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Methadone is not licensed for use in children though it can be employed for the management of neonatal opiate withdrawal syndrome. During the last 2 years, our laboratory has been asked to test for methadone and EDDP, its major metabolite, in hair from children that were admitted to hospital unconscious and where methadone had already been identified in a body fluid (4 cases) or where the children were deceased and evidence of methadone overdosage having already been established (2 cases). In all of these cases, segmental analysis revealed approximately the same amount of drug along the hair lock. As a consequence, contamination was considered as an issue and interpretation of the results was a challenge that deserves particular attention. After decontamination with dichloromethane and segmentation the hair was cut into small pieces, incubated overnight at 40 degrees C, liquid-liquid extracted and analysed with LC-MS/MS, using 2 transitions per compound. The LOQ for both methadone and EDDP was 10 pg/mg. In the first series involving children admitted to hospital, the following results were obtained: * case 1: 4 x 1 cm section, methadone at 0.05-0.08 ng/mg, no EDDP detected, * case 2: 4 x 1 cm section, methadone at 0.13-0.15 ng/mg, EDDP at 0.02 ng/mg, * case 3: 3 x 1.5 cm section, methadone at 0.07-0.09 ng/mg, EDDP at 0.01-0.03 ng/mg, * case 4: 6 x 2 cm section, methadone at 0.06-0.13 ng/mg, EDDP at 0.02-0.03 ng/mg. The following concentrations were obtained from the children who had died following a methadone overdose: * case 5: 2 x 2 cm section, methadone at 0.53-0.58 ng/mg, no EDDP detected, * case 6: 4 x 1 cm section, methadone at 0.44-0.77 ng/mg, EDDP at 0.04-0.06 ng/mg. The first observation is that all these concentrations are low by comparison with those observed in adults on methadone maintenance therapy. However, the more surprising observation is the relative homogenous concentrations along the hair locks in each specific case. This raises concerns around the possibility that contamination could have occurred prior to sampling and makes it hard to reach a conclusion regarding the possibility of repeated methadone exposure in the months prior the incidents. In these cases it was impossible to conclude that the children were deliberately administered methadone. The results of the analysis of hair could indicate that they were in an environment where methadone was being used and where the drug was not being handled and stored with appropriate care. The homogenous concentrations found on segmental analyses could be indicative of external contamination that may have arisen not only from direct contamination with the drug but also via contamination with body fluids at the post mortem or from sweat produced close to the time of the incident. In view of these results we concluded that a single determination should not be used firmly to discriminate long-term exposure to a drug., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

On March 2007, a police officer (46-year-old man) and a clerk (37-year-old woman) were arrested and subjected to investigation on the charges of drugs of abuse trafficking. The loving couple was exploiting their administrative positions to make money with the resale of seized drugs. The laboratory was requested to analyse their hair for drugs of abuse. Hair of the 2 subjects tested positive for heroin by GC-MS. A few days later, analysis of hair obtained from 11 other police officers of the same unit was requested, in order to compare the results, as external contamination was proposed to account for the positive results. The aim of the investigations was to demonstrate that passive contamination could not occur for persons dealing every day with drugs of abuse with minimal caution and hygiene, and that the measured concentrations in the arrested subjects correspond to personal abuse. All the narcotic team tested negative, irrespective of the compound., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

Dioxins and related compounds (furans) are persistent environmental contaminants that cause adverse biological effects. Their influence on humans is still unclear, except for accidental high-dose exposure. Chronic exposure to these compounds seems to be involved in cancer, endocrine disruption and neurobehavioral effects. For several years, a large concern about the potential health risks of dioxins is emerging in Europe and United States. The case of a 50-year-old man victim of an acute over-exposure to tetrachloro dibenzo-p-dioxin (TCDD or Seveso dioxin) is reported here with particular focus on hair investigations. The developed method involved the decontamination of the hair strand using picograde level methylene chloride, the homogenization of hair segments with scissors and their extraction in presence of 13C12-marked dioxin congeners under reflux of toluene using a Soxhlet, 8h at 130 degrees C. After reduction of the toluene fraction to 1 ml and addition of purification marker (37Cl4-2,3,7,8-TCDD), dioxins purification was achieved using three successive columns: silica, alumina/sodium sulfate and carbon/Celite columns. Finally, the toluene eluent was evaporated and the extract injected in the analytical system. After chromatographic separation, detection was achieved in single ion monitoring mode using a high resolution mass spectrometer operating in electron impact ionization mode (40 eV, minimal resolution of 10,000). The analysis of the first hair segment (0-6 cm) revealed the presence of 2,3,7,8-TCDD at 65 fg/mg when the distal one remained negative (LOQ=0.3 fg/mg). All other congeners (n=16) were in the range of those determined in the general population (0.62 and 0.89 fg/mg in the two hair segments, respectively). The extremely low dioxin levels generally found in hair specimens (low fg/mg range) lead us to analyze them using the very sensitive and specific gas chromatography-high resolution mass spectrometry apparatus. From the best of our knowledge, this is the first case of over-exposure to Seveso dioxin through hair analysis reported in the literature.

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.

Diphenhydramine is one of the first effective antihistamine agents to have been discovered. The compound is also used for its sedative and antiemetic effects. The first case involving repetitive sedation linked to the use of diphenhydramine as a drug-facilitated crime and subsequent impairment of a 9-year-old female victim is reported. Due to the long delay between the alleged crime and clinical examination, collection of blood or urine was of little value. Hence, the laboratory developed an original approach based on hair testing by LC-MS/MS. A single strand of hair from the victim was sampled about 7 weeks after the last suspected administration and was cut into small segments. After cutting into small pieces, about 20 mg of hair per segment was incubated overnight in a phosphate buffer (pH 8.4). The aqueous phase was extracted with 5 ml of a mixture of methylene chloride/diethyl ether (80/20), in presence of diazepam-d5, used as internal standard (IS). The hair extract was separated on an XTerra MS C18 column using a gradient of acetonitrile and formate buffer. Detection was based on two daughter ions: transitions m/z 256.2-152.1 and 167.1 and m/z 289.9-154.0 for diphenhydramine and the IS, respectively. In the hair of the child, diphenhydramine was detected at concentrations in the range 33-39 pg/mg, depending on the segment.

This work presents the validation of a new immunological assay, the One-Step enzyme-linked immunosorbent assay (ELISA) tests from International Diagnostic Systems Corp. for the screening of drugs of abuse (cannabis, amphetamines, opiates, and cocaine) in human hair, with subsequent GC-MS confirmation. After decontamination and segmentation into small pieces, 50 mg of hair sample were incubated in 1 ml of methanol during 16 h at 40 degrees C. A 100 microL aliquot was collected and evaporated to dryness in presence of 100 microL of methanol/hydrochloric acid (99:1, v/v) to avoid amphetamines loss. The dried extract was dissolved in 100 microL of the "sample and standard diluent" solution included in the kit. This solution was submitted to analysis according to the recommended instructions of the manufacturer. During the validation phase, GC-MS confirmations were conducted according to our fully validated and published methods for opiates, cocaine, cannabis, and amphetamines determinations in hair. In a last development step, these procedures were slightly modified to directly confirm ELISA results by GC-MS using the methanolic extract. Ninety-three specimens were simultaneously screened by the ELISA tests (103 for tetrahydrocannabinol (THC)) and confirmed by GC-MS. Twenty were found positive for cannabis (THC: 0.10-6.50 ng/mg), 21 for cocaine (0.50-55.20 ng/mg), 24 for opiates (6-acetylmorphine (6-AM): 0.20-11.60 ng/mg, MOR: 0.20-8.90 ng/mg, codeine (COD): 0.20-5.90 ng/mg), and 13 for amphetamines (AP: 0.20 and 0.27 ng/mg, methamphetamine (MAP): 0.30 and 1.10 ng/mg, methylenedioxymethamphetamine (MDMA): 0.22-17.80 ng/mg). No false negative results were observed according to the Society of Hair Testing's (SoHT) cutoffs (0.5 ng/mg for cocaine, 0.2 ng/mg for opiates and amphetamines, and 0.1 ng/mg for THC). The One-Step ELISA kits appear suitable due to their sensitivity and specificity for drug of abuse screening in hair. This technology should find interest in workplace drug testing or driving license regranting, especially when many samples have to be tested with a high rate of negative samples, as ELISA is an easy and high-throughput method.

Saliva or "oral fluid" has been presented as an alternative matrix to document drug use. The non-invasive collection of a saliva sample, which is relatively easy to perform and can be achieved under close supervision, is one of the most important benefits in a driving under the influence situation. Moreover, the presence of Delta9-tetrahydrocannabinol (THC) in oral fluid is a better indication of recent use than when 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) is detected in urine, so there is a higher probability that the subject is experiencing pharmacological effects at the time of sampling. In the first part of the study, 27 drug addicts were tested for the presence of THC using the OraLine IV s.a.t. device to establish the potential of this new on-site DOA detection technique. In parallel, oral fluid was collected with the Intercept DOA Oral Specimen Collection device and tested for THC by gas chromatography mass spectrometry (GC/MS) after methylation for THC (limit of quantification: 1 ng/mL). The OraLine device correctly identified nine saliva specimens positive for cannabis with THC concentrations ranging from 3 to 265 ng/mL, but remained negative in four other samples where low THC concentrations were detected by GC/MS (1-13 ng/mL). One false positive was noted. Secondly, two male subjects were screened in saliva using the OraLine and Intercept devices after consumption of a single cannabis cigarette containing 25mg of THC. Saliva was first tested with the OraLine device and then collected with the Intercept device for GC/MS confirmation. In one subject, the OraLine on-site test was positive for THC for 2 h following drug intake with THC concentrations decreasing from 196 to 16 ng/mL, while the test remained positive for 1.5 h for the second subject (THC concentrations ranging from 199 to 11 ng/mL). These preliminary results obtained with the OraLine IV s.a.t. device indicate more encouraging data for the detection of THC using on-site tests than previous evaluations.

The use of a drug to modify a person's behaviour for criminal gain is not a recent phenomenon. However, the recent increase in reports of drug-facilitated crimes (sexual assault, robbery) has caused alarm by the general public. Among the drugs that can be used, alprazolam (Xanax), an anxiolytic benzodiazepine, has been seldom observed. To document two cases involving this drug, we have developed an approach based on hair testing by LC-MS/MS. After pH 8.4 buffer incubation and extraction with methylene chloride/diethyl ether (80/20, v/v), hair extracts were separated on a XTerra MS C18 column using a gradient of acetonitrile and formate buffer. Alprazolam and diazepam-d5, used as internal standard, were detected by electrospray tandem mass spectrometry. In the first criminal case, alprazolam tested positive in two consecutive 2 cm hair segments at 4.9 and 2.4 pg/mg, from a 12-year-old girl, assaulted by her father who had sedated her three or four times. In the other case, alprazolam was detected in four consecutive 1cm hair segments at 3.1-0.4 pg/mg, obtained from an adolescent who had been forced to prostitute herself.

Chemical dependency is a disease that can affect all professions. Among the health care professionals, anesthesiologists represent a specific group. Numerous factors have been proposed to explain the high incidence of drug abuse among anesthesiologists. These include: easy access to potent drugs, particularly narcotics, highly addictive potential of agents with which they are in contact, and easy diversion of these agents since only small doses will initially provide an effect desired by the abuser. Opioids are the drugs of choice for anesthesiologists, and among them fentanyl and sufentanil are the most commonly used. Alcohol is mostly abused by older anesthesiologists. Propofol, ketamine, thiopental and midazolam are also abused. In fact, all but quaternary ammonium drugs can be observed. Signs and symptoms of addiction in the hospital workplace include: unusual changes in behavior, desire to work alone, refusal of lunch relief or breaks, volunteer for extra cases, call, come in early and leave late, frequent restroom breaks, weight loss and pale skin, malpractice, behind on charts .... Toxicological investigations are difficult, as the drugs of interest are difficult to test for. In most cases, half-lives of the compounds are short, and the circulating concentrations weak. It is, therefore, necessary to develop tandem mass spectrometry procedures to satisfy the criteria of identification and quantitation. In most cases, blood and/or urine analyses are not useful to document impairment, as these specimens are collected at inadequate moments. Hair analysis appears, therefore, as the unique choice to evidence chronic exposure. Depending the length of the hair shaft, it is possible to establish an historical record, associated to the pattern of drug use, considering a growth rate of about 1cm/month. An original procedure was developed to test for fentanyl derivatives. After decontamination with methylene chloride, drugs are extracted from the hair by liquid/liquid extraction after incubation in pH 8.4 phosphate buffer. Fentanyl derivatives are analyzed by GC-MS/MS. The following cases are included in this paper: Case 1: 50-year-old anesthetist, positive for fentanyl (644 pg/mg); Case 2: 42-year-old anesthetist, positive for fentanyl (101 pg/mg) and sufentanil (2 pg/mg); Case 3: 40-year-old anesthetist, positive for codeine (210 pg/mg), alfentanil (30 pg/mg) and midazolam (160 pg/mg); Case 4: 46-year-old nurse, found dead, positive for alfentanil (2 pg/mg) and fentanyl (8 pg/mg). In these cases, the combination of an alternative specimen (hair) and hyphenated analytical techniques (tandem mass spectrometry) appears to be a pre-requisite.

A procedure is presented for the screening of 17 benzodiazepines and hypnotics in oral fluid after collection with the Intercept(R) device by LC-MS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon, zopiclone and zolpidem). The method involves extraction of 0.5 mL of oral fluid (previously stored in the Intercept blue buffer) treated with 0.5 mL of phosphate buffer (pH 8.4) in the presence of 5 ng diazepam-d(5) used as internal standard, with 3 mL of diethyl ether/methylene chloride (50/50) and separation using liquid chromatography-tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.1 to 0.2 ng/mL. Linearity is observed from the limit of quantification of each compound to 20 ng/mL (r(2)>0.99). Coefficients of variation at 2 ng/mL, measured on 6 points range from 4 to 8% for all drugs, except zopiclone (34%). Extraction recovery, measured at the same concentration was higher than 90%. Ion suppression was evaluated for each compound and was lower than 10% for all drugs except zopiclone (93%). These results were found suitable to screen for 17 benzodiazepines in oral fluid and detect them at very low concentrations, making this method suitable for monitoring subjects under the influence.

The use of a drug to modify a person's behaviour for criminal gain is not a recent phenomenon. However, the recent increase in reports of drug-facilitated crimes (sexual assault, robbery) has caused alarm in the general public. The drugs involved can be difficult to detect due to low dosages or chemical instability. They possess amnesic properties and can be quickly cleared from the body fluids. In these situations, blood or even urine can be of poor interest. This is the reason why this laboratory developed an original approach based on hair testing by LC-MS/MS. Zopiclone (Imovane), due to its short half-life associated with rapid hypnotic activity, is considered as a compound of choice to sedate victims. To document the detection of zopiclone in hair, we first tested specimens obtained from two volunteers who had ingested a single 7.5 mg Imovane tablet, and from repetitive consumers of zopiclone. After pH 8.4 buffer incubation and extraction with methylene chloride/diethyl ether (80/20 (v/v)), hair extracts were separated on a Xterra MS C18 column using a gradient of acetonitrile and formate buffer. Zopiclone and diazepam-d5, used as internal standard, were detected by tandem mass spectrometry. A single exposure to zopiclone was detectable in the first hair segment of two volunteers at concentration of 5.4 and 9.0 pg/mg, respectively. Hair from repetitive consumers tested positive for zopiclone at concentrations of 37 and 66 pg/mg. Hair analysis was applied to two authentic criminal cases. In the first one, zopiclone tested positive in the corresponding hair segment at 4.2 pg/mg, in accordance with a single exposure to the drug. In the other expertise, zopiclone was detected in the two segments analyzed, at 21.3 and 21.5 pg/mg, making unlikely the hypothetical single exposure to zopiclone.

The purported lowering of sex opposition, coupled with a possible abrupt unconsciousness-inducing effect and ease of administration in spiked drinks have resulted in the use of hypnotics in cases of drug-facilitated offense. Among these compounds, lorazepam possesses amnesic properties and can impair an individual rapidly. The chances to detect this substance increase if the most sensitive methods are used and if the biological fluid which allows the longest possible detection time is available. In order to document the window of detection of lorazepam, we have orally administered 2.5 mg of the drug to three volunteers and collected oral fluid (n = l) over 8 h, urine (n = 2) over 144 h and hair (n = 3) 4 weeks after exposure. Lorazepam was analyzed by LC-MS/MS after alkalinisation (to pH 8.4 with phosphate buffer) and extraction by dichloromethane/diethyl ether in presence of diazepam-d5, used as internal standard. Reversed-phase separation on a XTerra C18 column was achieved in 12 min, under gradient conditions. Molecular ions (m/z 321 and 290 for lorazepam and the IS, respectively) were selected in Ql and the corresponding daughter ions (m/z 303 and 275 for lorazepam and m/z 154 and 198 for the IS) were detected in Q3 after collision with argon. Urine tested positive for lorazepam over 144 h (2-4 ng/ml), with a peak detected after 24 h exposure (411-880 ng/ml). Oral fluid tested positive for lorazepam over 8 h (0.7 ng/ml). Despite a limit of quantitation at 1 pg/mg, we were unable to detect a single lorazepam dose in hair, contrarily to most other benzodiazepines that are detectable. Therefore, in case of drug-facilitated crimes involving lorazepam, urine appears as the best specimen to document exposure, particularly if LC-MS/MS is used.

The number of reports on drug facilitated crimes is increasing these last years. Apart from ethanol and cannabis, benzodiazepines (BZD) and analogs are the most common drugs reported to be used probably due to their amnesic and sedative properties. We have developed a rapid and sensitive method using LC-MS/MS triple stage quadrupole (TSQ) for the determination of single exposure to bromazepam (Lexomil, 6 mg) and clonazepam (Rivotril, 2 mg) in urine and hair of healthy volunteers. Chromatography was carried out on a Uptisphere ODB 5 microm, 2.1 mm x 150 mm column (Interchim) with a gradient of acetonitrile and formate 2 mM buffer, pH 3. Urine was extracted with Toxitube A (Varian) and allowed the detection of bromazepam, 3-hydroxy-bromazepam, clonazepam and 7-Aminoclonazepam for more than 6 days. Head hair, collected 1 month after the exposure, was treated by incubation with Soerensen buffer pH 7.6, followed by liquid-liquid extraction with dichloromethane for common BZD. A specific pre-treatment for amino-BZD, with an incubation of 15 min at 95 degrees C in 0.1 N NaOH before liquid-liquid extraction with dichloromethane, gave better recoveries and repeatability. After single exposure, bromazepam was present in powdered hair at 28 pg/mg and 7-Aminoclonazepam at 22 pg/mg in the first 1-cm segment, while no clonazepam was detectable. This method was applied in two forensic cases. It allowed us to determine bromazepam in urine 3 days after the alleged offense and in cut head hair at a concentration of 6.7 pg/mg only in the 2-cm proximal segment. The other case showed the presence of clonazepam and 7-Aminoclonazepam in urine a few hours after the offense and the presence of 7-Aminoclonazepam at about 3.2 pg/mg in axillary hair 4 months later.

Since gamma-hydroxybutyrate (GHB) is present in both blood and urine of the general population as an endogenous compound, toxicologists must be able to discriminate between these endogenous levels and a concentration resulting from exogenous exposure. The implementation of a cut-off concentration must be done cautiously, due to the wide distribution of endogenous concentrations. To verify the accuracy of a proposed 50 mg/l postmortem blood cut-off, we tested 71 autopsy cases of subjects where the cause of death could exclude GHB exposure. The delay between death and autopsy ranged from 12 to 72 h. GHB was tested by gas chromatography-mass spectrometry (GC-MS) after precipitation. Briefly, 20 microl of blood, bile, or vitreous humor was pipetted in a glass tube, followed by 20 microl of GHB-d6 and 45 microl of acetonitrile. After vortexing and centrifugation, the supernatant was collected and evaporated to dryness. The residue was derivatized with bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) for 20 min at 70 degrees C. After injection on a 30 m HP5 MS capillary column, GHB (m/z 233, 204, and 147) and GHB-d6 (m/z 239) were identified by MS. GHB tested positive in all the 71 whole blood (cardiac) specimens, with concentrations in the range 0.4-409 mg/l, with a major distribution in the range 10-40 mg/l. A concentration >50 mg/l was observed in 14 cases. As there was no data to support GHB exposure, this was considered as postmortem formation. In order to discriminate this contamination, when available, femoral blood, bile or/and, vitreous humor were tested. The following results were obtained: cardiac blood (55-409 mg/l) versus bile (6.1-238 mg/l) in seven cases; cardiac blood (51-409 mg/l) versus femoral blood (17-44 mg/l) in five cases, and cardiac blood (51-409 mg/l) versus vitreous humor (3.9-2 mg/l) in six cases. It is obvious that bile does not fit the requirements for discrimination and that femoral blood and mostly vitreous humor can be of particular interest. These results demonstrate that a positive (>50 mg/l) postmortem blood GHB concentration cannot support alone drug exposure and that it is essential to document the case with other specimens, including peripheral blood and vitreous humor.

A LC-MS/MS method for the detection of zolpidem in hair was developed to detect this drug after a single dose in possible drug facilitated sexual assaults. To determine the window of detection of zolpidem in both urine and hair, three volunteers received a 10 mg dose. Urine specimens were collected each 12 h for 144 h. Hair was sampled 3-5 weeks after exposure. Hair and urine extracts were separated on a Xterra MS C18 column using a gradient of acetonitrile and formate buffer. For each compound, detection was related to two daughter ions. Zolpidem was detected for up to 60 h in urine with peak concentrations obtained at 12 h. A single exposure to zolpidem was detected in hair at concentrations ranging from 1.8 to 9.8 pg/mg. Hair analysis was applied to two possible criminal cases. In the first case, zolpidem tested positive in the corresponding hair segment at 4.4 pg/mg. In the other case, zolpidem was detected in all the segments analyzed, demonstrating likely previous drug use in addition to recent exposure associated with a positive blood result.

A solid-phase enzyme immunoassay involving microtiter plates was recently proposed by International Diagnostic Systems corporation (IDS) to screen for buprenorphine in human serum. The performance of the kit led us to investigate its applicability in other biological matrices such as urine or blood, and also hair specimens. Low concentrations of buprenorphine were detected with the ELISA test and confirmed by HPLC/MS (buprenorphine concentrations measured by HPLC/MS: 0.3 ng/mL in urine, 0.2 ng/mL in blood, and 40 pg/mg in hair). The intra-assay precision values were 8.7% at 1 ng/mL of urine (n = 8), 11.5% at 2 ng/mL in serum (n = 8), and 11.5% at 250 pg/mg of hair (n = 8), respectively. The immunoassay had no cross-reactivity with dihydrocodeine, ethylmorphine, 6-monoacetylmorphine, pholcodine, propoxyphene, dextromoramide, dextrometorphan at 1 and 10 mg/L, or codeine, morphine, methadone, and its metabolite EDDP. A 1% cross-reactivity was measured for a norbuprenorphine concentration of 50 ng/mL. Finally, the immunoassay was validated by comparing authentic specimens results with those of a validated HPLC/MS method. From the 136 urine samples tested, 93 were positive (68.4%) after the ELISA screening test (cutoff: 0.5 ng/mL) and confirmed by HPLC/MS (buprenorphine concentrations: 0.3-2036 ng/mL). From the 108 blood or serum samples screened, 27 were positive (25%) after the ELISA test with a cutoff value of 0.5 ng/mL (buprenorphine concentrations: 0.2-13.3 ng/mL). Eighteen hair specimens were positive (72%) after the screening (cutoff: 10 pg/mg) and confirmed by LC/MS (buprenorphine concentrations: 40-360 pg/mg). The ELISA method produced false positive results in less than 21% of the cases, but no false negative results were observed with the immunological test. Four potential adulterants (hypochloride 50 mL/L, sodium nitrite 50 g/L, liquid soap 50 mL/L, and sodium chloride 50 g/L) that were added to 10 positive urine specimens (buprenorphine concentrations in the range 5.3-15.6 ng/mL), did not cause a false negative response by the immunoassay.