顶空进样-双柱双检测器-气相色谱法分析人体血液中乙醇等12种挥发性化合物

doi:10.3724/SP.J.1123.2023.12015

色谱 ›› 2024, Vol. 42 ›› Issue (9): 909-917.DOI: 10.3724/SP.J.1123.2023.12015

顶空进样-双柱双检测器-气相色谱法分析人体血液中乙醇等12种挥发性化合物

郑琼英¹^,², 支瑜婕³, 段文佳², 吕敏³, 肖月³, 向平², 陈航²^,^*(), 贠克明¹^,^*()

1.山西医科大学法医学院,山西晋中 030600
2.司法鉴定科学研究院,司法部司法鉴定重点实验室, 上海市法医学重点实验室,上海市司法鉴定专业技术服务平台,上海市标准化创新中心(司法鉴定),上海 200063
3.中国药科大学药学院,江苏南京 210009

收稿日期:2023-12-14 出版日期:2024-09-08 发布日期:2024-08-29
通讯作者: *Tel:(021)52352955,E-mail:chenh@ssfjd.cn(陈航); E-mail:yunkeming5142@163.com(贠克明).
基金资助:
国家重点研发计划项目(2022YFC3302005-1);上海市法医学重点实验室(21DZ2270800)

A headspace injection double-column dual-detector gas chromatography system for the analysis of 12 volatile compounds such as ethanol in human blood

ZHENG Qiongying¹^,², ZHI Yujie³, DUAN Wenjia², LÜ Min³, XIAO Yue³, XIANG Ping², CHEN Hang²^,^*(), YUN Keming¹^,^*()

1. College of Forensic Medicine, Shanxi Medical University, Jinzhong 030600, China
2. Academy of Forensic Science, Key Laboratory of Forensic Science, Ministry of Justice, Shanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Shanghai Innovation Center of Standardization (Forensic Medicine), Shanghai 200063, China
3. School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China

Received:2023-12-14 Online:2024-09-08 Published:2024-08-29
Supported by:
National Key R&D Program of China(2022YFC3302005-1);Shanghai Key Laboratory of Forensic Medicine(21DZ2270800)

摘要/Abstract

摘要：

基于GB/T 42430-2023与GA/T 204-2019的技术方法,分别在两套不同的设备平台及色谱柱体系上建立了同时分析人体血液中乙醇等12种挥发性化合物的顶空进样-双柱双检测器-气相色谱分析方法。所建立方法中酮醇类、苯类物质色谱分离基本良好;乙醇等7种酮醇类化合物线性范围为0.10~3.00 g/L,线性相关系数(r)均大于0.997,检出限除正丙醇为0.005 g/L外,其余化合物均为0.05 g/L,定量限均为0.10 g/L,符合标准要求;苯等5种苯类化合物的线性范围为0.05~50 mg/L,r均大于0.995,检出限均为0.02 mg/L,定量限均为0.05 mg/L,符合标准要求;色谱柱J&W DB-BAC1 UI和色谱柱Rtx-BAC-PLUS 2上各化合物不同加标水平下的平均回收率为92.2%~111.6%,相对标准偏差(n=6)为0.4%~7.4%。对两套不同的设备平台及色谱柱体系上乙醇的测定进行不确定度评定,乙醇的不确定度主要来源于校准曲线,取置信概率95%(包含因子k=2),能力验证样品1在色谱柱J&W DB-BAC1 UI和色谱柱Rtx-BAC-PLUS 2的测定结果分别为(1.20±0.06) g/L和(1.20±0.07) g/L,样品2在色谱柱J&W DB-BAC1 UI和色谱柱Rtx-BAC-PLUS 2的测定结果分别为(0.78±0.04) g/L和(0.79±0.05) g/L。能力验证样本分析与z比分数评价展示了所建立方法的数据可靠性;甲醇及有机溶剂中毒患者样本分析结果表明,所建立方法适用于人体血液等体液中乙醇等7种酮醇类化合物和5种苯类化合物的高精度定量分析,可用于司法鉴定、中毒检测等实践场景。

关键词: 顶空进样, 双柱双检测器, 气相色谱法, 挥发性化合物, 人体血液, 标准化

Abstract:

Based on the technical methods of GB/T 42430-2023 and GA/T 204-2019, this study established an analytical method for headspace injection double-column dual-detector (hydrogen flame ion detector) gas chromatography for the simultaneous analysis of at least 12 volatile compounds, including ethanol, in human blood using two different equipment platforms and chromatographic columns. A 100 μL blood or urine sample and a 0.04 g/L tert-butanol working solution prepared as an internal standard are introduced into the headspace sample bottle and then sealed, mixed, and placed on the headspace sampler rack. Using different equipment platforms and columns, methodological parameters such as the limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy of the method were systematically evaluated. The chromatographic separation of acetone, alcohols and benzenes using the established method was satisfactory. The linear ranges, linear correlation coefficients (r), and LODs of acetone and six alcohols, including ethanol, were 0.10-3.00 g/L, >0.997, and 0.05 g/L, respectively. The LOQs were 0.10 g/L for all other compounds, excluding n-propanol (0.005 g/L). Additionally, the linear ranges, r values, LODs, and LOQs of benzene and four benzene derivatives were 0.05-50 mg/L, >0.995, 0.02 mg/L, and 0.05 mg/L, respectively (Column J&W DB-BAC1 UI and Column Rtx-BAC-PLUS 2). The average recoveries of compounds on J&W DB-BAC1 UI and Rtx-BAC-PLUS 2 columns ranged from 92.2% to 111.6%, and the relative standard deviations (RSDs, n=6) ranged from 0.4% to 7.4%. The LOD, LOQ, precision, accuracy, and linearity of the established method met the requirements of relevant standards, and no significant differences arose between the methodological parameters of the two platforms. CNAS-GL006 (2019) and JJF 1059.1-2012 were used as guides to evaluate the uncertainty of ethanol on two different sets of equipment platforms and chromatographic columns. The ethanol uncertainty was mainly derived from the calibration curve; however, the confidence probability was 95% (k=2). According to the analysis of the verification samples and real samples, the established method is suitable for the high-precision quantitative analysis of acetone and six alcohols and five benzene derivatives in human blood and other body fluids. It can be used in practical scenarios such as judicial identification and the detection of poisons.

Key words: headspace injection (HS), double-column dual-detector, gas chromatography (GC), volatile compounds, human blood, standardization

中图分类号:

O658

郑琼英, 支瑜婕, 段文佳, 吕敏, 肖月, 向平, 陈航, 贠克明. 顶空进样-双柱双检测器-气相色谱法分析人体血液中乙醇等12种挥发性化合物[J]. 色谱, 2024, 42(9): 909-917.

ZHENG Qiongying, ZHI Yujie, DUAN Wenjia, LÜ Min, XIAO Yue, XIANG Ping, CHEN Hang, YUN Keming. A headspace injection double-column dual-detector gas chromatography system for the analysis of 12 volatile compounds such as ethanol in human blood[J]. Chinese Journal of Chromatography, 2024, 42(9): 909-917.

图/表 6

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Compound	Level^#	Column a-Ⅰ		Column b-Ⅱ
Compound	Level^#	Recovery/%	RSD/%	Recovery/%	RSD/%
Methanol	0.20	100.0	5.2	106.2	4.7
	0.80	98.6	1.2	101.4	1.3
	2.00	96.7	3.7	99.6	1.8
	3.00	100.7	2.3	98.9	1.3
Ethanol	0.20	109.1	1.6	107.3	3.9
	0.80	96.7	0.4	101.1	1.6
	2.00	92.2	2.6	100.3	2.3
	3.00	100.7	1.0	99.0	0.5
Isopropanol	0.20	99.5	3.2	106.1	1.5
	0.80	97.4	2.0	99.7	0.6
	2.00	97.0	1.0	101.2	1.2
	3.00	99.4	1.6	100.0	0.9
Acetone	0.20	111.6	1.2	107.1	0.8
	0.80	98.9	1.0	96.0	4.4
	2.00	97.2	1.3	99.7	1.9
	3.00	100.7	1.7	98.9	2.2
N-Propanol	0.20	106.3	5.8	110.8	1.4
	0.80	98.8	1.9	101.0	0.8
	2.00	95.2	0.9	97.9	4.4
	3.00	102.8	0.9	99.4	0.4
Isobutanol	0.20	105.6	7.4	111.2	0.5
	0.80	97.8	1.4	101.2	1.0
	2.00	96.2	2.1	98.1	3.3
	3.00	102.3	0.9	99.6	0.9
N-Butanol	0.20	105.4	1.2	107.9	3.3
	0.80	97.9	1.1	100.6	0.4
	2.00	93.6	0.7	101.1	2.1
	3.00	101.3	0.4	99.9	0.7
Benzene	0.50	102.8	1.7	98.9	1.9
	1.00	95.8	4.2	99.6	3.2
	5.00	100.1	1.2	98.5	1.2
	25.00	98.7	1.2	98.5	1.0
Toluene	0.50	98.7	2.4	105.1	0.8
	1.00	101.7	2.6	103.2	2.3
	5.00	96.6	1.9	97.8	1.3
	25.00	99.7	1.8	99.8	1.7
p-Xylene	0.50	100.7	2.5	100.2	2.0
	1.00	101.0	4.0	94.2	4.0
	5.00	97.5	1.2	97.2	1.8
	25.00	101.0	3.3	101.7	2.4
m-Xylene	0.50	96.6	2.0	95.8	1.2
	1.00	100.0	4.7	97.5	1.0
	5.00	99.7	0.5	98.8	1.1
	25.00	98.1	2.4	101.8	2.5
o-Xylene	0.50	100.2	3.0	100.9	2.7
	1.00	106.4	3.0	99.2	2.5
	5.00	95.2	1.2	96.5	1.1
	25.00	98.9	4.5	101.2	1.2

Compound	Level^#	Column a-Ⅰ		Column b-Ⅱ
Compound	Level^#	Recovery/%	RSD/%	Recovery/%	RSD/%
Methanol	0.20	100.0	5.2	106.2	4.7
	0.80	98.6	1.2	101.4	1.3
	2.00	96.7	3.7	99.6	1.8
	3.00	100.7	2.3	98.9	1.3
Ethanol	0.20	109.1	1.6	107.3	3.9
	0.80	96.7	0.4	101.1	1.6
	2.00	92.2	2.6	100.3	2.3
	3.00	100.7	1.0	99.0	0.5
Isopropanol	0.20	99.5	3.2	106.1	1.5
	0.80	97.4	2.0	99.7	0.6
	2.00	97.0	1.0	101.2	1.2
	3.00	99.4	1.6	100.0	0.9
Acetone	0.20	111.6	1.2	107.1	0.8
	0.80	98.9	1.0	96.0	4.4
	2.00	97.2	1.3	99.7	1.9
	3.00	100.7	1.7	98.9	2.2
N-Propanol	0.20	106.3	5.8	110.8	1.4
	0.80	98.8	1.9	101.0	0.8
	2.00	95.2	0.9	97.9	4.4
	3.00	102.8	0.9	99.4	0.4
Isobutanol	0.20	105.6	7.4	111.2	0.5
	0.80	97.8	1.4	101.2	1.0
	2.00	96.2	2.1	98.1	3.3
	3.00	102.3	0.9	99.6	0.9
N-Butanol	0.20	105.4	1.2	107.9	3.3
	0.80	97.9	1.1	100.6	0.4
	2.00	93.6	0.7	101.1	2.1
	3.00	101.3	0.4	99.9	0.7
Benzene	0.50	102.8	1.7	98.9	1.9
	1.00	95.8	4.2	99.6	3.2
	5.00	100.1	1.2	98.5	1.2
	25.00	98.7	1.2	98.5	1.0
Toluene	0.50	98.7	2.4	105.1	0.8
	1.00	101.7	2.6	103.2	2.3
	5.00	96.6	1.9	97.8	1.3
	25.00	99.7	1.8	99.8	1.7
p-Xylene	0.50	100.7	2.5	100.2	2.0
	1.00	101.0	4.0	94.2	4.0
	5.00	97.5	1.2	97.2	1.8
	25.00	101.0	3.3	101.7	2.4
m-Xylene	0.50	96.6	2.0	95.8	1.2
	1.00	100.0	4.7	97.5	1.0
	5.00	99.7	0.5	98.8	1.1
	25.00	98.1	2.4	101.8	2.5
o-Xylene	0.50	100.2	3.0	100.9	2.7
	1.00	106.4	3.0	99.2	2.5
	5.00	95.2	1.2	96.5	1.1
	25.00	98.9	4.5	101.2	1.2

Item	Relative standard uncertainty components
	Source	Symbol	Numeric values
	Source	Symbol	Column a-Ⅰ	Column b-Ⅱ
A	determination repeatability	u_rel(A)	3.59×10^-3	5.03×10^-3
B₁	standard curve fitting	u_rel(B₁)	2.14×10^-2	2.30×10^-2
B₂	standard solution	u_rel(B₂)	2.49×10^-3	2.49×10^-3
B₃	pipette (50-250 μL)	u_rel(V₁₀₀)	4.08×10^-3	4.08×10^-3
B₃	pipette (100-1000 μL)	u_rel(V₅₀₀)	2.04×10^-3	2.04×10^-3
B₄	instrumental determination	u_rel(GC)	5.00×10^-3	5.00×10^-3

Item	Relative standard uncertainty components
	Source	Symbol	Numeric values
	Source	Symbol	Column a-Ⅰ	Column b-Ⅱ
A	determination repeatability	u_rel(A)	3.59×10^-3	5.03×10^-3
B₁	standard curve fitting	u_rel(B₁)	2.14×10^-2	2.30×10^-2
B₂	standard solution	u_rel(B₂)	2.49×10^-3	2.49×10^-3
B₃	pipette (50-250 μL)	u_rel(V₁₀₀)	4.08×10^-3	4.08×10^-3
B₃	pipette (100-1000 μL)	u_rel(V₅₀₀)	2.04×10^-3	2.04×10^-3
B₄	instrumental determination	u_rel(GC)	5.00×10^-3	5.00×10^-3

Sample	Column a-Ⅰ			Column b-Ⅱ
Sample	Compound	Assay result/(g/L)	RD/%	Compound	Assay result/(g/L)	RD/%
Blood	methanol	0.83	1.1	methanol	0.87	3.1
	ethanol	0.48	0.40	ethanol	0.52	2.8
	acetone	+	+	acetone	+	+
Urine	methanol	0.78	0.58	methanol	0.83	2.8
	ethanol	0.20	2.1	ethanol	0.24	0.16
	acetone	-	-	acetone	-	-

顶空进样-双柱双检测器-气相色谱法分析人体血液中乙醇等12种挥发性化合物

A headspace injection double-column dual-detector gas chromatography system for the analysis of 12 volatile compounds such as ethanol in human blood

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Metrics

Column	Compound	Linear range	Linear equation	r	LOD	LOQ
a-Ⅰ	methanol	0.10-3.00	y=0.6794x-0.0399	0.9992	0.05	0.10
	ethanol	0.10-3.00	y=0.9193x-0.0762	0.9990	0.05	0.10
	isopropanol	0.10-3.00	y=1.5593x-0.127	0.9992	0.05	0.10
	acetone	0.10-3.00	y=2.8565x-0.1656	0.9995	0.05	0.10
	N-propanol	0.10-3.00	y=2.2476x-0.1746	0.9986	0.005	0.10
	isobutanol	0.10-3.00	y=2.4088x-0.196	0.9992	0.05	0.10
	N-butanol	0.10-3.00	y=2.0575x-0.0503	0.9994	0.05	0.10
	benzene	0.05-50	y=1.1112x-3.5888	0.9991	0.02	0.05
	toluene	0.05-50	y=0.7995x-3.0549	0.9984	0.02	0.05
	p-xylene	0.05-50	y=1.121x-3.9122	0.9991	0.02	0.05
	m-xylene	0.05-50	y=1.0954x-3.7899	0.9990	0.02	0.05
	o-xylene	0.05-50	y=0.7076x-3.4284	0.9987	0.02	0.05
b-Ⅱ	methanol	0.10-3.00	y=0.7166x-0.0573	0.9986	0.05	0.10
	ethanol	0.10-3.00	y=1.4533x-0.1259	0.9988	0.05	0.10
	isopropanol	0.10-3.00	y=3.7507x-0.2053	0.9994	0.05	0.10
	acetone	0.10-3.00	y=3.8073x-0.2351	0.9992	0.05	0.10
	N-propanol	0.10-3.00	y=2.6454x-0.2184	0.9994	0.005	0.10
	isobutanol	0.10-3.00	y=4.2931x-0.4383	0.9989	0.05	0.10
	N-butanol	0.10-3.00	y=2.9704x-0.2727	0.9991	0.05	0.10
	benzene	0.05-50	y=0.9121x-3.6015	0.9989	0.02	0.05
	toluene	0.05-50	y=1.1417x-4.0097	0.9986	0.02	0.05
	p-xylene	0.05-50	y=0.7722x-3.6543	0.9988	0.02	0.05
	m-xylene	0.05-50	y=0.9910x-3.4859	0.9986	0.02	0.05
	o-xylene	0.05-50	y=1.1091x-4.3205	0.9979	0.02	0.05

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