# Nano-HPLC-HRMS Analysis to Evaluate Leptin Level in Milk Samples: A Pilot Study
## 4 Discussion
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Precursor Ion m/z [MH]\({}^{2+}\)** & **Product Ion m/z** & **ppm Error** & **Ions Type** \\ \hline
708.3599 & 960.4745 & +0.425 & y9\({}^{+}\) \\  & 699.8466 & +0.385 & [MH-NH\({}_{3}\)]\({}^{2+}\) \\  & 343.1612 & +0.265 & b3\({}^{+}\) \\  & 736.3836 & +0.475 & y7\({}^{+}\) \\  & 873.4452 & +0.397 & y8\({}^{+}\) \\  & 651.8179 & +0.463 & y12\({}^{2+}\) \\  & 321.1769 & +0.245 & y3\({}^{+}\) \\  & 1095.5429 & +0.423 & b10\({}^{+}\) \\  & 507.2773 & +0.368 & y5\({}^{+}\) \\  & 1073.5586 & +0.478 & y10\({}^{+}\) \\  & 1188.5855 & +0.386 & y11\({}^{+}\) \\
764.4099 & 960.4745 & +0.398 & y8\({}^{+}\) \\  & 568.3453 & +0.465 & b5\({}^{+}\) \\  & 657.8542 & +0.381 & y11\({}^{2+}\) \\  & 1201.6171 & +0.451 & y10\({}^{+}\) \\  & 1073.5586 & +0.421 & y9\({}^{+}\) \\  & 873.4425 & +0.369 & y7\({}^{+}\) \\  & 531.2885 & +0.436 & y4\({}^{+}\) \\  & 327.2027 & +0.298 & b3\({}^{+}\) \\  & 402.2459 & +0.236 & y3\({}^{+}\) \\  & 455.2613 & +0.312 & b4\({}^{+}\) \\  & 1353.7009 & +0.481 & b12\({}^{+}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Precursor protonated ions _m_% of selected peptides and monoisotopic mass of the product CID ions. As expected, y and b ions were the only formed with CID fragmentation. Immunofluorimetric assay with species-specific antibody was used by [PERSON] group [36] to quantify leptin in pig saliva with limit of detection of 1.047 ng mL\({}^{-1}\). Although by using ELISA, [PERSON] et al. [37] obtained a good sensitivity of 0.05 ng mL\({}^{-1}\), and other studies used this technique to detect leptin [38; 39], it is well known that ELISA normally suffers from cross-reactivity [40]. [PERSON] et al. [41] performed the quantitation of human leptin in human serum using a HPLC-tandem mass instrument and declared 15.63 ng mL\({}^{-1}\) as LOQ; the authors did not mention any LOD value. However, serum is a simpler matrix compared with milk, whose content is not only a significant amount of proteins, but above all a massive quantity of lipids. Nevertheless, leptin is primarily secreted from adipose tissue and here is present in cytoplasm in the interface between nuclei and rim surrounding lipid droplets [42; 43]. Consequently, it was not possible to precipitate the lipids with, for example, heptane solution, before the immunoaffinity extraction to avoid the precipitation of leptin too. Methods based on immunoaffinity approach retaining highly selectivity due to the use of the selected antibody; but the immobilization columns, since the loaded antibody cannot cover quantitatively the total surface of the resin, may bond to other molecules, such as lipids or sugars. We thought that this was the reason of our quite higher LLOQ vs. Wang limit (100 vs. 15.63 ng mL\({}^{-1}\) respectively [41]). However, the LLOQ (100 ng mL\({}^{-1}\)) obtained with this method based on immunoaffinity sample purification and nano-HPLC-HRMS was suitable to quantify the protein hormone leptin in milk samples. Quality control (QC) samples, consisting of bovine whole milk without leptin fortification, were analyzed and we did not detect endogenous bovine leptin. Using the immunoaffinity columns bound with polyclonal antibody anti-LEP (from rabbit, specific for Bos taurus species), the Mascot software did not recognize any bovine leptin in whole bovine milk. On the contrary, when bovine colostrum that did not undergo any treatment was analyzed, the software identified bovine leptin with a coverage percentage of 85%. Moreover, as mentioned above in \"Material and methods\", whole bovine milk samples were purchased in a local supermarket; they were commercially available products. After milking, bovine milk is subjected to industrial processes comprising pasteurization, homogenization, centrifugation and eventually skimming. During the centrifugation step fat globules are separated from aqueous milk component leaving three categories of milk: whole (fat content 3.5-4%), semi-skimmed (fat content between 1.5 and 1.8%), skimmed (fat content less than 0.5%).