrazepam; 14 – clona-zepam; 15 – estazolam; 16 – alprazolam; 17 – camazepam, 18 – triazolam.

TABLE II. ANALYTICAL PARAMETERS OF COMPOUNDS IDENTIFIED IN ANALYSEDSAMPLE OF URINE (SAMPLE No. 3025)

No. CompoundReten-

tion time[min]

FormulaElementary ions

[m/z] in massspectruma)

1 Amphetamine 3.86 C9H13N 44, 91, 65, 120

2 Nicotine 5.97 C10H14N2 84, 133, 161, 162

3 Nornicotine 6.68 C9H12N2 119, 70147, 80

4 Cotinine 9.08 C10H12N2O 98, 176, 118, 119

5 3-hydroxecotinine 9.93 C10H12N2O2 106, 192, 135, 93

6Metabolite ofcarbamazepineb) 11.64 C14H9NO 179, 207, 178, 151

7 Methadone 12.30 C21H27NO 72, 165, 223, 294

8Metabolite ofmethadonec) 11.57 C20H23N 277, 276, 262, 220

9Metabolite ofmethadoned) 12.52 C18H19NO 265, 193, 115, 130

10 Promethazine 13.38 C17H20N2S 72, 180, 198, 213

11Metabolite ofpromethazinee) 13.04 C16H18N2S 213, 58, 198, 180

12Metabolite ofpromethazinef) 16.02 C16H18N2OS 212, 58, 180, 229

13Metabolite ofpromethazineg) 16.57 C17H20N2OS 72, 58, 213, 198

14 Nordiazepamh) 15.31 C15H11ClN2O 242, 241, 269, 270

a) – peak with the highest intensity on MS spectrum is underlined; b) – 9-formylacrydyne;c) – EDDP: 3,3-diphenyl-1,5-dimethyl-2-ethylidenepirolidyne; d) – 3,3-diphenyl-1,5-dimethyl-2-pirolidynon; e) – product of dimethylization of promethazine-norpromethazine; f) – product ofhydroxylation of norpromethazine; g) – sulphoxide of promethazine; h) –metabolite of diaze-pam.

The dominant peak of tetrazepam and three peaks corresponding to theamide of nicotinic acid, caffeine and theobromine (Table III) can be observedin Figure 17. The peak of tetrazepam constitutes evidence of taking of thispreparation by the patient.


TABLE III. ANALYTICAL PARAMETERS OF COMPOUNDS IDENTIFIED IN ANALYSEDSAMPLE OF URINE (SAMPLE No. 3526)

No. CompoundRetentiontime [min]

FormulaElementary ions

[m/z] in massspectruma)

1Amideof nicotinic acid

8.41 C6H6N2O 122, 78, 106, 51

2 Caffeine 12.11 C8H10N4O2 194, 109, 55, 67

3 Theobromine 12.24 C7H8N4O2 180, 55, 67, 109

4 Tetrazepam 16.64 C16H17ClN2O 253, 288, 259, 225

a) – peak with the highest intensity on MS spectrum is underlined.

The distinct peak of nordiazepam and four peaks corresponding to nico-tine and its three metabolites can be observed in Figure 18 (Table IV).

CONCLUSIONS

The developed conditions of analysis of 18 benzodiazepines in urine al-low – in contrast to screening methods – identification of individual benzo-diazepines present in urine, thus not only showing that a patient has failedto observe abstinence, but also enabling determination of which benzodiaz-


Fig. 17. Chromatogram of the total ion current (TIC) of extract of patient urine (sam-ple no. 3526). Identified compounds are presented in Table III.

epine has been taken. This is an important factor in disciplining the patientto maintain abstinence.

TABLE IV. ANALYTICAL PARAMETERS OF COMPOUDS IDENTIFIED IN ANALYSEDSAMPLE OF URINE (SAMPLE No. 4287)

No. CompoundRetention

time

[min]Formula

Elementary ions[m/z] in mass

spectruma)

1 Nicotine 6.02 C10H14N2 84, 133, 161, 162

2 Nornicotine 6.69 C9H12N2 119, 70, 147, 80

3 Cotinine 9.11 C10H12N2O 98, 176, 118, 119

4 3-hydroxycotinine 9.94 C10H12N2O2 106, 192, 135, 93

5 Nordiazepam 15.31 C15H11ClN2O 242, 241, 269, 270

a) – peak with the highest intensity on MS spectrum is underlined.

The applied analytical procedure also allows detection of many other psy-choactive compounds, which – beside benzodiazepine – can be found in theurine of drug addicts who do not observe abstinence.


Fig. 18. Chromatogram of total ion current (TIC) of extract of patient urine (sampleno. 4287). Identified compounds are presented in Table IV.

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ZASTOSOWANIE PO£¥CZONEJ CHROMATOGRAFII GAZOWEJI SPEKTROMETRII MASOWEJ (GC/MS) DO ANALIZYBENZODIAZEPIN

Dariusz B£ACHUT, Marta BYKAS-STRÊKOWSKA, Ewa TARACHA,

Bogdan SZUKALSKI

WSTÊP

Oprócz typowych narkotyków, jak heroina, kokaina, przetwory konopi indyjskichi pochodne fenyloizopropyloaminy (amfetaminy), nadu¿ywane s¹ równie¿ szerokostosowane leki, które oprócz ukierunkowanego dzia³ania farmakologicznego wywo-³uj¹ efekty euforyzuj¹ce i mog¹ doprowadziæ do uzale¿nienia. Do tej grupy nale¿¹benzodiazepiny, które wywo³uj¹ dobre samopoczucie (stan euforii, wyciszenie, uspo-kojenie), wzmacniaj¹ efekty psychotropowe innych substancji psychoaktywnych (ko-kainy, heroiny, pochodnych fenyloizopropyloaminy, alkoholu), a niektóre z nichdzia³aj¹ odurzaj¹co i s¹ wykorzystywane w celach przestêpczych jako narkotyki typudate rape drugs [36] s³u¿¹ce do osza³amiania ofiary w celu dokonania czynu zabro-nionego, np. gwa³tu, podstêpnego uzyskania numeru PIN-u, konta bankowego itp.

W odró¿nieniu od typowych narkotyków, których Ÿród³em pozyskiwania mo¿ebyæ wy³¹cznie nielegalny rynek, benzodiazepiny s¹ dostêpne w obrocie farmaceu-tycznym i stosunkowo ³atwe do zdobycia. Konsekwencj¹ stosowania leków benzodia-zepinowych do celów pozamedycznych by³o wprowadzenie regulacji prawnych do-tycz¹cych ich obrotu i stosowania. W Polsce ustawa o przeciwdzia³aniu narkomanii[10] objê³a kontrol¹ 34 pochodnych benzodiazepiny, wymieniaj¹c je w wykazie sub-stancji psychotropowych w grupie VI-P. Ustawodawca polski bardziej restrykcyjniepotraktowa³ flunitrazepam, zaliczaj¹c go do grupy III-P.

W zwi¹zku z szerokim stosowaniem benzodiazepin zarówno w terapii, jak i w ce-lach niemedycznych, opracowano liczne metody ich wykrywania oraz oznaczania.O ile jednak badanie skonfiskowanego materia³u dowodowego w postaci tableteki kapsu³ek nie sprawia na ogó³ wiêkszych trudnoœci analitycznych, to wykrywaniei identyfikacja poszczególnych benzodiazepin w moczu i krwi jest zadaniem trud-nym, wymagaj¹cym kilkuetapowego postêpowania analitycznego.

Szybkie wykrywanie obecnoœci benzodiazepin w materiale biologicznym umo¿li-wiaj¹ metody homogeniczne [2, 12, 39], w tym metoda enzymatyczno-immunologicz-na (EMIT), metoda fluorescencyjno-polaryzacyjna (FPIA) oraz metoda radioimmu-nologiczna (RIA). Ich zalet¹ jest prostota i szybkoœæ wykonania testu, bez k³opotliwe-go etapu ekstrakcji, a niekiedy równie¿ mo¿liwoœæ okreœlenia przybli¿onego stê¿eniaanalitu. Wad¹ jest niska specyficznoœæ powodowana zjawiskiem reaktywnoœci krzy-¿owej ze zwi¹zkami o podobnej budowie. Metody te pozwalaj¹ jedynie na grupow¹identyfikacjê klasy zwi¹zków oraz bardzo przybli¿one okreœlenie stê¿enia, które jestwypadkow¹ stê¿eñ i reaktywnoœci wszystkich substancji reaguj¹cych z testem, niedaj¹ natomiast mo¿liwoœci identyfikacji poszczególnych zwi¹zków. Problemem jesttak¿e zbyt wysoki poziom limitu detekcji okreœlany jako cut-off value, bowiem mo¿e

on okazaæ siê niewystarczaj¹cy przy monitoringu benzodiazepin, których dawki tera-peutyczne s¹ bardzo ma³e. Nale¿¹ do nich m.in. triazolam (dawka 0,125–0,25 mg,stê¿enie w osoczu 2–20 ng/ml), flunitrazepam (dawka 0,5–1 mg, stê¿enie w osoczu5–15 ng/ml) i loprazolam (dawka 0,5–1 mg, stê¿enie w osoczu 3–10 ng/ml) [4].

W laboratoriach kryminalistycznych do identyfikacji benzodiazepin stosuje siêrutynowo metody chromatograficzne, w tym chromatografiê cienkowarstwow¹ (TLC),wysokosprawn¹ chromatografiê cieczow¹ (HPLC), chromatografiê gazow¹ (GC),a tak¿e metody spektroskopowe – spektrometriê mas (MS) oraz spektrometriêw podczerwieni (FTIR). Techniki chromatograficzne (GC i HPLC), które pozwalaj¹rozdzielaæ z³o¿one mieszaniny benzodiazepin, a sprzê¿one z technikami spektrosko-powymi w uk³ady GC/MS [11, 29, 38] oraz HPLC/MS [21, 25, 37, 40], umo¿liwiaj¹identyfikacjê poszczególnych benzodiazepin w materiale biologicznym (mocz, krew,œlina, w³osy) nie tylko w tabletkach, ale tak¿e w mieszaninach z innymi substancjami.

Zwiêkszon¹ czu³oœæ i selektywnoœæ w stosunku do klasycznej detekcji MS uzys-kuje siê przy zastosowaniu tandemowej spektrometrii mas (MS/MS) [19, 22, 28] orazjonizacji chemicznej jonów ujemnych (NICI). W³aœciwoœci strukturalne benzodiaze-pin, tj. relatywnie du¿a zawartoœæ atomów azotu oraz halogenu (zwiêkszaj¹ca powi-nowactwo cz¹steczki do niskoenergetycznych elektronów) a tak¿e obecnoœæ skonden-sowanych pierœcieni (stabilizuj¹ca ³adunek ujemny jonu molekularnego) czyni¹ tezwi¹zki idealnymi przedmiotami badañ technik¹ GC-NICI. Song i in. [33] w oparciuo powy¿sz¹ technikê oznaczali klonazepam w osoczu z limitem detekcji 0,25 ng/ml.W oznaczeniu flurazepamu i jego metabolitów w osoczu szczura ci sami autorzy [34]osi¹gnêli jeszcze ni¿szy limit detekcji wynosz¹cy 0,1 ng/ml. Selektywnoœæ i czu³oœætechniki GC-NICI okaza³a siê szczególnie przydatna w oznaczaniu œladowych iloœciró¿nych pochodnych benzodiazepin i ich metabolitów we w³osach [6, 7, 27].

W organizmie zachodz¹ procesy metaboliczne benzodiazepin, które polegaj¹ nahydroksylacji fragmentów alifatycznych oraz pierœcieni aromatycznych, dezalkila-cji, redukcji grupy nitrowej i acetylacji [35]. W koñcowej fazie przemian metabolity,a tak¿e niektóre zwi¹zki macierzyste, ulegaj¹ sprzêganiu z kwasem glukuronowym.Przed przyst¹pieniem do analizy benzodiazepin metod¹ GC konieczne jest uwol-nienie ich z tych po³¹czeñ. Wykonuje siê to w oparciu o hydrolizê kwaœn¹ lub enzyma-tyczn¹.

Pierwsza z nich prowadzi do powstania benzofenonów, które charakteryzuj¹ siêwzglêdn¹ lotnoœci¹ i dobrymi w³aœciwoœciami chromatograficznymi [16]. Warto do-daæ, ¿e w latach 60. i 70. XX stulecia, kiedy dysponowano wy³¹cznie pakowanymi ko-lumnami chromatograficznymi o niestabilnych termicznie fazach stacjonarnych,przeprowadzenie szeregu benzodiazepin o wy¿szej masie cz¹steczkowej w znacznielotniejsze benzofenony by³o jedynym sposobem ich detekcji technik¹ GC. Mimo pros-toty wykonania, metoda ma jeden powa¿ny mankament: w wyniku hydrolizy szere-gu benzodiazepin powstaje jeden benzofenon (rycina 1), co uniemo¿liwia identyfi-kacjê zwi¹zku macierzystego.

W celu rozk³adu glukuronianu i uwolnienia benzodiazepiny o niezmienionejstrukturze, stosuje siê hydrolizê enzymatyczn¹ za pomoc¹ �-glukuronidazy. Enzymten dzia³a wy³¹cznie na wi¹zania �-glikozydowe glukuronianów, uwalniaj¹c zwi¹zeksprzê¿ony z kwasem glukuronowym.

Istotnym elementem cyklu analitycznego przy badaniu materia³u biologicznegojest w³aœciwa procedura oddzielenia analitu od matrycy próbki. Do izolacji analitów

Zastosowanie po³¹czonej chromatografii ... 31

z matrycy stosuje siê nadal konwencjonaln¹ ekstrakcjê ciecz-ciecz (LLE) z u¿yciemró¿nych rozpuszczalników [1], ekstrakcjê do fazy sta³ej (SPE) na kolumnach z faz¹oktadecylow¹ [14, 32], oktylow¹ [13], fenylow¹, cykloheksylow¹ [5] oraz z fazamimieszanymi [15, 26], a tak¿e mikroekstrakcjê do fazy sta³ej (SPME) [30]. Przy szero-kim skryningu materia³u biologicznego korzystniej jest zastosowaæ ekstrakcjê typuciecz-ciecz. Natomiast ekstrakcja do fazy sta³ej daje z regu³y lepsze rezultaty (czys-toœæ ekstraktu, odzysk) w przypadku metodyki ukierunkowanej na oznaczenie wy-branej pochodnej benzodiazepiny.

Pozytywny wynik uzyskany metodami homogenicznymi musi byæ zweryfikowa-ny wybran¹ technikê konfirmacyjn¹ (ang. confirmatory test). Jej wybór spoœród tech-nik chromatograficznych (TLC, GC, HPLC, EC) oraz technik ³¹czonych (GC/MS,HPLC/MS, EC/MS) zale¿y od charakteru analizy, wyposa¿enia w odpowiedni¹ apa-raturê oraz od inwencji osoby wykonuj¹cej analizê.

Benzodiazepiny i ich metabolity ró¿ni¹ siê inaczej usytuowanymi grupami funk-cyjnymi, z których czêœæ jest pierwotnym elementem struktury zwi¹zku, a czêœæ wy-nikiem procesów metabolicznych. Warunkuje to, miêdzy innymi, ró¿n¹ stabilnoœætermiczn¹ benzodiazepin, o czym nale¿y pamiêtaæ przy interpretacji widm uzyska-nych dziêki analizie metod¹ GC/MS.

Analizê chromatograficzn¹ benzodiazepin poprzedza zwykle proces derywaty-zacji, polegaj¹cy na wymianie labilnego wodoru aktywnych grup funkcyjnych na gru-py trójmetylosilanowe [33, 38], acetylowe [3], heptafluorobutyrowe [6, 7] oraz alki-lowe [17, 24]. Otrzymane pochodne s¹ termicznie trwa³e, jednak wykazuj¹ (np. eterytrójmetylosilanowe) wra¿liwoœæ na œlady wody. Z tego wzglêdu do silanizacji stosujesiê odczynniki ze zmodyfikowanym, sferycznie rozbudowanym uk³adem alkilowym,które tworz¹ pochodne bardziej odporne na proces hydrolizy. Doskona³e w³asnoœcispektralne posiadaj¹ etery tert-butylodimetylosilanowe oksazepamu, temazepamu,lorazepamu, �-hydroksyalprazolamu, �-hydroksytriazolamu i 2-hydroksyetylflu-razepamu, których widma masowe charakteryzuj¹ siê obecnoœci¹ intensywnego jonuo M��-57 [9, 20].

Czytelników zainteresowanych metodyk¹ analizy benzodiazepin odsy³amy doopracowañ monograficznych [31] i przegl¹dowych [8, 23] oraz licznych prac oryginal-nych.

CEL PRACY

Celem pracy by³o opracowanie warunków rozdzia³u i identyfikacji 18 benzodiaze-pin przy u¿yciu technik chromatograficznych (GC/MS i GC-FID) i zastosowanie tejmetody do identyfikacji benzodiazepin w próbkach moczu pacjentów. Dodatkowymcelem by³a ocena trwa³oœci termicznej badanych benzodiazepin, poniewa¿ stwierdzo-no, ¿e czêœæ z nich nie daje sygna³u analitycznego w postaci pojedynczego piku, cowskazuje, ¿e w komorze nastrzykowej chromatografu, a tak¿e w trakcie elucji, do-chodzi do ich czêœciowej lub ca³kowitej ich dekompozycji. Zjawisko termicznegorozk³adu benzodiazepin opisano wczeœniej [18], jednak wydawa³o siê wskazaneokreœlenie iloœciowej charakterystyki tego zjawiska w zastosowanym w opisanychbadaniach uk³adzie GC/MS.

32 D. B³achut, M. Bykas-Strêkowska, E. Taracha i in.

MATERIA£ I METODY

Wzorce i odczynniki

Wzorce 7-aminoflunitrazepamu, alprazolamu, bromazepamu, kamazepamu,chlorodiazepoksydu, klobazamu, klonazepamu, nordiazepamu, diazepamu, estazo-lamu, flunitrazepamu, flurazepamu, klorazepatu dipotasowego, loprazolamu, lora-zepamu, lormetazepamu, medazepamu, nitrazepamu, oxazepamu, temazepamu, te-trazepamu i triazolamu pochodzi³y z firmy Sigma Chemicals Co. (St. Louis, StanyZjednoczone). Ich budowê przedstawiono na rycinie 2.

Rozpuszczalniki oraz inne odczynniki pochodzi³y z firm: Merck KgaA (Darm-stadt, Niemcy), Lancaster (Morecambe, Wielka Brytania) oraz Sigma Chemicals Co.(St. Louis, Stany Zjednoczone) i nie wymaga³y dodatkowego oczyszczania.

Wzorce benzodiazepin przygotowano w postaci roztworów o stê¿eniu 1 mg/mlw mieszaninie metanolu i octanu etylu (1:4).

Materia³

Materia³em u¿ytym do weryfikacji zastosowanej metodyki by³ mocz pacjentówOddzia³u Detoksykacyjnego (próbka nr 3025) oraz Kliniki Nerwic (próbki nr 3526i 4278) Instytutu Psychiatrii i Neurologii w Warszawie, u których badanie skrynin-gowe wykaza³o obecnoœæ substancji z grupy benzodiazepin.

Ekstrakcja do fazy sta³ej

Ekstrakcjê prowadzono przy u¿yciu kolumienek (Bond Elut Certify Extration Co-lumns – Varian) z sorbentem krzemionkowym, umieszczonych w urz¹dzeniu z regu-lowan¹ pró¿ni¹ (Analitychem Vac Elut SPS 24TM – Varian). Kolumienki przygoto-wywano, przepuszczaj¹c pod obni¿onym ciœnieniem 2 ml metanolu i 2 ml 0,1 M bufo-ru fosforanowego (pH = 6), a nastêpnie, nie dopuszczaj¹c do wyschniêcia fazy, nano-szono 5 ml odwirowanego moczu zmieszanego z 2 ml 0,1 M buforu fosforanowego. Ko-lumienki p³ukano 1 M kwasem octowym (1 ml), suszono przez 5 minut przy maksy-malnym przep³ywie powietrza, ponownie p³ukano metanolem (6 ml) i suszono przez2 minuty. Elucjê prowadzono 2% (v/v) roztworem amoniaku (25%) w octanie etylu(2 ml), eluat odparowywano do sucha w strumieniu azotu, a such¹ pozosta³oœæ roz-puszczano w 50 �l octanu etylu. Na kolumnê chromatograficzn¹ podawano 1 �l eks-traktu, co odpowiada³o 0,1 ml moczu.

Hydroliza kwaœna loprazolamu

Do 2 mg loprazolamu dodano 2 ml wody oraz 2 ml kwasu solnego i ogrzewanoprzez pó³ godziny pod ch³odnic¹ zwrotn¹. Roztwór zobojêtniano sta³ym wodorowêgla-nem sodu i poddano ekstrakcji chlorkiem metylenu. Ekstrakt odparowano do sucha,rozpuszczono w 30 �l octanu etylu i poddano analizie chromatograficznej.


Aparatura

Chromatograf gazowy HP 5890, seria II, z detektorem p³omieniowo-joniza-

cyjnym (FID)

Temperatura komory nastrzykowej wynosi³a 250oC, a detektora 280oC. Jako gaznoœny stosowano hel. Warunki analizy, zale¿ne od rodzaju u¿ytej kolumny kapilar-nej, przedstawiono w opisie tabeli I.

Chromatograf gazowy HP 6890 sprzê¿ony z detektorem masowym HP 5973

Do analizy próbek moczu u¿yto kolumny kapilarnej HP-5MS, 30 m � 0,25 mm �

0,25 �m. Temperatura komory nastrzykowej wynosi³a 250oC. Gazem noœnym by³ hel,objêtoœæ przep³ywu wynosi³a 0,6 ml/min. Rejestracjê ca³kowitego pr¹du jonowegoprowadzono w zakresie mas od 40 do 400 amu. Temperaturê pieca zaprogramowanonastêpuj¹co: pocz¹tkowa temperatura 110oC utrzymywa³a siê przez 2 minuty, nas-têpnie wzrasta³a o 15oC/min do 255oC i o 8oC/min do 300oC i tê temperaturê utrzymy-wano przez 5 minut. Czas trwania analizy wynosi³ ok. 23 minut. Analizê wzorcówprowadzono w opcji nastrzyku split 1:50, a ekstraktu moczu – w opcji nastrzyku split-

less. Objêtoœæ nastrzyku V wynosi³a 0,4 �l.Rodzaje u¿ytych kolumn oraz parametry chromatograficzne przedstawiono w opisie

tabeli I.

WYNIKI I ICH OMÓWIENIE

Dekompozycja benzodiazepin podczas analizy chromatograficznej

Podczas analizy nitrazepamu i klonazepamu, oprócz pików macierzystych zwi¹z-ków, obserwowano dodatkowe piki produktów ich czêœciowego rozk³adu termiczne-go: 7-aminonitrazepamu i 7-aminoklonazepamu. Chromatogram klonazepamu wrazz widmami MS zwi¹zku macierzystego i produktu jego rozk³adu przedstawiono narycinie 3.

Tak wiêc aromatyczne 7-nitrozwi¹zki (nitrazepam i klonazepam) ulegaj¹ pod-czas procesu chromatograficznego czêœciowej redukcji do odpowiednich amin w stop-niu zale¿nym od rodzaju kolumny oraz temperatury elucji (rycina 4). Wy¿sza tempe-ratura i d³u¿szy czas przebywania analizowanej substancji na kolumnie wywo³ujewzrost iloœci produktów degradacji.

W trakcie analizy chromatograficznej chlorodiazepoksydu otrzymano pojedynczypik, którego widmo masowe odpowiada zwi¹zkowi o masie cz¹steczkowej 283 a.j.m.Masa cz¹steczkowa chlorodiazepoksydu wynosi 299 a.j.m, a zatem jest to widmozwi¹zku pozbawionego atomu tlenu (rycina 5).

Wynika z tego, ¿e chlorodiazepoksyd zawieraj¹cy w strukturze uk³ad N-tlenkowyulega przebiegaj¹cej iloœciowo termicznej deoksydacji (rycina 6), która zachodziw komorze nastrzykowej podczas wprowadzania zwi¹zku na kolumnê chromatogra-ficzn¹.

Widmo masowe lorazepamu i oxazepamu charakteryzuj¹ siê jonem molekular-nym o m/z [ ]Moksazepam 18�

�� oraz m/z [ ]Mlorazepam 18�

��. Widma MS produktów dekompo-zycji obu zwi¹zków przedstawiono na rycinie 7 i 8.


Na podstawie uzyskanych widm mo¿na stwierdziæ, ¿e zwi¹zki o strukturze �-hy-droksyketonów (lorazepam i oxazepam) ulegaj¹ procesowi przegrupowania termicz-nego, w trakcie którego zachodzi dehydratacja prowadz¹ca do kontrakcji pierœcieniai utworzenia trwa³ej aromatycznej struktury (rycina 9).

Na chromatogramach lormetazepamu i temazepamu widoczne s¹ dwa dobrzerozdzielone piki. Jeden, bardziej intensywny, odpowiada produktowi przegrupowa-nia cz¹steczki macierzystej, a drugi, odpowiadaj¹cy produktowi z ugrupowaniem2,3-diketonu, jest efektem dekompozycji. Jego widmo masowe charakteryzuje siêwystêpowaniem jonu molekularnego mniejszego o 2 ajm od macierzystej benzodiaze-piny (rycina 10 i 11).

Lormetazepam i temazepam, zawieraj¹ce uk³ad N-metylo-�-hydroksyketonu,ulegaj¹ czêœciowo rozk³adowi, trac¹c cz¹steczkê wodoru z utworzeniem �,�-dike-tonów.

Temazepam i lormetazepam, pomimo pokrewieñstwa strukturalnego z ich N-de-metylowanymi analogami – oxazepamem i lorazepamem – ulegaj¹ dekompozycjiwed³ug innego mechanizmu (rycina 12).

Istotna jest iloœæ benzodiazepiny wprowadzonej na kolumnê chromatograficzn¹[18]. Przy niewielkich iloœciach kszta³t otrzymanego piku jest niesymetryczny, a wid-mo masowe produktu dekompozycji praktycznie niewidoczne.

Na uwagê zas³uguje analiza soli dwupotasowej klorazepatu, wymagaj¹cej izolacjize œrodowiska kwaœnego, w przeciwieñstwie do wszystkich analizowanych w tej pra-cy benzodiazepin. Sól dipotasowa kwasu 7-chloro-5-fenylo-2,2-dihydroksy-1H- 1,4-ben-zodiazepino-3-karboksylowego, ulegaj¹c procesowi dekarboksylacji i dehydratacjiw warunkach chromatografii gazowej, przekszta³ca siê w nordiazepam (rycina 13).

Loprazolam, ze wzglêdu na zbyt du¿¹ masê cz¹steczkow¹, nie daje sygna³u w pos-taci piku chromatograficznego na testowanych kolumnach. Natomiast produkt jegokwaœnej hydrolizy, 2-amino-5-nitro-2-chlorobenzofenon, daje sygna³ analitycznyw postaci piku chromatograficznego o charakterystycznym widmie masowym (ryci-na 14). Umo¿liwia to identyfikacjê loprazolamu. Nale¿y jednak pamiêtaæ, ¿e pow-sta³y produkt kwaœnej hydrolizy jest identyczny z produktem hydrolizy klonazepa-mu. Mo¿e to stanowiæ pu³apkê analityczn¹ dla osoby wykonuj¹cej analizê do celówkryminalistycznych.

Termiczny rozk³ad benzodiazepin podczas analizy chromatograficznej stanowiwa¿ny problem analityczny, który mo¿e prowadziæ do b³êdnej interpretacji wynikówekspertyz wykonywanych na zlecenie organów sprawiedliwoœci, a wiêc mog¹cychmieæ wp³yw na wynik postêpowania s¹dowego. W przypadku badañ metod¹ GC/MSpodstaw¹ identyfikacji jest wynik przeprowadzonej analizy instrumentalnej, którywskazuje na obecnoœæ okreœlonego zwi¹zku w badanym materiale dowodowym(w spektrometrii mas jest to widmo masowe). Nale¿y zawsze udowodniæ, ¿e produktrozk³adu termicznego analizowanej benzodiazepiny, zidentyfikowany przez detek-tor masowy, jest specyficzny dla tej benzodiazepiny, która zosta³a wprowadzona nakolumnê chromatograficzn¹. Ekspert wydaj¹cy opiniê musi pamiêtaæ, ¿e widmo ma-sowe produktu rozk³adu stanowi tylko poœredni dowód obecnoœci okreœlonej benzo-diazepiny w materiale dowodowym. Jest to szczególnie istotne w sytuacji, gdy wynikidentyfikacji danego zwi¹zku poci¹ga za sob¹ sankcje prawne wynikaj¹ce z ustawy.


Podsumowuj¹c, nale¿y stwierdziæ, i¿:1. Czêœciowa dekompozycja temazepamu, nitrazepamu, klonazepamu i oxazepa-

mu prowadzi do otrzymania chromatogramów zawieraj¹cych piki zwi¹zkumacierzystego oraz piki produktów rozk³adu. Wyniki s¹ najczêœciej niepowta-rzalne i zale¿¹ od rodzaju zwi¹zku i zastosowanych warunków chromatogra-ficznych.

2. Rozk³ad lub przegrupowanie chlorodizepoksydu, lorazepamu i kwasu kloraze-patowego prowadzi do stabilnego chromatograficznie produktu.

3. Aktywne grupy funkcyjne w strukturze analitu mog¹ powodowaæ nieodwra-caln¹ adsorpcjê na fazie stacjonarnej kolumny i w efekcie nieprawid³owykszta³t piku (np. „ogonowanie”). Dotyczy to pochodnych zawieraj¹cych grupyhydroksylowe oraz – w mniejszym stopniu – pierwszo- i drugorzêdowe grupyaminowe.

Rozdzia³ z³o¿onej mieszaniny benzodiazepin i identyfikacjabenzodiazepin w moczu

Efektywnoœæ rozdzia³u benzodiazepin badano na 5 kolumnach kapilarnych o ró¿-nych parametrach chromatograficznych: d³ugoœci, œrednicy wewnêtrznej oraz gru-boœci fazy stacjonarnej. Polarnoœæ faz zastosowanych kolumn wzrasta³a zgodniez nastêpuj¹cym szeregiem: SPB-Octyl < SPB-1 < HP-5MS < HP-35 < HP-30+. Bez-wzglêdne czasy retencji poszczególnych substancji lub ich produktów rozk³aduprzedstawiono w tabeli I.

W zastosowanych warunkach analizy, dziêki u¿yciu kolumn z niepolarn¹ faz¹stacjonarn¹ (SPB-1, SPB-Oktyl, HP-5MS), otrzymano chromatogramy wszystkichtermicznie stabilnych pochodnych; zbli¿one czasy retencji maj¹ nastêpuj¹ce zwi¹zkii produkty ich rozk³adu: nordiazepam/chlorodiazepoksyd oraz alprazolam/kamaze-pam na kolumnie z faz¹ HP-5MS, flunitrazepam/ bromazepam, kamazepam/alpra-zolam na kolumnie z faz¹ SPB-1 oraz flurazepam/nitrazepam na kolumnie z faz¹SPB-Oktyl. Wzrost polarnoœci fazy (HP-35 i HP-50+) wp³ywa³ ujemnie na w³aœciwoœ-ci chromatograficzne badanych zwi¹zków: wzrost szerokoœci po³ówkowej pików,znaczne obni¿enie ich intensywnoœci i wystêpowanie tzw. „ogonowania”.

Nie uzyskano satysfakcjonuj¹cych wyników analizy pochodnych z uk³adem tria-zolowym, tj. alprazolamu, triazolamu i estazolamu.

Chromatogram mieszaniny 18 benzodiazepin otrzymany przy u¿yciu metodyGC/MS przedstawiono na rycinie 15.

Zoptymalizowane warunki rozdzia³u wy¿ej wymienionych substancji wzorco-wych zastosowano do analizy materia³u biologicznego.

Chromatogram ekstraktu moczu pacjenta uczestnicz¹cego w programie leczeniametadonem (próbka nr 3025) przedstawiono na rycinie 16.

Zidentyfikowano pik nordiazepamu oraz amfetaminy, a tak¿e piki: metadonui jego metabolitów, nikotyny i jej metabolitów, prometazyny i jej metabolitów orazmetabolit karbamazepiny (tabela II). Obecnoœæ piku nordiazepamu i amfetaminyœwiadczy o z³amaniu abstynencji obowi¹zuj¹cej pacjentów uczestnicz¹cych w pro-gramie metadonowym, a obecnoœæ prometazyny i jej metabolitów – o samowolnymprzyjêciu przez pacjenta preparatu leczniczego nie zleconego przez lekarza. Obec-noœæ nikotyny oraz jej metabolitów dowodzi, ¿e pacjent jest palaczem tytoniu. Meta-don podawano pacjentowi w dawce 80 mg/dobê w ramach terapii uzale¿nienia


od opiatów, a karmazepinê, której metabolitem jest 9-formyloakrydyna, w dawce15 mg/kg m.c. na dobê w celu zapobie¿enia drgawkom.

Ryciny 17 i 18 przedstawiaj¹ dwa chromatogramy ekstraktów moczu pacjentówleczonych w Klinice Nerwic w Warszawie.

Na rycinie 17 widaæ dominuj¹cy pik tetrazepamu oraz cztery piki odpowiadaj¹ceamidowi kwasu nikotynowego, kofeinie i teobrominie (tabela III). Pik tetrazepamustanowi dowód przyjêcia tego preparatu przez badanego pacjenta.

Na rycinie 18 widaæ wyraŸny pik nordiazepamu oraz cztery piki odpowiadaj¹cenikotynie i jej trzem metabolitom: nornikotynie, kotyninie, 3-hydroksynikotynie (ta-bela IV).

WNIOSKI

Opracowane warunki analizy 18 benzodiazepin w moczu pozwalaj¹ – w odró¿-nieniu od metod skryningowych – zidentyfikowaæ poszczególne benzodiazepinyobecne w moczu, a wiêc nie tylko stwierdziæ fakt z³amania przez pacjenta abstynen-cji, ale równie¿ ustaliæ, jak¹ benzodiazepinê przyj¹³. Stanowi to wa¿ny czynnik dys-cyplinuj¹cy pacjenta do zachowania abstynencji.

Zastosowana procedura analityczna umo¿liwia równie¿ wykrycie wielu innychzwi¹zków psychoaktywnych, które – obok benzodiazepin – mog¹ znaleŸæ siê w moczunieprzestrzegaj¹cych abstynencji narkomanów.


application of gas chromatography/ mass … · application of gas chromatography/ mass spectrometry...

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