Hair analysis for cocaine: Factors in laboratory contamination studies and their relevance to proficiency sample preparation and hair testing practices

Hair samples were contaminated by rubbing with cocaine (COC) followed by sweat application, multiple shampoo treatments and storage. The samples were then washed with isopropanol for 15 min, followed by sequential aqueous washes totaling 3.5 h. The amount of drug in the last wash was used to calculate a wash criterion to determine whether samples were positive due to use or contamination. Analyses of cocaine and metabolites were done by LC/MS/MS. These procedures were applied to samples produced by a U.S. government-sponsored cooperative study, in which this laboratory participated, and to samples in a parallel in-house study. All contaminated samples in both studies were correctly identified as contaminated by cutoff, benzoylecgonine (BE) presence, BE ratio, and/or the wash criterion. A method for determining hair porosity was applied to samples in both studies, and porosity characteristics of hair are discussed as they relate to experimental and real-world contamination of hair, preparation of proficiency survey samples, and analysis of unknown hair samples.     

Removing and identifying drug contamination in the analysis of human hair

The procedure used in this laboratory for removing and identifying contamination of hair specimens with drugs is demonstrated by its application to hair contaminated by various experimental models. The models include soaking; coating with drug followed by sweat conditions for 6 h; and soaking in a very high concentration of cocaine followed by storage and multiple shampoo treatments. A multi-part wash procedure along with a wash criterion is applied to all samples containing drug above the cutoff. The failure of the wash criterion is a signal that the sample may be positive due to contamination rather than use, and in the absence of other over-riding evidence, the sample would be considered to be negative for drug use. This Wash Criterion has also been tested with hair from subjects demonstrated to be drug users by one or more drug-positive urines; in these studies, all hair samples from demonstrated users passed the Wash Criterion test.     

Hair combing to collect organic gunshot residues (OGSR)

A protocol is presented for the collection and analysis of gunshot residues (GSR) from hair. A fine-toothed comb is used for collection of the residues. A small zip-closure bag serves as a container for both sample storage and extraction of the characteristic organic powder additives. The success of this residue recovery approach was tested on simulated shooters and victims using mannequin-supported human wig hair as well as on human shooters. Residues were collected from four weapons: a revolver and semi-automatic pistol, rifle and shotgun. One characteristic additive, nitroglycerin, was detected by capillary electrophoresis (CE) in the majority of the collection experiments.     

The use of supercritical fluid extraction for the determination of amphetamines in hair

A laboratory study interested in the analysis of human hair for drugs-of-abuse was conducted to determine if drugs could be detected and quantified from hair. Supercritical fluid extraction (SFE) techniques followed by GC-MS analysis were applied to extract amphetamines from hair. The group of amphetamines included methylenedioxyamphetamine (MDA), methylenedioxymetamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and internal standard mephentermine (MP). To validate information on amphetamine use in hair, powdered hair samples free from drugs were collected and soaked in a known amphetamine standard solution. Authentic fortified case hair samples taken from known drug users known to have consumed amphetamines were also analyzed for amphetamine. Results from this study show that amphetamine use can be detected in spiked and authentic fortified human hair using SFE techniques for qualitative and quantitative reproducible results.     

The detection of cocaine in hair specimens using micro-plate enzyme immunoassay.

The analysis of hair for drugs of abuse is becoming increasingly popular and is under consideration by the Division of Health and Human Services as a possible alternative or adjunct to urinalysis in workplace programs. The detection of cocaine in human hair using a commercially available micro-plate enzyme immunoassay is described for the first time. Sample size and incubation time were the major variables in the optimization of the method. In order to validate the procedure, the method was applied to 105 consecutive hair samples routinely received into our laboratory. The samples were simultaneously analyzed by the Micro-Plate immunoassay (EIA), as well as our current fluorescence polarization immunoassay (FPIA) procedure and gas chromatography-mass spectrometry (GC/MS). The sensitivity of the EIA and FPIA assays were 75% and 67.8% respectively; specificity 97.4% and 80.5% respectively; and efficiency 91.4 and 77.1% respectively. The Micro-Plate EIA was shown to be a valid alternative to other immunoassay screening methods for the detection of cocaine in hair by demonstrating increased sensitivity, specificity and efficiency over our current technique.     

Differentiation of Morphologically Similar Human Head Hairs from Two Demographically Similar Individuals Using Amino Acid Ratios,

Human hair is frequently encountered as forensic evidence and can contribute valuable information to investigators. Conventional forensic hair analyses include microscopic hair comparison (MHC) and DNA analysis. However, MHC is not supported by statistics and DNA analysis cannot always be performed. Recent studies have demonstrated that evaluation of differences in the hair proteins may offer an alternate method to these analyses. In this study, an evaluation of the amino acids present in hair was investigated as an approach to differentiate morphologically indistinguishable hair samples from two demographically similar individuals. Proteins in the hair were digested using hydrochloric acid, and the resulting amino acids were derivatized with N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) for analysis using gas chromatography-mass spectrometry (GC-MS). Eight derivatized amino acids were detected and quantified relative to an internal standard, L-norvaline, and used to construct twenty-eight amino acid ratios. Hair samples were collected from four areas of the head on various days over the course of one month, and no significant differences in amino acid ratios (p-value > 0.05) were observed among the areas of the head, and the ratios were consistent over the time period of this study. Additionally, fifteen of these amino acid ratios were found to be significantly different between the two individuals when compared using a two-sample t-test (p-value ≤ 0.05). These data indicate that amino acid analysis was able to differentiate two morphologically similar hair samples from different individuals and demonstrates the applicability of this method to distinguish similar hair samples when DNA analysis cannot be performed.     

Identification of furosemide in hair in a post‐mortem case by UHPLC‐MS/MS with guidance on interpretation

Testing for drugs in hair raises several difficulties. Among them is the interpretation of the final concentration(s). In a post‐mortem case, analyses revealed the presence of furosemide (12 ng/mL) in femoral blood, although it was not part of the victim's treatment. The prosecutor requested our laboratory to undertake an additional analysis in hair to obtain information about the use of furosemide. A specific method was therefore developed and validated to identify and quantify furosemide in hair by UHPLC‐MS/MS. After decontamination of 30 mg of hair, incubation in acidic condition, extraction with ethyl acetate, the samples were analyzed by UHPLC‐MS/MS. Furosemide was found in the victim's hair at 225 pg/mg. However, it was not possible to interpret this concentration due to the absence of data in the literature. Therefore, the authors performed a controlled study in two parts. In order to establish the basis of interpretation, several volunteers were tested (four after a single 20 mg administration and twenty‐four under daily treatment). The first part indicated that a single dose is not detectable in hair using our method. The second part demonstrated concentrations ranging from 5 to 1110 pg/mg with no correlation between dosage and hair concentrations. The decedent's hair result was interpreted as repeated exposures. In the case of furosemide analysis, hair can provide information about its presence but cannot give information about dosage or frequency of use.     

Evaluating the discriminating power of amino acid ratios on distinguishing dark colored hair samples

Human hairs are one of the most commonly encountered items of trace evidence. Currently, conventional methods for hair analysis include microscopic comparison and DNA analysis (nuclear and mitochondrial). Each approach has its own drawbacks. Hair proteins are stable and offer an alternative to DNA testing, as demonstrated with proteomics for distinguishing humans. However, proteomics is complicated and requires identifying peptides to remain intact following harsh sample preparation methods. Alternatively, the actual amino acid content of a hair sample may also offer important identifying information and actually requires proteins and peptides to be broken down completely rather than remaining intact. This study evaluated the discriminating power of using hair amino acid ratios to differentiate hair samples from 10 unrelated individuals with dark colored hair. Hair proteins were digested, derivatized, and analyzed using gas chromatography–mass spectrometry. Amino acid ratios were calculated for each individual and comparisons using ANOVA and post-hoc pairwise t-test with Bonferroni correction were made with amino acid ratios for individuals. Overall, out of the 45 possible pairwise comparisons between all hair samples, 38 (84%) were differentiable. Out of the 36 possible pairwise comparisons between brown haired individuals, 32 (89%) were considered differentiable using univariate statistics. Multivariate statistics were also attempted but, overall, univariate models were sufficient for exclusionary purposes. These results indicate that amino acid ratio analysis can potentially be used as an exclusionary method using hair if DNA analysis cannot be performed, or to corroborate conclusions made following microscopic analysis.     

Methadone and EDDP in hair from human subjects following a maintenance program: results of a pilot study

A specific method has been developed for the quantitative determination of methadone (MTD) and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in hair.An amount of 50mg hair samples were incubated in 0.01M HCl overnight at 60 degrees C and deuterated internal standards of MTD and EDDP were added before extraction. Hydrolyzed solutions were extracted by automated solid-phase extraction procedure and analyzed on a gas chromatography (GC) coupled to a ion trap mass spectrometer (MS). Positive chemical ionization was used with acetonitrile as liquid reagent. The different validation parameters, linearity, repeatability, recovery and detection limits are presented. A relative standard deviation (R.S.D.) of 12 and 11% was obtained for the repeatability of MTD and EDDP, respectively. The limits of quantification (LOQ) was 0.05ng/mg for MTD and 0.2ng/mg for EDDP.A number of 26 hair samples from human subjects following a long-term MTD therapy were analyzed by this method. Blood samples of these subjects were analyzed with a routine method using a liquid-liquid extraction and GC/nitrogen phosphorus detector (NPD). MTD was quantified in blood and hair samples and EDDP found in 50% of the hair sample.A comparison was made between the concentrations found in blood or in hair and the dose administrated. This study could demonstrate that there is no relation between the administrated dose and MTD or EDDP concentrations in hair.     

A novel method for extraction and analysis of gunpowder residues on double-side adhesive coated stubs

A study was conducted to develop an efficient method for extraction and analysis of gunpowder (propellant) residues from double-side adhesive coated stubs, which are used for sampling suspects or their clothing for gunshot (primer) residues (GSR). Conductive and non-conductive double-side adhesives were examined, and the analysis was carried out by gas chromatography/thermal energy analyzer (GC/TEA) and ion mobility spectrometry (IMS). The optimal procedure for the extraction, as was developed in the present study, employs two stages: (1) extraction of the stubs with a mixture of 80% v/v aqueous solution of 0.1% w/v of sodium azide and 20% v/v of ethanol employing sonication at 80 degrees C for 15 min. and (2) residues from the obtained extract were further extracted with methylene chloride. The methylene chloride phase was concentrated by evaporation prior to analysis. Extraction efficiencies of 30-90% for nitroglycerine (NG) and for 2,4-dinitro toluene (2,4-DNT) were found. No significant interferences in the analysis were observed from the adhesives or skin. Interferences were observed in the analysis by the GC/TEA of the samples collected from hair. The method enables analysis of propellant residues on a double-side adhesive coated stub after it was examined for primer residues by scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX). Thus, the probative value of the evidence may be increased.     

Forensic Discrimination of Dyed Hair Color: I. UV‐Visible Microspectrophotometry*†

Abstract: Current protocols for examining hair do not attempt to differentiate hair color using instrumental analysis. In this study, hair samples treated with 55 different red hair dyes were analyzed using UV‐visible microspectrophotometry between 200 and 700 nm. Using air as a background reference gave the best results, although mounting media such as glycerin could also be used. The contribution of the hair substrate is predominantly observed in the range of 300–400 nm while the dye peak is evident in the range of 425–550 nm. It was found that the presence of hair dye reduces the overall intrasample variability of the hair color. In addition, visual inspection and spectral interpretation showed that dyed hair exhibits distinct and discernable shades. The color of all samples was stable during storage and while all hair dyes fade with washing, significant fading of the color was only evident after daily washing for 3 weeks.     

Detection of meperidine and its metabolites in the hair of meperidine addicts

The presence of meperidine and its metabolites in the hair of meperidine addicts was investigated using GC–MS (EI, PCI). Meperidine and its three metabolites – normeperidine, N-methoxy meperidine and acetyl normeperidine, were found in hair samples from addicted subjects. Methods for the simultaneous determination of meperidine and its metabolites by GC–MS-SIM were also established for human hair samples. After the addition of d₄-meperidine as an internal standard, hair samples weighing 5 mg were incubated in 0.1 M HCl at 45°C overnight, and the resulting digests were extracted with ether. The recoveries were greater than 80%, with coefficients of variation (CVs) between 4.48 and 8.31%. The calibration curves for meperidine and normeperidine in hair were linear over a concentration range of 1 to 500 ng per mg of hair, with correlation coefficients of r=0.9990 and r=0.9992, respectively. Values less than 0.25 ng/mg of hair were cut off. Hair samples obtained from 60 drug addicts were analyzed using this method, and the content of meperidine and normeperidine was determined to be 103±130 and 117±143 ng/mg, respectively. Sectional analysis revealed that meperidine was present and stable in hair for at least 20 months, but normeperidine content at the level of the hair root was higher compared to the tip of the hair shaft. The results also revealed that there was a correlation between the subject’s drug abuse history and the distribution of drug along the hair shaft, and between the doses of meperidine and drug content presented in hair.     

Tandem mass spectrometry: a helpful tool in hair analysis for the forensic expert

The Bavarian State Bureau of Investigation in Munich has the exclusive responsibility for investigation of criminal acts. One considerable expertise is that of hair analysis. According to the legal system in Germany, there is a special interest when some clients' hair tested positive for illicit drugs. An accused with a lot of drugs in his hair will be treated as a supposed addict and will be guaranteed extenuating circumstances. The instrumentation used for hair analysis is a powerful analytical tool: a Varian 3400 gas chromatograph linked to a Finnigan Tandem-MS (TSQ 700). The methanol extraction method is used for the detection of illegal drugs and metabolites: amphetamine, methamphetamine, MDA, MDMA (ecstasy), MDE, MBDB, methadone, THC, EDDP (metabolite of methadone), cocaine, benzoylecgonine, cocaethylene, opiates (dihydrocodeine, codeine, heroin, 6-monoacetylmorphine, morphine, acetylcodeine). For the detection of 9-carboxy-THC by negative chemical ionization the hair sample is hydrolyzed under alkaline conditions. Solid-phase extraction is used for clean-up. The LOQ for the determination of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic-acid is 0.16 pg/mg hair. An unsurpassed combination for rendering an expert opinion based on hair analysis may be: a forensic expert using diligence and experience, coupled with the performance of a sophisticated analytical instrument.     

基质辅助激光解吸飞行时间质谱分析生物样品中甲基苯丙胺

目的建立尿样和头发中甲基苯丙胺的基质辅助激光解吸飞行时间质谱（matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS）分析方法。方法尿样采用液液提取,头发经0.1mol/L盐酸水解后采用液液提取,以碳纳米管为基质应用MALDI-TOF-MS法检测。结果尿样中甲基苯丙胺的最低检测限（LOD）为0.5μg/mL,线线范围为线性范围为0.5～100μg/mL（R2=0.9970）;毛发中甲基苯丙胺的最低检测限（LOD）为0.4ng/mg,线性范围为0.4～60ng/mg（R2=0.9976）,对送检案例中尿样和头发检材进行检测,效果良好。结论本方法适用于尿样和头发中甲基苯丙胺的分析,与传统气相色谱质谱联用和液相色谱-质谱联用相比,分析速度更快,适合大批量样品同时分析。     

Clozapine dose-concentration relationships in plasma, hair and sweat specimens of schizophrenic patients

The aim of the present study was to establish an analytical method for the determination of clozapine in sweat and to determine whether the clozapine level in hair and sweat were correlated to the daily dose of clozapine delivered to patients. Twenty-six subjects treated with clozapine at 200-700 mg/day for refractory psychosis were included in the study. Clozapine was determined in plasma by liquid chromatography coupled to a diode array detection system, after extraction with an organic solvent at pH 9.5. Clozapine was extracted from hair and sweat patches specimens by incubation in methanol overnight at 40 degrees C. The residues were analyzed by gas chromatography coupled to mass spectrometry in the electronic impact mode of detection. It was possible to determine clozapine in concentrations ranging from 30 to 1016 ng/ml in plasma (n = 22), from 0.17 to 34.24 ng/mg in hair (n = 23) and from 49 to 5609 ng/patch in sweat (n = 20). Preliminary results suggest a lack of correlation between daily regimen of clozapine and plasma levels of the drug. Therefore, a better dose-concentration relationship was observed in our study between daily dose and hair concentration (r = 0.542, P < 7%) or between daily dose and sweat concentration (r = 0.589, P < 6%), but with wide variations for patients at the same posology. However, the idea of using quantitative drug measurements in hair or sweat to ascertain whether a patient has taken his treatment exactly as prescribed will remain inapplicable.     

氯胺酮滥用的毛发分析研究

目的建立毛发中氯胺酮及其代谢物的分析方法并探索氯胺酮进入毛发的机理。方法通过建立豚鼠连续给药(不同剂量)实验模型获取阳性头发和采集氯胺酮滥用者头发,经处理后用GC/MSscan和SIM法分析,以鉴别、确认毛发中氯胺酮及其代谢物。结果豚鼠毛发中氯胺酮的质量分数与给药剂量存在明显的正相关性。毛发中氯胺酮质量分数依白色、棕色、黑色毛发顺序随毛发中黑色素的质量分数增加而增加。豚鼠毛发中氯胺酮与代谢物NK质量分数之比为2.33～12.94,仅在高剂量组的豚鼠毛发中才检测到DHNK,其质量分数与NK接近。15名氯胺酮滥用者黑色头发中均检出原体和代谢物NK,但DHNK少见。豚鼠毛发中代谢物相对质量分数明显高于人。结论本实验结果很好地反映了药物进入毛发代谢过程与药物和黑色素亲和力以及药物的亲脂性密切相关这一规律,但人和动物在药物代谢及进入毛发的难易程度上存在差异。本方法可以用于法庭毒物分析领域头发中氯胺酮的检测。     

Ethyl glucuronide: unusual distribution between head hair and pubic hair

Ethyl glucuronide (EtG) is a minor metabolite of ethanol that can be detected in hair. In some specific situations, head hair can be missing, and therefore, alternative anatomical locations of hair are of interest. In this study, paired hair specimens (head hair and pubic hair) from eight social drinkers were analyzed for EtG. Each sample was decontaminated by two dichloromethane bathes (5 ml) for 2 min. After cutting into small pieces, about 50 mg of hair was incubated in 2 ml water in the presence of 10 ng of EtG-d5, used as internal standard and submitted to ultra-sonication for 2 h. The aqueous phase was extracted by SPE using Oasis MAX columns. The hair extract was separated on an ACQUITY BEH HILIC column using a gradient of acetonitrile and formate buffer. Detection was based on two daughter ions: transitions m/z 221-85 and 75 and m/z 226-75 for EtG and the IS, respectively. This laboratory is using a positive cut-off at 50 pg/mg. All eight head hair specimens were negative for EtG at a limit of quantitation fixed at 10 pg/mg. Surprisingly, EtG was identified at high concentrations in pubic hair, in the range 12-1370 pg/mg. It appears, therefore, that it is not possible to document the drinking status of a subject by simply switching from head hair to pubic hair.     

Diethyl phosphates accumulation in rabbits' hair as an indicator of long term exposure to diazinon and chlorpyrifos

Long term exposure to organophosphate pesticides can be evaluated by quantitative analysis of their non-specific metabolites in hair matrix. The aim of this study was to determine whether these metabolites can be internally incorporated into the hair of rabbits exposed to diazinon and chlorpyrifos. The influence of dose and dose duration of each pesticide dosage were investigated. Three groups of rabbits were exposed to different dosages of diazinon (3.0 and 6.0mg/kg/day) and chlorpyrifos (18.0mg/kg/day) via drinking water. Hair samples were collected every month and analyzed for diethyl phosphate (DEP) and diethyl thiophosphate (DETP) by gas chromatography-mass spectrometry (GC-MS). The mean concentrations of the low-dose treated group, ranged from 112 to 257pg/mg for DEP and from 295 to 515pg/mg for DETP in hair. The high-dose treated group demonstrated a range of mean concentrations from 142 to 585pg/mg for DEP and from 406 to 988pg/mg for DETP in hair. For the chlorpyrifos treated group, the concentrations ranged from 138 to 1070 for DEP and from 554 to 886pg/mg for DETP. Analysis revealed the incorporation of these metabolites into the rabbit hair in a dosage and dose duration-dependent manner. These data confirms the ability of using hair analysis for diethyl phosphates to assess long-term OP exposure.     

Sensitive Method for the Confident Identification of Genetically Variant Peptides in Human Hair Keratin

Recent reports have demonstrated that genetically variant peptides derived from human hair shaft proteins can be used to differentiate individuals of different biogeographic origins. We report a method involving direct extraction of hair shaft proteins more sensitive than previously published methods regarding GVP detection. It involves one step for protein extraction and was found to provide reproducible results. A detailed proteomic analysis of this data is presented that led to the following four results: (i) A peptide spectral library was created and made available for download. It contains all identified peptides from this work, including GVPs that, when appropriately expanded with diverse hair-derived peptides, can provide a routine, reliable, and sensitive means of analyzing hair digests; (ii) an analysis of artifact peptides arising from side reactions is also made using a new method for finding unexpected modifications; (iii) detailed analysis of the gel-based method employed clearly shows the high degree of cross-linking or protein association involved in hair digestion, with major GVPs eluting over a wide range of high molecular weights while others apparently arise from distinct non-cross-linked proteins; and (v) finally, we show that some of the specific GVP identifications depend on the sample preparation method.     

Preliminary study of hair form of Japanese head hairs using image analysis

The use of average curvature measurements for the forensic comparison of curly hairs has been reported, but a method, in which various types of hair form are quantitatively examined and objectively interpreted for hair comparison, has not been reported to date. In the present study, numerical data on hair form from Japanese subjects were obtained by image analysis and a morphological comparison of these head hairs was investigated. Head hairs obtained from eight Japanese males were measured for length (L), distance (D) and area (A) using a Kontron Imaging System KS400. From the three measurements mentioned above, three indexes, L/D, A/D and 2(A/L), were examined. The inter-individual variations for each value were investigated by a t-test and the availability of six values for the forensic comparison of hair form was evaluated by a stepwise linear discrimination analysis. Six values obtained from hair form by an image analysis showed large intra-individual variations. However, these six values were found to be useful for discriminating between two individuals, since the six values showed larger inter-individual variations than intra-individual variations. Discrimination on each comparison using a stepwise linear discrimination analysis was performed for some of the values and the results indicated conspicuous inter-individual variations between the two individuals. On 11 of 28 comparisons, 30 hairs from one individual could be completely distinguished from hairs of another individual, when a two-way comparison was employed. These results confirm that hair form could be quite useful in the forensic comparison of hair morphology, and suggest that numerical data obtained from hair form by image analysis are very important values for constructing a screening procedure for evidential hairs. The use of an objective measure of hair form will be especially useful for Japanese head hairs since they are generally thought to show very limited variation in morphological features.