| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1201528 | Journal of Chromatography A | 2013 | 8 Pages |
•An on-line 2DμSPE-OCD system coupled with HPLC–MS/MS was proposed.•A comprehensive method for trace plant BRs was established for the first time.•Low RSD (<10%), low LOD (~1 pg), and on-line processing time of 30 min were achieved.•In 2DμSPE, the broad elution band from PBA was sharply compressed on C18.•OCD was realized by elution of trapped derivatization reagent and analytes.
An on-line two-dimensional microscale solid phase extraction (2DμSPE)-on column derivatization (OCD)-high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) method was developed for quantification of brassinosteroids (BRs) in plant tissues. Five BRs with widest distribution in plant species and high bioactivity (24-epibrassinolide, 24-epicastasterone, 6-deoxo-24-epicastasterone, teasterone and typhastero) were selected as target analytes. 2DμSPE column packed sequentially with phenyl boronic acid silica sorbent (the first dimension) and C18 silica sorbent (the second dimension) was used to selectively extract and enrich BRs by 110–146 times. OCD was carried out on the second dimension of 2DμSPE column with m-aminophenylboronic acid (m-APBA) as a derivatization reagent, enhancing the sensitivity of MS/MS to BRs by 13–8437 times. It was also found that pre-trap of derivatization reagent on the C18 section of 2DμSPE column could increase reaction efficiency by 3–10 times. The whole process time of the on-line system was less than 30 min. The detection limits of the method for five BRs were between 1.4 and 6.6 pg with RSDs less than 10%. Endogeneous BRs in tomato leaves were analyzed by using this method. Owing to the high selectivity of this on-line 2DμSPE system, BRs in plant extracts could be quantified using matrix-free standard calibration method with relative recoveries in the range of 80–124%.
