Current Chromatography - Volume 8, Issue 1, 2021

Background: To prevent vitamin deficiencies or to restore their levels, multi-vitamin formulations are indicated for the elderly, adults, children, and infants. Objective: To provide a valid high-throughput method for simultaneous determination of vitamins in multi-vitamin formulations. Methods: A high-throughput UPLC-ESI-MS method was developed and validated for simultaneous determination of vitamins B1, B2, B3, B5, B6, B7, and B9. Analytes were eluted on an Acquity UPLC® BEH C18 1.7 μm, 2.1 x 50 mm column at 40 ± 5°C. Mobile phase containing acetonitrile (0.1% formic acid) and water (0.1% formic acid) in 30:70% ratio was pumped at 300 μL/min under isocratic control. Protonated ions of vitamin B1, B2, B3, B5, B6, B7, and B9 were monitored in single ion recording mode, using an electrospray ionization probe in positive mode. Results: The m/z ratios of positive ions of vitamin B1, B2, B3, B5, B6, B7, and B9 were 265.1, 377.2, 122.95, 220, 169.98, 244.9, and 442.1, respectively. The calibration curve of different linearity range (ng/ml) was prepared for each vitamin. Linearity range for vitamin B1, B2, B3, B5, B6, B7, and B9 were 60-1000, 25-1000, 75-5000, 30-1000, 30/1000, 25/1000 and 30-900 25-1000 ng/mL, respectively. Coefficient of variation for intra-day and inter-day precision for vitamin B1, B2, B3, B5, B6, B7, and B9 at the middle and higher limit of quantitation were less than 15%. Conclusion: The method was successfully developed and validated, and three different brands of multi-vitamin tablets were assayed for water-soluble vitamins.

Objective: To address the separation of interfering potential impurities associated with the drug is always a daunting task. We present the method validation and quantitative determination of sulfadoxine (SUL), an anti-malarial drug with the most important interfering impurities present in pharmaceutical dosages and bulk samples using HPLC-UV method. Methods: The UV detection was obtained at 270 nm and SUL is separated on Sunfire C18 (25 cm x 4.6 mm x 5 μm) column at 45°C with a flow rate of 1.0 mL/min in a mobile phase (CH3COOH: CH3CN). The stress testing (acidic/basic/oxidative) was performed using HPLC for SUL and its impurities, showing the highly efficient separation peaks between degradant and drug products. Results: The developed method was found to be highly accurate and sensitive in regulation with ICH guidelines. Also, it was found to be free from interference from degradation products which allows the stability indicating capability of developed HPLC-UV method for SUL for validation in bulk drugs. Conclusion: The main advantages of the present method; (a) Separation achieved in 30 minutes, (b) MS compatible mobile phase renders this developed method can be directly adapted to LC-MS without any major modifications in the near future, and (c) separation of twelve impurities on Sunfire C18 column. The CFs (correction factors) had been calculated for all the impurities. It was found to be 1.6 (IMP IX), 1.70 (IMP XI) and in between 0.8-1.3 for all other impurities. The LOD of the developed method for all the analytes was in the range of 0.05 to 0.11 μg/mL and the LOQ values were in the range of 0.17 to 0.36 μg/mL.

Aims: To develop RP-HPLC method for the simultaneous estimation of methotrexate (MTX) and minocycline (MNC). Background: Different HPLC methods were reported for the estimation of MTX/MNC individually, but there is no report for the simultaneous estimation of both MTX and MNC in a simple method. Objective: The objective of the developed method is to utilize the method for the estimation of MTX/MNC in different pharmaceutical formulations and in biological fluids. Methods: An HPLC method for the estimation of Methotrexate (MTX) and Minocycline (MNC) relevance to the evaluation of nanoparticulate formulations has been developed and validated. Chromatographic estimation was achieved using the mobile phase composition of sodium acetate buffer and acetonitrile (70:30% v/v) at pH 4.0 at a flow rate of 0.2 mL/min at 307 nm. Results: The calibration curve for MTX and MNC was found to be linear at nanogram (5 to 25 ng.mL-1) and microgram (5 to 25 μg.mL-1) levels at correlation coefficient range of 0.98 to 0.99 for both MTX/MNC. The lower limit of detection and limit of quantification were found to be 0.026 ng.mL-1 and 0.079 ng.mL-1 for MTX and MNC, respectively. The percentage relative standard deviation for validation parameters of both drugs was found to be less than 6.5%. The amount of MTX and MNC present within the nanoparticles was found to be MTX (0.84 mg/mL) and MNC (0.61 mg/mL). The in vitro release showed an immediate release pattern for MTX (64.95±2.08%) and MNC (90.90±1.78%) within 12 h. Conclusion: The developed analytical method for the simultaneous estimation of MTX and MNC was found to be simple, affordable, dynamic, low cost, rapid and easy to perform with good repeatability. This method is also time consuming, since the peaks were obtained within a moderate analysis time.

Background: Sulfated Polyborate, a novel inorganic material primarily designed as a catalyst, has shown properties such as high solubility in organic solvents, low U.V. cut-off, and pKa ≈2.0, which suggests its potential as a mobile phase buffer for reverse-phase liquid chromatography. Objective: This study aims to substantiate the role of Sulfated Polyborate as mobile phase buffer for reverse-phase liquid chromatographic analysis of basic drugs with high pKa values viz. Bisoprolol fumarate, Timolol maleate, Verapamil hydrochloride, and Carvedilol. Methods: Solubilities, U.V. cut-offs, and pKa of Sulfated Polyborate were first experimentally confirmed. The behaviour of Sulfated Polyborate as mobile phase buffer at pH 3.0 was ascertained by varying the buffer concentration, flow rates, and percent organic modifier for elution of the four basic drugs on a non-end capped octyl silyl (C8) column. Similarly, the study was performed with KH2PO4 as a reference buffer. The column performance and conductometric measurements ascertained the impact of Sulfated Polyborate on the stationary phase. Results: Sulfated Polyborate and KH2PO4 buffers showed correlation coefficients of 0.99 and 1.00 for analyte retention factors for variation of buffer concentration and organic modifier composition, respectively. Peak symmetries and the number of theoretical plates were improved from > 2.0 to < 2.0 and ≈1000 to ≈3000, respectively, for variation in buffer concentrations. Similar Van Deemter plots indicated equivalency of Sulfated Polyborate and KH2PO4 buffers. The column performance and conductometric measurements depicted no adsorption on the stationary phase. Conclusion: The present study demonstrates Sulfated Polyborate as a novel buffer for analytes with higher pKa on reverse-phase liquid chromatography.

Background: Metformin is the first-line drug to enhance glycemic control of type 2 DIABETES Mellitus (DM2) patients. Some reported methods to determine plasma metformin by HPLC-UV are not sensitive enough. Other methods require long extraction processes. Objective: The objective of this study was to develop and validate a simple and rapid analytical method to determine plasma metformin by HPLC-UV for application in a population pharmacokinetic study. Methods: Analyte was extracted from plasma by a simple protein precipitation technique using trichloroacetic acid (15%, w/v) as the precipitating agent. Plasma samples were analyzed using a C18 column (3.0 x 150 mm, 3.5 μm) under isocratic elution with 30 mM sodium hexansulfonate (pH 5) and acetonitrile (95: 5, v,v). Results: The limit of quantification (LOQ) was 0.1 μg mL^-1 and the calibration curve was linear up to 4 μg mL^-1 with a correlation coefficient >0.99. The mean recovery for metformin using this extraction procedure was 84.4 - 86.6%. The intra- and inter-day coefficients of variation and percent error values of the assayed method were <20% and <15% for LOQ and QCs, respectively. Metformin was stable in plasma samples by subjecting it to three freeze-thaw cycles and storing it up to 60 days at -80°C. This method was applied to determine plasma metformin concentrations in patients with type 2 diabetes mellitus treated with this drug. Conclusion: The HPLC-UV method developed is selective, accurate and precise for the quantification of metformin in plasma samples, since sample processing is fast and simple, in addition to being applicable in pharmacokinetic studies.