Acetonitrile Adducts of Tranexamic Acid as Sensitive Ions for Quantification at Residue Levels in Human Plasma by UHPLC-MS/MS

The quantitative analysis of pharmaceuticals in biomatrices by liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is often hampered by adduct formation. The use of the molecular ion resulting from solvent adducts for quantification is uncommon, even if formed in high abundance. In this work, we propose the use of a protonated acetonitrile adduct for the quantitative analysis of tranexamic acid (TXA) by LC-MS/MS. The high abundance of the protonated acetonitrile adduct [M + ACN + H]+ was found to be independent of source-dependent parameters and mobile phase composition. The results obtained for TXA analysis in clinical samples were comparable for both [M + ACN + H]+ and [M + H]+, and no statistically significant differences were observed. The relative stability and structure of the [M + ACN + H]+ ions were also studied by analyzing probable structures from an energetic point of view and by quantum chemical calculations. These findings, and the studied fragmentation pathways, allowed the definition of an acetimidium structure as the best ion to describe the observed acetonitrile protonated adduct of TXA.

Characterization and In-silico toxicity prediction of degradation products of felbamate

Background The objective of the work carried out was to assess the toxicity of the degradation products (DPs) for the drug felbamate. Stress studies were performed in the condition specified in the international council of harmonization (ICH) guideline Q1A (R2). Results The drug degraded under the alkaline stress conditions to generate two degradation products (DPs). They were separated on a Phenomenex C8 column (250 mm × 4.6 mm, 5 µm); mobile phase composition was 10 mM ammonium formate (pH adjusted to 3.7 with formic acid) and acetonitrile (80:20, v/v); flow rate and wavelength for recording absorbance were 1.0 ml/min and 206 nm, respectively. The structures of the degradation products were characterized by LC–MS/MS analysis. Conclusion The drug was prone to hydrolysis in the presence of alkali. It was found to be stable under other stress conditions, viz., acidic, neutral, thermal, photolytic and oxidative. The structures of the impurities were characterized by LC–MS/MS. The drug and the DPs were screened through ADME and toxicity prediction software’s like pkCSM, Toxtree and OSIRIS property explorer. Felbamate was flagged for possible hepatotoxicity.

A Rapid HPLC Method for the Concurrent Determination of Several Antihypertensive Drugs from Binary and Ternary Formulations

A rapid, synchronized liquid chromatographic method was established for the estimation of hydrochlorothiazide (HCZ), amlodipine (AMD), olmesartan (OLM), telmisartan (TEL), and irbesartan (IRB) in binary and ternary coformulations using the same chromatographic conditions. Five analytes were separated on a Zorbax C18 column using isocratic elution with a mobile phase consisting of acetonitrile, methanol, and 20 mM phosphate buffer (pH 3.5) in a ratio of 45:20:35% v/v. The analytes were detected at a wavelength of 230 nm at ambient temperature. Furthermore, the proposed liquid chromatographic procedure was validated for linearity, precision, accuracy, stability, and robustness using an experimental design. Analytes were separated with good resolution within 3.5 min. Analytes showed good linearity in a concentration satisfactory to analyze the different ratios of these analytes in the formulations. Pareto charts showed that the flow rate and mobile phase composition have a significant effect on the peak area of analytes and hence need to be carefully controlled, however, the method is robust. Finally, the different formulations consisting of HCZ, AMD, OLM, TEL, and IRB in different ratios were analyzed with high accuracy using an optimized HPLC method and compared with reported methods. Furthermore, the reported HPLC procedure is simple, rapid, and accurate and therefore can used for regular quality control of binary and ternary formulations using the same stationary and mobile phase.

Comparison of the Fitting Performance of Retention Models and Elution Strength Behaviour in Hydrophilic-Interaction and Reversed-Phase Liquid Chromatography

Hydrophilic interaction liquid chromatography (HILIC) is able to separate from polar to highly polar solutes, using similar eluents to those in the reversed-phase mode (RPLC) and a polar stationary phase, where water is adsorbed onto its surface. It is widely accepted that multiple modes of interaction take place in the HILIC environment, which can be far more complex than the interactions in an RPLC column. The behaviour in HILIC should be adequately modelled to predict the retention with optimisation purposes and improve the understanding on retention mechanisms, as is the case for RPLC. In this work, the prediction performance of several retention models is studied for seven HILIC columns (underivatised silica, and silica containing diol, amino and sulfobetaine functional groups, together with three columns recently manufactured with neutral, anionic, and cationic character), using uracil and six polar nucleosides (adenosine, cytidine, guanosine, thymidine, uridine, and xanthosine) as probe compounds. The results in HILIC are compared with those that were offered by the elution of several polar sulphonamides and diuretics analysed with two C18 columns (Chromolith Speed ROD and Zorbax Eclipse XDB). It is shown that eight retention models, which only consider partitioning or both partitioning and adsorption, give similar good accuracy in predictions for both HILIC and RPLC columns. However, the study on the elution strength behaviour, at varying mobile phase composition, reveals similarities (or differences) between RPLC and HILIC columns of diverse nature. The particular behaviour for the HILIC and RPLC columns was also revealed when the retention, in both modes, was fitted to a model that describes the change in the elution strength with the modifier concentration.

Evaluation and authentication for metformin HCL, Glimepride, atorvastatine by using bulk HPLC pharmaceutical dosage form

Reverse phase chromatographic techniques are used for analyzing drugs. It is advanced analytical instrumentation method used for analysis of drugs. A novel accurate, precise, simple, & selective RP- HPLC technique is established and authorized for stability representing RP – HPLC technique for simultaneous assessment of Metformin HCL, Glimepiride & Atorvastatin in bulk & pharmaceutical dosage form by RP– HPLC technique. The technique on Luna C 18, 250mm × 4.6 mm, 15 µm. Column with a mobile phase comprising of buffer and acetonitrile in ratio of (85:15 v/v) and 1.0ml/ minflow rate. The detection is 258 nm. The retention time for metformin HCL, Atorvastatin & Glimepiride are discovered to be 3.266, 5.237 and 8.457 correspondingly. The technique authorized accordingly to ICH rules for precision, specificity, LOQ, LOD, accuracy, robustness, & linearity. The technique demonstrates best recovery &reproducibility with % RSD lower than 2. The suggested technique is discovered to be precise, simple, accurate, specific, &linear. The results are within the acceptance limits of lower than 2% signified that suggested technique has best reproducibility. The toughness of developed technique is validated by altering mobile phase composition, flow rate, and changing wavelength. The % RSD was found within the limits i.e., should not be more than 2.0.

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-UPLC METHOD FOR THE QUANTIFICATION OF BALOXAVIR MARBOXIL IN TABLET FORMULATION

Objective: Aim of the present work is to develop a simple, accurate and precise stability-indicating method for the quantification of baloxavir marboxil (BLMX) in tablet dosage form by UPLC. Methods: Chromatographic elution was processed through a HSS C18 (100 x 2.1 mm, 1.8 mm) reverse phase column and the mobile phase composition of buffer 0.1% orthophosphoric acid and acetonitrile in the ratio of 50:50 was processed through a column at a flow rate of 0.3 ml/min. Column oven temperature was maintained at 30 °C and the detection wavelength was processed at 240 nm. Results: Retention time of BLMX was found to be 0.87 min. Repeatability of the method was determined in the form of %RSD and the value was 0.2. The percentage mean recovery of the method was found to be 99.47%. LOD, LOQ values obtained from the regression equation of BLMX were 0.69 and 2.10 mg/ml, respectively. Regression equation and correlation coefficient values of BLMX were y = 16994x+7179.2 and 0.9996. Drug was subjected for acid, peroxide, photolytic, alkali, neutral and thermal degradation studies and the results shown that the percentage of degradation was found between 5.96% and 9.55%. Conclusion: Retention time and total run time of the drug was decreased and the developed method was simple and economical. So, the developed method can be adopted in industries as a regular quality control test for the quantification of BLMX.

Purification of high purity docosahexaenoic acid from Schizochytrium sp. SH103 using preparative-scale HPLC

High purity polyunsaturated fatty acids (> 95%) are essential for the synthesis of specialized pro-resolving lipid mediators (SPMs), such as protectins, resolvins, and maresins, which are used for clinical application. To date, high purity (> 95%) eicosapentaenoic acid (EPA; C20:5n3) and docosahexaenoic acid (DHA; C22:6n3) have been produced through various manufacturing steps using fish oil. In this study, we optimized preparative high performance liquid chromatography (HPLC) process to purify high-purity DHA ethyl ester (DHAee; > 98%) from oleaginous microalgae Shizochytrium sp. SH103 containing at least 34% DHA content. The purity and yield of DHA were determined by reverse phase chromatography with changing the mobile phase velocity, loading amount, and mobile phase composition. On a semi-preparative scale, optimal DHA separation in isocratic elution was obtained with a mobile phase velocity of 0.5 mL/min, a loading amount of 10 mg/mL, and mobile phase composition of methanol/water (96:4, v/v), wherein the purity of DHA was 98.5%. This separation was scaled up to a preparative column, resulting in 99.0% DHA fraction with a yield of 79.8%. This result suggests that a large amount of high purity DHA can be produced from microalgae when scaling up a preparative column to an industrial column.

Development and validation of high-performance liquid chromatography method for the simultaneous monitoring of pantoprazole sodium sesquihydrate and domperidone maleate in plasma and its application to pharmacokinetic study

A sensitive, inexpensive high-performance liquid chromatography–ultraviolet detection (HPLC–UV) method has been developed and validated for the simultaneous monitoring of pantoprazole sodium sesquihydrate (PSS) and domperidone maleate (DM) in rabbit plasma on a C18 column with UV detection at 285 nm. Box–Behnken design was used with 3 independent variables, namely, flow rate (X1), mobile phase composition (X2), and phosphate buffer pH (X3), which were used to design mathematical models. Response surface design was applied to optimize the dependent variables, i.e., retention time (Y1 and Y2) and percentage recoveries (Y3 and Y4) of PSS and DM. The method was sensitive and reproducible over 1.562 to 25 μg/mL. The effect of the quadratic outcome of flow rate, mobile phase composition, and buffer pH on retention time (p ˂ 0.001) and percentage recoveries of PSS and DM (p = 0.0016) were significant. The regression values obtained from analytical curve of PSS and DM were 0.999 and 0.9994, respectively. The percentage recoveries of PSS and DM were ranged from 94.5 to 100.41% and 94.77 to 100.31%, respectively. The developed method was applied for studying the pharmacokinetics of PSS and DM. The Cmax of test and reference formulations were 48.06 ± 0.347 μg/mL and 46.31 ± 0.398 μg/mL for PSS, and 15.11 ± 1.608 μg/mL and 12.06 ± 1.234 μg/mL for DM, respectively.