Complementary approach for accurate determination of carbon isotopic compositions in γ-hydroxybutyric acid using gas chromatography/combustion-isotope ratio mass spectrometry.

RATIONALE: γ-Hydroxybutyric acid (GHB) is a naturally endogenous neurotransmitter that is popular as a recreational drug due to its sedative, hypnotic, and euphoric effects. GHB derived from endogenous production or exogenous ingestion has been effectively discriminated by carbon isotopic compositions (δ13 C values) through gas chromatography/combustion-isotope ratio mass spectrometry (GC/C-IRMS). However, an unintended uncertainty of isotopic signatures caused by a wide range of GHB quantities remains unsolved when using only single-isotope corrections of the di-TMS derivative. METHODS: The δ13 C values of the original GHB standard were first determined by elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). The δ13 C values of silylated GHB in concentrations from 10 to 500 ppm were determined by GC/C-IRMS. With respect to the silylated reaction products, the correction of δ13 C values for the introduced carbons was calculated from a stoichiometric mass balance equation. RESULTS: The results showed a significant quantity-dependent trend in δ13 C values of introduced carbon (δ13 Cdi-TMS values) with increased GHB standard concentrations (r2 = 0.70, p <0.05). We applied a logarithmic equation to determine isotopic data in low-GHB urine specimens from five healthy female volunteers. The δ13 CGHB values in urine samples corrected with quantity-dependent δ13 Cdi-TMS values were different by an average of 2.7‰ from those corrected with single δ13 Cdi-TMS values (p <0.05). CONCLUSIONS: Our results suggest that the overall residual amount-dependent isotope fractionation should be mathematically corrected by the logarithmic function and this may improve the reliability of isotopic analysis to evaluate the origin of GHB before applying the approach to routine toxicological and forensic studies.

Correction to: Differentiation of endogenous and exogenous γ-Hydroxybutyrate in rat and human urine by GC/C/IRMS.

The above article was published online with incorrect author names. The right spelling should be Dong-Hun Lee instead of Donghun Lee, Sanggil Choe instead of Sanggil Choi. The correct names are presented here. The original article has been corrected.

Differentiation of endogenous and exogenous γ-Hydroxybutyrate in rat and human urine by GC/C/IRMS.

Gamma (γ)-hydroxybutyric acid (GHB) has been reported to be an endogenous compound in the mammalian brain. It used to treat symptoms of alcohol, opioid, and drug withdrawal and cataplexy of narcolepsy. However, it is often used for criminal purposes because it is colorless, tasteless, and has short half-life. For this reason, there is a need for a method of distinguishing between endogenous and exogenous GHB administration. Therefore, urine from rat before administration of GHB and GHB urine after the single intraperitoneal injection of GHB as 30 mg/100 g were collected from Sprague-Dawley rats (7 weeks old, 10 males and females). Negative control urine, urine from individuals suspected of taking GHB, and urine from victims who were GHB-involved crime were collected. In urine samples, GHB was extracted with two-step SPE and collected fraction was derivatized and analyzed by GC/MS and GC/C/IRMS. In GC/MS and GC/C/IRMS analysis of rat urine, there was a statistically significant difference between urine from rat before administration of GHB and GHB rat urine (p < 0.05). In GC/MS analysis of human urine samples, there was no significant difference among human urine groups (negative control, suspects' urine, and victims' urine), but in GC/C/IRMS analysis of human urine samples, there was a statistically significant difference among human urine groups (p = 0.0001). Through these results, GC/C/IRMS can be more effective tool to identify endogenous and exogenous GHB in urine than GC/MS. This study can build a drug management system in forensic investigation agency and offer interpretation method to forensic science and court.

A fatal case of desvenlafaxine and paroxetine poisoning.

Desvenlafaxine (O-desmethylvenlafaxine) and paroxetine are antidepressants that inhibit serotonin reuptake. Despite their relatively safe profiles, several serious side effects, including serotonin syndrome, bleeding, mania, and high blood pressure, are observed. We report the confirmation of the death of a 41-year-old female, with an overdose of desvenlafaxine and paroxetine suspected as the main cause of death. To quantify the level of desvenlafaxine and paroxetine in whole blood and urine, solid phase extraction combined with liquid chromatography-tandem mass spectrometry was developed and validated. Calibration curves were linear with coefficients of determination (r2) >0.999 for desvenlafaxine and paroxetine. The limits of detection and the limits of quantification for both desvenlafaxine and paroxetine were 0.001 µg/mL and 0.02 µg/mL, respectively. Desvenlafaxine and paroxetine were detected in the postmortem samples, along with various psychiatric drugs, and the blood alcohol content level was below 0.010%. The concentrations of desvenlafaxine and paroxetine in the heart blood were 11.0 µg/mL and 2.1 µg/mL, respectively, indicating lethal concentrations. In the urine, the concentrations of desvenlafaxine and paroxetine were 87.7 µg/mL and 3.5 µg/mL, respectively. This is the first report to determine the blood concentration of desvenlafaxine in a fatal intoxication caused by an overdose of desvenlafaxine single formulation.

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.

Simultaneous analysis of synthetic cannabinoids in the materials seized during drug trafficking using GC-MS.

A rapid and simple gas chromatography-mass spectrometry (GC-MS) method was developed and validated to identify and quantify synthetic cannabinoids in the materials seized during drug trafficking. Accuracy and reproducibility of the method were improved by using deuterated JWH-018 and JWH-073 as internal standards. Validation results of the GC-MS method showed that it was suitable for simultaneous qualitative and quantitative analyses of synthetic cannabinoids, and we analyzed synthetic cannabinoids in seized materials using the validated GC-MS method. As a result of the analysis, ten species of synthetic cannabinoids were identified in dried leaves (n = 40), bulk powders (n = 6), and tablets (n = 14) seized in Korea during 2009-2012, as a single ingredient or as a mixture with other active co-ingredients. JWH-018 and JWH-073 were the most frequently identified compounds in the seized materials. Synthetic cannabinoids in the dried leaves showed broad concentration ranges, which may cause unexpected toxicity to abusers. The bulk powders were considered as raw materials used to prepare legal highs, and they contained single ingredient of JWH-073, JWH-019, or JWH-250 with the purity over 70 %. In contrast, JWH-018 and JWH-073 contents in the tablets were 7.1-13.8 and 3.0-10.2 mg/g, respectively. Relatively low contents in the tablets suggest that the synthetic cannabinoids may have been added to the tablets as supplements to other active co-ingredients.

Comprehensive evaluation of drug cases in Seoul and its metropolitan areas - 2022.

In order to prepare a response strategy for future drug analyses, the number and results of drug cases handled by the Seoul Institute of National Forensic Service were comprehensively evaluated, with a focus on Seoul and its metropolitan areas. In 2022, the Seoul Institute received approximately 12,150 requests for drug testing related to drug abuse and possession, and the urine samples were tested for approximately 16,000 drug species. The most frequently requested test was for cannabis (Δ-9-THC and Δ-8-THC), followed by methamphetamine, MDMA, ketamine, and synthetic cannabinoids. ADB-5'Br-BUTINACA and propyl butylone were newly emerging substances in 2022. These results were consistent with the main drug detection findings of the confiscated materials. During this period, 24 cases of drug-related deaths were reported, of which 6 were suspected to be the result of acute overdose poisoning caused by methamphetamine, MDMA, fentanyl, and heroin. In addition to the controlled substances regulated by the Narcotics Control Act, new psychoactive substances are being found to be circulating, and various measures are required to address this issue. This study is expected to improve future drug analyses methods and assist in establishing drug policies, and responding to future investigations.

Development of a target component extraction method from GC-MS data with an in-house program for metabolite profiling.

After gas chromatography-mass spectrometry (GC-MS) analysis, data processing, including retention time correction, spectral deconvolution, peak alignment, and normalization prior to statistical analysis, is an important step in metabolomics. Several commercial or free software packages have been introduced for data processing, but most of them are vendor dependent. To design a simple method for Agilent GC/MS data processing, we developed an in-house program, "CompExtractor", using Microsoft Visual Basic. We tailored the macro modules of an Agilent Chemstation and implanted them in the program. To verify the performance of CompExtractor processing, 30 samples from the three species of the genus Papaver were analyzed with Agilent 5973 MSD GC-MS. The results of CompExtractor processing were compared with those of AMDIS-SpectConnect processing by hierarchical cluster analysis (HCA) and principal component analysis (PCA). The two methods showed good classification according to their species in HCA. The PC1+PC2 scores were 54.32-63.62% for AMDIS-SpectConnect and 56.65-85.92% for CompExtractor in PCA. Although the CompExtractor processing method is an Agilent GC-MS-specific application and the target compounds must be selected first, it can extract the target compounds more precisely in the raw data file with batch mode and simultaneously assemble the matrix text file.

Rhododendric acid A, a new ursane-type PTP1B inhibitor from the endangered plant Rhododendron brachycarpum G. Don.

In spite of the critical role of the natural products in drug discovery, surprising little attention has been placed on endangered and rare plant species that could play a pivotal role in pharmaceutical and fiber development. Protein tyrosine phosphatase-1B (PTP1B), which blocks insulin signaling, has been gaining interest to be a promising therapeutic target for the treatment of type 2 diabetes mellitus. Bioassay-guided fractionation on the leaves of Rhododendron brachycarpum G. Don (Ericaceae) yielded seven PTP1B inhibitory triterpenoids, including a new triterpene, rhododendric acid A (1). Their PTP1B inhibitory potency and their lipophilicity were investigated to provide a feasible scaffold that may overcome the innate limitation of the previously reported PTP1B inhibitors.

Antioxidative oligostilbenes from Caragana sinica.

Two new oligostilbenes, caragasinins A (5) and B (10), and eight known compounds, kobophenol A (1), (+)-α-viniferin (2), (+)-ampelopsin F (3), pallidol (4), (+)-isoampelopsin F (6), miyabenol C (7), carasinaurone (8) and caraphenol B (9) were isolated from the ethylacetate-soluble extract of the roots of Caragana sinica. The structures of the isolates were determined on the basis of extensive spectroscopic analysis including 1D, 2D NMR and HRESI-MS. These compounds were assessed for antioxidant activities. Caragasinin A (5), caraphenol B (9), and caragasinin B (10) showed moderate DPPH scavenging activity and lipid peroxidation inhibitory activities with IC(50) values ranging from 34.7±1.0 to 89.1±2.3µM.

Identification of a novel bromoindazole synthetic cannabinoid analogue in seized e-cigarette liquid: ADB-BRINACA.

A wide variety of new synthetic cannabinoids have emerged around the world in recent years, and because of this rapid emergence, the detection and monitoring of this class of abused drugs remain a challenge. In this study, a new cannabimimetic indazole-3-carboxamide derivative, N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)- 5-bromo-1 H-indazole-3-carboxamide, was identified from seized e-cigarette liquid samples and newly named as ADB-BRINACA by referring to the names of other known indazole-class synthetic cannabinoids. Structure identification was accomplished based on gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-high resolution quadrupole mass spectrometry (HPLC-QTOF-MS/MS), and nuclear magnetic resonance spectrometry (NMR). The concentration range of ADB-BRINACA in six e-cigarette liquid samples was found to be 2228-4203 mg/L using ERETIC 2, a quantitative NMR method, which is advantageous in the absence of a reference material. As there have been no chemical or pharmaceutical reports on ADB-BRINACA until now, this is the first report presenting a comprehensive analytical characterization of ADB-BRINACA.

Development of diagnostic SNP markers and a novel SNP genotyping assay for distinguishing opium poppies.

The opium poppy acts as an important natural pain reliever but is also responsible for increased rates of severe drug abuse and addiction owing to its characteristic psychoactive effect. Non-medical illicit use of the poppy plant is markedly increasing worldwide, thereby highlighting the need for a robust species identification strategy. In this study, we identified SNPs within the region of two universal DNA barcodes, matK (maturase K) and the trnL-trnF (tRNA-Leu [3'exon]-tRNA-Phe [exon] intergenic spacer, that are forensically applicable for distinguishing opium poppy species based on a genetic analysis of 164 samples of family Papaveraceae obtained from locations spanning Jeolla-do and Jeju Island, Republic of Korea. A comparative analysis of the DNA barcode sequences for two narcotic types of the Papaver species (Papaver somniferum, Papaver somniferum subs. setigerum) to eight non-narcotic species revealed three unique nucleotide substitution events. Newly identified SNPs were located at position 255 of matK and at positions 305 and 306 of trnL-trnF; the narcotic species contained C, A, and T, whereas non-narcotic species contained T, G, and C at these positions. Phylogenetic analysis demonstrated that newly identified SNPs, which we named PsMAT255 and PsLF305/306, could be used to clearly differentiate between the narcotic and non-narcotic types of Papaver species based on the patterns of nucleotide variation. These results indicate that the nucleotide differences between the narcotic and non-narcotic species may influence genetic markers. We, therefore, developed a novel SNP-based allelic genotyping assay using the RT-PCR system that can reliably differentiate the narcotic type of the Papaver species. In summary, our findings suggest that the newly identified species-specific SNPs of both matK and trnL-trnF can be used as identification markers of narcotic Papaver species. Furthermore, a newly developed TaqMan allelic discrimination assay may be used as a practically applicable diagnostic method to survey several illicit narcotic specimens carrying the type-specific SNP.

Determination of etomidate and etomidate acid in hair using liquid chromatography-tandem mass spectrometry.

Etomidate, with efficacy similar to that of propofol, has been used as a propofol substitute because propofol is a designated narcotic drug, and an increase in the frequency of illegal distribution and misuse has been reported in Korea. Previous analytical studies on etomidate used blood and urine. For long-term use and timing estimation, a method for etomidate analysis using hair should be developed. Therefore, in this study, an analytical method using LC-MS/MS was developed to determine etomidate and its major metabolite in hair. Human hair samples were segmented after washing to eliminate possible contaminants on the hair and stirred with methanol. The LC-MS/MS conditions were optimized, and the chromatographic separation time was 10 min. Selectivity, linearity, limit of detection, limit of quantification, precision, accuracy, recovery, process efficiency, matrix effect, and stability were evaluated to validate the analytical method. The calibration curves ranged from 0.25 to 50 pg/mg for etomidate and 2-250 pg/mg for etomidate acid; the coefficients of determination were higher than 0.997. The intra- and inter-assay precision results for all the compounds were <15% and satisfied at recovery, process efficiency, matrix effect, and stability. In addition, this method was applied to the hair of 4 rats which are administered with etomidate to evaluate. The etomidate concentrations in the rat hair ranged from 2.60 to 8.50 pg/mg, and the etomidate acid concentrations were 2.06-7.13 pg/mg. Thus, this method can be used as basic data for monitoring etomidate in hair.

Dimeric ent-kaurane diterpenoids from Isodon excisus.

Five new dimeric ent-kauranoids, biexcisusins A-E (1-5), were isolated from the aerial parts of Isodon excisus. The structures and relative configurations of these compounds were determined on the basis of spectroscopic data interpretation. Of these, biexcisusins C-E (3-5) are dimeric ent-kaurane diterpenoids exhibiting an unprecedented linkage through a nine-membered lactone ring between two ent-kaurane subunits. Compounds 1-5 showed no inhibitory effects on the LPS-induced production of nitric oxide in murine macrophage RAW264.7 cells, up to a dose of 50 µM.

Metabolic analysis of the illegal analogues of anti-obesity drugs using LC-Q-TOF-MS/MS.

With an increase in the obese population, the indiscriminate demand for anti-obesity drugs for rapid weight loss or maintenance has grown. As a result, illegal substances that could induce unexpected negative health effects or fatal side effects are being produced and mixed into consumer products. In the present study, the metabolites of five major illegal anti-obesity drugs are analyzed for the first time. Our data can be utilized to identify related compounds and predict their toxicological effects. Didesmethylsibutramine, desmethylsibutramine, homosibutramine, chlorosibutramine, and benzylsibutramine were metabolized in in vitro and in vivo models, and the metabolites were identified using liquid chromatography quadrupole-time of flight mass spectrometry (LC-Q-TOF-MS) and tandem mass spectrometry (LC-Q-TOF-MS/MS). The in vivo metabolite analysis was carried out using urine and feces samples from rats, and the in vitro metabolite analysis was performed by incubating the analogues with human liver microsomes. We found that each sibutramine analogue was metabolized into several constituents: 2 (M1-2), 5 (M1-5), 11 (M1-11), 7 (N1-7), and 5 (O1-5). In conclusion, our metabolic study could be used for toxicological detection of illegal obesity treatments and metabolite identification in forensic cases.

A new minisatellite VNTR marker, Pscp1, discovered for the identification of opium poppy.

Opium poppy, a member of the Papaveraceae family, is an ancient herbaceous plant and well-known medical resource in the pharmaceutical industry. However, opium poppies are grown worldwide for producing illicit drugs, significantly increasing the incidence of narcotic drug abuse. Since the narcotic poppy has not yet been genetically investigated, we characterized a novel variable number tandem repeat (VNTR) marker of forensically important poppy species based on the genetic analysis of 164 samples collected from two locations spanning the Jeolla province and Jeju island of South Korea. Comparing analysis of the chloroplast (cp) genome sequences for four representative species of Papaver (Papaver somniferum, Papaver somniferum subs. setigerum, Papaver orientale, and Papaver rhoeas) revealed a unique region with 1-3 repeats for 16 nucleotide motifs in the genome inverted repeat A (IRA, positions 128,651 to 128,698) region. For 16 nucleotide motifs, 3 repeats were found in P. somniferum, and 2 repeats were found in P. somniferum subs. setigerum. Therefore, 10 known and the 133 unknown, seized Papaver species were compared to determine whether the species could be identified via variations in the repeat units. The sizes of a novel VNTR ranged from 181 to 252 bp between the species. Phylogenetic analysis confirmed that a novel VNTR, which we named Pscp1, could clearly distinguish between the narcotic and non-narcotic types of Papaver species based on the patterns of sequence variation. Interestingly, we found that Pscp1 could also distinguish between P. somniferum and P. somniferum subs. setigerum. The regions of eight non-narcotic species displayed similar patterns and also differences were found due to the nucleotide substitution and deletion events. The structural differences of Pscp1 were observed within the two narcotic species or between the narcotic and non-narcotic species, suggesting that these variations may act as a genetic marker. We, therefore, developed a new Pscp1 PCR-capillary electrophoresis (CE) method that can reliably identify the narcotic type of Papaver species. Taken together, our findings suggest that the newly developed Pscp1 can be used as an identification marker of opium poppy, and establish that the Pscp1 genotyping method by PCR-CE is an effective primary screening tool that can also contribute to species discrimination in the field of forensic diagnosis and applications.

A case of fatal intoxication by ingestion of an herbicide formulation containing fluroxypyr-meptyl and triclopyr.

Fluroxypyr-meptyl and triclopyr are synthetic auxin-like herbicides that are used to control woody and broadleaf weeds. Herein, we report a case of fatal intoxication involving fluroxypyr-meptyl and triclopyr. A 61-year-old man was found dead at his farm with several suicide notes, and a white plastic bottle and a plastic cup with traces of white emulsion were found next to him. The plastic bottle was labeled as an herbicide formulation containing fluroxypyr-meptyl and triclopyr. Forensic toxicological screening of the stomach contents revealed the presence of fluroxypyr-meptyl, fluroxypyr and triclopyr. However, no fluroxypyr-meptyl was detected in blood owing to its rapid hydrolysis to fluroxypyr. In this study, fluroxypyr and triclopyr in blood were extracted using solid-phase extraction, and analyzed by liquid chromatography-tandem mass spectrometry. The analytical method was validated in terms of linearity, precision, accuracy, recovery and matrix effect, and the acceptable criteria were satisfied. Toxicological analysis showed that fluroxypyr and triclopyr concentrations were 19.7 µg/mL and 137.4 µg/mL in peripheral blood and 16.5 µg/mL and 147.8 µg/mL in heart blood, respectively. Based on these toxicological results and autopsy findings, the cause of death was determined to be acute fatal intoxication by ingestion of the pesticide containing fluroxypyr-meptyl and triclopyr. This is the first report of the determination of fluroxypyr and triclopyr in a fatal intoxication case.

Fast and reliable analysis of veterinary metomidate and etomidate in human blood samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a postmortem case.

Metomidate and etomidate belong to the non-barbiturate imidazole family of sedative-hypnotics and elicit little analgesic action when used alone. Metomidate, in particular, has little analgesic activity in humans and is, therefore, used for veterinary purposes. In 2019, a Korean woman in her twenties was found unconscious in a motel bath and eventually died. Etomidate, alprazolam, escitalopram, and metomidate were detected in the postmortem specimens. To our knowledge, this is the first case of human metomidate abuse reported in the Republic of Korea. In this research, a simple and reliable method was developed for the analysis of metomidate and etomidate in human blood samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Blood samples were deproteinized with acetonitrile, filtered, and analyzed by LC-MS/MS. Linear calibration curves were obtained with six concentrations ranging from 1 to 50 ng/ml for metomidate and 10 to 500 ng/ml for etomidate. The method was validated by assessing the selectivity, linearity, limit of detection (LOD), limit of quantitation (LOQ), intra- and inter-day precision and accuracy, matrix effect, and stability and successfully applied to the analysis of metomidate and etomidate in human blood samples. In a postmortem case, the concentrations of metomidate and etomidate were found to be 8 and 110 ng/ml in femoral blood and 6 and 210 ng/ml in cardiac blood, respectively.

Metabolic study of vardenafil analogues: Pseudovardenafil and hydroxyvardenafil.

Vardenafil, a remedy for erectile dysfunction, is easily modified, facilitating the creation of analogues that have been illegally added to functional foods and counterfeit medications. However, the medical profile of these analogues, including their safety, efficacy, safe drug combinations, metabolism and excretion, has not been completely evaluated, which could cause serious health problems. In this study, two representative vardenafil analogues, pseudovardenafil and hydroxyvardenafil, were metabolized with in-vitro model (human liver microsome) and in-vivo model (rats). The metabolized samples were extracted and characterized, using liquid chromatography quadrupole-time of flight mass spectrometry (LC-Q-TOF-MS). Some imprecise interpretations were evaluated with tandem mass spectrometry (LC-Q-TOF-MS/MS) for mass fragmentation analysis. A total of 11 metabolites of pseudovardenafil and 13 metabolites of hydroxyvardenafil that were identified have never been reported. These new metabolites could be usefully applied to forensic science and other metabolic fields. Furthermore, they could serve as principal references for the toxicity, danger, and side effects of unlawful vardenafil counterfeits.

Monitoring urinary testosterone and epitestosterone levels, and their ratio, in Korean chemical castration subjects using liquid chromatography-tandem mass spectrometry.

In Europe, chemical castration has been adopted as a treatment for paraphilia since the 1930s. Among the various chemical castration agents, luteinizing hormone-releasing hormone (LHRH) agonists are now used widely because of their effectiveness and safety. In South Korea, a legislation of chemical castration to control the sexual impulses of sexual offenders was enforced in July 2011. Most of these subjects are treated with leuprorelin acetate, an LHRH agonist, for chemical castration. Despite this, there are few studies that address the long-term influence of LHRH agonists on testosterone (T) and epitestosterone (E) levels in chemical castration subjects. In order to analyze the urinary levels of T in chemical castration subjects, whose T levels are extremely low, we developed and validated an analytical method for the detection of both T and E in human urine using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The urine samples were hydrolyzed, extracted, and analyzed by LC-MS/MS with electrospray ionization in the positive-ion mode. The limits of detection were 0.02 ng/mL and the limits of quantitation were 0.05 ng/mL, which provided great sensitivity. The established method was applied to urine samples from chemical castration subjects and healthy male volunteers. The chemical castration subjects showed significantly lower urinary T levels than the control subjects. In addition, the urinary E levels were also lower in the chemical castration subjects; however, the T/E ratios were constant and did not show a notable decrease because of the simultaneous decrease in both urinary T and E. The urinary T levels and T/E ratio did not exceed the doping control criteria for exogenous T ingestion for any subject. This study shows the trend of urinary T and E levels in long-term treated chemical castration subjects by establishing a highly sensitive LC-MS/MS method, that provides useful information for monitoring chemical castration.