A method for the determination of non-lactose oligosaccharides (NLO) in milk using liquid chromatography has been developed. Oligosaccharides were labelled with a fluorescent tag, 2-aminobenzamide (2AB), and were identified by comparison of their retention times to those of oligosaccharide standards, their mass (as measured by mass spectrometry) and their fragmentation patterns in the mass spectrometer. The concentrations of the NLO in milk have been determined using 2 different approaches: (1) by preparing a calibration curve using genuine standards of each oligosaccharide. (2) by preparing a calibration curve using maltotriose as a universal standard for all NLO, and assuming all 2AB labelled oligosaccharides give an equimolar response in the detector. The accuracy of the method was assessed by spike-recovery experiments. Using genuine NLO standards for calibration, recoveries were in the range 96-114%. Using maltotriose as a universal calibrant, recoveries were in the range 86-120%. Method precision was assessed by determining the relative standard deviation of the results under repeatability (RSD(r)) and intermediate reproducibility (RSD(iR)) conditions. In most cases RSD(r) and RSD(iR) were below 5% irrespective of calibration method, but increased when NLO levels were close to LoQ.

中文翻译：

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非乳糖乳低聚糖的定量测定

开发了一种使用液相色谱法测定牛奶中非乳糖低聚糖 (NLO) 的方法。寡糖用荧光标签 2-氨基苯甲酰胺 (2AB) 进行标记，并通过将它们的保留时间与寡糖标准品的保留时间、它们的质量（通过质谱法测量）和它们在质谱仪中的碎片模式进行比较来进行鉴定。牛奶中 NLO 的浓度已使用 2 种不同的方法测定：(1) 使用每种寡糖的真正标准制备校准曲线。(2) 通过使用麦芽三糖作为所有 NLO 的通用标准制备校准曲线，并假设所有 2AB 标记的寡糖在检测器中给出等摩尔响应。该方法的准确性通过加标回收实验进行评估。使用真正的 NLO 标准进行校准，回收率在 96-114% 的范围内。使用麦芽三糖作为通用校准物，回收率在 86-120% 之间。通过确定在重复性 (RSD(r)) 和中间重现性 (RSD(iR)) 条件下结果的相对标准偏差来评估方法精度。在大多数情况下，无论采用何种校准方法，RSD(r) 和 RSD(iR) 均低于 5%，但当 NLO 水平接近 LoQ 时会增加。