Cristiana Stramesi

Determination of opiates in hair. Effects of extraction methods on recovery and on stability of analytes

In order to evaluate (i) the recovery of extraction of opiates from authentic hair samples and (ii) the extent of hydrolysis of acetylated opiates (6-acetylmorphine, acetylcodeine) occurring during sample preparation, three different methods of extraction commonly used for opiates have been compared. To this purpose a sample consisting of a pool of hair collected from several heroin overdose cases has been submitted alternately to (A) digestion in 2 M NaOH at 80 degrees C for 1 h (n = 5), (B) incubation in 0.1 M HCl at 45 degrees C for 18 h (n = 5) and (C) incubation in methanol at 37 degrees C for 18 h (n = 5). After pH adjustment of the different incubation media to 7-8, analytes have been isolated by means of SPE using Bond Elut certify columns and derivatized with MSTFA. Analyses have been performed by either GC-MS in the selected ion monitoring mode or, omitting SPE, by radioimmunoassay. The extent of hydrolysis of 6-acetylmorphine to morphine and of acetylcodeine to codeine have been determined by submitting blank hair samples spiked with the acetylated analytes to the different extraction methods and measuring the amount of morphine and codeine formed. Both the recovery of extraction of the total morphine fraction (6-acetylmorphine + morphine) and the rate of hydrolysis of 6-acetylmorphine were found to be in the order: A > B > C. Similar results were obtained for the total codeine fraction (acetylcodeine + codeine). These results clearly indicate that: (i) the concentration of opiates measured in hair depends on the extraction method used; (ii) ratios between different analytes (e.g. 6-acetylmorphine vs. morphine) may reflect the rate of hydrolysis during sample preparation rather than different types of exposure to opiates.

Immunological screening of drugs of abuse and gas chromatographic-mass spectrometric confirmation of opiates and cocaine in hair.

The work presents an analytical strategy to detect drugs of abuse in hair. It involves two sequential steps: a screening by a simple enzyme-linked immunosorbent assay (ELISA) methodology to detect opiates, cocaine and its metabolites, and benzodiacepines, followed by confirmation of opiates and cocaine metabolites in positive samples by gas chromatography coupled to mass spectrometry (GC-MS). In the same GC-MS run other drugs for substitution therapy (e.g. methadone and its main metabolite) can also be detected. After a double washing of hair samples with dichloromethane, hair specimens were cut into small pieces and 10 mg samples were incubated in 2 ml of methanol-trifluoroacetic acid (9:1) mixture, overnight at 37 degrees C. Aliquots of the extract were then evaporated, reconstituted in buffer and analysed according to the ELISA procedure. Confirmation involved solid-phase extraction of another fraction of the extract kept at -20 degrees C, derivatization with heptafluorobutyric anhydride and hexafluoroisopropanol and detection of cocaine, benzoylecgonine, ecgonine methylester, cocaethylene, morphine, codeine, 6-monoacetylmorphine, methadone and 2-ethylidene-1.5-dimethyl-3,3-diphenylpirrolidine (methadone metabolite) by selective ion monitoring after gas chromatographic separation. During the development of the method it was verified that no more than 10% of cocaine, opiates and benzodiacepines were lost when dichloromethane was used to wash real samples. The results also confirmed the increase of extractability power of TFA when it was added to methanol: the recovery for the analytes (cocaine and its metabolites and opiates) added to methanol-TFA alone was of the order of 90% except for benzoylecgonine (75%), and the recovery for the analytes added to methanol-TFA extract of drug-free hair was about 90% for all analytes except for benzoylecgonine and 6-MAM (around 70%). Regarding the stability of labile compounds, only small amounts of ecgonine methylester (2.3%) and morphine (7.2%) were produced, from cocaine and 6-MAM respectively, after the whole extraction procedure and two weeks of storage of methanol-TFA extracts at -20 degrees C. Satisfactory results were obtained when the procedures were applied to the analysis of external proficiency testing hair samples and actual specimens from drug addicts.

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 r2 > 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°C, when exposed to 3 freeze-thaw cycles, and when stored at −20°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.

Postnatal methadone withdrawal syndrome: hair analysis for detecting chronic exposure.

Sir, An infant was transferred to our clinic with a naso-gastric tube (NGT), because of feeding difficulties after cardiac surgery. On admission the NGT was removed as malposition was suspected. On visual inspection the distal part, estimated 10–15 cm, seemed missing and the end of the tube showed signs of tearing (Fig. 1A). The infant showed no signs of pain, obstructed intestinal passage or perforation. A plain abdominal X-ray showed the missing NGT part in the gastric cavity with one end near or in the gastric outlet (Fig. 1B). In the next days, the infant was fed with a new NGT. Stools were passed normally, but no distal part of the NGT was excreted. A second X-ray on day 7 showed the NGT was still positioned in the stomach. To prevent migration and perforation through the gastric wall, it was decided to retrieve the distal NGT part endoscopic. The retrieved part was combined with the proximal part and made a full length of an intact NGT. The referring hospital and manufacturer were contacted. A medicationadditive with NGT-dissolving properties or fabrication failure was suspected to be the cause of rupture.

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 patients 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 patients 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.

The standardization of results on hair testing for drugs of abuse: An interlaboratory exercise in Lombardy Region, Italy

Hair testing for drugs of abuse is performed in Lombardy by eleven analytical laboratories accredited for forensic purposes, the most frequent purposes being driving license regranting and workplace drug testing. Individuals undergoing hair testing for these purposes can choose the laboratory in which the analyses have to be carried out. The aim of our study was to perform an interlaboratory exercise in order to verify the level of standardization of hair testing for drugs of abuse in these accredited laboratories; nine out of the eleven laboratories participated in this exercise. Sixteen hair strands coming from different subjects were longitudinally divided in 3-4 aliquots and distributed to participating laboratories, which were requested to apply their routine methods. All the participants analyzed opiates (morphine and 6-acetylmorphine) and cocainics (cocaine and benzoylecgonine) while only six analyzed methadone and amphetamines (amphetamine, methamphetamine, MDMA, MDA and MDEA) and five Δ(9)-tetrahydrocannabinol (THC). The majority of the participants (seven labs) performed acidic hydrolysis to extract the drugs from the hair and analysis by GC-MS, while two labs used LC-MS/MS. Eight laboratories performed initial screening tests by Enzyme Multiplied Immunoassay Technique (EMIT), Enzyme-linked Immunosorbent Assay (ELISA) or Cloned Enzyme Donor Immunoassay (CEDIA). Results demonstrated a good qualitative performance for all the participants, since no false positive results were reported by any of them. Quantitative data were quite scattered, but less in samples with low concentrations of analytes than in those with higher concentrations. Results from this first regional interlaboratory exercise show that, on the one hand, individuals undergoing hair testing would have obtained the same qualitative results in any of the nine laboratories. On the other hand, the scatter in quantitative results could cause some inequalities if any interpretation of the data is required.

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

HighlightsWe developed and validated a GC–MS/MS method for ethyl glucuronide determination in hair.We compared GC–MS/MS and LC–MS/MS in analysis of real samples.We compared EtG concentration obtained after hair cutting and pulverization.Pulverization of hair increases the concentration of EtG, but some variability of EtG levels remains. ABSTRACT 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.