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A recent trend in urine drug testing in forensic and clinical toxicology has been the simultaneous determination of different chemical groups of target drugs, which are selected based on their local popularity. Rapid multiple drug analysis, made possible by the use of liquid chromatography-tandem mass spectrometry (LC/MS/MS), has become more widely used, especially in workplace drug testing. Therefore, in the present study, a method for simultaneously analyzing 35 drugs of abuse and relevant metabolites that are most prevalent in Korea, using LC/MS/MS with polarity switching electrospray ionization, was developed and validated. The drugs and metabolites in urine were extracted by using mixed mode strong cation exchange polymeric solid phase extraction cartridges after enzymatic hydrolysis and were then injected into the LC/MS/MS system. The validation results for selectivity, linearity, intra- and inter-assay precision and accuracy for this method were satisfactory, while the results for matrix effects and recovery showed significant variance among the urine samples from different sources. The limits of detection ranged from 0.1 to 10 ng/ml and the limits of quantification were from 1 to 10 ng/ml. To reduce the matrix effects in authentic samples, two different quantitative approaches were compared: quantification using calibration standards prepared by the drug-free pooled urine matrix and quantification using the standard addition. Of these, the latter method was found to be the most suitable. The method developed in this study will be very useful for forensic and clinical toxicology laboratories to adopt for monitoring the inappropriate use of controlled drugs.

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The transient receptor potential vanilloid 1 (TRPV1) channel, a nonselective Ca(2+) and Na(+) channel, is a molecular transducer of nociceptive stimuli. N-Arachidonoyl dopamine (NADA) and N-oleoyldopamine (OLDA), two unsaturated N-acyldopamines, are major activating endogenous TRPV1 ligands and their presence in mammalian brain tissue has been reported. However, the biological significance of NADA and OLDA remains unknown. To investigate their biological function in the nervous system, a sensitive and accurate quantitative method for determining endogenous NADA and OLDA in the brain is necessary. Thus, a column-switching liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to quantify NADA and OLDA in mouse striatum. Mouse cerebellum tissue in which neither NADA nor OLDA were detected was used as a surrogate matrix to prepare calibrators. NADA and OLDA were extracted from mouse brain tissue by solid-phase extraction and then filtered and analyzed by LC-MS-MS with electrospray ionization in the positive ion mode. The selectivity results and comparison of calibration curves prepared with mouse cerebellum and striatum established that the former was acceptable as the surrogate matrix of the latter for analyzing NADA and OLDA. The validation results of the matrix effect, linearity, precision, accuracy, and stability were satisfactory. The limits of detection and limits of quantification were 0.125 pg mg(-1) for both analytes. This method was sensitive and accurate enough to determine endogenous concentrations of these compounds in mouse striatum and will be very useful for further study of the biological functions of NADA and OLDA and other related factors in vivo.

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The chemical castration law, which targets child molesters with recidivism, was introduced in Korea in 2011. For this, leuprolide, a gonadotropin-releasing hormone agonist, is used to decrease testosterone production and suppress libido. In order to achieve efficient law enforcement, it is necessary to monitor intentional ingestion of drugs that antagonize the effect of leuprolide. Therefore, an analytical method for the simultaneous detection of mirodenafil, sildenafil, tadalafil, udenafil, vardenafil, icariin, alprostadil, and yohimbine, which are the major impotence treatment drugs, legitimately or otherwise, in Korea, as well as their selected metabolites, in human urine was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, different sample preparation methods, two solid-phase extractions with different cartridges and protein precipitation, were compared and protein precipitation was chosen for the entire study because it showed better matrix effects and recoveries. Thus, the drugs and metabolites in urine were extracted by protein precipitation and then filtered and analyzed by LC-MS/MS with polarity switching electrospray ionization. The validation results of selectivity, matrix effect, recovery, linearity, intra- and inter-assay precision and accuracy were satisfactory. The limits of detection ranged from 0.25 to 10 ng/mL, and the limits of quantification were 2.5 to 50 ng/mL. The drugs and metabolites in urine did not show any degradation under storage for 7 and 15 days at 4 and -20 °C as well as after three freeze-thaw cycles. The developed method will be very useful for monitoring the illegal use of impotence treatment drugs.

Background: The brain and gut are made up of a network of many neurons and are bidirectional through the vagus nerve. These two nervous systems adjust to each other when food is needed, stressed, and intestinal bacteria are out of balance. Trimethlamine N-oxide (TMAO) is produced by gut microbiota. Gut microbiota derived TMAO are known to be associated with poor outcome in various vascular diseases including coronary artery disease and peripheral artery obstructive disease. Therefore, increased TMAO may be also associated stroke outcome. We sought the association between TMAO level and infarction size in mice ischemic stroke model. Method: Five-week-old male C57BL / 6J mice are fed with Control, 1.2% choline and 0.12% TMAO for 6 weeks. At the 11th week, the transient middle cerebral artery occlusion model (MCAO) was performed. All assessments was blindly conducted. TMAO was measured with mass spectrometry. Infarction size was measured with 2,3,5-Triphenyltetrazolium staining at 1 day after MCAO. Neurological score was assessed with Longa score, Gracia's, and mNHSS. Results: During the 6 weeks of feeding, weighing was performed to confirm the diet feeding. Interestingly, the weight of TMAO group mice was generally high. The concentration of TMAO level also varied from group to group. TMAO and choline fed group had significant increase in the level of circulating TMAO compared to control diet fed group (P<0.0002). Therefore, it was found that the level of TMAO in the blood was elevated through dietary treatment with these different diets. We also evaluated infarction volume. Compared with the control group, the infarct volume was decreased in the group with higher TMAO level than the control group (control 18.9±7.4 vs. higher TMAO group 13.6±5.9, p=0.032). Conclusion: We found that the infarction volume was associated with TMAO level. Increased TMAO may be beneficial in acute ischemic stroke.