基于热解吸-气相色谱质谱法测定呼出气中27种挥发性有机物浓度
Determination of 27 volatile organic compounds in exhaled breath by thermal desorption-gas chromatography-mass spectrometry
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摘要:
背景 呼出气中的挥发性有机物（VOCs）与呼吸系统疾病密切相关，与体内多种代谢反应存在联系，建立其分析方法可为疾病中VOCs的研究提供技术支持。
目的 建立呼出气中27种VOCs的热解吸-气相色谱质谱（TD-GC-MS）检测方法。
方法 呼出气中VOCs使用Bio-VOC呼出气采样器进行采集，随后用Tenax TA热解吸管富集后进行TD-GC-MS分析。使用Tenax TA 60/80热解吸管采集标准气，对TD-GC-MS检测方法的热解吸条件和色谱、质谱条件进行优化：通过优化升温程序实现27种VOCs的分离，通过优化定量离子提高灵敏度，通过对热解吸时间和温度的优化提高VOCs解吸效率。采用空白气袋加标的方式对方法的检出限、定量限、准确度、精密度及稳定性进行检测，并采集20名健康志愿者呼出气样品进行方法的应用考察。
结果 本研究热解吸温度为280 ℃，解吸时间为6 min，选择VF-624ms色谱柱进行VOCs的分离，色谱柱升温程序：初始温度35 ℃，保持1 min；以3 ℃·min−1升至100 ℃保持1 min；以28 ℃·min−1升至210 ℃并保持5 min，选择单离子检测扫描（SIM）模式进行27种VOCs的定量分析。此检测条件下，27种VOCs在各自测定范围内线性良好，相关系数均>0.9990，方法检出限范围为0.01~0.13 nmol·mol−1，定量限范围为0.02~0.44 nmol·mol−1，方法加标回收率范围为80.1%~120.5%，批内、批间精密度分别≤18.8%、17.9%，所有VOCs在常温（23 ~28 ℃）条件下可保存7 d，在4 ℃条件下可保存14 d。应用本法对20名受试者呼出气样本进行检测，检出率≥80%（除反式-2-戊烯和癸烷），检出浓度范围为0.00~465.50 nmol·mol−1。
结论 本研究建立的呼出气中挥发性有机物TD-GC-MS定量法，具有灵敏度高、准确度好的特点，适用于呼出气中VOCs的定量检测，可为呼出气VOCs研究提供技术支持。
Abstract:Background Volatile organic compounds (VOCs) in exhaled breath are closely associated with respiratory diseases and are linked to various metabolic reactions in the human body. A quantitative analytical method can provide technical support for studying VOCs related to various diseases.
Objective To establish a thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) method for the determination of 27 VOCs in exhaled breath.
Methods VOCs in exhaled breath were collected using a Bio-VOC sampler and enriched with Tenax TA thermal desorption tubes before TD-GC-MS analysis. Standards were collected using thermal desorption tubes and optimized for thermal desorption conditions as well as chromatographic and mass spectrometric conditions: The separation of the 27 VOCs was achieved by an optimized temperature program, the improvement of sensitivity by optimizing quantitative ions, and the increase of VOCs desorption efficiency by optimizing thermal desorption time and temperature. Limit of detection, limit of quantification, accuracy, precision, and stability of the proposed method were investigated by spiking with a blank gas bag, and exhaled breath samples from 20 healthy individuals were collected for an application study of the proposed method.
Results The thermal desorption temperature was 280 ℃, and desorption time was 6 min. A VF-624ms chromatographic column was selected for the separation of target substances. The initial temperature of heating program was 35 ℃, maintained for 1 min, and then increased to 100 ℃ at a heating rate of 3 ℃·min−1 for 1 min, followed by increasing to 210 ℃ at a heating rate of 28 ℃·min−1 for 5 min. A quantitative analysis was conducted with a single ion monitoring (SIM) mode. Under these conditions, the 27 VOCs showed good linear relationships in their respective concentration ranges and the correlation coefficients were higher than 0.9990. The limits of detection of the method were in the range of 0.01-0.13 nmol·mol−1, the limits of quantification were in the range of 0.02-0.44 nmol·mol−1, and the spiked recoveries were in the range of 80.1%-120.5%, with intra-batch and inter-batch precision ≤ 18.8% and 17.9% respectively. All substances can be stored at room temperature (23-28 °C) for 7 d and at 4 °C for 14 d. The proposed method was applied to exhaled breath samples from 20 subjects with detection rates≥ 80% (except for trans-2-pentene and decane) and a concentration range of 0.00-465.50 nmol·mol−1.
Conclusion The established TD-GC-MS method for quantification of VOCs in exhaled breath is characterized by high sensitivity and good accuracy, and is suitable for quantitative determination of VOCs in exhaled breath, which can provide technical support for the study of exhaled breath VOCs.
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