Analytical procedures for determination of opiates in hair: a review

ELSEVIER

Forensic Science Intemationaf 70 (1995) 111-123

Forensic Science Internationid

Analytical procedures for determination of opiates in hair: a review C. Staub Institute of Forensic Medicine, University of Geneva, 1211-Geneva 4, Switzerland

Received8 May 1994;accepted13June 1994 Abstract This article reviews the analysis of opiates in hair. Hair matrix pretreatment, hydrolysis, extraction and detection procedures are presented amongst a study of over 70 bibliographic data. In addition, a new method for the extraction of opiates from hair, in which a powdered sample of hair is extracted directly by subcritical fluid, is presented. Keywords:

Hair analysis; Hair extraction; Review; Supercritical fluid extraction; Opiates

1. Inl.roduction At the end of 1970, Baumgartner et al. [l] presented a method for the analysis of opiates in hair. Since this initial study, a lot of literature data has been published, and it is sometimesdiffkult to choose the best method amongst the considerable variety of published analytical methods. The subject of this paper is the review of the published extraction and analysis techniques (70 publications) for opiates in hair. The way in which opiates accumulate in hair is a complex phenomenon and is still not fully understood. Several hypotheses [65] have been formulated and generally Abbreviations: AA, aceticanhydride; BSA, N,O-bis(trimethylsilyl)acetamide; BSTFA, N,O-bis(trimethylsilyl)triIluoroacetamide; CE, capillary electrophoresis; EIA, enzymeimmunoassay; EI, electronimpact ionization; FPIA, fluorescence polarisation immunoassay; GC-MS, gas-chromatography/mass spectrometry; HCI, hydrochloric acid; HFBA, heptafluorobutyric acid; HPLC, high performance liquid chromatography; HPTLC, high performance thin-layer chromatography; ITMS, ion trap massspectrometry; L, liquid-liquid extraction; 6-MAM, dmonoacetylmorphine; MBTFA, N-methylbistrifluoroacetamide; MeOH, methanol; mg, milligram; NCI, negative ion chemical ionization; ng, nanogram; PA, propionic anhydride; PFPA, pentafluoropropionic anhydride; pg, picogram; RIA, radio-immunoassay; SIM, selectedion monitoring; SP, solid-phase extraction; MS, massspectrometry; SFE, supercritical (subcritical) fluid extraction; TFAA, trifluoroacetic anhydride.

0379-0738/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0379-0738(94)01610-H

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it is assumed that drugs diffuse into the hair roots from the blood stream and are incorporated into the protein structure of the hair matrix. In order to simplify the study, we should essentially consider two distinct situations in which drugs could be present in hair: either by passive deposition from environmental contamination or by absorption from capillary blood circulating in the hair follicle following drug use. It is not possible to distinguish clearly between these two types of incorporation. Baumgartner et al. [ 1,6] have presented several washing procedures in order to eliminate passive deposition and therefore this discussion will focus on the extraction of opiates that have been accumulated into the hair by the blood after drug use. From a study of the literature data, it is evident that different methodological approaches have been proposed. These include pretreatment, ranging from simple elution with solvents, to complete dissolution of the protein structure by deep alkaline hydrolysis. After these pretreatments, different extraction techniques are described ranging from liquid-liquid extraction to so called solid-phase extraction. Finally, the detection methods are quite different, ranging from simple radio-immunoassay to very sophisticated techniques like MS-MS, via the very useful GC-MS technique. This review concentrates on this last technique because it is the most powerful tool for the identification and the quantification of opiates in hair. Finally, a new method for the extraction of opiates in hair by subcritical fluid (SFE) is presented. 2. Analytical

procedures for opiates in hair

2. I. Analysis of morphine

In 1980, Klug [2] presented a TLC method to detect morphine in the hair on heads of drug abusers. He dissolved the hair in sodium hydroxide, then hydrolysed the solution with hydrochloric acid and finally extracted the morphine with isoamyl alcohol making the identification by TLC. The first methods developed for the detection of morphine in human hair assumed that hair needed to be completely dissolved, thus allowing morphine to be extracted by solvent before its analysis. We find therefore in the literature (Table 1) 13 methods [4,7-9,14,15,23,28,29,43,44,63] using a pretreatment with a base like NaOH. The temperature varies from 45°C to 120°C and the concentration from 0.1 M to 1 M for a duration of between 5 min and 18 h. The most popular extraction technique is that of liquid-liquid, excepting that of immunoassays, which are generally applied directly to the neutralized basic solution. Solid-phase extractions range from various polar phases (silica like Extrelut), to combined phases containing reversed phase (Cs) and sulphonated cation exchange. These extraction procedures seem to give a cleaner extract for GC-MS determination [14,43]. An HPLC method to detect morphine in hair samples was reported by Marigo et al. [4], and afterwards, we find 14 methods [4,5,12,17,18,28,30,33,35,43,61,69] using a pretreatment with an acid like HCI. Here, the temperature varies from 45°C to 120°C and the concentration from 0.1 M to 2 M for a duration of between 30 min and 24 h. The liquid-liquid extraction method remains the most popular technique.

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Table 1 Analytical procedures for determination of morphine Pretreatment procedure Type Base Base Base Base Base Base Base Base Base Base Base Base Base Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid

Extraction technique

Detection

Reference

No

RIA

WI

No

RIA

171

No

RIA

1311

No

RIA

B91

No

FPIAIEIA

1631

No

FPIA

1231

L

HPLC

141

SP

GC-MS

[I41

L

GC-MS

171

L

GC-MS

[I51

L

GC-MS

WI

L

GC-MS

I'XW

SP

GC-MS

I431

No

RIA

WI

No

RIA

1331

L

RIA

WI

L

FPIA

[51

No

RIA

[691

L

HPLC

[41

L

HPLC

141

L

HPLC

u21

SP

CC-MS

11’31

L

CC-MS

WI1

SP

CC-MS

[431

Conditions NaOH 0.1 M 4YC, 18 h NaOH 1 M 8O”C, 30 min NaOH 1 M 8O”C, I h NaOH I M 100°C NaOH 1 M IOOYJ, 10 min NaOH 1 M IW’C, 1 h NaOH 0.6 M 120°C. 30 min NaOH 0.1 M WC, 18 h NaOH 1 M 8O”C, 30 min NaOH 1 M lOO”C, 5 min NaOH I M lWC, 10 min NaOH 1 M lOO“C, I h NaOH 1 M lOO“C, I h HCI 0.1 M 45”C, 18 h HCI 0.1 M 55”C, 12 h HCI 2 M 12O”C, 30 min HCI 0.1 M 45°C. 12 h HCI 0.1 M lOO”C, 3 h HCl 0.1 M 45”C, 12 h HCI 0.6 M lZO”C, 30 mitt HCI2M 120°C 30 min HCI 0.1 M 45”C, 24 h HCI 0.1 M 56T, 12 h HCI 2 M 12O”C, 30 min

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Table 1 (Continued) Pretreatment procedure Type Acid

Extraction technique

Detection

Reference

L

MS-MS

1171

L

C.E.

1351

L

ITMS

[301

No No L SP No SP SP

RIA RIA GC-MS GC-MS RIA GC-MS GC-MS

Ill 1281 1161

SP L

MS-MS TLC

I21

SP

HPTLC

1411

SP

GC-MS

19,11,511

SP

GC-MS

I131

Conditions HCl 0.1 M 12 h HCl 0.25 M 45”C, 12 h HCI 0.5 M 45”C, 16 h MeOH, 2 h MeGH 6O”C, 2 h MeGH 37°C 18 h MeOH 37°C 18 h Pronase Pronase Glucuronidase/arylsulfatase 45°C. 4 h Glucuronidase NaOH 3% HCI cont. NaOH HCI 80°C NaOH 3% HCI 25%, lOO”C, 30 min NaOH 2%, 30 min HCI2%, 12h 45T,

Acid Acid Solvent Solvent Solvent Solvent Enzyme Enzyme Enzyme Enzyme Mixed Mixed Mixed Mixed

1431 1311

P61 122,431

WI

At this time, three HPLC procedures are described; one from Staub and Robyr [ 121 using an electrochemical detector and two from Marigo et al. [4] using a fluorescence detector. For the latter types of detection, in spite of a higher recovery, alkaline pretreatment gave many spurious peaks which, though eluting after morphine, required extensive and time-consuming clean-up for the column. Acidic pretreatment therefore has been preferred for HPLC procedures. There are four methods [ 1,16,28,43]using a pretreatment with a solvent like MeOH. The temperature varies from 25°C to 60°C with a duration of between 2 and 18 h. In a comparison of methods [43], some preliminary results appear to show that recovery isn’t as good for morphine during a methanolic pretreatment. Four enzymatic methods are described [22,26,31,62], the most interesting appearing to be that of Ahrens et al. [22]. These authors mixed the pulverized hair sample with guanidine hydrochloride, in order to obtain denaturation of hair proteins and unfold the peptide chains, and then added 2-mercaptoethanol to obtain cleavage of the bisultide bonds before incubating with /3-glucuronidaselarylsulfatase at 45°C for 4 h. Solid-phase extraction gives very clean extracts for GC-MS analysis [43]. Finally, we find four different mixed methods that use a preliminary step with sodium hydroxide and a final step with hydrochloric acid. A high-performance thin-layer

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chromatographic technique (HPTLC) is described [41], where hair is incubated in sodium hydroxide and subsequently in hydrochloric acid at 80°C in both cases. After solid phase extraction on Extrelut, the solution is dansylated and quantified by densitometry. 2.2. Analysis of codeine (Table 2) The analysis of codeine is not problematic, and 13 procedures are given in Table 2. The chosen detection mode is mainly GC-MS (11/13 methods). Actually, there isn’t a specific radio-immunoassay for codeine available on the market. 2.3. Analysis of other opiates (Table 3) Heroin and 6-MAM. Goldberger et al. [ 161 have presented a gas chromatography/mass spectrometry (GUMS) assay for the detection and measurement of heroin and 6-acetylmorphine, Quantitation of each analyte is performed to determine the actual chemical form of heroin that is excreted into hair. The presence of heroin is difficult to confirm due to possible contamination by environmental exposure. Four procedures [16,22,43,61], can be found in the literature for the determinaTable 2 Analytical procedures for determination of codeine Pretreatment procedure Type

Conditions

Base

NaOH I M IWC, I h NaOH 0.1 M 6O”C, 18 h NaOH I M lOO”C, 5 min NaOH 1 M IWC, 1 h NaOH 1 M ICOT, 1 h HCI 0.1 M 45T, 24 h HCI 0.1 M 56T, 12 h HCI 2 M 120°C MeOH 37”C, 18 h MeOH 37”C, 18 h Glucuronidase/arylsulfatase 45”C, 4 h Glucuronidase NaOH 3% HCI 25%, lOO”C, 30 min

Base Base Base Base Acid Acid Acid Solvent Solvent Enzyme Enzyme Mixed

Extraction technique

Detection

Reference

No

FPIA

1231

SP

GC-MS

1141

L

GC-MS

(151

L

GC-MS

WY

SP

GC-MS

I431

SP

GC-MS

[I81

L

GC-MS

1611

SP

GC-MS

1431

L

GC-MS

1161

SP

GC-MS

I431

SP

GC-MS

I431

SP SP

MS-MS GC-MS

[621 HII

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Table 3 Analytical procedures for determination of other opiates Opiate

Pretreatment procedure Type

Conditions

Heroin 6-MAM CMAM 6-MAM

Solvent Solvent Solvent Enzyme

6-MAM

Acid

MeOH, 37°C 18 h MeOH, 37’C, 18 h MeOH, 37°C 18 h Glucuronidase/arylsulfatase 45”C, 4 h HCl O.lM 56°C. 12 h NaOH 1 M 100°C 1 h HCl 0.1 M 56°C. 12 h HC12 M 12O”C,30 min MeOH 37”C, 18 h Glucuronidase/arylsulfatase 45“C, 4 h HCl 30% NaOH NaOH

Ethylmorphine Base Ethylmorphine Acid Ethylmorphine Acid Ethylmorphine Solvent Ethylmorphine Enzyme Pholcodine

Mixed

Dihydrocodeine Base

Extraction technique

Detection mode

Reference

SP L SP SP

GC-MS GC-MS GC-MS GC-MS

(161

SP

GC-MS

I611

SP

GC-MS

1431

SP

GC-MS

[671

SP

GC-MS

[431

SP

GC-MS

[431

SP

GC-MS

[431

L

GC-MS

1271

L

GC-MS

WI

1161 [431

WWI

tion of 6,monoacetylmorphine (MAM), the first metabolite of heroin. Strong basic and acid incubations are poor procedures, because6-MAM is lost by hydrolysis and partially transformed into morphine. Methanolic and enzymatic incubations therefore are certainly the best procedures for the analysis of 6-MAM in hair. The procedure, given by the French Society of Analytical Toxicology [61], using a mild acidic (HCI, 0.1 M) incubation, seemsvery interesting. Furthermore, no data was found in the literature, demonstrating that 6-MAM is still not partially hydrolysed in these conditions. Ethylmorphine. Five procedures [43,67] are given for the analysis of ethylmorphine. There appears to be no preferred procedure, ethylmorphine being a sufficiently stable compound. Pholcodine and dihydrocodeine. One procedure for each compound is given in Table 3. 2.4. Derivatization

techniques for GC-MS detection (Table 4)

It is clearly shown in this review (Fig. I), that the GC-MS methods exceedby far, all other methods of detection used. Generally, derivatization steps are necessaryif relatively polar compounds, such opiates, are to be determined by GC-MS. In the papers reviewed, the following derivatization procedures are used: (1)

acetylation by acetic anhydride (AA);

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Table 4 CiC-MS methods for the analysis of opiates in hair Analyte

Derivative

Column

Ions detected (m/z)

Reference

Morphine Morphine Morphine Morphine Morphine Morphine Morphine Morphine Codeine Codeine Codeine Codeine Codeine Codeine Codeine Codeine 6-MAM 6-MAM 6-MAM 6-MAM Ethylmorphine Ethylmorphine Dihydrocodeine Pholcodine

AA BSTFA TFAA PA MBTFA PFPA HFBA BSA PFPA BSTFA BSA PA TFAA MBTFA HFBA AA MBTFA BSTFA PFPA PA PA BSTFA PFPA AA

ov-1 BP-5 DB-5 HP-5 HP-l DB-5 HP-l BP-5 DB-5 DB-5 HP-5 ov-1 HP-5 DB-5 HP-l DB-5 DB-5 DB-5 HP-l HP-l

369, 327, 429, 477, 397, 477, 577, 464,

[71 1151 1711 [431

(II) (III) (IV) (V) (VI) (VII)

268 236 364 341 364 414 207 429 445, 282 371, 234 371 355, 282 395, 282 395, 282 282, 225 341, 282 423, 364 399 473,414, 361 383, 327 369, 296 385 447, 284 Scan mode

1161

1221 1251

WI 1141 1151

1181 [431 1711 [161

1251 171 U61 1151

124 [431 [431 [671 1221

1271

propionylation by propionic anhydride (PA); this procedure is preferred to acetylation in order to discriminate morphine from 6-MAM; trifluoroacetylation by trifluoroacetic anhydride (TFAA); pentafluoropropionylation by pentafluoropropionic anhydride (PFPA); heptafluorobutylation by heptafluorobutyric acid (HFBA); silylation by N, O-bis(trimethylsily1) trifluoroacetamide (BSTFA) and by N, O-bis(trimethylsily1) acetamide (BSA); derivatization by N-methyl-bis-trifluoroacetamide (MBTFA).

Propionylation and acetylation, lead to stable derivatives with good GC properties. The derivatization mixture can be evaporated before analysis so that the resolution power of capillary columns does not decrease in contrast to other derivatization reagents (BSA, BSTFA). Furthermore, silylated polyfluoroacylated derivatives are unstable, especially in the presence of moisture. Halogenated derivatives would be useful if the opiates needed to be more sensitively detected by negative ion chemical ionization (NCI) mass spectrometry, but no paper dealing with opiates analysis in hair by NCI-MS was found in the literature data. 2.5. Subcritical fluid extraction of opiates All of the analytical procedures, presented in this review provide good results, but

118

40

35

30

25

20

15

10

5

0

C. Staub/Forensic Sri. ht. 70 (1995) III-123

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119

are long and fastidious. The use of subcritical fluids for the extraction of opiates in hair has been demonstrated by Edder et al. [43,70]. Subcritical fluid extraction (SFE) is generally faster (30-60 min) and milder than analytical procedures, including the incubation step. Furthermore, the conditions of extraction can be varied very simply by changes in pressure, temperature, or by the nature of the extractant phase (CO2 + polar modifiers). The SFE method using C0,/MeOH/Et,N/H20 (85:6:6:3 v/v) as an extractant phase is quantitative, is not 6-MAM destructive, and provides acceptable sensitivity and repeatability. The overall procedure is given in Fig. 2. In this method, the limit of detection (LOD) and quantification were determined from six replicates. The LOD and LOQ obtained for the four opiates tested [43] are the following: LOD is 30 pg/mg for codeine, ethylmorphine, morphine and 50 pg/mg for 6-MAM; LOQ is 0.1 ng/mg for codeine, ethylmorphine, morphine and 0.2 ng/mg for 6-MAM. These LOD and LOQ are comparable with those obtained by other techniques (601. pulverization

subcritical sznr, a -Y*eiction

______)

fluid (SFE)

______)

derivatization

-

GC-MS

_____)

Hair mill.

is pulverised

during

10 min.

CO+MeOH-ETsN-Hz0 (85/6/6/3) Pressure : 25 MPa Temperature : 40°C flow fate : 0.5-l mUmin. percolated in MeOH

with

a bait

v.v

100 pl propionic anhydride 100 pl pyridine 30 min. at 60-C evaporated and dissolved in 50 pl ethylacetate

SIM analysis

codeine mlz = 355,282 ethylmorphine m/z = 369,296 6-MAM m/z = 383,327 morphine m/z = 397,341 nalorphine (SC) m/z = 423,367

Fig. 2. Subcritical fluid extraction of opiates. Procedure from Edder et al. [43].

I20

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3. c0nc1usi0ns

This study of the literature data has shown that different methodological approaches are proposed. The choice of the best method is difficult. The most critical and important compound to be analysed however, is certainly 6-monoacetylmorphine (B-MAM). This is because 6-MAM might serve to determine heroin consumption, being the main metabolite of heroin found in hair and also because it is a labile compound that can easily be hydrolysed in strong basic and acid conditions. Methanolic and enzymatic incubations, therefore, are certainly the best procedures for hair pretreatment before extraction. Liquid-liquid and solid-phase extractions are comparable, but the latter might certainly be more easily automated and appears to give a cleaner extract for chromatographic analysis. Supercritical (or subcritical) fluid extraction (SFE), is a very attractive method which in the future could be standardised and should play an important role as a reference method. It would appear to be the method of choice. Coupled with GC-MS, it is a good technique for the detection of opiates in hair due to its specificity and sensitivity. Acknowledgements

The author thanks Emily Chapalay and Graham Lawrence for their assistance in preparation and typing of this Ifaper. References

111 A.M.

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1351 F. Tagliaro, Capillary electrophoresis for the investigation of illicit drugs in hair: determination of cocaine and morphine. J. Chromatogr., 638 (1993) 303-309. [361 A. Baumgartner and V. Hill, Hair analysis for drugs of abuse: decontamination issues. In I. Sunshine (ed.), Recent Development in Therapeutic Drug Monitoring and Clinical Toxicology, Marcel Dekker, New York, 1992, pp. 577-597.

122 [37]

1381 [39] [40] 1411 [42]

[43] [44] [45] [46] [47] [48] [49] 1501 [Sl] [52] [53] [54] (551 [56] [57] [58] [59] [60]

[61] [62]

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