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Smith-Lemli-Opitz syndrome is a birth defect caused by the deficiency of 7-dehydrocholesterol reductase in cholesterol biosynthesis pathway, which leads to accumulation of 7-dehydrocholesterol and reduction of cholesterol in body fluids. To effectively diagnose Smith-Lemli-Opitz syndrome and monitor therapy, a reliable method for simultaneous detection of 7-dehydrocholesterol and cholesterol is needed.In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), 50 μL of human plasma were hydrolyzed at 70℃ for 40 min with 1 M potassium hydroxide in 90% ethanol, and then 7-dehydrocholesterol and cholesterol were extracted by 600 μL of n-hexane for three times. After microwave-assisted derivatization with 70 μL of N,O-bis(trimethylsilyl)trifluoroacetamide at 460 W for 3 min, the analytes were measured by gas chromatography-mass spectrometry.The limits of detection were 100 ng/mL for 7-dehydrocholesterol and 300 ng/mL for cholesterol. Good linearity was obtained in the range of 1-600 μg/mL for 7-dehydrocholesterol and 10-600 μg/mL for cholesterol, which completely covered the biochemical levels of Smith-Lemli-Opitz syndrome patients that have been reported.A time-saving and accurate gas chromatography with mass spectrometry based method was developed for the determination of 7-dehydrocholesterol and cholesterol in human plasma, which also serves as a useful tool for Smith-Lemli-Opitz syndrome diagnosis, treatment, and research. more...

Accurate determination of a person's blood alcohol concentration (BAC) is an important task in forensic toxicology laboratories because of the existence of statutory limits for driving a motor vehicle and workplace alcohol testing regulations. However, making a correct interpretation of the BAC determined in postmortem (PM) specimens is complicated, owing to the possibility that ethanol was produced in the body after death by the action of various micro-organisms (e.g., Candida species) and fermentation processes. This article reviews various ways to establish the source of ethanol in PM blood, including collection and analysis of alternative specimens (e.g., bile, vitreous humor (VH), and bladder urine), the identification of non-oxidative metabolites of ethanol, ethyl glucuronide (EtG) and ethyl sulfate (EtS), the urinary metabolites of serotonin (5-HTOL/5-HIAA), and identification of n-propanol and n-butanol in blood, which are known putrefaction products. Practical utility of the various biomarkers including specificity and stability is discussed. more...

Identifying the source of ethanol in a decedent remained a complicated problem for forensic toxicologists because of postmortem ethanol formation. As ethanol’s non-oxidative metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS) have the potential to distinguish between antemortem ethanol consumption and postmortem ethanol formation, due to their high sensitivity and selectivity. In the current study, a simple and quick liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the determination of EtG and EtS in human whole blood and vitreous humor (VH). A total of 20 μL of the sample was precipitated by methanol, and the analytes were detected by LC-MS/MS in a run of 6 min. This method achieved high sensitivity (limits of detection: 2 ng/mL for both EtG and EtS), with linearity in the range of 5–10,000 ng/mL in both whole blood and VH. Deviations in accuracy, inter- and intra-day precision were all lower than 15% at three quality control levels. Subsequently, this method was applied to 62 real forensic cases. Only blood samples were available in 52 cases. Paired blood and VH samples were present in 10 cases. The concentrations of EtG and EtS in blood were in the range of 0–22,264.8 ng/mL and 0–2,126.0 ng/mL, respectively. In one case with both blood and VH, the blood ethanol concentration was 1.22 mg/mL, with EtG and EtS both below limits of quantification (5 ng/mL) in VH, and no EtG and EtS found in whole blood. The results suggested that EtG and EtS were useful markers for the interpretation of ethanol resource in postmortem blood and VH. more...

Alcohol is often found in the blood of the deceased. To cover up the true cause of victim's death, postmortem instillation of alcohol occurs in some criminal cases. Explaining the finding of alcohol is extremely vital in forensic practice. This study aims to evaluate whether ethyl glucuronide (EtG) and ethyl sulfate (EtS) in blood and vitreous humor (VH) can be used to distinguish alcoholic death and postmortem alcohol instillation. Saline or 12.6 g/kg ethanol (antemortem alcohol poisoning group) was introduced into rabbits' stomachs 2 h before sacrificed. Same amount of ethanol was introduced into rabbits' stomachs at 0 h, 0.5 h, 1 h and 2 h after death in four subgroups of postmortem alcohol instillation group, respectively. Cardiac blood and VH were collected at 10 min, 4 h, 10 h and 24 h after death in blank and antemortem alcohol poisoning group, and after instillation of alcohol in postmortem alcohol instillation group. Blood was also collected at 34 h. Ethanol and EtG levels in blood and VH and EtS in VH in antemortem alcohol poisoning group were overlapped with those in postmortem alcohol instillation group. The contents of EtG and EtS in blood in antemortem alcohol poisoning group (mean ≥ 7.833 μg/mL for EtG and ≥ 19.990 μg/mL for EtS) were much higher than those in postmortem alcohol instillation group (mean ≤ 0.118 μg/mL for EtG and ≤ 0.091 μg/mL for EtS), but apparent decomposition was observed in EtG, which might lead to misinterpretation. Blood EtS showed better stability and could be used to distinguish alcoholic death and postmortem alcohol instillation. more...

Problems of stability were found for biomarkers of alcohol consumption: ethyl glucuronide (EtG), ethyl sulfate (EtS), phosphatidylethanols (PEths), and fatty acid ethyl esters (FAEEs) in whole blood. The purpose of this study was to establish a method for the determination of these four kinds of ethanol's non-oxidative metabolites in dried blood spots (DBS) by liquid chromatography tandem mass spectrometry (LC-MS/MS), and to evaluate their stability. In this method, 50 μL of human blood was spotted onto a filter paper for DBS analysis. Samples were extracted by methanol, reconstituted by 2-propanol, and injected into the LC-MS/MS system. Limits of detection were among 0.5–50 ng/mL, and deviations in accuracy and precision were all lower than 15% at three quality control levels. The stability of the four kinds of ethanol non-oxidative metabolites in DBS was investigated during a 90-day range under three temperatures, −20 °C, 4 °C, and 25 °C. EtG and EtS showed a high level of stability in DBS in the 90-day range, regardless of the temperature. FAEEs were unstable after three days. PEths showed stability within 15 days in postmortem DBS and 60 days in antemortem DBS, respectively, at all temperatures. more...

Concentration ratio of ethanol/n-propanol has been employed to distinguish the source of ethanol in postmortem blood, though its reliability remains controversial. Forty-two postmortem human blood samples with ethanol levels in the range of 0.07–4.64 mg/mL were investigated. Ethanol and n-propanol were determined by head-space gas chromatography coupled with flame ionization detection, while ethyl glucuronide (EtG) and ethyl sulfate (EtS) were determined by liquid chromatography–tandem mass spectrometry. EtG and EtS were both negative in 26% of the investigated postmortem blood samples and 11% of n-propanol-negative postmortem samples, indicating that n-propanol was not a reliable marker of putrefaction. It was also found that the ratio of ethanol/n-propanol (supposed to be more...

N-ethylpentylone (NEP), a new synthetic cathinone, has been rising to be one of the most popular cathinone derivatives in recent years. However, research on NEP is rather limited. In this study, locomotor stimulation and sensitization, as well as anxiety-like behavior induced by NEP were studied in Sprague-Dawley rats, using the open field and elevated plus maze respectively. Rats were administered NEP (5, 20 or 50 mg/kg, intraperitoneal), with saline as the negative control and methamphetamine (5 mg/kg) as a positive control. Acute administration of NEP at all the doses tested significantly promoted locomotor activity, presenting an inverted U-shaped dose-effect curve. The highest activity was observed at the 20 mg/kg dose group, with the average distance traveled 18 times higher than the saline group. Repeated administration of NEP enhanced locomotor activity only at the 5 mg/kg dose group. After a week's withdrawal, re-challenge of NEP failed to induce marked behavioral sensitization. In elevated plus maze experiments, both acute and repeated administration of 20 mg/kg NEP induced anxiolytic-like effects, while no significant alteration was observed in the 5 and 50 mg/kg dose groups. In summary, acute administration of NEP caused significantly enhanced locomotor activity in rats at all the tested doses, while repeated NEP administration enhanced locomotor activity only at a low dose (5 mg/kg), while a high dose (20 mg/kg) of NEP induced anxiolytic-like effects after both acute and repeated administration. more...

Background: Dopamine transporter (DAT) and serotonin transporter (SERT) are targets for many psychoactive substances. Functional assays including uptake inhibition and release assays often involve radiolabeled compounds like [3H]-dopamine and [3H]-serotonin to assess drug activity at transporters, which have high requirements on handling radioactive samples. Aims: The aim of this study was to establish a label-free method to assess drug activity at DAT and SERT. Methods: A liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was established using transporter-transfected human embryonic kidney 293T (HEK293T) cells. This method was evaluated by testing the effects of amphetamine and cocaine in the assay procedure. Results: The limits of detection of this method were 0.2 nM for both dopamine (DA) and serotonin (5-HT), with good linearities in the range of 0.5–160 nM. Amphetamine and cocaine’s IC50 and EC50 on DAT and SERT determined by this method were consistent with previous reports. Conclusions: A rapid, reliable and label-free LC-MS/MS method for assessing drug activity was established, which affords an attractive alternative for those laboratories that do not have a radiation license or capabilities. more...

The lack of systematic studies on the stability of ethanol's non-oxidative metabolites in postmortem specimens restricts their use in forensic cases. This study aimed to compare the stability of ethyl glucuronide (EtG), ethyl sulfate (EtS), phosphatidylethanols (PEths) and fatty acid ethyl esters (FAEEs) in postmortem human blood. Three groups were established based on the level and source of ethanol: the blank group, the ethanol-spiked group and the ethanol-positive group. Each group contained six blood samples from different corpses. The samples in each group were placed at 37, 25, 4 and -20°C. Every 24 h for 7 days, 50 μL was collected from each sample. The levels of EtG, EtS, PEths and FAEEs were determined by liquid chromatography-mass spectrometry, and their stability was evaluated. EtG was not detected in the blank group, but it was found in samples in the ethanol-spiked group placed at 37°C, and it was degraded in the ethanol-positive group at 37 and 25°C. EtS showed no change in any of the groups. PEths were not detected in the blank group, but formation was found in the ethanol-spiked group at all temperatures. In the ethanol-positive group, PEth levels fluctuated at 37°C, decreased at 25°C and increased at -20°C. FAEEs were generated in the blank group and in the ethanol-spiked group at all temperatures. In the ethanol-positive group, FAEEs were degraded at 37 and 25°C but were generated at 4 and -20°C. EtS is a reliable biomarker of ethanol consumption, and EtG could be used as a biomarker at low temperatures (4 and -20°C), but PEths and FAEEs are not appropriate biomarkers of ethanol consumption. more...

The importance of ethanol non-oxidative metabolites as the specific biomarkers of alcohol consumption in clinical and forensic settings is increasingly acknowledged. Simultaneous determination of these metabolites can provide a wealth of information like drinking habit and history, but it was difficult to achieve because of their wide range of polarity. This work describes development and validation of a simple liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for 4 types of ethanol non-oxidative metabolites (ethyl glucuronide, ethyl sulfate, fatty acid ethyl esters and phosphatidylethanols) in 50 μL of human whole blood. Pretreatment method, column and MS conditions were optimized. For the first time, the four types of ethanol non-oxidative metabolites with enormous discrepancies of property were simultaneously extracted and analyzed in one run within 40 min. The limits of detections (LODs) were among 0.1–10 ng/mL, and good linearity was obtained. Deviations in precision and accuracy were all lower than 15% at three QC levels. This method was then applied to two forensic samples, resulting in information on drinking habits and drinking time which were very useful for the interpretation of the blood alcohol results. more...

N-Ethylpentylone (NEP) is a popular synthetic cathinone abused worldwide. To obtain more information about its pharmacokinetics and pharmacodynamics, a rapid, simple and sensitive liquid chromatography-tandem mass spectrometry method was developed for the determination of NEP, two important neurotransmitters, dopamine and serotonin, and their metabolites, including 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine and 5-hydroxyindole-3-acetic acid, in rat brain microdialysate. The analytes were separated on a Phnomenex Polar C18 column, with a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) under gradient elution to shorten the total chromatographic run time. A triple quadruple mass spectrometer coupled with an electrospray ionization source in both positive and negative ion mode was used to detect the analytes. This method showed excellent accuracy (87.4-113.5%) and precision (relative standard deviation more...

Ethyl sulfate (EtS) and ethyl glucuronide (EtG) in urine are biomarkers to monitor ethanol consumption. Due to their high polarity, severe matrix effects have been observed during analysis of EtS and EtG in urine by liquid chromatography tandem mass spectrometry (LC-MS/MS), which can lead to a loss of sensitivity and accuracy. In the present study, a novel and simple sample preparation approach based on fast-dried urine spot was established to reduce the matrix effect of EtS and EtG in urine. 20 μL of urine was dropped on the Whatman 903# paper and was subsequently dried by microwave in one minute. After ultrasonic assisted extraction with 500 μL of methanol, the analysis was conducted using an LC-MS/MS system. Limits of detection were 5 ng/mL and linear ranges were 10 ng/mL–10 μg/mL for both EtS and EtG. Matrix effects were in the range of 99.3–107.8% for EtS and 86.7–91.0% for EtG at three QC levels. Matrix effects for EtS and EtG were compared between the current method and other sample preparation methods including protein precipitation, and solid-phase extraction. The results showed that this fast-dried urine spot-based extraction method could eliminate matrix effects significantly in analysis of urine EtS and EtG by LC-MS/MS. more...

N-Ethylpentylone (NEP) is one of the most recent novel stimulants, and there is limited understanding of its toxicity. Here we employed zebrafish model for analyzing the effects of NEP on early embryos and cardiovascular and nervous systems at late developmental stages. We first observed multi-malformations in early embryos and larvae after NEP administration, together with significant deregulations of brain and heart development-associated genes (neurog1, her6, elavl3, nkx2.5, nppa, nppb, tnnt2a) at transcriptional level. Low-dosed NEP treatment induced an anxiety-like phenotype in zebrafish larvae, while higher doses of NEP exerted an inhibitory effect on locomotion and heart rate. Besides, the expression of th (tyrosine hydroxylase) and th2 (tyrosine hydroxylase 2), identifying dopamine (DA) release, were significantly increased during one-hour free swimming after effective low-dosed NEP administration, along with the upregulation of gene fosab and fosb related to stress and anxiety response. D1R antagonist SCH23390 and D2R antagonist sulpiride partially alleviated the aberrances of locomotion and heart rate, indicating dopaminergic receptors were involved in the bidirectional dosage-dependent pattern of NEP-induced performance. Meanwhile, sulpiride offset the upregulated expression of th, th2 and fosab in the group of 1.5 μM NEP, which highlighted the significant role of D2R in NEP-induced locomotive effects. This study systematically described the developmental, neuronal and cardiac toxicity of NEP in zebrafish, and identified the dopaminergic receptors as one of the downstream effectors of NEP administration. more...

Only sporadic data are available on hair concentrations of diazepam and some of its metabolites (nordazepam, oxazepam, and temazepam) following a single controlled dose. The aim of this study was to investigate the deposition of diazepam and its metabolites in human hair after eight healthy volunteers (four women and four men, ages 24-26, East Asian) consumed 10 mg of diazepam. Hair was collected from all volunteers 1 month after exposure, and also 2 months post-exposure from men and 10 months post-exposure from women. Diazepam and the complete metabolite profile, including oxazepam glucuronide and temazepam glucuronide, were measured by ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with limits of quantifications (LOQs) of 0.5-2.5 pg/mg for diazepam, nordazepam, oxazepam, and temazepam, and of 10 pg/mg for oxazepam glucuronide and temazepam glucuronide. There were no differences by gender in the amounts of diazepam or metabolites found. The concentration of the main metabolite nordazepam was consistently higher than that of diazepam at both 1 and 2 months after consumption. Oxazepam and temazepam traces were found in some volunteers' hair, but the glucuronides were not detected. Diazepam and nordazepam levels at 10 months post-exposure were extremely low (near the LOQ), indicating drug loss by personal hygiene and physical handling. To our knowledge, this is the first single-dose diazepam study using black hair and the first study to include measurements of oxazepam glucuronide and temazepam glucuronide in human hair. more...

A rapid, simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the qualitative and quantitative analysis of nine barbiturates (barbital, phenobarbital, pentobarbital, amobarbital, secobarbital, thiopental, butalbital, butabarbital, and hexobarbital) in human whole blood. Barbiturates were extracted from 100 μL of human whole blood samples using a simple liquid-liquid extraction (LLE) procedure, and detected by LC-MS/MS. An UPLC C18 (2.1 mm × 100 mm, 1.7 µm) column was used at 40 °C for the separation and acetonitrile/water system was used as the mobile phase with gradient elution. This method showed excellent accuracy (86–111%) and precision (relative standard deviation 0.99 over the range. This method achieved the separation and detection of pentobarbital and amobarbital at the same time in a convenient way. Moreover, it was both simple and sensitive for the determination of nine most commonly used barbiturate drugs, which was meaningful in the field of clinical and forensic toxicology. Copyright © 2016 John Wiley & Sons, Ltd. more...

A novel and simple online solid-phase extraction liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of diazepam and its five metabolites including nordazepam, oxazepam, temazepam, oxazepam glucuronide, and temazepam glucuronide in human oral fluid. Human oral fluid was obtained using the Salivette(®) collection device, and 100 μL of oral fluid samples were loaded onto HySphere Resin GP cartridge for extraction. Analytes were separated on a Waters Xterra C18 column and quantified by liquid chromatography with tandem mass spectrometry using the multiple reaction monitoring mode. The whole procedure was automatic, and the total run time was 21 min. The limit of detection was in the range of 0.05-0.1 ng/mL for all analytes. The linearity ranged from 0.25 to 250 ng/mL for oxazepam, and 0.1 to 100 ng/mL for the other five analytes. Intraday and interday precision for all analytes was 0.6-12.8 and 1.0-9.2%, respectively. Accuracy ranged from 95.6 to 114.7%. Method recoveries were in the range of 65.1-80.8%. This method was fully automated, simple, and sensitive. Authentic oral fluid samples collected from two volunteers after consuming a single oral dose of 10 mg diazepam were analyzed to demonstrate the applicability of this method. more...

A liquid chromatography with tandem mass spectrometry method was developed for the simultaneous screening of 34 drugs and poisons in forensic cases. Blood (0.5 mL, diluted 1:1 with water) or 1.0 mL of urine was purified by solid-phase extraction. Gastric contents (diluted 1:1 with water) were treated with acetonitrile, centrifuged, and supernatant injected. Detection was achieved using a Waters Alliance 2695/Quattro Premier XE liquid chromatography tandem mass spectrometry system equipped with electrospray ionization, operated in the multiple reaction monitoring modes. The method was validated for accuracy, precision, linearity, and recovery. The absolute recovery of drugs and toxic compounds in blood was greater than 51% with the limit of detection in the range of 0.02-20 ng/mL. The absolute recovery of drugs and toxic compounds in urine was greater than 61% with limit of detection in the range of 0.01-10 ng/mL. The matrix effect of drugs and toxic compounds in urine was 65-117% and 67-121% in blood. The limit of detection of drugs and toxic compounds in gastric content samples were in the range of 0.05-20 ng/mL. This method was applied to the routine analysis of drugs and toxic compounds in postmortem blood, urine, and gastric content samples. The method was applied to actual forensic cases with examples given. more...

We developed a simple and rapid analysis method for the determination of four amphetamines in human whole blood and urine using gas chromatography–mass spectrometry (GC–MS). During the course of sample preparation, the evaporation process was found to be the weak point for amphetamine and methamphetamine analysis. Liquid–liquid extraction in this study was conducted without the evaporation step, and the upper solvent layer was injected directly without derivatization, which effectively prevented the volatile amphetamines from vanishing during evaporation and guaranteed the linearity of the calibration curves. This method showed good selectivity, precision, and accuracy, and at the same time was simple and fast. The limits of detection were 5 ng/ml. As an application of this study, a whole blood sample and urine sample from a drug abuse suspect were analyzed. This method may be able to meet the need for rapid and accurate confirmation of positive immunoassay results as well as quantification in clinical and forensic toxicological cases. To our knowledge, no forensic toxicologists have noted that such a basic and simple method, without evaporation or derivatization, is useful for routine analysis of amphetamines using GC–MS. more...

A μElution solid-phase extraction (SPE) liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of diazepam, nordiazepam, oxazepam, oxazepam glucuronide, temazepam and temazepam glucuronide in human whole blood is presented. 200 μL of whole blood samples were loaded onto a Waters Oasis HLB 96-well μElution SPE plate using 75 μL of methanol as the elution solvent, and the eluents were injected into an Eclipse XDB C18 column. No hydrolysis, solvent transfer, evaporation or reconstitution was involved in the sample preparation procedures. Tandem mass spectrometric detection with Turbo Ion Spray was conducted via multiple reaction monitoring (MRM) under positive ionization mode. The method was validated and proved to be accurate (accuracy within 93-108%), precise (intra-day RSD more...

Increasing in cases involving drugs of abuse leads to heavy burden for law enforcement agencies, exacerbating demand for rapid screening technique. In this study, atmospheric pressure ionization technologies including direct analysis in real time (DART) ion source coupled to a time-of-flight mass spectrometer (DART-TOF-MS)as well asdopant-assisted positive photoionization ion mobility spectrometry (DAPP-IMS) without radioactivity were utilized together as the powerful analytical tool for the rapid screening and identification of 53 abused drugs.The limits of detection (LOD) were 0.05-2μg/mL when using DART-TOF-MS and 0.02-2μg when using DAPP-IMS which could satisfy the actual requirement in forensic science laboratory. The advantages of this method included fast response, high-throughput potential, high specificity, and minimal sample preparation. A screening library of reduced mobility (K0), accurate mass of informative precursor ion ([M+H]+) and fragment ions was established respectively by employing a bench-top DAPP-IMS and TOF-MS in-source collision induced dissociation (CID) mode. Then the standardized screening procedure was developed with criteria for the confirmation of positive result. A total of 50 seized drug samples provided by local forensic laboratory we reanalyzed to testify the utility of the method. This study suggests that a method combing DART-TOF-MS and DAPP-IMS is promising for the rapid screening and identification of abused drugs with minimal sample preparation and absence of chromatography. more...

The objective of this project was to investigate the incidence of alcohol consumption in fatal traffic deaths in Shanghai, one of the largest cities in China. A study was conducted on 803 individuals killed in road accidents during the period 2009-2011, in terms of alcohol-positive rate, mean blood alcohol content (BAC), gender, age, vehicle type, pedestrian alcohol problem, single-vehicle vs multiple-vehicle crashes, and time of day. It was found that 28.9% of the drivers involved had a BAC≥0.20mg/mL (limit of civil offense) and 21.8% had a BAC≥0.80mg/mL (limit of criminal offense). The mean BAC of alcohol-positive drivers (with a BAC≥0.20mg/mL) was 1.51mg/mL. The vast majority of the drivers involved were males. With regards to age, the largest group was of drivers aged between 40 and 49 years group in both alcohol-negative cases (26.8%) and alcohol-positive cases (26.2%). Motorcycles were most likely to be involved, representing 34.4% of alcohol-negative crashes and 51.6% of alcohol-positive crashes. Very high BACs were common among alcohol-positive pedestrians, yet all female pedestrians were alcohol-negative. Single-vehicle crashes were over-represented in alcohol-positive cases. Alcohol-negative crashes and alcohol-positive crashes most often happened during the time period of 17:00-18:59 and 19:00-20:59, respectively. more...

Recreational drugs have large impact on public health and security, and to monitor them is of urgent demand. In the present study, ultrasound-assisted dispersive liquid-liquid microextraction combined with the detection of gas chromatography-mass spectrometry was applied to the determination of seven common recreational drugs, including amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, meperidine, methadone and ketamine in 200μL of human whole blood. A series of factors which would affect the extraction efficiency were systematically investigated, including the nature and the volume of extraction and dispersing solvents, ultrasonication time, salting-out effect and pH value. The method consumed small amount of sample. The limits of detection and limits of quantification for each analyte were 10 and 40ng/mL, respectively, and the linearity was in the range of 0.04-25μg/mL (R2 higher than 0.99). Good specificity, precision (1.5-8.2% for the intra-day study and 2.6-12.8% for the inter-day study), satisfactory accuracy (85.0-117.1%) and extraction recovery (77.0-92.4%) were obtained, which makes it a high performance method for the determination of recreational drugs in human whole blood samples. more...

Fatty acid ethyl esters (FAEEs) have been widely studied as specific markers of ethanol intake and mediators of ethanol-induced diseases. In the present study, a simple and rapid gas chromatography-mass spectrometry (GC-MS) method was established for the qualitative and quantitative analysis of six fatty acid ethyl esters (FAEEs), including ethyl myristate, ethyl palmitate, ethyl stearate, ethyl oleate, ethyl linoleate, and ethyl arachidonate, in human whole blood. FAEEs were extracted from 200 μL of human whole blood by a modified liquid-liquid extraction, and the hexane layer was injected directly into GC-MS with ethyl heptadecanoate as the internal standard. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 5–50 ng/mL and 15–200 ng/mL, respectively. Linearity ranged up to 10 μg/mL with r 2 higher than 0.998. Accuracy was in the range of 90.3–109.7%, while intra-day and inter-day precision were 0.7–9.3% and 3.4–12.5%, respectively. This method was then applied to 38 real samples from forensic cases. Differences in the most common FAEEs between Chinese and Western subjects were discussed. The relationship of FAEE concentrations with age and gender was also investigated. more...

Chromatographic determination of glutathione disulfide (GSSG) without any preliminary reduction has been presented using GSSG derivatization by p-hydroxymercuribenzoate (pHMB) in strong alkaline medium followed by the determination of GS-pHMB complex by reversed phase chromatography coupled to chemical vapour generation and atomic fluorescence detector (RPC-CVGAFS). A detection limit of 35 nM for GSSG (corresponding to 1.8 pmol) detected as GS-pHMB species was achieved based on a signal-to-noise ratio of 3 in buffer and in blood. The proposed method was applied to the determination of GSSG in whole blood and validated by the classical determination of GSSG by derivatization after reduction with dithiothreitol (DTT). more...

A liquid chromatography method with mass spectrometric detection has been developed for the simultaneous determination of six thiols in the sulfur metabolic pathway, including cysteine (Cys), homocysteine (HCys), glutathione (GSH), cysteinyl-glycine (Cys-Gly), gamma-glutamyl-cysteine (Glu-Cys), and S-adenosyl-homocysteine (AdoHcy). Tris(2-carboxyethyl)phosphine (TCEP) was used as the reducing reagent and p-(hydroxymercuri)benzoate (PHMB) as the derivatization reagent. Thiols were extracted from 3 mg of yeast using water in an ultrasonic bath. The absolute detection limits for the compounds studied were in the subpicomole range. It was found that AdoHcy, Cys, GSH, Cys-Gly, Glu-Cys, and very little HCys were present in the selenium-enriched yeast sample studied, and GSH, Glu-Cys, very little AdoHcy, Cys, and Cys-Gly were present in three bakery yeasts. more...

Rapid and accurate determination of total ash and acid-insoluble ash of Chinese herbal medicine would be valuable for the quality illustration because current approaches are laborious and time-consuming. This study investigated the feasibility of near infrared spectroscopy in predicting simultaneously the total ash and acid-insoluble ash contents of Prunellae Spica (one of the most widely used Chinese herbal medicine). Samples were collected from ten different geographic regions and scanned under the near-infrared spectroscopy region (4000-12000 cm(-1)). Models between the spectral data and the results of reference analysis were developed, and subsequently validated. Calibration models based on partial least squares were accurate for the prediction of total ash (R2=0.914; standard errors of prediction=0.373), and acid-insoluble ash (R2=0.905; standard errors of prediction=0.452). more...

Acid value (AV) is an important parameter to illustrate the quality as well as degree of refining of peanut oil. A rapid near-infrared reflectance spectroscopy (NIRS) method was applied to determine AV in peanut oils. A partial least squares (PLS) regression model with a coefficient of determination ( R 2 ) of 0.9725 and a square error of cross-validation (SECV) of 0.308 was obtained. The prediction set gave a coefficient of determination ( r 2 ) and standard error of prediction (SEP) of 0.9379 and 0.333. Regarding qualitative evaluation, the classification of qualified peanut oil (with an acid value of less than or equal to 3 mg/g) and unqualified peanut oils (with an acid value of more than 3 mg/g) was conducted by using discriminant partial least squares analysis (DPLS). The results showed that DPLS technique was an effective method of classification model building, with a high correct percent of 96.55%. more...

Human acidic fibroblast growth factor (haFGF) stimulates repair and regeneration of central and peripheral nerves after various injuries. However, it is unable to cross the blood-brain barrier (BBB). To produce a therapeutic haFGF with cell-permeable activity, we fused the haFGF(19-154) gene with Tat-PTD. After its construction by a single-step insertion of a polymerase chain reaction (PCR)-amplified coding sequence, the vector pTat-haFGF(19-154)-His was expressed in Escherichia coli BL21 (DE3) cells. The optimal expression level of the soluble fusion protein was up to 36.7% of the total cellular protein. The recombinant Tat-haFGF(19-154)-His was purified by a combination of Ni-NTA affinity, Sephadex G-25, and heparin affinity chromatography to 95% as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final yield was 171 mg/l culture. Purified Tat-haFGF(19-154)-His had distinct mitogenic activity in Balb/c 3T3 cells, as measured by methylthiazoletetrazolium (MTT) assay and its ED(50) was 3.931 x 10(-4) micromol/l. Tat-haFGF(19-154)-His protein intravenously injected at the dose of 10 mg/kg could be detected in the pallium and hippocampi. more...

A rapid, simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the qualitative and quantitative analysis of nine barbiturates (barbital, phenobarbital, pentobarbital, amobarbital, secobarbital, thiopental, butalbital, butabarbital, and hexobarbital) in human whole blood. Barbiturates were extracted from 100 μL of human whole blood samples using a simple liquid-liquid extraction (LLE) procedure, and detected by LC-MS/MS. An UPLC C18 (2.1 mm × 100 mm, 1.7 µm) column was used at 40 °C for the separation and acetonitrile/water system was used as the mobile phase with gradient elution. This method showed excellent accuracy (86-111%) and precision (relative standard deviation15%). The limits of detection (LODs) were 0.2 ng/mL for barbital and secobarbital and 0.5 ng/mL for the other barbiturates. The linearity ranged from 2 ng/mL to 2000 ng/mL, with r more...

Co-administration of opioids with benzodiazepines is very common around the world. A semi-automated method was developed for the determination of four opioids and two benzodiazepines as well as their metabolites (including glucuronide metabolites) in human urine, based on on-line column-switching-solid-phase extraction (CS-SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The CS-SPE was performed by loading 200μL of urine sample to an Oasis HLB cartridge. Detection was achieved using a LC-MS/MS system equipped with an electrospray ionization source (ESI). For unequivocal identification and confirmation, two selected reaction monitoring transitions were registered for each compound, and no co-elution of interferences was observed at the expected retention time. Significant ion suppressions were observed for most analytes during chromatographic runs, but isotope-labeled internal standards (ISs) were used and found to be useful to compensate for the determination error caused by the matrix effect. The assay's linearity ranged from 1-20ng/mL to 800-1000ng/mL for 23 compounds, except for lorazepam (LOR), whose linearity was in the range of 1-100ng/mL. This method showed to be precise and accurate. The relative standard deviation (RSD) % values of within-run precision, between-run precision and total precision were not greater than 10.4% (n=3), 12.9% (n=5) and 15.1% (n=15), respectively. Accuracy values were in the range of 87.5-110%. Limits of detection (LODs) ranged from 0.2ng/mL to 5ng/mL, and limits of quantification (LOQs) ranged from 1ng/mL to 20ng/mL. The method was applied to the assay of 12 samples from forensic cases, which exemplified the co-administration of benzodiazepines (BZDs) by some heroin abusers. This method was of high sensitivity, selectivity and reliability, minimum sample manipulation, semi-automation, and fairly high throughput (analysis time per sample was 20min). The method developed will be useful for the detection of co-administrated drugs and the study of the interactions of BDZs with opioids. more...

A liquid chromatography with tandem mass spectrometry method was developed for the simultaneous screening of 34 drugs and poisons in forensic cases. Blood (0.5 mL, diluted 1:1 with water) or 1.0 mL of urine was purified by solid-phase extraction. Gastric contents (diluted 1:1 with water) were treated with acetonitrile, centrifuged, and supernatant injected. Detection was achieved using a Waters Alliance 2695/Quattro Premier XE liquid chromatography tandem mass spectrometry system equipped with electrospray ionization, operated in the multiple reaction monitoring modes. The method was validated for accuracy, precision, linearity, and recovery. The absolute recovery of drugs and toxic compounds in blood was greater than 51% with the limit of detection in the range of 0.02-20 ng/mL. The absolute recovery of drugs and toxic compounds in urine was greater than 61% with limit of detection in the range of 0.01-10 ng/mL. The matrix effect of drugs and toxic compounds in urine was 65-117% and 67-121% in blood. The limit of detection of drugs and toxic compounds in gastric content samples were in the range of 0.05-20 ng/mL. This method was applied to the routine analysis of drugs and toxic compounds in postmortem blood, urine, and gastric content samples. The method was applied to actual forensic cases with examples given. more...

A novel method for the screening of 151 drugs of abuse and toxic compounds in human whole blood has been developed and validated by online solid-phase extraction with liquid chromatography coupled to time-of-flight mass spectrometry. Analytes were extracted and separated by using a fully automated online solid-phase extraction liquid chromatography system with total chromatographic run time of 26 min. Time-of-flight mass spectrometry screening of 151 drugs of abuse and toxic compounds was performed in a full-scan (m/z 50-800) mode using an MS(E) acquisition of molecular ions and fragment ions data at two collision energies (one was 6 eV and another one was in the range of 5-45 eV). The compounds were identified based on retention times and exact mass of molecular ions and fragment ions. The limit of detection ranged from 1 to 100 ng/mL and the recovery of the method ranged from 6.3 to 163.5%. This method is proved to be a valuable screening method allowing fast and specific identification of drugs in human whole blood. more...

This study disproves the reliability of n-propanol as a biomarker to establish whether the ethanol found in postmortem blood is derived from antemortem ingestion or postmortem putrefactive processes. Two groups of rats were given ethanol or normal saline solution, respectively, and sacrificed 1.5 h later. After putrefaction, blood and, in a few cases, urine samples from the rats were analyzed for ethanol and n-propanol by head-space gas chromatography equipped with flame ionization detection. Although the concentration ratios of ethanol/n-propanol in the postmortem blood collected from the bodies without prior alcohol consumption were expected to be more...

Objective: The purpose of our study was to better characterize and evaluate drunk driving for governmental reference in order to further reduce alcohol-impaired driving.Method: This article reports the characteristics of 1226 alcohol-positive drivers with blood alcohol concentrations (BACs) at or over the legal limit of 0.20 mg/mL involved in nonfatal traffic accidents in Shanghai, China, from 2008 to 2011. The mean BAC, age, and gender of these drivers are discussed as well as the vehicle types and times of day when the crashes occurred.Results: The mean BAC was 1.41 mg/mL and the mean age was 38 years old, and the vast majority of drivers were male (96.9%). The mean BAC of male drivers (1.42 mg/mL) was higher than that of female drivers (1.20 mg/mL). The mean age of male drivers (38) was also higher than that of female drivers (33). Distributions of vehicle types involved were studied. Cars had the highest percentage of occurrences (56.4%), followed by motorcycles (32.8%), electric bicycles (6.8%), trucks (1.5%), and bicycles (0.8%). It was found that these alcohol-related traffic crashes most often occurred between 7:00 p.m. and 10:59 p.m., representing 48.1 percent (n = 590) of the 1226 cases. Single-vehicle crashes were overrepresented (70.3%) in all cases. The mean BAC in multivehicle crashes (1.69 mg/mL) was higher than that in single-vehicle crashes (1.30 mg/mL).Conclusions: The results indicated a notable need for more governmental attention that would prevent accidents caused by driving under the influence of alcohol. more...

Ecgonine is suggested to be a promising marker of cocaine (COC) ingestion. A combined mass spectrometry (MS) and tandem MS (MS/MS) method was developed to simultaneously determine ecgonine and seven other metabolites of cocaine in human urine and whole blood with ultra-high-pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The compounds were extracted from as little as 100 μL of sample by solid-phase extraction with a 96-well μElution solid-phase extraction plate. The protonated molecules or fragment ions at accurate mass acquired in MS mode were used to quantify specific analytes, following by dedicated MS/MS identification. The assay was linear in the range from 5 to 50-100 ng/mL for urine samples, except for ecgonine methyl ester (10-200 ng/mL) and ecgonine (40-400 ng/mL), and was linear from 1-2 to 50 ng/mL for whole blood samples, except for ecgonine methyl ester (20-1,000 ng/mL) and ecgonine (40-2,000 ng/mL). The correlation coefficients were all greater than 0.99. The limits of detection ranged from 0.2 to 16 ng/mL, and the lower limits of quantification ranged from 1 to 40 ng/mL. The repeatability and intermediate precision were 18.1 % or less. The accuracy was in the range from 80.0 to 122.9 %, process efficiencies were in the range from 8.6 to 177.4 %, matrix effects were in the range from 28.7 to 171.0 %, and extraction recoveries were in the range from 41.0 to 114.3 %, except for ecgonine (12.8 % and 9.3 % at low and high concentrations, respectively). This method was highly sensitive in comparison with previously published methods. The validated method was successfully applied to the analysis of real samples derived from forensic cases, and the results verified that, on the basis of data from four positive samples, ecgonine is a promising marker of cocaine ingestion. more...

A method for the simultaneous direct analysis of diazepam oxazepam glucuronide, temazepam glucuronide, oxazepam, nordiazepam, and temazepam in human urine was developed and validated. Urine sample was purified by solid phase extraction (SPE), and the analysis was achieved using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system equipped with an electrospray ionization source (ESI). Multiple reaction monitoring (MRM) mode was used to analyze the target compounds. Extraction recoveries were 65-122% for all the analytes. The method showed acceptable intra-assay and inter-assay precision (both relative standard deviation (RSD)≤11.2%) for quality control (QC) samples. The limits of detections (LODs) were in the range of 0.1-2 ng/mL. The present assay was applied to analyze the urine obtained from three volunteers after oral administration of a single dose 5mg of diazepam. The results showed that, the detection periods of oxazepam glucuronide and temazepam glucuronide were much longer than diazepam and other metabolites. more...

For the first time a liquid chromatography method with high resolution mass spectrometric detection has been developed for the simultaneous determination all key metabolites of the sulfur pathway in yeast, including all thiolic (cysteine (Cys), homocysteine (HCys), glutathione (GSH), cysteinyl-glycine (Cys-Gly), γ-glutamyl-cysteine (Glu-Cys)) and non-thiolic compounds (methionine (Met), s-adenosyl-methionine (AdoMet), s-adenosyl-homocysteine (AdoHcy), and cystathionine (Cysta)). The developed assay also permits the speciation and selective determination of reduced, oxidized and protein bound fractions of all of the five thiols. Iodoacetic acid (IAA) was chosen as the derivatizing reagent. Thiols were extracted from sub-mg quantities of yeast using hot 75% ethanol. The detection limits were in the range of 1-12 nmol L(-1) for standard solution (high femotomole, absolute), except AdoMet (116 nmol L(-1)), which was unstable. In freshly harvested yeast, most of the thiols were in the reduced forms and low levels of protein-bound GSH and Glu-Cys were found. In a selenium enriched yeast, the thiols were mainly in the oxidized forms, and a significant amount of protein-bound Cys, HCys, GSH, Cys-Gly and Glu-Cys were found. The method was also applied to the metabolic study of the adaptive response of Saccharomyces cerevisiae to hydrogen peroxide, cadmium, and arsenite, and the change in concentration of thiols in the sulfur pathway was monitored over a period of 4h. more...

A high-resolution mass spectrometric detection method is described for the identification of key metabolites in the selenium pathway in selenium enriched yeast. Iodoacetic acid (IAA) was used as the derivatizing reagent to stabilize the selenols. Oxidized forms of selenocysteine (Se-Cys), selenohomocystine (Se-HCys), selenoglutathione (Se-GSH), seleno-γ-glutamyl-cysteine (Se-Glu-Cys), N-(2,3-dihydroxy-1-oxopropyl)-selenocysteine (Se-DOP-Cys), N-(2,3-dihydroxy-1-oxopropyl)-selenohomocysteine (Se-DOP-HCys), selenomethionine (SeMet), seleno-S-adenosyl-homocysteine (Se-AdoHcy), the conjugate of glutathione and N-(2,3-dihydroxy-1-oxopropyl)-selenocysteine (GSH-Se-DOP-Cys), and the conjugate of glutathione and N-(2,3-dihydroxy-1-oxopropyl)-selenohomocysteine (GSH-Se-DOP-HCys) were found in the selenium enriched yeast certified reference material (SELM-1). Selenols were also derivatized with a mercury tag, p-hydroxymercurybenzoate (PHMB). The selenol-PHMB complexes showed the overlapped isotopic patterns of selenium and mercury, which provided supporting information for the identification of selenols. Both methods showed good agreement (4 ppm difference) between the theoretical masses of the target compounds and the measured masses in the yeast matrix. The method using IAA as the derivatizing reagent was used to study the response of Saccharomyces cerevisiae to three forms of selenium, Se-Met, Na(2)SeO(3) (Se(IV)), and Na(2)SeO(4)·10H(2)O (Se(VI)) (concentration of Se: 100 mg/L). The production of selenocompounds observed over a 6 h period was high in the Se-Met treated group compared to the groups treated with Se(IV) and Se(VI). more...