Simultaneous detection of amphetamine-like drugs with headspace solid-phase microextraction and gas chromatography-mass spectrometry

A headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) procedure for the simultaneous detection of methylen-dioxyamphetamine (MDA), methylen-dioxymethamphetamine (MDMA), methylen-dioxyethamphetamine (MDE) and N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB) in hair has been developed. This method is suitable for the separation of primary and secondary amines, is reproducible, is not time consuming, requires small quantities of sample and does not require any derivatization. It provides sufficient sensitivity and specificity, with limits of detection (LOD) and limits of quantitation (LOQ) for each substance of <0.7 and 1.90 ng/mg, respectively. Intra- and inter-day precision were within 2 and 10%, respectively. This method is suitable for routine clinical, epidemiological and forensic purposes and can be used for the preliminary screening of many other substances (amphetamine, methamphetamine, ketamine, ephedrine, nicotine, phencyclidine, methadone) in hair and other biological matrices such as saliva, urine and blood. We also describe the first application of this HS-SPME-GC-MS procedure to the analysis of hair and saliva samples from young people attending a disco in the Rome area. All positive hair samples were confirmed by the gas chromatography-mass-mass (GC-MS(2)) technique in positive chemical ionization (PCI) mode. Some examples of the use of the method in detecting different drugs are reported.     

Chromatographic procedures for determination of cannabinoids in biological samples, with special attention to blood and alternative matrices like hair, saliva, sweat and meconium

This paper reviews chromatographic procedures for determination of cannabinoids in biological samples. Special attention was focused on blood and alternative matrices like hair, saliva, sweat and meconium. Papers published from 1998 to the early beginning of 1999 were taken into consideration. Gas chromatographic and liquid chromatographic procedures with different detectors (e.g. mass spectrometer or diode array) were considered. Basic information about the biosample assayed, sample preparation, work-up, gas chromatography column or liquid chromatography column and mobile phase, detection mode, reference and validation data are summarized in tables.     

Simultaneous assay of cocaine, heroin and metabolites in hair, plasma, saliva and urine by gas chromatography—mass spectrometry

As part of an ongoing research program on the development of drug detection methodology, we developed an assay for the simultaneous measurement of cocaine, heroin and metabolites in plasma, saliva, urine and hair by solid-phase extraction (SPE) and gas chromatography—mass spectrometry (GC—MS). The analytes that could be measured by this assay were the following: anhydroecgonine methyl ester; ecgonine methyl ester; ecgonine ethyl ester; cocaine; cocaethylene; benzoylecgonine; cocaethylene; norcocaethylene; benzoylnorecgonine; codeine; morphine; norcodeine; 6-acetylmorphine; normorphine; and heroin. Liquid specimens were diluted, filtered and then extracted by SPE. Additional handling steps were necessary for the analysis of hair samples. An initial wash procedure was utilized to remove surface contaminants. Washed hair samples were extracted with methanol overnight at 40°C. Both wash and extract fractions were collected, evaporated and purified by SPE. All extracts were evaporated, derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and analyzed by GC—MS. The limit of detection (LOD) for cocaine, heroin and metabolites in biological specimens was approximately 1 ng/ml with the exception of norcodeine, normorphine and benzoylnorecgonine (LOD = 5 ng/ml). The LOD for cocaine, heroin and metabolites in hair was approximately 0.1 ng/mg of hair with the exception of norcodeine (LOD = 0.3 ng/mg) and normorphine and benzoylnorecgonine (LOD = 0.5 ng/mg). Coefficients of variation ranged from 3 to 26.5% in the hair assay. This assay has been successfully utilized in research on the disposition of cocaine, heroin and metabolites in hair, plasma, saliva and urine and in treatment studies.     

The disposition into hair of new designer drugs; methylone, MBDB and methcathinone

The disposition into hair of methylone and other new designer drugs, methcathinone and MBDB, was studied with the animal model. Moreover, the incorporation rates of these drugs were compared with those of their related eight compounds previously studied in order to evaluate their incorporation tendency into hair and the usefulness of hair specimens for the retrospective confirmation of the use of these drugs. When the ratio of hair concentration to AUC in plasma ([Hair]/AUC) was represented as an index of the incorporation rate of drugs into hair, the [Hair]/AUC of methylone was 14 times higher than that of methcathinone. It might support earlier findings that the methylenedioxy group on the benzene ring leads to considerably higher incorporation rates. However, [Hair]/AUC of methylone was five-sevenths times lower in comparison with that of MDMA. This suggested that the beta-carbonyl group leads to lower incorporation rates. Although methylone has both groups in its structure, the positive effect of the methylenedioxy group may be stronger than the negative effect of the beta-carbonyl group. On the other hand, the [Hair]/AUC of MBDB, which has methylenedioxyphenyl-2-butanamine structure, was higher than that of MDMA while that of methcathinone, having beta-ketone in its structure, was extremely low. In conclusion, as with MA and MDMA, the incorporation tendency of methylone and MBDB (except for methcathinone) into hair is relatively high, and a hair sample would be a good specimen for the confirmation of retrospective use of these drugs.     

Simultaneous enantioselective determination of amphetamine and congeners in hair specimens by negative chemical ionization gas chromatography-mass spectrometry

Enantioselective quantification of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) enantiomers in hair using gas chromatography-mass spectrometry (GC-MS) is described. Hair specimens were digested with 1M sodium hydroxide at 100 degrees C for 30 min and extracted by a solid phase procedure using Cleanscreen ZSDAU020. Extracted analytes were derivatised with (S)-heptafluorobutyrylprolyl chloride and the resulting diastereoisomers were quantified by GC-MS operating in the negative chemical ionization mode. Extraction yields were between 73.0 and 97.9%. Limits of detection varied in the range of 2.1-45.9 pg/mg hair, whereas the lowest limits of quantification varied between 4.3 and 91.8 pg/mg hair. Intra- and inter-assay precision and respective accuracy were acceptable. The enantiomeric ratios (R versus S) of AM, MA, MDA, MDMA and MDEA were determined in hair from suspected amphetamine abusers. Only MA and AM enantiomers were detectable in this collective and the quantification data showed in most cases higher concentrations of (R)-MA and (R)-AM than those of the corresponding (S)-enantiomers.     

The suitability of saliva for detection of glucose-6-phosphate dehydrogenase deficiency

Saliva was investigated for its suitability as a biopsy tissue for the determination of glucose-6-phosphate dehydrogenase deficiency. It appears that there is a significant difference between the activity of the enzyme in patients and controls. However, some controls have very low values making discrimination between patients and controls using a qualitative method impossible.Glucose-6-phosphate dehydrogenase deficiency is a relevant clinical problem in many rural areas in developing countries. Existing methods for determination of the deficiency in blood and hair follicles do not meet the criteria necessary for their large scale introduction in the areas of the world that are concerned by the problem. The present study shows that saliva is not a suitable alternative.Between the three biopsy tissues compared: blood, hair follicles and saliva, hair follicles remain most attractive since their isolation hardly involves the risk of infection. A simplified method for the detection of glucose-6-phosphate dehydrogenase activity in hair follicles that would allow health service workers in the field to determine the carrier status of pregnant women might form the basis for a future kernicterus prevention programme.     

Simultaneous determination of amphetamine,methamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine in human hair by gas chromatography-mass spectrometry

A procedure is presented for the simultaneous identification and quantification of amphetamine (AP), methamphetamine (MA), methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA) in human hair. The method involves decontamination of hair with dichloromethane and warm water, heat-alkaline hydrolysis in the presence of deuterated internal standards, liquid-liquid extraction and gas chromatography-mass spectrometry after derivatization with pentafluoropropionic anhydride-pentafluoropropanol. The limit of detection for AP, MA and MDA was 0.05 ng/mg using a 50-mg hair sample; for MDMA it was 0.1 ng/mg. Coefficients of variation ranged from 7 to 18%. This assay has been successfully utilized in the evaluation of the deposition of the drugs in hair obtained from various parts of the anatomy of a stimulant abuser.     

Determination of the volume of sweat accumulated in a sweat-patch using sodium and potassium as internal reference

In the present work, we assessed the suitability of sodium and potassium physiologically present in sweat, as internal reference allowing to re-calculate the corresponding volume of sweat collected on a PharmChek Patch. A method using capillary electrophoresis with indirect ultra-violet detection was developed for the determination of sodium and potassium in sweat. The concentrations determined in specimens collected from 12 females and 10 males, using a home-made system composed of polypropylene copolymer bag, were 1039+/-89 mg/L and 711+/-45 mg/L for sodium, and 489+/-293 mg/L and 474+/-196 mg/L for potassium, respectively. In parallel, for seven females and eight males, the comparison of the volume of sweat collected in the same way to the re-calculated volume of sweat accumulated in a patch using sodium as internal standard, gave an average agreement of 98.4+/-15.0%. Results demonstrated the usefulness of sodium as internal standard to determine the volume of sweat accumulated in a patch, and confirm the suitability of PharmChek patch for the collection and determination of cations in sweat.     

The measurement of ecstasy in human hair by triple phase directly suspended droplet microextraction prior to HPLC-DAD analysis

New pre-concentration technique, triple phase suspended droplet microextraction (SD-LPME) and liquid chromatography-photodiode array detection was applied to determine ecstasy, MDMA (3,4-methylendioxy-N-methylamphetamine) in hair samples. In this research MDMA in hair was digested and after treatment extracted. The effective parameters were investigated and method was evaluated. Under the optimal conditions, the MDMA was enriched by factor 98.11. Linearity (r = 0.9921), was obtained in the range of 10–15,000 ng mL^?1 and detection limit was 0.1 ng mL^?1.     

Testing hair for pharmaceuticals

More than hundred pharmaceuticals, drugs of abuse or doping agents have been reported to be detectable in human hair. This article reviews the analysis of 90 drugs and drug metabolites by chromatographic procedures, including the pretreatment steps, the extraction methods, the reported limits of detection and the measured concentrations in real human hair samples. Some progress is observed in the detection of low dose drugs, like fentanyl or flunitrazepam. The general tendency in the last years, to highly sophisticated techniques (GC–MS–NCI, HPLC–MS, GC–MS–MS) illustrates well this constant fight for sensitivity. Some new findings, based on the recent experience of the authors, are also added.     

Development and validation of a disk solid phase extraction and gas chromatography-mass spectrometry method for MDMA, MDA, HMMA, HMA, MDEA, methamphetamine and amphetamine in sweat

We describe the development and validation of a method for the simultaneous quantification of 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3-hydroxy-4-methoxymethamphetamine (HMMA), 3-hydroxy-4-methoxyamphetamine (HMA), 3,4-methylenedioxyethylamphetamine (MDEA), methamphetamine (MAMP) and amphetamine (AMP) in sweat. Drugs were eluted from PharmChek sweat patches with sodium acetate buffer, extracted with disk solid phase extraction and analyzed using GC/MS-EI with selected ion monitoring. Limits of quantification (LOQ) for MDMA, MDEA, MAMP and AMP were 2.5 ng/patch, and 5 ng/patch for MDA, HMA and HMMA. This fully validated procedure was more sensitive than previously published analytical methods and permitted the simultaneous analysis of multiple amphetamine analogs in human sweat.     

Ion chromatographic method for simultaneous determination of nitrate and nitrite in human saliva

A simple, rapid, accurate and sensitive method is proposed for the simultaneous determination of nitrite and nitrate in human saliva. Nitrite and nitrate present in the human saliva were determined after 10- to 100-fold dilution with ion chromatography (IC) using suppressed conductivity detection. Recoveries of nitrite and nitrate were found to be ranged between 95% and 101%. The method was linear (r²=0.9991) over the concentration working range. The detection limits were found to be 15.0 μg/l and 33.5 μg/l, for nitrite and nitrate, respectively. Ions that are present in human saliva and several other ions that are suspected to affect nitrite and nitrate determination were checked. It was found that most of the ions did not cause any interference in the determination. The method allows simultaneous determination of nitrite and nitrate in human saliva.     

Human saliva as a source of biochemical genetic markers. I. Techniques.

The number of genetic markers identified in human saliva is still small compared to known genetic markers of blood. Enzyme activities that can be detected in human saliva by spectrophotometric techniques are listed. The methodologies currently available for the detection of biochemical genetic markers by polyacrylamide-gel electrophoresis are summarized.     

Sensitive, rapid and validated gas chromatography/negative ion chemical ionization-mass spectrometry assay including derivatisation with a novel chiral agent for the enantioselective quantification of amphetamine-type stimulants in hair

A novel chiral derivatisation agent, (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride, was used for the indirect resolution of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) enantiomers using gas chromatography coupled to mass spectrometry operating in the negative-ion chemical ionization mode (GC/MS-NICI). This new chiral derivatisation reagent was readily obtained in optically pure form after a simple two-step synthesis. Optimal derivatisation was accomplished in 15 min at room temperature in a carbonate buffer and the resulting diastereoisomers were base line separated by GC in 12 min only. No racemization was observed during the derivatisation. The method was applied and fully validated for the enantiomeric quantification of amphetamines and methylenedioxylated amphetamines in hair. The analyses of 24 hair specimens from suspected ATS abusers showed that 24 cases were positive for MA and/or AM enantiomers and that in most cases the concentrations of (S)-MA and (S)-AM exceeded those of the corresponding (R)-enantiomers. One hair specimen was tested positive for both enantiomers of MDMA and MDA.     

Determination of MDMA, MDEA and MDA in urine by high performance liquid chromatography with fluorescence detection

This paper describes the development and validation of analytical methodology for the determination of the use of MDMA, MDEA and MDA in urine. After a simple liquid extraction, the analyses were carried out on a high performance liquid chromatography (HPLC) in an octadecyl column, with fluorescence detection. The mobile phase using a sodium dodecyl sulfate ion-pairing reagent allows good separation and efficiency. The method showed good linearity and precision. Recovery was between 85 and 102% and detection limits were 10, 15 and 20 ng/ml for MDA, MDMA and MDEA, respectively. No interfering substances were detected with fluorescence detection.     

A sex determination protocol for the Iberian desman (<Emphasis Type="Italic">Galemys pyrenaicus</Emphasis>) based on a three primer amplification of <Emphasis Type="Italic">DBX</Emphasis> and <Emphasis Type="Italic">DBY</Emphasis> fragments with non-invasive samples

We have sequenced partial fragments of DBX and DBY genes of the endangered Iberian desman (Galemys pyrenaicus). The sequences were used to design a sex determination protocol for non-invasive samples based on a PCR reaction, using only three primers. This protocol allows the simultaneous amplification of two fragments, one corresponding to the DBX gene and the other to the DBY gene, both differing in size. To increase sensitivity on the detection of positive amplifications and on the determination of fragment size we use a fluorescently labelled primer. The protocol has been tested in DNA samples from hair and stool, revealing major difficulties in sexing faecal samples, but unambiguous sexing of hair samples.     

Two optical coherence tomography systems detect topical gold nanoshells in hair follicles,sweat ducts and measure epidermis

Optical coherence tomography (OCT) is an established imaging technology for in vivo skin investigation. Topical application of gold nanoshells (GNS) provides contrast enhancement in OCT by generating a strong hyperreflective signal from hair follicles and sweat glands, which are the natural skin openings. This study explores the utility of 150 nm diameter GNS as contrast agent for OCT imaging. GNS was massaged into skin and examined in four skin areas of 11 healthy volunteers. A commercial OCT system and a prototype with 3 μm resolution (UHR‐OCT) were employed to detect potential benefits of increased resolution and variability in intensity generated by the GNS. In both OCT‐systems GNS enhanced contrast from hair follicles and sweat ducts. Highest average penetration depth of GNS was in armpit 0.64 mm ± SD 0.17, maximum penetration depth was 1.20 mm in hair follicles and 15 to 40 μm in sweat ducts. Pixel intensity generated from GNS in hair follicles was significantly higher in UHR‐OCT images (P = .002) and epidermal thickness significantly lower 0.14 vs 0.16 mm (P = .027). This study suggests that GNSs are interesting candidates for increasing sensitivity in OCT diagnosis of hair and sweat gland disorders and demonstrates that choice of OCT systems influences results.      

Multimode sensors as new tools for molecular recognition of testosterone,dihydrotestosterone and estradiol in children's saliva

Increased levels of testosterone (T₂), dihydrotestosterone (DHT) and estradiol (E₂) in children may be responsible for their early/delayed puberty and obesity conditions. Therefore, multimode sensors based on carbon matrices, such as graphite, graphene, fullerene C₆₀ and multiwall carbon nanotubes modified with maltodextrin, were designed to assess reliably T₂, DHT and E₂ in children saliva. The modes used for the assay of hormones were stochastic mode (for qualitative and quantitative determination of hormones) and differential pulse voltammetry mode (for quantitative determination of hormones). The advantage of this type of sensors, for hormone analysis, is their possibility to reach low concentration levels— are placed for children saliva under the detection limit of standard methods (e.g. ELISA used for the determination of these hormones in saliva). This made the multimode sensors an excellent tool for clinical analysis and especially for determination of substances of clinical importance in saliva samples. The proposed method is fast and simple, and no sampling of saliva is required. Copyright © 2014 John Wiley & Sons, Ltd.     

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT "point samples" obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair''s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.     

Methods used for analyses of “environmentally” damaged nucleic acids

In this review, we present various techniques, currently applied in many laboratories, which are useful in the detection of “environmentally”-induced damage to DNA. These techniques include: (a) chromatographic methods, which allow determination of chemical changes within DNA, be they formation of adducts with or oxidation of bases in DNA; (b) electrophoretic methods, which facilitate finding the site(s) in DNA where that chemical modification occurred; and (c) immunological assays, which help to detect DNA damage using externally produced antibodies that recognize the specific chemical changes in DNA or its fragments, as well as by detection of autoantibodies that develop in response to environmental exposures of animals and humans.