酸水解法提取-高效液相色谱-质谱法测定乳粉中胆碱的含量

Determination of Choline in Milk Powder by High Performance Liquid Chromatography-Mass Spectrometry with Acid Hydrolysis Extraction

取5g乳粉样品,用适量40~45℃水溶解后,用水稀释至100 mL。分取1 mL,与50μL 1 000mg·L^(-1)(以胆碱-1,1,2,2-d4计)氯化胆碱-1,1,2,2-d4内标溶液和10mL 1mol·L^(-1)盐酸溶液混合,于70℃加热3h。冷却后用500g·L^(-1)氢氧化钠溶液调节上述溶液酸度至pH(5.0±0.1),用水稀释至100mL。分取上述溶液1mL,用体积比95∶5的乙腈和10mmol·L^(-1)甲酸铵溶液的混合溶液稀释至10mL,过0.22μm有机滤膜,滤液供高效液相色谱-单四极杆质谱仪分析。在色谱分析时,以ACQUITY UPLC BEH HILIC色谱柱作固定相,以不同体积比的乙腈和10mmol·L^(-1)甲酸铵溶液-甲酸混合溶液(pH 5.0)的混合溶液作流动相进行梯度洗脱;在质谱分析时,以电喷雾离子源正离子(ESI+)模式电离,以选择离子监测(SIM)模式检测,内标法定量。结果显示:胆碱的质量浓度在10.0~200μg·L^(-1)内与对应峰面积与胆碱-1,1,2,2-d4峰面积的比值呈线性关系,检出限(3S/N)为1mg/100g;按照标准加入法进行回收试验,回收率为96.2%~100%;对3个实际样品进行日内、日间精密度试验,胆碱测定值的相对标准偏差分别为0.90%~3.3%(n=11)和1.6%~2.5%(n=9);方法用于和国家标准GB 5413.20-2013中酶比色法进行比对,15种实际乳粉样品中胆碱测定值的相对误差的绝对值均不大于8.0%。

The milk powder sample of 5 g was taken,dissolved by an appropriate amount of water at 40-45℃,and diluted to 100 mL by water.An aliquot(1 mL)was taken and mixed with 50μL of 1000 mg·L^(-1)(calculated by choline-1,1,2,2-d)internal standard solution of choline chloride-1,1,2,2-d4 and 10 mL of 1 mol·L^(-1) hydrochloric acid solution,and the mixted solution was heated at 70℃ for 3 h.After cooling,the acidity of the above solution was adjusted to pH(5.0±0.1)by 500 g·L^(-1) sodium hydroxide solution,and the solution obtained was diluted to 100 mL by water.An aliquot(1 mL)of the above solution was taken,diluted to 10 mL by mixed solution composed of acetonitrile and 10 mmol·L^(-1) ammonium formate solution at volume ratio of 95:5,and passed through a 0.22μm organic filter membrane.The filtrate was used for analysis by high performance liquid chromatograph-single quadrupole mass spectrometer.In chromatographic analysis,ACQUITY UPLC BEH HILIC column was used as the stationary phase,and mixed solutions composed of acetonitrile and 1o mmol·L^(-1) ammonium formate solution-formic acid mixed solution(pH 5.o)at different volume ratios were used as the mobile phase for gradient elution.In mass spectrometry analysis,ESI+mode was used for ionization,and SIM mode was used for detection.The internal standard method was used for quantification.It was shown by the results,linear relationship between values of the mass concentration and the peak area ratio of choline to choline-l,1,2,2-dy was kept in the range of 10.0-200μg·L-1,with detection limit(3S/N)of 1 mg/100 g.Test for recovery was made by standard addition method,giving recoveries in the range of 96.2%-100%.Tests for intra-day and inter-day precision were made on the 3 actual samples,and RSDs of determined values of choline were in the ranges of 0.90%-3.3%(n=11)and 1.6%-2.5%(n=9),respectively.The proposed method was applied to comparison with the enzyme colorimetric method in the national standard of GB 5413.20-2013,giving absolute values of relative errors between determined values of choline in the 15 actual milk powder samples not greater than 8.0%.