LC-MS Analysis of Serum for a Wide Analyte Range Using a Novel HLB SPE Phase

• Abstract
• Introduction
• Experimental Method
• Recovery and Ion Suppression/Enhancement
• Results & Discussion
• Conclusion

Abstract

In this study, a mixture of twenty compounds, the majority pharmaceuticals, with LogP values ranging from -0.89 to 4.65 were analyzed in calf serum, highlighting the broad utility of the new hydrophilic lipophilic balanced (HLB) SPE phase, Supel™ Swift HLB.

Introduction

The preparation of biological samples can have a large impact on the reproducibility and accuracy of their analytical results.¹ But, solid phase extraction (SPE) provides an opportunity to reduce matrix effects such as ion suppression and aid in the reliability of consistent results. Hydrophilic-Lipophilic Balanced (HLB) cartridges contain a sorbent offering good wettability for hydrophilic compounds, in addition to providing reverse phase retention.² These properties allow HLB cartridges to effectively handle a broad range of compounds with varying properties. In this study, a mixture of twenty example compounds with LogP values ranging from -0.89 to 4.65 were analyzed from spiked calf serum using a Supel™ Swift HLB SPE cartridge for cleanup and LC-MS determination.

Experimental Method

A series of twenty analytes and sixteen internal standards as listed in Tables 1& 2 were spiked into calf- serum and allowed to equilibrate for an hour. The calf-serum sample was diluted with an equal volume of 0.4% aqueous formic acid and mixed before the sample was loaded onto the Supel™ Swift HLB SPE cartridge (1 mL/30 mg), and another commercially available HLB cartridge (1 mL/30 mg) for comparison.

Recovery and Ion Suppression/Enhancement

The analytes were monitored at two different transitions of quantifier and qualifier (for confirmation), by a scheduled MRM method. Samples were analyzed against matrix-matched calibration curves. The external standard calibration used six concentrations of the analytes between 20 to 150 ng/mL and a fixed concentration of 50 ng/mL for the internal standards when applicable.

Analytes were spiked in triplicates into calf-serum at concentrations of 100 ng/mL, with a fixed internal standard of concentration 50 ng/mL, before their processing by either the 3-step or 5-step method as shown in Figure 1. The samples were dried and resuspended in starting mobile phase before duplicate analysis by LC-MS/MS on an Agilent 1290 Infinity LC attached to a Sciex 3200 QTRAP^® system. The LC conditions including the starting mobile phase are listed in Table 3. A representative trace from recovery is shown in Figure 2.

Results & Discussion

The recoveries of the 20 analytes following their SPE cleanup from both the 3-Step and 5-Step methods of the Supel™ Swift HLB SPE cartridges are presented in Table 4 and Figure 3. Overall, the 5-Step method showed better recovery (100.7 ±6.8%) compared to the 3-Step process (85.1 ±4.2%), partly because of the two earliest eluting compounds (amiloride and nizatidine, both below 70% recovery in 3-Step method). Under the 5-Step process, all twenty analytes had recoveries between 80% and 120%. For 16 of the 20 analytes, the 3-Step process achieved recoveries in the 80% to 120% range.

In addition to evaluating the performance of the Supel™ Swift HLB SPE cartridges, its performance was also compared with another commercially available HLB cartridge. Both cartridges contained 30 mg of resin and had a max sample volume of 1 mL. To compare the effectiveness of these cartridges, the 5-Step method was employed for the same set of analytes and under similar conditions. Figure 4 and Table 4 represent the recovery using the two cartridges. As previously mentioned, all analytes had a recovery in the range of 80-120% when using the Supel™ Swift HLB SPE cartridges. This is in contrast to the other commercially available cartridge where only 16 of the 20 analytes (80%) were recovered in the 80-120% range.

Beyond recovery rates, another important factor to consider when performing SPE is the impact of residual background on ion suppression and/or ion enhancement. The ion suppression/enhancement is an indication of how well the SPE step removes matrix components (in this case from calf-serum). As displayed in Figure 5, for 16 of the 20 analytes (80%) processed with Supel™ Swift HLB SPE cartridges, there was minimal impact on ionization (≤ +/- 10%). For the remaining 4 analytes, ion enhancement but no ion suppression was observed. This is in stark contrast to the other commercially available cartridge, where 14 of the 20 analytes (70%) showed an ionization suppression (13 of the 14) or enhancement (1 of 14) of more than 10%. To conclude, samples prepared using the Supel™ Swift HLB SPE cartridges showed less ion suppression/enhancement when compared to another commercially available cartridge.

Conclusion

Through the use of twenty analytes with various log P values, the Supel™ Swift HLB SPE cartridges demonstrated excellent recoveries (100% of analytes in the range of 80-120% with the 5-step method) and minimal effects on analyte ionization for 80% of the analytes, indicating a good matrix removal.

The advantages of the Supel™ Swift HLB SPE cartridges were further demonstrated on their comparison to a commercially available HLB cartridge under the same set of conditions.