On-line Monitoring of In-Vitro Oral Bioaccessibility Tests as Front-End to Liquid Chromatography for Determination of Chlorogenic Acid Isomers in Dietary Supplements

Abstract

A novel fully automated in-vitro oral dissolution test assay as a front-end to liquid chromatography has been developed and validated for on-line chemical profiling and monitoring of temporal release profiles of three caffeoylquinic acid (CQA) isomers,namely, 3-CQA,4-CQA and 5-CQA, known as chlorogenic acids, in dietary supplements. Tangential-flow filtration is harnessed as a sample processing approach for on-line handling of CQA containing extracts of hard gelatin capsules and introduction of protein-free samples into the liquid chromatograph. Oral bioaccessibility/dissolution test assays were performed at 37.0±0.5 °C as per US Pharmacopeia recommendations using pepsin with activity of ca. 749,000 USP units/L in 0.1 mol/L HCl as the extraction medium and a paddle apparatus stirred at 50 rpm. CQA release rates and steady-state dissolution conditions were determined accurately by fitting the chromatographic datasets, namely, the average cumulative concentrations of bioaccessible pools of every individual isomer monitored during 200 min, with temporal resolutions of ≥10 min, to a first-order dissolution kinetic model. Distinct solid-to-liquid phase ratios in the mimicry of physiological extraction conditions were assessed. Relative standard deviations for intra-day repeatability and inter-day intermediate precision of 5-CQA within the 5–40 µg/mL concentration range were <3.4% and <5.5%, respectively. Trueness of the automatic flow method for determination of 5-CQA released from dietary supplements in gastric fluid surrogate was demonstrated by spike recoveries, spanning from 91.5–104.0%, upon completion of the dissolution process. The proposed hyphenated setup was resorted for evaluating potential differences in dissolution profiles and content of the three most abundant chlorogenic acid isomers in dietary supplements from varied manufacturers.