Nano-HPLC-HRMS analysis to evaluate leptin level in milk samples: A pilot study

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.