Beijing Key Laboratory of Environmentally Harmful Chemical Analysis, College of Chemistry, Beijing University of Chemical Technology, Beijing, 100029, China.
Center for Advanced Measurement Science, National Institute of Metrology, Beijing, 100029, China.
Anal Bioanal Chem. 2023 Aug;415(19):4795-4804. doi: 10.1007/s00216-023-04772-x. Epub 2023 Jun 9.
Here we proposed a method for peptide purity analysis using gas chromatography-isotope dilution infrared spectroscopy. The principle and feasibility of the proposed measurement method were investigated. The derivatization, separation, and infrared detection conditions for amino acids were optimized, and the performance of the method was investigated. Then, the proposed method was used for assessment of [Glu]-fibrinopeptide B purity, and the results were compared with those obtained by high performance liquid chromatography-isotope dilution mass spectrometry. The average purity of six sub-samples using the proposed method was (0.755 ± 0.017) g/g, which agreed well with that obtained by isotope dilution mass spectrometry (0.754 ± 0.012) g/g. The repeatability of the proposed method was 2.2%, which was similar to that of isotope dilution mass spectrometry (1.7%). The proposed method has a similar principle and had similar accuracy, precision, and linearity to isotope dilution mass spectrometry; however, the developed method had higher limit of detection (LOD) and limit of quantitation (LOQ) values because of the low sensitivity of infrared detection. The results were also Système International d'Unités (SI) traceable. The developed method has the advantage of lower cost compared with isotope dilution mass spectrometry because only one isotope-labeled atom in an analog is required, and several infrared spectra can be extracted, averaged, and used for an amino acid calculation during one run, potentially leading to higher accuracy. This method could be easily expanded to the accurate quantitation of other organic compounds, including proteins. It is expected that the proposed method will be widely used in chemical and biological measurements as a new primary method.
在这里，我们提出了一种使用气相色谱-同位素稀释红外光谱法分析肽纯度的方法。研究了所提出的测量方法的原理和可行性。优化了氨基酸的衍生化、分离和红外检测条件，并研究了该方法的性能。然后，将该方法用于评估[Glu]-纤维蛋白肽 B 的纯度，并将结果与高效液相色谱-同位素稀释质谱法的结果进行比较。所提出的方法对六个亚样品的平均纯度为（0.755±0.017）g/g，与同位素稀释质谱法（0.754±0.012）g/g 获得的结果吻合良好。所提出的方法的重复性为 2.2%，与同位素稀释质谱法（1.7%）相似。所提出的方法具有与同位素稀释质谱法相似的原理，具有相似的准确度、精密度和线性度；然而，由于红外检测灵敏度较低，开发的方法具有更高的检测限（LOD）和定量限（LOQ）值。结果也具有与国际单位制（SI）的可溯源性。与同位素稀释质谱法相比，所开发的方法具有成本优势，因为仅需要在类似物中标记一个同位素原子，并且可以在一次运行中提取、平均和使用几个红外光谱来计算一个氨基酸，从而可能具有更高的准确性。该方法可以很容易地扩展到其他有机化合物（包括蛋白质）的准确定量。预计该方法将作为一种新的基本方法，在化学和生物学测量中得到广泛应用。
