Background and Objectives: Lysinoalanine (LAL) is created in the course of preparation of infant formula during the heat processing, alkaline pH, b elimination, dehydroalanine production and reaction with amines group. Produced LAL not only results in losses of necessary amino acids’ content, but also could cause nephrotoxic effects. So the formulated infant formula must be free from LAL. In this study, HPLC for detection and determination of LAL was developed and validated.
Materials and Methods: HPLC conditions were optimized (C18 Column, Fluorescence detector, Column temperature 30℃, flow rate 0.9 ml/min. Mobile phase consisted of mixed phosphate buffer, pH: 7, acetonitrile and pure acetonitrile in gradient elution), and LAL was dansylated by adding dansyl chloride. The method was validated according to the above conditions. 10 infant formula brands were analyzed according to the validated method.
- The calibration curve for concentration versus LAL peak area (R2= 0.9949) was linear in the range of 5_80 mg/L. LOD and LOQ were 2 and 5 mg/L respectively, and the accuracy result (recovery range) was within 83.6-87.7%. Assessment of precision showed that the relative standard deviation (RSD%) of concentration and area peak of spike samples in the intra-day study were less than 2.7% and 3.8% respectively, and in inter-day study were less than 7.4% and 5.2%, respectively. In the analyzed infant formula in seven samples, LAL was not detectable, and it was detected between LOD and LOQ only in three samples.
Conclusion: This method is a valuable validated way, available, reliable and cost benefit in LAL detection and determination in infant formula.
Keywords: Lysinoalanine, Infant formula, HPLC, Fluorescence detector |
