A multiplex assay of leptin, resistin, and adiponectin by immunoaffinity enrichment and targeted mass spectrometry

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Background: Leptin, resistin, and adiponectin are critical adipokines involved in the pathophysiology of obesity and its related disorders, including type 2 diabetes. Although these biomarkers have historically been quantified using immunoassays, the specificity of antibody-based methods has frequently been questioned. As a result, there is an increasing interest in developing reliable, multiplexed clinical assays that utilize mass spectrometry for improved accuracy. In this study, we present a multiplexed immunoaffinity liquid chromatography-tandem mass spectrometry (multi-IA-LC-MS/MS) assay designed for the sensitive and selective measurement of leptin, resistin, and adiponectin in human plasma. Methods: Leptin, resistin, and adiponectin were selectively enriched from plasma samples using an antibody cocktail composed of monoclonal antibodies targeting each respective adipokine. The enriched adipokines underwent enzymatic digestion, and the resulting tryptic peptides were quantified using LC-MS/MS. The validated assay was subsequently applied to plasma samples collected from a cohort of subjects representing various weight categories, including normal weight, overweight, and obesity. Results: The lower limits of quantification for the assay were determined to be 0.5 ng/mL for both leptin and resistin, and 50 ng/mL for adiponectin. Intra- day, inter- day, and total imprecision measurements were all < 15 %, while spike recovery consistently exceeded 83 %. Comparative analysis with individual immunoassays demonstrated strong correlation, with all correlation coefficients (r) being equal to or greater than 0.869. Notably, when comparing subjects with obesity to those with normal weight, there was an approximately nine-fold increase in circulating leptin levels and a ∼1.6-fold decrease in circulating adiponectin levels. Conclusions: A multi-IA-LC-MS/MS assay was developed for the simultaneous and sensitive measurement of leptin, resistin, and adiponectin in clinical samples. This quantitative method shows significant potential for applications related to obesity and could facilitate improved clinical management and understanding of obesity-related conditions.

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