Sunday, November 01, 2009
Figure 1 - UPLC (ESI–)/MS/MS MRM diagnostic signal of dexamethasone (451.4 > 361.3) obtained from a liver sample extract spiked at a) 1 ng/g, b) 0.5 ng/g, and c) 0.1 ng/g.
Figure 1 shows extracted multiple reaction monitoring (MRM) ion chromatograms of dexamethasone obtained from a liver sample spiked at various concentration levels. The figure clearly demonstrates the capability to unambiguously detect this target analyte at a concentration of more than 10 times lower than the MRL using Xevo TQ MS. Even if this level of sensitivity is not required for regulatory control purposes (efficient measurement at half MRL is sufficient), this is a very comfortable interpretation, and is clearly beneficial for the analyst. Moreover, it is an answer to the current lack of analytical methods dedicated to this particularly complex matrix, as highlighted by JECFA. Another advantage linked to this instrumental sensitivity is a reduction in the sample amount required for analysis, with subsequent reduction of matrix effects and direct positive impact on quantitative performance.
Figure 2 - UPLC (ESI–)/MS/MS MRM diagnostic signals of prednisoloned6 (I.S.), endogenous cortisol, and cortisone, and prednisolone, dexamethasone, and methylprednisolone, obtained from a liver sample extract spiked at 0.1 ng/g.
Figure 2 shows MRM chromatograms of the three target exogenous corticosteroids for which an MRL is imposed (dexamethasone, methylprednisolone, and prednisolone), as well as two endogenous corticosteroids (cortisol and cortisone) obtained from a liver sample spiked at 0.1 ng/g. The figure illustrates the capability to unambiguously identify target compounds according to EU/2002/657 requirements, as well as the good chromatographic resolution of cortisone and prednisolone using the ACQUITY UPLC system.
Instrument precision was assessed by injecting the same liver sample extract spiked at 0.5 ng/g six times. The obtained relative standard deviation (%RSD) of the absolute signal intensity was found to be lower than or equal to 5% for all the target analytes, indicating good analytical performance in heavy matrix.
Figure 3 - Specific extracted chromatograms of dexamethasone obtained from a liver sample extract spiked at 0.1 ng/g acquired in a) UPLC (ESI–)/MS/MS in MRM mode, b) UPLC (ESI–)/MS in neutral loss 90, and c) UPLC (ESI–)/MS in simultaneous full-scan “matrix monitor” modes.
Experiments were also performed using alternative acquisition modes, such as neutral loss scan. This mode is particularly adapted for corticosteroids due to a loss of formaldehyde observed in negative electrospray ionization (CH2O, 30 amu), which is a characteristic of this family.4,8 The results shown in Figure 3 demonstrate very good sensitivity in neutral loss mode, compared to other existing references, when expanding the scope of the analysis to unknown compounds belonging to the corticosteroid family. Additionally, Figure 3 shows the ability to monitor the background matrix with simultaneous MRM full-scan acquisition (dual-scan MRM matrix monitoring). This functionality allows real-time, qualitative information about the nature of the matrix to be acquired at the same time as routine quantitative analyses.
Figure 4 - IntelliStart’s automatic method development feature shows a) optimization of cone voltage, b) location of most abundant daughters, and (c) optimization of collision energy for clenbuterol.
Figure 5 - UPLC (ESI+)/MS/MS MRM diagnostic signal of clenbuterol (277.1 > 203.0) obtained for a urine sample extract spiked at a) 20 pg/mL, b) 10 pg/mL, and c) 1 pg/mL.
Initial instrument setup and MRM optimization of the ß-agonists were automatically performed using IntelliStart software. Compound information was entered and automatic method development was performed to generate multiple, fully optimized MRMs for each ß-agonist. Figure 4 gives the extracts from the IntelliStart-generated method development report that shows the optimization of cone voltage, location of most abundant product ions, and optimization of collision energy for clenbuterol. Figure 5 shows MRM chromatograms of clenbuterol obtained from a urine sample spiked at various concentration levels. These results indicate the clear capability to identify this target compound at concentrations as low as 10 pg/mL (ppt). These results, again, provide a comfortable determination, considering the minimum required performance levels (MRPLs) currently in place or being discussed at the European level. Moreover, it should be emphasized that the injected volume/final extract volume ratio in this case was equal to 2:50, which means there is potential for the sensitivity of the method to be improved by a factor of 5.