Major Metabolites In Rat Plasma Research Articles

A validated high‐performance liquid chromatography‐tandem mass spectrometry method for simultaneous determination of irinotecan and two major metabolites in rat plasma: Application for the pharmacokinetics study

AbstractA high‐performance liquid chromatography‐tandem mass spectrometry method has been developed for the determination of irinotecan, 7‐ethyl‐10‐hydroxycamptothecin (SN38), and SN38 glucuronide (SN38G) in rat plasma in the present study. The analytes were separated on a C18 column and a triple‐quadrupole mass spectrometry equipped with an electrospray ionization source was applied for detection. Irinotecan, SN38 and SN38G could be well retained in the C18 column after optimization of mobile phase. A simple protein precipitation was used to pretreat the plasma. The extraction recovery was above 90% and the matrix effect could be negligible. The method was linear over the concentration ranges of 3.46–3458.8 ng/mL for irinotecan, 2.53–2530.0 ng/mL for SN38 and 2.5–2500.0 ng/mL for SN38G. The precision and accuracy was within the acceptable limits. The simple and convenient method was validated and successfully applied to support the pharmacokinetic study and elucidate the mechanism of irinotecan‐induced diarrhea after irinotecan was intravenously injected to the Sprague‐Dawley rats.

Metabolite Study and Structural Authentication for the First-in-Human Use Sphingosine-1-phosphate Receptor 1 Radiotracer.

The sphingosine-1-phosphate receptor 1 (S1PR1) radiotracer [11C]CS1P1 has shown promise in proof-of-concept PET imaging of neuroinflammation in multiple sclerosis (MS). Our HPLC radiometabolite analysis of human plasma samples collected during PET scans with [11C]CS1P1 detected a radiometabolite peak that is more lipophilic than [11C]CS1P1. Radiolabeled metabolites that cross the blood-brain barrier complicate quantitative modeling of neuroimaging tracers; thus, characterizing such radiometabolites is important. Here, we report our detailed investigation of the metabolite profile of [11C]CS1P1 in rats, nonhuman primates, and humans. CS1P1 is a fluorine-containing ligand that we labeled with C-11 or F-18 for preclinical studies; the brain uptake was similar for both radiotracers. The same lipophilic radiometabolite found in human studies also was observed in plasma samples of rats and NHPs for CS1P1 labeled with either C-11 or F-18. We characterized the metabolite in detail using rats after injection of the nonradioactive CS1P1. To authenticate the molecular structure of this radiometabolite, we injected rats with 8 mg/kg of CS1P1 to collect plasma for solvent extraction and HPLC injection, followed by LC/MS analysis of the same metabolite. The LC/MS data indicated in vivo mono-oxidation of CS1P1 produces the metabolite. Subsequently, we synthesized three different mono-oxidized derivatives of CS1P1 for further investigation. Comparing the retention times of the mono-oxidized derivatives with the metabolite observed in rats injected with CS1P1 identified the metabolite as N-oxide 1, also named TZ82121. The MS fragmentation pattern of N-oxide 1 also matched that of the major metabolite in rat plasma. To confirm that metabolite TZ82121 does not enter the brain, we radiosynthesized [18F]TZ82121 by the oxidation of [18F]FS1P1. Radio-HPLC analysis confirmed that [18F]TZ82121 matched the radiometabolite observed in rat plasma post injection of [18F]FS1P1. Furthermore, the acute biodistribution study in SD rats and PET brain imaging in a nonhuman primate showed that [18F]TZ82121 does not enter the rat or nonhuman primate brain. Consequently, we concluded that the major lipophilic radiometabolite N-oxide [11C]TZ82121, detected in human plasma post injection of [11C]CS1P1, does not enter the brain to confound quantitative PET data analysis. [11C]CS1P1 is a promising S1PR1 radiotracer for detecting S1PR1 expression in the CNS.

Quantification of a promising JNK inhibitor and nitrovasodilator IQ-1 and its major metabolite in rat plasma byLC-MS/MS.

Background: IQ-1 is a promising c-Jun-N-terminal kinase inhibitor and nitrovasodilator. An LC-MS/MS method was validated to determine IQ-1 isomers andmajor metabolite IQ-18 in rat plasma. Materials & methods: The analytes were extracted using ethyl acetate. The chromatographic separation was performed on a C8 column (150×4.6mm, 5μm) under acetonitrile-water (5mM ammonium formate buffer, pH 2.93) gradient elution. Multiple reaction monitoring was used for MS/MS detection in the positive ion mode. Results: The method was fully validated over the range of 0.1-400ng/ml (Z-isomer), 0.9-3600ng/ml (E-isomer), 5.0-4000 (IQ-18). Conclusion: This method has been successfully applied to pharmacokinetic studies of IQ-1 and IQ-18 in rats after a single oral dose of IQ-1 (50mg/kg).

Development and validation of a novel UPLC‐MS/MS method for the simultaneous quantification of mycophenolic mofetil and its major metabolites in rat plasma and liver S9 hydrolysis assay in rats

A sensitive liquid chromatography‐tandem mass spectrometry method was developed and validated for the simultaneous quantification of mycophenolic mofetil (MMF) and its major metabolites, mycophenolic acid (MPA), mycophenolic acyl glucuronide (AcMPAG), and mycophenolic glucuronide (MPAG), in rat plasma. Upon administration, mycophenolic mofetil (MMF), a prodrug, undergoes extensive metabolism in the liver. Following hydrolyzation, the active metabolite MPA is produced. MPA can then be further metabolized to produce its glucuronide conjugated metabolites, AcMPAG and MPAG. This method can simultaneously determine both the parent compound and its major metabolites within biological samples. In addition, liver S9 fraction hydrolysis studies of MMF results in the production of MPA, demonstrating that the parent drug is first hydrolyzed within the liver. A novel UPLC method was developed to simultaneously quantify both compounds following S9 liver enzyme hydrolysis.A Shimadzu UHPLC system coupled to an AB Sciex QTrap 4000 mass spectrometer was used for the analysis. Separation was achieved using an Ultra Biphenyl 5µm column (100 × 2.1mm) with acetonitrile and 0.1% formic acid as the mobile phases. Analysis was performed under positive ionization mode using the multiple reaction monitoring (MRM) approach. Additionally, to determine the hydrolysis rate, different concentrations of MMF were incubated with liver S9 fraction (0.5µg/ml final concentration) for 30 mins, after which the reaction was terminated using 6% formic acid in acetonitrile. Samples were prepared and MPA concentrations were determined in the reaction system using UPLC for detection and quantification.The method was linear in the range of 9.77nM – 5000nM with correlation coefficient values > 0.99 for all components. The method has been shown to be reproducible, with intra‐ and inter‐day accuracy and precision ±12.3% of nominal values, for all analytes. The average extraction recovery rates for MMF, MPA, AcMPAG, and MPAG were 91.3%, 97.9%, 98.4%, and 90.5%, respectively. Matrix effect was in the acceptable range (<15%). The analytes in plasma were found to be stable under bench –top, freeze‐thaw, and storage (‐4°C) conditions. The metabolic studies showed that mycophenolic mofetil can be rapidly hydrolyzed by rat liver S9 fraction with Vmax and Km values of 0.66±0.78 nmol/min/µg and 80.06±123.1µM respectively.This novel UPLC‐MS/MS method can comprehensively evaluate and quantify the concentration of both mycophenolic mofetil and its major metabolites, mycophenolic acid, mycophenolic acyl glucuronide, and mycophenolic glucuronide in biological samples. This method can be used as a tool to further investigate and understand the extensive metabolism of mycophenolic mofetil in clinical practice.

Enhanced Intestinal Absorption and Pharmacokinetic Modulation of Berberine and Its Metabolites through the Inhibition of P-Glycoprotein and Intestinal Metabolism in Rats Using a Berberine Mixed Micelle Formulation

We aimed to develop a berberine formulation to enhance the intestinal absorption and plasma concentrations of berberine through the inhibition of P-glycoprotein (P-gp)-mediated efflux and the intestinal metabolism of berberine in rats. We used pluronic P85 (P85) and tween 80, which have the potential to inhibit P-gp and cytochrome P450s (i.e., CYP1A2, 2C9, 2C19, 2D6, and 3A4). A berberine-loaded mixed micelle formulation with ratios of berberine: P85: tween 80 of 1:5:0.5 (w/w/w) was developed. This berberine mixed micelle formulation had a mean size of 12 nm and increased the cellular accumulation of digoxin via P-gp inhibition. It also inhibited berberine metabolism in rat intestinal microsomes, without significant cytotoxicity, up to a berberine concentration of 100 μM. Next, we compared the pharmacokinetics of berberine and its major metabolites in rat plasma following the oral administration of the berberine formulation (50 mg/kg) in rats with the oral administration of berberine alone (50 mg/kg). The plasma exposure of berberine was significantly greater in rats administered the berberine formulation compared to rats administered only berberine, which could be attributed to the increased berberine absorption by inhibiting the P-gp-mediated berberine efflux and intestinal berberine metabolism by berberine formulation. In conclusion, we successfully prepared berberine mixed micelle formulation using P85 and tween 80 that has inhibitory potential for P-gp and CYPs (CYP2C19, 2D6, and 3A4) and increased the berberine plasma exposure. Therefore, a mixed micelle formulation strategy with P85 and tween 80 for drugs with high intestinal first-pass effects could be applied to increase the oral absorption and plasma concentrations of the drugs.

Preparation of a Major Metabolite of Iguratimod and Simultaneous Assay of Iguratimod and Its Metabolite by HPLC in Rat Plasma.

Iguratimod is a new synthetic disease-modifying antirheumatic drug intended to treat patients with rheumatoid arthritis. A new method using recombinant human CYP450s yeast cells containing c-DNA expressed P450s was applied to identify the metabolic pathways of iguratimod and to prepare its metabolite. The metabolite was isolated, and its structure was identified by quadrupole time-of-flight-mass spectrometry and nuclear magnetic resonance. Furthermore, a selective and sensitive high performance liquid chromatography (HPLC) method was developed for the simultaneous quantification of iguratimod and its major metabolite in rat plasma for the first time. The results indicated that iguratimod was mainly metabolized to a metabolite by CYP2C9 and CYP2C19 in in-vitro study. The structure of the metabolite was identified as M2 (N-[3-(acetamido)-4-oxo-6-phenoxy-4H-chromen-7-yl]methanesulfonamide). HPLC assay was achieved on a C18 column using methanol-water containing 0.1% trifluoroacetic acid (55:45 v/v) at a flow rate of 1 mL/min with UV detection at 257 nm. Standard calibration curves were obtained in the concentration range of 0.5–20 µg/mL for iguratimod and its metabolite M2. The lower limits of detection of iguratimod and M2 in rat plasma were 0.1 and 0.25 µg/mL, respectively. The intra- and inter-day precision (RSD%) were within 5% for the two analytes. The average recoveries of the analytes were greater than 90%. In conclusion, recombinant human CYP450s whole-yeast transformation system could be successfully used to identify and prepare the major metabolite of iguratimod. The HPLC method we developed could be successfully applied to evaluate pharmacokinetics of iguratimod and its metabolite M2 in rats.

Effect of Wuziyanzong pill on metabolism of dapoxetine in vivo and in vitro

In vitro incubation of rat liver microsomes with 30 μL of 100 μmol·L−1 dapoxetine and 30 μL of 10, 100, 250, 500, 1000, 2500, or 5000 μg·mL−1 Wuziyanzong pill was performed at 37 °C for 60 min. Dapoxetine concentration was analyzed by high performance liquid chromatography (HPLC). The half maximal inhibitory concentration (IC50) of Wuziyanzong pill on metabolism of dapoxetine was 296.10 μg mL−1in vitro. Twelve SD rats were randomly divided into 2 groups: Control group and Wuziyanzong pill group. The two groups were administrated with 10 mL·kg-1 saline (Control group) or 10 mL·kg-1 Wuziyanzong pill solution (Experimental group, solution contained 200 mg mL-1 Wuziyanzong pill) for 15 consecutive days. Following administration of saline or Wuziyanzong pill on the 15th day, 20 mg kg-1 dapoxetine was administered to all rats. Blood was collected from the tail vein (0.3 mL) at multiple time points, and ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was used to determine the concentration of dapoxetine and its main metabolites, dapoxetine-N-oxide and desmethyldapoxetine in rats. Pharmacokinetic analysis of dapoxetine showed that area under the concentration-time curve (AUC) and mean maximum plasma concentration (Cmax) of the Wuziyanzong pill group were decreased, while plasma clearance (CLz) was increased compared with control group (P < 0.01). The HPLC method for determination of dapoxetine in vitro was accurate and specific. The UHPLC-MS/MS method established for determination of dapoxetine and its major metabolites in rat plasma was rapid and specific, which met the requirements of pharmacokinetic guidelines. Wuziyanzong pill had a weak inhibitory effect on metabolism of dapoxetine in vitro, but had a very strong induction effect in vivo, suggesting the dosage of dapoxetine should be increased when administered in combination with Wuziyanzong pill.

An HPLC-MS/MS method for the simultaneous determination of luteolin and its major metabolites in rat plasma and its application to a pharmacokinetic study.

A selective and accurate HPLC-MS/MS method was established to simultaneously quantify luteolin and its active metabolites (diosmetin, chryseriol, and luteolin-7-O-glucuronide) in rat plasma. The analytes were separated on a C18 column with a mobile phase of water containing 0.5% formic acid and acetonitrile under gradient elution to shorten the total chromatographic run time and increase the resolution of diosmetin and chryseriol. A triple quadruple mass spectrometer coupled with an electrospray ionization source in the negative ion mode was used to detect the analytes. The multiple reaction monitoring transitions were of m/z 284.9→132.9 for luteolin, m/z 298.9→283.9 for diosmetin and chryseriol, m/z 461.1→284.9 for luteolin-7-O-glucuronide, and m/z 300.9→150.9 for the internal standard. The method was linear within the concentration ranges of 0.06-90 μg/mL for luteolin, 0.03-12 μg/mL for diosmetin, 0.015-4.8 μg/mL for chryseriol, and 0.06-60 μg/mL for luteolin-7-O-glucuronide. The intra- and interday precisions were all within 6.0%. Accuracy ranged from -3.2 to 6.4%. The matrix effect and instability were not observed during bioanalysis. This method was used to study the pharmacokinetic characteristics of luteolin and its metabolites in rats after treatment with luteolin.

Metabolites characterization of a novel DPP-4 inhibitor, imigliptin in humans and rats using ultra-high performance liquid chromatography coupled with synapt high-resolution mass spectrometry

Imigliptin has been reported as a novel dipeptidyl-peptidase-IV (DPP-4) inhibitor to treat type 2 Diabetes Mellitus (T2DM), and is currently being tested in clinical trials. In the first human clinical study, imigliptin was well tolerated and proved to be a potent DPP-4 inhibitor. Considering its potential therapeutic benefits and promising future, it is of great importance to study the metabolite profiles in the early stage of drug development. In the present study, a robust and reliable analytical method based on the ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) method combined with MassLynx software was established to investigate the characterization of metabolites of imigliptin in human and rat plasma, urine and feces after oral administration. As a result, a total of 9 metabolites were identified in humans, including 6, 9 and 8 metabolites in human plasma, urine, and feces, respectively. A total of 11 metabolites were identified in rats, including 7, 10 and 8 metabolites in rat plasma, urine, and feces, respectively. In addition, 6 of the metabolites detected in humans and rats were phase I metabolites, including demethylation, carboxylation, hydroxylation and dehydrogenation metabolites, and 5 of the metabolites were phase II metabolites, including acetylation and glucuronidation. There was no human metabolite detected compared to those in rats. The major metabolites detected in human plasma (M1 and M2) were products resulting from acetylation, and hydroxylation followed by dehydrogenation. M1 was the major metabolite in rat plasma. M2 and the parent drug were the major drug-related substances in human urine. The parent drug was the major drug-related substances in rat urine. M2, M5 (hydroxylation product) and M6 (2 × hydroxylation and acetylation product) were the predominant metabolites in human feces. M2 and M5 were the major metabolites in rat feces. In addition, renal clearance was the major route of excretion for imigliptin.

Simultaneous Determination of Deoxypodophyllotoxin and Its Major Metabolites in Rat Plasma by a Sensitive LC–MS/MS Method and Its Application in a Pharmacokinetic Study

The purpose of this study is to develop and validate a rapid and sensitive liquid chromatography-tandem mass spectrometry method for the quantification of deoxypodophyllotoxin (DPT) and its major metabolites (M1, M2, M7) in rat plasma. Diazepam was used as the internal standard. The method involves a simple liquid–liquid extraction with ethyl acetate. Chromatographic separation was accomplished on a Shim-pack VP-ODS analytical column (2.0 mm × 150 mm, 5 μm) with gradient elution using a mobile phase consisting of acetonitrile and water (containing 0.1 % formic acid) at a flow rate of 0.2 mL min−1. The detection was performed by multiple reaction monitoring mode via a positive electrospray ionization interface. The calibration curves were linear for all analytes over investigated range. The lower limits of quantification were 7.734 ng mL−1 for DPT, 1.973 ng mL−1 for M1, 7.969 ng mL−1 for M2, and 1.973 ng mL−1 for M7, respectively. The intra- and inter-day precision values were less than 10.59 %, and the accuracy ranged from −6.787 to 12.74 %. The extraction recoveries of the analytes were higher than 90 % and in this method no apparent matrix effect was observed. The validated method was successfully applied to pharmacokinetic study of DPT and its main metabolites in rat plasma.

An LC-MS/MS method for simultaneous determination of trantinterol and its major metabolite in rat plasma and its application to a comparative pharmacokinetic study.

Trantinterol is a novel β2-adrenoceptor agonist, currently undergoing clinical trials for the treatment of asthma. We developed and validated an liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of trantinterol and its major metabolite, 1-carbonyl trantinterol (SPFFCOOH), in rat plasma. Aliquots (100μL) of heparinized plasma samples were processed by protein precipitation with acetonitrile. Chromatographic separation used an Acquity UPLC BEH C18 column (2.1mm×50mm, 1.7μm) and acetonitrile-0.1% formic acid (20:80, v/v) as mobile phase, at a flow rate of 0.25mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple-reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The precursor-to-product ion transitions m/z 310.9→m/z 237.9 for trantinterol, m/z 324.9→m/z 251.9 for SPFFCOOH and m/z 368.0→m/z 294.0 for bambuterol (internal standard, IS) were used for quantification. The calibration curves were obtained in the concentration of 0.25-100ng/mL for both trantinterol and SPFFCOOH. The intra- and inter-day precision (relative standard deviations, RSD) values were below 15% and accuracy (relative error, RE) was from -4.3% to 6.6% at all quality control (QC) levels. The method was successfully applied to compare the pharmacokinetics of trantinterol and SPFFCOOH in male and female Wistar rats after a single oral administration of trantinterol.

Simultaneous determination of vasicine and its major metabolites in rat plasma by UPLC-MS/MS and its application to in vivo pharmacokinetic studies

An UPLC-MS/MS method was developed to simultaneously determinate vasicine and its main metabolites and applied to the pharmacokinetic study. In addition, the anti-butyrylcholinesterase activity of component in plasma was evaluatedin vitro.

Extensive intestinal first-pass metabolism of arctigenin: Evidenced by simultaneous monitoring of both parent drug and its major metabolites

The current study aims to investigate intestinal absorption and metabolism of arctigenin (AR) through simultaneous monitoring of AR and its major metabolites in rat plasma. An UPLC/MS/MS assay was developed with chromatographic separation of all analytes achieved by a C18 Column (3.9mm×150mm, 3.5μm) and a gradient elution with acetonitrile and 0.1% formic acid within 9min. Sample extraction with acetonitrile was optimized to achieve satisfactory recovery for both AR and its major metabolites. The lower limit of quantification (LLOQ) for all analytes was 25ng/ml. The intra-day and inter-day precision and accuracy of each analyte at LLOQ and three quality control (QC) concentrations (low, middle and high) in rat plasma was within 15.0% RSD and 15.0% bias. The extraction recoveries were within the range of 83.8–94.0% for all analytes. The developed and validated assay was then applied to the absorption study of AR in both Caco-2 cell monolayer model and in situ single-pass rat intestinal perfusion model. High absorption permeability of AR was demonstrated in both models with Papp of (1.76±0.48)×10−5 (A→B) (Caco-2) and Pblood of (8.6±3.0)×10−6cm/s (intestinal perfusion). Extensive first-pass metabolism of AR to arctigenic acid (AA) and arctigenin-4′-O-glucuronide (AG) was identified in rat intestinal perfusion study with Cummins's extraction ratios of 0.458±0.012 and 0.085±0.013, respectively. The current assay method demonstrated to be a practical tool for pharmacokinetics investigation of AR with complicated metabolism pathways and multiple metabolites.

Simultaneous determination of tectorigenin and its metabolites in rat plasma by ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Tectorigenin is a major isoflavone found in the flowers of Pueraria thomsonii Benth. and the rhizomes of Belamcanda chinensis (L.) DC. It possesses hepatoprotective, estrogenic, hypoglycemic and anti-inflammatory activities. In the present study, the plasma pharmacokinetic profile of tectorigenin in rats was evaluated. We developed a selective and accurate U-HPLC/Q-TOFMS method for the simultaneous characterization of nine tectorigenin metabolites, and quantitation of six major metabolites in rat plasma, including tectorigenin-7-O-glucuronide-4'-O-sulfate (Te-7G-4'S), tectorigenin-di-O-sulfate (Te-diS), tectorigenin-7-O-glucuronide (Te-7G), tectorigenin-4'-O-glucuronide (Te-4'G), tectorigenin-7-O-sulfate (Te-7S) and tectorigenin after oral administration of tectorigenin (130mg/kg). The plasma concentrations reached maximal values of 6.20±2.05μmol/L at 0.96±0.68h for Te-7G-4'S, 4.42±1.36μmol/L at 1.92±2.15h for Te-diS, 33.50±4.89μmol/L at 0.75±0.67h for Te-7G, 3.28±1.01μmol/L at 0.75±0.67h for Te-4'G, 12.80±2.80μmol/L at 0.85±1.54h for Te-7S, and 12.0±0.63μmol/L at 0.23±0.15h for tectorigenin, respectively. Enterohepatic recirculation resulted in double or triple peaks concentration curve/time profiles of the metabolites. Since the total plasma concentrations of tectorigenin conjugated metabolites were much higher than that of the tectorigenin aglycone, an extensive phase II metabolism plays an important role in the pharmacokinetics of tectorigenin in vivo.

Simultaneous quantification of seven plasma metabolites of sulfur mustard by ultra high performance liquid chromatography–tandem mass spectrometry

Sulfur mustard (SM) is a hazardous chemical warfare agent that has been used in several military conflicts. SM is also considered as a major threat to civilians because of its existing stockpiles and easy production. Analysis of exposure biomarkers in biological samples collected from suspected victims is a useful tool for early diagnosis of SM poisoning. In this study, a sensitive and rapid quantitative method with ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for simultaneous determination of seven SM plasma biomarkers, including its oxidative, hydrolysis and β-lyase metabolites. A simple one-step protein precipitation with acetonitrile-methanol (4:1) was used for sample preparation. A full validation was conducted with respect to specificity, linearity, recovery, matrix effect, precision, accuracy and stability. The lower limits of quantification for the seven metabolites ranged from 0.01μgL(-1) to 5μgL(-1). The intraday relative standard deviation was less than 7.0%, and the interday deviation was less than 9.1%. The recoveries varied in the range from 82.8% to 118%. This method has been successfully applied to a toxicokinetic study for obtaining the plasma profiles of seven metabolites in SM-exposed rats, following a single subcutaneous dose of 3.3mgkg(-1). All the targeted compounds were detected in rat plasma. bis-β-Chloroethyl sulfoxide (SMO), thiodiglycol (TDG), thiodiglycol sulfoxide (TDGO), 1,1'-sulfonylbis-[2-S-(N-acetylcysteinyl)ethane (SBSNAE), 1,1'-sulfonylbis-[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio)ethylsulfonyl]ethane (MSMTESE) were found to be the major metabolites in rat plasma. The time windows for the detection of these metabolites were varied in the range of 5min to 48h after exposure. The method provides a useful tool for short-term diagnosis of SM poisoning.

In vivo metabolites and plasma exposure of TongMai Keli analyzed by UHPLC/DAD/qTOF-MS and LC/MS/MS

Ethnopharmacological relevanceTongMai Keli (TM) is a widely used traditional Chinese medicine preparation for the treatment of cardiovascular and cerebrovascular diseases. It is composed of Puerariae Lobatae Radix (roots of Pueraria lobata (Willd.) Ohwi), Salviae Miltiorrhizae Radix (roots of Salvia miltiorrhiza Bge.), and Chuanxiong Rhizoma (rhizomes of Ligusticum chuanxiong Hort.). The aim of this study is to identify the in vivo metabolites of TM, and to elucidate the pharmacokinetics of TM constituents and their metabolites. Materials and methodsFor metabolites identification, TM was orally administered to rats (n=3), and the metabolites in plasma were identified by UHPLC/DAD/qTOF-MS analysis and β-glucuronidase hydrolysis. For pharmacokinetic study, rats (n=10) were treated with TM at a clinical dose, and the plasma was analyzed by LC/MS/MS. ResultsA total of 25 metabolites from TM were identified in rats plasma. Glucuronide and sulfate conjugations were the major metabolic reactions, and produced 14 metabolites. The analytical method for pharmacokinetic study was fully validated with good linearity (r>0.99), wide dynamic ranges (6–6000ng/mL), and low variations (<14.3%). The plasma concentration–time curves of puerarin and nine metabolites were profiled. ConclusionIsoflavones from Puerariae Lobatae Radix were the major metabolites in rat plasma after oral administration of TM. Puerarin and other isoflavone glycosides could reach their first Cmax within 30min, and were then rapidly eliminated, followed by their phase II metabolites.

Quantitation of cotinine and its metabolites in rat plasma and brain tissue by hydrophilic interaction chromatography tandem mass spectrometry (HILIC–MS/MS)

In this work, we developed a sensitive method to quantify cotinine (COT), norcotinine (NCOT), trans-3′-hydroxycotinine (OHCOT) and cotinine-N-oxide (COTNO) in rat plasma and brain tissue, using solid phase extraction (SPE), hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS/MS). The linear range was 1–100ng/mL for each analyte in rat plasma and brain homogenate (3–300ng/g brain tissue). The method was validated with precision within 15% relative standard deviation (RSD) and accuracy within 15% relative error (RE). Stable isotope-labeled internal standards (IS) were used for all the analytes to achieve good reproducibility, minimizing the influence of recovery and matrix effects. This method can be used in future studies to simultaneously determine the concentrations of COT and three major metabolites in rat plasma and brain tissue.

A Rapid HPLC Method for the Simultaneous Determination of Amiodarone and its Major Metabolite in Rat Plasma and Tissues: A Useful Tool for Pharmacokinetic Studies

A rapid and sensitive high-performance liquid chromatography (HPLC) method was developed and validated in rat plasma and tissue (heart, liver, kidney and lung) homogenates for the determination of amiodarone and its primary metabolite (desethylamiodarone), using tamoxifen as internal standard. Chromatographic separation was achieved within less than 5 min on a LiChroCART Purospher Star C18 column (55 × 4 mm, 3 µm). The mobile phase, consisting of phosphate buffer (50 mM) with 0.1% formic acid (pH 3.1)-methanol-acetonitrile (45:5:50, v/v/v), was pumped isocratically at a flow rate of 1.2 mL/min. The detection was conducted at 254 nm for all compounds. Calibration curves were linear (r(2) ≥ 0.995) in the range of 0.1-15 µg/mL for amiodarone and desethylamiodarone. The limits of quantification were established at 0.1 µg/mL for both analytes. The overall data of precision and accuracy were in accordance with international guidelines for bioanalytical method validation. Amiodarone and desethylamiodarone were extracted from rat matrices by a liquid-liquid extraction procedure and the mean recovery ranged from 59.9 to 97.6%. This novel HPLC method allows the fast and reliable determination of amiodarone and desethylamiodarone from several rat matrices (plasma, liver, kidneys, lungs and heart) and was successfully applied in a preliminary pharmacokinetic study.

Quantitative determination of aceclofenac and its three major metabolites in rat plasma by HPLC‐MS/MS

We developed a method for the simultaneous quantification of aceclofenac and its three major metabolites in rat plasma. After protein precipitation with acetonitrile including flufenamic acid as an internal standard (IS), aceclofenac, diclofenac, 4'-hydroxyaceclofenac, 4'-hydroxydiclofenac, and the IS were chromatographed on a reverse-phase C18 analytical column. The isocratic mobile phase of acetonitrile/0.1% formic acid (aq; 9:1 [v/v]) was eluted at 0.3 mL/min. Quantification was performed on a triple-quadrupole mass spectrometer using electrospray ionization, and the ion transitions were monitored in selective reaction-monitoring mode. The coefficient of variation in the assay precision was less than 8%, and the accuracy was 92-103%. This method was successfully used to measure the concentrations of aceclofenac and its three major metabolites in rat plasma following the oral administration of a single 20 mg/kg oral dose of aceclofenac.

Simultaneous determination of norisoboldine and its major metabolite in rat plasma by ultra‐performance liquid chromatography–mass spectrometry and its application in a pharmacokinetic study

Norisoboldine (NIB) is one of the main bioactive isoquinoline alkaloids in Linderae Radix. A rapid, selective and sensitive method using UPLC-ESI/MS was first developed for simultaneous determination of NIB and norisoboldine-9-O-α-glucuronide (NIB-Glu), its major metabolite in rat plasma. A one-step protein precipitation with methanol was employed as sample preparation technique. Chromatographic separation was carried out on an Acquity UPLC BEH C(18) column (50 × 2.1 mm, i.d. 1.7 µm) with a gradient mobile phase consisting of acetonitrile and water containing 0.1% formic acid. Detection and quantification were performed using a quadrupole mass spectrometer by selective ion reaction-monitoring mode. Good linearity was achieved using weighted (1/x(2) ) least squares linear regression over the concentration ranges 0.01-2 µg/mL for NIB and 0.025-25 µg/mL for NIB-Glu. The lower limit of quantification of NIB and NIB-Glu was 0.01 and 0.025 µg/mL, respectively. The intra- and inter-day precisions (relative standard deviations) of the assay at all three quality control levels were 4.6-14.1% for NIB, and 5.0-12.2% for NIB-Glu. The accuracies (relative error) were -13.5-8.1% for NIB and -12.8-7.6% for NIB-Glu, respectively. This developed method was successfully applied to an in vivo pharmacokinetic study in rats after a single intravenous dose of 10 mg/kg NIB.