Abuse In Hair Research Articles

Optimizing screening cutoffs for drugs of abuse in hair using immunoassay for forensic applications.

In forensic toxicology, positive immunoassay (IA) test results do not hold forensic validity and need to be confirmed with mass spectrometry (MS). On the other hand, a negative result is a strong indication that the drug and/or the drug metabolites are not present in the sample and that confirmatory analyses are not necessary. Consequently, a negative IA result must have forensic validity since it can be admitted in court during a trial. Screening cutoffs for the analysis of hair samples using immunoassays (IAs) were retrospectively optimized based on the Society of Hair Testing (SoHT) confirmation cutoffs and the utility of the test for forensic applications was discussed. Hair samples taken from 150 patients with a history of drug addiction were analyzed with ILab 650, Werfen (Milan, Italy) using DRI® reagents. Confirmatory analyses were subsequently performed using the ACQUITY UPLC® System, Waters Corporation (Milford, USA). Screening cutoffs were retrospectively optimized using receiver operating characteristic (ROC) analysis. A total of 162 single positive results were obtained for confirmatory analysis (10 for amphetamines/methamphetamines, 11 for MDMA, 37 for cocaine, 40 for THC, 33 for methadone, and 31 for opiates). The optimized screening cutoffs were 0.27 IA ng/mg for amphetamines, 0.51 IA ng/mg for MDMA, 0.59 IA ng/mg for cocaine, 0.14 IA ng/mg for cannabinoids, 0.63 IA ng/mg for methadone, and 0.26 IA ng/mg for opiates. An area under the curve (AUC) greater than 0.95 was obtained with very high sensitivity and specificity for all drugs. The presented screening method proved to be a useful technique on hair samples for the classes of drugs most commonly found in Italy and Europe and can be applied to forensic analysis.

Disappearance of codeine, morphine and 6-MAM in hair after cessation of abuse

Research on the determination of drugs of abuse in hair has established that drugs can be detected in hair even long after cessation of use. The purpose of this study was to analyze hair samples from chronic opioid users who were beginning a controlled drug cessation program. The study population (n = 15) is involved in a drug rehabilitation program in Santiago de Compostela, Spain. Over a 6-month period, subjects provided hair samples at 2-month intervals, with the first sample collected on the day they began the program. Codeine, morphine, and 6-MAM were analyzed by GC/MS (LOQ = 0.2 ng/mg). Hair tresses were divided into 1 cm segments and analyzed for all analytes 0–1 cm corresponding to the proximal portion to the scalp Following cessation of opioid use, traces of codeine, morphine, and 6-MAM still remained in the newly growing hair segments for a specified period. After 2 months, still 27 % of the users tested positive, and at 4 months, 20 % were positive but only for 6-MAM. However, after 6 months of abstinence, the results were negative for all analytes.

Design and development of a lanthanide-labeled immunochromatographic strip for simultaneous detection of morphine, methamphetamine and ketamine in hair.

Colloidal gold immunoassay is the most widely used method in the field of drug detection. However, this method often has poor quantitative identification ability and low analytical sensitivity, which is not suitable for the analysis of hair poisoning ingredients. In order to solve these limitations, we developed an immunochromatographic test strip for simultaneously screening multiple drugs in this study. This hand-held test strip used fluorescent nanoparticles loaded with lanthanide chelates as the signal carrier of fluorescence reading, and conducted quantitative testing of various drugs based on the competitive immune reaction between the analyte and antigen. Under the optimal conditions, the competition curves of morphine (MOP), methamphetamine (MET) and ketamine (KET) were obtained on a single band. The detection limit (LOD) of this analytical method was 100-1000 times lower than that of colloidal gold test strips. The detection limits of MOP, MET and KET were 0.06 ng mL-1, 0.1 ng mL-1 and 1.0 ng mL-1, respectively. No cross-reaction was observed when morphine, methamphetamine and ketamine were tested simultaneously with this method. And 184 hair samples were tested simultaneously, and the detected amount was very close to the results of LC-MS. The immunochromatographic strip showed good stability in repeated tests, and the coefficient of variation was less than 15%. Fluorescence immunochromatography strips and handheld strip readers have the characteristics of portability, speed, ease of operation and high sensitivity, and may become powerful tools for screening drug abuse in hair in forensic medicine.

Current status of hair analysis on narcotics in Korea

Since hair analysis of narcotics was first introduced to Korea in 1993, and it has been 30 years already. Although hair analysis in Korea started later than Europe or the United States, it has made remarkable progress in the field of forensic science. In this study, it was reviewed the current status of hair analysis on narcotics, mainly abused in Korea. In addition, the recent research trends also were investigated. Methamphetamine (MA) and cannabis, which are mainly abused narcotics in Korea, and other drugs such as ketamine (KT), synthetic cannabinoids (new psychoactive substances, NPS) and propofol were discussed here. The analytical methods for those drugs were developed by the National Forensic Service (NFS), mainly using gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). MA has been the most abused drug in Korea. Accordingly, its hair analysis was first introduced in 1993, earlier than any other drugs. In 2011, the NFS finally developed certified reference material (CRM) using Korean MA abusers’ hair. This achievement brought not only financial benefit as saving the cost of purchasing expensive standard products and it also contributed to the development of forensic science by providing free distribution to developing Asian countries. In addition, analytical method of cannabis in hair by gas chromatography-tandem mass spectrometry (GC-MS/MS) was also developed in 2011. It detects carboxytetrahydrocannabinol (THC-COOH), a metabolite of tetrahydrocannabinol (THC), following two steps using LC-MS system in two different acquisition modes (i.e., MRM and MS3). With using the method, it was found that both LOD and LOQ were 0.0001 ng/mg. After KT began to be regulated in Korea in 2005, the detection rates of KT in seized materials and urine during 2017–2020 were found 2.3 and 1.4%, respectively. Majority abusers of KT were young people in twenties. As a result of analyzing the hairs of 61 abusers, it was clear that KT was abused with other drugs such as methylenedioxymethamphetamine (MDMA) or MA rather than single administration. Since JWH-018 was first detected in Korea in 2009, the number of NPS has been rapidly increased also causing another a serious social problem. The most abused NPS is synthetic cannabinoids, accounting for 74% of the total. ABCHMINACA was the most detected synthetic cannabinoid in the hair of abusers from 2014 to May 2021. In February 2011, Korea designated propofol as a controlled drug and it was only country regulating it in the world. The detection method of the propofol in hair was finally established after two years later, 2013. For detection of the propofol in hair samples, LC-MS/MS is using to confirm propofol-glucuronide, major metabolite of the propofol. Though there is a cut-off value for each drug in hair recommended by the Society of Hair Testing (SoHT), each country has varied values due to the different analytical conditions such as method, equipment, and even cultural characteristics. Korea has been its own analytical method by referring to these international guidelines. The NFS is continuously developing new analytical method such as simultaneous analysis with increasing the number of abusers and drugs. Recently, research on hair metabolomics using animal is being actively conducted. One of findings was metabolic alterations in hair could provide insight into metabolic perturbation over a longer period than other conventional biological samples (i.e., plasma, urine). Thus, analytical techniques in hair metabolomics can be a significant approach with high potency for evaluating the animal and human pathological conditions.

Supramolecular solvent-based sample treatment workflow for determination of multi-class drugs of abuse in hair by liquid chromatography-tandem mass spectrometry

Hair is becoming a main matrix for forensic drug analyses due to its large detection window compared to traditional matrices (i.e. urine & blood) and the possibility of establishing the temporal pattern of drug consumption. However, the extremely time- and solvent-consuming nature of conventional sample treatments render it difficult for routine use of hair analysis in forensics. In this paper, this drawback was intended to be addressed by the use of hexanol-based supramolecular solvents (SUPRAS) with restricted-access properties. The aim was to develop a fast and interference-free sample treatment workflow for the determination of opioids, cocaine, amphetamines and their metabolites in human hair. The main variables affecting the extraction were optimized and the method was validated following the European Medical Agency guideline. Major advantages of the proposed method were the straightforward sample preparation, which combines a high extraction yield (93–107%) and matrix effect removal (93–102%SSE) in a single step, the high sample throughput, and the reduced volume of organic solvent required (100 µL of SUPRAS per sample), which makes sample treatment cost-effective and eco-friendly. Method quantification limits were lower enough for all the target drugs (0.5–1.1 pg mg−1) to allow their quantitation in human hair routine analyses. The method was successfully applied to the determination of drugs of abuse in a human hair control sample.

Towards a Comprehensive Sample Treatment Workflow for Determination of Multi-Class Drugs of Abuse in Hair by Liquid Chromatography-Tandem Mass Spectrometry

Towards a Comprehensive Sample Treatment Workflow for Determination of Multi-Class Drugs of Abuse in Hair by Liquid Chromatography-Tandem Mass Spectrometry

Determination of Drugs of Abuse in Hair by LC-MS-MS: Application to Suicide Attempts Investigation.

Considering that the use of psychoactive substances (PSs) is a risk factor to either higher intensity or frequency of suicidal behavior, hair analysis was conducted to investigate the most consumed PSs (opiates, amphetamine stimulants, marijuana, cocaine and heroin) in patients who attempted suicide and received urgent care at emergency service. Hair samples were extracted using methanol and sonicated under heating and then analyzed using liquid chromatography-tandem mass spectrometry. During validation, the method complied with international recommended criteria, with limits of detection between 0.0025 and 0.05 ng/mg and linearity between 0.1 and 4 ng/mg for methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), morphine, amphetamine, 6-acetylmorphine, 3,4-methylenedioxyamphetamine (MDA), fenproporex, diethylpropion and codeine; between 0.025 and 1 ng/mg for tetrahydrocannabinol (THC), benzoylecgonine and cocaethylene and between 0.25 and 10 ng/mg for cocaine and mazindol. A total of 109 hair samples were analyzed and segmented in 404 parts. Among all analyzed samples, 30.3% were positive for at least one PS (n = 33), such as cocaine (90.9%), codeine (12.1%), morphine (3.0%), MDMA (3.0%) and THC (3.0%). In segmental analysis of cocaine positive samples (n = 30), 76.7% of the samples indicated recent exposure to cocaine (<1 month). This same behavior was observed when analyzing codeine (n = 4) and morphine (n = 1). THC positive samples indicated exposure dated ∼4 months prior. In conclusion, the method was validated following international recommendations for the 12 most consumed PSs in Brazil, as well as two of the most common found metabolites.

Potential of High-Resolution Mass Spectrometry for the Detection of Drugs and Metabolites in Hair: Methoxetamine in a Real Forensic Case.

The analysis of drugs of abuse in hair and other biological matrices of forensic interest requires great selectivity and sensitivity. This is done traditionally through target analysis, with one or more analytical methods, or with different and specific preanalytical phases, and complex procedures performed by the toxicological laboratories, and there is no exception with ketamine-like compounds, such as methoxetamine, a new psychoactive substance whose use has increased in the last decades, and continues to grow quickly year by year. More validated methods of analysis are needed to detect these substances in low concentrations selectively. Reanalyzing the samples of a former case of a polydrug consumer accused of a crime against public health in Spain, five metabolites of methoxetamine (normethoxetamine, O-desmethylmethoxetamine, dehydromethoxetamine, dihydronormethoxetamine and hydroxynormethoxetamine) were tentatively detected using a high-resolution technique, that is, liquid chromatography coupled to high-resolution mass spectrometry (LC-HR-MS-MS). The highest analytical selectivity of LC-HR-MS-MS method together a universal and simpler pretreatment stages has demonstrated to allow faster analysis and more sensitivity than the one performed traditionally at the INTCF laboratories, which was gas chromatography coupled to mass spectrometry.

UHPLC-MS/MS method for simultaneously detecting 16 tryptamines and their metabolites in human hair and applications to real forensics cases

Tryptamines are hallucinogenic substances many of which have appeared recently as novel psychoactive substances (NPS). Herein, we describe the establishment of a rapid UHPLC-MS/MS quantitative method for the targeted screening of 16 tryptamines of abuse in hair. Twenty milligram pieces of hair were pulverized below 4 °C in 0.5 mL of deionized water containing 0.1% formic acid and an internal standard (2 ng/mL psilocin-d10 and psilocybin-d4). After subsequent centrifugation, 5 μL of the supernatant was injected into a LC–MS/MS system fitted with a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm). The column was gradient eluted at 0.3 mL/min with mobile phases composed of 20 mmol/L ammonium acetate, 5% acetonitrile, and 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). Limits of detection ranged between 0.1 and 20 pg/mg, with limits of quantitation ranging from 3 to 50 pg/mg. The calibration curves for all analytes were linear (r > 0.992). Accuracies varied between 91% and 114%, with intraday precision RSDs < 14% and interday precision RSDs of between 1.3% and 14%. The recoveries of all tryptamines were in the 85–115% range, with the matrix effect ranging from 95% to 112%. The validated method was successfully used to analyse 191 hair samples from suspected tryptamine users, 77 of which were 5-MeO-DiPT-positive, while the 16 tryptamines and their metabolites were not detected in the remaining 114 hair samples. 5-MeO-DiPT and its 5-MeO-NiPT, 5-OH-DiPT, and 4-OH-DiPT metabolites were concurrently detected in 34 hair samples. 5-MeO-DiPT, as the parent drug, was the parent substance found in the hair samples.

Development and validation of fast UHPLC-MS/MS screening method for 87 NPS and 32 other drugs of abuse in hair and nails: application to real cases.

Interest on keratinized matrix analysis for clinical and forensic purposes has been recently grown due to the wide temporary detection window for psychotropic and toxic substances entrapped after repeated consumption. The aim of this study was the development and full validation of an UHPLC-MS/MS screening method to quantify 119 molecules among most abused classic drugs and new psychoactive substances in hair and in nails, to assess the polyconsumption. Twenty-five milligrams of hair or nail samples, added with the internal standard mixture, were cut and incubated with 500μL M3® buffer reagent at controlled temperature. After cooling, 1μL supernatant was injected in the chromatographic system equipped with an Oasis HLB column. After the 10min chromatographic separation through a gradient mobile phase (aqueous ammonium formate, phase A; acetonitrile, phase B), the target compounds were detected in multiple reaction monitoring mode. The method was linear (r2 always better than 0.99) in a calibration range of LOQ 20000pg compound for milligram hair and of LOQ 1000pg compound per milligram nail. Process efficiency of analytes under investigation was always better than 65% and no significant ion suppression due to matrix effect was observed. Intra-assay and inter-assay precision and accuracy were always better than 15%. The applicability and trueness of the method were examined by analysing real samples of hair and nail from users of psychoactive drugs in recreational contexts. Both classic drugs and new psychoactive substances could be determined as result of single or repeated use and accumulation in keratin matrices. Graphical abstract.

Combination of pressurized liquid extraction with dispersive liquid liquid micro extraction for the determination of sixty drugs of abuse in hair

The increasing phenomenon of drug addiction and the introduction of New Psychoactive Substances (NPS) have led to a progressive growth of research in the field of forensic analytical toxicology, with the need to develop modern and faster analytical procedures. Hair testing has gained increasing attention and recognition as a complement to blood and urine analysis, since it is a unique material for the retrospective detection of drugs, due to its large detection window.In this paper, a multiclass method for the simultaneous extraction, identification and quantification of sixty drugs of abuse belonging to different chemical classes in hair is proposed. This method can provide a valid, fast, simple and low-cost alternative to common tests; at the same time, it provides quantitative results, to concurrently confirm the assumption of one or more illicit substances. Both the decontamination step and the extraction of the analytes from the inner core of the hair were carried out by means of pressurized liquid extraction (PLE) while the clean-up was performed by dispersive liquid/liquid microextraction (dLLME), giving the great advantage of a high enrichment factor. The selected chromatographic conditions allowed a satisfying separation of the 60 analytes in 14 min, while the detection was conducted with a high-resolution mass spectrometer with Orbitrap technology. This multiclass method was suitable for analytes with different chemical characteristics allowing to reduce time and cost of analysis, organic solvent volume and the amount of sample required for analysis. The whole method was fully validated as confirmatory method following SWGTOX guidelines.

Toxicological hair analysis: Pre-analytical, analytical and interpretive aspects.

Background and aims Hair analysis for drug detection is one of the widely accepted imperative techniques in the field of forensic toxicology. The current study was designed to investigate the efficacy of chromatography for detection of drugs of abuse in hair. Method A comprehensive review of articles from last two decades on hair analyses via PubMed and similar resources was performed. Issues concerning collection, decontamination and analytical techniques are summarised. Physiochemical nature of hair, mechanism of drug incorporation and its stability in hair are briefly discussed. Furthermore, various factors affecting results and interpretation are elucidated. Result A hair sample is chosen over traditional biological samples such blood, urine, saliva or tissues due to its inimitable ability to provide a longer time frame for drug detection. Its collection is almost non-invasive, less cumbersome and does not involve any specialised training/expertise. Recent advances in analytical technology have resulted in better sensitivity, reproducibility and accuracy, thus providing a new arena of scientific understanding and test interpretation. Conclusion Though recent studies have yielded many insights into drug binding and drug incorporation in hair, the major challenge in hair analysis lies in the interpretation of results, which may be affected by external contamination and thus lead to false-positives. Therefore, there is a need for more sensitive and selective analysis methods to be developed in order to minimise factors that induce the effect of melanin, age and so on, and this would certainly provide a new dimension to hair analysis and its applications.

Determination of hydroxy metabolites of cocaine in hair samples for proof of consumption.

Although hair is widely used to identify drug use, there is a risk of false positives due to environmental contamination. This especially applies to cocaine (COC). Several strategies such as detection of norcocaine (NCOC) or cocaethylene, metabolite concentration ratios or intricate washing procedures have been proposed to differentiate actual use from contamination. The aim of the present study was to identify hydroxy metabolites of COC in hair specimens, thus enabling unambiguous prove of ingestion. A suspect screening of 41 COC-positive samples for these compounds was performed by liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS). Once identified, mass transitions for o-, p- and m-isomers of hydroxy COC as well as p- and m-isomers of hydroxy benzoylecgonine (BE) and hydroxy NCOC were introduced into a routine procedure for testing drugs of abuse in hair by liquid chromatography-tandem mass spectrometry (LC-MS/MS) which was applied to 576 hair samples. Hydroxy metabolites were present in 92.2% of COC-positive hair samples; their detection rate exceeded that of cocaethylene and NCOC. Moreover, p-OH-BE, m-OH-BE as well as p-OH-NCOC and m-OH-NCOC have been identified for the first time in COC-positive hair specimens. Hydroxy cocainics could be detected in samples having a negative conclusion on drug use applying hitherto established criteria. We suggest a more conclusive interpretation outcome including detection of hydroxy metabolites into the evaluation of COC-positive hair samples.

Sample preparation method for the combined extraction of ethyl glucuronide and drugs of abuse in hair.

Often in hair analysis, a small hair sample is available while the analysis of a multitude of structurally diverse substances with different concentration ranges is demanded. The analysis of the different substances often requires different sample preparation methods, increasing the amount of required hair sample. When segmental hair analysis is necessary, the amount of hair sample needed is further increased. Therefore, the required sample amount for a full analysis can quickly exceed what is available. To combat this problem, a method for the combined hair sample preparation using a single extraction procedure for analysis of ethyl glucuronide with liquid chromatography-multistage fragmentation mass spectrometry/multiple reaction monitoring (LC-MS3 /MRM) and common drugs of abuse with LC-MRM was developed. The combined sample preparation is achieved by separating ethyl glucuronide from the drugs of abuse into separate extracts by fractionation in the solid-phase extraction step during sample clean-up. A full validation for all substances for the parameters selectivity, linearity, limit of detection, limit of quantification, accuracy, precision, matrix effects, and recovery was successfully completed. The following drugs of abuse were included in the method: Amphetamine; methamphetamine; 3,4-methylenedioxy-N-methylamphetamine (MDMA); 3,4-methylenedioxyamphetamine (MDA); 3,4-methylenedioxy-N-ethylamphetamine (MDE); morphine; 6-monoacetylmorphine; codeine; acetylcodeine; cocaine; benzoylecgonine; norcocaine; cocaethylene; methadone; 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and methylphenidate. In conclusion, as only 1 sample preparation is needed with 1 aliquot of hair, the presented sample preparation allows an optimal analysis of both ethyl glucuronide and of the drugs of abuse, even when the sample amount is a limiting factor.

Determination of AB-CHMINACA and its metabolites in human hair and their deposition in hair of abusers

Despite global efforts to control the abuse of synthetic cannabinoids, the high-level of turnover from the market impedes regulation, endangering public health. N-[(1S)-1-(aminocarbonyl)-2-methylpropyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (AB-CHMINACA) is the most popular synthetic cannabinoid in South Korea since its introduction in 2014. Nonetheless, few studies have been carried out on AB-CHMINACA and its metabolites, and its deposition in human hair. The purpose of this study was to develop and validate an analytical method for detection of AB-CHMINACA and its six metabolites in hair using a liquid chromatography tandem mass spectrometry (LC–MS/MS) system, for forensic applications. The methanol extracts of hair samples were evaporated, filtered, and analyzed by LC–MS/MS with electrospray ionization in positive ion mode. The limits of detection and quantification ranged from 0.5 to 10pg/mg and 2 to 50pg/mg, respectively. Good linearity was achieved within the range of 5–1000pg/mg or 10–1000pg/mg depending on the analyte. Intra- and inter-assay precision and accuracy values were below 15%. No significant variation was observed using different sources of hair matrices. These validation results proved the selectivity, accuracy and reproducibility of the method. The established method was applied to 37 authentic samples from suspected synthetic cannabinoid users. AB-CHMINACA and its two metabolites, AB-CHMINACA M2 and AB-CHMINACA M4, were detected. The concentration of the parent drug was much higher than those of its metabolites, and the amount of AB-CHMINACA M2 was greater than that of AB-CHMINACA M4 in all samples. No other metabolites were detected in the samples.

Mass Spectrometry Imaging of Drugs of Abuse in Hair.

Hair testing is a powerful tool routinely used for the detection of drugs of abuse. The analysis of hair is highly advantageous as it can provide prolonged drug detectability versus that in biological fluids and chronological information about drug intake based on the average growth of hair. However, current methodology requires large amounts of hair samples and involves complex time-consuming sample preparation followed by gas or liquid chromatography coupled with mass spectrometry. Mass spectrometry imaging is increasingly being used for the analysis of single hair samples, as it provides more accurate and visual chronological information in single hair samples.Here, two methods for the preparation of single hair samples for mass spectrometry imaging are presented.The first uses an in-house built cutting apparatus to prepare longitudinal sections, the second is a method for embedding and cryo-sectioning hair samples in order to prepare cross-sections all along the hair sample.

Influence of chemical straightening on the stability of drugs of abuse in hair.

Chemical straightening, also known as a relaxer, is ubiquitously used among African American women to obtain straighter hair compared with their natural tresses. This study focused on the stability of drugs of abuse in hair after a single application of the relaxer. Commercially available 'Lye' or 'No-Lye' chemical straightening products (Silk Elements™) were applied in vitro to drug-fortified hair (standard reference materials (SRM) 2379 and 2380) and hairs clipped from established drug users. Target analytes (cocaine (COC), benzoylecgonine (BZE), cocaethylene (CE), phencyclidine and tetrahydrocannabinol) were isolated using solid-phase extraction and then analyzed with isotope dilution gas chromatography-mass spectrometry with selective ion monitoring. After either treatment, drug concentrations were significantly (P < 0.05) decreased in both the SRM sample and the hair from authentic abusers. In the SRM groups, 6-67% of the original concentration remained after a single chemical treatment. Similarly, only 5-30% of the original concentration remained in authentic drug hairs that had formerly tested positive for COC, BZE and CE.

A quantitative, selective and fast ultra-high performance liquid chromatography tandem mass spectrometry method for the simultaneous analysis of 33 basic drugs in hair (amphetamines, cocaine, opiates, opioids and metabolites).

Forensic testing for drugs of abuse in hair has become a useful diagnostic tool in determining chronic drug use as well as examining long-term drug history thorough segmental analysis. However, sensitive and specific analytical methods are needed. A simple, rapid and highly sensitive and specific method for the extraction and quantification of 33 opioids, opiates, cocaine, and amphetamines is presented. The method was fully validated according to international guidelines. Twenty milligrams of hair sample was pulverized and then incubated in the same disposable tube with methanol (under sonication at 45°C) during 4h. After centrifugation the supernatant was evaporated up to about 100 μL and a solid phase extraction (SPE) followed by separation and quantification using ultra performance liquid chromatography-tandem mass spectrometry (UHLC-MS/MS) were carried out. Chromatographic separation was achieved using a BEH phenyl column eluted with 0.1% formic acid: methanol (0.1% formic acid). Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions. Good intra-assay and inter-assay precision (relative standard deviations (RSDs) were observed (<15%) for most of the compounds. The lower limit of quantification was fixed at the lowest calibrator in the linearity experiments and it ranged from 0.006 to 0.063 ng/mg. No instability was observed in processed samples. Extraction efficiency varied from 37 to 107% (except for EDDP with a recovery of 5%) and matrix effects ranged from 52 to 160%, and for most of the compounds it was compensated by the internal standard (IS). The method was subsequently applied to authentic hair samples obtained from forensic and toxicology cases and to proficiency test (obtaining z-scores lower than 1 for most of the compounds). The validation and actual sample analysis results show that this method is rugged, precise, accurate, and well-suited for routine hair analysis.

A study on photodegradation of methadone, EDDP, and other drugs of abuse in hair exposed to controlled UVB radiation.

The drug content of hair may be affected by washing, chemical or thermal treatments, the use of cosmetics, or exposure to the environment. Knowledge concerning the effect of natural or artificial light on drug content in hair can be helpful to the forensic toxicologist, in particular when investigating drug concentrations above or below pre-determined cut-offs. The photodegradation of methadone and its metabolite, 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was studied in authentic positive hair samples by comparing drug concentrations determined by liquid chromatrography-high resolution mass spectrometry before and after exposure to UVB light (in vivo study). The same approach was applied in order to investigate the light sensitivity of opiates (6-monoacetylmorphine and morphine) and cocainics (cocaine and benzoylecgonine) in true positive hair. The yields of photodegradation were calculated for each drug class in eight different positive hair samples irradiated by UVB at 300 J/cm(2) obtaining averages, ranges and standard deviations. In parallel, the photostability of all the compounds as 10(-5) -10(-4) M standard solutions in methanol were examined by means of UVB light irradiation in the range 0-100 J/cm(2) followed by UV/Vis spectroscopic analysis and direct infusion electrospray ionization-high resolution mass spectrometry (in vitro study). In hair, methadone was shown to be significantly affected by light (photodegradation of 55% on average), while its metabolite EDDP proved to be more photostable (17%). 6-monoacetylmorphine, morphine, benzoylecgonine, and cocaine were more photostable than methadone in vivo (on average, 21%, 17%, 20%, and 11% of degradation, respectively). When irradiated in standard solutions, the target molecules exhibited a larger photodegradation than in vivo with the exception of cocaine (photodegradation for methadone up to 70%, 6-monoacetylmorphine and morphine up to 90%, benzoylecgonine up to 67%, cocaine up to 15%). Some factors possibly affecting the yields of photodegradation in hair and partially explaining the differences observed between the in vivo and the in vitro studies were also investigated, such as the colour of hair (the role of melanin) and the integrity of the keratin matrix.

Rapid extraction, identification and quantification of drugs of abuse in hair by immunoassay and ultra-performance liquid chromatography tandem mass spectrometry

Drug testing in hair is a unique analysis in pharmacotoxicology for establishing a past repeated history of consumption or passive exposure to psychotropic substances. A rather lengthy sample treatment is usually required before parent drugs and eventual metabolites are amenable to quali-quantitative analysis. We evaluated a high throughput screening and confirmation analysis of drugs of abuse in hair by immunoassay and a validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) after applying a rapid digestion of the keratin matrix with VMA-T reagent before screening assay and M3 reagent before confirmatory analysis. Samples digestion with VMA-T reagent and immunometric screening analysis of hair calibrators, controls and clinical samples for a total of 150 samples was completed in 4 h. No false-positive and -negative results were found for the control material. UPLC-MS/MS analysis confirmed all of the 31 adult hair samples positive to the screening test using internationally established cut-offs, and identified and quantified drugs of abuse in 32 pediatric hair samples, applying lower limits of quantification from 0.01 to 0.1 ng analyte per mg hair. Analytical recovery was between 70.9% and 100.7%. Intra- and inter-assay imprecision and inaccuracy were always lower than 10%. Rapid extraction, identification and quantification of drugs of abuse in hair by immunoassay and UPLC-MS/MS was tested for its feasibility in clinical samples and provided excellent results for rapid and effective drug testing in hair in epidemiological studies.