Use of headspace solid-phase microextraction (HS-SPME) in hair analysis for organic compounds

Forensic Science International 107 (2000) 129–148 www.elsevier.com / locate / forsciint

Use of headspace solid-phase microextraction (HS-SPME) in hair analysis for organic compounds F. Sporkert, F. Pragst* Institute of Legal Medicine, Humboldt-University, Hannoversche Straße 6, D-10115 Berlin, Germany

Abstract Headspace solid phase microextraction (HS-SPME) has advantages of high purity of the extract, avoidance of organic solvents and simple technical manipulation and can be used in combination with gas chromatography–mass spectrometry (GC-MS) in the hair analysis of a number of drugs. HS-SPME coupled with the hydrolysis of the hair matrix by 4% sodium hydroxide in the presence of excess sodium sulphate and of a suitable internal standard proved to be a convenient one-step method for the measurement of many lipophilic basic drugs such as nicotine, amphetamine derivatives, local anaesthetics, phencyclidine, ketamine, methadone, diphenhydramine, tramadol, tricyclic antidepressants and phenothiazines. Detection limits were between 0.05 and 1.0 ng / mg. From spiked 10-mg hair samples absolute recoveries between 0.04 and 5.7% were found. These recoveries decreased considerably if larger sample amounts were used, perhaps due to increased drug solubility in the aqueous phase or to elevated viscosity in the presence of dissolved hair proteins. Because of the phenolic hydroxyl group a change of pH after alkaline hair digestion (by adding excess orthophosphoric acid) was necessary for the detection of D9 -tetrahydrocannabinol (D9 -THC), cannabinol (CBN) and cannabidiol (CBD) by HS-SPME. Nevertheless, the detection limits were such that only CBN could be detected in hair of a consumer. Clomethiazole, a compound hydrolysed in alkali, was measured by HS-SPME after extraction with aqueous buffer. The detection limit was 0.5 ng / mg. Cocaine could not be detected by HS-SPME. The application of HS-SPME to hair samples from several forensic and clinical cases is described.  2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Drug concentrations in hair; Hair analysis for drugs; Headspace solid phase microextraction (HS-SPME); Sample preparation for hair analysis; Solid phase microextraction (SPME)

1. Introduction Methods used in sample preparation for analysis of illicit or therapeutic drugs in hair *Corresponding author. 0379-0738 / 00 / $ – see front matter  2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0379-0738( 99 )00158-9

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generally involve extraction or digestion of the hair matrix and subsequent clean up by solid phase extraction [1]. Such methods involve several steps and a high degree of experience is needed to obtain reproducible results. On the other hand, in headspace gas chromatography the analyte is separated from the biological matrix in a simple way, but this method is limited to volatile compounds and there are problems in attempting to combine it with mass spectrometry because of the relatively large amount of air injected with the sample. Headspace-solid phase microextraction (HS-SPME) invented by Pawliszyn and coworkers [2,3] has proved to be a simple way of avoiding this problem and increasing sensitivity. The principle of HS-SPME is shown in Fig. 1. A fused silica fibre coated with a 7–100-mm layer of, for example, polyacrylate (PA), polydimethylsiloxan / polydivinylbenzene (PDMS / DVB) or polydivinylbenzene / polyethylenglycol (DVB / Carbowax) and protected in a stainless steel injection needle (SPME-device) is placed in the vapour phase of the headspace vial. The fibre is then exposed for a certain time at a certain temperature (adsorption time and adsorption temperature) and substances evaporated from the liquid or solid sample are absorbed into or adsorbed onto the layer. After that the fibre is retracted into the needle, which is injected into the injection port of the GC-MS. There the fiber is exposed again, and at the high temperature the substances are immediately desorbed for separation and identification. In toxicological analysis HS-SPME has been used in several cases for the detection and quantitation of volatile compounds from blood or urine [4–6]. But, surprisingly,

Fig. 1. Principle of headspace solid phase microextraction (HS-SPME) in hair analysis.

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compounds with a low volatility (‘semivolatile compounds’ [7,8]) such as amphetamines [9–11], phencyclidine [12], tri- and tetracyclic antidepressants [13,14], local anaesthetics [15,16], phenothiazines [17], diphenylmethane antihistamines [18] and some pesticides [19,20] can be measured by HS-SPME in body fluids with high sensitivity. SPME was used in hair analysis by Strano-Rossi and Chiarotti to detect cannabinoids, methadone and cocaine [21], not in the headspace mode but by direct immersion of the SPME fibre in the solution remaining after alkaline or enzymatic hair digestion. However, the analysis of amphetamine and methamphetamine from hair by HS-SPME was described by Koide et al. [22]. In a previous investigation [16] we found that the local anaesthetic lidocaine (lignocaine), which is frequently used as an adulterant of cocaine and cocaine–heroin mixtures, can be measured easily in 10-mg hair samples from drug consumers with a detection limit of less than 0.1 ng / mg using HS-SPME after alkaline hydrolysis of the matrix in the headspace vessel. In order to examine to what extent HS-SPME can be used as a general method in hair analysis for organic compounds, hair samples were investigated for a series of drugs, which were either naturally present from incorporation after intake or were added by spiking.

2. Materials and methods

2.1. Reference substances and reagents Therapeutic drugs used as reference substances were generously donated by the corresponding manufacturers. Samples of illicit drugs and deuterated standards were from Sigma or Promochem. All solvents and reagents were obtained from Merck / Darmstadt (Germany) in analytical purity.

2.2. Hair samples For the investigations with spiked samples a pool was prepared of head hair from volunteers who had not taken illicit or therapeutic drugs. The hair was washed (5 min) with deionised water and with acetone in an ultrasonic bath, dried, cut into pieces of 2–5 mm length and carefully mixed. The hair samples used for the analysis of drugs incorporated after intake were collected from volunteers who were under treatment with these drugs, or from forensic cases in which the history of drug ingestion was established from the case history and the results of the toxicological analysis. The samples were cleaned as described above. This investigation was approved by the ethics commission of the university hospital ´ Charite.

2.3. Sample preparation and solid phase microextraction The HS-SPME was carried out using a PDMS fibre (65 mm), a Carbowax / DVB fibre (65 mm) or a PA fibre (85 mm) and a fibre holder (Supelco). A heating block thermostat (Liebisch GmbH, Bielefeld, Germany) was used for temperature control of hair

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digestion and HS-SPME adsorption. Hair washing and ultrasonic extraction were performed in an ultrasonic bath Sonorex TK 52 of the firm Bandelin (Berlin). All HS-SPME experiments were performed in 4-ml vials with screw caps (Hewlett-Packard) and PTFE / silicone septa (Supelco). The measurements were carried out according to the following procedures.

2.3.1. HS-SPME with alkaline hair digestion Ten mg of washed and dried hair, 100 ng of internal standard, 0.5 g sodium sulphate and 1 ml 1 M sodium hydroxide were added to the headspace vessel. This was tightly closed and heated (30 min, 70–908C). The adsorption temperature was then adjusted to 60–908C depending on the analyte, the SPME needle was introduced and the fibre exposed for 15 or 20 min. After that the fibre was withdrawn into the needle and the SPME-device was transferred to the GC injection port. The desorption time and temperature were 5 min at 250 or 2908C, depending on the fibre used. During this time the fibre was fully regenerated for the next measurement and no carryover was observed. 2.3.2. Cannabinoid detection To detect D9 -tetrahydrocannabinol (THC), cannabinol, or cannabidiol the digestion of the hair sample was performed (30 min, 908C) as described above using D 3 -THC as internal standard but without addition of sodium sulphate. Then between 32 and 70 ml concentrated orthophosphoric acid were added to adjust the pH to 4–9 and a stream of nitrogen was passed through the solution for 5 min to remove hydrogen sulphide. After adding 0.5 g sodium sulphate, adsorption onto a PDMS / DVB fibre was carried out for 30 min at 1008C. 2.3.3. Clomethiazole After addition of N,N-diethylaniline (10 ng / mg, internal standard) 10 mg hair were extracted with 1 ml phosphate buffer (pH 6) in an ultrasonic bath (12 h). Then 0.5 g sodium sulphate were added and HS-SPME was carried out (608C, 15 min). 2.4. GC /MS The Hewlett-Packard GC-MS consisted of a HP 6890 series GC system and a HP 5973 mass selective detector. A HP capillary column (30 m30.25 mm i.d., 0.25 mm film, 95% dimethylsiloxane / 5% diphenylsiloxane) was used with a helium flow rate of 1 ml / min and one of the following temperature programs: I: hold at 508C for 2 min, then to 3008C at 208C / min, then hold at 3008C for 5 min — used for clomethiazole and nicotine; II: hold at 608C for 2 min, then to 2508C at 208C / min, then hold at 2508C for 7 min — used for lidocaine and etidocaine III: hold at 1008C for 2 min, then to 2058C at 308C / min, then to 2408C at 2.58C / min, then to 2908C at 308C / min — used for the general investigations about the effect of sample amount and drug structure (Sections 3.1 and 3.2) and for amitriptyline, diphenhydramine, doxepin, methadone, tramadol and trimipramine.

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Depending on the fibre, the injector temperature (5desorption temperature) was 2508C (Carbowax–DVB) or 2908C (PA). The temperatures of the GC / MS interface, of the ion source and of the quadrupole were 280, 230 and 1068C, respectively. The injection port was fitted with predrilled GC septa ‘Thermogreen LB2’ (Supelco). The mass-to-charge ratio values chosen from the mass spectra for the selected ion monitoring (SIM) of the drugs are given in Table 1. For calibration of the methods used for quantification of some drugs (Section 3.3) 10 mg of the hair pool were spiked with five drug concentrations between 0.5 and 10 ng / mg and 10 ng / mg of the corresponding internal standard. For nicotine and clomethiazole the concentration range was between 1 and 100 ng / mg and 1 and 300 ng / mg, respectively. In all cases linear calibration curves were obtained (r 2 50.9948 to 0.9993). The limits of detection and the limits of quantification were estimated from the signal-to-noise ratios of about 3:1 and 10:1 in the analysis of spiked samples. Table 1 Detection of drugs by HS-SPME and GC / MS from spiked hair samples a Compound

Nicotine Metamfepramone Phendimetracine Amfepramone Benzfetamine Diphenhydramine Ketamine Phencyclidine Doxylamine Tramadol Lidocaine Ethylbenzhydramine Chlorphenamine Methadone Methaqualone Amitriptyline Trimipramine Imipramine Doxepine Promethazine Dimetacrine D 9 -Methadone (ISTD) Clomethiazole N,N-Diethylaniline (ISTD) a

Retention time, min b

5.49 5.63 6.14 6.13 8.85 9.11 9.11 9.51 9.68 10.07 15.06 c 10.69 10.88 13.05 13.38 13.98 14.45 14.52 14.61 15.63 16.50 12.90 7.81 d 7.85 d

Detected masses, base peak first

84, 133, 161, 162 72, 120, 175 57, 85, 191 100, 72, 77 91, 148 58, 165, 73 180, 182, 209 200, 91, 243 58, 71, 167 58, 135, 263 86, 234 86, 58, 152, 165, 167 203, 58, 167 72, 223, 295 235, 233, 250 58, 202, 215 58, 234, 249 58, 193, 234, 280 58, 234 72, 180, 213, 284 58, 86, 279, 294 78, 303 112, 85, 161, 163 134, 77, 106, 149

Detection in 10 mg hair after spiking with ng / mg 0.1

0.3

1.0

3.0

10.0

2 1 1 1 1 1 1 2 2 1 1 1 2 1 2 1 2 2 2 2 2

2 1 1 1 1 1 1 1 1 1 1 1 2 1 2 1 1 1 1 1 2

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2

2

1

1

1

1, GC / MS-SIM signal is above the threefold noise of the base line; 2, not detected. GC temperature program III. c GC temperature program II. d GC temperature program I. b

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The within assay precision of the method was assessed using lidocaine as an example with etidocaine as the internal standard at 8.3% by repeated measurements with portions of the same homogenized hair sample (c510.9 ng / mg, n55).

3. Results and discussion Digestion of the hair matrix in alkaline solution, for example with 1 M sodium hydroxide, is one of the most efficient and convenient sample preparation methods if the analyte(s) are stable under these conditions. Examples are carbamazepine [23], amphetamines [24], and tricyclic antidepressants [25]. However, esters such as heroin or cocaine are hydrolysed under these conditions. An advantage of HS-SPME is that the dissolution of the hair matrix and the extraction can be combined in the same vessel without additional treatment. However, a general prerequisite for the use of HS-SPME from aqueous solutions is that the drug exists in a non-ionized state. In alkaline medium this is the case for basic and neutral drugs. Compounds containing –COOH, phenolic –OH or –SH groups are generally excluded from the one-step digestion / HS-SPME method and need pH adjustment prior to HS-SPME.

3.1. Optimization of experimental conditions 3.1.1. Composition of the solution for hair digestion Besides the hydrolysis of the hair matrix any added reagent should also increase the volatility of the drug by a salting-out effect. Therefore the 4% w / v (1 M) sodium hydroxide solution generally used for hair dissolution was compared with 30% sodium hydroxide and 4% sodium hydroxide1sodium sulphate (0.5 g / 1 ml) using lidocaine, etidocaine and propipocaine as test compounds. The results are shown in Fig. 2. Adding excess sodium sulphate to 4% sodium hydroxide gave a higher extraction yield than increasing the NaOH concentration to 30%. The addition of excess ammonium sulphate, sodium chloride, potassium carbonate, sodium bicarbonate and other salts has been described as an effective way of improving recoveries from blood or urine samples [8]. Ammonium sulphate should lead to a decrease of pH and is therefore not useful for stronger basic compounds. Perhaps for this reason Watanabe et al. [15] found a decrease of the recovery of local anaesthetics after adding ammonium sulphate to the alkaline blood sample. Therefore, ammonium sulphate should not be as generally advantageous as sodium sulphate, and all further experiments were performed using the 4% sodium hydroxide / sodium sulphate combination. In order to examine if the dissolution of hair is hampered in the presence of sodium sulphate, a hair sample with lidocaine incorporated from regular intake was analyzed with addition of the salt (i) before and (ii) after sodium hydroxide treatment. There was no significant difference between both measurements. Therefore in all further investigations both reagents were added at the beginning of the procedure. 3.1.2. Sample amount In SPME the partition equilibrium between the analyte and the coating of the fibre depends in part on the solubility of the analyte in the sample matrix, which can be

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Fig. 2. Effect of the reagents used for hair digestion and HS-SPME on the GC / MS abundance of the local anaesthetics lidocaine, propipocaine and etidocaine. HS-SPME conditions: fiber carbowax–DVB 65 mm, adsorption: 15 min at 708C, m /e-values measured in GC / MS-SIM: propipocaine 121, 163 and 178, lidocaine 86 and 234, etidocaine 86, 128, 247.

strongly affected by other components of the solution [2]. Therefore the amount of the organic products formed after alkaline hydrolysis of hair might have an important effect on the extraction yield. In order to examine this effect, the compounds given in Table 1 were analyzed in the absence and in the presence of 10 mg and of 50 mg of the hair pool. The results are shown in Fig. 3 for 10 substances, which were ordered by GC

Fig. 3. Effect of the hair sample amount on the GC / MS peak area of some drugs after HS-SPME from 1 ml 4% NaOH10.5 g Na 2 SO 4 . All samples were spiked with 50 ng of each drug. 100%5peak area in absence of hair. HS-SPME conditions: fiber PA 85 mm, adsorption 15 min at 708C.

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retention time. With the exception of metamfepramone, amfepramone and phendimetrazine, in the presence of 10 mg hair the peak area was decreased to 8–60% of its value in the absence of hair (100%), and with 50 mg hair for all compounds only between 2 and 38% of the original peak area was found. Thus, the use of 50 mg rather than 10 mg hair does not lead to the expected increase of the GC-MS signal by a factor of 5 but at maximum only by a factor of 2, and for diphenhydramine, chlorphenamine, methadone and imipramine a decrease of the peak area to between 0.6 and 0.75 of its value with 10 mg hair sample was found. Furthermore it is obvious from Fig. 3 that the effect of the hair matrix increases with increasing retention time, i.e. with decreasing volatility of the compounds. A similar effect of the biological sample material was described also by Nagasawa et al. [10], who found a decrease of the recovery of amphetamine and methamphetamine from blood to 27 to 58% when compared to aqueous solution. Thus, it seems that the solubility of the drugs in the sample matrix even in the presence of excess sodium sulphate is increased by the products of hair hydrolysis. But there may be also kinetic reasons, since the viscosity of the solution is noticeably higher after hair hydrolysis. From the analytical point of view the sample amount used should be as small as possible. Koide et al. measured amphetamine and methamphetamine by HS-SPME from about 1 mg hair with detection limits of 0.1 and 0.4 ng / mg, respectively [22]. For a systematic investigation of the method 10 mg seems to be a reasonable sample amount. Furthermore it follows that calibration and analysis must always be carried out with the same sample amount since analyte and internal standard may be affected by the matrix to a different extent. However, this should be negligible with deuterated standards.

3.1.3. Adsorption temperature and adsorption time The adsorption temperature and the adsorption time are important influences on the sensitivity of the HS-SPME method. On one hand an increased temperature leads to a higher concentration of the drugs in the vapour phase. On the other hand the adsorption at the fibre is diminished, and hydrolysis of the analyte(s) in the alkaline medium becomes more probable. In Fig. 4 the effect of the temperature on the GC / MS abundance is shown for the measurement of lidocaine with etidocaine as internal standard and of amitriptyline with dimetacrine as internal standard. For lidocaine, 708C was chosen as the optimum temperature, whereas for amitriptyline because of its lower volatility a steady increase up to 1208C was found. In the literature adsorption temperatures between 558C for amphetamines [22] and 1408C for tetracyclic antidepressants [14] were described. We found that at 1208C the measurement was strongly disturbed by the high pressure in the headspace vial. For this reason we chose to use 908C for tricyclic antidepressant assay. The effect of adsorption time on the GC / MS abundance is shown for the same two substance pairs in Fig. 5. In the case of the more volatile lidocaine and etidocaine, after 10 min no significant increase in recovery was found, and 15 min was chosen as the incubation time. However, for the less volatile amitriptyline and dimetracine a steady increase up to 50 min occurred. Namera et al. described an increase up to 60 min for tetracyclic antidepressants [14]. In such cases a compromise between sensitivity and time efficiency of the method is necessary. For tricyclic antidepressants an adsorption

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Fig. 4. Effect of adsorption temperature on the GC / MS-SIM abundance in HS-SPME sample preparation. (A) Lidocaine with etidocaine as internal standard, fiber 65 mm Carbowax–DVB, adsorption time 15 min. (B) Amitriptyline with dimetacrine as internal standard, fiber 85 mm PA, adsorption time 15 min.

time of 20 min (908C) was chosen and appeared to be adequate, although under these conditions equilibrium is not attained.

3.2. Effect of drug structure A mixture of drugs (Table 1) at five concentrations (0.1, 0.3, 1.0, 3.0 and 10.0 ng / mg hair) was added to 10 mg of the hair pool and the analysis was carried out according to the procedure given in the experimental part. For comparison the drug mixture was also analyzed in the absence of hair (10 ng of each drug, corresponding to the 1.0 ng / mg measurement). Furthermore, for the measurement of the absolute HS-SPME recoveries the mixture (10 ng of each drug) was directly injected into the GC column.

Fig. 5. Effect of adsorption time on the GC / MS-SIM abundance in HS-SPME sample preparation. (A) Lidocaine with etidocaine as internal standard, fiber 65 mm Carbowax–DVB, adsorption temperature 708C. (B) Amitriptyline with dimetacrine as internal standard, fiber 85 mm PA, adsorption temperature 908C.

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Mass spectrometric detection was carried out in nine time windows in the selected ion monitoring (SIM) mode. The mass-to-charge values of the ions used for the detection as well as the retention times are given in Table 1 together with data showing the concentrations at which each drug was detected with a signal / noise ratio .3. The chromatogram obtained from the 1.0 ng / mg spiked solution is shown in Fig. 6. Most compounds were detected down to 0.3 or 0.1 ng / mg. Methaqualone and dimetacrine were detected only at 1.0 ng / mg. But there are some compounds, not listed in Table 1, which were not detected in preliminary experiments from a solution of 100 ng / ml in the absence of hair (corresponding to 10 ng / mg): carbamazepine, phenazone, caffeine and maprotiline. Maprotiline is a secondary amine and is poorly eluted unless derivatized on the GC column used. In principle, secondary amines should also be detected with high sensitivity by HS-SPME, as was shown for blood samples by Namera et al. [14], who used a more suitable GC column. Carbamazepine and caffeine are too hydrophilic for good recoveries to be obtained with HS-SPME. The absolute recovery of the HS-SPME measurement assesses the efficiency of the extraction. It is calculated by comparison of the peak area from the HS-SPME experiment with that obtained from the direct injection of the compound into the chromatographic system. The results for 17 compounds obtained in a single measurement in the absence of hair and in four measurements at four different concentrations (0.3, 1.0, 3.0 and 10 ng / mg) in the presence of 10 mg of the hair pool are shown in Table 2 together with literature data about the boiling point at reduced pressure. The recoveries from the spiked hair samples were between 0.04 and 5.7%. For some compounds considerable differences were found between the four values. Possible reasons for this are that within the measurement series the extraction ability of the fibre decreased or that due to the manual performance of HS-SPME the experimental conditions (position of the fibre in the vessel, agitation of the solution, temperature) were

Fig. 6. GC / MS-SIM chromatogram of a 10-mg hair sample (non-smoker) spiked with 16 drugs after HS-SPME sample preparation. Concentrations: 1 ng / mg, ethylbenzhydramine (peak 11, internal standard) 10 ng / mg, nicotine not added. Fiber: PA 85 mm. Adsorption: 15 min at 708C. SIM measurement in nine time windows using the masses given in Table 1.

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Table 2 Absolute HS-SPME recovery of some drugs from spiked solutions in the absence and in the presence of 10 mg hair Drug

Metamfepramone Phendimetracine Amfepramone Benzfetamine Diphenhydramine Ketamine Phencyclidine Doxylamine Tramadol Chlorphenamine Methadone Methaqualone Amitriptyline Imipramine Promethazine

Absolute recovery, % a

Boiling temperature (8C) at reduced pressure [38]

Without hair b

With 10 mg hair pool c

1.3 2.7 1.9 18 5.6 0.2 15 14.5 18 9.9 4.5 0.07 6.2 5.0 3.3

1.7–2.6 2.4–3.4 1.2–2.8 4.8–5.7 0.7–1.7 0.17–0.32 1.8–3.4 1.5–2.5 1.7–2.1 0.8–1.2 1.4–2.0 0.04–0.06 0.4–2.3 0.9–1.4 0.4–0.8

126 (1.73 kPa) 123 (1.07 kPa) No data 127 (2.7 Pa) 150–165 (266 Pa) No data 135–137 (133 Pa) 137–141 (66 Pa) No data 142 (133 Pa) No data No data No data 160 (13 Pa) 190 (400 Pa)

a

Calculated by comparison with direct injection of the same amount. One measurement. c Four measurements (concentrations 0.3, 1.0, 3.0 and 10 ng / mg). b

not held constant. Variation of the experimental conditions could have particularly serious effects on the absolute recovery for those compounds where the extraction equilibrium was not attained at 708C within the 15 min applied in this comparative study. Because of this variation of the absolute recoveries HS-SPME cannot be used for quantitative work without an internal standard. But by using an internal standard with a similar chemical structure the relative recoveries were found to be quite reproducible, as shown by the good linearity of the calibration curves for the compounds investigated in Section 3.3. The absolute recoveries found in this study are similar to those described in the literature for HS-SPME analysis of blood and urine. Watanabe et al. found recoveries between 0.6 and 8.5% for five local anaesthetics [15] and Namera et al. described absolute extraction yields between 0.12 and 0.53% for tetracyclic antidepressants [14]. Koida et al. reported 48 to 62% recovery in HS-SPME analysis of amphetamine and methamphetamine from hair [22]. Such absolute headspace extraction yields are surprisingly high since the boiling temperature of the majority of the compounds after extrapolation to normal atmospheric pressure should be well above 3008C. Perhaps transport of compounds in droplets facilitates analyte equilibration with the fibre coating. From the structural point of view, most of the semi-volatile compounds with a good HS-SPME recovery from aqueous medium described in the literature or in this paper have an aliphatic or an alicyclic group in the molecule which may contribute to both lipophilicity and surface activity.

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3.3. Application to hair samples from forensic or clinical cases The method described above for spiked hair samples was applied to clinical and forensic cases, in which a regular abuse or a regular therapeutic intake of the drugs was suspected. Some examples of the use of the method for different drugs are presented. The results of all cases are given in Table 3. All of these compounds are tertiary amines, which are known to be metabolized to the corresponding N-desalkyl metabolites. Most of these metabolites have been found in hair in previous investigations by GC / MS of derivatized extracts [25]. However, these secondary amines were not detected after HS-SPME in any case. We assume that this was due to the low sensitivity of (underivatized) GC / MS detection rather than a decrease in the HS-SPME recovery. Amphetamine, methamphetamine and the ecstasy drugs MDMA and MDE are effectively deposited in hair and are regularly controlled by hair analysis for drug abuse [24]. In principle these compounds can be measured by HS-SPME as was shown for blood and urine by Yashaki et al. [9], Nagasawe et al. [10] and Centini et al. [11]. Koide et al. measured amphetamine and methamphetamine with a detection limit of 0.1 and 0.4 ng / mg, respectively, from only 1 mg hair after digestion in 200 ml 5 M sodium hydroxide and 20 min headspace adsorption at 558C with a 100-mm PDMS fibre [22]. In our preliminary investigations the detection of these compounds by HS-SPME and GC-MS was confirmed, but the chromatographic conditions were not suitable for separation of these primary or secondary amines without derivatization. Table 3 Results of the hair analysis of some forensic or clinical cases with known consumption of drugs. Analysis by HS-SPME from 10 mg hair in 1 ml 4% NaOH10.5 g Na 2 SO 4 and GC / MS-SIM Drug

Internal standard

HS-SPME conditions

LOD/LOQ a ng/mg

Case no.

Age, sex

Hair concentration, ng/mg

Amitriptyline

Dimetacrine

PA, 908C, 20 min

0.05/0.15

Clomethiazole

N,N-Diethylaniline

0.5/1.7

Diphenhydramine Doxepine

Ethylbenzhydramine

PDMS/DVB 608C, 15 min PA, 808C, 20 min

Dimetacrine

PA, 908C, 20 min

0.2/0.7

31f 53f 56f 44m 48m 57m 28m 60f 37f

Lidocaine

Etidocaine

0.1/0.4

Methadone

D 9 -Methadone

Carbowax–DVB, 708C, 15 min PA, 808C, 20 min

Nicotine

N,N-Diethylaniline

PA, 608C, 15 min

1/3.5

Tramadol

Ethylbenzhydramine

PA, 908C, 20 min

0.1/0.4

Trimipramine

Dimetacrine

PA, 908C, 20 min

0.2/0.7

T336/97 T380/97 T164/99 T156/99 T167/99 T098/97 T025/97 Z999/99 T141/99 49 drug fatalities T096/97 T264/97 T096/97 T264/97 T463/96 T035/96 T463/96 T051/97 T164/99

4.84 0.22 0.29 15.3 115 2.0 3.7 6.1 3.2 0.4–675 in 32 cases, see Fig. 8 20.1 9.9 135 334 19.6 0.78 1.14 0.56 10.4

a

0.05/0.15

0.1/0.4

LOD, limit of detection; LOQ, limit of quantification.

38m 29m 38m 29m 81f 50m 81f 69f 56f

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3.3.1. Nicotine Hair analysis for nicotine and cotinine has been used to differentiate between smokers, passive smokers and non-smokers [26,27] or to identify exposure of neonates to nicotine in utero [28,29]. Hair concentrations of nicotine in smokers were between 0.4 and 63.5 ng / mg. Use of HS-SPME for measuring nicotine and cotinine in urine was described by Yashaki et al. [30]. In our investigations N,N-diethylaniline was used as the internal standard. The SIM chromatogram of the hair sample of a smoker (135 ng / mg) and a passive smoker (19.6 ng / mg) are shown in Fig. 7. A detection limit of 1 ng / mg was found with 10 mg hair. This is relatively high compared to the other less volatile compounds (Table 3) and is caused by a higher background in the first part of the chromatograms. Cotinine was not detected either in these samples or in spiked samples in the presence or in the absence of hair, although it was found to be stable in sodium hydroxide solution. Therefore, it must be assumed that it is too hydrophilic for HS-SPME analysis. 3.3.2. Lidocaine The analysis of the hair samples of 49 drug fatalities for lidocaine by HS-SPME was described in detail elsewhere [16]. As internal standard etidocaine proved to be most suitable, and a detection limit of 0.1 ng / mg was obtained using a 10-mg sample. Lidocaine was found in 32 of the 49 cases with concentrations between 0.4 and 300 ng / mg (and 675 ng / mg in one case) (Fig. 8). The comparison with the concentrations of cocaine and opiates showed that lidocaine was very often consumed as an adulterant not only in cocaine, but also in cocaine–heroin mixtures. In Berlin between 1994 and 1998 several deaths were caused primarily by a bolus injection of lidocaine [31]. Like lidocaine and etidocaine, propipocaine and several other local anaesthetics of the carboxylamide type can be measured by HS-SPME, as shown for blood samples by Watanabe et al. [15]. Normally, the amide group strongly increases the hydrophilic

Fig. 7. GC / MS-SIM chromatogram of the hair samples of a smoker and a passive smoker after HS-SPME with N,N-diethylaniline (100 ng / mg) as internal standard. m /z values cf. Table 1.

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Fig. 8. Hair concentrations of lidocaine in 49 drug fatalities determined by HS-SPME and GC / MS with etidocaine as internal standard [16]. HS-SPME conditions: fiber 65 mm Carbowax–DVB, adsorption 15 min at 708C.

properties and should decrease the volatility. Obviously in these compounds the neighboring substituents diminish the solvatation by shielding the amide group.

3.3.3. Diphenhydramine In Germany diphenhydramine is frequently used as a mild hypnotic and it can be obtained without prescription. There are no literature data about the analysis of this compound in hair. For the HS-SPME ethylbenzhydramine is a suitable internal standard since it has a very similar structure to diphenhydramine, and it is not available for therapeutic use. In two hair samples from fatalities, for which the intake of diphenhydramine was known from the case history, hair concentrations of 2.0 ng / mg and 3.7 ng / mg were found. The SIM chromatogram of one of these samples is shown in Fig. 9A. In the SIM mode the detection limit was 0.05 ng / mg. 3.3.4. Tramadol Tramadol is an opioid analgesic prescribed for pain control, e.g. for cancer patients. Rickert and Daldrup found it by methanol extraction, derivatization with MSTFA and GC-MS in the hair sample of a case with supposed heavy abuse (80 ng / mg) and in a case with therapeutic treatment (0.22 ng / mg) [32]. Tramadol has an alicyclic hydroxyl group, but perhaps due to steric effects this does not prevent the use of HS-SPME. Hair samples from two cases in which a chronic intake of tramadol was expected from the case history were analyzed by HS-SPME with ethylbenzhydramine as internal standard. Tramadol was found at concentrations of 0.78 and 1.14 ng / mg, respectively. The SIM chromatogram of one of these cases is shown in Fig. 9B. 3.3.5. Methadone Methadone is frequently prescribed in substitution therapy of heroin addicts. In this way it gets on the black market and is abused by others. Hair concentrations of

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Fig. 9. GC / MS-SIM chromatograms of some drugs from hair after sample preparation by alkaline hydrolysis and HS-SPME. Sample amount: 10 mg; internal standards: ethylbenzhydramine (A and B, 10 ng / mg) or D 9 -methadone (C, 10 ng / mg); fiber: PA 85 mm, adsorption 20 min at 808C (A and C) or at 908C (B), m /z values used in SIM see Table 1. (A) Subject (T098 / 97) with frequent intake of diphenhydramine. (B) Subject (T035 / 96) with frequent intake of tramadol. (C) Subject (T264 / 97) with regular intake of methadone.

substitution patients have been measured several times [33,34] and are between 0.8 and 42 ng / mg. Hair samples from two drug-related deaths who were known to have been treated with methadone were investigated by HS-SPME using D 9 -methadone as internal standard. The methadone concentrations found (20.1 and 9.9 ng / mg) were slightly higher than those found using conventional SPE after buffer extraction (15.7 and 8.0 ng / mg). The SIM chromatogram of one of these samples after HS-SPME is shown in Fig. 9C. Because of the high number of deuterium atoms the peaks are almost completely separated. The detection limit of 0.1 ng / mg using a 10 mg sample is quite sufficient for routine investigations.

3.3.6. Tricyclic antidepressants The analysis of the tricyclic antidepressants amitriptyline, clomipramine, doxepin and imipramine together with their desmethyl metabolites by alkaline hydrolysis of the hair matrix has been described by several authors [25,35]. Our HS-SPME investigation using dimetacrine as internal standard was carried out at 908C and 20 min adsorption time for amitriptyline (three cases), doxepin (two cases) and trimipramine (two cases). The analyte concentrations were found to be between 0.22 and 10.4 ng / mg. The chromatograms from two cases are shown in Fig. 10A and B. The detection limits with a sample amount of 10 mg were between 0.05 and 0.2 ng / mg. 3.4. Experiments for detection of cannabinoids by HS-SPME For the detection of marijuana or hashish abuse D9 -THC, D9 -THC-COOH, cannabinol (CBN) and cannabidiol (CBD) are measured in routine hair analysis [36,37]. Sample preparation is carried out by methanol extraction in an ultrasonic bath [37] or

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Fig. 10. GC / MS-SIM chromatograms of tricyclic antidepressants from hair after sample preparation by alkaline hydrolysis and HS-SPME. Sample amount: 10 mg; internal standard: dimetacrine (10 ng / mg); fiber: PA 85 mm, adsorption 20 min at 908C, m /z values used in SIM see Table 1. (A) Subject (T164 / 99) with regular intake of amitriptyline and trimipramine. (B) Subject (T149 / 99) with regular intake of doxepine.

preferentially by alkaline hair hydrolysis and subsequent solid phase column extraction [36]. SPME by directly dipping of a PDMS coated fibre into the solution of hair digestion in 1 M sodium hydroxide and subsequent pH adjustment at 7.5 by hydrochloric acid and phosphate buffer was described by Strano-Rossi and Chiarotti [21]. Detection limits were 0.1 ng / mg for D9 -THC and CBN and 0.2 ng / mg for CBD. D9 -THC, CBN and CBD also fulfil the prerequisites for headspace sampling: lipophilic and semi-volatile, but the boiling point of D9 -THC is very high (2008C at 2.7 Pa [38]). Because of the phenolic hydroxyl group (pKa 510.6) the extraction must be carried out under neutral or acidic conditions. Therefore some preliminary HS-SPME experiments with 4% sodium hydroxide10.5 g sodium sulphate with different amounts of orthophosphoric acid added and spiked with 200 ng of each of the three compounds were performed. The best results were obtained in the pH range 4–7, where all three compounds were clearly seen (Fig. 11A). These conditions were applied to the hair sample (30 mg) of a cannabis consumer. This was dissolved in 1 ml 4% sodium hydroxide, and the pH was then adjusted to 6 by adding orthophosphoric acid and 0.5 g sodium sulphate (see experimental part and chromatogram 11B). Only CBN was detected, whereas D9 -THC (1.3 ng / mg, determined by conventional methods) was not seen. The sensitivity is still far too low to compete with the liquid extraction methods, and more optimization work has to be done.

3.5. Measurement of clomethiazole by HS-SPME after buffer extraction Clomethiazole is a hypnotic but is mainly prescribed against withdrawal symptoms in alcoholics. No data about deposition and concentrations in hair were found in the literature. Chlomethiazole free base is an oily substance with a relatively high volatility (boiling temperature 928C at 933 Pa [38]) but it is a very weak base (pKa 3.2). Nevertheless, it was not detected in spiked hair samples after alkaline digestion, since

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Fig. 11. Detection of cannabinoids by HS-SPME and GC / MS-SIM. (A) 200 ng of cannabidiol, D9 tetrahydrocannabinol (THC) and cannabinol in 1 ml 4% NaOH after addition of 50 ml 85% H 3 PO 4 . (B) 30 mg hair of a cannabis consumer after 30 min hydrolysis in 1 ml 4% NaOH at 1008C and addition of 50 ml 85% H 3 PO 4 .

the b-chloroethyl group is hydrolysed to a b-hydroxyethyl moiety in a more hydrophilic product. In pH 6.0 buffer clomethiazole is stable and can be analyzed by HS-SPME from spiked hair samples with a detection limit of 0.5 ng / mg (adsorption temperature 608C, adsorption time 15 min) using diethylaniline as internal standard. In two hair samples from alcoholics, who were known to have treated themselves with this drug, chlormethiazole (15.3 and 115 ng / mg, respectively) was found after 12 h buffer extraction in an ultrasonic bath. The SIM chromatogram of one case is shown in Fig. 12. In a similar way also other drugs sensitive to alkaline hydrolysis should be accessible to HS-SPME. As an example the detection of cocaine (pKa 58.6, b.p. 1878C at 13 Pa) was tried from aqueous solution in the presence of 0.5 g disodium hydrogen orthophosphate (pH¯9.8). At this pH cocaine should predominantly be non-ionized but also not yet hydrolysed. Nevertheless the experiments were unsuccessful.

4. Conclusions HS-SPME can be applied with advantage to hair analysis in a convenient one-step method for a wide variety of basic drugs including frequently abused compounds such as phencyclidine, ketamine and methadone. Some of the results presented here are preliminary, and the methods are not fully evaluated. By adaptation of the conditions to the special properties of a particular substance an extension to some other drugs and a further decrease in detection limits are possible. Practical advantages of the method are easy handling, short investigation time, saving of organic solvents and small sample amount.

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Fig. 12. GC / MS-SIM chromatogram of the hair sample (10 mg) of an alcoholic with regular clomethiazole intake. Sample preparation: 12 h extraction in an ultrasonic bath with 1 ml buffer pH 6.0, subsequent addition of 0.5 g Na 2 SO 4 and HS-SPME. Internal standard: N,N-diethylaniline (10 ng / mg); fiber: PA 85 mm, adsorption 15 min at 608C, m /z values used in SIM cf. Table 1.

In comparison to other sample preparation methods the chromatographic background is very low. Therefore also the low m /z values such as 58 or 72, which form the base peaks of many drugs, but are normally not usable because of high matrix burden, can successfully be used in quantitative GC / MS-SIM methods with consequent improvement in sensitivity. At the present time the HS-SPME method is restricted to lipophilic compounds with a certain volatility and suitable gas chromatographic properties without derivatization.

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