Optimization Of The Detection Method For Casein Phosphopeptides In Milk Powder

In China,casein phosphopeptide(CPP)is widely added to milk powder,especially infant formula as nutritional fortifier,and the limit of CPP is specified in the national standard(GB 14880-2012).With China’s increasingly stringent requirements for formula milk powder,especially infant formula milk powder,establishing a highly effective CPP detection method is the key to control the amount of CPP added.In this paper,the CPP detection method was optimized using the extraction rate as an indicator.The CPP in the sample was extracted with water as the solvent by ultrasonic treatment.The extract solution was adjusted to p H 4.6 with hydrochloric acid for protein precipitation.After mixed with acetonitrile containing 0.5%formic acid for a certain volume,the solution was filtered with 0.22μm film and separated by liquid chromatography-tandem mass spectrometry.The external reference method was used for quantitative analysis.Two specific peptides were determined using Quadrupole-flight time series mass spectrometer(QTOF)then the HPLC-MS/MS was used as an analytical method to determine the content of CPP in milk powder.The feasibility analysis of the method was verified through evaluating the recovery rate,precision,linear range and matrix effect of HPLC-MS/MS.The linear range of CPP was 5 to 100μg/m L via the regression equation from CPP standard curve,and the correlation coefficient R~2was between 0.9991 and 0.9997.The quantitation limit in this study was 100 mg/kg.The recovery test validation was used to evaluate the recovery rate using the blank sample contained artificially added CPP.The average recovery rate of the milk powder samples ranged from 79.7%to 97.3%,the average standard recovery rate was 87.7%,and the relative standard deviation RSD ranged from 2.2%to 12.2%.The experimental results show that this method has the advantages of high extraction efficiency,simple operation,short analysis time,accurate qualitative and quantitative analysis,and high reliability.It provides reliable technical support for the detection of CPP from different manufacturers with potential application prospects.