Development and validation of LC-MS/MS methods for the determination of EVT201 and its five metabolites in human urine: Application to a mass balance study.

In this study, two simple and accurate LC-MS/MS methods were firstly developed and validated to quantify EVT201, a new partial GABAA receptor agonist used for the treatment of insomnia, and its metabolites comprising M1, M2, M3, M4 and M6 in human urine. The analytes in urine samples were determined after simple dilution, and ideal chromatographic separations were obtained on C18 columns using gradient elution. The assays were performed in MRM mode on AB QTRAP 5500 tandem mass spectrometry (ESI+). The concentration ranges (ng/mL) of analytes in human urine were as follows: EVT201, 1.00 to 36.0; M1, 1.40 to 308; M2, 2.00 to 72.0; M3, 5.00 to 1100; M4, 2.00 to 300; and M6, 2.80 to 420. The methods were fully validated including selectivity, carryover, matrix effect, recovery, linearity, accuracy, precision, dilution integrity and stability, and acceptable criteria were obtained. The methods were successfully applied to a mass balance study of EVT201. The results showed that the total cumulative urinary excretion rate of EVT201 and its five metabolites was 74.25 ± 6.50%, which suggested that EVT201 had high oral bioavailability, and urinary elimination was its major excretion pathway in human.

Response characteristics of highland barley under freeze-thaw, drought and artemisinin stresses.

The freeze-thaw of early spring in China's Qinghai-Tibet Plateau is often accompanied by severe droughts. Artemisia annua, widely distributed in China, releases allelopathic substances, mainly artemisinin, to the environment and exerts a wide range of effects on crops. This paper studied the physiological effects of highland barley under freeze-thaw, drought, and artemisinin stress through indoor simulation experiments. The physiological response characteristics of superoxide dismutase (SOD) activity, catalase (POD) activity, net photosynthetic rate, relative water content (RWC), relative electrical conductivity, malondialdehyde (MDA) content, and soluble protein content in highland barley were analyzed. The results showed that artemisinin and drought contributed to the increase of SOD activity and the decrease of POD activity. Under the freeze-thaw stress, the SOD and POD activities both decreased firstly and then increased, but the effect of compound stress on POD was more complicated. Either artemisinin, drought, or low temperature could reduce the net photosynthetic rate of highland barley. Low temperature had more significant impacts on photosynthesis, and compound stress would show a single stress superimposed effect. Artemisinin, drought, and low temperature could reduce the RWC of highland barley, and increase the relative electrical conductivity and the concentration of soluble protein (except for low temperature stress above zero, which reduces the concentration of soluble protein). However, the effect of compound stress on soluble protein is more complex. The single stress of artemisinin and drought had no obvious effect on MDA content, while the MDA content was increased significantly under the freeze-thaw stress and the compound stress of artemisinin and drought, and the MDA content reached its peak at T1. The results are helpful to explore the effects of freeze-thaw, drought and artemisinin stress on the growth of highland barley under the background of the aridification of the Qinghai-Tibet Plateau, and provide ideas for rational agricultural management.

Pharmacokinetics and bioequivalence of two strontium ranelate formulations after single oral administration in healthy Chinese subjects.

Pharmacokinetics of exogenous strontium (Sr) and bioequivalence of a new oral formulation of strontium ranelate compared with the brand-name drug in healthy Chinese subjects was evaluated. A balanced, randomized, single-dose, two-treatment parallel study was conducted in 36 healthy Chinese subjects. Subjects were randomly allocated into two groups of 18 to receive a single oral dose of test formulation and reference formulation under a fasting state, respectively. Blood samples were collected at 19 designated time points up to 240-h post-dose. Serum concentrations of Sr were quantified by ICP-MS. A total of 36 subjects were enrolled and completed the study. Nine mild adverse events in 6 subjects were reported. The Cmax, AUC0-72 h, AUC0-t, and AUC0-∞ of test and reference formulations shown as mean ± SD were 6.97 ± 1.78 and 6.78 ± 1.80 µg/mL, 199 ± 51 and 187 ± 38 µg·h/mL, 303 ± 89 and 278 ± 54 µg·h/mL, and 337 ± 109 and 305 ± 60 µg·h/mL, respectively. Two formulations were bioequivalent, and both were generally well tolerated.

Pharmacokinetic study of methylnaltrexone after single and multiple subcutaneous administrations in healthy Chinese subjects.

1. Pharmacokinetics of methylnaltrexone (MNTX) were evaluated after subcutaneous administrations (s.c.) in healthy Chinese subjects. 2. In a cross-over single dose study, 12 subjects were given 0.075, 0.15, and 0.3 mg/kg of MNTX bromide injection. In a multiple doses study, another 12 subjects subcutaneously received 0.15 mg/kg of MNTX bromide injection every 48 h, in total five administrations. The concentrations of MNTX in plasma were quantified by LC-MS/MS. 3. After single s.c. administrations of 0.075, 0.15, and 0.3 mg/kg of MNTX bromide, Cmax values of MNTX were 93.5 ± 28.6, 191 ± 37, and 364 ± 54 ng/mL, respectively, and AUC0-∞ were 88.8 ± 8.8, 181 ± 16, and 357 ± 34 ng⋅h/mL, respectively. The t1/2 of MNTX was about 7.7 h. After multiple doses administration, the Cmax, Cav, AUCss, and MRT0-∞ values were 191 ± 50, 3.79 ± 0.40 ng/mL, 182 ± 19 ng⋅h/mL, and 3.56 ± 1.17 h, respectively. 4. Methylnaltrexone bromide displayed dose-proportional pharmacokinetics in the dose range of 0.075-0.3 mg/kg. After multiple doses administration, t1/2 was slightly prolonged, with the cumulative factor of 1.02. This study provides a pharmacokinetic reference after a single dose and multiple doses of MNTX bromide in Chinese subjects.

A chiral LC-MS/MS method for the stereospecific determination of efonidipine in human plasma.

Efonidipine hydrochloride is a new generation dihydropyridine Ca(2+) channel blocker designed to inhibit both T-type and L-type Ca(2+) channels. Efonidipine possesses a chiral carbon and is clinically administered as a racemate. In the present study, an enantioselective and sensitive LC-MS/MS method of determining efonidipine enantiomers in human plasma was developed and validated to characterize the stereoselective pharmacokinetics. Plasma samples were processed by liquid-liquid extraction (LLE). Chiral separation was optimized on a CHIRALPAK(®) ID column using an isocratic mobile phase of acetonitrile/water (60:40, v/v). Detection was using MS in multiple reaction monitoring (MRM) mode, using the transitions of m/z 632.3→91.1 for efonidipine enantiomers, and m/z 493.3→117.2 for cilnidipine (internal standard). The calibration curves were linear over 0.100-20.0 ng/mL for each enantiomer. The lower limit of quantification (LLOQ) for each enantiomer was established at 0.100 ng/mL. Intra- and inter-day precisions were less than 12.1% for each enantiomer in terms of relative standard deviation (RSD), and accuracies were between -5.0% and 5.0% in terms of relative error (RE) for each enantiomer. No chiral inversion was observed during sample storage, preparation procedure and analysis. The validated method was successfully applied to a stereoselective pharmacokinetic study of efonidipine in healthy subjects after oral administration of 40 mg (20 mg × 2) efonidipine hydrochloride tablets.

An LC-MS/MS method for simultaneous determination of cefprozil diastereomers in human plasma and its application for the bioequivalence study of two cefprozil tablets in healthy Chinese volunteers.

A rapid and sensitive liquid chromatography-tandem mass spectrometric method was developed for the first time and validated for the determination of cefprozil diastereomers in human plasma. The plasma samples were prepared by protein precipitation using acetonitrile. Detection was performed using an electronic spray ion source in the negative ion mode, operating in the multiple reaction monitoring of the transitions m/z 388.0 to m/z 205.0 for cefprozil diastereomers and m/z 346.1 to m/z 268.1 for cephalexin (the internal standard). The calibration curves of cis-cefprozil and trans-cefprozil were linear in the ranges 0.125-16.0 µg/mL and 0.0403-1.72 µg/mL, respectively. The lower limits of quantification of cis- and trans-cefprozil were 0.125 and 0.0403 µg/mL in human plasma, respectively. The intra- and inter-day precisions of cis- and trans-cefprozil were all <9.7%, and the accuracy ranged from 99.2 to 104.7% and from 100.6 to 102.2%, respectively. The validated method was successfully applied to a bioequivalence study of two cefprozil formulations in 24 healthy Chinese volunteers. The two cefprozil tablets were bioequivalent by measurement of cis-, trans- and total cefprozil. We suggest that the bioequivalence of cefprozil formulations can be evaluated only using cis-cefprozil as the analyte in future studies.

Pharmacokinetics of peramivir after single intravenous doses in healthy Chinese subjects.

1.The aim of the study was to evaluate the pharmacokinetics of peramivir after single intravenous (i.v.) doses in healthy Chinese subjects. 2.In a cross-over study, 12 subjects were given 300 and 600 mg peramivir by i.v. infusion. Blood and urine samples were collected at 17 designated time points and 7 designated intervals up to 36 h post-dose. Plasma and urine concentrations of peramivir were quantified by LC-MS/MS. 3.After single i.v. doses of 300 and 600 mg peramivir, Cmax and AUC0-t of peramivir were 21.4 ± 3.7, 41.1 ± 5.3 mgcL(-1) and 55.90 ± 10.62, 112.1 ± 13.2 mgch L(-1), respectively. Cmax and AUC increased in proportion to the dose. Within 12 h, accumulative urinary recoveries of peramivir after single i.v. doses of 300 and 600 mg peramivir were 84.31 ± 11.75% and 88.10 ± 7.39%, respectively. 4.In healthy Chinese subjects, peramivir displayed linear pharmacokinetics in the range of 300-600 mg, and was primarily excreted via urine as unchanged drug.

Determination of efonidipine in human plasma by LC-MS/MS for pharmacokinetic applications.

Efonidipine hydrochloride is a new generation dihydropyridine calcium channel blocker designed to inhibit both T-type and L-type calcium channels. For the first time, a simple and robust LC-MS/MS method was developed for the determination of efonidipine in human plasma over the range of 0.100-20.0ng/mL. Efonidipine was extracted from plasma by an LLE procedure, separated by LC and detected by MS/MS in positive mode ESI. The method was validated for selectivity, carryover, sensitivity, extraction recovery, matrix effects, linearity, accuracy and precision, dilution integrity and stability studies. The calibration curves were linear over 0.100-20.0ng/mL (r≥0.9980). The lower limit of quantification (LLOQ) was established at 0.100ng/mL. Intra- and inter-day precisions (LLOQ, low-QC, mid-QC, high-QC and ultra-high QC) were less than 12.5% in terms of relative standard deviation (RSD), and accuracies were between -5.0% and 5.0% in terms of relative error (RE). Matrix effect was acceptable (105.6-110.2%) and extraction recovery was reproducible (85.8-91.3%, RSD≤10.0%). Efonidipine was stable in the investigated conditions. The method was applied to the pharmacokinetics of efonidipine in human subject.

Quantification of strontium in human serum by ICP-MS using alternate analyte-free matrix and its application to a pilot bioequivalence study of two strontium ranelate oral formulations in healthy Chinese subjects.

A rapid, sensitive and accurate ICP-MS method using alternate analyte-free matrix for calibration standards preparation and a rapid direct dilution procedure for sample preparation was developed and validated for the quantification of exogenous strontium (Sr) from the drug in human serum. Serum was prepared by direct dilution (1:29, v/v) in an acidic solution consisting of nitric acid (0.1%) and germanium (Ge) added as internal standard (IS), to obtain simple and high-throughput preparation procedure with minimized matrix effect, and good repeatability. ICP-MS analysis was performed using collision cell technology (CCT) mode. Alternate matrix method by using distilled water as an alternate analyte-free matrix for the preparation of calibration standards (CS) was used to avoid the influence of endogenous Sr in serum on the quantification. The method was validated in terms of selectivity, carry-over, matrix effects, lower limit of quantification (LLOQ), linearity, precision and accuracy, and stability. Instrumental linearity was verified in the range of 1.00-500ng/mL, corresponding to a concentration range of 0.0300-15.0µg/mL in 50µL sample of serum matrix and alternate matrix. Intra- and inter-day precision as relative standard deviation (RSD) were less than 8.0% and accuracy as relative error (RE) was within ±3.0%. The method allowed a high sample throughput, and was sensitive and accurate enough for a pilot bioequivalence study in healthy male Chinese subjects following single oral administration of two strontium ranelate formulations containing 2g strontium ranelate.

Regulation of lovastatin on a key inflammation-related microRNA in myocardial cells.

BACKGROUND: Advances in the understanding of cardiovascular pathogenesis have highlighted that inflammation plays a central role in atherosclerotic coronary heart disease. Therefore, exploring pharmacologically based anti-inflammatory treatments to be used in cardiovascular therapeutics is worthwhile to promote the discovery of novel ways of treating cardiovascular disorders. METHODS: The myocardial cell line H9c2(2-1) was exposed to lipopolysaccharide (LPS) in culture and resulted in a cellular pro-inflammation status. miR-21 microRNA levels were detected using quantitative real-time polymerase chain reaction (Q-RT-PCR). The influence of lovastatin on miR-21 under normal and pro-inflammatory conditions was tested after being added to the cell culture mixture for 24 hours. Conditional gene function of two predicted cardiovascular system relevant downstream targets of miR-21, protein phosphatase 1 regulatory subunit 3A (PPP1R3A) and signal transducer and activator of transcription 3 (STAT3), were analyzed with immunoblotting. RESULTS: Forty-eight hours of LPS treatment significantly increased the miR-21 to 170.71%± 34.32% of control levels (P = 0.002). Co-treatment with lovastatin for 24 hours before harvesting attenuated the up-regulation of miR-21 (P = 0.013). Twenty-four hours of lovastatin exposure up-regulated PPP1R3A to 143.85%± 21.89% of control levels in cardiomyocytes (P = 0.023). Lovastatin up-regulated the phosphorylation level of STAT3 compared to the background LPS pretreatment (P = 0.0077), this effect was significantly (P = 0.018) blunted when miR-21 was functionally inhibited. CONCLUSIONS: miR-21 plays a major role in the regulation of the cellular anti-inflammation effects of lovastatin.

LC-MS/MS method for the determination of agomelatine in human plasma and its application to a pharmacokinetic study.

A sensitive and selective LC-MS/MS method for the determination of agomelatine in human plasma was developed and validated. After simple liquid-liquid extraction, the analytes were separated on a Zorbax SB-C18 column (150 × 2.1 mm i.d., 5 µm) with an isocratic mobile phase consisting of 5 mm ammonium acetate solution (containing 0.1% formic acid) and methanol (30:70, v/v) at a flow-rate of 0.3 mL/min. The MS acquisition was performed in multiple reaction monitoring mode with a positive electrospray ionization source. The mass transitions monitored were m/z 244.1 → 185.3 and m/z 285.2 → 193.2 for agomelatine and internal standard, respectively. The methods were validated for selectivity, carry-over, matrix effects, calibration curves, accuracy and precision, extraction recoveries, dilution integrity and stability. The validated method was successfully applied to a pharmacokinetic study of agomelatine in Chinese volunteers following a single oral dose of 25 mg agomelatine tablet.

Gender differences in pharmacokinetics of a combination tablet of niacin extended-release/simvastatin in healthy Chinese volunteers.

The gender differences in pharmacokinetics of a combination tablet of niacin extended-release/simvastatin were evaluated in healthy Chinese volunteers. Thirty-six healthy male and female volunteers were enrolled in the study receiving a single oral dose of niacin extended-release/simvastatin 1,000/20 mg. The results indicated that the systemic exposure of simvastatin hydroxy acid and the total urine excretion of niacin were significantly higher for females compared with those for males, and the T max of niacin in plasma was significantly shorter for males than that for females. There were no significant differences in the systemic exposure of simvastatin, niacin, and NUA in plasma between males and females.

Pharmacokinetics of lansoprazole and its main metabolites after single and multiple intravenous doses in healthy Chinese subjects.

The aim of the study was to evaluate and compare the pharmacokinetics of lansoprazole (LPZ) and its main metabolites, 5'-hydroxy lansoprazole (HLPZ) and lansoprazole sulfone (LPZS), after single and multiple intravenous (i.v.) doses of LPZ in healthy Chinese subjects. Twelve subjects (six males and six females) were given a single dose of LPZ by i.v. infusion on day 1, and multiple doses from day 2 to day 6. Blood samples were collected at designated time points for analysis of plasma concentrations of LPZ, HLPZ and LPZS by an LC-MS/MS method. LPZ was generally well tolerated in healthy Chinese subjects. After single and multiple i.v. doses of 30 mg LPZ, the C max values of LPZ, HLPZ and LPZS were 1490 ± 290 and 1450 ± 280, 175 ± 71 and 154 ± 56, and 51.3 ± 82.9 and 74.1 ± 158.7 ng/mL, with the AUC0-t values 3280 ± 2550 and 4260 ± 3880, 381 ± 128 and 389 ± 111, and 389 ± 1204 and 700 ± 2255 ng h/mL, respectively. The t 1/2 and CL values of LPZ after single and multiple i.v. doses were 1.48 ± 1.03 and 2.19 ± 1.03 h, and 11.67 ± 4.49 and 9.56 ± 4.08 L/h, respectively. Compared with the pharmacokinetics of LPZ after a single dose, t 1/2 increased markedly, CL decreased significantly and AUC increased by over 20 % after multiple doses. The results indicated that there was drug accumulation of LPZ after multiple i.v. doses, and there was no gender-related difference in pharmacokinetics of LPZ and its two metabolites.

Simultaneous quantification of niacin and its three main metabolites in human plasma by LC-MS/MS.

A sensitive and specific LC-MS/MS method for the simultaneous quantification of niacin (NA) and its three main metabolites nicotinamide (NAM), nicotinuric acid (NUA) and N-methyl-2-pyridone-5-carboxamide (2-Pyr) in human plasma has been developed and validated. Plasma samples (200 µL) were prepared by deproteinization with acetonitrile (500 µL), then the supernatant after centrifugation was evaporated and reconstituted. Chromatography was performed on a phenomenex synergi hydro-RP column with an isocratic elution of methanol-0.1% formic acid (5:95, v/v). The full separation of all analytes was achieved within 9 min. Multiple-reaction monitoring (MRM) using the fragmentation transitions of m/z 124.1 → 80.1, 123.1 → 80.0, 181.0 → 79.0 and 153.1 → 110.2 in positive electrospray ionization (ESI) mode was performed to quantify NA, NAM, NUA and 2-Pyr, respectively. The calibration curves were linear over the concentration range of 2.0-3000 ng/mL for NA and NUA, 10.0-1600 ng/mL for NAM and 50.0-5000 ng/mL for 2-Pyr. This method has been validated in accordance with the US FDA guidelines for bioanalytical method development and applied to the determination of NA and its three main metabolites in Chinese subjects following a single oral dose of niacin extended-release and simvastatin 1000 mg/20mg. In particular, because of the endogenous NAM and 2-Pyr in human plasma, the concentrations of NAM and 2-Pyr in human plasma after dosing were determined by subtracting blank values of them.

Pharmacokinetics of terazosin enantiomers in healthy Chinese male subjects.

The purpose of this study was to elucidate the pharmacokinetics of terazosin enantiomers in healthy Chinese male subjects. After a single oral dose of 2-mg terazosin, the plasma concentrations of terazosin enantiomers were measured over the course of 48 h in 12 healthy subjects. The plasma concentrations of (+)-(R)-terazosin at all time points were higher than those of (-)-(S)-terazosin. The area under the plasma concentration-time curve (AUC(0-∞) ) and maximum plasma concentration of (+)-(R)-terazosin were significantly greater than those of the (-)-(S)-terazosin (P < 0.01, respectively). The R/S ratio of AUC(0-∞) of terazosin was 1.68. For the first time, it was proven that the pharmacokinetics of terazosin was stereoselective in healthy Chinese male subjects.

Pharmacokinetics of lansoprazole and its main metabolites after single intravenous doses in healthy Chinese subjects.

The aim of the study was to evaluate the pharmacokinetics (PK) of lansoprazole (LPZ) and its main metabolites 5'-hydroxy lansoprazole (HLPZ) and lansoprazole sulphone (LPZS) after single intravenous (i.v.) doses of LPZ in healthy Chinese subjects, and the relationship between the cytochrome P450 (CYP) 2C19 phenotypes and the plasma concentrations of LPZS at the time-points in the elimination phase of LPZ. Twelve subjects were given lansoprazole by i.v. infusion. Blood samples were collected at designated time points up to 24 h. Plasma concentrations of LPZ, HLPZ and LPZS were quantified by a selective and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method. After single i.v. doses of 15, 30 and 60 mg LPZ, C(max) and area under the plasma concentration-time curve (AUC(0-t)) of LPZ were 725 ± 151, 1480 ± 190, 3130 ± 480 µg · L(-1) and 1690 ± 1210, 3630 ± 2530, 8080 ± 4550 µg · h · L(-1), respectively. LPZ was generally well tolerated in healthy Chinese subjects, and displayed linear PK in the range of 15-60 mg. There were significant differences in the elimination of LPZ and the formation of LPZS between the single CYP2C19 poor metabolizer (PM) and the CYP2C19 extensive metabolizers (EM). The concentration of LPZS at the time-points in the elimination phase of LPZ could be monitored for CYP2C19 phenotyping. As a probe drug for CYP2C19 phenotyping, LPZ for injection might be more suitable than LPZ oral formulations.

Pharmacokinetics of ibuprofen enantiomers in rats after intravenous and oral administration of ibuprofen arginate.

The pharmacokinetics of ibuprofen enantiomers were studied in rats after intravenous and oral administration of ibuprofen arginate by means of a chiral HPLC method. The pharmacokinetics of ibuprofen was stereoselective after intravenous and oral administration of ibuprofen arginate. The pharmacokinetic stereoselectivity was higher after oral administration than that after intravenous administration. The systematic (R)-(-)-to-(S)-(+) inversion might be more important than the presystematic one in the stereoselective pharmacokinetics after oral administration. Oral administration of ibuprofen arginate resulted in a very rapid absorption of (S)-(+)-ibuprofen (eutomer), and the absolute bioavailabilities of (S)-(+)-ibuprofen and (R)-(-)-ibuprofen were about 100% and 80%, respectively. Based on the systemic exposure of (S)-(+)-ibuprofen, it could be concluded that the pharmacological actions might be similar when ibuprofen arginate was given orally and intravenously, except some differences in the onset of action.

Effects of CYP2C19 polymorphism on the pharmacokinetics of lansoprazole and its main metabolites in healthy Chinese subjects.

The aim of the study was to determine the pharmacokinetics of lansoprazole and its main metabolites (5'-hydroxy lansoprazole and lansoprazole sulphone) after administration of enteric-coated tablet in healthy Chinese subjects classified by CYP2C19 genotypes, and evaluate the effects of CYP2C19 genotypes on the pharmacokinetics of the three compounds. A single oral dose of 30 mg lansoprazole was administrated to 24 healthy Chinese male volunteers in different CYP2C19 genotype groups. Blood samples were collected from pre-dose up to 14-h post-dose. Plasma concentration of lansoprazole and its main metabolites were quantified by liquid chromatography-tandem mass spectrometry. CYP2C19 polymorphism had significant effects on the pharmacokinetics of lansoprazole and its main metabolites. The differences in the pharmacokinetics between CYP2C19 extensive metabolizers (Ems) (homo-EMs and hete-EMs) and PMs were more significant for lansoprazole sulphone than for 5'-hydroxy lansoprazole. The results indicate that the monitoring of lansoprazole and its main metabolites in plasma at the time-points in the elimination phase for lansoprazole could reflect the activity of CYP2C19. Simultaneously monitored with lansoprazole sulphone, lansoprazole might be a useful probe drug for CYP2C19.

[Metabolic pathways of dipfluzine in rats].

AIM: To investigate the metabolic pathways of dipfluzine in rats. METHODS: After an oral dose of dipfluzine (80 mg x kg(-1)) to rats, urine was collected for 12 h. The metabolites of dipfluzine in urine were chromatographed and identified by LC/DAD/MS methods. RESULTS: In the rat urine, there were 1-(4-fluorophenyl)-4-piperazinylbutanone and its glucuronide, 4-hydroxybenzophenone and its glucuronide, 4-fluoro-gamma-hydroxybenzenebutanoic acid and its glucuronide and sulfate, diphenylmethanol and its glucuronide, dipfluzine, and benzophenone. CONCLUSION: In rats, dipfluzine was mainly metabolized in the pathways of N-desalkylation at 1- and 4-positions of piperazine ring. Some of metabolites were further conjugated with glucuronic acid and/or sulfuric acid.