Retention behaviour of analytes in reversed-phase high-performance liquid chromatography-A review.

The understanding of principles that drive the separation in reversed-phase chromatography plays an important role in the prediction of the elution of solutes in RP-HPLC. The separation in RP-HPLC is based on the principle of adsorption and partition. In addition, the logP value, the pKa value of the drug and chromatographic parameters like mobile phase pH, buffer concentration, organic modifier and mobile phase additives also influence the retention and selectivity of the analyte. It was found that hydrophobic, electrostatic, hydrogen bonding and other specific interactions between the stationary phase and the solutes, along with the hydrophobicity of an analyte molecule (logP), modify the retention behaviour of the analytes. This article gives special attention to the influence of ionization and ion interaction on the separation of analytes. The drug molecules with different logP values containing protonated and deprotonated acids, bases and zwitterions are selected as examples and this article addresses various issues related to the method development, relationships between analyte retention and mobile phase pH and the pKa value of the analyte. The advances in this regard, with highlights on topics such as mechanisms of retention and various factors that influence the retention behaviour of analytes, are also updated with suitable examples.

Estimation of minoxidil in human plasma using UHPLC-MS/MS and its application in pharmacokinetic study.

Measurement of drug concentration in biological matrices (such as serum, plasma, blood, urine, and saliva) is important to determine Bioavailability (BA) and/or Bioequivalence (BE) of a drug product which are required during the drug product development and approval process to support applications for new active substances (INDs, NDAs) and generic (ANDAs) drug products to make critical decisions on safety and efficacy. Because of their vital role, bioanalytical methods should be well-characterized, fully validated and documented to yield reliable results. In present work, a simple, specific, high throughput, accurate and sensitive UHPLC-MS/MS method has been developed and validated for quantification of Minoxidil in human plasma. The analyte and the internal standard were extracted from plasma by Liquid-Liquid Extraction using ethyl acetate. The chromatographic separation was achieved on Thermo Hypersil Gold column (4.6x50mm, 5µm) using acetonitrile-0.1% formic acid in water (60:40, v/v) at a flow rate of 0.400 ml/min. Detection by turbospray positive ionization mass spectrometry in the multiple reaction monitoring mode with a mass transition ion-pair of m/z 210.152 → 163.965 (Minoxidil) and m/z 220.267 → 169.089 (Internal Standard-Minoxidil D10) was found to be linear over the concentration range of 1.280 to 151.075 ng/ml. The method was fully validated as per USFDA guidelines and the results were within regulatory limits. The inter and intra-day precision ranged from 5.42 to 9.27% and 2.55-9.42% respectively. The inter and intra-day accuracy ranged from 89.2 to 98.9% and 102-105% respectively. The method was successfully applied to a BE study involving human volunteers.