Validated Reversed-Phase Liquid Chromatographic Method with Gradient Elution for Simultaneous Determination of the Antiviral Agents: Sofosbuvir, Ledipasvir, Daclatasvir, and Simeprevir in Their Dosage Forms.

A simple, rapid, sensitive, and precise reversed-phase liquid chromatographic method was developed and validated for the simultaneous determination of four direct-acting antivirals, sofosbuvir (SF), ledipasvir (LD), declatasvir (DC), and simeprevir (SM), in their respective pharmaceutical formulations. Effective chromatographic separation was achieved on an Agilent Eclipse plus C8 column (250 mm × 4.6 mm, 5 µm) at 40 °C with gradient elution using a mobile phase composed of acetonitrile:phosphate buffer (pH 6.5). The quantification of SF and DC was based on peak area measurements at 260 nm, while the quantification of LD and SM was achieved at 330 nm. The linearity was acceptable from 1.0 to 20.0 µg/mL for the studied drugs, with correlation coefficients >0.999. The analytical performance of the newly proposed HPLC procedure was thoroughly validated according to ICH guidelines in terms of linearity, precision (RSD%, 0.39-1.57), accuracy (98.05-101.90%), specificity, limit of detection (LOD) (0.022-0.039 µg/mL), limit of quantification (LOQ) (0.067-0.118 µg/mL), and robustness. The validated HPLC method was successfully used to analyze the abovementioned drugs in their pure and dosage forms without interference from common excipients present in commercial formulations.

Development and Validation of an RP-HPLC Method for the Determination of Vinpocetine and Folic Acid in the Presence of a Vinpocetine Alkaline Degradation Product in Bulk and in Capsule Form.

An alkaline-forced degradation hydrolytic product of vinpocetine was prepared and characterized by 1H-NMR, FTIR spectroscopy, and MS. Subsequently, a simple, selective, and validated reversed-phase HPLC method was developed for the simultaneous estimation of vinpocetine and folic acid in the presence of a vinpocetine alkaline degradation product. Chromatographic separation was achieved using an isocratic mobile phase consisting of acetonitrile-0.02 M KH2PO4 [containing 0.2% (v/v) triethylamine and adjusted to pH 6 with orthophosphoric acid; (80 + 20, v/v)] at a flow rate of 0.9 mL/min at ambient temperature on a Eurospher II C18 (250 × 4.6 mm, 5 µm) column, with UV detection at 280 nm for folic acid and 230 nm for vinpocetine and its alkaline hydrolytic product. Linearity, accuracy, and precision were found to be acceptable over a concentration range of 12.5-200 µg/mL for vinpocetine and 1-16 µg/mL for folic acid. The proposed method was successfully applied for the determination of both drugs and a vinpocetine hydrolysis product in a laboratory-prepared mixture and in a capsule containing both drugs.

UPLC-Tandem Mass Spectrometry Method for Simultaneous Determination of Fluoxetine, Risperidone, and Its Active Metabolite 9-Hydroxyrisperidone in Plasma: Application to Pharmacokinetics Study in Rats.

Risperidone (RIS) is used as an antipsychotic drug alone or with other drugs, like fluoxetine (FLX). A simple method was developed and validated for the determination of both RIS and its metabolite 9-hydroxyrisperidone (9-OH-RIS), FLX, and olanzapine (OLA) as an internal standard in rat's plasma using UPLC-MS/MS. FLX, RIS, 9-OH-RIS, and OLA were purified using acetonitrile as a protein precipitating agent. Separation was performed on an ACQUITY™ "UPLC BEH™" C18 column (50 mm × 2.1 mm i.d., 1.7 µm; Waters Corp., USA). The ranges of the calibration curves were 1.0-1000.0 ng/mL for FLX and 0.2-1000.0 ng/mL for RIS and 9-OH-RIS. Linearity, recovery, precision, and stability were within the acceptable range. This method is rapid, fast, and precise for the determination of RIS and FLX in plasma and is applicable in pharmacokinetic studies.

Spectrophotometric Methods for Simultaneous Determination of Sofosbuvir and Ledipasvir (HARVONI Tablet): Comparative Study with Two Generic Products.

Sofosbuvir and ledipasvir are the first drugs in a combination pill to treat chronic hepatitis C virus. Simple, sensitive, and rapid spectrophotometric methods are presented for the determination of sofosbuvir and ledipasvir in their combined dosage form. These methods were based on direct measurement of ledipasvir at 333 nm (due to the lack of interference of sofosbuvir) over a concentration range of 4.0-14.0 µg/mL, with a mean recovery of 100.78 ± 0.64%. Sofosbuvir was determined, without prior separation, by third-derivative values at 281 nm; derivative ratio values at 265.8 nm utilizing 5.0 µg/mL ledipasvir as a divisor; the ratio difference method using values at 270 and 250 nm using 5.0 µg/mL ledipasvir as a divisor; and the ratio subtraction method using values at 261 nm. These methods were found to be linear for sofosbuvir over a concentration range of 5.0-35.0 µg/mL. The suggested methods were validated according to International Conference on Harmonization guidelines. Statistical analysis of the results showed no significant difference between the proposed methods and the manufacturer's LC method of determination with respect to accuracy and precision. These methods were used to compare the equivalence of an innovator drug dosage form and two generic drug dosage forms of the same strength.

Development and validation of LC/MS/MS method for the simultaneous determination of montelukast, gliclazide, and nifedipine and its application to a pharmacokinetic study.

BACKGROUND: Montelukast is a leukotriene receptor antagonist for treatment of asthma, gliclazide is an oral hypoglycemic antidiabetic agent, and nifedipine is a calcium channel blocker used for treatment of angina pectoris and hypertension. These drugs may be prescribed to patients suffering from these chronic diseases. A survey of the literature reveals that there is no reported method for the simultaneous determination of montelukast, gliclazide, and nifedipine in pharmaceutical preparations or biological fluids. RESULTS: A simple, sensitive, and rapid method for the simultaneous quantification of montelukast, gliclazide, and nifedipine in human plasma was developed and validated. Montelukast, gliclazide, and nifedipine were resolved using rapid resolution LC/MS/MS Agilent system and SB-C18 (50 × 4.6 mm) 1.8 µm particle size column. The mobile phase consisted of acetonitrile: 0.1% formic acid (84:16). The three drugs were simultaneously extracted from plasma by protein precipitation with acetonitrile using zaferolukast as an internal standard. The method was validated according to FDA guidelines with good reproducibility and linearity of 0.999 and the limits of quantification were 0.11, 0.04, and 0.07 ng/mL for montelukast, gliclazide, and nifedipine, respectively. The accuracies of the three QCs for the three drugs were 99.48% (montelukast), 106.53% (gliclazide), and 108.03% (nifedipine) in human plasma. The validated method was applied to a pharmacokinetic study in human volunteers after oral administration of the three drugs. The applied LC/MS/MS method was shown to be sufficiently sensitive and suitable for pharmacokinetic studies. CONCLUSION: The LC/MS/MS method was validated and successfully applied for the determination of montelukast, gliclazide, and nifedipine concentrations in human plasma.