Rat Urine Samples Research Articles

Identification of metabolites of Radix Paeoniae Alba extract in rat bile, plasma and urine by ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry

Ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC–Q-TOF/MS) was developed to identify the absorbed parent components and metabolites in rat bile, plasma and urine after oral administration of Radix Paeoniae Alba extract (RPAE). A total of 65 compounds were detected in rat bile, plasma and urine samples, including 11 parent compounds and 54 metabolites. The results indicated that glucuronidation, hydroxylation and methylation were the major metabolic pathways of the components of RPAE. Furthermore, the results of this work demonstrated that UPLC–Q-TOF/MS combined with MetaboLynx™ software and mass defect filtering (MDF) could provide unique high throughput capabilities for drug metabolism study, with excellent MS mass accuracy and enhanced MSE data acquisition. With the MSE technique, both precursor and fragment mass spectra can be simultaneously acquired by alternating between high and low collision energy during a single chromatographic run.

Simultaneous determination of trantinterol and its metabolites in rat urine and feces by liquid chromatography–tandem mass spectrometry

A highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of trantinterol (SPFF) and its major metabolites for the first time. The analytes were extracted from rat urine and feces samples by liquid-liquid extraction (LLE) and determined in multiple reaction monitoring (MRM) mode with clenbuterol as the internal standard. Chromatographic separation was achieved on a Venusil ASB C8 column (2.1mm×100mm, 3μm), with the mobile phase consisted of methanol-0.2% formic acid (30:70, v/v) at the flow rate of 0.2mL/min. Each sample was chromatographed within 5min. This method has a lower limit of quantification (LLOQ) of 0.450, 1.05, 1.35, 0.904 and 1.36ng/mL for trantinterol (SPFF), arylhydroxylamine trantinterol (N-OH-SPFF), tert-butyl hydroxylated trantinterol (Tert-OH-SPFF), 1-carbonyl trantinterol (SPFF-COOH) and 3-methyl sulfone-dechloro-trantinterol (SPFF-SO2CH3) in rat urine, and 0.450, 1.35 and 0.904ng/mL for SPFF, Tert-OH-SPFF and SPFF-COOH in rat feces, respectively. The linear correlation coefficients were greater than 0.990. The intra- and inter-day precision (relative standard deviation, RSD) values were below 15% and the accuracy (relative error, RE) was -9.9% to 11% at three quality control levels. The method has been successfully applied to the excretion study following an oral administration of 1mg/kg trantinterol to rats.

Simultaneous and sensitive determination of eight coumarins in rat bile and urine after oral administration ofRadix Angelicae Dahuricaeextract by liquid chromatography-electrospray ionization-mass spectrometry

A selective and sensitive liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method was developed and validated for analysis of xanthotoxol (1), xanthotoxin (2), isoimpinellin (3), bergapten (4), oxypeucedanin (5), imperatorin (6), cnidilin (7), and isoimperatorin (8) in rat bile and urine using pimpinellin as an internal standard (IS). An Agilent 1200 liquid chromatography system (Agilent Technologies, USA) equipped with a quaternary pump, an autosampler, and a column compartment was used for all analyses. Chromatographic separations were performed on a Sapphire C18 column (150 mm × 4.6 mm, 5 µm), and the column temperature was maintained at 30°C; the sample injection volume was 10 µL. The specificity, linearity, accuracy, precision, recovery, matrix effect, and several stabilities were validated for all analytes in the rat bile and urine samples. The method was successfully applied in monitoring the concentrations of eight coumarins in rat bile and urine after a single oral administration of Radix Angelicae Dahuricae extract with a dosage of 8.0 mL/kg. In the bile samples, the eight coumarins excreted completely in twenty-four hours. The average percentages of coumarins (1-8) excreted were 0.045%, 0.019%, 0.177%, 0.105%, 0.337%, 0.023%, 0.024%, 0.021%. In the urine samples, the eight coumarins excreted completely in seventy-two hours. The average percentages of coumarins (1-8) excreted were 1.78%, 0.095%, 0.130%, 0.292%, 0.082%, 0.008%, 0.005%, 0.004%. The method is robust and specific and it can successfully complete the requirements of the excretion study of the eight coumarins in Radix Angelicae Dahuricae.

High-Performance Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry for Metabolism Study of Timosaponin AIII

A sensitive method based on high-performance liquid chromatography-electrospray ionization tandem mass spectrometry was developed for the determination of timosaponin AIII (TA3) and its in vivo and in vitro metabolites. The rat plasma, urine, feces and tissue samples were collected after oral administration of TA3 at a single dose of 300 mg/kg. TA3 was incubated into artificial gastric juice and artificial intestinal juice. The in vivo and in vitro samples were purified by using liquid-liquid extraction. The structures of metabolites were elucidated by comparing their molecular weights, retention times and tandem mass spectrometric spectra with those of the parent drug. As a result, four metabolites (deglycosylated TA3, two hydroxylated TA3 and timosaponin BII) and the parent drug were found in in vivo and in vitro samples. In addition to the parent drug, one, one and two metabolites were identified in heart, urine and feces, respectively. Only the parent drug was detected in plasma, liver and kidney. One hydroxylation metabolite and TA3 were identified from incubation samples with AGJ, whereas two hydroxylation metabolites and TA3 were detected from the incubation with AIJ. This is the first systematic metabolism study of TA3. The biotransformation pathways of TA3 primarily included deglycosylation, hydroxylation and glycosylation.

The biotransformation of oleuropein in rats

A highly sensitive and specific LC-MS/MS method was developed to investigate the in vivo bio-transformation of oleuropein in rat. Rat feces and urine samples collected after oral administration were determined by liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative-ion mode. The assay procedure involves a simple liquid-liquid extraction of parent oleuropein and the metabolite from rat feces and urine with ethyl acetate. Chromatographic separation was operated with 0.1% formic acid aqueous and methanol in gradient program at a flow rate of 0.50 mL/min on an RP-C18 column with a total run time of 31 min. This method was successfully applied to simultaneous determination of oleuropein and its metabolites in rat feces and urine. De-glucosylation, hydrolysis, oxygenation and methylation were found to comprise the major metabolic pathway of oleuropein in rat gastrointestinal tract and three metabolites were absorbed into the blood circulatory system within 24 h after oral administration.

Investigation of in vivo metabolic profile of Abelmoschus Manihot based on pattern recognition analysis

Ethnopharmacological relevance:Abelmoschus manihot (L.) Medik. var. manihot is one of the most commonly used Chinese medicines and has played an important role in treating chronic glomerulonephritis and diabetic nephropathy. Aim of the study:Metabolites identification of traditional Chinese medicine (TCM) is a complex and time-consuming process due to the complicity of TCM and subsequent large number of detected ions. In this paper, UPLC–MS combined with pattern recognition analysis approach were used to simplify and quicken the identification of the metabolites of Abelmoschus Manihot. Materials and methods:Rat urine samples were collected before (as control sample) and after Abelmoschus Manihot administration. Pattern recognition analysis method was used to differentiate components between Abelmoschus Manihot-treated group and its controlled comparison. These components could be considered as Abelmoschus Manihot-related metabolites in vivo. Results:LC–MS based metabolomics could be an advanced tool to help us find metabolites with regards to its capacity of processing large datasets, differentiating and classifying of sample groups, as well as its indiscriminative nature of biomarker and metabolite identification. Using this method, seven metabolites were identified, which are flavonoid aglycone glucuronidation, sulfatation, and methylation metabolites. Conclusion:Our results showed that UPLC–MS based- pattern recognition analysis approach can be used to quickly identify Abelmoschus Manihot related metabolites in biological fluids. Furthermore, this work demonstrates the potential application of combining the UPLC–MS approach with the metabolomics approach in identifying the metabolites of TCM.

An integrated strategy for in vivo metabolite profiling using high-resolution mass spectrometry based data processing techniques

An ongoing challenge of drug metabolite profiling is to detect and identify unknown or low-level metabolites in complex biological matrices. Here we present a generic strategy for metabolite detection using multiple accurate-mass-based data processing tools via the analysis of rat samples of two model drug candidates, AZD6280 and AZ12488024. First, the function of isotopic pattern recognition was proved to be highly effective in the detection of metabolites derived from [14C]-AZD6280 that possesses a distinct isotopic pattern. The metabolites revealed using this approach were in excellent qualitative correlation to those observed in radiochromatograms. Second, the effectiveness of accurate mass based untargeted data mining tools such as background subtraction, mass defect filtering, or a data mining package (MZmine) used for metabolomic analysis in detection of metabolites of [14C]-AZ12488024 in rat urine, feces, bile and plasma samples was examined and a total of 33 metabolites of AZ12488024 were detected. Among them, at least 16 metabolites were only detected by the aid of the data mining packages and not via radiochromatograms. New metabolic pathways such as S-oxidation and thiomethylation reactions occurring on the thiazole ring were proposed based on the processed data. The results of these experiments also demonstrated that accurate mass-based mass defect filtering (MDF) and data mining techniques used in metabolomics are complementary and can be valuable tools for delineating low-level metabolites in complex matrices. Furthermore, the application of distinct multiple data-mining algorithms in parallel, or in tandem, can be effective for rapidly profiling in vivo drug metabolites.

Discovery of safety biomarkers for realgar in rat urine using UFLC-IT-TOF/MS and 1H NMR based metabolomics

As an arsenical, realgar (As4S4) is known as a poison and paradoxically as a therapeutic agent. However, a complete understanding of the precise biochemical alterations accompanying the toxicity and therapy effects of realgar is lacking. Using a combined ultrafast liquid chromatography (UFLC) coupled with ion trap time-of-flight mass spectrometry (IT-TOF/MS) and (1)H NMR spectroscopy based metabolomics approach, we were able to delineate significantly altered metabolites in the urine samples of realgar-treated rats. The platform stability of the liquid chromatography LC/MS and NMR techniques was systematically investigated, and the data processing method was carefully optimized. Our results indicate significant perturbations in amino acid metabolism, citric acid cycle, choline metabolism, and porphyrin metabolism. Thirty-six metabolites were proposed as potential safety biomarkers related to disturbances caused by realgar, and glycine and serine are expected to serve as the central contacts in the metabolic pathways related to realgar-induced disturbance. The LC/MS and NMR based metabolomics approach established provided a systematic and holistic view of the biochemical effects of realgar on rats, and might be employed to investigate other drugs or xenobiotics in the future.

Irreversible Renal Damage after Transient Renin-Angiotensin System Stimulation: Involvement of an AT1-Receptor Mediated Immune Response

Transient activation of the renin-angiotensin system (RAS) induces irreversible renal damage causing sustained elevation in blood pressure (BP) in Cyp1a1-Ren2 transgenic rats. In our current study we hypothesized that activation of the AT1-receptor (AT1R) leads to a T-cell response causing irreversible impairment of renal function and hypertension. Cyp1a1-Ren2 rats harbor a construct for activation of the RAS by indole-3-carbinol (I3C). Rats were fed a I3C diet between 4–8 weeks of age to induce hypertension. Next, I3C was withdrawn and rats were followed-up for another 12 weeks. Additional groups received losartan (20 mg/kg/day) or hydralazine (100 mg/kg/day) treatment between 4–8 weeks. Rats were placed for 24h in metabolic cages before determining BP at week 8, 12 and 20. At these ages, subsets of animals were sacrificed and the presence of kidney T-cell subpopulations was investigated by immunohistochemistry and molecular marker analysis. The development of sustained hypertension was completely prevented by losartan, whereas hydralazine only caused a partial decrease in BP. Markers of renal damage: KIM-1 and osteopontin were highly expressed in urine and kidney samples of I3C-treated rats, even until 20 weeks of age. Additionally, renal expression of regulatory-T cells (Tregs) was highly increased in I3C-treated rats, whereas the expression of T-helper 1 (Th1) cells demonstrated a strong decrease. Losartan prevented these effects completely, whereas hydralazine was unable to affect these changes. In young Cyp1a1-Ren2 rats AT1R activation leads to induction of an immune response, causing a shift from Th1-cells to Tregs, contributing to the development of irreversible renal damage and hypertension.

Pharmacokinetics of 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐D‐glucoside in rat using ultra‐performance LC‐quadrupole TOF‐MS

2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside (THSG) from Polygoni multiflori has been demonstrated to possess a variety of pharmacological activities, including antioxidant, anti-inflammatory and hepatoprotective activities. Ultra-performance LC-quadrupole TOF-MS with MS Elevated Energy data collection technique and rapid resolution LC with diode array detection and ESI multistage MS(n) methods were developed for the pharmacokinetics, tissue distribution, metabolism, and excretion studies of THSG in rats following a single intravenous or oral dose. The three metabolites were identified by rapid resolution LC-MS(n). The concentrations of the THSG in rat plasma, bile, urine, feces, or tissue samples were determined by ultra-performance LC-MS. The results showed that THSG was rapidly distributed and eliminated from rat plasma. After the intravenous administration, THSG was mainly distributing in the liver, heart, and lung. For the rat, the major distribution tissues after oral administration were heart, kidney, liver, and lung. There was no long-term storage of THSG in rat tissues. Total recoveries of THSG within 24 h were low (0.1% in bile, 0.007% in urine, and 0.063% in feces) and THSG was excreted mainly in the forms of metabolites, which may resulted from biotransformation in the liver.

PHARMACOKINETICS, BIOAVAILABILITY, AND METABOLISM OF 2,3,5,4′-TETRAHYDROXYSTILBENE-2-O-β-D-GLUCOSIDE IN RATS BY ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY–QUADRUPOLE TIME-OF-FLIGHT MASS SPECTROMETRY AND HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-ULTRAVIOLET DETECTION

2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (THSG), a natural product purified from the Chinese medical herb Polygoni multiflori Radix, has been demonstrated to possess many pharmacological activities, including antioxidant, anti-inflammatory, hepatoprotective, and antitumor properties. Ultra-performance liquid chromatography–time-of-flight mass spectrometry (UPLC-Q-TOF/MS) and high-performance liquid chromatography-ultraviolet detection (HPLC-UV) methods were developed for the pharmacokinetics, bioavailability, absorption, and metabolism studies of THSG in rats following a single intravenous or oral administration. The metabolites (M1 and M2) were identified in plasma by UPLC-Q-TOF/MS. The concentrations of the THSG in rat plasma, and bile, urine, or feces samples were determined by HPLC-UV. The results show THSG was rapidly absorbed within 30 min in plasma. Absolute bioavailability of THSG was 40%. Total recovery of unchanged THSG within 24 hr were low (0.041% in bile, 0.06% in feces), whereas the amount of unchanged THSG excreted in the urine within 24 hr was lower than LLOQ.

2-Methiopropamine, a thiophene analogue of methamphetamine: studies on its metabolism and detectability in the rat and human using GC-MS and LC-(HR)-MS techniques

2-Methiopropamine [1-(thiophen-2-yl)-2-methylaminopropane, 2-MPA], a thiophene analogue of methamphetamine, is available from online vendors selling "research chemicals." The first samples were seized by the German police in 2011. As it is a recreational stimulant, its inclusion in routine drug screening protocols should be required. The aims of this study were to identify the phase I and II metabolites of 2-MPA in rat and human urine and to identify the human cytochrome-P450 (CYP) isoenzymes involved in its phase I metabolism. In addition, the detectability of 2-MPA in urine samples using the authors' well-established gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-linear ion trap-mass spectrometry (LC-MS(n)) screening protocols was also evaluated. The metabolites were isolated from rat and human urine samples by solid-phase extraction without or following enzymatic cleavage of conjugates. The phase I metabolites, following acetylation, were separated and identified by GC-MS and/or liquid chromatography-high-resolution linear ion trap mass spectrometry (LC-HR-MS(n)) and the phase II metabolites by LC-HR-MS(n). The following major metabolic pathways were proposed: N-demethylation, hydroxylation at the side chain and at the thiophene ring, and combination of these transformations followed by glucuronidation and/or sulfation. CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were identified as the major phase I metabolizing enzymes. They were also involved in the N-demethylation of the analogue methamphetamine and CYP2C19, CYP2D6, and CYP3A4 in its ring hydroxylation. Following the administration of a typical user's dose, 2-MPA and its metabolites were identified in rat urine using the authors' GC-MS and the LC-MS(n) screening approaches. Ingestion of 2-MPA could also be detected by both protocols in an authentic human urine sample.

In vivo and in vitro metabolism of a novel β2-adrenoceptor agonist, trantinterol: metabolites isolation and identification by LC-MS/MS and NMR

Trantinterol is a novel β(2)-adrenoceptor agonist used for the treatment of asthma. The aim of this study is to identify the metabolites of trantinterol using liquid chromatography tandem mass spectrometry (LC-MS/MS), to isolate the main metabolites, and confirm their structures by nuclear magnetic resonance (NMR). Urine, feces, bile, and blood samples of rats were obtained and analyzed. Reference standards of six metabolites were achieved with the combination of chemical synthesis, microbial transformation, and the model systems of rats. Moreover, in order to investigate the phase I metabolism of trantinterol in humans and to study the species differences between rats and humans, incubations with liver microsomes were performed. The biotransformation by a microbial model Cunninghamella blakesleana AS 3.970 was also studied. A total of 18 metabolites were identified in vivo and in vitro together, 13 of which were newly detected. Three phase I metabolites were detected in vivo and in vitro as well as in the microbial model, including the arylhydroxylamine (M1), the tert-butyl hydroxylated trantinterol (M2) and the 1-carbonyltrantinterol (M3). Another important pathway in rats is glutathione conjugation and further catabolism and oxidation to form consecutive derivatives (M4 through M10). Other metabolites include glucuronide, glucoside, and sulfate conjugates. The results of in vitro experiments indicate no species difference exists among rats, humans, and C. blakesleana AS 3.970 on the phase I metabolism of trantinterol. Our study provided the most comprehensive picture for trantinterol in vivo and in vitro metabolism to this day, and may predict its metabolism in humans.

Metabolite profiling and identification of L41, a novel cyclin-dependent kinase 1 inhibitor, in rat plasma and urine by liquid chromatography–tandem mass spectrometry

(E)-3-(2-Chlorophenyl)-1-(2-hydroxy-4,6-dimethoxy-3-(1,2,3,6-tetrahydropyridine-4-yl)phenyl)-2-propylene-1-one (L41) is a novel synthetic cyclin-dependent kinase 1 inhibitor. In this study, we proposed a strategy to systematically characterize associated phase I and phase II metabolites of L41 using rat plasma and urine samples. Biological samples were treated with an optimized sample preparation approach involving protein precipitation, liquid–liquid extraction and solid-phase extraction prior to LC–MS/MS analysis. Both an Agilent LC/MSD Trap VL and an AB SCIEX API 5000 triple quadrupole mass spectrometer were used for LC–MS/MS analysis. Based on the proposed strategy, 3 phase I and 4 phase II metabolites were detected by a highly sensitive MRM method. The major metabolites of L41 were found to be demethylation metabolites (M5, M6), C-4′,6′-di-demethylation metabolites followed by a C-2′ (OH) methylation metabolite (M7), and were shown to undergo subsequent glucuronidation processes. These results provide evidence for in vivo L41 metabolism, and the method may be applied to the further analysis of L41 and its metabolites in biological samples.

Determination of stanozolol and 3′-hydroxystanozolol in rat hair, urine and serum using liquid chromatography tandem mass spectrometry

BackgroundAnabolic androgenic steroids, such as stanozolol, are typically misused by athletes during preparation for competition. Out-of-competition testing presents a unique challenge in the current anti-doping detection system owing to logistic reasons. Analysing hair for the presence of a prohibited drug offers a feasible solution for covering the wider window in out-of-competition testing. To assist in vivo studies aiming to establish a relationship between drug levels detected in hair, urine and blood, sensitive methods for the determination of stanozolol and its major metabolite 3′-hydroxystanozolol were developed in pigmented hair, urine and serum, using brown Norway rats as a model system and liquid chromatography tandem mass spectrometry (LC-MS/MS).ResultsFor method development, spiked drug free rat hair, blood and urine samples were used. The newly developed method was then applied to hair, urine and serum samples from five brown Norway rats after treatment (intraperitoneal) with stanozolol for six consecutive days at 5.0 mg/kg/day. The assay for each matrix was linear within the quantification range with determination coefficient (r2) values above 0.995. The respective assay was capable of detecting 0.125 pg/mg stanozolol and 0.25 pg/mg 3′-hydroxystanozolol with 50 mg hair; 0.063 ng/mL stanozolol and 0.125 ng/mL 3′-hydroxystanozolol with 100 μL of urine or serum. The accuracy, precision and extraction recoveries of the assays were satisfactory for the detection of both compounds in all three matrices. The average concentrations of stanozolol and 3′-hydroxystanozolol, were as follows: hair = 70.18 ± 22.32 pg/mg and 13.01 ± 3.43 pg/mg; urine = 4.34 ± 6.54 ng/mL and 9.39 ± 7.42 ng/mL; serum = 7.75 ± 3.58 ng/mL and 7.16 ± 1.97 ng/mL, respectively.ConclusionsThe developed methods are sensitive, specific and reproducible for the determination of stanozolol and 3′-hydroxystanozolol in rat hair, urine and serum. These methods can be used for in vivo studies further investigating stanozolol metabolism, but also could be extended for doping testing. Owing to the complementary nature of these tests, with urine and serum giving information on recent drug use and hair providing retrospective information on habitual use, it is suggested that blood or urine tests could accompany hair analysis and thus avoid false doping results.

Metabolism studies of benzbromarone in rats by high performance liquid chromatography–quadrupole time of flight mass spectrometry

A high performance liquid chromatography–quadrupole time of flight mass spectrometry (HPLC–QTOF-MS) method was employed in investigation of benzbromarone metabolites in rat plasma, urine, feces and bile samples. Meanwhile, the metabolic pathways of benzbromarone in rats were discussed. The identification was achieved on a reversed-phase C18 column with mobile phase gradient method. The QTOF-MS was operated under full scan of MS or MS/MS in negative mode. The fragments were acquired by raising collision induced dissociation (CID) energy for speculating the structures of parent ions. According to the information from the chromatograms and mass spectra, 17 metabolites were obtained. Among them, the deoxidized phase I metabolites and an array of phase II metabolites—sulfate conjugates detected in the biological samples made the work more significant.

704 IS THERE AN ESSENTIAL ROLE FOR ADAPTIVE IMMUNITY UNDERLYING RENIN-ANGIOTENSIN-INDUCED RENAL DAMAGE?

Objective: Transient activation of the renin-angiotensin system (RAS) induces irreversible renal damage causing sustained elevation in blood pressure (BP) in Cyp1a1-mRen2 transgenic rats. We hypothesize that activation of the AT1-receptor induces an adaptive immune response underlying this irreversible impairment of renal function and pathology. Methods: Cyp1a1-mRen2 rats harbor a construct for activation of the RAS by indole-3-carbinol (I3C). Rats were fed a I3C diet between 4-8 weeks of age to induce hypertension. Next, I3C was withdrawn and rats were followed-up for another 12 weeks. Additional groups received losartan or hydralazine treatment between 4-8 weeks. Rats were placed for 24 h in metabolic cages before determining BP at week 8, 12 and 20. At these ages, subsets of animals were sacrificed and the presence of kidney T-cell subpopulations was investigated by immunohistochemistry and molecular marker analysis. Results: Losartan prevented the development of sustained hypertension, whereas hydralazine only caused a partial decrease in BP. KIM-1 and osteopontin, markers of renal damage linked to inflammation, were highly expressed in urine and kidney samples of I3C-treated rats, even till 20 weeks. Furthermore, renal expression of regulatory-T cells (Tregs) was highly increased in I3C-treated rats, whereas expression of T-helper1 (Th1) cells demonstrated a strong decrease. Losartan prevented these effects completely, whereas hydralazine was ineffective. Conclusion: In young Cyp1a1-mRen2 rats AT1-receptor activation leads to induction of an adaptive immune response, a shift from Th1-cells to Tregs, contributing to the development of irreversible renal damage and hypertension. This study was performed within the framework of TI-Pharma projectT2-301.

A Sensitive and Selective LC-MS Method for the Determination of Lurasidone in Rat Plasma, Bile, and Urine

A liquid chromatography-mass spectrometry (LC-MS) assay was developed and validated for the quantification of lurasidone, an atypical antipsychotic drug, in rat plasma, bile, and urine. Rat plasma, bile, or urine samples were processed by liquid–liquid extraction and injected onto an LC-MS system for the quantification of lurasidone and ziprasidone (an internal standard). Lurasidone and ziprasidone were separated from endogenous substances using a Gemini C6-Phenyl column with mixture of acetonitrile and 0.1 % formic acid (80:20, v/v) as the mobile phase. Quantification was performed using the selected ion monitoring mode at m/z 493 for lurasidone and m/z 413 for the IS. The detector response was specific and linear for lurasidone in the concentration range 5–5,000 ng mL−1 The intra- and inter-day accuracy and precision of the method were determined to be within the acceptable criteria for assay validation guidelines. In addition, lurasidone was stable under a variety of processing and handling conditions. Lurasidone concentrations could be readily measured in rat plasma, bile, and urine samples up to 24 h after an intravenous or oral administration, suggesting that the assay can be used in pharmacokinetic studies of lurasidone in rats.

A HILIC–MS/MS method for the simultaneous determination of seven organic acids in rat urine as biomarkers of exposure to realgar

Realgar (As4S4) is a traditional medicine used in China and Europe for thousands of years. As an arsenical, the toxicity from realgar has raised public concern. Several organic acids in urine are found to be potential biomarkers of realgar exposure, including taurine, citric, glutamic, lactic, pyruvic, succinic and uric acid. In this study, using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS), a rapid and sensitive method was developed to separate and quantify these compounds in urine. A ZIC®-HILIC column was used for the separation at an isocratic condition of acetonitrile and 10mM ammonium acetate in water. Analytes were detected in multiple-reaction monitoring with negative ionization mode, using ibuprofen as internal standard. Good line arities (R2>0.996) were obtained for all analytes with the limits of detection from 0.2 to 0.7μg/mL. The intra-day and inter-day accuracy ranged from 89.1 to 104.4% and the relative standard deviation (RSD) did not exceed 15.0%. The recovery was more than 80%with RSD less than 14.0%. The validated method was applied to analyze the urine samples of control and reaglar treated rats, and significant changes of these organic acids were observed.

A simple and sensitive HPLC‐UV determination of 7‐O‐succinyl macrolactin A in rat plasma and urine and its application to a pharmacokinetic study

A simple, sensitive and reproducible isocratic reversed-phase (C(18) ) high-performance liquid chromatography (HPLC) method was developed to determine 7-O-succinyl macrolactin A (SMA) in rat plasma and urine samples using UV detector set at 230 nm. Lamotrigine was used as internal standards (IS) to ensure the precision and accuracy of the method. The retention times of SMA and IS for the plasma sample were 9.2 and 4.4 min, respectively, and those for the urine samples were 7.9 and 4.3 min, respectively. The intra- and inter-day variations of the analytical responses, expressed in terms of relative standard deviation, were less than 14.9%. The accuracy, in terms of average analytical recovery, ranged from 90.4 to 119%. The lower limits of quantification of SMA in rat plasma and urine samples were 0.02 and 0.1 µg/mL, respectively. This method is applicable for the pharmacokinetic studies of SMA in rats.