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.     

Gas chromatographic-mass spectrometric analysis of buflomedil hydrochloride in biological samples after acute intoxication.

Two cases of acute intoxication of buflomedil hydrochloride are reported. The analysis of this compound was performed by an Extrelut extraction followed by a gas chromatographic-mass spectrometric determination. Analytical parameters of linearity, reproducibility and specificity were satisfactory.     

A measurement of human hair oxidation by Fourier transform infrared spectroscopy

Human scalp hair samples were oxidized to determine the sulfonic acid absorption peak. This peak was monitored at 1044 cm-1 by Fourier transform infrared spectroscopy (FTIR) in hair samples from 135 whites and found to provide a degree of discrimination in treated and untreated hairs. The effects of moisture, laboratory storage, natural hair color, and variation over time were also studied.     

Gas chromatographic and gas chromatographic-mass spectrometric determination of gasoline in a case of gasoline vapor and alcohol poisoning

A case of fatal poisoning due to the combined effect of alcohol and gasoline following an automobile accident is described. Toxicological analyses by means of gas chromatography and gas chromatography-mass spectrometry permitted the identification and quantitation of alcohol and several hydrocarbons in the heart blood and in the gas in the lung. Great variation was found in the estimates of blood gasoline concentration, depending on which of six constituents of gasoline was chosen for quantitation. The cause of this variation is discussed, together with the possible mechanisms leading to death.     

Determination of cocaine in human hair by gas chromatography/mass spectrometry

A qualitative method for the determination of cocaine alone without its metabolites in human hair by gas chromatography/mass spectrometry (GC/MS) was developed. The assay used helium as carrier gas, a 30-m bonded phase fused silica OV-1 capillary column, and solid injection at 290 degrees C evaporator temperature. The cocaine concentrations in hair were determined also by radioimmunoassay (RIA). The values obtained are the sum of cocaine and its metabolites. Both GC/MS and RIA meet the requirements for the determination of drug abuse by two different methods in forensic science.     

Detection of methadone in human hair by gas chromatography/mass spectrometry

Determination of methadone in human hair by gas chromatography/mass spectrometry was described. Helium as carrier gas, a 30-m bonded phase-fused silica DB-1 capillary column and splitless injection at 230 degree C temperature were used. The concentrations of methadone and its metabolites were measured in addition by radioimmunoassay (RIA). Both methods GC/MS and RIA showed the presence of methadone in human hair.     

Identification of ten corticosteroids in human hair by liquid chromatography-ionspray mass spectrometry

This paper describes a screening procedure based upon high-performance liquid chromatography-ionspray mass spectrometry for the identification of ten corticosteroids in human hair: triamcinolone, prednisolone, prednisone, methylprednisolone, cortisone, cortisol, beta- and dexamethasone, flumethasone and beclomethasone. Hair strands were washed in methylene chloride, pulverized in a ball mill and 50 mg of the powdered hair were incubated in 1 ml Soerensen buffer, pH 7.6 for 16 h at 40 degrees C, in presence of 50 ng cortisol-d3 used as internal standard. Purification of the incubation medium was achieved on SPE C18 Isolute extraction columns. The eluates were evaporated to dryness and resuspended in 30 microliters MeOH before analysis by HPLC-IS-MS in positive and negative modes of detection. The validation parameters were found satisfactory for a corticosteroid screening procedure. The correlation coefficient of the calibration curve ranged from 0.939 to 0.997, showing linearity between 0.1 and 10 ng/mg, excepted for beclomethasone which was between 0.2 and 10 ng/mg. Extraction recovery at 4 ng/mg ranged from 43.2 to 85.7%. Repeatability (CV values) at 4 ng/mg ranged from 6.1 to 17.5%. The limits of detection ranged from 0.03 to 0.17 ng/mg for a signal-to-noise ratio of 2. The detection of prednisone and beclomethasone in three hair specimens obtained from forensic and clinical cases have documented corticosteroids incorporation into human hair.     

Validation of an ion-trap gas chromatographic-mass spectrometric method for the determination of cocaine and metabolites and cocaethylene in post mortem whole blood

The coingestion of cocaine (COC) and ethanol is a very frequent occurrence and is known to increase the risk of morbidity and mortality. The formation occurs of a transesterification product, the cocaethylene (CE), which is even more toxic than cocaine. In order to study the role of ethanol as an agent of interaction in lethal cocaine intoxication, and to establish its influence in post mortem cocaine concentrations, an ion-trap gas chromatographic-mass spectrometric method (GC-MS) was validated to quantify simultaneously the agent and its biotransformation products, benzoylecgonine (BE), ecgoninemethylester (EME) and the 'biomarker' of the interaction, the CE present in whole blood. Deuterated internal standards were added to 2 ml of post mortem whole blood and extracted in Bond Elut Certify columns. The residues were evaporated and derivatized with N-methyl-N-t-butyldimethylsilyltrifluoroacetamide (MTBSTFA). Detection was performed by electron impact ionization. The monitored ions were m/z 82/85 for EME-tert-butyldimethylsilyl (TBDMS)/EME-d3-TBDMS; m/z 182/185 for COC/COC-d3; m/z 196/199 for CE/CE-d3 and m/z 282/285 for BE-TBDMS/BE-d3-TBDMS. The limits of detection and quantification were found to be 25 ng and 50 ng ml(-1), respectively, for COC and CE, and 50 and 100 ng ml(-1) for BE and EME. Accuracy was different for each of the compounds, varying from 65 to 98%. The dynamic range of the assay was 50-2000 ng ml(-1).     

Solid-phase microextraction for the determination of cocaine and cocaethylene in human hair by gas chromatography-mass spectrometry

A method for the simultaneous determination of cocaine (COC) and cocaethylene (CE) in human hair was developed, using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) as analytical technique to identify and quantify the drugs. Selected ion monitoring (SIM) mode was used to obtain higher sensitivity. The deuterated-labeled analogues were used as internal standards. The detector response was linear for the drugs studied over the range 0.4-15 ng/mg, with correlation coefficients higher than 0.995. The coefficients of variation oscillated between 0.65% and 14.18% and the accuracy was in the range from 0.73% to 11.20%. The limits of quantitation and detection were found to be acceptable. Finally, this method was applied to 15 hair samples from cocaine users, obtaining positive results in all cases. The mean concentrations were 5.39 ng/mg (range: 0.43-8.98 ng/mg) for cocaine and 1.11 ng/mg (range: 0.42-2.23 ng/mg) for cocaethylene.     

Enzyme typing of human hair roots.

Sufficient phosphoglucomutase activity was found to be present in plucked hair noses bearing either fragmentary or complete outer root sheaths to enable typing of individual roots by starch-gel electrophoresis. Hair roots collected by brushing were found to contain very little PGM activity. Other isoenzyme systems were detected in hair roots but in insufficient quantities to make typing feasible.     

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.     

Determination of methadone in human hair by radioimmunoassay

The concentrations of methadone in human hair were measured. The washed hair was cut in 1-mm pieces approximately, then incubated overnight at 45 degrees C with 0.1 m HCl. The extracts were alcalized by 1 m NaOH and diluted by phosphate buffer, pH 7.4. The methadone concentrations were determined by radioimmunoassay. The method is simple, rapid, and practicable for routine determination.     

Drugs in prehistory: chemical analysis of ancient human hair

Concern about drug abuse in modern populations has led to the development of specific methods for identification of cocaine, opiates and cannabis in human hair. Drug use in prehistory can provide indirect evidence of interpopulational contact and social stratification. This paper reports drug evaluation in nineteen ancient hair samples from archaeological sites in northern Chile. Each sample was tested for the presence of traces of cocaine, opiates and cannabis, in order to establish a standard methodology for studies of drug use among prehistoric groups. Although results are negative, this absence of evidence could be due to two main causes: (1) the individuals evaluated did not use any drugs, which does not mean that other members of their cultural group did, or (2) the wide range of known drugs studied did not consider some group specific drugs, derived from local or imported plants, thus meaning that a greater drug range must be tested. In any case, our study confirms that drug testing in prehistoric samples is viable. However, in order to determine what kind of substances were used in prehistoric times new patterns that incorporate all drugs which are not part of the western pharmacopeia must be created. Finally, a methodology for the study of drug use among prehistoric groups using ancient hair samples is described.     

Quantitative determination of amphetamines, cocaine, and opiates in human hair by gas chromatography/mass spectrometry

Hair of young subjects (N = 36) suspected for drug abuse was analysed for morphine, codeine, heroin, 6-acetylmorphine, cocaine, methadone, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA). The analysis of morphine, codeine, heroin, 6-acetylmorphine, cocaine, and methadone in hair included incubation in methanol, solid-phase extraction, derivatisation by the mixture of propionic acid anhydride and pyridine, and gas chromatography/mass spectrometry (GC/MS). For amphetamine, methamphetamine, MDA, MDMA, and MDEA analysis, hair samples were incubated in 1M sodium hydroxide, extracted with ethyl acetate, derivatised with heptafluorobutyric acid anhydride (HFBA), and assayed by GC/MS. The methods were reproducible (R.S.D. = 5.0-16.1%), accurate (85.1-100.6%), and sensitive (LoD = 0.05-0.30ng/mg). The applied methods confirmed consumption of heroin in 18 subjects based on positive 6-acetylmorphine. Among these 18 heroin consumers, methadone was found in four, MDMA in two, and cocaine in two subjects. Cocaine only was present in two, methadone only in two, methamphetamine only in two, and MDMA only in seven of the 36 subjects. In two out of nine coloured and bleached hair samples, no drug was found. Despite the small number of subjects, this study has been able to indicate the trend in drug abuse among young people in Croatia.     

Detection of methamphetamine and amphetamine in a single human hair by gas chromatography/chemical ionization mass spectrometry

A detailed procedure of an extremely sensitive method for quantitation of methamphetamine and amphetamine in human hair by gas chromatography (GC)/chemical ionization (CI) mass spectrometry (MS) is presented. N-methylbenzylamine was used as an internal standard. The samples, after extraction with an organic solvent, were derivatized with trifluoroacetic anhydride before the GC/MS analysis. Quantitation was made with quasi-molecular ions of the derivatives by selected ion monitoring in the CI mode. The detection limit was about 10 pg in an injected volume. The high sensitivity enabled us to measure both stimulants in a single human hair in actual cases.     

Determination of chronic methamphetamine abuse by hair analysis

A 30-year-old male truck driver, known to be asthmatic, was found dead at the roadside lying near his car. A nebulizer bottle of Berotec (fenoterol hydrobromide) was found near his hand. The anatomic cause of death was suspected to be asthma. Toxicological screening of urine using Triage demonstrated the presence of methamphetamine. The blood concentration of methamphetamine was 0.4 microg/ml, and fenoterol was not detected. Hair analysis clearly indicated chronic methamphetamine abuse and medium dependency during the 2 months before death. We conclude that death might have been induced by the interaction of fenoterol and methamphetamine.     

Detection of drugs in human hair using Abbott ADx, with confirmation by gas chromatography/mass spectrometry (GC/MS).

The authors suggest use of the fluorescence polarization immunoassay (FPIA) technique in evaluation of chronic drug abuse using human hair. Hair was decontaminated in 5 mL of ethanol for 15 min at 37 degrees C and then incubated in 3 mL of 1M sodium hydroxide (NaOH) for 1 h at 100 degrees C. Afterwards, the aliquots were neutralized and analyzed using Abbott ADx for a negative or positive response for the following drugs: benzodiazepines, barbiturates, antidepressants, opiates, cocaine, amphetamine, and cannabis. All the positive samples were confirmed by gas chromatography/mass spectrometry (GC/MS). Only one false positive was detected (caused by interference of a phenothiazine with the antidepressants kit), clearly demonstrating the capability of ADx for toxicological screening of human hair.     

Removing and identifying drug contamination in the analysis of human hair

The procedure used in this laboratory for removing and identifying contamination of hair specimens with drugs is demonstrated by its application to hair contaminated by various experimental models. The models include soaking; coating with drug followed by sweat conditions for 6 h; and soaking in a very high concentration of cocaine followed by storage and multiple shampoo treatments. A multi-part wash procedure along with a wash criterion is applied to all samples containing drug above the cutoff. The failure of the wash criterion is a signal that the sample may be positive due to contamination rather than use, and in the absence of other over-riding evidence, the sample would be considered to be negative for drug use. This Wash Criterion has also been tested with hair from subjects demonstrated to be drug users by one or more drug-positive urines; in these studies, all hair samples from demonstrated users passed the Wash Criterion test.