Poly(2-methyl-2-oxazoline)Coating By Thermally Induced Immobilization For Determination Of Bovine Lactoferrin In Infant Formula

Lactoferrin(Lf)is a safe functional protein and one of the four major proteins in human milk.Lf plays a key role in the host's first line of defense,as it can respond to physiological and environmental change.The structural characteristics of Lf provide many functions,such as antimicrobial activity,immunomodulatory and anti-inflammatory activity,anti-carcinogenic activities and enzymatic functions.Lf is the vital nutrition for both infants and adults,which exists in human milk whereas only minor amounts in bovine milk.Thus it is extremely important to strengthen Lf in infant formula to make the nutritional ingredients relatively close to breast milk.Hence,quality control on Lf strengthening infant formula is becoming particularly important,and some analytical methods have been developed to perform this quality control through the detection of quantities of Lf in the infant formula.Lf has been quantified using the techniques such as radial immunodiffusion(RID),enzyme linked immunosorbent assay(ELISA),reversed phase high-performance liquid chromatography(RP-HPLC),and capillary electrophoresis(CE).RID has been used because of its simple instrumentation and ease of operation while its precision is poor.There are few researches have been reported for the analysis of Lf in infant formula using ELISA due to the existence of matrix interferences.The resolution of HPLC is insufficient because of the inherently poor interaction of the analytes with the stationary phase.CE has been widely used in chemical industry,environment,medicine and bioscience due to its low operation costs,saving of solvent,simplicity and accurate quantification.In this work,a one-step coating procedure by a simple annealing protocol of poly(2-methyl-2-oxazoline)-random-glycidyl methacrylate(PMOXA-r-GMA)copolymer was used to yield covalent and cross-linked PMOXA-based antifouling coating on a fused-silica capillary inner surface,which was used to determine the bovine lactoferrin(Lf)in infant formula by capillary electrophoresis with ultraviolet(CE-UV)detection.Firstly,PMOXA-MA macromonomer was synthesized by the cationic ring-opening polymerization(CROP)of MOXA terminated with MAA and NEt3.The PMOXA-r-GMA copolymers were synthesized via free radical polymerization with a varied feed ratio of PMOXA-MA macromonomer to GMA.The PMOXA-r-GMA coated capillary was prepared by a simple annealing protocol.The influences of different ratios of PMOXA-MA to GMA and different polymer concentration for four basic proteins(cytochrome c,lysozyme,ribonuclease A,and?-chymotrypsinogen A)were studied.The result suggested that 20 mg/mL PMOXA-r3/1-GMA coated capillary were chosen as the optimum since its excellent protein separation performance.Therefore,the PMOXA-r-GMA coated capillary was used to determine the quantity of Lf in infant formula.Under the optimal conditions,the peak area(A)and the concentration of Lf showed a good linear relationship within the range of 10-500 ?g/mL with a linear correlation coefficient of 0.9995.The limit of detection(LOD)and limit of quantitation(LOQ)for Lf were 5.0 ?g/mL and 16.7 ?g/mL,respectively.The recoveries at spiked concentrations of 0.20 and 0.40 mg/mL Lf in infant formula were 97.1 ± 5.5%-97.8 ± 5.1%.The determined values of Lf in infant formula samples with Lf were consistent with the nominal values,indicating that our CE method could be successfully applied to the quantitative analysis of Lf in commercial infant formula.