The standardization of results on hair testing for drugs of abuse: An interlaboratory exercise in Lombardy Region, Italy.

Hair testing for drugs of abuse is performed in Lombardy by eleven analytical laboratories accredited for forensic purposes, the most frequent purposes being driving license regranting and workplace drug testing. Individuals undergoing hair testing for these purposes can choose the laboratory in which the analyses have to be carried out. The aim of our study was to perform an interlaboratory exercise in order to verify the level of standardization of hair testing for drugs of abuse in these accredited laboratories; nine out of the eleven laboratories participated in this exercise. Sixteen hair strands coming from different subjects were longitudinally divided in 3-4 aliquots and distributed to participating laboratories, which were requested to apply their routine methods. All the participants analyzed opiates (morphine and 6-acetylmorphine) and cocainics (cocaine and benzoylecgonine) while only six analyzed methadone and amphetamines (amphetamine, methamphetamine, MDMA, MDA and MDEA) and five Δ(9)-tetrahydrocannabinol (THC). The majority of the participants (seven labs) performed acidic hydrolysis to extract the drugs from the hair and analysis by GC-MS, while two labs used LC-MS/MS. Eight laboratories performed initial screening tests by Enzyme Multiplied Immunoassay Technique (EMIT), Enzyme-linked Immunosorbent Assay (ELISA) or Cloned Enzyme Donor Immunoassay (CEDIA). Results demonstrated a good qualitative performance for all the participants, since no false positive results were reported by any of them. Quantitative data were quite scattered, but less in samples with low concentrations of analytes than in those with higher concentrations. Results from this first regional interlaboratory exercise show that, on the one hand, individuals undergoing hair testing would have obtained the same qualitative results in any of the nine laboratories. On the other hand, the scatter in quantitative results could cause some inequalities if any interpretation of the data is required.

Hair testing is superior to urine to disclose cocaine consumption in driver's licence regranting.

In driver licence regranting, subjects with a history of cocaine use are requested to undergo laboratory testing to verify both current and past abstinence from the drug. Identification of cocaine use based only on urinalysis may miss some cases because of the short elimination half-life of the drug. Moreover, many abusers know how to time their cocaine consumption in such a way that they can "beat" the urinalysis, having a series of negative urine tests. We report on the use of hair testing to disclose sporadic cocaine consumption in seven subjects attending the Local Medical Commission to reobtain driver licence, with constant negative urinalysis. Even with one or two weekly negative urine screens along several months, all the subjects were positive using hair testing for cocaine and benzoylecgonine, above the internationally recommended limit of quantification: 0.5 ng/mg and 0.05 ng/mg for cocaine and benzoylecgonine, respectively (concentration range for cocaine: 0.51-2.23 ng/mg hair; concentration range for benzoylecgonine: 0.08-1.70 ng/mg hair). The obtained results support strongly the use of hair testing for cocaine in drug addicts and occasional abusers applying for regranting of driver licence in order to minimize social risk behaviours.

Segmental hair analysis in order to evaluate driving performance.

On the 31st of July 2002 the Lombardy local government issued a memorandum, C.R. 35/SAN, providing "guidelines to investigate drugs of abuse addiction in order to judge driving performance". About hair samples, this memorandum advises that the proximal lock of 6 cm-length would be analysed for opiates, cocaine, cannabinoids, amphetamine and derivatives, divided into two segments of 3 cm each. The Local Medical Driving Licence Commissions (CML) can decide whether or not to enforce these instructions; from our survey it resulted that most CMLs do not abide by the memorandum, not requiring segmental analysis. The purpose of our study was to verify whether this procedural discordance could affect analytical results and, consequently, the evaluation of the subject's driving performance. We analysed hair samples taken from subjects who were requesting the renewal of their driving licence in our Laboratory during the period from 1 August 2002 to 31 December 2006. We divided samples into two groups: (1) samples previously analysed in one single segment which resulted positive for at least one analyte, but under the cut-off (0.5 ng/mg), were re-analysed in accordance with the guidelines; (2) samples previously processed following guidelines which resulted positive in one of the segments were newly analysed in a single segment. Comparing the new results with the original ones, an increase of positive results emerged in the first group. The second set of results fully supported the first ones. These results underscore the importance of the 35/SAN memorandum, so if the guidelines had been followed there would have been a larger amount of driving licence renewal denied.

HAIRVEQ 2006: evolution of laboratories' performance after different educational actions.

HAIRVEQ is a proficiency testing program for hair analysis of illicit drugs organized by the Istituto Superiore di Sanità (Rome, Italy) and the Institut Municipal d'Investigació Mèdica (Barcelona, Spain). The aim of the three exercises performed in 2006 was the evaluation of 32 laboratories' performance when analyzing the same hair sample containing opiates, cocaine and methadone, after carrying out some specific educational interventions. In the first round, the sample was sent to be analyzed following laboratory routine methodology. In the second round, standard operating procedures (SOP) for hair testing including sample preparation, method validation and qualitative and quantitative data evaluation, and an open hair sample for SOP training were also sent together with other hair samples including the one used for performance evaluation. After the second round, a workshop was held with participant laboratories to discuss methodological issues and interpretation of obtained results. An additional amount of open samples was distributed to the laboratories for implementing the SOPs. In the third round, the same unknown sample containing opiates, cocaine and methadone was resent for the final evaluation of laboratory performance. In the first round, 11 incorrect qualitative results (10 false negative and 1 false positive) were reported by seven laboratories (22%), in the second round, a reduction in the number of incorrect results was observed (4 false negatives and 1 false positive were reported by four laboratories, 13%) and in the third round, 5 false positives and 5 false negatives were reported by seven laboratories (22%). Concerning quantitative results, the scatter was similar between the three rounds and similar to the ones reported by other proficiency tests in hair analysis. More educational actions should be addressed to a group of laboratories, which did not yet show satisfying qualitative and quantitative results.

Hair analysis for opiates, cocaine and metabolites. Evaluation of a method by interlaboratory comparison.

The aim of this study was to evaluate the performance of a technique for the simultaneous testing of opiates, cocaine and metabolites in hair by interlaboratory comparison. Sixteen forensic and clinical laboratories with different degrees of experience in hair analysis participated voluntarily in the study (no selection criteria were applied). The suggested analytical procedure, the one routinely used in our laboratory, consisted of incubation in HCl 0.1N (45 degrees C, overnight), solid phase extraction (with Bond Elut Certify) cartridges), derivatisation (trimethylsilyl (TMS) derivatives) and GC-MS analysis. Three different mixtures of finely cut (1 mm or less) hair were prepared using drug-users' and drug-free hair: one 'negative' sample (<0.1 ng/mg for morphine, 6-acetylmorphine (6AM), cocaine and benzoylecgonine (BE)), one 'low concentration' sample (between 0.5 and 2 ng/mg) and one 'high concentration' sample (>3 ng/mg). Accuracy and precision (CV% lower than 5.1, 9.9, 5.2, 3.8, 7.3 and 8.3% for morphine, 6AM, codeine, cocaine, BE, and methylecgonine (ME), respectively; range 0.5-5 ng/mg) of the method and homogeneity of the mixtures were evaluated in our laboratory by intraday (CV% lower than 12% for all analytes) and interday analyses (CV% lower than 17% for all analytes except 6AM, 25%). Participants in the study were grouped into: (1) laboratories (n = 6) obtaining the best qualitative and quantitative values, corresponding to those with long experience in hair analysis; (2) laboratories (n = 5) with no reported false positive and/or false negatives; (3) laboratories (n = 5) with one or more reported false positives/false negatives. The results obtained by the labs of the first group were used as reference values. The scatter of data was similar to those obtained in other published studies.

Simultaneous hair testing for opiates, cocaine, and metabolites by GC-MS: a survey of applicants for driving licenses with a history of drug use.

A sensitive GC-MS method for the simultaneous determination of opiates, cocaine, and metabolites in hair at a cut-off level of 0.1 ng/mg was adopted to assess past exposure to these drugs in applicants for driving licenses with a history of drug use. The sampling protocol consisted of collection of one hair (sample A, 5-cm length) and one urine sample. When hair and urine (EMIT Syva, cut-off levels: 0.3 mg/l for opiates, 0.15 mg/l for cocaine, GC-MS confirmation of positives) were both positive or negative the protocol was concluded. In the other cases, the assessment of 'current exposure' to drugs was carried out, in order to avoid seriated random urinalysis, by collecting a second hair sample (sample B) 6 weeks later and analysing the proximal 1-cm segment. Out of the 214 'A' hair samples analyzed, 14 (6.5%) tested positive for morphine and/or 6-acetylmorphine (6AM), and 26 (12%) for cocaine and/or benzoylecgonine (BE), whereas none of the samples tested positive for both drugs. Levels between 0.1 and 1 ng/mg of the single analytes were found in eight out of the 14 morphine-6AM positives (57%) and in 18 out of the 26 cocaine-BE positives (69%). The time course of positive cases showed a progressive decrease of morphine-6AM positives and a corresponding increase of cocaine-BE positives within the study period September 1995-February 1999. No cases with positive urine and negative hair were observed. Among the 40 positive cases, seven (four and three for opiates and cocaine, respectively) were found to be 'currently exposed to drug', four by urinalysis (three and one) and three by analysis of the hair sample B (1 and 2).

Immunological screening of drugs of abuse and gas chromatographic-mass spectrometric confirmation of opiates and cocaine in hair.

The work presents an analytical strategy to detect drugs of abuse in hair. It involves two sequential steps: a screening by a simple enzyme-linked immunosorbent assay (ELISA) methodology to detect opiates, cocaine and its metabolites, and benzodiacepines, followed by confirmation of opiates and cocaine metabolites in positive samples by gas chromatography coupled to mass spectrometry (GC-MS). In the same GC-MS run other drugs for substitution therapy (e.g. methadone and its main metabolite) can also be detected. After a double washing of hair samples with dichloromethane, hair specimens were cut into small pieces and 10 mg samples were incubated in 2 ml of methanol-trifluoroacetic acid (9:1) mixture, overnight at 37 degrees C. Aliquots of the extract were then evaporated, reconstituted in buffer and analysed according to the ELISA procedure. Confirmation involved solid-phase extraction of another fraction of the extract kept at -20 degrees C, derivatization with heptafluorobutyric anhydride and hexafluoroisopropanol and detection of cocaine, benzoylecgonine, ecgonine methylester, cocaethylene, morphine, codeine, 6-monoacetylmorphine, methadone and 2-ethylidene-1.5-dimethyl-3,3-diphenylpirrolidine (methadone metabolite) by selective ion monitoring after gas chromatographic separation. During the development of the method it was verified that no more than 10% of cocaine, opiates and benzodiacepines were lost when dichloromethane was used to wash real samples. The results also confirmed the increase of extractability power of TFA when it was added to methanol: the recovery for the analytes (cocaine and its metabolites and opiates) added to methanol-TFA alone was of the order of 90% except for benzoylecgonine (75%), and the recovery for the analytes added to methanol-TFA extract of drug-free hair was about 90% for all analytes except for benzoylecgonine and 6-MAM (around 70%). Regarding the stability of labile compounds, only small amounts of ecgonine methylester (2.3%) and morphine (7.2%) were produced, from cocaine and 6-MAM respectively, after the whole extraction procedure and two weeks of storage of methanol-TFA extracts at -20 degrees C. Satisfactory results were obtained when the procedures were applied to the analysis of external proficiency testing hair samples and actual specimens from drug addicts.

Determination of opiates in hair. Effects of extraction methods on recovery and on stability of analytes.

In order to evaluate (i) the recovery of extraction of opiates from authentic hair samples and (ii) the extent of hydrolysis of acetylated opiates (6-acetylmorphine, acetylcodeine) occurring during sample preparation, three different methods of extraction commonly used for opiates have been compared. To this purpose a sample consisting of a pool of hair collected from several heroin overdose cases has been submitted alternately to (A) digestion in 2 M NaOH at 80 degrees C for 1 h (n = 5), (B) incubation in 0.1 M HCl at 45 degrees C for 18 h (n = 5) and (C) incubation in methanol at 37 degrees C for 18 h (n = 5). After pH adjustment of the different incubation media to 7-8, analytes have been isolated by means of SPE using Bond Elut certify columns and derivatized with MSTFA. Analyses have been performed by either GC-MS in the selected ion monitoring mode or, omitting SPE, by radioimmunoassay. The extent of hydrolysis of 6-acetylmorphine to morphine and of acetylcodeine to codeine have been determined by submitting blank hair samples spiked with the acetylated analytes to the different extraction methods and measuring the amount of morphine and codeine formed. Both the recovery of extraction of the total morphine fraction (6-acetylmorphine + morphine) and the rate of hydrolysis of 6-acetylmorphine were found to be in the order: A > B > C. Similar results were obtained for the total codeine fraction (acetylcodeine + codeine). These results clearly indicate that: (i) the concentration of opiates measured in hair depends on the extraction method used; (ii) ratios between different analytes (e.g. 6-acetylmorphine vs. morphine) may reflect the rate of hydrolysis during sample preparation rather than different types of exposure to opiates.