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.

A Simple Validated LC-UV Method for the Simultaneous Determination of Brimonidine and Brinzolamide in the Presence of Brinzolamide's Degradation Product (Major Metabolite) in Rabbit Aqueous Humor.

A sensitive, specific, reliable and low-cost LC-UV method has been developed and validated for simultaneous quantification of brimonidine tartrate (BM) and brinzolamide (BZ) in rabbit aqueous humor (AH) in the presence of N-desethyl-brinzolamide (NDBZ); BZ is a major degradation product, and it is also considered to be its major metabolite. Dorzolamide hydrochloride (DZ) was used as an internal standard (IS). The analytes were extracted from rabbit AH samples by a simple pre-treatment utilizing ZnSO4.7H2O as a deproteinizing agent. The analytes were separated on a cyanopropyl Waters column (4.6 × 200 mm, 5 µm) with an isocratic mobile phase consisting of 25 mM ammonium acetate pH 4.5 (adjusted with 85% phosphoric acid):methanol:acetonitrile (94:4.5:1.5, v/v) at a flow rate of 1.0 mL min-1. The detection was done at 254 nm. The lower limit of quantification in rabbit AH was 100.0 ng mL-1. The method was validated according to EMA guidelines. The method was confirmed to be accurate, precise and linear over a range of 100.0-1000.0 ng mL-1 for BM and BZ. The method developed herein is simple, safe, eco-friendly, rapid and accurately applied for the quantification of BM and BZ, along with the successful separation of NDBZ, which is considered as a potential irritant degradation product of BZ.

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.

New Modified UPLC/Tandem Mass Spectrometry Method for Determination of Risperidone and Its Active Metabolite 9-Hydroxyrisperidone in Plasma: Application to Dose-Dependent Pharmacokinetic Study in Sprague-Dawley Rats.

Sensitive and specific liquid-chromatography tandem mass spectrometry (UPLC-MS/MS) assay has been developed and validated for simultaneous quantification of risperidone (RIS) and its active metabolite 9-hydroxyrisperidone (9-OH-RIS) in rat plasma using olanzapine (OLA) as internal standard (IS). Pharmacokinetics of risperidone and its active metabolite 9-hydroxyrisperidone was compared across different doses (0.3, 1.0, and 6.0 mg/kg). Serial blood sample was collected over a time of 48 hours and analyzed for risperidone and its active metabolite 9-hydroxyrisperidone. The pharmacokinetics parameters including Cmax, tmax, and AUC were determined for risperidone and its active ingredient. The method was linear in the concentration range of 0.2-500 ng/mL for risperidone and 9-OH-risperidone, with coefficients of determination greater than 0.998 and lower limit of quantitation of 0.2 ng/mL. Blood levels of risperidone and its active metabolite were roughly dose-proportional. The method developed herein is simple and rapid and was successfully applied for dose-dependent pharmacokinetic study.

Novel ratio difference at coabsorptive point spectrophotometric method for determination of components with wide variation in their absorptivities.

Different methods have been introduced to enhance selectivity of UV-spectrophotometry thus enabling accurate determination of co-formulated components, however mixtures whose components exhibit wide variation in absorptivities has been an obstacle against application of UV-spectrophotometry. The developed ratio difference at coabsorptive point method (RDC) represents a simple effective solution for the mentioned problem, where the additive property of light absorbance enabled the consideration of the two components as multiples of the lower absorptivity component at certain wavelength (coabsorptive point), at which their total concentration multiples could be determined, whereas the other component was selectively determined by applying the ratio difference method in a single step. Mixture of perindopril arginine (PA) and amlodipine besylate (AM) figures that problem, where the low absorptivity of PA relative to AM hinders selective spectrophotometric determination of PA. The developed method successfully determined both components in the overlapped region of their spectra with accuracy 99.39±1.60 and 100.51±1.21, for PA and AM, respectively. The method was validated as per the USP guidelines and showed no significant difference upon statistical comparison with reported chromatographic method.

Novel liquid chromatographic methods for the determination of varenicline tartrate.

Two simple, sensitive, rapid, and stability-indicating liquid chromatographic (LC) methods have been developed for the determination of varenicline tartrate. They comprised the determination of varenicline (VRC) in the presence of its oxidative degradates and related impurity (N-formyl varenicline) (NFV). The first method was a LC with diode array detection (DAD) at 235 nm using Ristek-Ultra® C18 column (100 mm × 2.1 mm, 5 µm). Isocratic elution of VRC was employed using a mobile phase consisting of buffer mixture (1.2% potassium dihydrogen phosphate and 0.08% octane sulphonic acid): acetonitrile (86:14, v/v), pH (5.0). In the second method; a fluorimetric detection technique was developed, based on precolumn derivatization of VRC using 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl). The fluorescence detector (FLD) was operated at 474 nm for excitation and 539 nm for emission. Isocratic elution was applied with a mobile phase consisting of methanol-distilled water (70:30, v/v). Separation was achieved using Symmetry® Waters C18 column (150 mm × 4.6 mm, 5 µm). Linearity, accuracy and precision were found to be acceptable over the concentration ranges of 0.5-20.0 µg mL(-1) and 0.2-20.0 µg mL(-1) with the first and the second method, respectively. The optimized methods were validated and proved to be specific, simple, and accurate for the quality control of the drug in its pharmaceutical preparation.

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.