Pharmacokinetic studies of a novel trioxane antimalarial (99/411) in rats and monkeys using LC–MS/MS

The pharmacokinetic profile of 99/411, a novel anti‐malarial drug, was established in rats (12 mg/kg of body weight) and monkeys (20 mg/kg of body weight). Following oral administration, the presence of 99/411 was rapidly determined in rat plasma, tissues, urine, feces and monkey plasma using a validated LC–MS/MS method. The tissue distribution studies in rats indicated that the drug was partially distributed in all major tissues and plasma, and peak concentration levels were achieved within 0.5–4 h. Area under the curve in different rat tissues and plasma was found in order of blood > lung > intestine > heart > muscle > brain > kidney > spleen > liver. The total recoveries (within 86 h) of 99/411 were <0.0017% and <0.08% in urine and feces, respectively. The peak plasma concentration was 3499 ng/mL in rats after ~2 h of oral administration and 697–767 ng/mL in monkeys after ~6 h of oral administration. No plasma accumulation was observed in both male and female monkeys, even after multiple dosing. The preclinical pharmacokinetic profile and tissue distribution data are expected to assist in future clinical explorations of 99/411 as a promising anti‐malarial agent.     

A new metabolite of nodakenetin by rat liver microsomes and its quantification by RP‐HPLC method

The biotransformation of nodakenetin (NANI) by rat liver microsomes in vitro was investigated. Two major polar metabolites were produced by liver microsomes from phenobarbital‐pretreated rats and detected by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) analysis. The chemical structures of two metabolites were firmly identified as 3′(R)‐hydroxy‐nodakenetin‐3′‐ol and 3′(S)‐hydroxy‐nodakenetin‐3′‐ol, respectively, on the basis of their ¹H‐NMR, MS and optical rotation analysis. The latter was a new compound. A sensitive, selective and simple RP‐HPLC method has been developed for the simultaneous determination of NANI and its two major metabolites in rat liver microsomes. Chromatographic conditions comprise a C₁₈ column, a mobile phase with MeOH‐H₂O (40 : 60, v/v), a total run time of 40 min, and ultraviolet absorbance detection at 330 nm. In the rat heat‐inactivated liver microsomal supernatant, the lower limits of detection and quantification of metabolite I, metabolite II and NANI were 5.0, 2.0, 10.0 ng/mL and 20.0, 5.0, 50.0 ng/mL, respectively, and their calibration curves were linear over the concentration range 50–400, 20–120 and 150–24000 ng/mL, respectively. The results provided a firm basis for further evaluating the pharmacokinetics and clinical efficacy of NANI. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of antiviral nucleoside analogues AM365 and AM188 in perfusate and bile of the isolated perfused rat liver using HPLC

Development, validation and application of an HPLC assay for new antiviral nucleoside analogues AM365 and AM188 in isolated perfused rat liver perfusate and bile were performed. An analytical column (Phenosphere-NEXT, 250 x 4.6 mm, C(18), 4 microm, Phenomenex) was used in tandem with a guard column (4 x 3 mm, C(18), Phenomenex) and operated at 25 degrees C. The mobile phase [methanol:10 mmol/L sodium orthophosphate buffer (pH 7.0), 15:85, v/v] was pumped at 1 mL/min. The signal from a diode array detector was collected from 190 to 300 nm. The chromatogram was processed at 220 and 252 nm for AM365 and AM188, respectively. The HPLC method was validated by six intraday and seven interday runs. Standard curves were linear in the range 0.125-8.00 microg/mL for AM365 and AM188, and the lower limit of quantification for AM365 and AM188 was 0.125 microg/mL. Mean interday precision and accuracy of IPL perfusate quality control samples were within 8.8%, and mean intraday precision and accuracy were within 13.1%. The assay has been successfully used in the study of metabolism and disposition of AM365 in the isolated perfused rat liver.     

Metabolic profiles of ribociclib in rat and human liver microsomes using liquid chromatography combined with electrospray ionization high-resolution mass spectrometry

Ribociclib is a highly specific CDK4/6 inhibitor. Determination of the metabolism of ribociclib is required during the drug development stage. In this study, metabolic profiles of ribociclib were investigated using rat and human liver microsomes. Metabolites were structurally identified by liquid chromatography electrospray ionization high-resolution mass spectrometry operated in positive-ion mode. The metabolites were characterized by accurate masses, MS² spectra and retention times. With rat and human liver microsomes, a total of 10 metabolites were detected and further identified. No human-specific metabolites were detected. The metabolic pathways of ribociclib were oxygenation, demethylation and dealkylation. Most importantly, two glutathione (GSH) adducts were identified in human liver microsomes fortified with GSH. The formation of the GSH adducts was hypothesized to be through the oxidation of electron-rich 1,4-benzenediamine to a 1,4-diiminoquinone intermediate, which is highly reactive and can be trapped by GSH to form stable metabolites. The current study provides an overview of the metabolic profiles of ribociclib in vitro, which will be of great help in understanding the efficacy and toxicity of this drug.     

A simple validated RP‐HPLC bioanalytical method for the quantitative determination of a novel valproic acid arylamide derivative in rat hepatic microsomes

A simple and specific bioanalytical method based on reversed‐phase high‐performance liquid chromatography (RP‐HPLC) coupled with ultraviolet detection was developed and validated for the determination of a novel valproic acid arylamide, N‐(2‐hydroxyphenyl)‐2‐propylpentanamide (HO‐AAVPA) in rat hepatic microsomes (a subcellular fraction containing phase I enzymes, especially cytochrome P450). The chromatographic separation was achieved using a reversed‐phase Zorbax SB‐C₁₈ column and a mobile phase of acetic acid in water (0.2% v/v) and acetonitrile (40:60 v/v) with a flow rate of 0.5 mL/min. The calibration curve was linear over the range of 882–7060 ng/mL (r² = 0.9987), and the lower limit of quantification and the lower limit of determination were found to be 882 and 127.99 ng/mL, respectively. The method was validated with excellent sensitivity, and intra‐day accuracy and precision varied from 93.79 to 93.12%, and from 2.12 to 4.36%, respectively. The inter‐day accuracy and precision ranged from 93.29 to 97.30% and from 0.68 to 3.60%, respectively. The recovery of HO‐AAVPA was measured between 91.36 and 97.98%. The assay was successfully applied to the analysis of kinetic metabolism and pharmacokinetic parameters in vitro by a substrate depletion approach. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of piceatannol in rat serum and liver microsomes: pharmacokinetics and phase I and II biotransformation

A method of analysis of piceatannol in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and determine its concentration in foodstuffs. A novel and simple high-performance liquid chromatographic method was developed for simultaneous determination of piceatannol and products of its metabolism in rat serum and liver microsomes. Serum, or microsomes (0.1 mL), were precipitated with acetonitrile after addition of the internal standard, 4-methylumbelliferone. Separation was achieved on a phenomenex C(18) column (250 x 4.6 mm i.d., 5 microm) equipped with a phenomenex C(18) (4 x 3.0 mm i.d., 5 microm) guardcolumn with fluorescence excitation at 320 nm and emission at 420 nm. Separation was also possible with UV detection at 310 nm. The fluorescent calibration curves were linear ranging from 0.05 to 100 microg/mL. The mean extraction efficiency was >95%. Precision of the assay was <10% (coefficient of variation), and was within 10% at the limit of quantitation (0.05 ng/mL). Bias of the assay was lower than 7%. The limit of detection was 50 ng/mL for a 0.1 mL sample. The assay was applied successfully to the in vitro kinetic study of metabolism of piceatannol in rat liver microsomes and pharmacokinetics in rats. Three metabolites of piceatannol have been identified. .     

Determination of the active metabolites of sibutramine in rat serum using column-switching HPLC

A simple and direct analysis using column-switching HPLC method was developed and validated for the quantification of active metabolites of sibutramine, N-mono-desmethyl metabolite (metabolite 1, M1) and N-di-desmethyl metabolite (metabolite 2, M2) in the serum of rats administered sibutramine HCl (5.0 mg/kg, p.o.). Rat serum was directly injected onto the precolumn without sample prepreparation step following dilution with mobile phase A, i. e., methanol-ACN-20 mM ammonium phosphate buffer (pH 6.0 with phosphoric acid) (8.3:4.5:87.2 by volume). After the endogenous serum components were eluted to waste, the system was switched and the analytes were eluted to the trap column. Active metabolites M1 and M2 were then back-flushed to the analytical column for separation with mobile phase B, i. e., methanol-ACN-20 mM ammonium phosphate buffer (pH 6.0 with phosphoric acid) (35.8:19.2:45 by volume) and detected at 223 nm. The calibration curves of active metabolites M1 and M2 were linear in the range of 0.1-1.0 microg/mL and 0.15-1.8 microg/mL. This method was fully validated and shown to be specific, accurate (10.4-10.7% error), and precise (1.97-8.79% CV). This simple and rapid analytical method using column-switching appears to be useful for the pharmacokinetic study of active metabolites (M1 and M2) of sibutramine.     

Simultaneous determination of D- and L-serine in rat brain microdialysis sample using a column-switching HPLC with fluorimetric detection

Both D- and L-serine in rat brain microdialysis sample were simultaneously determined by pre-column fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), separation of the derivatives on ODS column, TSKgel ODS-80TsQA, followed by Pirkle type chiral columns, Sumichiral OA-2500 (S), which gave a sufficient enantiomeric separation of NBD-D-serine and NBD-L-serine, and fluorimetric detection at a wavelength of 540 nm with an excitation wavelength of 470 nm. The peaks of NBD-D-serine and NBD-L-serine in the rat brain microdialysis sample were clearly found, and the validation study showed satisfactory results; the precision and accuracy were within 5.14 and 109%, respectively. Using the proposed HPLC method, the time-course profile of D-serine concentration in rat prefrontal cortex following intraperitoneal administration of D-serine was investigated. As a consequence, D-serine appeared to be rapidly distributed in the brain, and then decreased gradually with time in the extracellular fluid of the rat prefrontal cortex. The proposed HPLC method will be useful for in vivo studies on D-serine, which acts as a coagonist for N-methyl-D-aspartate receptor, to the extracellular fluid of rat brain.     

Development of HPLC with fluorescent detection using NBD‐F for the quantification of colistin sulfate in rat plasma and its pharmacokinetic applications

Colistin sulfate, composed of a mixture of colistin A sulfate (CLA) and colistin B sulfate (CLB), is available for treating life‐threatening infections caused by multidrug‐resistant Gram‐negative bacteria. In this study, the CLA and CLB were quantified separately. Colistin sulfate was extracted from rat plasma with a solid‐phase extraction C₁₈ cartridge and reacted with 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F), and the fluorescent derivatives were subjected to reversed‐phase high‐performance liquid chromatography analysis and used to investigate the pharmacokinetics of CLA and CLB in rat plasma. The recovery rates of CLA and CLB were 41.2 ± 4.4 and 45.5 ± 3.1%, respectively. The recovery rate calculated from the total area of CLA and CLB was 43.9 ± 3.6%. When 2 mm NBD‐F and 10 mm boric acid buffer (pH 9.5) were added to colistin sulfate, the highest recovery rate was obtained. The best heating time was 5 min at 60°C. The lower limits of quantification for CLA, CLB and the total area of CLA and CLB were 0.05, 0.05 and 0.1 μg/mL; the coefficients of variations were 13.5, 14.5 and 14.1%, respectively. This method was found to have acceptable linearity, precision and accuracy, and has been successfully applied to a pharmacokinetic study in rat plasma.     

Determination of rifampicin in rat plasma by modified large‐volume direct injection RAM‐HPLC and its application to a pharmcokinetic study

A direct large volume injection high‐performance liquid chromatography (HPLC) method with homemade restricted‐access media (RAM) pre‐column and combined with a column‐switching valve was established and developed for determination rifampicin (RIP) in rat plasma. The rat plasma samples (100 μL) were injected directly onto pre‐column, where RIP was retained and pre‐concentrated, while proteins were washed to waste using a methanol–water (5:95) as the mobile phase at a flow rate of 1 mL/min. Then, by rotation of the switching valve at 5 min, the RIP were eluted from the pre‐column and transferred to an Luna C₁₈ analytical column by the chromatographic mobile phase consisting of methanol–acetonitrile–10 mm ammonium format (60:5:35) at a flow rate of 1 mL/min. The total analytical run time was 15 min with UV detection wavelength at 254 nm. Carbamazepine was used as the internal standard. Excellent linear correlation (r = 0.9993) was obtained in the range of 0.25–8 µg/mL for rat plasma. The intra‐day and inter‐day precisions of RIP were all <5.0%. The recoveries were in the range of from 99.98–113.66% for plasma. This on‐line RAM‐HPLC method was successfully applied to the pharmacokinetic study of RIP in rat plasma. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolic profile of glyburide in human liver microsomes using LC‐DAD‐Q‐TRAP‐MS/MS

The sulfonylurea urea drug glyburide (glibenclamide) is widely used for the treatment of diabetes milletus and gestational diabetes. In previous studies monohydroxylated metabolites were identified and characterized for glyburide in different species, but the metabolite owing to the loss of cyclohexyl ring was identified only in mouse. Glyburide upon incubation with hepatic microsomes resulted in 10 metabolites for human. The current study identifies new metabolites of glyburide along with the hydroxylated metabolites that were reported earlier. The newly identified drug metabolites are dihydroxylated metabolites, a metabolite owing to the loss of cyclohexyl ring and one owing to hydroxylation with dehydrogenation. Among the 10 identified metabolites, there were six monohydroxylated metabolites, one dihydroxylated metabolite, two metabolites owing to hydroxylation and dehydrogenation, and one metabolite owing to the loss of cyclohexyl ring. New metabolites of glyburide were identified and characterized using liquid chromatography–diode array detector–quadruple‐ion trap–mass spectrometry/mass spectrometry (LC‐DAD‐Q‐TRAP‐MS/MS). An enhanced mass scan–enhanced product ion scan with information‐dependent acquisition mode in a Q‐TRAP‐MS/MS system was used to characterize the metabolites. Liquid chromatography with diode array detection was used as a complimentary technique to confirm and identify the metabolites. Metabolites formed in higher amounts were detected in both diode array detection and mass spectrometry detection. Copyright © 2012 John Wiley & Sons, Ltd.     

Studies on metabolites and metabolic pathways of bulleyaconitine A in rat liver microsomes using LC‐MSn combined with specific inhibitors

Bulleyaconitine A (BLA) from Aconitum bulleyanum plants is usually used as anti‐inflammatory drug in some Asian countries. It has a variety of bioactivities, and at the same time some toxicities. Since the bioactivities and toxicities of BLA are closely related to its metabolism, the metabolites and the metabolic pathways of BLA in rat liver microsomes were investigated by HPLC–MSⁿ. In this research, the 12 metabolites of BLA were identified according to the results of HPLC‐MSⁿ data and the relevant literature. The results showed that there are multiple metabolites of BLA in rat liver microsomes, including demethylation, deacetylation, dehydrogenation deacetylation and hydroxylation. The major metabolic pathways of BLA in rat liver microsomes were clarified by HPLC‐MS combined with specific inhibitors of CYP450 isoforms. As a result, CYP3A and 2C were found to be the principal CYP isoforms contributing to the metabolism of BLA. Moreover, CYP2D6 and 2E1 are also more important CYP isoforms for the metabolism of BLA. While CYP1A2 only affected the formation rate of M11, its effect on the metabolism of BLA is very small. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of gymnemagenin in rat plasma using high‐performance liquid chromatography–tandem mass spectrometry: application to pharmacokinetics after oral administration of Gymnema sylvestre extract

A sensitive and rapid high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) method has been developed and validated for the determination of gymnemagenin (GMG), a triterpene sapogenin from Gymnema sylvestre, in rat plasma using withaferin A as the internal standard (IS). Plasma samples were simply extracted using liquid–liquid extraction with tetra‐butyl methyl ether. Chromatographic separation was performed on Luna C₁₈ column using gradient elution of water and methanol (with 0.1% formic acid and 0.3% ammonia) at a flow rate of 0.8 mL/min. GMG and IS were eluted at 4.64 and 4.36 min, ionized in negative and positive mode, respectively, and quantitatively estimated using multiple reaction monitoring (MRM) mode. Two MRM transitions were selected at m/z 505.70 → 455.5 and m/z 471.50 → 281.3 for GMG and IS, respectively. The assay was linear over the concentration range of 5.280–300.920 ng/mL. The mean plasma extraction recoveries for GMG and IS were found to be 80.92 ± 8.70 and 55.63 ± 0.76%, respectively. The method was successfully applied for the determination of pharmacokinetic parameters of GMG after oral administration of G. sylvestre extract. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of cnidilin and its two metabolites in rat bile and stool after oral administration by HPLC/electrospray ionization tandem mass spectrometry

A simple, fast and sensitive method for the simultaneous determination of cnidilin and its two metabolites (M1 and M2) in rat bile and stool using HPLC coupled with electrospray ionization tandem mass spectrometry (HPLC‐ESI‐MS/MS) has been developed. The sample pretreatment was simple, because methanol was the only additive used for dilution of bile and ultrasound of stool. Pimpinellin was used as internal standard (IS). The separation was performed on a reverse phase C₁₈ column with gradient elution consisting of 0.5‰ aqueous formic acid and methanol (containing 0.5‰ formic acid). The detection was in the multiple‐reaction monitoring mode within 7 min. All the analytes were in accordance with the requirement of the validation of the method in vivo (linearity, precision, accuracy, limit of detection and limit of quantification). After oral administrating 24 mg/kg of the prototype drug cnidilin, M1 and M2 were determined in bile within 36 h, and in stool within 60 h. Cnidilin in bile was completely excreted in 24 h, and the main excretive amount of cnidilin was 80% in the first 6 h, but the drug recovery in bile within 24 h was <1.95%. In stool, the main excretive amount of cnidilin was 95.8% in the first 24 h, and the drug recovery within 48 h was lower than 1.48%. Copyright © 2012 John Wiley & Sons, Ltd.     

Multi‐analysis strategy for metabolism of Andrographis paniculata in rat using liquid chromatography/quadrupole time‐of‐flight mass spectrometry

Compared with chemical drugs, it is a huge challenge to identify active ingredients of multicomponent traditional Chinese medicine (TCM). For most TCMs, metabolism investigation of absorbed constituents is a feasible way to clarify the active material basis. Although Andrographis paniculata (AP) has been extensively researched by domestic and foreign scholars, its metabolism has seldom been fully addressed to date. In this paper, high‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry was applied to analysis and characterization of AP metabolism in rat urine and feces samples after oral administration of ethanol extract. The differences in metabolites and metabolic pathways between the two biological samples were further compared. The chemical structures of 20 components were tentatively identified from drug‐treated biological samples, including six prototype components and 14 metabolites, which underwent such main metabolic pathways as hydrolyzation, hydrogenation, dehydroxylation, deoxygenation, methylation, glucuronidation, sulfonation and sulfation. Two co‐existing components were found in urine and feces samples, suggesting that some ingredients' metabolic processes were not unique. This study provides a comprehensive report on the metabolism of AP in rats, which will be helpful for understanding its mechanism. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of a hydrophilic paclitaxel derivative, 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma by LC–MS/MS

A LC‐MS/MS method for the determination of a hydrophilic paclitaxel derivative 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma was developed to evaluate the pharmacokinetics of 7‐xylosyl‐10‐deacetylpaclitaxel in the rats. 7‐Xylosyl‐10‐deacetylpaclitaxel and docetaxel (IS for 7‐xylosyl‐10‐deacetylpaclitaxel) were extracted from rat plasma with acetic ether and analyzed on a Hypersil C₁₈ column (4.6 × 150 mm i.d., particle size 5 µm) with the mobile phase of ACN/0.05% formic acid (50:50, v/v). The analytes were detected using an ESI MS/MS in the multiple reaction monitoring mode. The standard curves for 7‐xylosyl‐10‐deacetylpaclitaxel in plasma were linear (r >0.999) over the concentration range of 2.0–1000 ng/mL with a weighting of 1/concentration². The method showed a satisfactory sensitivity (2.0 ng/mL using 50 µL plasma), precision (CV ≤ 10.1%), accuracy (relative error ?12.4 to 12.0%), and selectivity. This method was successfully applied to the pharmacokinetic study of 7‐xylosyl‐10‐deacetylpaclitaxel in rat plasma after intravenous administration of 7‐xylosyl‐10‐deacetylpaclitaxel to female Wistar rats. Copyright © 2008 John Wiley & Sons, Ltd.     

Development and validation of a HPLC method for quantification of rivastigmine in rat urine and identification of a novel metabolite in urine by LC‐MS/MS

A sensitive, specific and accurate HPLC method for the quantification of rivastigmine (RSM) in rat urine was developed and validated. The method involves the simple liquid–liquid extraction of RSM and pyridostigmine as an internal standard (IS) from rat urine with tertiary methyl butyl ether. The chromatographic separation of RSM and IS was achieved with 20 mm ammonium acetate buffer (pH 6.5) and acetonitrile (65:35, v/v) delivered at flow‐rate of 1 mL/min on a Kromasil KR‐100. The method was in linear range from 50 to 5000 ng/mL. The validation was done as per FDA guidelines and the results met the acceptance criteria. The method was successfully applied for the quantification of RSM in rat urine. Besides method validation, we have identified two metabolites of RSM in urine. Both the metabolites were characterized by HPLC‐PDA and LC‐MS/MS and it was found that one metabolite is novel. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination and tissue distribution studies of dantrolene sodium with hydroxypropyl‐β‐cyclodextrin in rat tissue by HPLC/MS/MS

The established analytical method for determining the concentration of dantrolene sodium (Da) in rat tissues by HPLC/MS/MS technique was successfully applied to tissue distribution studies of Da in rats. Tissue homogenate samples were pretreated by protein precipitation with pre‐cooled methanol. Chromatographic separation was achieved on an Acquity HPLC column (Kromat Universil XB‐C₁₈, 2.1 × 150 mm, 3 μm). Mass spectrometry was conducted with an electrospray ionization interface in negative ionization mode and multiple reaction monitoring was used for quantitative analysis. The results showed that Da was rapidly and widely distributed in tissues and reached the maximum concentration within 0.5 h in all tissues after oral administration of Da–hydroxypropyl‐β‐cyclodextrin (DHC). It was then metabolized by liver and finally excreted from kidney,which indicated that DHC inclusion complex has better absorption and higher oral bioavailability than Da. The results also provided evidence for the safety and effectiveness of drug clinical application.