Establishment of the isotope dilution-gas chromatography-tandem mass spectrometry (ID-GC/MS) method for accurate quantification of phenylalanine and its application to value assignment of external quality assessment samples

IF 3.2 3区医学 Q2 MEDICAL LABORATORY TECHNOLOGY

Clinica Chimica Acta Pub Date : 2025-03-26 DOI:10.1016/j.cca.2025.120262

Zhizheng Li , Zhonggan Jin , Ming Zong

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引用次数: 0

Abstract

Objectives

Phenylalanine (Phe) was an essential amino acid that was crucial for diagnosing phenylketonuria (PKU) and managing their blood Phe levels. Currently, there was no JCTLM-recognized reference method for measuring Phe internationally. To standardize the measurement of serum Phe, our primary goal was to establish an accurate quantitative method based on isotope dilution combined with gas chromatography-mass spectrometry (ID-GC/MS) for the quantitative detection of Phe in human serum, and its performance was thoroughly validated.

Methods

Serum samples were pre-treated using acetonitrile protein precipitation. After derivatization with N-(Methyl)-N-(trimethylsilyl)trifluoroacetamide (MSTFA), serum Phe content was quantitatively detected using the ID-GC/MS method. Mass spectrometry analysis was conducted using the SIM monitoring mode. The analytical performance and application of the new method were investigated. External quality assessment (EQA) samples were tested using the established method, and their accuracy was compared with clinical laboratory results.

Results

The total detection time for Phe using the established method was 7 min. This method was capable of quantifying Phe within the range of 7.39 to 1188.81 μmol/L, with a limit of quantitation of 3.03 μmol/L. The intra-assay and inter-assay coefficients of variation (CV) were both ≤2.5 %, and the spike recovery rate ranged from 97.64 % to 100.72 %, and a relative expanded uncertainty was ≤3.0 % (k = 2). The method was free from interference, matrix effect and carry-over. Moreover, the ID-GC/MS measurement procedure was successfully applied to measure Phe in serum samples and to assign value of EQA samples.

Conclusions

This study successfully established an accurate quantitative method for detecting serum Phe concentrations using ID-GC/MS technology and it could support EQA management for Phe testing, providing traceable target values for samples.

查看原文本刊更多论文

同位素稀释-气相色谱-串联质谱法（ID-GC/MS）苯丙氨酸准确定量方法的建立及其在外部质量评价样品定值中的应用

目的：苯丙氨酸（Phe）是一种必需氨基酸，对诊断苯丙酮尿症（PKU）和控制其血液Phe水平至关重要。目前，国际上尚无jctlm认可的Phe测量参考方法。为了规范血清Phe的测定，我们的主要目的是建立一种基于同位素稀释结合气相色谱-质谱（ID-GC/MS）的准确定量检测人血清中Phe的方法，并对其性能进行了充分验证。方法：采用乙腈蛋白沉淀法对血清样品进行预处理。用N-（甲基）-N-（三甲基硅基）三氟乙酰胺（MSTFA）衍生后，采用ID-GC/MS法定量检测血清Phe含量。质谱分析采用SIM监测模式。研究了新方法的分析性能和应用。采用建立的方法对外部质量评价（EQA）样品进行检测，并与临床实验室结果进行准确性比较。结果：建立的方法对Phe的总检测时间为7 min。该方法定量范围为7.39 ~ 1188.81 μmol/L，定量限为3.03 μmol/L。测定内、间变异系数(CV)≤2.5 %，峰回收率为97.64 % ~ 100.72 %，相对扩展不确定度≤3.0 % （k = 2）。该方法不受干扰、矩阵效应和结转的影响。此外，还成功地将ID-GC/MS测定程序应用于血清样品中Phe的测定和EQA样品的定值。结论：本研究成功建立了一种利用ID-GC/MS技术准确定量检测血清Phe浓度的方法，可支持Phe检测的EQA管理，为样品提供可追溯的目标值。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Clinica Chimica Acta 医学-医学实验技术

CiteScore

10.10

自引率

2.00%

发文量

1268

审稿时长

23 days

期刊介绍： The Official Journal of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Clinica Chimica Acta is a high-quality journal which publishes original Research Communications in the field of clinical chemistry and laboratory medicine, defined as the diagnostic application of chemistry, biochemistry, immunochemistry, biochemical aspects of hematology, toxicology, and molecular biology to the study of human disease in body fluids and cells. The objective of the journal is to publish novel information leading to a better understanding of biological mechanisms of human diseases, their prevention, diagnosis, and patient management. Reports of an applied clinical character are also welcome. Papers concerned with normal metabolic processes or with constituents of normal cells or body fluids, such as reports of experimental or clinical studies in animals, are only considered when they are clearly and directly relevant to human disease. Evaluation of commercial products have a low priority for publication, unless they are novel or represent a technological breakthrough. Studies dealing with effects of drugs and natural products and studies dealing with the redox status in various diseases are not within the journal''s scope. Development and evaluation of novel analytical methodologies where applicable to diagnostic clinical chemistry and laboratory medicine, including point-of-care testing, and topics on laboratory management and informatics will also be considered. Studies focused on emerging diagnostic technologies and (big) data analysis procedures including digitalization, mobile Health, and artificial Intelligence applied to Laboratory Medicine are also of interest.