# Nano-HPLC-HRMS Analysis to Evaluate Leptin Level in Milk Samples: A Pilot Study
## 4 Discussion  
Before the quantitation of leptin in milk samples, a deep investigation about MS/MS fragmentation of selected peptides was conducted. With the orbitrap fusion high resolution mass spectrometry analyzer we performed the precursor ion fragmentation both with CID, HCD (high collision induced dissociation) and ETD (electron transfer dissociation). The precursor ions _m/z_ ratio were 708.4 (quantifier) and 764.4 (qualifier). With CID we obtained the best sensitivity. Figure 2 shows the liquid chromatographic separation and tandem mass spectra of the protonated peptides. Product ions are listed in Table 4; as expected, the y and b ions were the main species formed by CID fragmentation. Figure 2: Chromatographic separation of human leptin analytical standard at concentration of 250 ng mL\({}^{-1}\) digested peptides and MS/MS fragmentation products of quantifier (_n_/z= 708.3599, z = 2) and qualifier (_m/z_ = 764.4099, z = 2) ions. \begin{table}
\begin{tabular}{c c} \hline
**Milk Sample** & **Leptin Concentration (ng mL\({}^{-1}\))** \\ \hline Human breast milk sample 1 & \(7.1\pm 1.06\) \\ Human breast milk sample 2 & \(6.4\pm 1.28\) \\ Human breast milk sample 3 & \(7.5\pm 1.12\) \\ Human breast milk sample 4 & \(5.8\pm 1.16\) \\ \hline Cow milk sample 1 & Nd \\ Cow milk sample 2 & Nd \\ Cow milk sample 3 & Nd \\ Cow milk sample 4 & Nd \\ \hline Liquid infant formula sample 1 & Nd \\ Liquid infant formula sample 2 & Nd \\ Liquid infant formula sample 3 & Nd \\ Liquid infant formula sample 4 & Nd \\ \hline \end{tabular}
\end{table} 
Table 3: Concentration value of leptin in human breast milk, cow milk and liquid infant formula samples. The amount is expressed in ng\(\cdot\)mL\({}^{-1}\). Nd, not detectable. Once obtained the mass spectrum information, bovine milk samples were used for calibration curve. As previously reported, we gained a LLOQ and LOD of 100 and 27 mg mL\({}^{-1}\) respectively. The LLOQ was quite higher if compared with other analytical methodologies developed for leptin quantitation. These methods were based on chemiluminescent or enzyme immunoassay (CLEIA and EIA), radioimmunoassay (RIA), immunofluorimetric assay (IFMA), enzyme-linked immunosorbent assay (ELISA) and HPLC-MS and reported very low limit of quantitation [30; 31; 32; 33; 34; 35; 36; 37; 38; 39; 40]. CLEIA and EIA leptin analysis attained by the research groups of [PERSON] [30] and [PERSON] [31] detected the protein in serum. [PERSON] and co-workers with their developed chemiluminescent immunosensor gained a LOD of 1.9 pg mL\({}^{-1}\)[30]. Using the anti-leptin polyclonal antibody with a chemiluminescent enzyme immunoassay [PERSON] team measured leptin in human serum with a LOD of 0.1 pg mL\({}^{-1}\)[31]. RIA was used to quantify leptin concentration in serum and breast milk [15; 32; 33; 34]. [PERSON] research group measured with RIA leptin concentrations in 36 serum of healthy terms infant exclusively breast feeding and in 24 samples of breast milk obtaining a median concentration of 3.42 ng mL\({}^{-1}\) in infant serum and 0.51 ng mL\({}^{-1}\) in breast milk [32]. In a similar experiment performed in 2016 with RIA detection of leptin, the group confirmed the obtained results testing 58 samples of infant and mother serum, and mother milk [15]. Quite comparable results were found by [PERSON] et al. [33] with RIA detection of leptin in 23 healthy, lactating women and their newborn infants. RIA and ELISA assays were also used by [PERSON] and co-workers to quantify leptin in mice serum exposed to high fat diet [34]. Leptin was quantified both with RIA and ELISA with a sensitivity of 25 and 46.7 pg mL\({}^{-1}\) respectively. All these studies were conducted with a commercially available radioimmunoassay with a declared sensitivity of 0.04 ng mL\({}^{-1}\)[15; 32] or 0.25 pg mL\({}^{-1}\)[15]. However, RIA presents some important weaknesses, such as the presence of radioisotope and rather high cost of reagents and assay [35].