Schimpf Karen J, Thompson Linda D, Pan Shang-Jing
Abbott Nutrition, 3300 Stelzer Rd, Columbus, OH 43219.
J AOAC Int. 2018 Jan 1;101(1):264-276. doi: 10.5740/jaoacint.17-0287. Epub 2017 Sep 12.
This reversed-phase HPLC method uses C30 chromatography and UV-Vis spectroscopy to determine cis and trans isomers of lutein, β-carotene, and lycopene in infant, pediatric, and adult nutritionals. Samples are saponified with a mixture of potassium hydroxide, tetrahydrofuran, and methanol, and carotenoids are extracted from saponified samples with 75 + 25 hexane-methyl tertiary butyl ether (MtBE). After extraction, a portion of the organic layer is evaporated to dryness, and the residue is dissolved in 75 + 25 10% butylated hydroxytoluene in methanol-MtBE. Prepared samples are injected into a C30 HPLC column where cis and trans isomers of lutein, β-carotene, and lycopene are separated with a methanol-MtBE gradient and detected with UV-Vis spectroscopy at 445 nm. Total carotenoid concentrations are calculated by comparison of sample peak areas with the areas of trans carotenoid standards of known concentration. During a single-laboratory validation of this method, total lutein repeatability and intermediate precision ranged from 1.89 to 14.9 and 1.93 to 14.0%, respectively, in infant and adult nutritional matrixes with concentrations >1 μg/100 g. Total β-carotene repeatability and intermediate precision ranged from 1.81 to 6.77 and 3.07 to 16.2%, respectively, in infant and adult nutritional matrixes with concentrations >1 μg/100 g, and total lycopene repeatability and intermediate precision ranged from 3.01 to 6.37 and 4.29 to 10.3%, respectively, in infant and adult nutritional matrixes with concentrations >1 μg/100 g. Mean overspike recoveries ranged from 90.3 to 95.3, 89.3 to 108, and 97.3 to 109% for lutein, β-carotene, and lycopene, respectively. The method also demonstrated good linearity. For lutein, r averaged 0.99991 over a standard range of approximately 10-250 µg/L trans-lutein. with average calibration errors of <1%. For β-carotene and lycopene, r averaged 0.99993 and 0.9998 over standard ranges of approximately 25-500 and 5-100 µg/L with calibration errors of <1 and <1.5%, respectively. Lutein, β-carotene, and lycopene LOQs in ready-to-feed nutritionals were estimated to be 0.4, 0.1, and 0.3 µg/100 g, respectively. This method met AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals Standard Method Performance Requirements and was approved as a First Action Official Method at the AOAC INTERNATIONAL 2017 midyear meeting.
这种反相高效液相色谱法采用C30色谱和紫外 - 可见光谱法，用于测定婴儿、儿童和成人营养产品中叶黄素、β - 胡萝卜素和番茄红素的顺式和反式异构体。样品用氢氧化钾、四氢呋喃和甲醇的混合物进行皂化，然后用75 + 25的己烷 - 甲基叔丁基醚（MtBE）从皂化样品中提取类胡萝卜素。提取后，将一部分有机层蒸发至干，残留物溶解于75 + 25的含10%丁基化羟基甲苯的甲醇 - MtBE中。将制备好的样品注入C30高效液相色谱柱，在该柱中，叶黄素、β - 胡萝卜素和番茄红素的顺式和反式异构体通过甲醇 - MtBE梯度进行分离，并在445 nm处用紫外 - 可见光谱法进行检测。通过将样品峰面积与已知浓度的反式类胡萝卜素标准品的面积进行比较，计算总类胡萝卜素浓度。在该方法的单实验室验证过程中，在浓度>1 μg/100 g的婴儿和成人营养基质中，总叶黄素的重复性和中间精密度分别为1.89%至14.9%和1.93%至14.0%。在浓度>1 μg/100 g的婴儿和成人营养基质中，总β - 胡萝卜素的重复性和中间精密度分别为1.81%至6.77%和3.07%至16.2%，在浓度>1 μg/100 g的婴儿和成人营养基质中，总番茄红素的重复性和中间精密度分别为3.01%至6.37%和4.29%至10.3%。叶黄素、β - 胡萝卜素和番茄红素的平均加标回收率分别为90.3%至95.3%、89.3%至108%和97.3%至109%。该方法还显示出良好的线性。对于叶黄素，在约10 - 250 µg/L反式叶黄素的标准范围内，r平均为0.99991，平均校准误差<1%。对于β - 胡萝卜素和番茄红素，在约25 - 500和5 - 100 µg/L的标准范围内，r平均分别为0.99993和0.9998，校准误差分别<1%和<1.5%。即食营养产品中叶黄素、β - 胡萝卜素和番茄红素的定量限估计分别为0.4、0.1和0.3 µg/100 g。该方法符合AOAC婴儿配方奶粉和成人营养产品利益相关者小组的标准方法性能要求，并在AOAC INTERNATIONAL 2017年年中会议上被批准为首次行动官方方法。
