Urinary excretion profile of higenamine in females after oral administration of supplements - Doping scenario.

In 2017, higenamine was added to the World Antidoping Agency's (WADA) Prohibited list under group S3: beta-2 agonists and it is banned for athletes both in - and out of competition. Aim of this study was to characterize the urinary excretion profile of higenamine and its metabolite coclaurine after oral administration of multiple doses of higenamine capsules. For this purpose, an administration study including female basketball players was performed. For the detection of higenamine and cocalurine in the collected urine samples, a new, fast, and highly sensitive quantitative on-line SPE LC HRMS method was developed and validated. The method was applied for the quantification of higenamine and cocalurine in urine and their excretion pattern was defined. Results obtained show substantial inter-individual differences in the excretion profile of higenamine and coclaurine. For higenamine, half-lives were estimated to be between 4 and 27 h, and for coclaurine between 5 and 25 h. Furthermore, the data indicate that the elimination of coclaurine is rate-limited by its formation. Higenamine could be detected at a urine concentration above 10 ng/mL for at least 20 h after the last application for all study participants.

Novel proposed cutoff values for anatabine and anabasine in differentiating smokers from non-smokers.

OBJECTIVES: Anatabine and anabasine are two tobacco alkaloids used to differentiate between tobacco users and abstainers, including users of nicotine replacement therapy. Cutoff values (>2 ng/mL for both alkaloids) have not been revised since their implementation in 2002. These values may be too high, leading to increased likelihood of misclassification between smokers and abstainers. This results in major consequences, especially adverse outcomes of transplantation when smokers were incorrectly identified as being abstinent. This study proposes that a lower threshold for anatabine and anabasine will better distinguish tobacco users from non-users and thereby improve patients' care. DESIGN AND METHODS: A new and more sensitive analytical method by liquid chromatography-mass detection was developed to allow the quantification of low concentrations. Anatabine and anabasine were measured in urine samples of 116 self-reported daily smokers and 47 long-term non-smokers (confirmed by the analysis of nicotine and its metabolites). The best compromise between sensitivity and specificity allowed us to determine new cutoff values. RESULTS: The thresholds >0.097 ng/mL for anatabine and >0.236 ng/mL for anabasine were associated with a sensitivity of 97% (anatabine) and 89% (anabasine) and a specificity of 98% for both alkaloids. These cutoff values greatly increased the sensitivity given that it dropped to 75% (anatabine) and 47% (anabasine) when using the reference value (>2 ng/mL). CONCLUSIONS: The cutoff values >0.097 ng/mL for anatabine and >0.236 ng/mL for anabasine appear to better differentiate tobacco users from abstainers than the current reference threshold (>2 ng/mL for both alkaloids). It may considerably impact patients' care, especially in transplantation settings in which smoking abstinence is essential to avoid adverse outcomes of transplantation.

Presence of higenamine in beetroot containing 'foodstuffs' and the implication for WADA-relevant anti-doping testing.

Higenamine is an alkaloid found within plant species including some that are used in traditional Asian and Chinese herbal medicines. Identified as having mixed mode adrenergic receptor activity, higenamine is present within some nutritional supplements marketed for stimulant and/or weight loss. Its inclusion within nutritional supplements can be via its natural presence within botanical ingredients or as a synthetic additive, often added in mg amounts. The World Anti-doping Agency (WADA) prohibited list has contained higenamine since 2017 as banned at all times in the beta-2 agonist (S3) category, with a reporting level of 10 ng/ml for the free parent form in urine. In this study, an investigation into the content of beetroot or beetroot-containing foodstuffs and supplement products was conducted. Higenamine was confirmed as present within the majority of foodstuffs and supplements, with experimental evidence that higenamine can arise within beetroot extracts through heating. The results in this paper demonstrate the first reported evidence of a link between beetroot and this WADA prohibited substance. To investigate the link between intake and excretion, concentrated beetroot drinks were consumed by six individuals and higenamine quantified in their urine. Free higenamine was detected in the urine of all individuals, with maximum measured concentration in samples of less than 1% of the current WADA reporting limit. Although the risk of an inadvertent doping violation by consumption of the foodstuffs and products investigated in this study is low, beetroot as a source of higenamine should be considered by athletes.

Comparative pharmacokinetics, intestinal absorption and urinary excretion of six alkaloids from herb pair Phellodendri Chinensis cortex-Atractylodis Rhizoma.

Phellodendri Chinensis Cortex (PCC) and Atractylodis Rhizoma (AR) are frequently used as herb pair to treat eczema and gout owing to their synergistic effects. Alkaloids are the major ingredients from PCC and the effect of their combination on the in vivo processing of alkaloids remains unclear. In this study, a simple and reliable UPLC-MS/MS method for simultaneous determination of six alkaloids in rat plasma was developed. This method was applied to a comparative pharmacokinetic study between PCC and PCC-AR in rats. Effect of AR on absorption of alkaloids was investigated by a single-pass intestinal perfusion study. The effect of AR on urinary excretion of alkaloids was studied. Pharmacokinetic studies showed that the values of rea under the concentration-time curve of phellodendrine, magnoflorine and palmatine were greater in the PCC-AR group than in the PCC group. The intestinal absorptive parameters absorption rate constant and effective permeability of phellodendrine and jatrorrhizine in PCC-AR groups were higher than those in the PCC group. Urinary excretion studies revealed that the excreted amount of alkaloids in the PCC-AR group was lower than that in the PCC group. The results revealed that the combination of PCC and AR improves intestinal absorption of alkaloids and reduces their urinary excretion, which enhances their systemic exposure. This study may explain the synergetic effects of PCC and AR in clinical applications.

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.

Rapid detection of nine synthetic cathinones in blood and urine by direct analysis in real-time-tandem mass spectrometry.

RATIONALE: Designer drugs of cathinone, a kind of hallucinogen, were abused in the recent years. They were also known as bath salts, plant fertilizers, and zombie potions in drug market. The abuse of synthetic cathinones caused many bad effects on social order. Rapid detection of synthetic cathinones became an important subject of study in forensic science. METHODS: Direct analysis in real-time-tandem mass spectrometry (DART-MS/MS) was used to develop an effective method on nine synthetic cathinones in human whole blood and urine. The reference materials with 100 ng/mL were prepared for mass spectrometry optimization with electrospray ionization (ESI) probe tandem QTRAP 4000 mass spectrometer. The temperature of DART ion source was optimized to 400°C. The volumes of 4/1 (v/v) MeCN/MeOH with 0.69 mL were selected for the preparation of 0.31 mL blood and urine samples, respectively. Then the spiked analytes were prepared for detection by the DART 12Dip-it autosampler module. RESULTS: The results showed that the linearities range between 0.1 and 5 µg/mL, the correlation coefficients (r2 ) ranged from 0.99 to 1, the limits of detection (LODs) were all between 0.5 and 50 ng/mL, and the relative standard deviations (RSDs) of repeatability, intra-day and inter-day precisions were all below 13% and 14% in blood and urine, respectively. CONCLUSION: The results indicated that the method could meet the needs of rapid screening of samples that may contain synthetic cathinones. In addition, the method developed has many advantages, such as efficient, fast sample preparation, and environmental protection. Therefore, the DART-MS/MS method would provide effective data support for rapid screening of synthetic cathinones in forensic science.

Pepper Alkaloids and Processed Meat Intake: Results from a Randomized Trial and the European Prospective Investigation into Cancer and Nutrition (EPIC) Cohort.

SCOPE: Processed meat intake has been associated with adverse health outcomes. However, little is known about the type of processed meat more particularly responsible for these effects. This study aims to identify novel biomarkers for processed meat intake. METHODS AND RESULTS: In a controlled randomized cross-over dietary intervention study, 12 healthy volunteers consume different processed and non-processed meats for 3 consecutive days each. Metabolomics analyses are applied on post-intervention fasting blood and urine samples to identify discriminating molecular features of processed meat intake. Nine and five pepper alkaloid metabolites, including piperine, are identified as major discriminants of salami intake in urine and plasma, respectively. The associations with processed meat intake are tested for replication in a cross-sectional study (n = 418) embedded within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Three of the serum metabolites including piperine are associated with habitual intake of sausages and to a lesser extent of total processed meat. CONCLUSION: Pepper alkaloids are major discriminants of intake for sausages that contain high levels of pepper used as ingredient. Further work is needed to assess if pepper alkaloids in combination with other metabolites may serve as biomarkers of processed meat intake.

A sensitive HPLC-MS/MS method for the detection, resolution and quantitation of cathinone enantiomers in horse blood plasma and urine.

Resolution of cathinone enantiomers in equine anti-doping analysis is becoming more important to distinguish the inadvertent ingestion of plant-based products from those of deliberate administration of designer synthetic analogs. With this in mind, a rapid and sensitive method was developed and validated for the detection, resolution and quantitative determination of cathinone enantiomers in horse blood plasma and urine. The analytes were recovered from the blood plasma and urine matrices by using a liquid-liquid extraction after adjusting the pH to 9. The recovered analytes were derivatized with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide, a chiral derivatizing agent analogous to Marfey's reagent. The resulting diastereoisomers were baseline resolved under a reversed-phase liquid chromatographic condition. Derivatization of the analytes not only allowed the separation of the enantiomers using cost-effective traditional liquid chromatography conditions and reversed-phase columns but also increased the sensitivity, at least to an order of magnitude, when tandem mass spectrometry is used for the detection. A limit of detection of 0.05 ng/mL was achieved for cathinone enantiomers for both matrices. Acceptable intraday and interday precision and accuracy along with satisfactory dilution accuracy and precision were observed during the method validation. The method suitability was tested using the post administration urine samples collected after single doses of cathinone and ephedrine as single-enantiomeric form and methcathinone as racemic form. Finally, a proof of concept of the isomeric ratio in urine samples to distinguish the presence of cathinone as a result of accidental ingestion of plant-based product from that of an illicit use of a designer product is demonstrated. To the best of our knowledge, this is the first such work where cathinone enantiomers were resolved and quantified in horse blood plasma and urine at sub nanogram levels.

Optimization and validation of a new approach based on CE-HRMS for the screening analysis of novel psychoactive substances (cathinones, phenethylamines, and tryptamines) in urine.

The continuous introduction in the market of new psychoactive drugs (NPS) represents a well-known international emergency. Indeed, the European Monitoring Centre for Drugs and Drug Addiction and the United Nations Office on Drugs and Crime are paying great attention to the spread of NPS. In addition to the traditional analytical approaches based on GC-MS and HPLC-MS, also CE coupled with MS has proved to be a precious tool for the toxicological screening of biosamples. On these grounds, the aim of the present work was to test the application of CE-HRMS as a new screening tool for the rapid detection of these novel drugs in urine. Separations were performed in an uncoated fused-silica capillary with id of 75 µm with a total length of 100 cm, by applying a constant voltage of 15 kV. The QTOF-MS was implemented with an electrospray ion source operating in positive ionization full scan mode in the range of 100-1000 m/z. Under these conditions, different NPS has been tested, including eight cathinones, five phenethylamine, and seven tryptamines. The method was validated after optimization of the following analytical parameters: BGE composition and pH, separation voltage, sheath liquid composition, and flow rate and ESI source settings. The applicability of the method was successfully tested by analyzing a series of real urine samples obtained from drug users.

Stability of synthetic cathinones in clinical and forensic toxicological analysis-Where are we now?

Understanding the stability of analyzed drugs in biological samples is a crucial part for an appropriate interpretation of the analytical findings. Synthetic cathinones, as psychoactive stimulants, belong to a major class of new psychoactive substances. As they are subject to several degradation pathways, they are known to clinical and forensic toxicologists as unstable analytes in biological samples. When interpreting analytical data of synthetic cathinones in biological samples, analysts must be aware that the concentration of analytes may not accurately reflect the levels at the time they were acquired owing to many factors. This review provides (i) an overview of the current scientific knowledge on the stability of synthetic cathinones and/or metabolites in various human biological samples with a focus on factors that may deteriorate their stability-such as storage temperature, length of storage, matrix, pH, type of preservatives, concentration of analytes, and the chemistry of the analytes-and (ii) possible solutions on how to avoid such degradation. The PubMed database as well as Google Scholar was thoroughly searched to find published studies on the stability of synthetic cathinones since 2007 by searching specific keywords. A total of 23 articles met the inclusion criteria and were included in this review. Synthetic cathinones that carry methylenedioxy or N-pyrrolidine ring showed higher degradation resistance over other substituted groups. Acidification of samples pH plays a crucial role at increasing the stability of cathinones even with analytes that were frequently considered as poorly stable. This review also provides several recommendations for best practice in planning the experimental design, preservation, and storage conditions in order to minimize synthetic cathinones' degradation in human biological samples.

GC-MS analysis of underivatised new psychoactive substances in whole blood and urine.

Herein a method was develop and validated for the detection and quantification of five new psychoactive substances (NPS) belonging to three categories: synthetic cathinones (mephedrone, 3,4-MDPV), opioids (AH-7921) and cannabinoids (JWH-018, AM-2201) by EI GC-MS. Target analytes were quantified in whole blood; in urine the same compounds plus methylone were detected. Liquid-liquid extraction by MTBE - butyl acetate (1:1, v/v) in blood and butyl acetate in urine was applied for the recovery of analytes, while no derivatization was necessary for their sensitive detection and quantification. The method showed good linearity for all analytes within a concentration range from 0.25 to 2 µg/mL for mephedrone, from 0.02 to 0.16 µg/mL for 3,4-MDPV and AH-7921 and from 0.005 to 0.04 µg/mL for JWH-018 and AM-2201. LOD ranged from 0.002 µg/mL (JWH-018 and AM-2201 in blood and urine), to 0.08 µg/mL (mephedrone in urine). LOQ in blood ranged from 0.005 µg/mL for JWH-018 and AM-2201 to 0.25 µg/mL for mephedrone. Accuracy was within acceptable limits with % bias ranging from +20% to -17.98% for intra-assay study and from +18.87% to -11.16% for inter-assay study. Precision was found to be between 2.60% and 17.17% (CV%) for intra-assay study and from 6.03% to 13.72% (CV%) for inter-assay study. An intra laboratory comparison provided proof of the method robustness. The developed method can be used for the reliable and fast quantification of five NPS in blood and the detection of six NPS in urine within the practice of a clinical or forensic toxicology laboratory.

Absorption, Pharmacokinetics, and Urinary Excretion of Pyridines After Consumption of Coffee and Cocoa-Based Products Containing Coffee in a Repeated Dose, Crossover Human Intervention Study.

SCOPE: The present study assesses the absorption, pharmacokinetics, and urinary excretion of coffee pyridines and their metabolites after daily regular exposure to specific dosages of coffee or cocoa-based products containing coffee (CBPCC), considering different patterns of consumption. METHODS AND RESULTS: In a three-arm, crossover, randomized trial, 21 volunteers are requested to randomly consume for 1 month: one cup of espresso coffee per day, three cups of espresso coffee per day, or one cup of espresso coffee plus two CBPCC twice per day. The last day of the one-month treatment, blood and urine samples are collected for 24 h. Trigonelline, N-methylpyridinium, N-methylnicotinamide, and N-methyl-4-pyridone-5-carboxamide are quantified. Trigonelline and N-methylpyridinium absorption curves and 24-h urinary excretion reflect the daily consumption of different servings of coffee or CBPCC, showing also significant differences in main pharmacokinetic parameters. Moreover, inter-subject variability due to sex and smoking is assessed, showing sex-related differences in the metabolism of trigonelline and smoking-related ones for N-methylpyridinium. CONCLUSION: The daily exposure to coffee pyridines after consumption of different coffee dosages in a real-life setting is established. This data will be useful for future studies aiming at evaluating the bioactivity of coffee-derived circulating metabolites in cell experiments, mimicking more realistic experimental conditions.

A LC-MS/MS method for determination of 73 synthetic cathinones and related metabolites in urine.

Synthetic cathinones, which are a group of ß-keto analogs of phenethylamine, have been reported as the most emerging new psychoactive substances in the past decade. The quantity and variety of synthetic cathinones have continued to increase, which poses considerable risks to public health and social security. In this study, an analytical method based on liquid chromatography-tandem mass spectrometry (LCMS/MS) was established for the simultaneous determination of 73 synthetic cathinones and related metabolites in urine. The chromatographic analysis was performed using a Kinetex® Biphenyl column (10 cm ×2.1 mm, 1.7 µm), applying a gradient mobile phase, comprising 0.1 % formic acid aqueous solution with 5 mM ammonium acetate and 0.1 % formic acid methanolic solution; the entire run time of the analysis was within 8 min. The multiple reaction monitoring (MRM) mode was employed to collect the monitoring and quantitative ion pairs. Intra-day/inter-day precision and accuracy were less than 10 % for all the studied analytes. The limits of detection and quantification for all the analytes were 0.1-0.5 ng/mL and 0.5-1.0 ng/mL, respectively. The matrix effect was satisfactory for all the analytes, with a deviation lower than 20 %. The present method was further applied to 67 authentic urine samples in which 13 different synthetic cathinones were detected from 32 positive samples. The abuse of poly-synthetic cathinones was examined that up to seven items was detected in one case from authentic samples in this study.

Tentative identification of the phase I and II metabolites of two synthetic cathinones, MDPHP and α-PBP, in human urine.

Cathinone derivatives are one of the more prominent groups of new psychoactive substances in terms of the number of forensic case reports and the variety of chemical structures available. These substances often sold as "bath salts" are classified as psychostimulants. Using liquid chromatography-high resolution mass spectrometry, the metabolites of two pyrrolidine cathinone derivatives, α-PBP and the less common MDPHP, were tentatively identified in urine samples collected from patients admitted to hospital following drug intoxications. The major metabolic pathways for α-PBP and MDPHP were similar to those of their more common analogs (α-PVP and MDPV). Metabolites arising from hydroxylation, reduction of the carbonyl group to an alcohol, oxidation to form a lactam and subsequent ring-opening, and a combination of these processes were identified. In addition, biotransformations of the benzodioxole moiety in MDPHP included demethylenation with subsequent methylation and carboxylation of the butyl group. The majority of the hydroxylated metabolites of α-PBP and MDPHP were found to be glucuronidated. Both α-PBP and MDPHP undergo extensive metabolism and the chromatographic peak areas of the metabolites were found to be comparable to or exceeded those of the parent substances. Metabolites resulting from demethylenation and subsequent methylation (MDPHP), reduction of carbonyl group (α-PBP), and oxidation to form a lactam combined with ring-opening (α-PBP and MDPHP) were found to be the most useful target analytes for the confirmation of ingestion.

Anabasine-based measurement of cigarette consumption using wastewater analysis.

Community tobacco use can be monitored over time using wastewater-based epidemiological approaches by estimating the mass loads of nicotine and its metabolites, cotinine, or hydroxycotinine, in wastewater. However, due to the use of nicotine in smoking cessation products, other sources of nicotine contribute to cotinine and hydroxycotinine loads. The use of nicotine replacement therapies could vary in space and time and mask the true rates of tobacco consumption. Therefore, this work evaluated the content of tobacco specific markers, anatabine and anabasine, in cigarettes, in urine of smokers, and in wastewater. The results indicated that the anabasine content in both licit and illicit cigarettes in Australia is less variable than anatabine and is therefore considered a better measure of tobacco consumption. A study determining the excretion of tobacco-specific alkaloids of smoking and non-smoking volunteers gave an average urinary mass load of anabasine of 4.38 µg/L/person and a daily mass load of 1.13 µg/day/person. Finally, this was compared with the mass loads of anabasine from wastewater-based epidemiology data of 3 µg/day/person to estimate cigarette rates in a South Australian city: equivalent to 2.6 cigarettes/person/day. The rate of decline of cigarette use was greater when using anabasine as a measure of consumption compared with cotinine. This is the first study to estimate the rate of anabasine excretion, which can be used to estimate tobacco use independent of therapeutically prescribed nicotine.

Mass Spectrometry-Based Analysis of Urinary Biomarkers for Dietary Tomato Intake.

SCOPE: In this study, the applicability of several ß-carboline, imidazole, and steroidal alkaloids as biomarkers for tomato juice intake is evaluated. METHODS AND RESULTS: Over the course of a 2-week crossover dietary intervention study, 14 volunteers were given low and high doses of tomato juice after 3 days of avoiding tomato-based products. On the day of consumption and the following days, volunteers provided urine samples that were quantitatively analyzed by high-performance liquid chromatography-tandem mass spectrometry. Herein, glucose-derived ß-carboline alkaloids are determined as supporting, yet non-specific dietary biomarkers for tomato juice intake. Several imidazole alkaloids represent further biomarkers, which are shown to specifically indicate consumption of tomato juice for 24 h and partly >24 h. Additionally, steroidal alkaloids derived from esculeogenin B are determined to be specific biomarkers for tomato juice detectable for at least 48 h after consumption. The intake of low and high amounts of tomato juice is significantly distinguishable based on the urinary excretion of all determined biomarkers as well. CONCLUSIONS: The dietary intake of tomato juice is conclusively traceable based on urinary excretion of multiple ß-carboline, imidazole, and steroidal alkaloids, and can be determined for up to 48 h after consumption. Furthermore, different intake doses can clearly be distinguished based on their urinary excretion.

Discovery of Intake Biomarkers of Lentils, Chickpeas, and White Beans by Untargeted LC-MS Metabolomics in Serum and Urine.

SCOPE: To identify reliable biomarkers of food intake (BFIs) of pulses. METHODS AND RESULTS: A randomized crossover postprandial intervention study is conducted on 11 volunteers who consumed lentils, chickpeas, and white beans. Urine and serum samples are collected at distinct postprandial time points up to 48 h, and analyzed by LC-HR-MS untargeted metabolomics. Hypaphorine, trigonelline, several small peptides, and polyphenol-derived metabolites prove to be the most discriminating urinary metabolites. Two arginine-related compounds, dopamine sulfate and epicatechin metabolites, with their microbial derivatives, are identified only after intake of lentils, whereas protocatechuic acid is identified only after consumption of chickpeas. Urinary hydroxyjasmonic and hydroxydihydrojasmonic acids, as well as serum pipecolic acid and methylcysteine, are found after white bean consumption. Most of the metabolites identified in the postprandial study are replicated as discriminants in 24 h urine samples, demonstrating that in this case the use of a single, noninvasive sample is suitable for revealing the consumption of pulses. CONCLUSIONS: The results of the present untargeted metabolomics work reveals a broad list of metabolites that are candidates for use as biomarkers of pulse intake. Further studies are needed to validate these BFIs and to find the best combinations of them to boost their specificity.

Field-amplified sample injection combined with CE for the enantiodetermination of cathinones in urine samples.

This work presents a capillary electrophoresis methodology for the enantiodetermination of cathinones in urine employing a liquid-liquid extraction sample pretreatment. The cathinones were enantioseparated by adding a mixture of 8 mM 2-hydroxypropyl ß-cyclodextrin and 5 mM ß-cyclodextrin to the background electrolyte, which consists of 70 mM of monosodium phosphate aqueous solution at pH 2.5. Field-amplified sample injection was used as preconcentration strategy to improve the sensitivity. We studied various parameters that affect this stacking strategy, in particular, the sample solvent and its pH, the presence or absence of a low conductivity solvent plug introduced before the sample injection, the nature and volume of this plug, and the voltage and time of the electrokinetic injection of the sample. The optimum conditions were achieved by injecting a plug of isopropanol:H2 O 50/50 at 50 mbar for 5 s prior to the electrokinetic injection of the sample prepared in an aqueous solution of HCl 10-6  M. The sensitivity enhancement factors were from 562 to 601 in terms of peak area and from 444 to 472 in terms of peak height. The method was validated by analyzing spiked urine samples, obtaining a linear range of 25 to 1000 ng/mL and limits of detection ranging from 15 to 45 ng/mL.

Metabolic profile of alkaloids in Rhizoma Coptidis in rat plasma, urine and feces after oral administration using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

RATIONALE: Rhizoma Coptidis (RC) has been used to treat diabetes, pertussis, bacillary dysentery, sore throat, eczema, and aphtha for thousands of years. Alkaloids are the major components in RC, and its curative effect is achieved by oral administration. However, information on its composition in vivo is weak. METHODS: In this study, ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/QTOF-MS) was used to analyze the major active components and their metabolites in rat plasma, urine and feces after oral administration of RC extract. RESULTS: A total of 96 compounds including 8 prototype compounds and 88 metabolites were identified, and hydroxylation, reduction, demethylenation, demethylation, dehydrogenation, sulfation, glucuronidation and methylation were the major metabolic pathways. CONCLUSIONS: This study analyzed metabolic processes of the major active components in RC in vivo, which provided important information for its active composition and in vivo mechanism research. Meanwhile, metabolic profile studies on representative compounds provided valuable reference materials to elucidate the full-scale metabolites of RC.

Determination of synthetic cathinone α-pyrrolidinovalero-phenone and its metabolite in urine using solid-phase extraction and gas chromatography-mass spectrometry.

RATIONALE: The presence of α-pyrrolidinovalerophenone (α-PVP) and its metabolites in urine is evidence of the administration of α-PVP. A toxicological challenge is that the metabolites of α-PVP exhibit amphoteric properties, which make them unsuitable for detection using gas chromatography-mass spectrometry (GC/MS). In the study reported, proper derivatization and sample extraction were essential for improving the sensitivity for GC/MS analysis. METHODS: An automated solid-phase extraction (SPE) method has been developed and optimized. The derivatization efficiency was tested using longer reaction time and the addition of polar pyridine into a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane. Method validation, including linearity, limit of detection, precision, accuracy, and recovery, was evaluated using automatic SPE and GC/MS. RESULTS: The results suggested that adding pyridine to BSTFA (1:1, v/v) significantly improved derivatization efficiency and precision. After optimization, the linear range was from 25 to 1000 ng mL-1 with R2 > 0.9950. The limit of detection was 5 ng mL-1 for α-PVP and 25 ng mL-1 for OH-α-PVP. The recovery for SPE was over 88%. The inter-day and intra-day precisions were less than 15%. A forensic sample has been found containing α-PVP (67.3 ng mL-1 ) and OH-α-PVP (560.2 ng mL-1 ). CONCLUSIONS: This study is the first to validate an auto-SPE-GC/MS method for the quantification and qualification of α-PVP and OH-α-PVP in urine. We have successfully improved the derivatization efficiency and developed a sensitive and semi-automatic approach. This approach is desirable for the detection of synthetic cathinone at trace levels in biological samples.