Rapid analysis of amphetamines in blood using head space-solid phase microextraction and selected ion monitoring

A simple and rapid method for analysis of methamphetamine (MA) and amphetamine (AP) in blood was developed using head space-solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry/electron impact ionization-selected ion monitoring (GC-MS/EI-SIM). A vial containing a blood sample, sodium hydroxide, and pentadeuterated methamphetamine as an internal standard, was heated at 80 °C for 20 min. The extraction fiber of the SPME was exposed for 5 min in the head space of the vial. First, heptafluorobutyric anhydride solution was injected into the injection port of the GC-MS to make heptafluorobutyramide (HFB) derivatives of amphetamines, and compounds absorbed on the fiber were detached by exposing the fiber in the injection port. Straight calibration curves of MA and AP were obtained from 0.01 to 2 μg/g in blood, respectively. No interfering substances were found, and the time for analysis was 30 min for one sample.     

Highly sensitive analysis of methamphetamine and amphetamine in human whole blood using headspace solid-phase microextraction and gas chromatography-mass spectrometry

A simple and highly sensitive method for analysis of derivatized methamphetamine (MA) and amphetamine (AM) in whole blood was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry electron impact ionization selected ion monitoring (GC-MS-EI-SIM). A whole blood sample, deuterated-MA (d(5)-MA), as an internal standard (IS), tri-n-propylamine and pentafluorobenzyl bromide were placed in a vial. The vial was heated and stirred at 90 degrees C for 30min. Then the extraction fiber of the SPME was exposed at 90 degrees C for 30min in the headspace of the vial while being stirred. The derivatives adsorbed on the fiber were desorbed by exposing the fiber in the injection port of a GC-MS. The calibration curves showed linearity in the range of 0.5-1000ng/g for both MA and AM. The time for analysis was about 80min per sample. In addition, this proposed method was applied to two autopsy cases where MA ingestion was suspected. In one case, MA and AM concentrations in the mixed left and right heart blood were 165 and 36.9ng/g, respectively. In the other case, MA and AM concentrations were 1.79 and 0.119 microg/g in the left heart blood, and 1.27 and 0.074 microg/g in the right heart blood, respectively.     

Simple and simultaneous analysis of fenfluramine, amphetamine and methamphetamine in whole blood by gas chromatography-mass spectrometry after headspace-solid phase microextraction and derivatization

A simple and sensitive method for the simultaneous analysis of fenfluramine, amphetamine and methamphetamine in whole blood was developed using a headspace-solid phase microextraction (SPME) and derivatization. A 0.5 g whole blood sample, 5 microl d(5)-methamphetamine (50 micrig/ml) as an internal standard, and 0.5 ml sodium hydroxide (1 M) were placed into a 12 ml vial, and sealed rapidly with a silicone septum and an aluminum cap. Immediately after the vial was heated to 70 degrees C in an aluminium block heater, the needle of the SPME device was inserted through the septum of the vial, and the extraction fiber was exposed in the headspace for 15 min. First, heptafluorobutyric anhydride was injected into the injection port of the GC-MS, and the compounds extracted by the fiber were then desorbed and derivatized simultaneously by exposing the fiber in the injection port. The calibration curves, using an internal standard method, demonstrated good linearity throughout the concentration range from 0.01 to 1.0 microg/g. The detection limits of this method were 5.0 ng/g for fenfluramine and methamphetamine, and 10 ng/g for amphetamine. No interferences were found, and the time for analysis was about 30 min for one sample. This method was applied to a suicide case in which the victim ingested fenfluramine. Fenfluramine was detected in the blood sample collected from the victim at the concentration of 7.7 microg/g.     

Simple analysis of arylamide herbicides in serum using headspace-solid phase microextraction and GC/MS

A simple and sensitive method for analysis of four arylamide herbicides (butachlor, propanil, diphenamide and propyzamide) in serum was developed using a headspace–solid phase microextraction (SPME) and a gas chromatograph–mass spectrometer (GC–MS). A vial containing a serum sample and sodium chloride was heated at 90°C. The extraction fiber of the SPME was exposed for 45 min in the headspace of the vial. The compounds adsorbed on the fiber were desorbed by exposing the fiber in the injection port of the GC–MS. The calibration curves, using an internal standard method, demonstrated good linearity throughout the concentration range from 0.25 to 10.0 μg/ml. Propyzamide was used for an internal standard. The limit of detection was 0.10, 0.05, and 0.25 μg/ml for butachlor, diphenamide, and propanil, respectively. No interferences were found, and the time for analysis was 60 min for one sample. In addition, this proposed method was applied to a suicide case in which the patient ingested Kusanon A^®, a herbicide. Propanil, which was the main ingredient in the herbicide, was detected in the eight serum samples collected from the patient during the hospitalization at the concentration range from 26.7 to 1.1 μg/ml.     

Aqueous phase hexylchloroformate derivatization and solid phase microextraction: determination of benzoylecgonine in urine by gas chromatography-quadrupole ion trap mass spectrometry.

A derivatization/solid phase microextraction (SPME) method for the determination of benzoylecgonine in urine was developed. The derivatization is conducted directly in 1 mL of urine while sonicating for 3 min with 12 microL of hexyl chloroformate and 70 microL of a mixture containing acetonitrile:water:hexanol:2-dimethylaminopyridine (5:2:2:1 v/v), yielding benzoylecgonine hexyl ester (BHE) as the product. After the 3 min period, an aliquot of 250 microL is transferred to a vial for SPME. After the desired extraction time the 100 microns polydimethylsiloxane SPME fiber was transferred to the GC-MS for separation and analysis with a quadrupole ion trap mass spectrometer. The hexyl chloroformate derivatization and SPME procedures were optimized for compatibility and sensitivity. The method was found linear for 0.10 to 20.0 micrograms/mL (r2 = 0.999) of benzoylecgonine in urine using benzoylecgonine-d3 as an internal standard (1.5 micrograms/mL). Intra-day precisions were 8.8 and 6.8% RSD for 0.30 microgram/mL and 17 micrograms/mL benzoylecgonine standards in urine (n = 6), respectively. Inter-day precision (n = 3) were < or = 3.3% RSD, indicating good reproducibility. A detection limit of 0.03 microgram/mL (S/N = 3) was achieved, thus making the SPME method a simplified alternative to SPE for GC-MS confirmation after EMIT tests for benzoylecgonine which have a cutoff of 0.30 microgram/mL. Quantitative results by SPME and SPE of two clinical urine specimens known positive for cocaine by EMIT were in excellent agreement. Benzoylecgonine was detected by the derivatization/SPME method in 22 out of 22 other urine specimens known positive for cocaine.     

SPME-GC\NPD法快速分析尿液中苯丙胺类化合物

建立了ＳＰＭＥ－ＧＣ／ＮＰＤ法同时分析尿液中九种苯丙胺类化合物的方法。取１ｍｌ尿样以４－苯基丁胺为内标,在碱性条件下,将１００μｍＰＤＭＳ纤维头浸入尿液中２０ｍｉｎ,气相色谱进样后热解吸３ｍｉｎ,用ＧＣ／ＮＰＤ进行测定。九种苯丙胺类化合物及内标完全分离,五种苯丙胺类化合物分别在０．０５～１５、０．１～１５、０．２～１５μｇ／ｍｌ浓度范围内线性良好,ｒ为０．９９２８～０．９９９５,变异系数均小于１０％。方法简便、快速、准确,可用于尿液中苯丙胺类违禁药物的检测     

Identification of impurities and the statistical classification of methamphetamine using headspace solid phase microextraction and gas chromatography-mass spectrometry

The profiling of impurities in methamphetamine (MA) using headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) is described. The extraction of the impurities with an SPME fiber was examined under varying conditions. Optimal chromatograms were obtained when a 50 mg MA sample at 85 degrees C for 30 min was extracted using a fiber coated with divinylbenzene/carboxen/polydimethylsiloxane. MA samples from nine different origins were analyzed under optimized extraction conditions. Compounds related to MA such as benzaldehyde, benzyl alcohol, amphetamine, benzyl methyl ketone, cis- and trans-1,2-dimethyl-3-phenylaziridine, dimethylamphetamine, N-acetylamphetamine, N-acetylmethamphetamine and N-formylmethamphetamine were detected in the chromatograms. Trace amounts of ethanol, diethyl ether and acetic acid were also detected in some of the chromatograms. The numbers and intensities of the peaks detected were different, depending on the sample. After the areas of the eight principal peaks were converted to their square root and logarithm, similarities among the samples were evaluated by Euclidian distance, cosine distance and correlation coefficient. The results showed that a combination of logarithmic conversion and cosine distance was the most suitable for discriminating and classifying the samples. HS-SPME/GC-MS is a simple and effective method for the extraction and identification of impurities. The present method, in combination with an appropriate statistical analysis, would be useful for developing a profile of impurities in MA.     

Automated determination of carboxyhemoglobin contents in autopsy materials using head-space gas chromatography/mass spectrometry

To establish a method for the routine analysis of carboxyhemoglobin (COHb) in autopsy materials including those which have undergone postmortem changes, e.g. thermo-coagulation, putrifaction and contamination, an automated head-space gas chromatography/mass spectrometry (GC/MS) analysis was utilized. The procedure consisted of preparation of the sample in a vial and a carbon monoxide (CO) saturated sample, for estimation of hemoglobin content, in another vial, the addition of n-octanol, potassium ferricyanide and an internal standard (t-butanol), GC separation and determination of CO using a GC/MS system equipped with an automated head-space gas sampler. The method was practical not only with the blood and bone marrow aspirates to confirm the findings on the CO-oximeter system, but also with the thermo-coagulated and putrified blood.     

Time since discharge of shotgun shells

A technique for the estimation of time since discharge of a given class of spent shotgun shells is presented. The technique involved the use of SPME (Solid Phase Microextraction) sampling from the atmosphere inside spent shotgun shells. Compounds absorbed on the SPME fiber were desorbed and analyzed with GC-MS. The decrease in concentration of the combustion product naphthalene was monitored in all shells over a two-month period. Three conditions were examined to prevent naphthalene from escaping prior to testing the shells. A glass vial was successfully incorporated to halt the dissipation of naphthalene. At room temperature the naphthalene peak can be measured for several months and the curve-fitting data can lead to an estimation of time since discharge.     

Fatal Intoxication with Methoxetamine

Methoxetamine (MXE) is a new synthetic drug of abuse structurally related to ketamine and phencyclidine. A case of a 29-year-old male with acute toxicity related to the analytically confirmed use of MXE is reported. The man was found dead at his residence. Biological material was analyzed using liquid chromatography–tandem mass spectrometry. The concentration of MXE in urine of the deceased was 85 μg/mL. Despite the vial containing the blood sample being destroyed during transportation and the blood leaking out into the cardboard packaging, the blood level of MXE was estimated. After determination of the cardboard grammage (approx. 400 g/m³) and the mean mass of the blood obtained after drying (0.1785 ± 0.0173 g per 1 mL), the estimated blood concentration of MXE was found to be 5.8 μg/mL. The high concentration of MXE in blood and urine and the circumstances of the case indicate an unintentional, fatal intoxication with this substance.     

Automated headspace solid-phase dynamic extraction for the determination of cannabinoids in hair samples

This article describes a fully automated procedure for detecting cannabinoids in human hair samples. The procedure uses alkaline hydrolysis and headspace solid-phase dynamic extraction (HS-SPDE), followed by on-coating derivatization and gas chromatography-mass spectrometry (GC-MS). SPDE is a further development of solid-phase microextraction (SPME), based on an inside needle capillary absorption trap. It uses a hollow needle with an internal coating of polydimethylsiloxane as extraction and pre-concentration medium.Ten mg of hair were washed with deionised water, petroleum ether and dichloromethane. After adding deuterated internal standards, the sample was hydrolyzed with sodium hydroxide and directly submitted to HS-SPDE. After absorption of analytes for an on-coating derivatization procedure, the SPDE-needle was directly placed into the headspace of a second vial containing N-methyl-N-trimethylsilyl-trifluoroacetamide before GC-MS analysis. The limit of detection was 0.14 ng/mg for Delta(9)-tetrahydrocannabinol, 0.09 ng/mg for cannabidiol, and 0.12ng/mg for cannabinol. Absolute recoveries were in the range of 0.6 to 8.4%. Linearity was verified over a range from 0.2 to 20 ng/mg, with coefficients of correlation between 0.998 and 0.999. Intra- and inter-day precision were determined at two different concentrations and resulted in ranges between 2.3 and 6.0% (intra-day) and 3.3 and 7.6% (inter-day). Compared with conventional methods of hair analysis, this automated HS-SPDE-GC-MS procedure is substantially faster. It is easy to perform without using solvents and with minimal sample quantities, and it yields the same sensitivity and reproducibility. Compared to SPME, we found a higher extraction rate, coupled with a faster automated operation and greater stability of the device.     

A fatal case of oleandrin poisoning

The study presents a case of fatal poisoning with oleander leaves in an adult diabetic male. After repeated vomiting, and gastrointestinal distress the patient was admitted at the hospital with cardiac symptoms 1h after the ingestion. Urine samples were assayed immunochemically and by GC-MS for drugs of abuse and for general toxicological screen. Blood was analyzed for alcohol and volatiles by static head space GC-MS. Blood and oleander leaves were analyzed by LC-MS/MS for oleandrin and related compounds, the main cardiac glycosides of Nerium oleander. Oleandrin was detected by LC-MS/MS in the blood sample at a concentration of approximately 10 ng/ml. Another cardiac glycoside with pseudo-molecular ion of m/z 577, a likely structural isomer of oleandrin, was also detected in the blood and oleander leaves. However, by using the response as a function of concentration for oleandrin, this cardiac glycoside was roughly estimated at a concentration of approximately 10 ng/ml in the deceased blood. This would give a total fatal blood concentration of cardiac glycosides of about approximately 20 ng/ml in the deceased blood.     

Pesticide levels in head hair samples of Cretan population as an indicator of present and past exposure

In the present work we assessed chronic exposure of different working population groups of Messara and Sitia districts, Crete, Greece, to common currently used pesticides (diazinon, fenthion, methyl parathion and malathion) and two banned pesticides hexachlorocyclohexane (HCH) and DDT. The study population (211 persons, 110 females and 101 males) was divided to three groups; people working in greenhouses, animal breeders and people working in open cultivations. Methanolic extraction of pulverized hair was used for organophosphate pesticides extraction, followed by liquid-liquid extraction with water-ethyl acetate as a clean up step. The extraction of organochlorine pollutants was performed by acidic hydrolysis of the hair matrix followed by liquid-liquid and solid phase extraction. The levels of the aforementioned pesticides were measured by GC-ECD and gas chromatography-mass spectrometry (GC-MS). The median concentrations of a-HCH, HCB, lindane, opDDE, ppDDE, opDDD, ppDDD + opDDT and ppDDT were determined at 7.2, 2.2, 70.2, 2.7, 5.7, 3.1, 2.6 and 23.2 pg/mg. The median concentration of total HCHs and DDTs detected in the three working groups were: 95.0 and 8.9 pg/mg for the greenhouse workers, 38.2 and 3.3 pg/mg for the animal breeders and 24.1 and 5.2 pg/mg for the open cultivation group. Ten head hair samples were positive for diazinon at a mean concentration of 2.8 pg/mg. Fenthion, methyl parathion and malathion were not detected. Our results demonstrated the ability to assess chronic human past pesticides exposure, offering valuable information to epidemiological clinical studies.     

Detection of thiopental and pentobarbital in head and pubic hair in a case of drug-facilitated sexual assault

The quali-quantitative determination of two barbiturates, thiopental and its metabolite pentobarbital, in head and pubic hair samples of a woman who had been sexually assaulted during hospitalisation, is reported. Hair was analysed by means of solid-phase microextraction (SPME) and gas chromatography-multiple mass spectrometry (GC-MS-MS), in chemical ionisation conditions. Thiopental and pentobarbital were found in three proximal head hair segments (sample 1A: 0.30 and 0.40 ng/mg; sample 1B: 0.20 and 0.20 ng/mg; sample 3: 0.15 and 0.20 ng/mg) and pubic hair sample. Two distal head hair segments were negative for both barbiturates. Despite the lack of collection and toxicological analysis of blood or urine samples within the hospital setting, analytical findings from hair revealed the use of the anaesthetic agent thiopental to sedate the victim quickly and deeply and commit sexual assault.     

Detection of organophosphate poisoning in a putrefying body by analyzing arthropod larvae

Deaths as a result of organophosphate poisoning are usually detected by analysis of body fluids and tissues for the presence of the toxic agent. These procedures present particular difficulties when performed on remains in an advanced stage of decomposition. Malathion poisoning was suspected in a case in which the remains were in an advanced stage of decomposition and the presence of malathion was confirmed by analyses of gastric content and body fat. Two species of fly larvae, Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart), were present on the remains. A sample of these larvae was analyzed for malathion content. A total of 574 micrograms of malathion was detected in 0.26 g of pooled larvae, or a level corresponding to 2050 micrograms/g of larvae. Presence of organophosphates in arthropod larvae has not been documented previously and the analysis of larvae from decomposing remains may prove a useful technique for detection of these toxicants in decomposing remains.     

GC-MS法测定血中1-甲基海因含量及其法医学应用

目的建立全血中1-甲基海因的气相色谱-质谱(GC-MS)分析方法,为1-甲基海因相关法医学鉴定案件提供技术支持。方法取0.5 m L血样,加入500 ng内标盐酸双苯戊二氨酯(SKF_(525A))、0.01 mol/L稀盐酸溶液2 m L,加入碳酸铵0.5 g调节p H值为9,加入乙酸乙酯2 m L,然后离心,取有机溶剂层,吹干后进行GC-MS分析。结果血液中1-甲基海因在0.5~50 ng/m L范围内线性关系良好,线性回归方程为y=0.015 51 x+0.007 26(R~2=0.999 7),最低检出限为0.1 ng/m L,回收率为93.02%~108.12%,日内、日间精密度分别小于6.07%和13.37%。结论本方法测定所得结果准确,重现性好,可用于血样中1-甲基海因含量的测定。     

Α Simple Method for the Determination of Lacosamide in Blood by GC-MS

Lacosamide is a functionalized amino acid with antiepileptic function. Therapeutic drug monitoring (TDM) in patients for lacosamide is critical as it allows clinicians to control epileptic seizures. A single liquid–liquid extraction step was applied for the extraction of lacosamide from whole blood samples which were thereafter analyzed by GC-MS. Optimum extraction conditions were selected on the basis of experiments with various solvents at different pHs, indicating ethyl acetate at pH 12 as the most efficient parameters for the extraction of lacosamide. Method exhibited linearity from 2 to 100 μg/mL with R² = 0.998. Accuracy and precision were evaluated at three concentrations and found to be within acceptable limits. LOD and LOQ were determined at 0.1 and 0.5 μg/mL, respectively. Lacosamide was found to be stable at storage conditions. The developed method was applied successfully in clinical samples and postmortem blood sample from an overdose case.     

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.     

Enzyme-linked immunosorbent assay and latex agglutination inhibition reaction test for cocaine and benzoylecgonine in urine

An enzyme-linked immunosorbent assay (ELISA) and a latex agglutination inhibition reaction test (LAIRT) for cocaine and benzoylecgonine have been established. In ELISA with polystyrene microtiter wells coated with anti-benzoylecgonine antibody and alkaline phosphatase (ALP)-labeled benzoylecgonine, the activity of antibody-bound ALP was measured with the enzyme cycling method. The range of benzoylecgonine measurable by ELISA was 12 pg-25 ng/well; the analysis time for 96 wells was 90 min. In LAIRT, the agglutination reaction with anti-benzoylecgonine antibody-coated latex and benzoylecgonine-rabbit serum albumin (RSA) conjugate-coated latex was inhibited by 0.1 μg benzoylecgonine/ml urine; the analysis time for six samples on one glass slide was 20 min. The urine samples of 47 abusers were analyzed by ELISA and LAIRT. From the comparison with results of the enzyme-multiplied immunoassay technique (EMIT^®) and gas chromatography-mass spectrometry (GC-MS), it was clarified that both ELISA and LAIRT were suitable for the screening method of urine samples.