Nano-HPLC-HRMS Analysis to Evaluate Leptin Level in Milk Samples: A Pilot Study

Abstract

Leptin is a 16 kDa lipophilic protein hormone secreted by adipocytes and its most significant function is to inform the brain with negative feedback that regulates food intake. Recently the protein found in human breast milk was related to breast feeding and onset of obesity, and the evidence of a low probability to develop pediatric obesity in children fed with breast milk was also confirmed. Since leptin could have a critical role, its quantitation both in human breast, bovine milk and in infant formula products is interesting. For this reason, we developed an analytical method based on immunoaffinity purification followed by an analysis with nano-High Pressure Liquid Chromatography coupled with High Resolution Mass Spectrometry analyzer (nano-HPLC-HRMS) to identify and quantify leptin in milk samples and performed a pilot study using samples of human breast milk, bovine milk and infant formulas. With an obtained lower limit of quantitation (LLOQ) of 100 ng mL−1 we quantified leptin in human breast milk finding an average of 6.70 ng mL−1. Our results show that leptin was under LLOQ both in bovine milk and in infant formula products. In conclusion, the developed analytical method here described was suitable to quantify leptin in milk samples with a good sensitivity and selectivity, and without the use of radioactive reagents.

Human Milk Metabolic Hormones: Analytical Methods and Current Understanding

“…The nano-HPLC-HRMS reported a LOD of 27 ng/mL, which was substantially higher than the LOD of RIA and ELISA (0.017 and 0.05 ng/mL, respectively) [18,44]. Further, the average concentration of HM leptin reported by nano-HPLC-HRMS (6.7 ng/mL) is much higher than values reported by RIA and ELISA and below the detection limit reported by the authors (LOD = 27 ng/mL) [65].…”

“…It has been previously assumed that higher whole milk leptin concentrations are due to the lipophilic nature of leptin, however, leptin concentrations reportedly have no association with HM fat content and do not differ between pre-and post-feed samples despite increasing HM fat content over the course of a breastfeed [48,70]. Nevertheless, precipitation of HM lipids, before the immunoaffinity extraction was not achieved without precipitating leptin [65], which warrants further investigation.…”

“…Another recently developed analytical method is based on immunoaffinity purification followed by analysis with nano-high pressure liquid chromatography coupled with a highresolution mass spectrometry analyser (nano-HPLC-HRMS) [65]. The nano-HPLC-HRMS reported a LOD of 27 ng/mL, which was substantially higher than the LOD of RIA and ELISA (0.017 and 0.05 ng/mL, respectively) [18,44].…”

Isocratic Reverse-Phase HPLC Method for Determination of Aspirin, Paracetamol, and Naproxen

“…In combination with mass spectrometry, chromatography has been an essential part of proteomic analysis. [8][9][10][11][12][13]. High-performance liquid chromatography (HPLC) has been adapted to analyze multi-component drugs by adjusting the affinity between the surfactant of column beads and the drug molecules according to size and electric charges.…”

Analysis of Chemical Biomarkers and Contaminants in Food

“…The development of an analytical method based on nano-high performance liquid chromatography (nanoHPLC) coupled with high-resolution mass spectrometry (HRMS) to detect trace levels of leptin in milk samples [4]. Leptin is a fundamental protein related to adipose tissue and food intake, and it was demonstrated that its intake during the early lactation period diminished the probability of developing infant obesity.…”