High throughput and very specific screening of anabolic-androgenic steroid adulterants in healthy foods based on stable isotope labelling and flow injection analysis-tandem mass spectrometry with simultaneous monitoring proton adduct ions and chloride adduct ions.

In this work, a stable isotope labelling-flow injection analysis-tandem mass spectrometry (SIL-FIA-MS/MS) with simultaneous monitoring [M+H] and [M+Cl] method was developed for very specific and high throughput screening of anabolic-androgenic steroids (AAS) illegally added to healthy foods. Initially, a simple centrifugation step was carried out for liquid samples, and for solid samples, a solid-liquid extraction step was conducted. Afterwards, batch chemical derivatization was carried out. After adding a certain amount of C-3-NPH labelled AAS standards as the internal standards, it can be directly transferred for FIA-MS/MS analysis based on the no MS response characteristics of 3-NPH. The 3-NPH labelled AAS showed dual-polarity property, observing chloride adduct ion ([M+Cl]) in negative ion mode and proton adduct ion ([M+H]) in positive ion mode. The average time cost for pretreatment of each sample was less than 1 min by carrying out batch processing. The subsequent FIA-MS/MS detection enabled rapid and high throughput detection. The addition of C-3-NPH-labelled AAS as internal standards can correct the matrix effect to achieve accurate quantitative analysis. The detection sensitivity was also improved by 2-5 folds after 3-NPH labelling. The limits of detection (LODs) in positive MRM mode were in ranges of 0.1-0.3 ng/mL. The validated method with simultaneous monitoring [M+H] and [M+Cl] was validated in the range of 6.0-1000 ng/mL with the linear coefficient (R) greater than 0.997. Satisfactory recoveries were found to be in ranges of 93.0-108.7%. The intra-day and inter-day RSDs were in the range of 3.5-9.9% and 5.1-14.1%, respectively. No changes in detection sensitivity of the mass spectrometry and no carry-over effects were found after numerous consecutive injections of AAS derivates. Compared with previously reported methods, the proposed method proved accurate, very specific, high throughput with good sensitivity.