Forensic determination of hair deposition time in crime scenes using electron paramagnetic resonance

Several types of biological samples, including hair strands, are found at crime scenes. Apart from the identification of the value and the contributor of the probative evidence, it is important to prove that the time of shedding of hair belonging to a suspect or victim matches the crime window. To this end, to estimate the ex vivo aging of hair, we evaluated time‐dependent changes in melanin‐derived free radicals in blond, brown, and black hairs by using electron paramagnetic resonance spectroscopy (EPR). Hair strands aged under controlled conditions (humidity 40%, temperature 20–22°C, indirect light, with 12/12 hour of light/darkness cycles) showed a time‐dependent decay of melanin‐derived radicals. The half‐life of eumelanin‐derived radicals in hair under our experimental settings was estimated at 22 ± 2 days whereas that of pheomelanin was about 2 days suggesting better stabilization of unpaired electrons by eumelanin. Taken together, this study provides a reference for future forensic studies on determination of degradation of shed hair in a crime scene by following eumelanin radicals by utilizing the non‐invasive, non‐destructive, and highly specific EPR technique.     

FEAR OF CRIME IN RESIDENTIAL COMMUNITIES

Three variables were hypothesized to cause a fear of crime and a potential change in behavior. These were: (1) crimes against a person rather than crimes against property; (2) a crime committed in an area frequented rather than a crime occurring in an area one never entered; (3) a recurring crime rather than a crime that occurred once. Two different samples of female subjects (n = 249) were approached at their residences and were asked to read one of a number of fictitious crime stories that the news media supposedly had not reported and to complete two scales measuring: (1) an emotional response to crime and (2) a potential behavioral response to crime. The results indicate that a physical assault produces both more fear and more potential behavioral change than a burglary. A crime that occurs eight times causes people to consider taking precautions in comparison to a crime that occurs once. There is some evidence that a crime in an area one frequents causes more fear than a crime occurring in an area one never enters.     

Forensic DNA-typing of dog hair: DNA-extraction and PCR amplification

The forensic application of DNA-typing for the identification of dog hair provides objective evidence in the characterisation of traces found at crime scenes. During the past few years forensic dog identity testing has been improved considerably using multiplex PCR systems. However, DNA-typing from samples of one up to 10 dog hairs is often problematic in forensic science. A single dog hair contains very small quantities of DNA or the hair sample consists of hairs with roots of bad quality or even of broken hairshafts without roots. Here we describe an experimental study about dog hairs by means of a Ca(2+) improved DNA-extraction method, quantification and amplification.     

Disappearance of cocaine from human hair after abstinence

In this work the study of the disappearance of cocaine in hair is reported. The subject of the study is a woman who stopped the consumption of cocaine after a period of drug abuse of over 1 year. Hair samples were collected over a period of 10 months. During this time the absence of cocaine intake was monitored by the toxicological analysis of urine, performed every 2 days. After decontamination with methanol, the hair sample, cut in two segments (0-1.5 and 1.5-3 cm from the hair root) was added with cocaine-D(3) (internal standard), hydrolyzed and extracted with chloroform/isopropanol (9:1). The extract was evaporated to dryness, reconstituted in 25 microl of ethyl acetate and analyzed by GC-MS in SIM mode. The obtained results show that the incorporation of cocaine in hair decreased during the first 3 months after the last consumption and after this period of time no cocaine was found in the hair sections closest to the root.     

Practical experiences in application of hair fatty acid ethyl esters and ethyl glucuronide for detection of chronic alcohol abuse in forensic cases

This article presents results from 1872 hair samples, which were analyzed for fatty acid ethyl esters (FAEEs) and ethyl glucuronide (EtG). The results were evaluated in the context of self-reported drinking behavior, the use of hair cosmetics, the gender of the sample donors and hair sample length. For comparison, CDT and GGT in serum were available in 477 and 454 cases, respectively. A number of alcohol abstainers or low moderate drinkers and excessive drinkers were selected for assessment of cut-offs for FAEEs in the proximal 6cm hair segments and for EtG in the proximal 3cm hair segments. Cut-off values were assessed by ROC analysis. It was found that the cut-offs of 1.0ng/mg FAEE and 30pg/mg EtG presently used for excessive drinking lead to a low portion of false positives (4% and 3% respectively) but to a higher portion of false negatives (23% and 25% respectively). Comparison of the mean and medium concentrations in samples without any reported hair cosmetics (N=1079) and in samples with reported use of hair spray (N=79) showed an increase by the factor of about two for FAEE but no significant difference for EtG. Mean values of EtG were decreased by 80% in bleached samples (N=164) and by 63% in dyed samples (N=96). There was no significant effect of bleaching and dyeing on FAEE. Hair gel and hair wax, oil or grease showed no significant effect on both FAEE and EtG. With respect to gender and investigated hair length ambiguous results were obtained because of major differences in the compared subpopulations of male with higher alcohol consumption and mainly shorter hair, and less drinking female with longer hair. For excessive drinkers FAEEs in the 0-6cm hair segment and EtG in the 0-3cm segment decreased with increasing time of reported abstinence before sample collection. These drinkers attain the level of teetotalers only after more than 10 months of abstinence. In comparison to scalp hair, FAEEs recovered from armpit hair and leg hair were lower and from chest hair were higher. EtG in armpit hair was lower and in leg hair higher than in scalp hair. It is concluded that the combined use of FAEE and EtG essentially increases the accuracy of interpretation since both markers complement each other by a different sensitivity to sources of error.     

Do drug users use less alcohol than non-drug users? A comparison of ethyl glucuronide concentrations in hair between the two groups in medico-legal cases

Two groups were selected from the remainder of hair samples that had been tested for drugs at TrichoTech for medico-legal cases: samples that tested negative (drug-negative group; N=42, age 33.4+/-7.2 years) and samples that tested positive for drugs (drug-positive group; N=57, age 32.5+/-8.8 years). A rapid, simple method to detect the ethanol metabolite, ethyl glucuronide (EtG) in hair has been developed. The hair samples were sectioned, and then submitted to overnight sonication in water. Samples then underwent SPE using anion exchange cartridges, followed by derivatisation with N,O-bis[trimethylsilyl]trifluoroacetamide (BSTFA), before confirmation by GC-MS/MS. The assay produced excellent linearity and sensitivity over the calibration range 0.02-1.0 ng/mg, assuming a 10 mg hair sample. The mean age of the two groups was not statistically different (p=0.575, Student t-test), indicating a homogeneous group. Twelve of the 57 (21.0%) hair samples of the drug-positive group tested positive for EtG, and 17 of the 42 (40.5%) hair samples of the drug-negative group tested positive for EtG. The mean concentration of EtG in the drug-positive group was 0.011 ng/mg compared to 0.107 ng/mg in the drug-negative group. When the full results of this study were subjected to statistical analysis it was shown that EtG levels in the drug-negative group were statistically higher than those found in the drug-positive group (p<0.05). This preliminary finding may be of use in the study of addiction and adds valuable data to previous studies regarding the use of EtG as a valuable marker for alcohol levels in hair.     

Interpretation problems in a forensic case of abstinence determination using alcohol markers in hair

In a child custody case a mother with a longstanding history of alcohol misuse had to show absolute abstinence for one year. She entered a residential rehabilitation for six months and was tested two months later by way of a hair test for ethyl glucuronide (EtG) with the result of 22 pg/mg in the proximal 0-1cm segment and the segments 1-2 cm and 2-3 cm being negative. This was interpreted as a minimum alcohol intake of 20-50 units per week in the month before sampling. Since the mother denied any alcohol intake a second hair sample was collected seven weeks after the first and analyzed for fatty acid ethyl esters (FAEEs) by a second laboratory. A low concentration of 0.03 ng/mg was measured within the 0-6 cm segment of recently bleached hair and was interpreted as showing no evidence of alcohol use during the last six months. Three further hair samples were analyzed during the next nine months with low EtG values (<2.4-3.3 pg/mg, 0-3 cm segment) and low FAEE values (0.27-0.53 ng/mg, 0-6 cm segment). These findings were summarized as indicating continued low alcohol consumption over the past one year period. As a consequence of the conflicting results, the case was dealt with in a hearing before the Family Division of the High Court of London. It was concluded in the judgment that the evidence did not indicate that the mother had consumed alcohol in the period tested by the hair samples. It was stated that the evidence in this case highlighted the need for the exercise of considerable caution when hair tests for alcohol are being interpreted and relied upon, both generally and particularly in isolation, and that this case is a proper reminder of the need for expert evidence to be given in a manner according to the Practice Direction.     

Evaluation and quantification of nuclear DNA from human telogen hairs

Nuclear DNA was extracted from human telogen hairs from 60 individuals. Six to nine hairs from each individual were individually extracted. The amount of DNA recovered from each individual varied greatly, and most samples yielded a quantity of 550 pg or less per hair. A selective extraction buffer was used to remove epithelial cell DNA and the amount of exogenous DNA was determined. DNA was also quantified by real time PCR using three different sized amplicons targeting an Alu sequence. The results were used to determine the state of degradation of the extracted DNA. Different quantities of sample (<100 pg, 100-500 pg, >500 pg) were amplified with the Miniplex kits to determine the minimum DNA template required for successful amplification. DNA recovered from hair showed degradation; however, partial profiles were obtained for those samples containing at least 60 pg using MiniSTRs.     

A study on the concentrations of 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in hair root and whole hair

In this study, we investigated the patterns of cannabis users (n=412) according to their sex, age, and the results of urinalysis and hair analysis, and classified the concentrations of THCCOOH in hair into three categories to examine the levels of cannabis use. We also compared the concentrations of THCCOOH in hair root, hair without the hair root and whole hair and examined the relationship among them according to the results of urinalysis. The hair samples were washed, digested with 1ml of 1M NaOH at 85°C for 30min and extracted with 2ml of n-hexane:ethyl acetate (9:1) two times after adding 1ml of 0.1N sodium acetate buffer (pH 4.5) and 200μl of acetic acid. The final mixture was derivatized with 50μl of PFPA and 25μl of PFPOH for 30min at 70°C. The solution was evaporated, and the residue was reconstituted in 40μl of ethyl acetate and transferred to an autosampler vial. One microlitre was injected into the GC/MS/MS-NCI system. The concentrations of THCCOOH ranged from 0.06 to 33.44pg/mg (mean 2.96; median 1.32) in hair from cannabis users who had positive urine results and ranged from 0.05 to 7.24pg/mg (mean 1.35; median 0.37) in hair from cannabis users who had negative urine results. The average concentration of THCCOOH in hair from cannabis users who had positive urine results was higher than that from cannabis users who had negative urine results. Male cannabis users in their forties were predominant. We classified the concentrations of THCCOOH in hair into three groups (low, medium and high), and could use the grouping of THCCOOH in hair as a guide for determining the level of use. The low, medium and high concentration ranges for THCCOOH in hair were 0.05-0.24, 0.25-2.60 and 2.63-33.44pg/mg, respectively. We also investigated 28 hair samples with the root. The highest concentrations of THCCOOH were seen in the hair root from 18 out of the 28 hair samples. The average concentrations of THCCOOH in hair root, hair without hair root and whole hair from cannabis users who had positive urine results were higher than those who had negative urine results.     

Analysis of the fluorescence of body fluids on different surfaces and times

The use of screening techniques, such as an alternative light source (ALS), is important for finding biological evidence at a crime scene. The objective of this study was to evaluate whether biological fluid (blood, semen, saliva, and urine) deposited on different surfaces changes as a function of the age of the sample. Stains were illuminated with a Megamaxx™ ALS System and photographed with a Canon EOS Utility™ camera. Adobe Photoshop™ was utilized to prepare photographs for analysis, and then ImageJ™ was used to record the brightness values of pixels in the images. Data were submitted to analysis of variance using a generalized linear mixed model with two fixed effects (surface and fluid). Time was treated as a random effect (through repeated measures) with a first-order autoregressive covariance structure. Means of significant effects were compared by the Tukey test. The fluorescence of the analyzed biological material varied depending on the age of the sample. Fluorescence was lower when the samples were moist. Fluorescence remained constant when the sample was dry, up to the maximum period analyzed (60 days), independent of the substrate on which the fluid was deposited, showing the novelty of this study. Therefore, the forensic expert can detect biological fluids at the crime scene using an ALS even several days after a crime has occurred.     

Deposition of 7-aminoflunitrazepam and flunitrazepam in hair after a single dose of Rohypnol

In recent years, there has been a notable increase in the number of reports on drug-facilitated sexual assault. Benzodiazepines are the most common so-called "date-rape" drugs, with flunitrazepam (Rohypnol) being one of the most frequently mentioned. The aim of this study was to determine whether flunitrazepam and its major metabolite 7-aminoflunitrazepam could be detected in hair collected from ten healthy volunteers after receiving a single 2 mg dose of Rohypnol using solid phase extraction and NCI-GC-MS. Such data would be of great importance to law enforcement agencies trying to determine the best time interval for hair collection from a victim of drug-facilitated sexual assault in order to reveal drug use. Ten healthy volunteers (eight women and two men, 21 to 49 years old) participated in the study. The following hair samples were collected from each volunteer: one before flunitrazepam administration, and 1, 3, 5, 14, 21, and 28 days after. In five volunteers, 7-aminoflunitrazepam was detected 24 h after flunitrazepam administration and remained in hair throughout the entire 28-day study period (0.6-8.0 pg/mg). In two cases, 7-aminoflunitrazepam appeared in hair 21 days after drug intake (0.5-2.7 pg/mg), and in two subjects 14 days later (0.5-5.4 pg/mg). In one volunteer, 7-aminoflunitrazepam was detected on day 14 and 21 but concentrations were below the quantitation limit. Flunitrazepam was detected in some samples but all concentrations were below the quantitation limit (0.5-2.3 pg/mg).     

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.     

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.     

USGS42 and USGS43: human-hair stable hydrogen and oxygen isotopic reference materials and analytical methods for forensic science and implications for published measurement results

Because there are no internationally distributed stable hydrogen and oxygen isotopic reference materials of human hair, the U.S. Geological Survey (USGS) has prepared two such materials, USGS42 and USGS43. These reference materials span values commonly encountered in human hair stable isotope analysis and are isotopically homogeneous at sample sizes larger than 0.2 mg. USGS42 and USGS43 human-hair isotopic reference materials are intended for calibration of δ(2)H and δ(18)O measurements of unknown human hair by quantifying (1) drift with time, (2) mass-dependent isotopic fractionation, and (3) isotope-ratio-scale contraction. While they are intended for measurements of the stable isotopes of hydrogen and oxygen, they also are suitable for measurements of the stable isotopes of carbon, nitrogen, and sulfur in human and mammalian hair. Preliminary isotopic compositions of the non-exchangeable fractions of these materials are USGS42(Tibetan hair)δ(2)H(VSMOW-SLAP) = -78.5 ± 2.3‰ (n = 62) and δ(18)O(VSMOW-SLAP) = +8.56 ± 0.10‰ (n = 18) USGS42(Indian hair)δ(2)H(VSMOW-SLAP) = -50.3 ± 2.8‰ (n = 64) and δ(18)O(VSMOW-SLAP) = +14.11 ± 0.10‰ (n = 18). Using recommended analytical protocols presented herein for δ(2)H(VSMOW-SLAP) and δ(18)O(VSMOW-SLAP) measurements, the least squares fit regression of 11 human hair reference materials is δ(2)H(VSMOW-SLAP) = 6.085δ(2)O(VSMOW-SLAP) - 136.0‰ with an R-square value of 0.95. The δ(2)H difference between the calibrated results of human hair in this investigation and a commonly accepted human-hair relationship is a remarkable 34‰. It is critical that readers pay attention to the δ(2)H(VSMOW-SLAP) and δ(18)O(VSMOW-SLAP) of isotopic reference materials in publications, and they need to adjust the δ(2)H(VSMOW-SLAP) and δ(18)O(VSMOW-SLAP) measurement results of human hair in previous publications, as needed, to ensure all results on are on the same scales.     

Testing human hair for drugs of abuse. IV. Environmental cocaine contamination and washing effects

Active cocaine use results in sequestration of parent drug in hair. In addition, hair has unique physicochemical properties that permit absorption of cocaine from the environment. When hair is tested for evidence of cocaine, it is important to consider whether the positive test resulted from active drug use or environmental contamination. In a series of laboratory experiments, it was found that exposure of ‘cut’ hair to cocaine vapor (‘crack’ smoke) and to aqueous solutions of cocaine hydrochloride resulted in significant contamination of hair samples. Similar results were obtained with two subjects who were exposed to cocaine vapor in an unventilated room. The amount of contamination adsorbed by hair depended upon both time and extent of exposure. Washing the hair samples with methanol removed >70% of the cocaine contaminant after cocaine vapor exposure, but was less effective (<50%) following contamination with aqueous cocaine. Shampoo treatment cycles (overnight soaking) progressively removed increasing amounts of cocaine from the contaminated hair, but residual cocaine remained after 10 cycles. Studies were also performed to determine the usefulness of benzoylecgonine as a marker of active cocaine administration. Small amounts of benzoylecgonine (ca. 1 ng/mg) were formed in hair as a result of environmental contamination with cocaine. Also, it was found that benzoylecgonine could be adsorbed from illicit cocaine contaminated with benzoylecgonine. It was concluded that positive hair test results should be interpreted cautiously due to the possibility of environmental contamination from cocaine and related constituents.     

A comprehensive ATR-FTIR spectroscopic analysis for the identification and differentiation of lip balms

Lip balm may be encountered as physical evidence in cases involving sexual assaults, homicides, and kidnappings. Lip balm can be used as corroborative evidence by providing a potential link between the victim, accused, and the crime scene. For lip balms to be used as evidence, it is important to understand the diversity and their aging process under different conditions. Therefore, in this study, ATR-FTIR spectroscopy in conjunction with chemometric tools such as principal component analysis (PCA) and linear discriminant analysis (LDA) has been used for the objective identification and differentiation of 20 brands of lip balms. Moreover, lip balms on different substrates and wearing effects over time were also investigated. The results show that the PCA-LDA training accuracy was 92.5%, whereas the validation accuracy comes out to be 83.33%. A blind study using pristine samples was also performed which resulted in 80% PCA-LDA accuracy. PCA-LDA prediction of samples on various substrates showed a higher chemometric prediction accuracy for nonporous substrates (glass, plastic, and steel), than for porous substrates (cotton cloth, cotton swab stick, dry tissue paper, and white paper) for samples kept in room temperature and under sunlight for 15 days. The substrate study showed that the samples from various substrates could effectively generate respective spectra which can help in brand-level identification even after several days. The present method demonstrates a potential for lip balm samples to be used in forensic casework applications.     

The study of metabolite-to-parent drug ratios of methamphetamine and methylenedioxymethamphetamine in hair

The metabolite-to-parent drug ratios were determined in the hair of 2444 methamphetamine (MA) abusers who had produced MA-positive hair results from 2001 to May 2005 and in the hair of 53 ecstasy abusers who had produced positive methylenedioxymethamphetamine (MDMA) hair results from 2002 to May 2005. For the hair analyses, hair strands were washed, cut into small pieces and extracted for 20 h in 1 mL methanol containing 1% HCl. Drugs in the extract were determined by gas chromatography-mass spectrometry (GC-MS) using selective ion monitoring after derivatization with trifluoroacetic anhydride. The six range groups were divided as follows on the basis of MA concentrations in hair (n = 2389): 0.5-5 ng/mg (n = 950), 5-10 ng/mg (n = 582), 10-20 ng/mg (n = 503), 20-30 ng/mg (n = 160), 30-40 ng/mg (n = 80), more than 40 ng/mg (n = 114) to assess the correlations between MA concentrations and metabolite-to-parent drug ratios. In groups of higher MA concentrations, lower ratios of AP/MA were found, and there was a statistically significant difference among six range groups. Comparisons of age groups (tens, twenties, thirties, forties, fifties, and sixties) and male and female subjects for the ratios of AP/MA showed a statistically significant difference. The detection of metabolites and the parent drug with reasonable ratios was found to be a useful indicator for distinguishing internal drug incorporation from external contamination. In our study, MA users can produce 0.4-116% (mean = 9%) of amphetamine (AP) concentrations in hair, and ecstasy users 1-110% (mean = 12%) of methylenedioxyamphetamine (MDA) in appropriately washed hair samples.     

Analysis of fatty acid ethyl esters in hair as possible markers of chronically elevated alcohol consumption by headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS)

Fatty acid ethyl esters (FAEE) are products of the nonoxidative ethanol metabolism, which are known to be detectable in blood only about 24h after the last alcohol intake. After deposition in hair they should be suitable long-term markers of chronically elevated alcohol consumption. Therefore, a method for the analysis of ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate from hair was developed based on the extraction of the hair sample by a dimethylsulphoxide (DMSO)/n-hexane mixture, separation and evaporation of the n-hexane phase and application of headspace solid-phase microextraction (HS-SPME) in combination with gas chromatography-mass spectrometry (GC-MS) to the extract. For use as internal standards, the corresponding D(5)-ethyl esters were prepared. The HS-SPME/GC-MS measurements were automatically performed using a multi-purpose sampler. The detection limits of the FAEE were between 0.01 and 0.04ng/mg and the reproducibility was between 3.5 and 16%. By application of the method to hair samples of 21 fatalities with known heavy alcohol abuse 0.045-2.4ng/mg ethyl myristate, 0.35-13.5ng/mg ethyl palmitate, 0.25-7.7ng/mg ethyl oleate and 0.05-3.85ng/mg ethyl stearate were measured. For social drinkers (30-60g ethanol per week), the concentrations were about one order of magnitude smaller. For 10 teetotalers negative results or traces of ethyl palmitate were found. It was shown by supplementary investigations in single cases that FAEE are also present in sebum, that there is no strong difference in their concentrations between pubic, chest and scalp hair, and that they are detectable in hair segments after a 2 months period of abstinence. From the results follows that the measurement of FAEE concentrations in hair is a useful way for a retrospective detection of alcohol abuse.     

Development and validation of a liquid chromatography-tandem mass spectrometry assay for hair analysis of atomoxetine and its metabolites: Application in clinical practice

Atomoxetine (ATX) is a potent inhibitor of the noradrenaline reuptake transporter approved since 2002 for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children, adolescents, and adults as alternative treatment to methylphenidate. A procedure based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of ATX and its main metabolites (4-hydroxyatomoxetine - 4 hydroxyATX - and N-desmethylatomoxetine - des-methylATX) in hair of one treated child and five treated adolescents. Since hair samples can be easily collected without the need for specials skills and exposing a patient to discomfort, hair testing of ATX and eventually of its metabolites should be useful, especially in case of pediatric patients, to check compliance in a wider time-window. After addition of duloxetine as internal standard, hair samples were overnight digested with 2ml 1M NaOH at 45°C. Then, analytes were extracted from alkaline solution with two different 2ml aliquots of tert-butyl methyl ether. Chromatographic separation was achieved at ambient temperature using a reverse-phase column and a mobile phase of 40% of water-60% 5mM ammonium acetate, 50mM formic acid, 4mM trifluoroacetic acid in acetonitrile-water (85:15, v/v). The mass spectrometer was operated in positive ion mode using multiple reaction monitoring. The method was linear over the concentration range 0.2-50ng/mg hair for the all analytes under investigation, with an intra- and inter-assay imprecision and inaccuracy always less than 20% and an analytical recovery between 33.1% and 76.1%, depending on the considered analyte. Only ATX and 4-hydroxyATX were detected in hair samples with concentrations varying from 0.2 to 2.0ng/mg hair and from 0.3 to 1.0ng/mg, respectively. Notwithstanding the absence of any dose-hair concentration relationship, hair monitoring of ATX and concomitant medications commonly administrated in ADHD children and adolescents can be crucial in verifying long-term compliance to prescribed medication in individuals displaying a non negligible tendency to refuse drugs and to lie on the adherence to therapy as a specific symptom of the disease.