Detection of methadone in human hair by gas chromatography/mass spectrometry

Summary Determination of methadone in human hair by gas chromatography/mass spectrometry was described. Helium as carrier gas, a 30-m bonded phasefused silica DB-1 capillary column and splitless injection at 230°C temperature were used. The concentrations of methadone and its metabolites were measured in addition by radioimmunoassay (RIA). Both methods GC/MS and RIA showed the presence of methadone in human hair.     

Interpretation of forensic cases based on empirical data

Over the course of the last years the importance of hair analysis increased obviously. For example work place testing, driving licence cases, so-called health checks, and especially in criminal acts the analysis of illicit substances is common. With the modern multiplex analytical methods (GC/MS, GC/MS/MS, LC/MS) the routine analytical probe of hair is in general unproblematic. But one of the major problems in hair analysis is the interpretation of the results. To solve this difficult and challenging task statistical data as a source of information could be a helpful tool. Examination of more than 1,000 hair segments within the past 2 years using gas chromatography/tandem-mass spectrometry (GC/MS/MS) as the preferred method for conducting the daily routine detection of the most prevalently abused drugs and metabolites, such as cannabinoids, cocaine, some synthetic drugs from the amphetamine group, and opiates serve as the basis for the statistics. The quantity of analytical results and additional essential facts have been documented in the publication to present our sources of information and supplementary data to prepare forensic reports. We hereby state that we do not have a significant financial interest or other relationship with any product manufacturer or provider of services discussed in this article.     

Zum Suchtmittelnachweis in Haaren

Hair samples collected from drug addicts (n=25) were analysed for opiates and cocaine by GC/MS. A decrease in drug concentration was observed even after a single bleaching or permanent wave application. The stability of the drug molecules in hair seemed to be different and cocaine was less affected than morphine or 6-acetylmorphine. When drug concentrations were low, the drug content often declined below the detection limit after cosmetic treatment. In hair samples which showed a drug content above 2 ng/ mg hair the qualitative detection of drugs was regularly successful. However, a distinct rule could not be established mainly due to influences of the individual hair matrix.     

Deposition of cocaine in tissue after lethal and repeated sublethal administration to sheep

Summary The deposition of cocaine in tissue after lethal administration to sheep was investigated. In addition, the presence of cocaine in tissue obtained from sheep treated for 30 days with a sublethal dose (2.4 mg/kg b.wt.) after 1-day, 1-week, or 1-month withdrawal was studied. The determination of cocaine was performed by radioimmunoassay. The concentrations measured represent the sum of cocaine and its metabolites. The presence of cocaine was also qualitatively proven by gas chromatography/mass spectrometry. After a lethal dose administration cocaine was found in all investigated organs. The highest concentrations were present in liver, bile, and kidney. In tissue obtained from the sheep treated daily with a sublethal dose and killed after 1-week withdrawal, the concentrations found were significantly lower. After 1-month withdrawal, cocaine was not to be discovered in tissue.Diese Arbeit ist Herrn Prof. Dr. G. Dotzauer zu seinem 75. Geburtstag gewidmet     

Quantitation of sibutramine in human hair using gas chromatography–isotope dilution tandem mass spectrometry

Forensic Toxicology - An analytical method for quantitation of sibutramine in human hair using gas chromatography (GC)–isotope dilution tandem mass spectrometry (MS/MS) was newly established....     

Zum Suchtmittelnachweis in Haaren. VII. Untersuchungen bei Methadonpatienten unter konstanter Wirkstoffeinnahme

Hair samples and perspiration collected from long term drug addicts (n = 18) under constant methadone treatment were investigated. Hair segments representing the individual hair growth of 4 weeks were analyzed for methadone, opiates and cocaine by GC/MS. The results obtained from the transdermal collection devices confirmed the presence of drug substances in variable amounts on the skin surface, methadone being the main analyte. The perspiration test period lasted for 4 days. For six persons illicit drugs similar to those in the corresponding hair samples were found. Overall, a poor relationship (r = 0,602) was found between the methadone dose under steady state conditions and the methadone level in hair. The results are discussed in the light of pharmacokinetic aspects, drug uptake by perspiration and forensic interpretation.     

Detection and quantification of psychotropic drug etaqualone in human hair using GC–MS/MS

In this study, sensitive analytical procedure for detection and quantification of etaqualone in human hair samples using gas chromatography tandem mass spectrometry (GC–MS/MS) was newly established, and applied it to authentic human samples obtained from an abuser. In this method, the hair samples were treated with hydrochloric acid and then extracted with ethyl ether. The ether layer was dried in a warm water bath, and the residue was reconstituted in ethyl acetate, followed by GC–MS/MS analysis. Multiple reaction monitoring (MRM) mode was used for data collection, and quantitative analysis was performed using internal standard method. Good linear relationship within the concentration range of 1–100 pg/mg were obtained in calibrators for the hair samples showing its correlation coefficient value was 0.9993. The lower limit of quantitation in this study was 1 pg/mg and the recovery rate examined ranged from 100.4% to 108.5%. The intra-day precision and accuracy were less than 5.0% and 5.8%, respectively. The inter-day precision and accuracy were lower than 6.4% and 4.6%, respectively. Using this established method, etaqualone could be detected in the hair sample obtained from a suspected user to be level of 65.2 pg/mg. It should be expected that the method established in this study would contribute to rapid detection and identification of psychotropic drug etaqualone among multiple fields including forensic investigation, clinical application and of course public health matters.     

Comparison of the Cozart DDS 801 on-site drug test device and gas chromatography/mass spectrometry (GC/MS) confirmation results of cannabis and cocaine in oral fluid specimens

Driving under the influence of drugs (DUID) is a problem around the world. The objective of this study is to assess the reliability of the oral fluid screening device the Cozart DDS 801 by comparing the on-site results with confirmatory gas chromatography/mass spectrometry (GC/MS) oral fluid analysis. The study was carried out in Catalonia (Spain) with a sample of 2180 oral fluid specimens taken from subjects suspected of driving under the influence of drugs of abuse, and collected by police officers during 2009–2010. Statistical parameters of the tests were determined for cannabis and cocaine. The sensitivity, specificity, predictive positive value, predictive negative value, likelihood positive ratio and likelihood negative ratio for cocaine were 92%, 90%, 95%, 85%, 9.44, and 0.09 respectively. Sensitivity, specificity, predictive positive value, predictive negative value, likelihood positive ratio and likelihood negative ratio for cannabis were 87%, 86%, 94%, 73%, 6.15 and 0.6 respectively. Accuracy was 91% for cocaine and 86% for cannabis. The Cozart DDS 801 drug test system is a simple to use screening tool for cocaine and cannabis in oral fluid, at initial screening cut-off established by the manufacturer, confirmed with a GC/MS analysis. The system has demonstrated its acceptable performance.     

Radioimmunological determination of cocaine in human hair

Summary A simple procedure for the determination of cocaine in human hair was described. After washing hair samples were crushed in 0.1m HCl and incubated overnight at 45°C. The acid extracts were neutralized with 1m NaOH. Phosphate buffer (pH 7.4) was added to the extracts. The cocaine concentrations were measured by radioimmunoassay. Detection in hair was achieved in all hair samples obtained from cocaine users. This method appears to be suitable for the routine determination of cocaine.     

Toxicological detection of pholcodine and its metabolites in urine and hair using radio immunoassay,fluorescence polarisation immunoassay,enzyme immunoassay and gas chromatography-mass spectrometry

Summary Pholcodine (3-O-(2-morpholinoethyl)-morphine) is used in many countries as an antitussive without analgesic or addictive properties. It is of forensic relevance that pholcodine interferes with opiate immunoassays. In this paper a gas chromatographic-mass spectrometric (GC-MS) procedure for the precise and sensitive detection of pholcodine and its metabolites in urine and hair, after acid hydrolysis, extraction and acetylation, is presented. Furthermore, detection of pholcodine using radio immunoassay (RIA), fluorescence polarisation immunoassay (FPIA) and enzyme immunoassay (EIA) for opiates is described. Using GC-MS, unmodified pholcodine could be detected in urine samples 4–7 weeks after ingestion of a single therapeutic dose of 50 mg of pholcodine, the desmorpholinohydroxy metabolite for 1–2 weeks and the other metabolites (nor-, nordesmorpholinohydroxy-, hydroxy-, oxo- and noroxo-pholcodine) only during the first few hours. Morphine could also be detected in urine samples for the first few days. It was however mainly formed artificially during acid hydrolysis and only in trace amounts by metabolism. All the immunoassays tested gave positive results in urine samples during the first week taking the cut-off values recommended by the manufacturer into consideration. If values between the cut-off and the detection limit were taken into consideration, RIA and FPIA gave positive results for 2–4 weeks and EIA up to 2 weeks. Pholcodine could also be detected by RIA and GC-MS in samples of head hair clipped 10 weeks after ingestion of 50 mg and in daily shaved samples of beard hair over a period of three weeks after ingestion of three doses of 60 mg. It can be concluded that the widely used immunoassays for opiates show positive results in urine and hair samples for a long time after ingestion of the non-opioid pholcodine and that these results can be confirmed by the GC-MS procedure described in this paper.     

Detection of malathion in a victim by gas chromatography/negative ion chemical ionization mass spectrometry

Summary We experienced an autopsy case in which a 53-year-old woman committed suicide by ingesting allegedly a certain agricultural chemical. The blood and stomach contents, after extraction with acetonitrile and chloroform, were subjected to analysis by gas chromatography (GC)/negative ion chemical ionization (CI) mass spectrometry (MS). By total ion monitoring in the negative CI mode, a large peak appeared. The mass spectrum of the peak showed a strong anion at m/z 157, suggesting the presence of an organophosphorus pesticide. By measuring its spectrum in the positive electron impact (EI) mode, it was identified as malathion. The selected ion monitoring in the negative CI mode showed that the malathion peak was not interfered with by any impurities, and its background was very low. The sensitivity in the negative CI mode was about 5–10 times higher than that in the positive EI mode. Our data show that the GC/negative ion CI MS is useful for both screening and sensitive quantitation of organophosphorus pesticides.     

Effect of the shampoo Ultra Clean on drug concentrations in human hair

The influence of the special shampoo Ultra Clean (Zydot Unlimited, Tulsa, Oklahoma) on the results of hair analyses was investigated. Hair samples from persons (n = 14) with a known history of drug abuse were collected at autopsy. The hair samples were divided into separate strands which were analyzed both after washing with Ultra Clean and without treatment. Hair analyses were performed by methanol extraction under sonication, purification by solid phase extraction and GC/MS in SIM mode according to routine procedures for tetrahydrocannabinol (THC), cocaine, amphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDE), heroin, 6-monoacetylmorphine (6-MAM), morphine, codeine, dihydrocodeine and methadone. All drugs originally present in the hair fibers were still detected after a single application of Ultra Clean. However, a slight decrease in drug concentrations could mostly be observed e.g. cocaine (n = 10) -5%, 6-MAM (n = 12) -9%, morphine (n = 12) -26%, THC (n = 4) -36%. The findings clearly demonstrated that drug substances had not been sufficiently removed from human hair by a single Ultra Clean treatment to drop their concentrations below the limit of detection of the analytical method applied.     

Automated solid-phase extraction and two-step derivatisation for simultaneous analysis of basic illicit drugs in serum by GC/MS

A combination of automated solid-phase extraction (SPE) and subsequent two-step derivatisation has been developed for the simultaneous analysis of basic drugs of abuse and cocaine metabolites in serum samples. Substances included in this procedure are morphine, codeine, methadone, cocaine, benzoylecgonine, methylecgonine, amphetamine, methamphetamine, MDMA, MDEA and MDA. SPE with mixed-mode cartridges (RP-C8 and cation-exchange) was fully automated with a Zymark RapidTrace SPE robot. GC/MS analysis was performed after derivatisation with a new two-step reaction by trifluoroacetic anhydride and 2,2,3,3,3-pentafluoropropanol. High recoveries (> 85%) with high reproducibility (CV 1.1-3.8%) were found for all drugs. High correlation coefficients (r > 0.998) were obtained due to the addition of deuterated standards prior to extraction. Experience obtained over 2 years of applying this method to drug analysis in serum is discussed.     

Applicability of an on-site test for its use in post-mortem blood

The number of deaths related to drugs of abuse makes necessary the use of an on-site test for those cases in which a rapid detection of the consumed drug is required. Cozart® DDS test provides a simple, fast and reliable tool for the qualitative on-site analysis in post-mortem blood. Owing that this test is prepared for oral fluid samples, a validation becomes essential in order to use it for a different matrix than the established one. According to that, results obtained by Cozart® DDS test used in post-mortem blood samples have been compared with a qualitative gas chromatography–mass spectrometry (GC/MS/MS). Positive results for cocaine family compounds (COC-F) were 43.75%, for opiates family compounds (OPI-F) 25.78%, and for cannabis family compounds (THC-F) 2.34%. Negative results were 28.13%. No amphetamines (AMP) or methamphetamines (MA) were found. Sensitivity and specificity was available for cocaine and opiates but not for cannabis because only five cases were detected. Sensitivity, specificity, predictive positive value and predictive negative value for cocaine were 98%, 91%, 88% and 99%, respectively. Sensivilty, specificity, predictive positive value (PPV) and predictive negative value (NPV) for opiates were 93%, 92%, 76% and 98%, respectively. Likelihood positive ratios for cocaine and opiates have been 10.92 and 11.69, respectively, while likelihood negative ratios have been 0.02 and 0.08, respectively. Results show the suitability of Cozart® DDS test for the qualitative detection of cocaine and opiates in post-mortem blood.     

Death of a female addict due to heroin and cocaine overdoses: a case report with multiparameter evaluation

This study undertook a multiparameter evaluation of the death of a 21-year-old woman known to be an abuser of heroin and cocaine. The toxicological analysis of multiple postmortem specimens such as blood and hair was carried out using liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS-MS). The blood specimens of the deceased showed the presence of opium components such as morphine and its glucuronides together with cocaine and benzoylecgonine. The detected xenobiotic levels probably explained the cause of her death resulting from combined action of unintentional illicit drug overdose. By analysis of four 2-cm long hair segments, a heroin-cocaine addiction for at least 8 months antemortem was able to be documented; the presence of 6-monoacetylmorphine (6-MAM), cocaine, and benzoylecgonine was demonstrated. The histopathological findings of lesions of the internal organs of the deceased were consistent with long heroin and cocaine abuse. The use of multiple parameters, such as blood and hair segments as matrices and drug metabolites such as 6-MAM, morphine, glucuronides, and benzoylecgonine as target compounds, gave a well-defined outline of her death.     

Determination of antidepressants in human postmortem blood,brain tissue,and hair using gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric (GC–MS) method in positive ion chemical ionization mode in combination with a solid phase extraction was optimized for new-generation antidepressants and their metabolites in postmortem blood, brain tissue, and hair. Twelve antidepressants and their active metabolites (i.e., mirtazapine, viloxazine, venlafaxine, citalopram, mianserin, reboxetine, fluoxetine, fluvoxamine, sertraline, maprotiline, melitracen, paroxetine, desmethylfluoxetine, desmethylmianserin, desmethylmirtazapine, desmethylsertraline, desmethylmaprotiline, desmethylcitalopram, and didesmethylcitalopram) could be quantified. In this article, in addition to the validation of the GC–MS method, four postmortem cases are discussed to demonstrate the usefulness of the described method in forensic toxicology. In these cases, sertraline, fluoxetine, citalopram, and trazodone in combination with their active metabolites were quantified. Blood concentrations ranged from subtherapeutic to toxic concentrations, while brain to plasma ratios ranged from 0.8 to 17. Hair concentrations ranged from 0.4 to 2.5 ng/mg depending on the compound and hair segment.     

Cocaine and opiate concentrations in hair from subjects in a heroin maintenance program in comparison to a methadone substituted group

One month before (T-1) and 12 months after (T12) controlled i.v. administration of pharmaceutical heroin–HCl (10–100 mg/day) in the context of a heroin maintenance program (HMP), concentrations of opiates and cocaine as well as its metabolites were determined in head hair (n = 46) using a validated gas chromatographic–mass spectrometric method. In addition, a patient collective of a methadone maintenance program (MMP, daily doses 15–260 mg) was examined (n = 35). The incidence of additional cocaine consumption decreased in both groups during the study period (T-1 to T12): in HMP from 64.6% to 45.8% and in MMP from 71.4% to 60.0%. A significant reduction of cocaine consumption was defined as an at least 30% reduction of analyte concentrations in hair (Δc > 30%). Accordingly, in HMP, a decrease in 45.8% of initially (T-1) cocaine-positive patients was determined; in MMP, the reduction was 48.6%. In 22.9% of HMP and 37.1% of MMP, an increase of cocaine concentrations was detected. Codeine and acetylcodeine were found in 50.0% and 43.5% (T-1) and 13.0% and 10.9% (T12) of the samples of the HMP, as well as in 45.7% and 25.7% (T-1) and 17.1% and 5.7% (T12) in MMP, respectively. The missing of acetylcodeine, in particular at T-1, questions its applicability as a characteristic marker of a preceding consumption of illicit heroin in hair analysis.     

Treatments against hair loss may hinder cocaine and metabolites detection

Recently, some of the hair samples that we routinely analyse for drugs of abuse did not produce valid results for cocaine and metabolites. A series of very intense interfering peaks with ion fragments common to cocaine (CO), and benzoylecgonine (BE) were found to cover up the “cocaine” region of the chromatogram. In one of these cases the subject declared he had used a lotion containing Minoxidil in order to prevent hair loss. Starting from this observation we found that the interfering peaks belonged to four different TMS derivatives of Minoxidil. Minoxidil interference was further investigated by applying Tricoxidil^®, a Minoxidil solution, to the hair of CO-free volunteers and to a CO-positive hair strand dipped into Tricoxidil. Hair were analysed before and after treatment. In both cases interfering peaks were absent in the chromatograms of untreated hair and appeared in treated hair. In the CO-positive hair detection of CO, BE and internal standard was completely hindered after treatment with Minoxidil. Attempts to separate interfering peaks from CO and metabolites by modifying the temperature programme failed. None of the hair washing methods tested (methanol; dichloromethane; sodium dodecyl sulphate water solution, 1% w/v followed by methanol; phosphate buffer 0.1 M, pH 6 followed by methanol) succeeded in removing Minoxidil interference. However, a simple solution to partially overcome the problem was to dry up the derivatised extract, reconstitute it in methanol (in order to switch back Minoxidil derivatives to the native molecule), and re-inject it: owing to the higher polarity, underivatised Minoxidil does not interfere any more with the chromatography of CO, at the expense of the disappearance of BE and ecgonine methyl ester both producing TMS derivatives. This strategy was applied to four real cases where Minoxidil interference was recognised: in two of these cases CO was detected. The problem of Minoxidil interference on CO detection may be limited to procedures involving trimethylsilylation, which is probably the most commonly adopted derivatisation in laboratories performing hair analysis for drugs of abuse.     

Analysis of fentanyl and sufentanil in hair by GC/MS/MS

Fentanyl and sufentanil are potent narcotic analgesics used only in hospitals as anaesthetic agents. The dependence potential of fentanyl is known. As they are given in doses at the microgram level and their elimination half-life is in the order of a few hours, detection in body fluids is possible only for a short time after administration. Radioimmunological methods are the only ones capable of detecting fentanyl in hair, as normal GC/MS methods for hair analysis are not sensitive enough to detect the drugs after doses in the order of micrograms. We therefore chose GC/MS/MS to analyse fentanyl and sufentanil in two cases where the drugs were given under controlled conditions over several days. The concentration was in the order of less than 100 pg/mg hair.     

On-site testing of saliva and sweat with Drugwipe and determination of concentrations of drugs of abuse in saliva, plasma and urine of suspected users

Potential drug users participated voluntarily in a Belgian study on the usefulness of the non-instrumental immunoassay Drugwipe (Securetec, Germany) for the screening of cocaine, opiates, amphetamine and cannabinoids in saliva and sweat. If one of the screening assays (urine, oral fluid, sweat) showed a positive result, blood and saliva were collected. The on-site Drugwipe results were correlated with the Drugwipe results for saliva in the laboratory and with the GC/MS results of the corresponding saliva, plasma and urine samples and pharmacological effects at the time of sampling. The Drugwipe assay proved to be sufficiently sensitive for the detection of recent cocaine (n = 6) and amphetamine (n = 15) abuse, whether the device was wiped on the tongue or on the surface of the body, or when a saliva sample was applied to the wiping part. In five of the six potential cocaine users, the saliva concentrations of cocaine exceeded 1000 ng/ml. In the amphetamine group, the saliva concentrations of amphetamine, MDMA or both were high (> 1000 ng/ml) in 13 subjects. For cocaine and amphetamine, the positive scores for Drugwipe matched the GC/MS results for the three body fluids. Recent heroin abuse (n = 5) could be demonstrated to some extent with Drugwipe on samples from the tongue but only the two subjects with the highest saliva concentrations of MAM (> 500 ng/ml) and morphine (> 500 ng/ml) were positive. If the legal cut-off value for driving under the influence of opiates in Belgium (20 ng/ml of free morphine in plasma) was taken into account, only three subjects would have been legally positive. For cannabinoids (n = 15), false negatives and even some false positives were observed. Saliva can be considered as a useful analytical matrix for the detection of drugs of abuse after recent abuse when analysed with GC/MS. Received: 26 January 1999 / Received in revised form: 19 May 1999 / Accepted: 17 June 1999