Detection of antidepressant and antipsychotic drugs in human hair

The presence of therapeutic drugs and their metabolites in the hair of psychiatric patients was investigated using gas chromatography (GC)-mass spectroscopy (MS)-electron ionization (EI) and GC-MS-chemical ionization (CI). In hair samples tested from 35 subjects, carbamazepine, amitriptyline, doxepin, trihexyphenidyl, chlorpromazine, chlorprothixene, trifluoperazine, clozapine and haloperidol were detected, with maximal concentrations of 22.5, 57.7, 183.3, 15.6, 68.2, 30.0, 36.8, 59.2 and 20.1 ng/mg of hair sample, respectively. Chlorpromazine and clozapine concentrations in the hair were found to be dependent on the dosage used and their correlation coefficients were 0.8047 (P<0.001, n=16) and 0.7097 (P<0.001, n=16), respectively. Segmental analysis demonstrated that there was a correlation between the history of subject's drug exposure and the distribution of drug along the hair shaft. Our results also show that drug analysis in hair may provide useful information about drug treatment and the history of usage, and that drugs can be detected in normally kept hair for at least 16 months after intake.     

Hair analysis for drug abuse: I. Determination of methamphetamine and amphetamine in hair by stable isotope dilution gas chromatography/mass spectrometry method

Determination of methamphetamine and amphetamine in hair was performed by gas chromatography/mass spectrometry using stable isotope-labeled internal standards, 2-methylamino-1-phenylpropane-2,3,3,3-d4 and 2-amino-1-phenylpropane-2,3,3,3-d4. Extraction of hair with methanol/5M hydrochloric acid (20:1) using ultrasonication was chosen as the standard method. The calibration curves for amphetamines in the hair were linear from 1 to 100 ng/mg (r greater than 0.99). The detection limit was 0.5 ng/mg at the 95% confidence level. The coefficients of variation (CV) (n = 8) of analysis using the spiked hair with methamphetamine were from 0.7 to 6%. The CV (n = 8) of analysis of the methamphetamine abuser's hair was 17.5%. Sectional analysis of monkey and human hair after methamphetamine ingestion suggested a good correlation between the duration of drug use and drug distribution in the hair.     

Clozapine--a dangerous drug in a clozapine-naïve subject

Clozapine is a uniquely effective antipsychotic, but is very toxic in clozapine-na?ve subjects. A 34-year-old male patient in a mental health facility, who was not prescribed clozapine, took 350 mg clozapine obtained from another patient at night. He was found dead the next morning. The presence of cardiomegaly related to obesity may have increased the risk of suffering an acute cardiac event after ingestion of clozapine. The medication prescribed to the patient was not thought to have contributed to the fatal outcome. Post mortem femoral blood clozapine and norclozapine concentrations were 0.48 and 0.20mg/L, respectively. By way of comparison, audit of 104,127 plasma samples (26,796 patients) assayed for therapeutic drug monitoring purposes 1993-2007, showed plasma clozapine 0.35 mg/L or more in 57.5% samples (8.4% 1mg/L or more). Those involved in the investigation of clozapine-associated deaths need to be aware that that death in an adult may occur after a single 'therapeutic' dose. A diagnosis of fatal clozapine poisoning cannot be made solely on the basis of a post mortem blood clozapine measurement.     

Effect of murine retroviral infection on hair and serum levels of cocaine and morphine.

LP-BM5 retrovirally infected female C57BL/6J mice were administered cocaine, morphine or both by daily intraperitoneal injection for 9 weeks. Drug concentrations were measured by radioimmunoassay in serum and in hair extracts. Hair samples obtained from all drug-treated mice were positive for the drug injected, while none of the saline-treated mice had detectable drug levels in hair or serum. The average morphine concentration obtained from non-infected mice was 11 ng/mg hair whereas the amount found in the LP-BM5-infected mice was significantly higher (20 ng/mg hair). Mice injected with both morphine and cocaine were given 50% of the regular dose of each drug and drug levels in the hair of these animals were approximately half that of mice injected with the full dose of the single drug. Non-infected mice treated with both drugs had a mean value of 7 ng morphine/mg hair and 374 ng cocaine/mg hair while retrovirus-infected mice had significantly higher concentrations, 10 ng morphine/mg hair and 1160 ng cocaine/mg hair (P less than 0.001). Serum concentrations of cocaine and morphine were significantly higher (P less than 0.01) in the retrovirus-infected animals from 40 min to 1.5 h. The increased concentrations of cocaine and morphine in serum during retrovirus infection are accompanied by a significant increase in the amount of drug incorporated into the hair matrix. This change indicates that retroviral infection may influence the disposition of these drugs in the systemic circulation.     

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.     

Comparison of daily urine,sweat, and skin swabs among cocaine users

This study (1) compares urine, skin swabs, and PharmChek sweat patches for monitoring drug use; (2) measures possible environmental contamination in recent cocaine (COC) users; and (3) evaluates various immunoassays (IA) for screening COC in diverse matrices. Unique aspects include daily urine monitoring of 10 participants for 4 weeks, multiple monitoring methods, analysis for all specimens by IA and gas chromatography (GC)/mass spectrometry (MS), and the potential for continued illicit drug use by participants. Urine served as the "gold standard" specimen for determining drug use. Only cocaine and related substances were detected.Trace amounts of drugs were found on the skin (<50 ng per swab) of urine-negative participants' hands or forehead. In contrast, larger quantities of COC were found on the skin of individuals with BE-positive urines or individuals living with drug users (up to 20 microg per swab). Patch COC amounts among the three regular users (250-9000, 0-240, 160-22,000 ng per patch) exceeded BE (50-950, none, 30-2200 ng per patch). Pre-swabs, valuable for interpreting the source or time frame of positive patch results, contained substantial COC (38-1160, 0-152, 34-762 ng per swab) prior to patch application; therefore, patch results may represent current use, prior use, contamination, or a combination. In three individuals with no indication of cocaine use, false positives (defined as sweat patch positive when urine specimens were <300ng BE/ml) occurred at a 7% rate. Proposed cut-off concentrations of 75 ng cocaine per patch and 300 ng BE/ml urine curtail the incidence of false positives in this limited population. Three immunoassays were compared to screen specimens for cocaine: a modified, manual Microgenics CEDIA; a Cozart ELISA; and an OraSure ELISA. CEDIA's limit of detection (LOD) was 81ng/ml, compared with LODs of 4 ng/ml for the Cozart ELISA and 1.5 ng/ml for the OraSure ELISA. Cozart correlated with OraSure results for COC concentrations <2000 ng per swab (n=117), r(2)=0.79.     

毛发中GHB的检测及评价

目的探索毛发中外源性GHB的检测及判断的可行性,为涉GHB的鉴定提供方法和依据。方法建立毛发中GHB的GC/MS分析方法,并通过动物实验,考察毛发中内源性GHB的质量分数范围、外源性GHB在毛发中的时间过程以及给药剂量、毛发颜色与毛发中GHB的质量分数关系。结果豚鼠和中国人黑色毛发中内源性GHB质量分数分别为(3.01±1.41)ng/mg(n=28)和(1.02±0.27)ng/mg(n=20);摄GHB后毛发中GHB质量分数明显增加且与给药剂量呈正相关性;GHB在毛干中呈窄带分布;深色毛发中GHB质量分数高于浅色毛发。结论毛发中GHB的检测适用于GHB滥用和中毒的法医毒物学鉴定;根据毛发中的GHB质量分数和毛发分段分析可判断GHB的来源。     

Plasma,oral fluid and sweat wipe ecstasy concentrations in controlled and real life conditions

In a double-blind placebo controlled study on psychomotor skills important for car driving (Study 1), a 75 mg dose of +/- 3,4-methylenedioxymethamphetamine (MDMA) was administered orally to 12 healthy volunteers who were known to be recreational MDMA-users. Toxicokinetic data were gathered by analysis of blood, urine, oral fluid and sweat wipes collected during the first 5h after administration. Resultant plasma concentrations varied from 21 to 295 ng/ml, with an average peak concentration of 178 ng/ml observed between 2 and 4h after administration. MDA concentrations never exceeded 20 ng/ml. Corresponding MDMA concentrations in oral fluid, as measured with a specific LC-MS/MS method (which required only 50 microl of oral fluid), generally exceeded those in plasma and peaked at an average concentration of 1215 ng/ml. A substantial intra- and inter-subject variability was observed with this matrix, and values ranged from 50 to 6982 ng/ml MDMA. Somewhat surprisingly, even 4-5h after ingestion, the MDMA levels in sweat only averaged 25 ng/wipe. In addition to this controlled study, data were collected from 19 MDMA-users who participated in a driving simulator study (Study 2), comparing sober non-drug conditions with MDMA-only and multiple drug use conditions. In this particular study, urine samples were used for general drug screening and oral fluid was collected as an alternative to blood sampling. Analysis of oral fluid samples by LC-MS/MS revealed an average MDMA/MDEA concentration of 1121 ng/ml in the MDMA-only condition, with large inter-subject variability. This was also the case in the multiple drug condition, where generally, significantly higher concentrations of MDMA, MDEA and/or amphetamine were detected in the oral fluid samples. Urine screening revealed the presence of combinations such as MDMA, MDEA, amph, cannabis, cocaine, LSD and psilocine in the multiple-drug condition.     

Metal and metalloid multi-elementary ICP-MS validation in whole blood, plasma, urine and hair. Reference values

Four multi-elementary metal and metalloid quantification methods using inductively coupled plasma mass spectrometry (ICP-MS) were developed and validated in human whole blood, plasma, urine and hair by means of a single preparation procedure for each sample. The ICP-MS measurements were performed using a Thermo Elemental X7CCT series and PlasmaLab software without a dynamic reaction cell. With this procedure 27-32 elements can be simultaneously quantified in biological matrices: Li, Be, B, Al, V, Cr, Mn, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Rb, Sr, Mo, Pd, Ag, Cd, Sn, Sb, Te, Ba, W, Pt, Hg, Tl, Pb, Bi, U. Whole blood, plasma and urine samples (0.4 ml each) were diluted with purified water, acid, triton X100 and butanol. Rhodium was used as internal standard. The urine sample results were corrected for enzymatic creatinine determination. Twenty-five milligrams hair samples were acid mineralized after a decontamination procedure and diluted as previously described for biological fluids. To be validated, each element had to show linearity with a correlation coefficient higher than 0.99. The intra-assay and inter-assay inaccuracy, measured as the variation coefficient, were below 5 and 10% respectively. Global performance was assessed by a quality control program. Our laboratory is a registered participant of the Institut National de Santé Publique du Québec (Sainte-Foy, Canada) inter-laboratory comparison program for whole blood, urine, and beard hair of non-occupationally exposed individuals spiked with selected elements. In our study multi-element metal and metalloid analysis was assessed for 27 elements in whole blood, 27 elements in plasma, 30 elements in urine and 32 elements in hair, from 0 to 25, or 250 to 1000 ng/ml, depending on the element. Quantification limits ranged from 0.002 ng/ml (U) to 8.1 ng/ml (Al) for whole blood, from 0.002 ng/ml (U) to 7.7 ng/ml (Al) for plasma, from 0.001 ng/ml (U) to 2.2 ng/ml (Se) for urine, and from 0.2 pg/mg (Tl) to 0.5 ng/mg (B) for hair. Normal values were determined in whole blood (n=100), plasma (n=100), urine (n=100), and hair (n=45) of healthy volunteers, leading to approximately 10,000 analyses. All results are presented and discussed. Clinical toxicology and forensic toxicology applications are also reported. ICP-MS has made significant advances in the field of clinical biology, particularly in toxicological analysis. This is due to the use of extremely effective equipment that permits better clinical and forensic toxicological analysis of metal and metalloid status of each individual patient.     

Deposition of cannabinoids in hair after long-term use of cannabis

Hair analysis has shown great potential in the detection and control of drug use. Whether an assay is of quantitative value roughly corresponding to the amount of drug consumed, is still a matter of debate. The present investigation was aimed at a possible relationship between the cannabinoid concentration in hair and the cumulative dose in regular users of cannabis. Hair samples from the vertex region of the scalp were obtained from 12 male regular users of cannabis, and 10 male subjects with no experience of cannabis served as controls. None of the subjects had his hair permed, bleached or colored. Cannabis users provided information on drug use such as the current cannabis dose per day, the cumulative cannabis dose of the last 3 months, as well as the frequency of cannabis use during the last year. The concentration of delta-9-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD) in hair was determined using gas chromatography-mass spectrometry. Cannabinoids were present in any hair sample of cannabis users, but were not detectable in control specimens. An increase in the amount of cannabinoids in hair with increasing dose was evident. The concentration of major cannabinoids (sum of THC, CBD and CBN) was significantly correlated to either the reported cumulative cannabis dose during the last 3 months or to the cannabis use during the last 3 months estimated from the daily dose and the frequency per year (r=0.68 or 0.71, p=0.023 or 0.014). A significant relationship between THC and the amount of cannabis used could not be established. As a conclusion, the sum of major cannabinoids in hair of regular users may provide a better measure of drug use than THC.     

Suspected clozapine poisoning in the UK/Eire, 1992-2003

OBJECTIVE: Toxicological analyses are often performed to investigate suspected poisoning, but the interpretation of results may not be straightforward. We studied suspected poisoning cases 1992-2003 where blood clozapine and N-desmethylclozapine (norclozapine) were measured in order to assess the relationship of these parameters to outcome. METHODS: Samples were referred from clinicians, pathologists/coroners, or via the Clozaril Patient Monitoring Service (CPMS, Novartis). Information was gathered from clinical, post-mortem, or coroners' reports. RESULTS: There were seven fatal [five male, two female; median (range) age 28 (24-41) year] and five non-fatal [four male, one female; median age 35 (26-41) year] clozapine overdoses. The median post-mortem blood clozapine and norclozapine concentrations were 8.2 (3.7-12) and 1.9 (1.4-2.4)mg/L, respectively [median clozapine:norclozapine ratio 4.4 (2.9-5.1)]. The median plasma clozapine and norclozapine concentrations (first or only sample) were 3.9 (1.7-7.0) and 0.40 (0.30-0.70)mg/L, respectively [median clozapine:norclozapine ratio 7.6 (5.3-18)] in the remainder. These overdoses were in patients who were poorly or non-adherent to clozapine, or who had taken tablets prescribed for someone else. In 54 further people who died whilst receiving clozapine [38 male, 16 female; median age 41 (22-70) year], the median post-mortem blood clozapine and norclozapine concentrations were 1.9 (0-7.7, n = 43) and 1.4 (0-6.0, n = 39)mg/L, respectively [median clozapine:norclozapine ratio 1.5 (0.4-7.6, n = 38)]. The median post-mortem increase in blood clozapine and norclozapine as compared to the most recent ante-mortem measurement was 489 (98-5,350)% and 371 (139-831)%, respectively [median sample time before death 14 (0-30, n = 21) days]. CONCLUSION: Clozapine poisoning cannot be diagnosed on the basis of blood clozapine and norclozapine concentrations alone. The analysis of ante-mortem blood specimens collected originally for white cell count monitoring and the blood clozapine:norclozapine ratio may provide additional interpretative information.     

Effect of post-mortem changes on peripheral and central whole blood and tissue clozapine and norclozapine concentrations in the domestic pig (Sus scrofa)

Interpretation of the results of psychoactive or other drug measurements in post-mortem blood specimens may not be straightforward, in part because analyte concentrations in blood may change after death. There is also the issue of comparability of plasma (or serum) results to those obtained in whole blood. To investigate these problems with respect to clozapine, this drug (10mg/kg daily) was given orally to two pigs. Blood was collected 3h post-dose on day 7, the animals were sacrificed, and blood taken from central and peripheral veins for up to 48 h after death. Tissue samples were also collected immediately after death and at 48 h. Ante-mortem whole blood clozapine/N-desmethylclozapine (norclozapine) concentrations were 0.86/1.07 and 1.11/1.15 mg/l in pigs 1 and 2, respectively. Blood clozapine and norclozapine concentrations generally increased after death (central vein: clozapine up to 300%, norclozapine up to 460%; peripheral vein: clozapine up to 155%, norclozapine up to 185%). Initial blood and kidney clozapine and norclozapine concentrations were comparable in both animals, but were some two-fold higher in heart, liver and striated muscle in pig 2. In both animals, the heart and striated muscle clozapine and norclozapine concentrations had increased some two- to three-fold at 48 h, whilst the liver and kidney concentrations were essentially unchanged. The reason for the increase in heart and striated muscle concentrations at 48 h is unclear, but could be simple variation in sample site. The plasma:whole blood distribution of clozapine and norclozapine was studied in vitro. In human blood (one volunteer donor, haematocrit 0.50) the plots of plasma versus whole blood concentration were linear for both analytes across the range 0.1-1.5mg/l, although clozapine favoured plasma (plasma:whole blood ratio=1.12), whereas norclozapine favoured whole blood (ratio 0.68). In pig blood, the plots of plasma versus whole blood were non-linear in both cases, although clozapine favoured plasma to a greater extent than norclozapine. This may be due to lower plasma clozapine and norclozapine protein binding capacity in the pig as compared to man.     

Blood, brain, and hair GHB concentrations following fatal ingestion

Despite the increasing incidence of illicit use of gamma-hydroxybutyrate (GHB), little information is available documenting levels of the drug in GHB fatalities. We measured GHB levels in postmortem blood, brain and hair specimens from a suspected overdose case by gas chromatography/mass spectrometry (GC/MS) following solid phase extraction (SPE) and derivatization with bis(trimethyl-silyl) trifluoroacetamide (BSTFA). Examination found 330 microg/mL GHB in femoral blood and 221 ng/mg GHB in frontal cortex brain tissue, values higher than those typically reported in the literature. The hair shaft was negative for GHB whereas the plucked root bulbs with outer root sheath attached (2,221 ng/mg) and root bulbs after washing and removal of the outer root sheath (47 ng/mg) contained the drug. Our results are consistent with an acute single dose of GHB and, as the toxicology screen was negative for other drugs of abuse, emphasize the significant danger of this drug.     

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.     

Ofloxacin in human hair determined by high performance liquid chromatography.

A procedure is presented for quantitating ofloxacin (OFLX) in human scalp hair by high performance liquid chromatography (HPLC) with a fluorescence detector. An octadecylsilane (ODS) column was used and the mobile phase was a mixture of potassium phosphate buffer (pH 2.6) and acetonitrile. The recovery of OFLX was 90.9-93.8% and within- and between-run precisions were 0.35-1.41% and 1.41-5.49% as the coefficient of variation (CV), respectively, when 5-50 ng OFLX was added to 1 mg blank hair. The calibration curve was linear in the range of 0.5-50 ng/tube (0.5 ml). Interference with other quinolone derivatives could be avoided according to the difference in their retention times or fluorescence spectra. Several pieces of hair were obtained from each of twelve healthy male volunteers, who had taken OFLX (100, 300 or 900 mg total dose) over a 1-3 day period 2 weeks before the hair sampling. In all hair samples except one obtained from a volunteer, who had taken the lowest dose, the 2-cm long segments nearest the scalp contained OFLX (5-45 ng/mg hair), while the upper segments did not. A highly significant positive correlation was observed between the total dose and the concentration of OFLX in the 2-cm long hair segments. Such a positive correlation was also revealed in rat hair sampled after repeated i.p. administration of OFLX over a 5-week period. These results suggest that the measurement of OFLX in hair by the present method would be useful for testing patient compliance in clinical pharmacology as well as for application to forensic science.     

度冷丁滥用者毛发分段分析及其结果评价

以度冷丁滥用者为研究对象,在度冷丁滥用者毛发中检出度冷丁及代谢物去甲度冷丁、N一羟甲基度冷丁和N-乙酰度冷丁。60例度冷丁滥用者头发中度冷丁和去甲度冷丁的含量分别为103±130ng／mg和117±143ng／mg。度冷丁稳定地存在于头发中,检出时限至少为药后20个月,而去甲度冷丁则随着离头发根距离的增加而降低。头发分段分析揭示度冷丁在毛干中的分布和滥用史、剂量和含量存在相关性。     

Testing for GHB in hair by GC/MS/MS after a single exposure. Application to document sexual assault

Gamma-hydroxybutyric acid, or GHB, is a substance naturally present within mammal species. Properties of neurotransmitter or neuromodulator are generally given to this substance. GHB is therapeutically used as an anesthetic, but can be used for criminal offenses (date-rape drug). It appears that the window of detection of GHB is very short in both blood and urine, and therefore its presence is very difficult to prove after a rape case. In order to document single exposure, we investigated the use of hair. Hair was collected one month after the allegated event in order to sample the corresponding period after regular growing. After rapid (2 min) decontamination with dichloromethane, the hair shaft was cut into 3-mm segments. They were overnight incubated in 0.01 N NaOH in the presence of GHB-d6, followed by neutralization and extraction in ethyl acetate under acidic conditions. GHB (precursor ion m/z 233, product ions m/z 147 and 148) was tested by GC/MS/MS (Finnigan TSQ 700) after derivatization with BSTFA + 1% TMCS. Physiological concentrations (n = 24) were in the range 0.5 to 12.0 ng/mg, with no influence due to hair color. No variation of concentrations was observed along the hair shaft in controlled subjects, except for the proximal segment, due to an incorporation through sweat. This demonstrates that endogenous levels for each single subject are constant during hair growth. A controlled human administration of 25 mg/kg to a volunteer demonstrated that a single exposure to GHB is detectable in hair after segmentation. In a case of rape under influence, a clear increase of the corresponding segment (about 2.4 ng/mg) in time was observed, in comparison with the other segments (0.6 to 0.8 ng/mg). This study demonstrates that a single exposure to GHB in a case of sexual assault can be documented by hair analysis when collected about one month after the crime.     

Determination of opiates and cocaine in hair using automated enzyme immunoassay screening methodologies followed by gas chromatographic-mass spectrometric (GC-MS) confirmation

The objective of this study was to develop a two-step strategy for analysis of opiates and cocaine in hair samples involving an immunological screening procedure followed by confirmation of results using gas chromatography-mass spectrometry (GC-MS). A semi-quantitative automated competitive enzyme-linked immunosorbent assay (ELISA) methodology using Oral Fluid Micro-Plate Enzyme Immunoassays (Orasure Technologies, Inc.) was developed and validated. Applicability was proven by analysis of authentic head hair samples from drug users (n=103) and from opiate associated fatalities (n=21). The optimum cutoff values for the ELISA tests were 0.1 ng cocaine-equivalents/mg hair and 0.05 ng morphine-equivalents/mg hair using a 50 mg hair sample. Both ELISA tests had a sensitivity of 100%, the specificity was 66% for cocaine-equivalents and 42% for morphine-equivalents. The intraassay precision was 11% for the cocaine and 3% for the opiates ELISA, while interassay precision was 12% for the cocaine and 4% for the opiates ELISA test. The actual analyte concentrations in the hair samples were determined using GC-MS and were between 0.04 and 5.20 ng/mg for heroin (HER), between 0.04 and 30.01 ng/mg for 6-monoacetylmorphine (MAM), between 0.03 and 11.87 ng/mg for morphine (MOR), between 0.02 and 1.84 ng/mg for codeine (COD), between 0.02 and 2.48 ng/mg for acetylcodeine (AC), between 0.01 and 21.37 ng/mg for cocaine (COC), between 0.03 and 10.51 ng/mg for benzoylecgonine (BE) and between 0.05 and 1.26 ng/mg for cocaethylene (CE). The automated ELISA tests were proven to be valid screening procedures for the detection of cocaine and opiates in hair as confirmed by GC-MS. Screening methods provide rapid and inexpensive automated pre-test procedures to detect drugs in hair or other matrices. For forensic purposes screening therefore represents an ideal complement to routinely applied GC-MS procedures.     

A fatal dothiepin overdose

High pressure liquid chromatography coupled to photodiode array detector and capillary gas chromatography coupled to mass spectrometry were employed to quantify dothiepin in biological fluids, tissues and hair in a death attributed to oral dothiepin (Prothiaden^R) ingestion. The blood concentration of dothiepin was 5.75 mg/l. Hair analysis clearly indicated a chronic antidepressant exposure, with a dothiepin concentration of 1.89 ng/mg hair. Results are discussed in the light of the existing literature.     

Segmental hair analysis can demonstrate external contamination in postmortem cases

Excluding laboratory mistakes, a false positive hair result can be observed in case of contamination from environmental pollution (external contamination) or after drug incorporation into the hair from the individual body fluids, such as sweat or putrefactive fluid (post mortem artifact). From our 20 years experience of hair testing, it appears that artifact(s) cannot be excluded in some post mortem cases, despite a decontamination procedure. As a consequence, interpretation of the results is a challenge that deserves particular attention. Our strategy will be reviewed in this paper, based on six cases. In all cases, a decontamination procedure with two washes of 5 ml of dichloromethane for 5 min was performed and the last dichloromethane wash was negative for each target drug. From the histories, there was no suspicion of chronic drug use. In all six cases, the concentrations detected were similar along the hair shaft, irrespective of the tested segment. We have considered this as indicative of external contamination and suggested to the forces or the judges that it is not possible to indicate exposure before death. In contrast to smoke, it seems that contamination due to aqueous matrices (sweat, putrefactive fluid, blood) is much more difficult to remove. To explain potential incorporation of 7-aminoflunitrazepam via putrefactive material, the author incubated negative hair strands in blood spiked at 100 ng/ml and stored at +4°C, room temperature and +40 °C for 7, 14 and 28 days. After routine decontamination, 7-aminoflunitrazepam tested positive in hair, irrespective of the incubation temperature, as early as after 7 days (233-401 pg/mg). In all periods, maximum concentrations were observed after incubation at room temperature. The highest concentration (742 pg/mg) was observed after 28 days incubation at room temperature. It is concluded that a standard decontamination procedure is not able to completely remove external contamination in case of post mortem specimens. Homogenous segmental analyses can be probably indicative of external contamination and therefore a single hair result should not be used to discriminate long-term exposure to a drug. Nor should the presence of a metabolite be considered as a discrimination tool, as it can also be present in putrefactive material.