The involvement of calcium in the toxic effect of 4-methylethcathinone on SH-SY5Y cells.

Neurotoxicity induced by psychoactive substances is often accompanied by an imbalance of intracellular calcium ions. It is unclear whether calcium ions play a role in the toxicity induced by psychoactive substances. In the present study, we aimed to evaluate the occurrence of calcium dysregulation and its contribution to cytotoxicity in human neurotypic SH-SY5Y cells challenged with a recently developed psychoactive substance 4-methylethcathinone (4-MEC). An increase in the intracellular calcium was detected by inductively coupled plasma atomic emission spectrometry and Fluo-3 AM dye in SH-SY5Y cells after being treated with 4-MEC. The increase of intracellular Ca2+ level mediated G0/G1 cell cycle arrest and ROS/endoplasmic reticulum stress-autophagy signaling pathways to achieve the toxicity of 4-MEC. In particular, N-acetyl-L-cysteine, a classical antioxidant, was found to be a potential treatment for 4-MEC-induced toxicity. Taken together, our results demonstrate that an increase in intracellular calcium content is one of the mechanisms of 4-MEC-induced toxicity. This study provides a molecular basis for the toxicity mechanism and therapeutic intervention of psychoactive substances.

The correlation between CpG island methylation of hTERT promoter and human age prediction.

DNA methylation is an epigenetic modification that occurs during the life cycle of individuals. Its degree is closely associated with the methylation status of CpG sites in its promoter region. Based on the previous screening that the hTERT methylation is both related to tumors and age, we suspected that the age inference based on hTERT methylation would be disturbed by the disease of the tested person. Herein, eight CpG sites in the hTERT promoter region were analyzed by real-time methylation-specific PCR, and we found that CpG2, CpG5, and CpG8 were closely related to the tumor (P < 0.05). The remaining five CpG sites had a large error in predicting age alone. Combining them to establish a model yielded better results, with an average age error of 4.35 years. This study provides a reliable and accurate detection method for the DNA methylation status of multiple CpG sites on the hTERT gene promoter, which can be used for the prediction of forensic age and assistant diagnosis of clinical diseases.

Clioquinol induces autophagy by down-regulation of calreticulin in human neurotypic SH-SY5Y cells.

Clioquinol (CQ) is considered as a promising drug of neurodegenerative diseases. However, the underlying mechanism is unclear. Our previous study has proved that CQ induces S-phase cell cycle arrest through the elevation of intracellular calcium concentration ([Ca2+]i) with high levels of SERCA2. Furthermore, it could induce autophagy in an intracellular calcium independent manner in human neurotypic SH-SY5Y cells. In this study, the involvement of calreticulin (CRT) in autophagy induced by CQ was investigated. Our results illustrated the endoplasmic reticulum (ER) stress induced by CQ and DTT led to the cell death in different manners. DTT, an ER stress positive control, induced UPR accompanied with up-regulation of CRT and apoptosis, while CQ inhibited UPR accompanied with down-regulation of CRT，resulting in autophagy. Then, overexpression of CRT was shown to cause UPR and decrease [Ca2+]i, leading to cell apoptosis and inhibition of S-phase arrest induced by CQ. While the UPR was alleviated and autophagy was further enhanced in CRT deficient cells by using targeted siRNA. Meanwhile, down-regulation of CRT resulted in [Ca2+]i overload and induction of S-phase arrest. Finally, we found that the effect of CQ on the HT22 cells was similar to that on the SH-SY5Y cells. Our data showed for the first time that CQ decreased expression of CRT, leading to autophagy, an increase of [Ca2+]i, and cell S-phase arrest in the neurotypic cells. The present study describes the cellular signal pathways regulating autophagy by CQ and highlights the potential therapeutic application of CQ in neurodegenerative disorders.

Identification of three novel new psychoactive substances 4F-AB-BUTINACA, AB-PHETINACA, and 2F-NENDCK.

The identification of new psychoactive substances (NPS) is an active and cutting-edge topic in forensic science. With the emergence of a large number of NPS, their timely identification to prevent spread can pose a challenge to clinical and forensic toxicology laboratories. Three emerging NPS had been identified in recently seized materials, including two synthetic cannabinoids [N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobutyl)-1H-indazole-3-carboxamide (4F-AB-BUTINACA) and N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-phenethyl-1H-indazole-3-carboxamide (AB-PHETINACA)] and a ketamine-like substance [2-(2-fluorophenyl)-2-(ethylamino) cyclohexan-1-one(2F-NENDCK)]. The three compounds were first identified by Fourier transform infrared spectrometry (FT-IR), gas chromatography-mass spectrometry (GC-MS), ultrahigh-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (UHPLC-QTOF-MS), and nuclear magnetic resonance (NMR). These data may assist forensic analysts in analyzing the same substances or their homologous compounds.

Antimicrobial and anticancer activities of Hainan dry noni fruit alcoholic extracts and their novel compounds identification using UPLC-Q-Exactive Obitrap-MS/MS.

Morinda citrifolia Linn (noni) is an important plant in the Pacific Asian region. The fruit has been used as a food source and has shown therapeutical benefits for health. Recently, it has become a source for bioactive compounds. In this study, we investigated the antimicrobial and anticancer activities of alcoholic extracts of Hainan dry noni fruit with machinery assistance and identified their novel compounds by UPLC-Q-Exactive Obitrap-MS/MS. By IE extractor aided method, the extraction of both NFE (Noni Fruit Ethanol) and NFM (Noni Fruit Methanol) solvent crude sample extracts were obtained with recovery yields of 98.48% and 71.65%, respectively. The antimicrobial effect of the crude extracts was subjected to disc diffusion test screening against two microbial strains bacterium SA (Staphylococcus aureus) and, fungal CA (Candida albicans). The MIC values of SA and CA were 35.34 and 47.80 mg/mL for NFE, 117.40 and 108.01 mg/mL for NFM, respectively. Further on, cell viability assay showed that IC50 values of extract NFE and NFM on human UMUC-3 bladder carcinogenic cells were 865.1 and 789.1 µg/mL with less effect to human SVHUC-1 normal cell line for 72hr incubation. Using UPLC-Q-exactive Orbitrap-MS/MS, ten compounds were identified in the noni extracts and confirmed from the HMDB and FooDB. Five known bioactive compounds had been used for treatments in anti-cancer, anti-obesity, and Covid-19 patients. The remaining five compounds were found novel in noni fruit. They were Cyanidin 3-(2 G-xylosylrutinoside), Inulobiose, Clausarinol, Pectachol, and 4,7-Megastigmadien-9-ol. The potential bioactivities of these novel compounds will be studied in the near future. These findings form a basis on screening natural medicinal plant extracts for beneficial use as a food and health source.

Effect of berberine on copper and zinc levels in chickens infected with Eimeria tenella.

Berberine, a traditional Chinese medicine, was found to exhibit anticoccidial activity. However, its mechanism is unclear. Trace metals such as copper and zinc are extremely low (less than 0.01% of the total weight of the body) but play a vital role in organisms. In the present study, we investigated the effect of berberine on copper and zinc levels in chickens infected with Eimeria tenella. Firstly, our data confirmed that infected chickens with E. tenella exhibited classic impairment on the 8th day of post infection, such as weight loss and increased feed conversion. Further study showed that E. tenella infection decreased the contents of copper and zinc in the liver and serum of chickens. Berberine was similar to amprolium and significantly improved the pathogenic conditions. Berberine could restore copper and zinc imbalance caused by E. tenella in chickens to a large extent. Studies on the development of cecum lesions demonstrated that the protective effect of berberine on the intestinal cecum was similar to that of the Cu/Zn mixture. Additionally, the mRNA expression of several metal transport related genes of the chick small intestine, including zinc transporter 1, copper transporter 1 and divalent metal ion transporter 1, was elevated by the treatment with berberine. Taken together, we speculate that the anticoccidial activity of berberine may be related to the maintenance of certain metals (Cu/Zn) homeostasis by affecting mRNA expression of their transport genes. However, the mode of action of BBR on these vital metals in the chicks infected with E. tenella still needs to be further studied.

Rapid quantitation of three synthetic cathinones in urine by magnetic dispersive solid-phase extraction combined with DART-HRMS.

For the rapid quantitation of three synthetic cathinones, namely 1-(4-chlorophenyl)-2-(1-pyrrolidinyl)pentan-1-one (4-Cl-α-PVP), 1-(4-methylphenyl)-2-(methylamino)pentan-1-one (4-MPD), and 1-(5,6,7,8-tetrahydronaphthalen-2-yl)-2-(1-pyrrolidinyl)pentan-1-one (ß-TH-naphyrone), in urine, a new method was established using magnetic dispersive solid-phase extraction (MDSPE) combined with direct analysis in real time and high-resolution mass spectrometry (DART-HRMS). Methcathinone-D3 and proadifen (SKF525A) were used as the internal standards. Hydrophobic magnetic adsorbents were used and consisted of hydrophobic functional group (divinylbenzene) and hydrophilic functional group (vinylpyrrolidone) at a ratio of 3 : 1, and NaH2PO4//NaOH buffer (0.2 M, pH 7) was used in MDSPE. Detection was conducted by DART-HRMS in less than 1 min. For 4-Cl-α-PVP, 4-MPD and ß-TH-Naphyrone, the limits of detection were 0.1 ng mL-1, 0.05 ng mL-1 and 0.1 ng mL-1, and the linear ranges were 0.5-100 ng mL-1, 0.2-100 ng mL-1 and 0.2-100 ng mL-1, respectively. The correlation coefficients were all greater than 0.99. The precision and deviation of accuracy were all within ±15%, and the stability of the samples was high under various conditions. The method was successfully applied to detect 4-Cl-α-PVP, 4-MPD and ß-TH-naphyrone in rat urine after subcutaneous administration. In summary, a fast and convenient detection method was established, providing new and effective technical support for the rapid quantitation of three synthetic cathinones (4-Cl-α-PVP, 4-MPD and ß-TH-Naphyrone) for forensic purposes.

BSC2 enhances cell resistance to AmB by inhibiting oxidative damage in Saccharomyces cerevisiae.

Amphotericin B has been the gold standard for the treatment of invasive mycosis for many years. Its resistance mechanisms are reported to be mainly related to the decrease of ergosterol content or the changes of cell wall. Previous study has shown that Saccharomyces cerevisiae strain lack of BSC2 was sensitive significantly to Amphotericin B. In the present study, the role of BSC2 on Amphotericin B resistance were investigated. We found that BSC2 enhanced the resistance of yeast cells to Amphotericin B, which was not related to cellular ergosterol content. BSC2 can maintain the permeability of mitochondrial membrane and cell membrane integrity by inhibiting the accumulation of intercellular reactive oxygen species and alleviating the production of lipid peroxidation and superoxide radical. These alterations were attributed to the enhancement of the activities of superoxide dismutase, catalase and glutathione peroxidase, and the increased glutathione content. Taken together, BSC2 inhibits oxidative damage induced by Amphotericin B through increasing activities of antioxidant enzymes and levels of GSH to alleviate the accumulation of reactive oxygen species, lipid peroxidation and superoxide radical, resulting in the maintenance of mitochondrial membrane potential and cell membrane integrity. However, Amphotericin B resistance mediated by BSC2 is independent of Yap1p, GSH1 and Hog1p. The results demonstrate for the first time that BSC2 enhances cell resistance to Amphotericin B by inhibiting oxidative damage in yeast. Our findings improve current understanding of the mechanism of Amphotericin B resistance and provide potential strategy for reducing Amphotericin B resistance.

Effect of cadmium on mRNA mistranslation in Saccharomyces cerevisiae.

Although highly accurate molecular processes and various messenger RNA (mRNA) quality control and ribosome proofreading mechanisms are used by organisms to transcribe their genes and maintain the fidelity of genetic information, errors are inherent in all biological systems. Low-level translation errors caused by an imbalance of homologous and nonhomologous amino acids caused by stress conditions are particularly common. Paradoxically, advantageous phenotypic diversity can be generated by such errors in eukaryotes through unknown molecular processes. Here, we found that the significant cadmium-resistant phenotype was correlated with an increased mistranslation rate of the mRNA in Saccharomyces cerevisiae. This phenotypic change was also related to endogenous sulfur amino acid starvation. Compared with the control, the mistranslation rate caused by cadmium was significantly increased (p < .01). With the increase of cysteine contents in medium, the mistranslation rate of WT(BY4742a) decreased significantly (p < .01). This demonstrates that cadmium treatment and sulfur amino acid starvation both can induce translation errors. Although cadmium uptake is independent of the Sul1 transporter, cadmium-induced mRNA mistranslation is dependent on the sulfate uptake of the Sul1p transporter. Furthermore, cadmium-induced translation errors depend on methionine biosynthesis. Taken together, cadmium causes endogenous sulfur starvation, leading to an increase in the mRNA mistranslation, which contributes to the resistance of yeast cells to cadmium. We provide a new pathway mediating the toxicity of cadmium, and we propose that altering mRNA mistranslation may portray a different form of environmental adaptation.

Succession of oral microbiota community as a tool to estimate postmortem interval.

The establishment of postmortem interval is one of the most important aspects of forensic expertise. Microbes may provide a novel way to estimate the postmortem intervals in order to avoid many of these limitations. The oral cavity harbors one of the most diverse microbiomes that play a key role in the decomposition of corpses. In this study, the oral bacterial community showed obvious changes in relative abundance during the process of mice decomposition. Meanwhile, at different taxonomic levels, specific bacteria were found to be significantly correlated with the postmortem interval. Linear regression models between relative abundance and the postmortem interval were constructed. Among these species, Gamma-proteobacteria and Proteus were the best ones that can be used to infer the postmortem interval, especially late postmortem interval. Therefore, we suggest that succession of oral microbial community can be developed as a forensic tool for estimating the postmortem interval.

Studies of hTERT DNA methylation assays on the human age prediction.

As an important aspect of epigenetics, DNA methylation has been proven to be suitable for forensic DNA analysis. By detecting changes in DNA methylation, it is desirable to construct a model of age patterns associated with it to infer the age of the individual. The hTERT gene methylation is closely related to tumors, but there are few reports on the relationship between hTERT gene promoter methylation and age. In this study, we utilized the methylation-specific polymerase chain reaction and real-time PCR (relative quantification and absolute quantification) approach to explore the connection between hTERT DNA methylation and age prediction. We fit three models for age prediction based on methylation assay for 90 blood samples from donors aged 1-79 years old. Among them, the model of absolute quantification of real-time enabled the age prediction with R2 = 0.9634. We verified the linear regression model with a validation set of 30 blood samples where prediction average error was 4.29 years. Generally, this reliable method improves the DNA methylation analysis of forensic samples.

Thermo-Responsive Graphene Oxide/Poly(Ethyl Ethylene Phosphate) Nanocomposite via Ring Opening Polymerization.

An efficient strategy for growing thermo-sensitive polymers from the surface of exfoliated graphene oxide (GO) is reported in this article. GO sheets with hydroxyls and epoxy groups on the surface were first prepared by modified Hummer's method. Epoxy groups on GO sheets can be easily modified through ring-opening reactions, involving nucleophilic attack by tris(hydroxymethyl) aminomethane (TRIS). The resulting GO-TRIS sheets became a more versatile precursor for next ring opening polymerization (ROP) of ethyl ethylene phosphate (EEP), leading to GO-TRIS/poly(ethyl ethylene phosphate) (GO-TRIS-PEEP) nanocomposite. The nanocomposite was characterized by ¹H NMR, Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA), differential thermal gravity (DTG), transmission electron microscopy (TEM) and atomic force microscopy (AFM). Since hydrophilic PEEP chains make the composite separate into single layers through hydrogen bonding interaction, the dispersity of the functionalized GO sheets in water is significantly improved. Meanwhile, the aqueous dispersion of GO-TRIS-PEEP nanocomposite shows reversible temperature switching self-assembly and disassembly behavior. Such a smart graphene oxide-based hybrid material is promising for applications in the biomedical field.

An enzyme-free FRET nanoprobe for ultrasensitive ketamine detection based on ATP-fueled target recycling.

Ketamine is a commonly abused drug due to its stimulant, dissociative and hallucinogenic effects. An overdose of ketamine has been found to cause a variety of side effects. Therefore, the identification and quantification of ketamine are of significant importance for clinical purposes and drug seizing. However, conventional methods for ketamine detection possess some disadvantages such as sophisticated procedures, expensive instruments and low sensitivity. Herein, we develop a novel fluorescent nanoprobe for ultrasensitive ketamine detection with signal amplification based on Adenosine Triphosphate (ATP)-fueled target recycling and FRET (fluorescence resonance energy transfer) occurring between the FAM (Fluorescein, tagged with Y-shape DNA) and AuNPs. Based on the combination of FRET and signals circle amplification, the gold nanospheres functionalized with Y-motif DNA (Y@AuNPs) nanoprobe was utilized for effective ketamine detection with the limit of detection (LOD) down to 3 pg mL-1, which was lower than previously reported. Furthermore, the high sensitivity of Y@AuNPs facilitated quantitative analysis in biological media and practical samples.

A lateral flow strip based on gold nanoparticles to detect 6-monoacetylmorphine in oral fluid.

We used lateral flow strips based on gold nanoparticles to detect 6-monoacetylmorphine (6-MAM; heroin's unique metabolite) in oral fluid samples. In this competitive lateral chromatographic immunoassay, the 6-MAM was chemically synthesized and conjugated to bovine serum albumin. The results were qualitatively detected via the colour change of the test line. By using a proper sample pad, a suitable nitrocellulose membrane and a customized sponge device adsorbed the oral fluid directly from the mouth; the total test time was 3 min. The sensitivity of the assay was 4.0 ng ml-1 without any cross-reactivity with 10 normal drugs, which are widely subject to abuse, including morphine and codeine. This test could be easily used on site to detect heroin in oral fluid, and it could be a promising product in the future including for driving under the influence.

Iron depletion participates in the suppression of cell proliferation induced by lipin1 overexpression.

Lipin1 participates in numerous cellular processes, including in the dephosphorylation of phosphatidic acid to diacylglycerol and as a co-transcriptional regulator. Iron is also essential in various critical biological processes. Previous studies have shown that compared to normal tissue cells, lipin1 expression and iron metabolism are abnormal in cancer cells. However, the involvement of lipin1 in the regulation of iron metabolism is unknown. In this study, we compared the contents of eight metal ions (potassium, calcium, sodium, magnesium, manganese, zinc, iron and copper) in human hepatoma carcinoma BEL7402 control cells as well as stable cells overexpressing lipin1 by using ICP-AES. Our results showed that only intracellular iron content was significantly decreased by lipin1 overexpression. Meanwhile, we observed that lipin1 overexpression could inhibit cell proliferation, similar to iron chelator deferoxamine. Western blotting showed that the up-regulation of p53-p21-p27 elicited cell cycle G0/G1 arrest in the stable cells overexpressing lipin1. Conversely, after lipin1 was down regulated with siRNA, we found that cell proliferation was promoted, accompanied by an increase in iron content, and the downregulation of p53 and p21. Our data indicate that lipin1 overexpression may cause reduction of intracellular iron content, which could activate the p53-p21-p27 signaling pathways, leading to cell cycle arrest at the G0/G1 phase in the hepatic carcinoma cells. Subsequently, we identified the putative cause for the decrease of the intracellular iron content induced by lipin1 overexpression. Our results suggested that the intracellular iron reduction was due to the increase in the expression of ferroportin, an iron export protein in the stable cells overexpressing lipin1. In contrast, after transfection with lipin1 siRNA, the decreased expression of ferroportin contributed to an increase in the iron content in BEL7402 cells. It was further confirmed that the intracellular iron content was increased after ferroportin was knocked down by siRNA in BEL7402 cells. Taken together, our findings demonstrate for the first time that lipin1 participates in the regulation of iron metabolism in human hepatic carcinoma cells. This suggests that lipin1 may play an important protective role in inhibiting the development of cancer through the reduction of iron content in tumors, which further demonstrates that iron reduction could be a potential strategy of cancer prevention and treatment.

Two Fatal Intoxications with Cyanohydrins.

Cyanohydrins, also be called cyanoalcohols, are important industrial precursors to carboxylic acids and some amino acids. Acetone cyanohydrin (ACH) and formaldehyde cyanohydrin (glycolonitrile, FCH), which are the typical examples of cyanohydrins, are classified as extremely hazardous substances. As the cyanohydrins can readily decompose, and it is hard to find cyanohydrins in gastric contents and heart blood, the determination study in biological samples can be divided into two parts: the first is the determination of HCN by using a Prussian blue reaction and the HS-GC-MSD after derivatization by chloramine-T. The second is the determination of acetone or formaldehyde. In this part, headspace gas chromatography with flame ionization detector (HS-GC-FID) and solid phase microextraction (SPME)-gas chromatography with mass spectrometric detectors (GC-MSD) had been used. In this report, we reported two fatal intoxication cases of ACH and FCH; one person was killed by his wife by poisoning his food and the other was suicide by poison. Two real cases of ACH and FCH in human blood and gastric contents have been analyzed by using the above-mentioned method. The Prussian blue reaction was positive in the two cases. The peaks of acetone with retention times of 0.998 min appear in specimens of the deceased are consistent with the retention times of pure acetone. The peaks of formaldehyde with a retention time of 1.658 min appear in heart blood of the deceased, and the retention time of formaldehyde of the liquid is 1.674 min, which are consistent with the retention times of pure formaldehyde (1.673 min).

In vitro selection of DNA aptamers binding pesticide fluoroacetamide.

Fluoroacetamide (Mw = 77.06) is a lethal rodenticide to humans and animals which is still frequently abused in food storage somewhere in China. The production of antibodies for fluoroacetamide is difficult due to its high toxicity to animals, which limits the application of immunoassay method in poison detection. In this work, aptamers targeting N-fluoroacetyl glycine as an analog of fluoroacetamide were selected by a specific systematic evolution of ligands by exponential enrichment (SELEX) strategy. The binding ability of the selected aptamers to fluoroacetamide was identified using surface plasmon resonance (SPR)-based assay. The estimated KD values in the low micromolar range showed a good affinity of these aptamers to the target. Our work verified that the SELEX strategy has the potential for developing aptamers targeted to small molecular toxicants and aptamers can be employed as new recognition elements instead of antibodies for poison detection.

[DNA aptamer selection in vitro for determining ketamine by FluMag-SELEX].

OBJECTIVE: To select specific DNA aptamer for determining ketamine by FluMag-SELEX. METHODS: Based on magnetic beads with tosyl surface modification as solid carrier and ketamine as target, a random ssDNA library with total length of 78 bp in vitro was compounded. After 13 rounds screening, DNA cloning and sequencing were done. Primary and secondary, structures were analyzed. The affinity, specificity and Kd values of selected aptamer were measured by monitoring the fluorescence intensity. RESULTS: Two ssDNA aptamers (Apt#4 and Apt#8) were successfully selected with high and specific abilities to bind ketamine as target with Kd value of 0.59 and 0.66 µmol/L. The prediction of secondary structure was main stem-loop and G-tetramer. The stem was the basis of stability of aptamer's structure. And loop and G-tetramer was the key of specific binding of ketamine. CONCLUSION: FluMag-SELEX can greatly improve the selection efficiency of the aptamer, obtain the ketamine-binding DNA aptamer, and develop a new method for rapid detection of ketamine.

Determination of ecgonine and seven other cocaine metabolites in human urine and whole blood by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry.

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.

Extending the detection window of diazepam by directly analyzing its glucuronide metabolites in human urine using liquid chromatography-tandem mass spectrometry.

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.