Determination of bromadiolone in whole blood by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A rapid, sensitive and selective high-performance liquid chromatography tandem mass spectrometric method (HPLC/MS-MS) has been developed and validated for the determination of bromadiolone in whole blood using warfarin as an internal standard (IS). Bromadiolone was extracted from the whole blood samples by liquid-liquid extraction with ethyl acetate. Multiple-reaction monitoring (MRM) was used to detect bromadiolone and IS, using precursor --> product ion combinations at m/z 527 --> 465 and 307 --> 161, respectively. The calibration curve was linear (r2=0.998) in the concentration range of 0.5-100.0 ng/mL with a lower limit of quantification of 0.5 ng/mL in whole blood. Intra- and inter-day relative standard deviations (R.S.D.s) were less than 7.5 and 11.9%, respectively. Recoveries of bromadiolone ranged from 82.1 to 85.2%. This method is found to be determined trace bromadiolone in whole blood and can be used in the diagnosis of the poisoned human beings.     

稻田水中莠去津及其代谢物的硅烷基衍生化-气质联用分析法

目的建立稻田水中莠去津及其代谢物去乙基莠去津（DEA）、去异丙基莠去津（DIA）和双去烃基莠去津（DDA）的灵敏的气质联用分析方法。方法稻田水样品用GDX501大孔树脂进行固相萃取,萃取物用N,O-双三甲硅烷基乙酰胺（BSA）进行三甲硅烷基衍生化,衍生物用选择离子监测的气质联用法分析。结果稻田水中4种分析物的检出限和测量限都不高于2ng/mL。结论该方法可用于刑事案件中莠去津毁坏稻田案件的检验。     

血中安定及其代谢物的酶水解研究

目的考察木瓜蛋白酶水解血中安定及其代谢物蛋白结合物的酶解条件,提高安定的提取率。方法采用正交试验确定酶解的最优条件,检材经蛋白酶水解,固相萃取后,应用LC-MS／MS方法进行检测,运用保留时间和MRM（多离子反应监测）方式来对血中安定及其代谢物进行定性定量分析。结果安定、去甲安定、去甲异安定、羟基安定和去甲羟基安定的最佳酶解条件分别为55℃,2．5h,pH7．0,8000U;50℃,1h,pH7．5,8000U;50℃,1h,pH7．5,8000U;50℃,1．5h,pH7．5,8000U;50℃,1．5h,pH7．5,8000U。结论酶水解后的血液中安定及其代谢物检出量明显增加。     

Rapid determination of flunitrazepam in alcoholic beverages by desorption electrospray ionization-mass spectrometry

Desorption electrospray ionization-mass spectrometry (DESI-MS), a novel ambient ionization technique, was used in this study for determining flunitrazepam in various alcoholic beverages. Using this technique, no pretreatment of the samples was necessary and identification of the drug was accomplished in individual samples in minutes. In addition, the acquired mass spectra provide the information of the identity of the drink based on the detected characteristic ions from the matrices. This study also demonstrates the capability of DESI-MS to perform quantitative analysis of simulated evidence samples with a limit of quantification of 3μg/mL. Furthermore it has been shown that this method can be used for high-throughput analysis whereby six samples were analyzed in a row within 6 minutes and no observable sample carry-over was noted. DESI-MS shows potential as a rapid, sensitive, and selective technique for forensic analysis of spiked beverages which are typical evidence of drug facilitated sexual assault and robbery cases.     

A method for screening for various sedative-hypnotics in serum by liquid chromatography/single quadrupole mass spectrometry

A screening method for the detection of sedative-hypnotics in serum is described. The target drugs, which include practically all the sedative-hypnotics distributed in Japan, consisted of 5 barbiturates, 30 benzodiazepine-related drugs and 11 other sedative-hypnotics (i.e., apronalide, bromisovalum, chloral hydrate, triclofos, chlorpromazine, promethazine, diphenhydramine, hydroxyzine, zopiclone, zolpidem and tandospirone). Thirty-nine analytes, selected in terms of the pharmacokinetics of the target drugs, in human serum were screened using a combination of mixed-mode solid-phase extraction and liquid chromatography/electrospray-ionization single-quadrupole mass spectrometry. The detection limits (non-basic analytes, 1-50 ng/ml; basic analytes, 0.1-5 ng/ml) were sufficient to permit the screening of a single therapeutic administration of a target drug.     

Identification of inorganic anions by gas chromatography/mass spectrometry

Inorganic anions were identified by using gas chromatography/mass spectrometry (GC/MS). Derivatization of the anions was achieved with pentafluorobenzyl p-toluenesulphonate (PFB-Tos) as the reaction reagent and a crown ether as a phase transfer catalyst. When PFB-Br was used as the reaction reagent, the retention time of it was close to those of the derivatized inorganic anions and interfered with the analysis. In contrast, the retention time of PFB-Tos differed greatly from the PFB derivatives of the inorganic anions and the compounds of interest could be detected without interference. Although the PFB derivatives of SO4, S2O3, CO3, ClO4, and ClO3 could not be detected, the derivatives of F, Cl, Br, I, CN, OCN, SCN, N3, NO3, and NO2 were detected using PFB-Tos as the derivatizing reagent. The inorganic anions were detectable within 30 ng approximately, which is of sufficient sensitivity for use in forensic chemistry. Accurate mass number was measured for each PFB derivative by high-resolution mass spectrometry (HRMS) within a measurement error of 2 millimass units (mmu), which allowed determination of the compositional formula from the mass number. In addition, actual analysis was performed successively by our method using trial samples of matrix.     

Fatal Datura poisoning: identification of atropine and scopolamine by high performance liquid chromatography/photodiode array/mass spectrometry

A forensic method comprising solid phase extraction and HPLC analysis was developed for the detection and confirmation of atropine and scopolamine, the main toxic alkaloids of Datura stramonium and Datura ferox. This method allowed the direct coupling of an electrospray (ZMD) mass selective detector to the HPLC system. Under these conditions, atropine and scopolamine were well separated from other components and detected on the PDA (LOD = 1 microg/ml) and ZMD (LOD(atropine) = 10 pg/ml; LOD(scopolamine) = 100 pg/ml) detectors. Four geographically isolated populations of each of D. stramonium and D. ferox were analysed for seed alkaloids and it was found that the two species were diagnostically different in their atropine-scopolamine ratios. The optimised HPLC method was used to analyse three viscera samples of an adult Caucasian male whose death was ascribed to a fatal heart attack. Atropine and scopolamine were detected in the stomach and its contents, which contained Datura seeds. The chemical profile of the seeds found in the stomach contents was similar to those from four geographically different D. ferox plants.     

Accidental fatal poisoning by Nicotiana glauca: identification of anabasine by high performance liquid chromatography/photodiode array/mass spectrometry

A method, based on reversed phase high performance liquid chromatography (HPLC) was developed for the detection and quantification of anabasine, the toxic alkaloid of Nicotiana glauca, in forensic applications. A standard solid phase extraction (SPE) method was used for the extraction of anabasine from viscera, but was optimized for the extraction of this alkaloid from plant material. The careful selection of mobile phase components allowed the direct coupling of electron impact (EI) and Z spray mass selective detector (ZMD) of the HPLC. Under these conditions, anabasine was well separated from nicotine and could be detected on the PDA (limit of detection, LOD = 250 ng/ml), TMD (LOD = 10 microg/ml) and ZMD (LOD =1 ng/ml) detectors. Three geographically isolated N. glauca trees were analyzed for alkaloid content and it was found that both the leaves and the flowers contain anabasine. The optimized HPLC method was used to analyze two viscera samples (the stomach and contents of a mother and child who putatively died from food poisoning) and a flower exhibit. Anabasine was detected in both the viscera samples, supporting the finding that these fatalities were due to the ingestion of N. glauca accidentally collected with traditional spinach (marog). The alkaloid profile of the flower exhibit submitted with the viscera samples was similar to those obtained from flowers collected from three different N. glauca trees. The results show that anabasine and/or N. glauca poisoning can easily be confirmed using the forensic methodology described.     

Trace detection of meglumine and diatrizoate from Bacillus spore samples using liquid chromatography/mass spectrometry

Following the September 11, 2001 terrorist attacks, letters containing Bacillus anthracis were distributed through the United States postal system killing five people. A complex forensic investigation commenced to identify the perpetrator of these mailings. A novel liquid chromatography/mass spectrometry protocol for the qualitative detection of trace levels of meglumine and diatrizoate in dried spore preparations of B. anthracis was developed. Meglumine and diatrizoate are components of radiographic imaging products that have been used to purify bacterial spores. Two separate chromatographic assays using multiple mass spectrometric analyses were developed for the detection of meglumine and diatrizoate. The assays achieved limits of detection for meglumine and diatrizoate of 1.00 and 10.0 ng/mL, respectively. Bacillus cereus T strain spores were effectively used as a surrogate for B. anthracis spores during method development and validation. This protocol was successfully applied to limited evidentiary B. anthracis spore material, providing probative information to the investigators.     

Unconjugated morphine in blood by radioimmunoassay and gas chromatography/mass spectrometry

Morphine, the active metabolite of heroin, is rapidly inactivated by glucuronidation at the 3 carbon. Unconjugated (pharmacologically active) morphine was measured in postmortem blood by radioimmunoassay using an antibody-coated tube kit. The kit shows less than 0.2% cross-reactivity with codeine and morphine-glucuronide. Unconjugated morphine concentrations were confirmed by gas chromatography/mass spectrometry (GC/MS) using deuterated morphine as the internal standard. The blood was precipitated with 10% trichloroacetic acid (TCA) and concentrated hydrochloric acid (HCl), centrifuged, and decanted. The supernatant was then either diluted (unhydrolyzed) or heated to 100 degrees C, 30 min (hydrolyzed), followed by a wash with 4:1 chloroform:isopropranol. The upper aqueous layer was then saturated with sodium bicarbonate (NaHCO3) and extracted with 4:1 chloroform:isopropranol. The organic layer was evaporated, derivatized with trifluoroacetic anhydride (TFA), and analyzed by selected ion monitoring (SIM) GC/MS. Comparison of the results for unconjugated morphine by radioimmunoassay and unhydrolyzed morphine by GC/MS gave a correlation coefficient of r = 0.98, n = 100. Unconjugated morphine ranged from 0 to 100% of total morphine with a mean of 42%, n = 200, for heroin or morphine involved deaths. Review of 56 putative rapid deaths gave a mean of 68% unconjugated morphine with a range of 26 to 100%. The ratio of unconjugated to total morphine was found to be stable in postmortem blood after more than a year of storage at room temperature, within the precision of the method.     

Surface-sampling and analysis of TATP by swabbing and gas chromatography/mass spectrometry

The method of sample recovery for trace detection and identification of explosives plays a critical role in several criminal investigations. After bombing, there can be difficulties in sending big objects to a laboratory for analysis. Traces can also be searched for on large surfaces, on hands of suspects or on surfaces where the explosive was placed during preparatory phases (e.g. places where an IED was assembled, vehicles used for transportation, etc.).In this work, triacetone triperoxide (TATP) was synthesized from commercial precursors following reported methods. Several portions of about 6 mg of TATP were then spread on different surfaces (e.g. floors, tables, etc.) or used in handling tests. Three different swabbing systems were used: a commercial swab, pre-wetted with propan-2-ol (isopropanol) and water (7:3), dry paper swabs, and cotton swabs wetted with propan-2-ol. Paper and commercial swabs were also used to sample a metal plate, where a small charge of about 4 g of TATP was detonated. Swabs were sealed in small glass jars with screw caps and Parafilm^® M and sent to the laboratory for analysis. Swabs were extracted and analysed several weeks later by gas chromatography/mass spectrometry. All the three systems gave positive results, but wetted swabs collected higher amounts of TATP. The developed procedure showed its suitability for use in real cases, allowing TATP detection in several simulations, including a situation in which people wash their hands after handling the explosive.     

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.     

Simultaneous determination of creatinine and uric acid in urine by liquid chromatography-tandem mass spectrometry with polarity switching electrospray ionization

A simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated to simultaneously determine creatinine (Cr) and uric acid (UA) levels as a confirmatory method for adulteration or dilution of urine. Centrifuged urine samples (10μL) were diluted with 390μL of distilled water. 30μL of internal standard solution (Cr-d(3), 5μg/mL) and 10μL of acetonitrile were added to 20μL aliquots of diluted urine samples and filtered. The samples (1μL) were introduced into LC-MS/MS with no further pretreatment. Cr and UA were separated on a multi-mode ODS column (Scherzo SM-C18, 75mm×2.0mm I.D., 3μm) and quantified by LC-MS/MS with polarity-switching electrospray ionization. Cr requires the positive-ion mode, whereas the negative-ion mode is required for the analysis of UA. The linear ranges were 1.0-300mg/dL for Cr and 0.5-300mg/dL for UA, with good determination coefficients (R(2)≥0.9988). The intra-day and inter-day precision of the analytes was within 13.0% and 14.4%, respectively. The intra-day and inter-day accuracy was -8.8 to 3.7% and -0.3 to 6.6%, respectively. The lower limits of detection (LLODs) were 0.3mg/dL for Cr and 0.07mg/dL for UA. The applicability of the developed method was examined by analyzing urine samples from suspected drug abusers (n=46).     

气相色谱/质谱检验合成大麻素K3中AKB48

目的建立较为快速准确的合成大麻素K3中AKB48的气相色谱/质谱检验方法。方法对进样口温度、初始柱温、柱流速及质谱采样率等4项色谱及质谱实验参数进行考察优化。结果 GC/MS检验合成大麻素K3中AKB48的优化条件为:进样口温度280℃,柱初始温度80℃,柱流速为2.0ml/min,质谱采样率为2。结论该方法具有快速、准确、灵敏等优点,可用于K3中AKB48的定性检验鉴定。     

Development and validation of a liquid chromatography–mass spectrometry assay for hair analysis of methylphenidate

Methylphenidate (MPH) is a phenethylamine derivative used in the treatment of childhood attention-deficit hyperactivity disorder. MPH is biotransformed in the body by the hydrolysis of the methyl ester linkage to its metabolite, ritalinic acid. Whereas both compounds are usually measured in plasma and urine, preliminary observations show that only the parent compound is present in hair from treated individuals.     

Determination of nimetazepam and 7-aminonimetazepam in human urine by using liquid chromatography–tandem mass spectrometry

We report determination of metabolites of popular drugs of abuse, including nimetazepam and nitrazepam, in urine by using liquid chromatography/mass spectrometry. Nimetazepam and its metabolites, 7-aminonimetazepam and nitrazepam, were extracted by solid-phase extraction using a DAU cartridge. An ammonium acetate buffer solution (pH 4) and a Luna polar-RP column were selected as the mobile and stationary phase, respectively, for liquid chromatography. Mass spectrometry was used for analysis and was optimized for operation in the positive mode for all analytes. The urine specimens were screened for the presence of nimetazepam and its metabolites nitrazepam and 7-aminonimetazepam at a concentration of 0.1 ng/mL. Presence of 7-aminonimetazepam in the urine was an indicator of the subject being a probable abuser of nimetazepam.     

Simultaneous screening and quantitation of 18 antihistamine drugs in blood by liquid chromatography ionspray tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is presented for the simultaneous screening and quantitation of 18 antihistamine drugs in blood samples. Sample pretreatment involved liquid-liquid extraction of the basic antihistamines followed by a second extraction of the acidic antihistamines. The recoveries were 43-113% for basic drugs and 23-66% for acidic drugs. The combined extracts were run by LC on C(18) reversed phase column using acetonitrile-ammonium acetate mobile phase at pH 3.2. The mass spectrometric analysis was performed with a triple stage quadrupole mass analyzer. Screening was performed using multiple reaction monitoring (MRM) and any compounds tentatively identified as antihistamine drugs were then automatedly verified by their Product Ion Spectra in a subsequent MS/MS run.Quantitation was based on the MRM data from the screening step. In validation tests, the method showed good linearity at the relevant concentrations. The attained limits of quantitation varied between 0.0005 and 0.01mg/l in blood and were lower than the therapeutic concentrations (C(max)). The limits for identification by Product Ion Spectra were also lower than C(max), except for clemastine, which has exceptionally low concentrations in blood. The intra-assay relative standard deviations were better than 10% and the inaccuracy varied between 39% for levocabastine and 5% for cyclizine, the majority of the values being <20%.     

Toxicological analysis of thiamylal in biological materials by gas chromatography/mass spectrometry

A reliable and sensitive method to analyze thiamylal in biological materials was developed, using gas chromatography/mass spectrometry (GC/MS). A quantitative determination was made by use of mass fragmentography with the lower detection limit of 0.01 microgram/g. Thiopental was used as the internal standard. Distribution of the drug in the blood and body tissues of rats was examined. The method was then used to detect thiamylal in tissues from an autopsied patient and concentration of this drug in the body materials was evaluated, from medico-legal aspects.     

Forensic identification of dyes extracted from textile fibers by liquid chromatography mass spectrometry (LC-MS)

LC-MS is used for the identification of dyes extracted from textile fibers and the utility of the method for forensic trace analysis is demonstrated. The technique is shown to provide a high degree of chemical structural information, making dye identification highly specific in comparison to optical and/or chromatographic methods of dye analysis. A UV-visible absorbance detector, placed in series before the MS detector, facilitates monitoring the elution of dyes in the presence of other non-dye components extracted from colored textile fibers. In this way, dye identification becomes practical, even when a dye standard is not available for comparison. A set of 22 reference dyestuffs and 10 dyes extracted from textile fibers were analyzed to demonstrate the utility of the method. Six of the extracted dyes corresponded to dyes also contained in the set of 22 reference dyestuffs. Reference dyestuffs were not available for four of the extracted dyes. Triethylamine (TEA) was shown to increase the electrospray ionization-mass spectrometry (ESI-MS) response of dyes containing multiple sulfonated groups.