Analytical and Bioanalytical Chemistry ( IF 3.8 ) Pub Date : 2023-06-09 , DOI: 10.1007/s00216-023-04772-x Jiayi Song 1 , Dongmei Zhou 1 , Liqing Wu 2 , Ziliang Wang 3 , Xue Jiang 1 , Ping Su 1 , Yi Yang 1
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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 [Glu1]-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.
Graphical Abstract
中文翻译：
气相色谱-同位素稀释红外光谱法分析肽纯度的潜在主要方法
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。对氨基酸的衍生、分离和红外检测条件进行了优化，并考察了方法的性能。然后，采用该方法对[Glu 1 ]-纤维蛋白肽B纯度进行了评价，并将结果与高效液相色谱-同位素稀释质谱法的结果进行了比较。采用该方法得到的6个子样品的平均纯度为（0.755±0.017）g/g，与同位素稀释质谱法得到的平均纯度（0.754±0.012）g/g吻合较好。该方法的重复性为2.2%，与同位素稀释质谱法的重复性（1.7%）相似。该方法与同位素稀释质谱法原理相似，具有相似的准确度、精密度和线性度；然而，由于红外检测的灵敏度较低，所开发的方法具有较高的检测限（LOD）和定量限（LOQ）值。结果也可通过国际统一系统 (SI) 进行追踪。与同位素稀释质谱相比，该方法具有成本更低的优点，因为在类似物中只需要一个同位素标记的原子，并且可以在一次运行中提取、平均并用于氨基酸计算的多个红外光谱，潜在地从而带来更高的准确度。该方法可以轻松扩展到其他有机化合物（包括蛋白质）的精确定量。预计该方法将作为一种新的主要方法广泛应用于化学和生物测量。
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