Method development for HILIC assays

Abstract

In this review, method development for hydrophilic interaction LC (HILIC) is highlighted. HILIC is a chromatographic technique that uses aqueous-organic solvent mobile phases with a high organic-solvent fraction, and a hydrophilic stationary phase. It is mainly applied for the separation of polar and hydrophilic compounds. Method development, in general, can be done uni- or multivariately. In the univariate approach, the factors that are expected to potentially affect the separation of the compounds will be examined sequentially and one-at-a-time. All HILIC methods found in the literature were developed in this way. For these methods, the analytes, the considered factors, the selected responses, and the finally chosen experimental conditions are discussed in this review. Where examined, the method validation and the comparison with other analytical assay methods is also described. For the multivariate method-development approach, which is based on the use of experimental designs, only a possible strategy is presented, because of the lack of relevant publications in the literature.

Bioanalytical Method for the Determination of Hydroxyproline in Mouse Kidney by High-Performance Liquid Chromatography with Tandem Mass Spectrometric Detection

“…12,19,20 In this separation mode, a deterioration of the HILIC column has been observed due to direct consecutive injections of strongly acidic hydrolyzed samples. In order to prevent column damage, the hydrolyzed kidney samples had to undergo evaporation and neutralization before injection of the assay solution to the HPLC.…”

Mise au point et validation d’une méthode de dosage simultané de la morphine et de ses métabolites glucuronoconjugués totaux par chromatographie liquide d’interactions hydrophiles

“…Le mécanisme de rétention principal en mode HILIC repose sur le système de partition liquide-liquide créé entre la couche fortement aqueuse formée par la phase mobile à la surface de la phase stationnaire d'une part, et la phase mobile faiblement aqueuse d'autre part. En fonction de la nature de la phase stationnaire, d'autres méca-nismes de rétention contribuent à la séparation des composés hydrophiles polaires comme les liaisons hydrogène, les interactions électrostatiques fortes ou les échanges d'ions [15,16]. Dans le cadre du protocole expérimental pour lequel nous avons développé la méthode décrite dans cet article, nous avons choisi de doser les métabolites glucuronoconjugués de la morphine de manière globale, sans séparer spécifiquement la M3G et la M6G.…”

Translating innovative pharmacokinetic sampling techniques into improved antibiotic dosing regimens in critically ill patients

“…Despite the low injection volumes being used, 0.1 μL for plasma and 0.5 μL for urine, we conclude that the changes in the retention times and quality of the chromatography observed may have been due to the sensitivity of the interaction of fosfomycin with the stationary phase to changes in ionic strength and pH of the buffer [128,129], as well as the age of the analytical column. Considering each matrix separately, with each being validated separately several months apart, the robustness of the chromatography for each matrix is evidenced by very low variability in retention times (less than 1.0% relative standard deviation) as seen across the validation and clinical samples for each matrix.…”

“…We conclude that the changes in the retention times and quality of the chromatography observed may have been due to the and pH of the buffer [128,129], as well as the age of the analytical column. The VAMS extractions were performed many months after those performed in plasma and urine [136], on different columns, and after a substantial reconfiguration of instrumentation.…”

“…Retention of the analyte by the stationary phase is caused by hydrophilic partitioning within an aqueous-enriched liquid layer and/or with the positive or negative charges on the HILIC stationary phase [129]. The balance of the partition is provided by the aqueous content and pH of the mobile phase.…”