[Ultra-high performance liquid chromatography-orbitrap high-resolution mass spectrometry for rapid screening and identification of 32 illegally added drugs in slimming and anti-impotence health foods]

Se Pu. 2022 Jun;40(6):531-540. doi: 10.3724/SP.J.1123.2021.12009.
[Article in Chinese]


A novel method based on ultra-high performance liquid chromatography-orbitrap high-resolution mass spectrometry (UHPLC-Orbitrap HRMS) was developed for the rapid screening and confirmation of 32 illegally added drugs in slimming and anti-impotence health foods. In addition, the key points of the database establishment and application are summarized. This research focused on the derivatives of illegally added drugs. An HRMS database was established by comparing the response intensity of each compound in the positive and negative modes. The experimental conditions such as the type of extraction solvent and chromatographic column temperature were explored in detail. The analytes were separated on a Hypersil gold vanquish column (100 mm×2.1 mm, 1.9 μm) by gradient elution with acetonitrile/water (containing 0.1%(v/v) formic acid) as the mobile phase at a flow rate of 0.3 mL/min. Positive and negative ion full scanning/data-dependent secondary scanning mode was used to collect the 32 target compounds within 17 min, and TraceFinder software was used to screen the fragment ions. All the 32 compounds could be well separated within 17 min. The measured and theoretical values of the exact mass of the 32 compounds in the two matrix-spiked solutions were within an error of 5×10-6, and the MS2 fragment ions were within an error of 1×10-5. All the compounds showed an excellent linear relationship, with correlation coefficients (r2) above 0.99. Except dapoxetine, hydroxythiohomo sildenafil, thiohomo sildenafil, thiosildenafil, desmethyl thiosildenafil, the recoveries ranged from 50.5% to 84.5% in the solid matrix, with the relative standard deviations (RSDs) ranging from 1.2% to 13%. The recoveries were 60.4% to 109.3% in the liquid matrix, with the RSDs ranging from 0.77% to 8.2%. The matrix effect (ME) values of the 32 compounds ranged from 0.61 to 0.95 in the solid matrix and from 0.73 to 1.09 in the liquid matrix. Thiohomo sildenafil, desmethyl thiosildenafil, and chlorpretadalafil exhibited strong matrix inhibitory effects in the solid matrix. Therefore, solid and liquid negative matrix extracts were used to prepare a series of mixed standard solutions in order to reduce the ME values. The limits of detection (LODs) were 0.02 mg/kg for the 32 drugs in the liquid sample and 0.02 mg/kg for 29 compounds in the solid sample; the LODs for chlorothalidone, udenafil, and desmethyl thiosildenafil in the solid sample were 0.04 mg/kg. When the retention time in the self-built database matches the sample collection method, it should be used as one of the screening conditions. As for the selection of the matching mode, if the identify mode is selected, the retention time is a necessary condition for compound confirmation. When the retention time does not meet the requirements, subsequent screening of the fragment ions and isotope abundance ratios will not be performed. If the confirm mode is selected, the retention time is the optional condition for compound confirmation. When the retention time does not meet these requirements, subsequent matching of other conditions such as fragment ions and isotope information is required. Isotope information is very important in HRMS and is an effective supplement to the first-order extracted mass. Therefore, its use is recommended, but the isotope abundance ratio will be even lower when the target content is very low in the complex matrix, which may affect isotope matching. In addition, if the fragment ions are not detected in the screening results of the TraceFinder software but can be extracted in the data browser, their intensity threshold in the screening conditions can be further reduced to find the corresponding fragment ions. One positive sample was detected among 48 healthy food samples, with a detection rate of 2.08%. This method has the advantages of simple operation and high accuracy. It can be used for the rapid screening and confirmation of 32 illegally added drugs in slimming and anti-impotence health foods.

建立了基于超高效液相色谱-Orbitrap高分辨质谱的快速筛查及确证减肥和壮阳类保健食品中32种非法添加药物的分析方法,并总结了数据库建立和应用的相关要点。研究对象聚焦于非法添加药物的衍生物,在对比正负离子模式下各化合物响应强度的基础上建立了高分辨质谱信息库,对提取溶剂、色谱柱温度等实验条件进行了详细探究,尽可能给出了较宽的标准曲线线性范围。使用Hypersil gold vanquish色谱柱(100 mm×2.1 mm, 1.9 μm),梯度洗脱,流量0.3 mL/min,正、负离子全扫描/数据依赖的二级扫描模式,在17 min内完成32种目标化合物的数据采集,通过TraceFinder软件进行快速定性筛查和定量。结果显示在17 min内32种化合物能得到较好分离;2种基质加标溶液中32种化合物的一级质谱离子精确质量数的实测值与理论值均在5×10-6误差之内,二级碎片离子质量数的实测值与理论值均在1×10-5误差之内;方法学验证结果表明,所有化合物均显示出优异的线性关系,相关系数(r2)均大于0.99;固体基质中除达泊西汀、羟基硫代豪莫西地那非、硫代豪莫西地那非、硫代西地那非、去甲基硫代西地那非的回收率较低外,其余27种化合物的回收率为50.5%~84.5%,相对标准偏差(RSD)为1.2%~13%,液体基质中32种化合物的回收率范围为60.4%~109.3%, RSD为0.77%~8.2%;在48份减肥及壮阳类保健食品中检出1份阳性样品,检出率2.08%。该方法操作简单,结果定性准确,可用于减肥及壮阳类保健食品中32种非法添加药物的快速筛查及确证。

Keywords: health foods; high-resolution mass spectrometry (HRMS); illegally added drugs; rapid screening; ultra-high performance liquid chromatography (UHPLC).

MeSH terms

  • Chromatography, High Pressure Liquid
  • Limit of Detection
  • Sildenafil Citrate
  • Tandem Mass Spectrometry*


  • Sildenafil Citrate