Comparison of Two Immunoassay Screening Methods and a LC-MS/MS in Detecting Traditional and Designer Benzodiazepines in Urine.

Sensitive and specific immunoassay screening methods for the detection of benzodiazepines in urine represent an important prerequisite for routine analysis in clinical and forensic toxicology. Moreover, emerging designer benzodiazepines force labs to keep their methodologies updated, in order to evaluate the reliability of the immunochemical techniques. This study aimed at evaluating the sensitivity and specificity of two different immunoassay methods for the detection of benzodiazepines in urine, through a comparison with the results obtained by a newly developed liquid chromatographic tandem mass spectrometric (LC-MS/MS) procedure. A cohort of authentic urine samples (N = 501) were processed, before and after a hydrolysis procedure, through two immunoassays and an LC-MS/MS method. The LC-MS/MS target procedure was optimized for monitoring 25 different molecules, among traditional and designer benzodiazepines, including some metabolites. At least one of the monitored substances was detected in 100 out of the 501 samples. A good specificity was observed for the two immunoassays (>0.99), independently of the cut-offs and the sample hydrolysis. The new kit demonstrated a fairly higher sensitivity, always higher than 0.90; in particular, a high cross-reactivity of the new immunoassay was observed for samples that tested positive for lorazepam and 7-aminoclonazepam. The two immunoassays appeared adequate to monitor not only traditional benzodiazepines but also new designer ones.

Variability on ethyl glucuronide concentrations in hair depending on sample pretreatment, using a new developed GC-MS/MS method.

Quantitative determination of ethyl glucuronide in keratin matrix, particularly in hair samples, provides a significant contribution to the evaluation of the extent of ethanol intake. The first-choice method to carry out this analysis is LC-MS/MS, but other techniques may be used. The aim of this work is: a) to develop and validate a GC-MS/MS method for ethyl glucuronide determination in hair; b) to compare GC-MS/MS and LC-MS/MS in analysis of real samples; c) to compare EtG concentration obtained after hair cutting and pulverization. About 30 mg hair samples were washed, pulverized and soaked in 1 ml deionized water. After incubation, the solution was purified through a SPE anion exchange cartridge; the eluate was dried under nitrogen stream, derivatized with PFPA and reconstituted in n-hexane. Then, the sample was injected in the GC-MS/MS system, operating in negative chemical ionization mode and in selected reaction monitoring. The two most intense transitions were used to monitor ethyl glucuronide and deuterated internal standard. All the validation parameters fulfilled the international acceptance criteria. LOD and LOQ were set at 2.0 and 3.0 pg/mg respectively. This method was applied to 194 hair samples collected from teetotallers and alcohol consumers and represents a suitable alternative to LC-MS/MS for the determination of EtG in hair samples, in particular when scarce quantity of hair is available. This study confirmed that pulverization of hair increases the concentration of EtG, but some variability of EtG levels remains probably due to the presence of non-homogeneous material even though pulverization.

Methadone-related deaths. A ten year overview.

Over the last 10 years we have registered in our district (about 500,000 inhabitants) 36 cases of fatal methadone poisoning, involving both patients on treatment and naive subjects: this is a significant increase of deaths due to methadone use, misuse or abuse compared with previous years. Twenty-four patients (66.7%) were on methadone maintenance programs for heroin detoxification, while 12 (33.3%) were taking the drug without a medical prescription. The average blood concentration of methadone in patients undergoing a maintenance program was 1.06 mg/L (0.21-3.37 mg/L), against 0.79 mg/L (0.2-3.15 mg/L) in those taking the non-prescribed drug. Since 111 heroin-related deaths were recorded in our district in the same period, the fact that there appear to be many methadone deaths (about a third of heroin-related deaths) cannot be overlooked. The aim of this work is to understand the possible reasons for such a large number of methadone-related deaths. On this subject, we have noticed that risks associated with methadone intake are often underestimated by clinicians prescribing the drug: sometimes methadone is prescribed without taking into account patient's tolerance to opiates, and a large number of subjects enrolled in methadone maintenance programs in Italy, have also been given take-home doses, thus increasing the risk of abuse and diversion.

Workplace drug testing in Italy: findings about second-stage testing.

Workplace Drug Testing (WDT) in Italy includes two levels of monitoring: a first stage concerning drug testing on urine samples and a second involving both urine and hair analysis. The second stage is performed only on workers who tested positive at the first level. We analyzed urine and hair specimens from 120 workers undergoing second-level testing between 2009 and 2012. Eighty percent of them had tested positive for cannabinoids during the first level analysis, and 15.8% for cocaine. Both urine and hair samples were analyzed in order to find the following drugs of abuse: amphetamines, buprenorphine, cannabinoids, cocaine, ecstasy, methadone, and opiates. Urine analyses were performed by immunological screening (EMIT); urine confirmatory tests and hair analyses were performed by gas chromatography-mass spectrometry (GC-MS). As regards second-stage testing on urine samples, 71.2% of workers were always negative, whereas 23.9% tested positive at least once for cannabinoids and 2.5% for cocaine. Hair analyses produced surprising results: 61.9% of hair samples tested negative, only 6.2% tested positive for cannabinoids, whereas 28.8% tested positive for cocaine. These findings confirm that second-level surveillance of WDT, which includes hair analysis, is very effective because it highlights drug intake - sometimes heavy - that cannot be revealed only through urine analyses. The employees for whom drug addiction is proved can begin rehabilitation, while keeping their job. Eventually, our results confirmed the widespread and undeclared use of cocaine in Italy.

Distribution of venlafaxine and O-desmethylvenlafaxine in a fatal case.

Venlafaxine is an extensively used antidepressant drug; it is considered to be quite safe and only a few pure cases of fatal poisoning have been reported. Here we describe a fatal case of venlafaxine self-poisoning including detailed tissue distribution of the drug and its metabolite O-desmethylvenlafaxine and the exact time sequence of events, as reported in the patient's clinical record. Qualitative analyses were performed by GC-MS while quantitative analyses were carried out by LC-MS/MS. We then compared our results with those of previously published cases. Fatal venlafaxine poisoning often occurs after the intake of an extremely elevated number of tablets, corresponding to tens of grams of the drug, or it can be due to interaction between the drug and other substances. In the present case, no other drugs or ethanol were found and death occurred 12h after ingesting only 3g of venlafaxine, despite timely medical treatment.

Workplace drug testing in Italy - critical considerations.

Workplace drug testing (WDT) was established in Italy on 30 October 2007. Two tiers of survey are required: the first tier concerns drug testing on urine samples, the second involves both urine and hair analysis. Between July 2008 and December 2011, 10 598 workers' urine samples and 72 hair samples for opiates, cocaine, cannabinoids, amphetamines, methylenedioxyamphetamines, methadone, and buprenorphine were tested in our laboratory. Urine analyses were performed by immunological screening (EMIT); hair analysis and confirmation tests in urine were performed by gas chromatography-mass spectrometry (GC-MS). Employees tested positive in urine for drugs of abuse numbered 2.8% in 2008, 2.03% in 2009, 1.62% in 2010, and 1.43% in 2011. As regards the second level of analysis, we observed that only one-third of the workers who had been tested positive for drugs of abuse were referred to an Addiction Treatment Unit in order to verify drug addiction. Our experience shows that, four years after approval of the law on WDT, the percentage of workers positive for drugs of abuse in urine has reduced in comparison to the first year. Moreover, our data show that most of the times employees who tested positive are tardily referred or not referred at all to a Public Addiction Treatment Unit to verify drug addiction. This makes us believe that the legal provisions are widely disregarded not paying the right tribute to the fact that Italy is one of few European countries with legislation on WDT.

Hair testing and self-report of cocaine use.

Hair analysis is a useful tool in both clinical and forensic fields: it allows information about drugs of abuse (DOA) consumption to be obtained. However, in spite of analytical results, sometimes patients continue to deny using drugs or, on the contrary, insist on describing themselves as severe drug addicts; indeed there are often considerable difficulties in getting truthful statements about the real amount of drugs used. In this study we have tried to compare cocaine concentration in hair samples with self-reported drug intake. We enrolled 113 subjects (61 Africans, 52 Caucasians) who had been recently sent to jail. They were asked to tell about their use of illicit drugs during the last three months and then submitted to hair analysis. Hair segments (3 cm) were analyzed by GC-MS for amphetamines, cocaine and opiates. Useful data was obtained from 82 subjects, separated into two main groups on account of ethnic origin (African or Caucasian) and divided further into daily, weekly and monthly users. The results showed qualitative results and self-reported consumption to be in good agreement, although the correlation between frequency of consumption and concentration in hair revealed sometimes higher concentrations in contrast with the admission of low consumption. There was a definite separation between occasional and daily use (especially in Caucasian people), while concentrations found where weekly use was reported were more variable. Concentrations of cocaine measured in Africans' hair were much higher than in Caucasians'. Even if this study is exclusively based on self-report, it provides some interesting information in order to differentiate the frequency of consumption, and especially underlines the great importance of ethnic bias on hair analysis.

Liquid chromatography with tandem mass spectrometric detection for the measurement of ethyl glucuronide and ethyl sulfate in meconium: new biomarkers of gestational ethanol exposure?

A liquid chromatography tandem mass spectrometric (LC-MS/MS) method with postcolumn addition of acetonitrile for the determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in meconium was developed and validated using pentadeuterated EtG and pentadeuterated EtS as internal standards. The analytes were extracted from the matrix by acetonitrile, concentrated by solid phase extraction, separated using a reversed-phase chromatographic column, and quantified within 9 minutes. Lower limits of quantification were 5 and 1 ng/g meconium for EtG and EtS, respectively. Calibration curves were linear from lower limits of quantifications to 500 ng/g, with a minimum r > 0.999. At 3 concentrations spanning the linear dynamic range of the assay, mean recoveries ranged between 78.7% and 96.8% for EtG and between 72.1% and 95.6% for EtS. Inaccuracy was better than 8.1%, with intra-assay and interassay imprecision better than 7.2% and 10.5%, respectively. Matrix effects (ion suppression/enhancement) were found to be negligible. The analytes of interest were stable at room temperature, at 4 degrees C, when exposed to 3 freeze-thaw cycles, and when stored at -20 degrees C for up to 6 months. This sensitive and specific method was used to assess the presence of these alcohol biomarkers in meconium samples from 2 different city cohorts.

Treatments against hair loss may hinder cocaine and metabolites detection.

Recently, some of the hair samples that we routinely analyse for drugs of abuse did not produce valid results for cocaine and metabolites. A series of very intense interfering peaks with ion fragments common to cocaine (CO), and benzoylecgonine (BE) were found to cover up the "cocaine" region of the chromatogram. In one of these cases the subject declared he had used a lotion containing Minoxidil in order to prevent hair loss. Starting from this observation we found that the interfering peaks belonged to four different TMS derivatives of Minoxidil. Minoxidil interference was further investigated by applying Tricoxidil(®), a Minoxidil solution, to the hair of CO-free volunteers and to a CO-positive hair strand dipped into Tricoxidil. Hair were analysed before and after treatment. In both cases interfering peaks were absent in the chromatograms of untreated hair and appeared in treated hair. In the CO-positive hair detection of CO, BE and internal standard was completely hindered after treatment with Minoxidil. Attempts to separate interfering peaks from CO and metabolites by modifying the temperature programme failed. None of the hair washing methods tested (methanol; dichloromethane; sodium dodecyl sulphate water solution, 1% w/v followed by methanol; phosphate buffer 0.1 M, pH 6 followed by methanol) succeeded in removing Minoxidil interference. However, a simple solution to partially overcome the problem was to dry up the derivatised extract, reconstitute it in methanol (in order to switch back Minoxidil derivatives to the native molecule), and re-inject it: owing to the higher polarity, underivatised Minoxidil does not interfere any more with the chromatography of CO, at the expense of the disappearance of BE and ecgonine methyl ester both producing TMS derivatives. This strategy was applied to four real cases where Minoxidil interference was recognised: in two of these cases CO was detected. The problem of Minoxidil interference on CO detection may be limited to procedures involving trimethylsilylation, which is probably the most commonly adopted derivatisation in laboratories performing hair analysis for drugs of abuse.

Markers of chronic alcohol use in hair: comparison of ethyl glucuronide and cocaethylene in cocaine users.

Two direct ethanol metabolites, namely ethyl glucuronide (EtG) and cocaethylene (CE), in the hair of cocaine (COC) users were compared in this study. Hair samples (n=68) were submitted to the determination of EtG (by liquid chromatography-electrospray-tandem mass spectrometry) and of COC and metabolites, including CE (by gas chromatography-mass spectrometry). Quantitative and qualitative results were compared. No quantitative correlation was found between EtG and CE, as well as between EtG and the cocaethylene concentration divided by the concentration of COC and its metabolites (benzoylecgonine and ecgonine methylester, as COC equivalents). Nevertheless, many factors are supposed to affect the amount of the two substances incorporated in the hair matrix, such as the subject's habits in ethanol and COC use, genetic variability in the metabolism of both substances, and the different chemical and physical properties of EtG and CE. When establishing a cut-off of 4 pg/mg for EtG and of 200 pg/mg for CE, 47 samples tested positive for EtG and 41 samples tested positive for CE; 12 samples out of the 47 EtG-positives tested negative for CE (25%), whereas 6 samples out of the 41 CE-positives tested negative for EtG (15%). According to these data, EtG appears to be a more sensitive and specific marker of non-moderate alcohol users than CE.

Determination of ethyl glucuronide in hair samples by liquid chromatography/electrospray tandem mass spectrometry.

A method for the determination of ethyl glucuronide (EtG) in hair samples, using liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS), was developed and validated. The treatment of hair samples was as follows: to 100 mg of washed (dichloromethane followed by methanol, 1 ml each) and cut (1-2 mm) material, 700 microl of water, 20 microl of internal standard solution (pentadeuterated EtG, D(5)-EtG, 500 microg/l) and 20 microl of methanol were added. Samples were incubated at 25 degrees C overnight and then ultrasonicated for 2 h. Finally, 8 microl of the centrifuged solution (13,000 rpm) were analyzed by LC/ESI-MS/MS in negative ion mode. The surviving ions of EtG and D(5)-EtG were monitored together with the following MRM transitions: m/z 221 --> 75, m/z 221 --> 85 (EtG) and m/z 226 --> 75, m/z 226 --> 85 (D(5)-EtG). The method exhibited a mean correlation coefficient better than 0.9998 over the dynamic range (3-2000 pg/mg). The lower limit of quantification (LLOQ) and the limit of detection (LOD) were 3 and 2 pg/mg respectively. The intra- and interday precision and accuracy were studied at four different concentration levels (3, 5, 56 and 160 pg/mg) and were always better than 7% (n = 5). Matrix effects did not exceed 20%. The method was applied to several hair samples taken from autopsies of known alcoholics, from patients in withdrawal treatment, from social drinkers, from adult teetotalers and from children not exposed to ethanol, with EtG concentrations globally ranging from < or =2 to 4180 pg/mg.

The role of cocaine in heroin-related deaths. Hypothesis on the interaction between heroin and cocaine.

In recent years, there has been an increase in cocaine-related deaths at the Department of Legal Medicine and Public Health of Pavia, probably reflecting the rising trend in cocaine use in Western Europe. Deaths from cocaine alone have increased from 6 cases in 1979-1991 (1.5% of drug-of-abuse deaths) to 13 in 1992-2002 (3.2%) and comparing the same periods, heroin-related deaths (HRDs) involving cocaine more than doubled from 8 (1.9%) to 22 (5.4%). In an attempt to investigate the role of cocaine in HRDs, acute narcotic death cases testing positive for cocaine use (blood cocaine or metabolite concentration >0.01 mg/l, COC+) were examined. Only cases from 1997 to 2001 were considered as in this period all data were obtained using the same analytical procedures (free morphine and total morphine by DPC Coat-A-Count radioimmunoassay before and after enzymatic hydrolysis, cocaine and metabolites in blood by SPE, TMS derivatization and gas chromatography-mass spectrometry (GC-MS)). The median, minimum and maximum concentrations of free morphine in blood (FM) and total morphine in blood (TM), urine (UM) and bile (BM) in the COC+ group (n = 9) were compared with those calculated in the group of "pure" HRDs (no other drugs detected in blood, COC-, n = 30). Differences among the medians in the two groups were statistically evaluated using the two-tailed Mann-Whitney U-Test. Statistical analysis was also carried out including in both groups cases with a blood alcohol concentration (BAC) > 20 mg/L (COC+, n = 19; COC-, n = 76). For the COC+ group, median TM was lower (0.32 mg/l versus 0.90 mg/l, P = 0.0214), median FM was lower, but not statistically significant (0.08 mg/l versus 0.28 mg/l, P = 0.1064), FM/TM ratio was similar (0.33 and 0.35), UM was also similar (21.0 mg/l and 18.0 mg/l), and BM was higher (90.0 mg/l versus 49.0 mg/l, P = 0.0268). Similar comparison results were obtained by repeating statistical analyses after including in the two groups cases with positive BAC. The picture observed for HRD cases involving cocaine is very different from what was previously observed for HRD cases involving ethanol [A. Polettini, A. Groppi, M. Montagna, The role of alcohol abuse in the etiology of heroin related deaths: evidence for pharmacokinetic interactions between heroin and alcohol, J. Anal. Toxicol. 23 (1999) 570-576], and updated with more recent data; in the high-ethanol (HE, BAC > 1000 mg/l) group, TM was lower than in the low-ethanol (LE, BAC