Application of hygrine and cuscohygrine as possible markers to distinguish coca chewing from cocaine abuse on WDT and forensic cases.

The objectives of present work are twofold. First, we want to verify that hygrine and cuscohygrine are good markers to distinguish between chewing coca leaves and cocaine abuse. Secondly, we try to develop a quick and easy qualitative method to determine the two mentioned markers. We analyzed two kinds of urine samples: the first group consisted of twenty-four (24) subjects: urine samples were obtained from various types of workers (e.g. doctors, chemists, nurses, technicians, painters, contractors, employees and some retired persons) who admitted chewing coca leaves. Frequency of the habit of chewing coca leaves was variable. They practiced "coqueo" between two (2) and forty-four (44) years. Sixteen (16) of them used alkaline substances to enhance the extraction of cocaine from the leaves The second group of urine samples consisted on thirty-eight (38) cocaine abusers, from forensic cases from Spain and Argentina. A GC/MS qualitative method, performed after liquid-liquid extraction, was developed and validated (the parameters studied were selectivity/specificity, LOD and stability), and then applied to the urine samples. Hygrine and cuscohygrine are good markers to distinguish between chewing coca leaves and cocaine abuse, and the qualitative method presented can be used successfully in workplace drug testing and forensic cases.

Hygrine and cuscohygrine as possible markers to distinguish coca chewing from cocaine abuse in workplace drug testing.

Cocaine abuse is widespread all over the world, and is performed generally by sniffing, injecting or smoking cocaine or crack. The distinction between the recreational use of cocaine from the practice of the so called "coqueo" is still an issue in those countries where this habit is diffused and where it is not considered an addiction, by this reason is necessary to develop a method for to distinguish the coca chewers and cocaine abusers. The use of an unique marker to distinguish between cocaine abuse and chewing of coca leaves is of fundamental importance in those countries where this habit is diffused. Certain alkaloids of the leaves of Erythroxylum coca are lost during the process of extraction/purification of cocaine and it is not possible to find them neither in seizures of chlorhidrate of cocaine nor urine samples of cocaine abusers. These markers are the hygrine and cuscohygrine that are present in the leaves of E. coca. A fast GC/MS method involving a liquid:liquid extraction procedure with tertbutylmethylether (TBME) is proposed for the determination of some alkaloids in cocaine leaves, cocaine seizures and biological samples. All specimens were alkalinized to pH 9 with a carbonate/bicarbonate buffer and then extracted with TBME. The analysis was carry out by GC/MS with electron impact at 70 eV and in full scan mode. The results demonstrate that hygrine and cuscohygrine are not found neither in the urine of cocaine abusers nor in cocaine seizures. For this reason this compounds could be considered as markers of coca chewing. This developed method permits to distinguish coca chewing from cocaine abuse in workplace drug testing through the analysis of urine samples.

Analytical techniques in androgen anabolic steroids (AASs) analysis for antidoping and forensic purposes.

A survey on the main analytical challenges related to the analysis of Androgen Anabolic Steroids (AASs) is reported. AASs analysis is an issue regarding antidoping analyses as well as forensic toxicology applications. This paper reports an overview of the more recent literature regarding various aspects of sample preparation, analytical techniques and interpretation of results for AASs identification in biological samples. New analytical approaches, mainly for their application to the antidoping field, are reported. The application of AASs analysis in forensic cases is also described, taking into consideration mainly the different biological samples that can be analysed for forensic purposes. Particular attention was played on the application of hair analysis as alternative biological specimen for the determination of AASs abuse.

Use of solid-phase microextraction (SPME) for the determination of methadone and EDDP in human hair by GC-MS.

Solid-phase microextraction (SPME) is a new extraction technique with many advantages: small sample volume, simplicity, quickness and solvent-free. It is mainly applied to environmental analysis, but is also useful for the extraction of drugs from biological samples. In this paper the use of SPME is proposed for the determination of methadone and its main metabolite EDDP in hair by GC-MS. The hair samples were washed, cut into 1-mm segments, and incubated with Pronase E for 12 h. A 100-micron polydimethylsiloxane (PDMS) film fibre was submerged for 30 min in a diluted solution of the hydrolysis liquid (1:4 with borax buffer) containing methadone-d3 and EDDP-d3 as internal standards. Once the microextraction was concluded the fibre was directly inserted into the CG injection port. Linearity was found for methadone and EDDP in the range studied, 1.0-50 ng/mg hair, with correlation coefficients higher than 0.99. Interassay relative standard deviation (R.S.D) was determined to be less than 13.30% for methadone and less than 8.94% for EDDP, at 3.0 and 30.0 ng/mg. Analytical recoveries were close to 100% for both compounds on spiked samples. The method was applied to the analysis of real hair samples from eight patients of a methadone maintenance programme. The concentration of methadone in hair ranged from 2.45 to 78.10 ng/mg, and for EDDP from 0.98 to 7.76 ng/mg of hair.

Evaluation of cocaine, amphetamines and cannabis use in university students through hair analysis: preliminary results.

The evaluation of drug abuse in a defined population was performed through toxicological hair analysis. Hair samples from university students ranging from 18 to 25 years of age were anonymously collected and screened for cocaine, amphetamines and cannabinoids by radioimmunoassay (RIA). Positive results (cut-off values adopted were 2 ng/mg for cocaine and amphetamines and 0.5 ng/mg for cannabinoids) were confirmed by GC/MS. Preliminary results showed 19% of positive results for cocaine on 200 samples analysed. No confirmed positive results were obtained for amphetamine analysis. RIA technique demonstrated its unsuitability for cannabinoids preliminary screening on hair, giving a high percent of false positive results.

Methods used to detect drug abuse in pregnancy: a brief review.

A brief review of different methods adopted to assess foetal drug exposure is reported here. Various operative procedures have been described in the literature to detect drug abuse in pregnancy. All protocols can be clustered into two main groups: self-report and interview procedures and analytical methods. The latter can be performed on tissues and/or fluids withdrawn from the mother, from the new-born or both. The aim of this review is to analyse the capability of these different techniques and the suitability of the biological specimens in view of the lack of consensus on a method of choice. The results obtained and the conclusions are different between various authors. A brief discussion on the capability of the different techniques and biological specimens adopted for this purpose is presented.

Solid-phase microextraction for cannabinoids analysis in hair and its possible application to other drugs.

This paper describes the application of solid-phase microextraction (SPME) to cannabis testing in hair. Fifty milligrams of hair was washed with petroleum ether, hydrolyzed with NaOH, neutralized, deuterated internal standard was added and directly submitted to SPME. The SPME was analyzed by GC-MS. The limit of detection was 0.1 ng/mg for cannabinol (CBN) and delta9-tetrahydrocannabinol (THC) and 0.2 ng/mg for cannabidiol (CBD). THC was detected in a range spanning from 0.1 to 0.7 ng/mg. CBD concentrations ranged from 0.7 to 14.1 ng/mg, and CBN concentrations ranged from 0.4 to 0.7 ng/mg. The effectiveness of different decontamination procedures was also studied on passively contaminated hair. The proposed method is also suitable for the analysis of methadone in hair; cocaine and cocaethylene can be detected in hair with SPME extraction after enzymatic hydrolysis.

Application of hair analysis to document coercive heroin administration to a child.

This paper describes the application of hair analysis in a judicial case to document coercive heroin administration to a 5-year-old child, who was admitted with overdose symptoms to an intensive care division. Segmental hair analysis was performed in order to determine if drug consumption had continued. Quantitative results showed the presence of morphine and O-6-monoacetylmorphine (MAM) in increasing amounts from the second to the distal segment of hair, each approximately corresponding to a period of time of one month. MAM concentrations ranged from 0.2 ng/mg in the second segment of hair to 0.6 ng/mg in the distal segment; morphine concentrations ranged from 0.1 to 0.3 ng/mg. Limits of detection were 0.1 ng/mg for morphine and 0.2 ng/mg for MAM. The proximal segment of hair, which corresponded to the period that the child was housed in a social center, was negative for opiates.

Evaluation of cocaine use during pregnancy through toxicological analysis of hair.

Toxicological analysis of hair was used to detect cocaine use in a group of 615 pregnant women. Hair samples were washed, enzymatically digested, and analyzed by radioimmunoassay for cocaine. Positive results were confirmed by gas chromatography-mass spectrometry after a solid-phase extraction. Benzoylecgonine (BZE) and cocaine were detected after derivatization with pentafluoropropionic-anhydride-pentafluoropropanol. Deuterated cocaine and BZE were used as internal standards. This study demonstrated a mean frequency of 1.9% cases positive for cocaine in all the hair samples examined. The positive rate was 6% among women admitted for spontaneous abortion (66 cases), and the positive rate was 1.4% for those who carried to full term (549 cases). These data underline the usefulness of hair analysis for the diagnosis of drug abuse and demonstrate that there is significant use of cocaine in the Italian population.

Cocaine abuse in pregnancy: its evaluation through hair analysis of pathological new-borns.

Cocaine abuse in pregnancy has been evaluated through toxicological analysis of hair from 123 pathological new-borns admitted in an intensive care division. The new-borns were affected with malformations, low gestational age, low birth weight, respiratory distress. A control group of 39 healthy new-borns was also analysed. Hair samples (about 50 mg) were enzymatically digested and directly analysed by RIA. Samples positive to the preliminary screening were extracted by SPE columns and injected in GC/MS. Results obtained showed 3 positive samples (2.4%). All cocaine babies had low gestational age, low birth weight, two of them showed heart malformation, one kidney malformation and one genital malformation. None of the control new-born resulted positive for cocaine. These results show a worrying trend of cocaine diffusion in the Italian population.

Preparation of Hair Samples for Drug Analysis.

Toxicological hair analysis has attracted substantial attention because of its promising diagnostic power. Analysis of drugs in hair, even though widely studied since 1970, is still not standardized; many different sample preparation and analytical procedures are practiced. To obtain reliable results, hair analysis requires preparation steps that are not necessary for conventional biological samples such as blood or urine. Pretreatment procedures are mainly conducted to decontaminate hair samples and to isolate drugs from the solid hair structure before instrumental analysis. Many types of sample preparation procedures have been reported in the literature. Selecting an appropriate one can be difficult because the choice depends on different factors, including decontamination efficiency, analytical recovery, and chemical stability of the target drug and its metabolites. This review discusses the main aspects of hair sample preparation procedures through a critical evaluation of literature data, focusing on the procedures, reliability, advantages, and result interpretation.

Hair and urine analysis: relative distribution of drugs and their metabolites.

This work studies the distribution of cocaine and heroin metabolites in hair and urine of living polidrug abusers. Cocaine, benzoylecgonine (BEG), ecgonine methyl ester (EME), morphine, codeine and 6-monoacetylmorphine (6-MAM) were simultaneously extracted and analyzed by GC/MS in SIM mode. The results obtained show a different distribution of heroin and cocaine metabolites in urine and hair. In urine, we generally find BEG and EME for cocaine abuse, and morphine for heroin abuse. In hair, we detect cocaine and MAM as major metabolites for cocaine and heroin abuse, respectively.

Segmental hair analysis for cocaine and heroin abuse determination.

Segmental hair analysis was performed to obtain information about the history of drug abuse of subjects in a rehabilitation programme. The analytical data from hair samples were correlated, when possible, with urine analysis and to toxicological anamnesis. Toxicological analysis of hair seems to be a valid tool in this specific field.

Simultaneous detection of cocaine and heroin metabolites in urine by solid-phase extraction and gas chromatography-mass spectrometry.

The present paper reports a method for the simultaneous extraction of cocaine, heroin and their metabolites from small amounts of urine (0.5 ml), using deuterated internal standards. Solid-phase extraction (SPE) on C18 columns followed by chromatographic separation coupled with mass spectrometry allowed the detection of all the substances after their derivatization. Mass spectrometry was performed in the electron-impact selected-ion monitoring (EI-SIM) mode. The limit of detection was found to be as low as 50 ng/ml for all the analytes; for reproducibility the C.V. was always better than 7%; the method was found to be linear with correlation coefficients between 0.989 and 1.00.

Drug distribution in the head, axillary and pubic hair of chronic addicts.

This study investigates the distribution of some drugs in hair samples taken from different parts of the body (head, pubis and axillae). Samples drawn from 15 subjects who died from drug overdose were analysed. The concentrations of the drugs detected in the biological fluids did not appear to be correlated with those present in hair. The highest drug levels were observed in pubic hair. The concentration differences observed in the various types of hair can hypothetically be ascribed to a likely incorporation of the drugs from the outside.

Improved enzymatic hydrolysis of hair.

An enzymatic hair extraction method is proposed for drug analysis. Pronase digestion of various aliquots of hair from a cocaine abuser was preceded by a 2-h incubation with a dithiothreitol solution. The extraction solution was tested to identify possible interferences in the radioimmunoassay and was compared with other hydrolysis methods to assess the results of extraction.