High-throughput analysis of biological samples by direct coupling of solid-phase (micro-)extraction and mass spectrometry

Abstract

High-throughput analysis of biological samples is becoming increasingly important. On-line sample pretreatment and separation increases the speed and reliability of the analysis. In order to increase the speed even more, solid-phase extraction (SPE) and solid-phase microextraction (SPME) have been directly coupled to an ion-trap mass spectrometer (ITMS). As a result, analysis times can be reduced to a few minutes.If SPE is directly coupled to the ITMS it is very important to obtain clean extracts to prevent detection problems, e.g. ion suppression. With a non-selective stationary phase, the removal of interfering matrix compounds requires an efficient washing step in which no elution of the analyte occurs. The choice of single MS vs. multiple-stage MS is also dependent on this step. Special attention has been paid to differences in chromatograms of blank samples and to ion-suppression effects. Extended AbstractMultiple-stage MS is very interesting for a reduction of matrix effects, and so a considerable increase of selectivity and sensitivity was obtained. Multiple-stage MS cannot eliminate ion suppression. SPE-MS methods with atmospheric pressure chemical ionisation (APCI) have been developed for the determination of clenbuterol in urine [1] and prednisolone in serum using a polydivinylbenzene (PDVB) and C18 stationary phase, respectively.A PDVB cartridge (10 • 2mm i. d.) allowed effective washing of the SPE phase, i.e. 50% (v/v) methanol could be used without elution of clenbuterol from the cartridge. This resulted in relatively clean extracts. Elution was performed with a flow-rate of 1.0mLmin 1 using a methanol gradient (50 to 70% in 2.5 min). However, for sensitive analysis MS-MS had to be applied. Consequently, a detection limit (LOD) of about0.5 ngmL 1 was observed for clenbuterol in urine. Despite the washing procedure severe ion suppression (45%) was observed for low levels of clenbuterol (0.5 10ngmL 1), whereas the ion suppression was less prominent for higher concentrations of clenbuterol (suppression about 4%). Even though the suppression was concentration dependent, good linearity was observed (R>0.99, range 1.0 250ngmL 1). The total analysis time was about 8.5 min, which is still relatively long due to rather low flow-rates that were applied during the different SPE steps.Prednisolone was extracted from serum by a C18 stationary phase (10 • 2 mm i.d.). Due to the apolar ring structure of the analyte it was possible to wash the stationary phase with 25% (v/v) methanol. Elution was performed using a methanol gradient (25 to 50% in 1.5 min with a 1-mL min 1 flow-rate). Subsequently, MS-MS was used for the analyis. The total set-up resulted in an LOD of 10 ng mL 1 of prednisolone in serum (Figure 1). The relatively high LOD is due to MS fragmentation of prednisolone. The total analysis time including sample pretreatment is about 5 min. No ion suppression was observed during the determination of prednisolone. Good linearity (R > 0.99) was observed over the range 10 250 ngmL 1.A first attempt to couple SP...