Zhan-Zhang Wang

Simultaneous determination of glimepiride and pioglitazone in human plasma by liquid chromatography–tandem mass spectrometry and its application to pharmacokinetic study

The rapid, sensitive, and selective liquid chromatography-electrospray ionization-tandem mass spectrometry method (LC-ESI-MS/MS) for the simultaneous estimation and pharmacokinetic investigation of glimepiride and pioglitazone in human plasma has been developed and fully validated. Glimepiride and pioglitazone, compounds which exert synergistic effects on blood glucose control, were investigated in human plasma using deuterium-labeled analogs as internal standards (IS). Liquid-liquid extraction was carried out on 0.2 mL of human plasma using ethyl acetate, and chromatographic separation was performed on an Agilent Eclipse plus C18 column (4.6 mm × 100 mm, 3.5 μm) using a mobile phase consisting of methanol-water-formic acid (95:5:0.1, v/v/v, plus 5mM ammonium acetate) at a flow rate of 0.8 mL/min. To quantify glimepiride, pioglitazone and their IS, multiple reaction monitoring (MRM) transitions of m/z 491.2→352.2, m/z 496.2→357.2, m/z 357.2→134.2 and m/z 361.2→138.2 were performed in positive mode. The total run time was 3.0 min and the elution time was about 2.4 min. The method exhibited good separation of analytes, without interference from endogenous substances. The linear calibration curves were 0.2-250 ng/mL for glimepiride and 0.2-1,250 ng/mL for pioglitazone; the lower limit of quantification (LLOQ) was 0.2 ng/mL for both analytes. Intra- and inter-day reproducibility was less than 10% for glimepiride and less than 5% for pioglitazone, with relative errors ranging from -8.00% to 2.80% at the three concentrations of analytes used for quality control (QC). The matrix effect was negligible and recoveries were similar for each analyte and its IS. Glimepiride and pioglitazone were found to be stable under the assay conditions and the method was successfully applied to the evaluation of pharmacokinetic studies of glimepiride and pioglitazone, following oral doses of 2mg glimepiride tablets and 15 mg pioglitazone tablets to 16 healthy volunteers.

Effect of grapefruit juice and food on the pharmacokinetics of pirfenidone in healthy Chinese volunteers: a diet–drug interaction study

Abstract 1. Ingestion of grapefruit juice and food could be factors affecting the pharmacokinetics of pirfenidone, a promising drug for treatment of idiopathic pulmonary fibrosis. 2. A randomized, open-label, three-period crossover study was carried out in 12 healthy Chinese male volunteers who were randomized to one of the three treatments: pirfenidone tablets (0.4 g) were orally administered to fasted or fed subjects, or with grapefruit juice. The washout period was 7 d. 3. Significantly reduced maximum plasma concentration (Cmax, 5.0 5 ± 1.39 versus 10.9 0 ± 2.94 mg·L− 1), modestly affected area-under-the-plasma concentration–time curve (AUC) from time zero to 12 h post dosing (AUC0–12 h, 21.8 9 ± 6.47 versus 26.1 6 ± 7.32 mg·h·L− 1) and delayed time to reach Cmax (Tmax) were observed in fed group compared with fasted group. Similar effects on Cmax (5.8 2 ± 1.23 versus 10.9 0 ± 2.94 mg·L− 1) and AUC0–12 h (modest but not statistically significant, 24.4 4 ± 7.40 versus 26.1 6 ± 7.32 mg·h·L− 1) were observed for grapefruit juice compared to fasted subjects. 4. Co-administration of pirfenidone with grapefruit juice resulted in modestly reduced overall oral absorption and significantly reduced peak concentrations compared to fasting, which was similar to effect of food ingestion. No adverse events were observed in the study, but relatively dramatic reduction of peak concentrations should raise concerns for clinical efficacy and safety.

A rapid LC–MS/MS quantification of peramivir using a simple and inexpensive sample precipitation: application to PK

AIM Peramivir is a newly approved selective neuraminidase inhibitor designed to inhibit influenza virus infection. METHODOLOGY/RESULTS We report a robust and sensitive method utilizing simple precipitation extraction with LC-MS/MS for the high-throughput quantification. Addition of 0.06 M of ammonium formate and 0.1% formic acid in mobile phase could help reduce the matrix effect. This method uses 100 µl of plasma and covers a linear concentration range from 5 to 10,000 ng/ml. Other validation parameters are also evaluated and meet regulatory expectations by US FDA guidelines. CONCLUSION The developed HPLC-MS/MS method has been successfully applied to support a clinical pharmacokinetic study. The strategy presented here can be applied elsewhere and may be useful for other amphiphilic drugs.

The effect of food on the pharmacokinetic properties and bioequivalence of two formulations of pitavastatin calcium in healthy Chinese male subjects

Abstract 1. Pitavastatin is an effective treatment for primary hyperlipidemia and mixed dyslipidemia. The aim of the present study was to investigate the effect of food on the pharmacokinetic properties and bioequivalence of the original, branded, formulation of pitavastatin calcium and a new generic formulation in healthy Chinese male subjects under fasting and fed conditions. 2. Under fasting and fed conditions, 90% CIs of the geometric mean of generic/branded AUC0–48 h ratios were 92.2–102.4%, 93.1–104.5%, the ratios of ln(AUC0–∞) were 92.6–103.7%, 93.2–103.5%, and ln(Cmax) ratios were 90.7–110.3%, 84.7–100.8%, respectively. The generic and branded formulations were bioequivalent in terms of rate and extent of absorption under both the conditions. The average values of AUC0–48 h, AUC0–∞ and Cmax decreased noticeably following a high-fat breakfast. Values for AUC0–48 h were 87.69% and 83.7%, values for AUC0–∞ were 87.5% and 84.6%, and values for Cmax were 45.0% and 50.4% in subjects given the generic and branded preparations, respectively. The absorption of pitavastatin calcium tablets was delayed following a high-fat meal, with Tmax increasing by up to 2.43-fold. 3. Both formulations were generally well tolerated, with no serious adverse reactions reported. The newly developed generic formulation may provide a reliable alternative to the branded tablets for patients with primary hyperlipidemia or mixed dyslipidemia.

Development and validation of a sensitive LC-MS/MS assay for the quantification of nizatidine in human plasma and urine and its application to pharmacokinetic study.

We developed and validated a high performance liquid chromatographic method coupled with triple quadrupole mass spectrometry for analysis of nizatidine in human plasma and urine. The biological samples were precipitated with methanol before separation on an Agilent Eclipse Plus C18 column (100mm×46mm, 5μm) with a mixture of methanol and water (95:5, plus 5mM ammonium formate) as the mobile phase at 0.5mL/min. Detection was performed using multiple reaction monitoring modes via electrospray ionization (ESI) at m/z 332.1→155.1 (for nizatidine) and m/z 335.1→155.1 (for [(2)H3]-nizatidine, the internal standard). The linear response range was 5-2000ng/mL and 0.5-80μg/mL for human plasma and urine, with the lower limits of quantification of 5ng/mL and 0.5μg/mL, respectively. The method was validated according to the biological method validation guidelines of the Food and Drug Administration and proved acceptable. This newly developed analytical method was successfully applied in a pharmacokinetic study following single oral administration of a 150mg nizatidine capsule in to 16 healthy Chinese subjects. Maximum and endpoint concentrations in plasma and urine were quantifiable, suggesting our method is appropriate for routine pharmacokinetic analysis.

Simultaneous determination of pirfenidone and its metabolite in human plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study.

A simple and rapid analytical method for the simultaneous determination of pirfenidone and its metabolite, 5-carboxy-pirfenidone, in human plasma using liquid chromatography-tandem mass spectrometry has been developed and validated. Aliquots of plasma (0.1 mL) containing pirfenidone and 5-carboxy-pirfenidone, as well as deuterium-labeled internal standards (ISs), were deproteinized using acetonitrile. An Agilent Zorbax Plus C18 column was used for the chromatography, with isocratic elution. The mobile phase was a mixture of acetonitrile and aqueous ammonium formate solution (5 mM) containing 0.1% formic acid (60 : 40, v/v). Using multiple reaction monitoring in positive ionization mode, transitions m/z 186.1 → 65.1, m/z 216.0 → 77.0, m/z 191.1 → 65.1 and m/z 221.0 → 81.0 were chosen to quantify pirfenidone, 5-carboxy-pirfenidone and the two ISs, respectively. The time of analysis was <3 min. The calibration curve was linear over the concentration ranges 0.005-25 μg/mL for pirfenidone, and 0.005-15 μg/mL for 5-carboxy-pirfenidone. The lower limit of quantification for both analytes was 0.005 μg/mL. The intra- and interday precision and relative errors in quality control samples were between -11.7 and 1.3% for pirfenidone and between -5.6 and 2.5% for 5-carboxy-pirfenidone, with mean recoveries ≥90%. The method that has been developed is easy to carry out, sensitive and rapid, and has been successfully used to investigate the pharmacokinetics of pirfenidone in healthy human volunteers.

Simultaneous analysis of olanzapine, fluoxetine, and norfluoxetine in human plasma using liquid chromatography-mass spectrometry and its application to a pharmacokinetic study

Adjunctive therapy with olanzapine and fluoxetine has been shown to be beneficial in treatment-resistant depression and the depressive phase of bipolar disorder. Consensus guidelines issued by the Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie strongly recommend that patients taking olanzapine undergo therapeutic drug monitoring (TDM), and suggest that TDM is useful for patients taking fluoxetine. The aim of the current study was to develop and validate a sensitive, practical, and robust liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for simultaneous determination of olanzapine, fluoxetine, and norfluoxetine in human plasma for routine TDM. Simple liquid-liquid extraction using ethyl acetate was used to extract olanzapine, fluoxetine, and norfluoxetine from 200 μL of pre-basified human plasma. Analytes were separated on an Agilent Eclipse Plus C18 column (4.6 × 100 mm, 5 μm) eluted with a mobile phase consisting of methanol:20 mM ammonium formate buffer (82.5:17.5, v/v), and then quantified using an electrospray ionization source operated in positive ion multiple reaction monitoring mode. The linear range for the analytes was 0.2-25 ng/mL, covering the vast majority of levels encountered in real-life samples. A weighting factor of 1/x2 best fit the calibration curves. The mean internal standard-normalized matrix effects for all analytes were 99.5%-110%. The extraction recoveries were 75%-85% for olanzapine and olanzapine‑d3, and 58%-69% for fluoxetine, norfluoxetine, and their deuterated internal standards. Accuracy and precision values also met the acceptance criteria. The stability assessments showed that QC samples containing the three analytes were stable for at least 1 d at room temperature, 21 d at -70 °C, and through three freeze-thaw cycles. Post-preparation storage for 2 d in the autosampler did not cause obvious degradation of the investigated compounds. This validated high performance LC-MS/MS method was successfully applied to a pharmacokinetic study in healthy male volunteers.

Pharmacokinetics of guaifenesin, pseudoephedrine and hydrocodone in a combination oral liquid formulation, administered as single and multiple doses in healthy Chinese volunteers, and comparison with data for individual compounds formulated as Antuss®.

Abstract 1. A new oral liquid formulation combining guaifenesin, pseudoephedrine and hydrocodone is effective in improving the symptoms of common cold. The pharmacokinetic properties of the individual components were evaluated in a randomized, open-label, four-period study in 12 healthy Chinese volunteers following single and multiple doses. The data were compared with data for the individual ingredients in Antuss®. 2. In the single-dose period, exposure levels (AUC and Cmax) for guaifenesin, pseudoephedrine and hydrocodone increased directly as the dose of the oral liquid formulation increased from 5 to 15 mL. Only minor amounts of guaifenesin and hydrocodone were excreted in urine (∼0.10% and 4.66%, respectively). Pseudoephedrine was mainly excreted unchanged, with 44.95% of the dose excreted in urine within 24 h. After multiple dosing, there was no obvious accumulation of any drug, as assessed by AUC. When considering Cmax, there was a trend toward accumulation of hydrocodone and pseudoephedrine. The pharmacokinetic profiles of guaifenesin and pseudoephedrine in the oral liquid formulation were similar to those in the branded preparation, Antuss®. 3. The newly developed oral liquid formulation combining guaifenesin, pseudoephedrine and hydrocodone was safe and well tolerated and might provide a reliable alternative to the branded formulation for patients with common colds.

Development and validation of an HILIC–MS/MS method by one-step precipitation for chloroquine in miniature pig plasma

Background: Quantification of polar compounds such as chloroquine by revered-phase LC is a challenge because of poor retention and silanol interactions with stationary phase. Strong ion-pairing reagents added to mobile phases to improve reversed-phase retention and improve peak shape can be harmful for MS. Results: This new approach provides a rapid and sensitive method for the detection of chloroquine using hydrophilic interaction LC coupled to MS/MS (HILIC–MS/MS). Ammonium formate and formic acid were added to mobile phase to attain good peak shapes and the salified chloroquine as well retained in an HILIC column. Linearity, intra- and inter-day precision, accuracy, recovery, matrix effect and stability were evaluated during the validation process. Conclusion: The validated method has been successfully used in a PK study in miniature pigs, and paves way for future development.