Analytical methods for detecting butane, propane, and their metabolites in biological samples: Implications for inhalant abuse detection.

Worldwide, various inhalants are widely abused for recreational purposes, with butane and propane emerging as among the most commonly misused volatile substances, posing a significant risk of sudden death. The rapid elimination and oxidation of these highly volatile compounds upon inhalation necessitate the identification of butane and propane along with their metabolites in biological samples. Hence, the primary objective of this study is twofold: firstly, to establish a method for analyzing butane, propane, and metabolites, and secondly, to demonstrate the detection window and exposure indicators associated with the inhalation of butane and propane. In pursuit of this objective, we developed analytical methods for the determination of isobutane, n-butane, propane, and their nine metabolites in both blood and urine. Headspace-gas chromatography-mass spectrometry (GC-MS) and solid-phase microextraction-GC-MS were employed for the analyses, demonstrating acceptable precision and accuracy. An animal study revealed that isobutane and n-butane were only detectable below the limit of quantification (LOQ) in rat blood 5 min after exposure. Meanwhile, the three major metabolites-2-methyl-2-propanol, 2-butanol, and 2-butanone-were observed 5 min after exposure but persisted in rat urine even 5 h post-exposure. Additionally, human urine samples identified other metabolites, including acetone, acetoin, and 2,3-butanediol isomers. The presence of specific metabolites corresponding to each inhalant confirmed the abuse of butane and propane. This comprehensive approach provides valuable insights into the detection and assessment of inhalation to these volatile substances.

Detection of l-Methamphetamine and l-Amphetamine as Selegiline Metabolites.

Selegiline (SE) is a selective, irreversible monoamine oxidase-B inhibitor, used for reducing symptoms in early-stage Parkinson's disease. The metabolites of SE include l-methamphetamine, l-amphetamine and desmethylselegiline (DSE). The stereoisomers of SE metabolites, d-methamphetamine and d-amphetamine are highly addictive psychostimulants and some of the most abused drugs in South Korea. In order to differentiate medical SE users form illicit methamphetamine abusers, it is important to distinguish between the l-isomers and d-isomers in urine samples. A 52-year-old male, seemingly under the influence of intoxication and demonstrating abnormal behavior, was reported to the police. The initial urine test using a methamphetamine detection kit demonstrated a positive result. Given the initial results, the police officer requested a further analysis of the urine sample. The urine sample was screened using headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Both methamphetamine and amphetamine were detected, in addition to SE and DSE. To quantitate methamphetamine and amphetamine by HS-SPME-GC-MS, we performed a standard addition method due to the matrix effect of the case sample. Consistent with previous studies, our results indicated that the ratio of amphetamine to methamphetamine was 0.27, which was in the range of SE ingestion. Furthermore, we confirmed l-methamphetamine and l-amphetamine by chiral derivatization using (R)-(-)-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride.

Determination of Ketamine and Norketamine in Hair and Evaluation of Polydrug Use in Ketamine Abusers Using Hair Analysis in Korea.

This study evaluated hair samples from 28 subjects who tested positive for ketamine at Seoul Institute National Forensic Service in Korea between 2016 and 2017. Ketamine in the hair was extracted using a solution of 1% hydrochloric acid in methanol for 16 h. Extracts were analyzed using gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS-MS). The LC-MS-MS method was validated by determining the limit of detection (LOD), limit of quantification (LOQ), linearity, intra- and inter-accuracy, precision and matrix effect. In 59 ketamine-positive hair or hair segments from 28 ketamine abusers, the ketamine concentration was found to be in the range of 0.011-335.8 ng/mg (mean, 13.6; median, 1.8), and the norketamine concentration was found to be in the range of 0.001-35.7 ng/mg (mean, 7.5; median, 0.44). The ratio of norketamine to ketamine concentrations in hair was in the range of 0.01-1.46 (mean, 0.34; median, 0.26). The distribution of ketamine concentration in hair samples was as follows: 0.01-0.1 ng/mg in 11 samples (18.6%), 0.1-5 ng/mg in 33 samples (55.9%), 5-10 ng/mg in 4 samples (6.8%), 10-15 ng/mg in 2 samples (3.4%), 15-20 ng/mg in 4 samples (6.8%), 40-45 ng/mg in 2 samples (3.4%), 45-50 ng/mg in 1 sample (1.7%) and >100 ng/mg in only 2 samples (3.4%). In the hair of ketamine abusers, 26 of 28 subjects were detected simultaneously ketamine with other drugs, including methylenedioxymethamphetamine (MDMA; n = 9), methamphetamine (MA; n = 3), MDMA/MA (n = 3), MDMA/para-methoxyamphetamine (PMA; n = 3), MDMA/PMA/MA (n = 2), cocaine (n = 1) and other drugs (n = 5, propofol, zolpidem or benzodiazepines). Along with ketamine, other controlled drugs were detected in most of the hair samples: MDMA (60.7%), MA (28.6%), PMA (17.9%), zolpidem (17.9%) and propofol (14.3%) in the frequency of abuse. In conclusion, most of the ketamine abusers (92.9%) were polydrug abusers, who were concomitantly abusing other controlled substances.

Simultaneous determination of methylphenidate and ritalinic acid in hair using LC-MS/MS.

Methylphenidate (MPH) is one of the most commonly prescribed stimulants for attention deficit hyperactivity disorder and its abuse is on the rise with its growing availability. Some analytical methods have been reported for the detection of MPH in hair. However, the concentration range of MPH as well as its metabolite, ritalinic acid (RA) in the hair of MPH abuse cases has not been reported. In this study, a sensitive and reliable liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of MPH and RA in hair. Sample preparation was carried out by a simple methanol extraction using 10mg of hair. Limits of detection for MPH and RA in hair were 0.5pg/mg and 1pg/mg, respectively, and the limits of quantification (LOQs) were 1pg/mg for both the analytes. Validation results showed good linearity in the range of 1-100pg/mg with acceptable precision and accuracy. The developed method was applied to real hair samples obtained from ten drug users who obtained MPH illegally without a prescription. MPH concentrations in the hair samples ranged from 1.0pg/mg to 265.0pg/mg, and RA was present at concentrations

A comprehensive and sensitive method for hair analysis in drug-facilitated crimes and incorporation of zolazepam and tiletamine into hair after a single exposure.

Hair is a highly relevant specimen that is used to verify drug exposure in victims of drug-facilitated crime (DFC) cases. In the present study, a new analytical method involving ultrahigh-performance liquid chromatography-tandem mass spectrometry was developed for determining the presence of model drugs, including zolazepam and tiletamine and their metabolites in hair specimens from DFCs. The incorporation of zolazepam and tiletamine into hair after a single exposure was investigated in Long-Evans rats with the ratio of the hair concentration to the area under the curve. For rapid and simple sample preparation, methanol extraction and protein precipitation were performed for hair and plasma, respectively. No interference was observed in drug-free hair or plasma, except for hair-derived diphenhydramine in blank hair. The coefficients of variance of the matrix effects were below 12%, and the recoveries of the analytes exceeded 70% in all of the matrices. The precision and accuracy results were satisfactory. The limits of quantification ranged from 20 to 50 pg in 10 mg of hair. The drug incorporation rates were 0.03 ± 0.01% for zolazepam and 2.09 ± 0.51% for tiletamine in pigmented hair. We applied the present method to real hair samples in order to determine the drug that was used in seven cases. These results suggest that this comprehensive and sensitive hair analysis method can successfully verify a drug after a single exposure in crimes and can be applied in forensic and clinical toxicology laboratories.

Determination of major metabolites of MAM-2201 and JWH-122 in in vitro and in vivo studies to distinguish their intake.

Abuse of fluorinated synthetic cannabinoid analogs to avoid existing legal regulations has increased globally. The fluorinated JWH-122 analog, MAM-2201, was first reported in September 2012 as an ingredient in herbal mixtures in Korea. MAM-2201 is more potent than JWH-122 and a fatal intoxication case has been reported. In this study, we identified major MAM-2201 and JWH-122 metabolites from in vitro metabolism studies using human liver microsomes and compared the results with those of urine specimens from suspected MAM-2201 or JWH-122 users. MAM-2201 and JWH-122 produced common metabolites, N-5-hydroxylated, N-4-hydroxylated and carboxylated JWH-122 metabolites. Trace amounts of an N-4-hydroxylated MAM-2201 metabolite, a characteristic MAM-2201 metabolite, was detected in only a few urine specimens from MAM-2201 users. Both in vitro and in vivo studies demonstrated that N-5-hydroxylated JWH-122 metabolite was the primary metabolite of MAM-2201, whereas N-4-hydroxylated JWH-122 metabolite was predominant in JWH-122 metabolism. Based on these results, relative concentrations of N-5- and N-4-hydroxylated JWH-122 metabolites should be considered to verify MAM-2201 or JWH-122 users.

Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters.

We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

Determination of AM-2201 metabolites in urine and comparison with JWH-018 abuse.

With respect to the continuous emergence of new synthetic cannabinoids on the market since 2008, evaluation of the metabolism of these compounds and the development of analytical methods for the detection of these drugs including their respective metabolites in biological fluids have become essential. Other than JWH-018 or JWH-073, AM-2201 is one of the frequently identified synthetic cannabinoids in Korea. Recently, in our laboratory, several JWH-018 metabolites have been detected in some urine samples obtained from subjects who were arrested for the possession of herbal mixtures containing only AM-2201 or from those who confessed AM-2201 abuse. In the present study, we identified major urinary metabolites of AM-2201 and several metabolites of JWH-018, i.e., N-5-hydroxylated and carboxylated metabolites from rats administered AM-2201 and found that the metabolic profile in rats was similar to those in human subjects in this study. Analytical results of the urine samples from suspects who had a considerable possibility of AM-2201 or JWH-018 intake were also compared to distinguish between AM-2201 and JWH-018 abuse. The presence of 6-indole hydroxylated metabolites of each drug and N-4-hydroxy metabolite of AM-2201 was found to contribute to the decisive differences in the metabolic patterns of the two drugs. In addition, the concentration ratio of the N-(5-hydroxypentyl) metabolite to the N-(4-hydroxypentyl) metabolite of JWH-018 may be used as a criterion to differentiate between AM-2201 and JWH-018 abuse.

H-Ras-specific activation of Rac-MKK3/6-p38 pathway: its critical role in invasion and migration of breast epithelial cells.

Human tumors frequently exhibit constitutively activated Ras signaling, which contributes to the malignant phenotype. Mounting evidence suggests unique roles of the Ras family members, H-Ras, N-Ras and K-Ras, in normal and pathological conditions. In an effort to dissect distinct Ras isoform-specific functions in malignant phenotypic changes, we previously established H-Ras- and N-Ras-activated MCF10A human breast epithelial cell lines. Using these, we showed that p38 kinase is a key signaling molecule differentially regulated between H-Ras and N-Ras, leading to H-Ras-specific induction of invasive and migrative phenotypes. The present study is to further investigate H-Ras- and N-Ras-mediated signaling pathways and to unveil how these pathways are integrated for regulation of invasive/migrative phenotypic conversion of human breast epithelial cells. Here we report that the Rac-MAPK kinase (MKK)3/6-p38 pathway is a unique signaling pathway activated by H-Ras, leading to the invasive/migrative phenotype. In contrast, Raf-MEK-ERK and phosphatidylinositol 3-kinase-Akt pathways, which are fundamental to proliferation and differentiation, are activated by both H-Ras and N-Ras. A significant role for p38 in cell invasion is further supported by the observation that p38 activation by MKK6 transfection is sufficient to induce invasive and migrative phenotypes in MCF10A cells. Activation of the MKK6-p38 pathway results in a marked induction of matrix metalloproteinase (MMP)-2, whereas it had little effect on MMP-9, suggesting MMP-2 up-regulation by MKK6-p38 pathway as a key step for H-Ras-induced invasion and migration. We also provide evidence for cross-talk among the Rac, Raf, and phosphatidylinositol 3-kinase pathways critical for regulation of MMP-2 and MMP-9 expression and invasive phenotype. Taken together, the present study elucidated the role of the Rac-MKK3/6-p38 pathway leading to H-Ras-specific induction of malignant progression in breast epithelial cells, providing implications for developing therapeutic strategies for mammary carcinoma to target Ras downstream signaling molecules required for malignant cancer cell behavior but less critical for normal cell functions.

Chemopreventive allylthiopyridazines inhibit invasion, migration and angiogenesis in hepatocarcinoma cells.

Numerous studies have revealed the chemopreventive and hepatoprotective activities of dietary and synthetic organosulfur compounds. We previously showed that synthetic allylthiopyridazine derivatives, designated as K compounds, induced apoptosis in SK-Hep-1 hepatocarcinoma cells. In order to extend our program to pursue the chemopreventive potential of these compounds, we investigated the effects of the K compounds on invasive and migrative properties of the SK-Hep-1 cells in this study. Here, we show that 3-methoxy-6-allylthiopyridazine (K6) and 3-propoxy-6-allylthiopyridazine (K17) efficiently inhibit SK-Hep-1 cell invasion and migration. A prominent downregulation of matrix metalloproteinase (MMP)-2, but not MMP-9, was observed, presenting MMP-2 as a potential target molecule for the anti-invasive and anti-migrative activities of the compounds. Since hepatocarcinoma is characterized as a hypervascular tumor, we examined the effect of the compounds on angiogenesis of human umbilical vein endothelial cells (HUVECs). The K compounds exerted anti-angiogenic activity, supporting that the development of these compounds would be a promising approach for treatment of hepatocarcinoma. Taken in conjunction with the fact that hepatocellular carcinoma is one of the most lethal malignancies, our findings may be critical to the chemopreventive potential of these synthetic organosulfur compounds for hepatocarcinoma.