UPLC-Q/Orbitrap HRMS测定口服液中6种西地那非衍生物的不确定度分析

张申平; 侯超; 张妮; 杜茹芸; 周静; 刘洋

doi:10.13386/j.issn1002-0306.2022060278

UPLC-Q/Orbitrap HRMS测定口服液中6种西地那非衍生物的不确定度分析

doi: 10.13386/j.issn1002-0306.2022060278

上海市质量监督检验技术研究院，国家市场监管重点实验室（乳及乳制品检测与监控技术），上海 200233

基金项目: 上海市食品质量安全检测与评价专业技术服务平台（18DZ2292400）。

详细信息

作者简介:
张申平（1992−），男，博士，工程师，研究方向：高分辨质谱技术应用，E-mail：zhangsp1@sqi.org.cn

通讯作者:
杜茹芸（1982−），女，博士，正高级工程师，研究方向：食品化妆品安全检测研究，E-mail：dury@sqi.org.cn

中图分类号: TS207.3
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- 文章访问数: 80
- HTML全文浏览量: 20
- PDF下载量: 6
- 被引次数: 0
出版历程
- 收稿日期: 2022-06-28
- 网络出版日期: 2023-04-18
- 刊出日期: 2023-06-01

Uncertainty Evaluation for Determination of 6 Sildenafil Derivatives in Oral Liquid by UPLC-Q/Orbitrap HRMS

Shanghai Institute of Quality Inspection and Technical Research, Key Laboratory of Milk and Dairy Products Detection and Monitoring Technology for State Market Regulation, Shanghai 200233, China

摘要

摘要: 为了保证高分辨质谱法测定壮阳类口服液中西地那非类似物的结果准确性和可靠性，对测定结果进行不确定度评定是十分必要的。本研究采用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱法（UPLC-Q/Orbitrap HRMS）测定口服液保健食品中6种西地那非衍生物的含量，依据JJF 1059.1-2012《测量不确定度评定与表示》，建立数学模型，进行测量不确定度评定。当口服液中N-去甲基西地那非、去甲基硫代西地那非、二甲基西地那非、羟基硫代豪莫西地那非、硫代豪莫西地那非、硫代西地那非的含量为0.90、1.04、1.22、2.01、1.51、1.14 mg/kg时，扩展不确定度分别为0.07、0.09、0.10、0.16、0.13、0.09 mg/kg（k=2）。评定结果表明，不确定度主要来自UPLC-Q/Orbitrap HRMS仪器、标准溶液配制、回收率和随机效应。
- UPLC-Q/Orbitrap HRMS /
- 口服液 /
- 西地那非衍生物 /
- 不确定度 /
- 扩展不确定度
Abstract: In order to ensure the accuracy and reliability of high resolution mass spectrometry for the determination of sildenafil derivatives in aphrodisiac oral liquid, it is necessary to evaluate the uncertainty of the determination results. In this study, the content of 6 sildenafil derivatives in oral liquid health food was determined by ultra-high performance liquid chromatography with quadrupole/electrostatic field Orbitrap high resolution mass spectrometry (UPLC-Q/Orbitrap HRMS). The mathematical model was established and the measurement uncertainty was evaluated according to JJF 1059.1-2012 “Evaluation and Expression of Measurement Uncertainty”. When the contents of N-demethyl sildenafil, dimethyl thiosildenafil, dimethyl sildenafil, hydroxythiocarmo sildenafil, thiocarmo sildenafil and thiosildenafil in oral solution were 0.90, 1.04, 1.22, 2.01, 1.51 and 1.14 mg/kg, the extended uncertainties were 0.07, 0.09, 0.10, 0.16, 0.13 and 0.09 mg/kg (k=2), respectively. The results show that the uncertainty mainly comes from UPLC-Q/Orbitrap HRMS, standard solution preparation, recovery and random effect.
- UPLC-Q/Orbitrap HRMS /
- oral liquid /
- sildenafil derivatives /
- uncertainty /
- the expanded uncertainty

HTML全文

图 1 6种西地那非衍生物定量离子的提取离子色谱图

Figure 1. The extracted ion chromatogram of quantitative ions of six sildenafil derivatives

注：a：N-去甲基西地那非；b：去甲基硫代西地那非；c：二甲基西地那非；d：羟基硫代豪莫西地那非；e：硫代豪莫西地那非；f：硫代西地那非。

下载: 全尺寸图片幻灯片

图 2 口服液中西地那非衍生物测定不确定度来源分析

Figure 2. Analysis of source of uncertainty in sildenafil derivatives of oral liquid

下载: 全尺寸图片幻灯片

图 3 口服液中6种西地那非衍生物的不确定度分量

Figure 3. Uncertainty components of six sildenafil derivatives in oral liquid

下载: 全尺寸图片幻灯片

表 1 梯度洗脱程序

Table 1. Gradient elution program

时间（min）	流速（mL/min）	A：0.1%甲酸/水（%）	B：0.1%甲酸/乙腈（%）
0	0.3	90	10
2	0.3	90	10
8	0.3	10	90
10	0.3	10	90
11	0.3	90	10
14	0.3	90	10

下载: 导出CSV

表 2 6种西地那非衍生物的质谱参数

Table 2. Mass spectrometry parameters of 6 sildenafil derivatives

化合物	分子式	保留时间（min）	母离子（m/z）	碎片离子（m/z）	加合方式
N-去甲基西地那非	C₂₁H₂₈N₆O₄S	5.94	461.19626	283.11868^*，255.12376	[M+H]⁺
去甲基硫代西地那非	C₂₁H₂₈N₆O₃S₂	6.69	477.17288	299.09586^*，315.09033	[M+H]⁺
二甲基西地那非	C₂₃H₃₂N₆O₄S	6.05	489.22803	283.1192^*，344.14856	[M+H]⁺
羟基硫代豪莫西地那非	C₂₃H₃₂N₆O₄S₂	6.65	521.19958	299.09589^*，327.12759	[M+H]⁺
硫代豪莫西地那非	C₂₃H₃₂N₆O₃S₂	6.81	505.20401	299.09607^*，113.10741	[M+H]⁺
硫代西地那非	C₂₂H₃₀N₆O₃S₂	6.72	491.18918	341.14389^*，407.12027	[M+H]⁺
注：*为定量离子。

下载: 导出CSV

表 3 标准物质的纯度及不确定度

Table 3. Purity and uncertainty of standard materials

标准物质	纯度（%）	扩展不确定度U_p（%）	包含因子k	标准不确定度（%）	相对标准不确定度（%）
N-去甲基西地那非	99.9	0.5	2	0.25	0.00250
去甲基硫代西地那非	99.9	0.5	2	0.25	0.00250
二甲基西地那非	99.8	0.5	2	0.25	0.00250
羟基硫代豪莫西地那非	98.5	0.5	2	0.25	0.00254
硫代豪莫西地那非	99.6	0.5	2	0.25	0.00251
硫代西地那非	98.0	0.5	2	0.25	0.00255

下载: 导出CSV

表 4 标准溶液稀释过程中由量器引入的相对不确定度

Table 4. Relative uncertainty introduced by instruments during standard solution dilution

量器规格（次数）	最大允差	标准不确定度	单次相对标准不确定度
200 µL移液器（5次）	±3.0 µL	\setlength{\voffset}{0.5pt}$ \dfrac{3.0}{\sqrt{6}} $	\setlength{\voffset}{0.5pt}$ \dfrac{3.0}{200\times \sqrt{6}}=0.00612 $
1000 µL移液器（6次）	±10.0 µL	$ \dfrac{10.0}{\sqrt{6}} $	$ \dfrac{10.0}{1000\times \sqrt{6}}=0.00408 $
5 mL A级容量瓶（1次）	±0.020 mL	$ \dfrac{0.020}{\sqrt{6}} $	$ \dfrac{0.020}{5\times \sqrt{6}}=0.00163 $
10 mL A级容量瓶（5次）	±0.020 mL	$ \dfrac{0.020}{\sqrt{6}} $	$ \dfrac{0.020}{10\times \sqrt{6}}=0.000816 $

下载: 导出CSV

表 5 最小二乘法拟合标准曲线

Table 5. Standard curve data by fitting least square curve

标准曲线浓度c（µg/L）	平均峰面积y	线性回归方程	线性相关系数r
5	4421764	y=872031c+331764	1
10	8835701
50	44072074
100	88264579
200	174355919

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表 6 不确定度分量汇总及6种西地那非衍生物测定不确定度评定结果

Table 6. Summary of each uncertainty component and uncertainty evaluation result for determination of six sildenafil derivatives

不确定度分量	N-去甲基西地那非	去甲基硫代西地那非	二甲基西地那非	羟基硫代豪莫西地那非	硫代豪莫西地那非	硫代西地那非
$ {\mathrm{u}}_{\mathrm{r}\mathrm{e}\mathrm{l}}\left(\mathrm{s}\mathrm{t}\mathrm{d}\right) $	0.00250	0.00250	0.00250	0.00254	0.00251	0.00255
$ {\mathrm{u}}_{\mathrm{r}\mathrm{e}\mathrm{l}}\left({\mathrm{V}}_{5}\right) $	0.00188
$ {\mathrm{u}}_{\mathrm{r}\mathrm{e}\mathrm{l}}\left(\mathrm{V}\right) $	0.01398
$ {\mathrm{u}}_{\mathrm{r}\mathrm{e}\mathrm{l}}\left(\mathrm{c}\right) $	0.00481	0.00445	0.00837	0.00669	0.00441	0.00343
$ {\mathrm{u}}_{\mathrm{r}\mathrm{e}\mathrm{l}}\left(\mathrm{m}\right) $	0.00082
$ {\mathrm{u}}_{\mathrm{r}\mathrm{e}\mathrm{l}}\left(\mathrm{M}\mathrm{S}\right) $	0.035
$ {\mathrm{u}}_{\mathrm{r}\mathrm{e}\mathrm{l}}\left(\mathrm{R}\right) $	0.00881	0.01445	0.00837	0.00969	0.01041	0.00343
$ {\mathrm{u}}_{\mathrm{r}\mathrm{e}\mathrm{l}}\left(\mathrm{A}\right) $	0.00666	0.00840	0.00587	0.00669	0.01441	0.00531
合成相对不确定度	0.03970	0.04159	0.04007	0.04018	0.04203	0.03851
实测值（mg/kg）	0.90	1.04	1.22	2.01	1.51	1.14
扩展不确定度U（k=2，mg/kg）	0.07	0.09	0.10	0.16	0.13	0.09
测定结果（k=2，mg/kg）	0.90±0.07	1.04±0.09	1.22±0.10	2.01±0.16	1.51±0.13	1.14±0.09

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