恩诺沙星粉中未知添加物的确证

doi:10.3724/SP.J.1123.2020.09007

摘要/Abstract

摘要：

应用非靶向分析技术,筛查、分析和确证恩诺沙星粉(水产用)中的非法添加物。分别制备甲酸酸化、碳酸钠碱化的恩诺沙星粉供试品溶液,经超高效液相色谱-二极管阵列检测器(UPLC-PDA)检测初筛,获取未知物色谱图。应用超高效液相色谱-飞行时间高分辨质谱(UPLC-TOF-HRMS),在正、负离子模式下对酸化、碱化样液进一步检测,获得未知物母离子和二级特征碎片的精确质荷比、同位素信息,并应用SCIEX OS软件进行分析推导。最后取疑似化合物对照品进行确证研究。UPLC-PDA初筛结果显示:酸化样液在1.870 min、5.122 min,碱化样液在5.122 min,均发现高响应未知色谱峰。对吸收波长和峰面积进行分析,推测含2个未知物,且未知物1(1.870 min)与未知物2(5.122 min)在酸性/碱性条件下可能发生转换。SCIEX OS软件分析推导结果显示:未知物2,母离子分子式拟合为C₁₁H₈O₂,二级碎片结构解析含1个苯环、2个羰基和1个通过成环连接的丙烯结构,推测为甲萘醌;未知物1的分子离子峰为C₁₁H₉O₅S^-,二级碎片仅采集到HSO₃^-,丢失部分与未知物2一致,结合甲萘醌常见衍生物类型,推测为亚硫酸氢钠甲萘醌。取甲萘醌、亚硫酸氢钠甲萘醌对照品进行对比研究,UPLC-PDA检测结果显示:未知物1与亚硫酸氢钠甲萘醌、未知物2与甲萘醌,保留时间和紫外光谱一致;向供试溶液中添加对照品溶液后检测,未知物为单一峰。UPLC-TOF-HRMS检测发现:未知物1与亚硫酸氢钠甲萘醌保留时间一致,一级质谱质量偏差为1.0×10^-6,二级谱库匹配度为100%;未知物2与甲萘醌保留时间一致,一级质谱质量偏差为0.6×10^-6,二级谱库匹配度为99.7%。未知物1和2的结构得以确证。亚硫酸氢钠甲萘醌可止血,与恩诺沙星治疗败血症的适应证相应,佐证试验结果。随着对兽药非法添加行为的严格监管和严厉打击,非法添加物和添加手段愈发隐蔽,常规靶向分析难以满足监控需求。该文详述的使用UPLC-PDA结合UPLC-TOF-HRMS对未知物进行非靶向分析的技术,可为药品、食品、保健品、化妆品及农药等产品中非法添加物的筛查和确证提供思路和技术参考。

关键词: 超高效液相色谱, 二极管阵列检测, 飞行时间高分辨质谱, 亚硫酸氢钠甲萘醌, 恩诺沙星粉(水产用), 非法添加

Abstract:

In order to ensure the safety of animal food and regulate the application of veterinary drugs, it is necessary to strictly monitor their content, and to constantly improve the methods used to detect non-specific, illegally added substances in veterinary drugs. A study about the screening, analysis, and confirmation of illegal additives in enrofloxacin powder (used for aquaculture) using non-targeted analysis technology was introduced. First, an enrofloxacin powder test solution under acidic conditions was prepared by adding formic acid, and an enrofloxacin powder test solution under alkaline conditions was prepared by adding sodium carbonate. An ultra-performance liquid chromatography with photodiode array detector (UPLC-PDA) was used to assay the test solutions for the presence of unknown additives. Results revealed two high-response unknown peaks in the acidified test solution, with retention times of 1.870 min and 5.122 min respectively. In the alkalized test solution, only one high-response unknown peak was found, with a retention time of 5.122 min. The ultraviolet spectrum characteristic peaks at 5.122 min in acidified and alkalized test solutions were similar, but the peak area in the alkalized test solution was almost ten times that in the acidified solution. Two potential unknown substances were detected. Unknown substance 1 (1.870 min) and unknown substance 2 (5.122 min) may transform under acidic or alkaline conditions. Ultra-performance liquid chromatography-time of flight high resolution mass spectrometry (UPLC-TOF-HRMS) was used to analyze the unknown compounds in more detail. The acidified and alkalized test solutions were detected in the positive and negative ion modes of mass spectrometry, respectively. Accurate mass of the precursor ion, characteristics of secondary ion fragments, and isotopic intensity ratio of the two unknown substances were collected. This information was imported into SCIEX OS software. The molecular formula of the parent ion of unknown substance 2 was found to fit to C₁₁H₈O₂, and its secondary fragment structure may contain a benzene ring and two carbonyl groups, with a propylene structure connected to them through ring formation. From this, unknown substance 2 was presumed to be a menadione. The molecular ion peak of unknown substance 1 was found to fit to C₁₁H₉O₅S^-, only HSO₃^- was collected in the secondary fragments, and the missing part was consistent with unknown substance 2. Considering the most common derivatives of menadione, unknown substance 1 can be proposed to be menadione sodium bisulfite. Finally, we used menadione and menadione sodium bisulfite as reference substances in a comparative study. The same treatment method was used to prepare menadione, menadione sodium bisulfite reference solution, and enrofloxacin powder test solution. After UPLC-PDA detection, unknown substance 1 and menadione sodium bisulfite, unknown substance 2 and menadione, were found to have similar retention times and UV spectra. When the reference solution was added to the enrofloxacin powder test solution, the peak purity of the unknown substance did not change, and were all single peaks. UPLC-TOF-HRMS analysis revealed that the retention time of unknown substance 1 was consistent with that of sodium menadione bisulfite: compared to its accurate mass number in theory, the mass accuracy error was 1.0×10^-6, and the matching degree of fragmentation information in the library was 100%. The retention time of unknown substance 2 was same as the menadione: compared to its accurate mass number in theory, the mass accuracy error was 0.6×10^-6, and the matching degree of fragmentation information in the library was 99.7%. The structures of unknown substances 1 and 2 were confirmed.
Menadione sodium bisulfite is known to participate in the synthesis of thrombin in the liver, and also promotes the formation of prothrombin, and accelerates coagulation. The indication of enrofloxacin powder (used for aquaculture) is the treatment of hemorrhage and sepsis in aquaculture animals such as fish and eel. The pharmacological effects of the two drugs correspond to each other, which can cause producers to take risks and add them illegally. With the strict supervision and severe restrictions on the addition of veterinary drugs, illegal additives are becoming more and more subtle. Conventional targeted analysis does not always meet the monitoring requirements. In this paper, the non-targeted analysis of unknown substances using UPLC-PDA combined with UPLC-TOF-HRMS is described in detail. The results may provide a technical reference for screening and identifying illegal additives in drugs, food, health care products, cosmetics, and pesticides.

Key words: ultra-performance liquid chromatography (UPLC), photo diode array detection (PAD), time of flight high resolution mass spectrometry (TOF-HRMS), menadione sodium bisulfite, enrofloxacin powder (used for aquaculture), illegal addition

中图分类号:

O658

熊玥, 汪澄, 刘建晖, 石慧慧, 汪云花, 孙瑶, 于洁. 恩诺沙星粉中未知添加物的确证[J]. 色谱, 2021, 39(6): 633-641.

XIONG Yue, WANG Cheng, LIU Jianhui, SHI Huihui, WANG Yunhua, SUN Yao, YU Jie. Confirmation of unknown additives in enrofloxacin powder[J]. Chinese Journal of Chromatography, 2021, 39(6): 633-641.

图/表 12

图 1 酸性初筛溶液和碱性初筛溶液的色谱图及紫外光谱图

Fig. 1 Chromatograms and UV spectra of acid test solution and alkaline test solution

图 2 碱性推导溶液正离子模式下的(a)BPC图及(b)紫外色谱图

Fig. 2 (a) Base peak chromatogram (BPC) and (b) UV chromatogram of alkaline test solution in positive ion mode

图 3 未知物2的(a)精确质荷比及同位素分布图和(b)二级碎片图

Fig. 3 (a)Precise m/z, natural isotope distribution and (b) fragment ion spectrum of unknown compound 2

图 4 未知物2结构推测

Fig. 4 Structure prediction of unknown compound 2

图 5 酸性推导溶液负离子模式下的(a)BPC图及(b)紫外色谱图

Fig. 5 (a) BPC and (b) UV chromatogram of acid test solution in negative ion mode

图 6 未知物1的(a)精确质荷比及同位素分布图和(b)二级碎片图

Fig. 6 (a)Precise m/z, natural isotope distribution and (b) fragment ion spectrum of unknown compound 1

图 7 未知物1的裂解途径

Fig. 7 Fragmentation pathway of unknown compound 1

图 8 未知物2的裂解途径

Fig. 8 Fragmentation pathway of unknown compound 2

图 9 对照品溶液中亚硫酸氢钠甲萘醌和甲萘醌的色谱图及紫外光谱图

Fig. 9 Chromatograms and UV spectra of menadione sodium bisulfite and menadione standards

表 1 未知物1峰纯度检查与光谱相似度检查结果

Table 1 Results of peak purity test and spectral similarity test of unknown compound 1

Analyte	Additive proportion	Purity angle	Purity threshold	Results of peak purity test	PDA matching angle	PDA matching threshold	Results of spectra similarity test
Acid test solution	-	0.225	0.282	All of purity angles are less than the threshold, which prove it is a peak of pure compound.	0.435	1.158	All of the PDA matching angles are less than the threshold, which prove its spectra is similar to the standard’s.
Acid test solution-standard solution of menadione sodium bisulfite	1∶4	0.084	0.287		0.091	1.158
	1∶1	0.108	0.284		0.221	1.158
	4∶1	0.228	0.297		0.328	1.158

图 10 (a)未知物1与(b)谱库中亚硫酸氢钠甲萘醌对照品的匹配图

Fig. 10 Matching diagrams between (a) unknown compound 1 and (b) menadione sodium bisulfite reference substance in the library

图 11 (a)未知物2与(b)谱库中甲萘醌对照品的匹配图

Fig. 11 Matching diagrams between (a) unknown compound 2 and (b) menadione reference substance in the library

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