Unlocking Chromatographic Quantification of Serum Lactate Dehydrogenase via Ionic-Liquid-Based Aqueous Biphasic System Pretreatment

Lactate Dehydrogenase (LDH) is a key biomarker for various diseases, but its conventional analysis using colorimetric assays frequently faces reliability issues due to variations in enzyme activity. Additionally, its low serum concentration relative to high-abundance proteins hinders accurate quantification due to matrix effects. This work pioneers the integration of ionic-liquid-based aqueous biphasic systems (IL-ABS) as a sample pretreatment step for the chromatographic quantification of LDH in human serum. ABS composed of phosphonium-based ILs and citrate buffer (K 3 C 6 H 5 O 7 /C 6 H 8 O 7 , pH 7) enable LDH quantification based on concentration rather than enzyme activity determined through colorimetric assays. Proper design, focusing on the IL anion and mixture composition, ensures that IL-ABS completely deplete high-abundance proteins, while extracting LDH from human serum with minimal loss in a single step. Under optimal conditions, LDH levels can be determined using size-exclusion high-performance liquid chromatography with diode array detection (SE-HPLC-DAD), with a relative error of 8%. Remarkably, IL-ABS overcome the interfering effects of high-abundance serum proteins, unlocking LDH analysis via chromatography and potentially improving accuracy and practicality over colorimetric assays. This analytical strategy could enhance the clinical significance of LDH as a biomarker, aiding in diagnosis, prognosis, and medical decisions.