Determination of the R- and S-enantiomers of methylone and ethylone in seized drugs by enantioselective liquid chromatography tandem mass spectrometry analysis.

Synthetic cathinones constitute a major class of the new psychoactive substances (NPS) that have come to dominate recreational drug abuse in recent years. As synthetic cathinones are all chiral molecules, the use of chiral analysis to determine their enantiomeric ratios (ER) in seized drug samples can assist in the identification of the precursor chemicals and manufacturing processes used in production, thereby contributing to drug intelligence. In addition, as different enantiomers of synthetic cathinones vary in biological activity and toxicology, chiral analysis can support research into the physiological properties and side effects of seized drugs, thus enabling better treatment for overdose or addiction. Therefore, suitable methods for chiral separation and quantitative analysis of synthetic cathinones in forensic laboratories are needed. This study describes the separation of R- and S-enantiomers of methylone and ethylone, two of the most commonly abused synthetic cathinones, by liquid chromatography (LC) using a Lux AMP polysaccharide-based chiral column, followed by tandem mass spectrometry analysis. Enantiomeric separation was achieved in 13.4min, and the precision, accuracy, carryover, detection limit, and quantification limit of this method were determined. A total of 39 seized drug samples were analyzed in this study, and the results yielded ER of 0.97-1.09 for methylone and 0.87-1.17 for ethylone. The purity of methylone or ethylone was lower in instant coffee bags than in other package types. The ER and purity profiles can be useful for forensic science and drug intelligence.

Tyrosine kinase inhibitors modulate dendritic cell activity via confining c-Kit signaling and tryptophan metabolism.

Dendritic cell (DC)-based vaccine has been established in tumor immunotherapy. Importantly, the efficiency of anti-tumor T-cells in draining lymph nodes is dependent on the status of DCs surrounding in tumors. It has been shown that Indoleamine 2,3-dioxygenase (IDO) plays a key role to induce tolerogenic DCs in tumor microenvironment, and tyrosine kinase inhibitors (TKIs) can suppress the function of IDO in DCs. However, the stimulatory effect of TKI-modified DCs on T cells remains unclear. In this report, we found that one type of TKI-dasatinib can modify DCs to increasing the activation of allogenic T cells. These TKI-modified DCs delayed the onset of B16 melanoma progression in mice. In mechanistic studies, TKIs did not increase the maturation but reduce the expression and phosphorylation levels of IDO and IDO mediated tryptophan metabolism in DCs. In addition, the suppressive effect of TKIs on tryptophan metabolism may be caused by blocking c-Kit pathway in DCs. Furthermore, the increased phosphorylation of general control nonderepressible (GCN2) and decreased expression of aryl hydrocarbon receptor (AhR)/aryl hydrocarbon receptor nuclear translocator (ARNT) were observed in the T cells activated by TKI-modified DCs, suggesting the enhancement of effector function of T cells. These results indicate that TKI could be used to modulate DC immunogenic activity and may potentially be applied in DC-based cancer immunotherapy.

Effects of Copayment in Long-Term Care Insurance on Long-Term Care and Medical Care Expenditure.

OBJECTIVE: This study aimed to clarify the difference in (1) long-term care (LTC) usage and expenditure and (2) medical care service usage and expenditure before and after the change in the copayment limit for qualifying individuals from 10% to 20%. SETTING AND PARTICIPANTS: This quasi-experimental longitudinal design used the database from 1 prefecture of Japan that included 570,434 person-month records of 23,879 insured individuals (in August 2014) who used LTC services between August 2014 and July 2015 and were aged 65 years and older on August 1, 2014. METHODS: We conducted difference-in-difference estimations to compare "before" and "after" outcome differences between insured individuals whose LTC copayment increased to 20% and those whose copayment remained at 10%. Sex, age, Care Needs Level, subsidy, and public assistance were adjusted in the models, along with robustness checks. RESULTS: Differences in both insurer's payment and insured's copayment indicated statistical significance between those whose copayment increased and those whose copayment did not increase. We found no significant difference in the number of minutes of home care service use, days of facility care service use, and LTC expenditures among those with copayment increases as well as those with no increase in copayment following the insured's copayment increase policy implementation. In contrast, the policy implementation caused significant differences in the number of days of hospitalization, medical care expenditures, and total expenditures. CONCLUSIONS AND IMPLICATIONS: The increase in insured individuals' copayment decreased LTC insurer's payment. However, total LTC expenditure increased over time although the increase trend slowed down in the treatment group after the copayment increase policy implemented. Besides, medical care expenditure increased consistently among insured individuals whose copayment increased. As there appears to be a "balloon effect" between LTC and medical care services, it is important to discuss the medical care system while considering the LTC insurance system comprehensively.

The Cholesterol-Modulating Effect of Methanol Extract of Pigeon Pea (Cajanus cajan (L.) Millsp.) Leaves on Regulating LDLR and PCSK9 Expression in HepG2 Cells.

Pigeon pea (Cajanus cajan (L.) Millsp.) is a legume crop consumed as an indigenous vegetable in the human diet and a traditional medicinal plant with therapeutic properties. The current study highlights the cholesterol-modulating effect and underlying mechanisms of the methanol extract of Cajanus cajan L. leaves (MECC) in HepG2 cells. We found that MECC increased the LDLR expression, the cell-surface LDLR levels and the LDL uptake activity in HepG2 cells. We further demonstrated that MECC suppressed the proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and protein expression, but not affected the expression of other cholesterol or lipid metabolism-related genes including inducible degrader of LDLR (IDOL), HMG-CoA reductase (HMGCR), fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC1), and liver X receptor-α (LXR-α) in HepG2 cells. Furthermore, we demonstrated that MECC down-regulated the PCSK9 gene expression through reducing the amount of nuclear hepatocyte nuclear factor-1α (HNF-1α), a major transcriptional regulator for activation of PCSK9 promoter, but not that of nuclear sterol-responsive element binding protein-2 (SREBP-2) in HepG2 cells. Finally, we identified the cajaninstilbene acid, a main bioactive stilbene component in MECC, which significantly modulated the LDLR and PCSK9 expression in HepG2 cells. Our current data suggest that the cajaninstilbene acid may contribute to the hypocholesterolemic activity of Cajanus cajan L. leaves. Our findings support that the extract of Cajanus cajan L. leaves may serve as a cholesterol-lowering agent.

Systematic Evaluation and Validation of Benzodiazepines Confirmation Assay Using LC-MS-MS.

Benzodiazepines is a large drug group used extensively to treat sleep disorders and anxiety; these drugs are frequently associated with various crimes such as murder and drug-facilitated sexual assault. With growing use and misuse of these compounds, confirmatory assays are increasingly required in clinical laboratories. In this study, 4 ß-glucuronidase enzymes were systematically evaluated for their hydrolysis efficiencies. Additionally, the matrix effects and extraction recoveries of three protein precipitation plates were systematically evaluated. The recombinant IMCSzyme ß-glucuronidase enzyme showed higher hydrolysis efficiency than did ß-glucuronidase from abalone, Patella vulgata and Helix pomatia. Lower ion suppression and more favorable overall recovery were observed in the Supelco protein precipitation plate than in the two other plates. Analytes were separated on a superficially porous particle column (ultra biphenyl) within 4.5 min and characterized through electrospray ionization-tandem mass spectrometry in the positive ion multiple-reaction monitoring mode. The accuracy, carryover, extraction recovery, detection limit, matrix effect, quantification limit and precision of the method were validated. Sixty opioid-positive urine samples were analyzed; a high incidence of benzodiazepines was detected in these samples. The proposed technique is robust and suitable for efficiently monitoring the numerous benzodiazepines that occur in urine samples.

Biological roles of indole-3-acetic acid in Acinetobacter baumannii.

Indole-3-acetic acid (IAA) is an important plant hormone, and many types of bacteria interact with plants by producing or degrading IAA in the rhizosphere. The iac (indole-3-acetic acid catabolism) gene locus in Acinetobacter baumannii ATCC19606 was previously associated with IAA degradative capability, and in this study, transcriptome analysis results derived from A. baumannii cultured with IAA showed that the expression of catechol-degrading and phenylacetate-degrading genes was elevated, indicating that IAA is likely degraded through these pathways. This study further found that A. baumannii also has IAA productive capability, primarily involving the ipdC gene, and transcriptome and spent media analysis of wild-type and mutant cultures grown in minimal media revealed that A. baumannii likely produces IAA through the indole-3-pyruvic acid (IPyA) pathway. Exogenously applied IAA improved tolerance against oxidative stress in wild-type A. baumannii and iacA mutants unable to degrade IAA, but not in ipdC mutants incapable of producing IAA, suggesting that endogenous IAA is important for stress tolerance. Meanwhile, ipdC mutants also had reduced virulence against human A549 epithelial cells as compared to wild-type. Endogenously produced IAA was found to enhance root growth in A. baumannii and kidney bean plant co-cultures, indicating that A. baumannii can interact with plants through the production and degradation of IAA. Taken together, this study sheds light on the biosynthesis pathways and functional significance of IAA in A. baumannii, and may be useful in exploring other IAA-mediated plant-microbe interactions as well.

Development of a risk score for the prediction of incident dementia in older adults using a frailty index and health checkup data: The JAGES longitudinal study.

In Japan, the prevalence of dementia is expected to reach 4.7 million by 2025. This study aimed to develop a risk score for the prediction of incident dementia in community-dwelling older adults. In this longitudinal observational study, we used data from the Japan Gerontological Evaluation Study (JAGES) conducted in K City. We performed Cox regression analyses to develop three risk score models for the prediction of incident dementia in older adults using a frailty index and health checkup data. Analyses of the area under the receiver operating characteristic curve were conducted to compare the models' predictive abilities. We identified 6656 (9.2%) individuals who developed incident dementia during the observation period. The C-statistics of the risk scores ranged from 0.733 to 0.790. The risk score models were able to predict incident dementia in older adults and may help non-medical professionals detect dementia risk at an early stage.

Survival analysis of increases in care needs associated with dementia and living alone among older long-term care service users in Japan.

BACKGROUND: Japan is known for its long life expectancy and rapidly aging society that there are various demands of older adults need to be fulfilled with, and one of them is long-term care needs. Therefore, Japan implemented the Long-Term Care Insurance in year 2000 for citizens who are above 65-year old and citizens who are above 40-year old in needs of long-term care services. This study was undertaken to longitudinally examine the influence of dementia and living alone on care needs increases among older long-term care insurance service users in Japan. METHODS: Long-term care insurance claims data were used to identify enrollees who applied for long-term care services between October 2010 and September 2011, and subjects were tracked until March 2015. A Kaplan-Meier survival analysis was conducted to examine increases in care needs over time in months. Cox regression models were used to examine the effects of dementia and living alone on care needs increases. RESULTS: The cumulative survival rates before care needs increased over the 4.5-year observation period were 17.6% in the dementia group and 31.9% in the non-dementia group. After adjusting for age, sex, care needs level, and status of living alone, the risk of care needs increases was found to be 1.5 times higher in the dementia group. Living alone was not a significant risk factor of care needs increases, but people with dementia who lived alone had a higher risk of care needs increases than those without dementia. CONCLUSION: Dementia, older age, the female sex, and lower care needs levels were associated with a higher risk of care needs increases over the study period. Among these variables, dementia had the strongest impact on care needs increases, especially in persons who lived alone.

Rapid and sensitive detection of carbapenemase activity in Acinetobacter baumannii using superficially porous liquid chromatography-tandem mass spectrometry.

BACKGROUND: The emergence and spread of carbapenem-resistant Acinetobacter baumannii poses a challenge for optimizing antibiotic therapies and preventing outbreaks. Traditional phenotypic assays such as the modified Hodge test (MHT) or polymerase chain reaction (PCR)-based detection of the carbapenemase genes are time-consuming and complicated. Therefore, new approaches for the efficient detection of carbapenemase-producing A. baumannii are urgently required. METHODS: In this study, we used the superficially porous liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure carbapenem hydrolysis in a solution spiked with test strains of A. baumannii. The rate of carbapenem hydrolysis during incubation was expressed as the ratio of the carbapenem peak area of the test A. baumannii strains to the noncarbapenemase-producing A. baumannii ATCC 17978. This method can accurately measure the carbapenem hydrolysis rate and, therefore, can effectively identify carbapenemase-producing strains within 75 minutes. RESULTS: A total of 112 A. baumannii strains were used in this study, including 103 clinical isolates with 68 carbapenem-resistant strains and 35 carbapenem-susceptible strains, seven ATCC strains and two selected mutants. The results of the superficially porous LC-MS/MS assay showed higher detection sensitivity compared to the results of the MHT. CONCLUSION: Our results demonstrate the ability of the former method to routinely detect carbapenemase-producing A. baumannii.

Highly Selective Dissociation of a Peptide Bond Following Excitation of Core Electrons.

The controlled breaking of a specific chemical bond with photons in complex molecules remains a major challenge in chemistry. In principle, using the K-edge absorption of a particular atomic element, one might excite selectively a specific atomic entity in a molecule. We report here highly selective dissociation of the peptide bonds in N-methylformamide and N-methylacetamide on tuning the X-ray wavelength to the K-edge absorption of the atoms connected to (or near) the peptide bond. The high selectivity (56-71%) of this cleavage arises from the large energy shift of X-ray absorption, a large overlap of the 1s orbital and the valence π* orbital that is highly localized on a peptide bond with antibonding character, and the relatively low bond energy of the peptide bonds. These characteristics indicate that the high selectivity on bond dissociation following core excitation could be a general feature for molecules containing peptide bonds.

Systematic profiling of indole-3-acetic acid biosynthesis in bacteria using LC-MS/MS.

Indole-3-acetic acid (IAA) is produced from tryptophan through five synthesis pathways. A comprehensive method for the quantification of IAA and biosynthesis-related intermediates in a culture medium was developed. Sample preparation was simple with protein precipitation. The analytes were separated on a superficially porous C18 silica column and detected by electrospray ionization-tandem mass spectrometry in the positive ion multiple reaction monitoring mode. The limit of detection was 0.05 µM, and the lower limits of quantification ranged from 0.05 to 2 µM. The intra-day and inter-day precision and accuracy were less than 13.96%. Ion suppression was observed, and the deuterated internal standards were used to compensate for the matrix effect. The method was applied to analyze changes in tryptophan catabolism in a culture medium of Pseudomonas putida. The proposed method is robust and suitable for the systematic profiling of IAA biosynthesis in culture supernatant.

The effects of dementia and long-term care services on the deterioration of care-needs levels of the elderly in Japan.

To investigate the associations between dementia, the use of long-term care (LTC) services, and the deterioration of care-needs levels of elderly persons in Japan. Using a retrospective cohort study, we analyzed 50,268 insurance beneficiaries aged 65 years and older who had utilized LTC services between 2010 and 2011 in Kyoto prefecture, Japan. Logistic regression analyses were used to identify predictors of care-needs level deterioration. Dementia, facility care services, the male sex, older age, and lower baseline care-needs levels were associated with care-needs level deterioration. The disparity between odds ratios of home care services, dementia diagnoses, and facility care services on care-needs level deterioration diminished with increasing baseline care-needs levels. The other risk factors of care-needs level deterioration showed stronger associations as care-needs levels and age increased. The effects of baseline care-needs levels and dementia should be considered when developing LTC policies.

Transcriptome profiling in imipenem-selected Acinetobacter baumannii.

BACKGROUND: Carbapenem-resistance in Acinetobacter baumannii has gradually become a global challenge. To identify the genes involved in carbapenem resistance in A. baumannii, the transcriptomic responses of the completely sequenced strain ATCC 17978 selected with 0.5 mg/L (IPM-2 m) and 2 mg/L (IPM-8 m) imipenem were investigated using RNA-sequencing to identify differences in the gene expression patterns. RESULTS: A total of 88 and 68 genes were differentially expressed in response to IPM-2 m and IPM-8 m selection, respectively. Among the expressed genes, 50 genes were highly expressed in IPM-2 m, 30 genes were highly expressed in IPM-8 m, and 38 genes were expressed common in both strains. Six groups of genes were simultaneously expressed in IPM-2 m and IPM-8 m mutants. The three gene groups involved in DNA recombination were up-regulated, including recombinase, transposase and DNA repair, and beta-lactamase OXA-95 and homologous recombination. The remaining gene groups involved in biofilm formation were down-regulated, including quorum sensing, secretion systems, and the csu operon. The antibiotic resistance determinants, including RND efflux transporters and multidrug resistance pumps, were over-expressed in response to IPM-2 m selection, followed by a decrease in response to IPM-8 m selection. Among the genes over-expressed in both strains, blaOXA-95, previously clustered with the blaOXA-51-like family, showed 14-fold (IPM-2 m) to 330-fold (IPM-8 m) over-expression. The expression of blaOXA-95 in IPM-2 m and IPM-8 m cells was positively correlated with the rate of imipenem hydrolysis, as demonstrated through Liquid Chromatography-Mass Spectrometry/Mass Spectrometry, suggesting that blaOXA-95 plays a critical role in conferring carbapenem resistance. In addition, A. baumannii shows an inverse relationship between carbapenem resistance and biofilm production. CONCLUSION: Gene recombination and blaOXA-95 play critical roles in carbapenem resistance in A. baumannii. Taken together, the results of the present study provide a foundation for future studies of the network systems associated with carbapenem resistance.

Improved identification of multiple drugs of abuse and relative metabolites in urine samples using liquid chromatography/triple quadrupole mass spectrometry coupled with a library search.

RATIONALE: Although two multiple reaction monitoring (MRM) transitions per compound are used for identification performed using liquid chromatography/triple quadrupole mass spectrometry (LC/QqQ-MS/MS), differences in identification criteria among several regulations may lead to misidentification. We demonstrated that the use of two MRM transitions and product ion spectra improves compound identification. METHODS: The scan cycle time was reduced using time-scheduled MRM (tMRM), data-dependent product ion scanning, and dynamic exclusion. The quantification and identification performance for 13 drugs of abuse and their metabolites were evaluated. RESULTS: Deuterated internal standards compensated for ion suppression. All analytes exhibited intra- and interday precision <12.11%, accuracy of -10.31% to +10.10%, and no carryover. The LC/QqQ-MS/MS and reference gas chromatography/MS methods were equally precise, accurate, and specific. Several regulatory organizations include two MRM transitions, their ratio, and retention time as identification criteria. In 28 samples, the relative ion ratio variation was >10% and product ion spectral matches with >94% probabilities improved drug and metabolite identification. CONCLUSIONS: The LC/QqQ-MS/MS method is a comprehensive assay in which tMRM and the product ion scan are combined in a single run by using a QqQ mass analyzer to simultaneously quantify amphetamine, ketamine, morphine, and their relative metabolites in urine. The proposed method can be applied in forensic science.

Simultaneous quantification of amphetamine, opiates, ketamine and relative metabolites in urine for confirmatory analysis by liquid chromatography tandem mass spectrometry.

The rise in amphetamine, ketamine and opiates abuse in Taiwan has created a need for a reliable confirmatory assay. A method that combines superficially porous liquid chromatography tandem mass spectrometry (LC-MS/MS) with solid-phase extraction (SPE) was developed for the simultaneous quantification of amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), ketamine, opiates, and their corresponding metabolites in urine. The total run time of the method was 6.7min including equilibration time. The method was validated in accordance with the European Commission (EC) Decision 2002/642/EC. The within- and between-day precision was below 13.6% and the accuracy ranged from -17.1% to +9.9% for all analytes. Ion suppression was observed but compensated by using deuterated internal standards. No carryover was detected and the analytes were stable at room temperature for 16h, and for 72h at 4°C, and three-thaw cycles. The method was further validated by comparison with a reference gas chromatography-mass spectrometry (GC-MS) method, using 52 authentic urine samples. The results indicated that for the target analytes studied, the LC-MS/MS analysis was as precise, accurate, and specific as the GC-MS method. In conclusion, the present LC-MS/MS method is robust and reliable, and suitable for use as a confirmation assay in the simultaneous urine drug testing and quantification of amphetamines, ketamines, and opiates.

Simultaneous determination of opiates, methadone, buprenorphine and metabolites in human urine by superficially porous liquid chromatography tandem mass spectrometry.

For monitoring compliance of methadone or buprenorphine maintenance patient, a method for the simultaneous determination of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), buprenorphine, norbuprenorphine, opiates (morphine, codeine, 6-monoacetylmorphine) in urine by superficially porous liquid chromatography tandem mass spectrometry was developed and validated. After enzyme digestion and liquid-liquid extraction, reverse-phase separation was achieved in 5.2 min and quantification was performed by multiple reaction monitoring. Chromatographic separation was performed at 40 °C on a reversed phase Poroshell column with gradient elution. The mobile phase consisted of water and methanol, each containing 0.1% formic acid, at a flow rate of 0.32 mL/min. Intra-day and inter-day precision were less than 12.1% and accuracy was between -9.8% and 13.7%. Extraction efficiencies were more than 68%. Although ion suppression was detected, deuterated internal standards compensated for these effects. Carryover was minimal, less than 0.20%. All analytes were stable at room temperature for 16 h, 4 °C for 72 h, and after three freeze-thaw cycles. The assay also fulfilled compound identification criteria in accordance with the European Commission Decision 2002/657/EC. We analyzed 62 urine samples from patients received maintenance therapy and found that 54.8% of the patient samples tested were detected for morphine, codeine, or 6-monoacetylmorphine. This method provides a reliable and simultaneous quantification of opiates, maintenance drugs, and their metabolites in urine samples. It facilitates the routine monitoring in individuals prescribed the drug to ensure compliance and help therapeutic process.

Dual regulation by ethanol of the inhibitory effects of ketamine on spinal NMDA-induced pressor responses in rats.

BACKGROUND: Acute exposure of ethanol (alcohol) inhibits NMDA receptor function. Our previous study showed that acute ethanol inhibited the pressor responses induced by NMDA applied intrathecally; however, prolonged ethanol exposure may increase the levels of phosphorylated NMDA receptor subunits leading to changes in ethanol inhibitory potency on NMDA-induced responses. The present study was carried out to examine whether acute ethanol exposure influences the effects of ketamine, a noncompetitive NMDA receptor antagonist, on spinal NMDA-induced pressor responses. METHODS: The blood pressure responses induced by intrathecal injection of NMDA were recorded in urethane-anesthetized rats weighing 250-275 g. The levels of several phosphorylated residues on NMDA receptor GluN1 subunits were determined by western blot analysis. RESULTS: Intravenous injection of ethanol or ketamine inhibited spinal NMDA-induced pressor responses in a dose-dependent and reversible manner. Ketamine inhibition of NMDA-induced responses was synergistically potentiated by ethanol when ethanol was applied just before ketamine. However, ketamine inhibition was significantly reduced when applied at 10 min after ethanol administration. Western blot analysis showed that intravenous ethanol increased the levels of phosphoserine 897 on GluN1 subunits (pGluN1-serine 897), selectively phosphorylated by protein kinase A (PKA), in the lateral horn regions of spinal cord at 10 min after administration. Intrathecal administration of cAMPS-Sp, a PKA activator, at doses elevating the levels of pGluN1-serine 897, significantly blocked ketamine inhibition of spinal NMDA-induced responses. CONCLUSIONS: The results suggest that ethanol may differentially regulate ketamine inhibition of spinal NMDA receptor function depending on ethanol exposure time and the resulting changes in the levels of pGluN1-serine 897.

Synthesis of deuterium labeled ketamine metabolite dehydronorketamine-d4.

A convenient synthesis of ketamine metabolite dehydronorketamine-d(4), starting from commercially available deuterium labeled bromochlorobenzene, was achieved. Key steps include Grignard reaction, regioselective hydroxybromination, Staudinger reduction, and dehydrohalogenation.

Transactivation of protein expression by rice HSP101 in planta and using Hsp101 as a selection marker for transformation.

Plant HSP101 has dual activities, first, in conferring thermotolerance, and secondly, in serving as a translational activator. In this study, we introduced Oryza sativa Hsp101 (osHsp101) cDNA into tobacco by Agrobacterium-mediated transformation. Stable integration and expression of the transgene into the tobacco genome was demonstrated by Southern and Western blot analysis. Overexpression of osHSP101 had no noticeable effect on growth or development of the transgenic plants. Homozygous T(2) transgenic plants with overexpressed osHSP101 survived heat treatment better than untransformed control plants. In addition, taking advantage of conferring basal thermotolerance by plant HSP101, we were able to demonstrate the feasibility of using osHsp101 as a selection marker and select the transformants under high temperature in tobacco leaf disc transformation mediated by Agrobacterium. Furthermore, transgenic tobacco plants with overexpressed osHSP101 were able to enhance luciferase expression up to 2.9-fold more than untransformed plants in the progeny of reciprocally crossed with omega-luciferase reporter lines.

Simultaneous determination of amphetamines and ketamines in urine by gas chromatography/mass spectrometry.

A method for the simultaneous determination of amphetamines and ketamines (ketamine, norketamine and dehydronorketamine) in urine samples by gas chromatography/mass spectrometry was developed and validated. Urine samples were extracted with organic solvent and derivatized with trifluoroacetic anhydride (TFAA). The limits of detection and limits of quantification for each analyte were lower than 19 and 30 ng/mL, respectively. Within-day and between-day precisions were within 0.5% and 10.6%, respectively. Biases for three levels of control samples were within -10.6% and +7.8%. The concentration of dehydronorketamine was greater than those of ketamine or norketamine in 19 of 35 ketamine-positive samples. A group of 110 human urine samples previously determined to contain at least one of the target analytes was analyzed using the new method, and excellent agreement was observed with previous results.