伏马毒素B<sub>1</sub>纯度标准物质的研制

郭文博; 孟佳佳; 牛雪珂; 王欣艺; 聂冬霞; 赵志辉; 韩铮

doi:10.12338/j.issn.2096-9015.2023.0284

伏马毒素B₁纯度标准物质的研制

doi: 10.12338/j.issn.2096-9015.2023.0284

郭文博^{1, 2,},
孟佳佳¹,
牛雪珂^{1, 2},
王欣艺¹,
聂冬霞¹,
赵志辉¹,
韩铮^1, ,

1.
上海市农业科学院，上海 201403
2.
上海科立特农产品检测技术服务有限公司，上海 201403

基金项目: 上海市科技兴农技术创新项目（沪农科创字（2012）第3-2号）；上海市“科技创新行动计划”启明星项目（21QB1403300）。

详细信息

作者简介:
郭文博（1988-），上海市农业科学院助理研究员，研究方向：真菌毒素标准物质研制与应用，邮箱：guo1103bo@126.com

通讯作者:
韩铮（1983-），上海市农业科学院研究员，研究方向：农产品质量安全，邮箱：hanzheng@saas.sh.cn

中图分类号: TB99
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出版历程
- 收稿日期: 2023-11-17
- 录用日期: 2023-11-20
- 修回日期: 2023-11-29
- 网络出版日期: 2023-12-06

Development of a Certified Reference Material for Fumonisin B₁ Purity

GUO Wenbo^{1, 2
,},
MENG Jiajia¹,
NIU Xueke^{1, 2},
WANG Xinyi¹,
NIE Dongxia¹,
ZHAO Zhihui¹,
HAN Zheng^{1
, ,}

1.
Shanghai Academy of Agricultural Sciences, Shanghai 201403, China
2.
Shanghai Kelite Agricultural Product Testing Technology Service Co., Ltd., Shanghai 201403, China

摘要

摘要: 为更好的满足农产品质量安全检测需求，保证检测结果的准确性、可靠性和可溯源性，研制了伏马毒素B₁（FB₁）纯度标准物质。以实验室自主分离纯化获得的FB₁为候选物，通过质谱、红外光谱、核磁共振谱等对其进行定性鉴定。采用质量平衡法和定量核磁法两种不同原理的方法对候选物的纯度进行定值，其中质量平衡法中采用面积归一化法、卡尔费休法、气相色谱串联质谱法和电感耦合等离子体质谱法，分别对候选物中主成分（FB₁）、水分、挥发性杂质和非挥发性杂质含量进行测定。此外，开展了均匀性检验、稳定性考察，并对研制过程中产生的不确定度进行了系统的评定。结果表明：FB₁纯度标准物质量值为99.1%，扩展不确定度为0.3%（k=2），均匀性良好，且满足12个月的稳定性要求。研制的FB₁纯度标准物质获得国家二级标准物质证书，编号为GBW（E）100550，能够用于农产品中FB₁的定性和定量检测、方法评价以及FB₁量值溯源体系的建立。
- 计量学 /
- 伏马毒素B₁ /
- 纯度标准物质 /
- 质量平衡法 /
- 定量核磁法 /
- 不确定度评定
Abstract: To enhance the accuracy, reliability, and traceability of agricultural product quality safety testing, a certified reference material (CRM) for fumonisin B₁ (FB₁) purity was developed. The CRM candidate, obtained through laboratory purification of FB₁, was qualitatively identified using mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The purity of the candidate was quantified using both mass balance and quantitative nuclear magnetic resonance methods. The mass balance method included area normalization, the Karl Fischer method, gas chromatography-tandem mass spectrometry, and inductively coupled plasma mass spectrometry to determine the main component, moisture, volatile and non-volatile impurity contents. Furthermore, homogeneity tests and stability studies were conducted, along with systematic uncertainty evaluations. The results indicated that the FB₁ CRM has a purity value of 99.1% with an extended uncertainty of 0.3% (k=2), exhibiting good homogeneity and fulfilling a 12-month stability requirement. The developed FB₁ CRM, certified as a national secondary standard material (GBW (E) 100550), is suitable for qualitative and quantitative detection, method evaluation, and establishing a traceability system for FB₁ in agricultural products
- metrology /
- fumonisin B₁ /
- purity certified reference material /
- mass balance method /
- quantitative nuclear magnetic method /
- uncertainty evaluation

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图 1 FB₁标准物质候选物的红外光谱图

Figure 1. Infrared spectroscopy of FB₁ certified reference material candidate

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图 2 FB₁标准物质候选物的高分辨质谱图

Figure 2. Time-of-flight mass spectrometry (TOF/MS-MS) of the FB₁ certified reference material candidate

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图 3 FB₁标准物质候选物的UHPLC-CAD色谱图

Figure 3. Ultra-high performance liquid chromatography with charged aerosol detection (UHPLC-CAD) chromatograms of the FB₁ certified reference material candidate

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图 4 FB₁标准物质候选物和苯甲酸混合样品在氘代甲醇中的¹H-NMR 谱图

Figure 4. Proton nuclear magnetic resonance (¹H-NMR) spectra of mixed samples of FB₁ certified reference material candidate and benzoic acid in deuterated methanol

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表 1 FB₁标准物质候选物的质量平衡法定值结果

Table 1. Quantitative determination of the FB₁ certified reference material candidate using the mass balance method (%)

项目	结果	平均值	标准偏差
主成分纯度	99.31	99.31	0.031
	99.29
	99.32
	99.31
	99.29
	99.31
水分含量	0.143	0.139	0.003
	0.136
	0.134
	0.141
	0.143
	0.137
无机离子杂质	0.043	0.050	0.005
	0.057
	0.051
	0.044
	0.055
	0.050
纯度	99.12

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表 2 FB₁物质候选物均匀性数据与检验结果

Table 2. Data analysis and homogeneity testing (F-test) of the FB₁ certified reference material candidate (%)

瓶号	1		2	3
1	99.35		99.34	99.31
2	99.35		99.33	99.34
3	99.29		99.29	99.32
4	99.34		99.28	99.31
5	99.32		99.27	99.31
6	99.30		99.32	99.29
7	99.26		99.32	99.34
8	99.28		99.31	99.32
9	99.29		99.32	99.33
10	99.30		99.29	99.25
11	99.34		99.29	99.31
12	99.34		99.28	99.37
13	99.34		99.29	99.31
14	99.29		99.31	99.32
15	99.32		99.31	99.28
平均值		99.31
组间方差（M_between）		0.000671
组内方差（M_within）		0.000667
F (M_between/M_within)		1.01
F_0.05（14, 30）		2.04

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表 3 FB₁物质候选物稳定性数据与检验结果

Table 3. Analysis of short-term and long-term stability (t-test) of the FB₁ certified reference material candidate (%)

时间（天）	短期稳定性			时间（月）	长期稳定性
时间（天）	−20 ℃	4 ℃	60 ℃	时间（月）	−20 ℃
0	99.34	99.31	99.29	0	99.32
2	99.31	99.29	99.31	1	99.29
4	99.32	99.33	99.32	3	99.31
6	99.29	99.30	99.31	6	99.33
8	99.31	99.31	99.30	12	99.29
β₁	−0.0040	0.0005	0.0010	β₁	−0.0010
β₀	99.330	99.306	99.302	β₀	99.313
s	0.0151	0.0170	0.0126	s	0.0198
s(β₁)	0.0024	0.0027	0.0020	s(β₁)	0.0021
t_{0.95, 3}	3.18	3.18	3.18	t_{0.95, 3}	3.18
s(β₁)·t_0.95,3	0.0076	0.0086	0.0064	s(β₁)·t_{0.95, 3}	0.0065

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表 4 FB₁标准物质候选物不确定度评定结果

Table 4. Evaluation of uncertainties associated with the developed FB₁ certified reference material candidate

来源	公式	不确定度/%
均匀性（u_bb）	$ {{u}}_{{{\mathrm{bb}}}}{=}\sqrt{\dfrac{{{M}}_{{{\mathrm{between}}}}{-}{{M}}_{{{\mathrm{within}}}}}{{n}}} $	0.0012
长期稳定性（u_lst）	$ {{u}}_{{{\mathrm{lts}}}}={s(}{{\beta }}_{{1}}{)}\times {t} $	0.0247
短期稳定性（u_sts）	$ {{u}}_{{{\mathrm{sts}}}}={s(}{{\beta }}_{{1}}{)}\times {t} $	0.0215
质量平衡法（u_MB）	$ {{u}}_{{{\mathrm{MB}}}}{}{=}{P}_{{{\mathrm{MB}}}}\times \sqrt{{\left[\dfrac{{u}\left({{P}}_{{0}}\right)}{{{P}}_{{0}}}\right]}^{{2}}{+}\dfrac{{{[}{u}{(}{{X}}_{{{\mathrm{W}}}}{)]}}^{{2}}{+}{{[}{u}{(}{{X}}_{{{\mathrm{NV}}}}{)]}}^{{2}}}{{{(1-}{{X}}_{{{\mathrm{W}}}}{-}{{X}}_{{{\mathrm{NV}}}}{)}}^{{2}}}} $	0.0513
定量核磁法（u_qNMR）	$ {{u}}_{{ {\mathrm{}}}}={{P}}_{{{\mathrm{FB1}}}}\sqrt{{\left[\dfrac{{u}{(}{{I}}_{{{\mathrm{FB1}}}}/{{I}}_{{{\mathrm{std}}}}{)}}{{{I}}_{{{\mathrm{FB1}}}}{/}{{I}}_{{{\mathrm{std}}}}}\right]}^{{2}}{+}{\left[\dfrac{{u}{(}{{M}}_{{{\mathrm{FB1}}}}{)}}{{{M}}_{{{\mathrm{FB1}}}}}\right]}^{{2}}{+}{\left[\dfrac{{u}{(}{{M}}_{{{\mathrm{std}}}}{)}}{{{M}}_{{{\mathrm{std}}}}}\right]}^{{2}}{+}{\left[\dfrac{{u}{(}{{m}}_{{{\mathrm{std}}}}{)}}{{{m}}_{{{\mathrm{std}}}}}\right]}^{{2}}{+}{\left[\dfrac{{u}{(}{{m}}_{{{\mathrm{FB1}}}}{)}}{{{m}}_{{{\mathrm{FB1}}}}}\right]}^{{2}}{+}{\left[\dfrac{{u}{(}{{P}}_{{{\mathrm{std}}}}{)}}{{{P}}_{{{\mathrm{std}}}}}\right]}^{{2}}} $	0.2821
定值合成不确定度（u_char）	$ {{u}}_{{{\mathrm{char}}}}{=}\dfrac{\sqrt{{{u}}_{{{\mathrm{MB}}}}^{{2}}{+}{{u}}_{{{\mathrm{qNMR}}}}^{{2}}}}{{2}} $	0.1434
合成不确定度（u_CRM）	$ {{u}}_{{{\mathrm{CRM}}}}{=}\sqrt{{{u}}_{{{\mathrm{char}}}}^{{2}}{+}{{u}}_{{{\mathrm{bb}}}}^{{2}}{+}{{u}}_{{{\mathrm{sts}}}}^{{2}}{+}{{u}}_{{{\mathrm{lst}}}}^{{2}}} $	0.1471
扩展不确定度（U）	U=k×u_CRM, k=2	0.3

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