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Improve Sample Prep Selectivity using 96-well SPE Method Development Plates

By: An Trinh, Reporter EU Volume 25

An Trinh
atrinh@sial.com

In pharmaceutical bioanalysis, researchers are charged with the responsibility of developing and running assays to quantitate drugs, pharmaceutical candidates, and their metabolites in biological fl uids such as serum and plasma. With recent advances in combinatorial chemistry, genomics and proteomics, knowledge of drug mechanisms are increasing resulting in drug designs structurally catered to endogenous biomolecules. Such drugs are often more potent allowing for smaller dosages which results in smaller concentrations of the drugs and their metabolites in biological fl uids. Although advances in LC-MS technology have reaped overwhelming benefi ts in terms of increased throughput and sensitivity, good sample preparation has and continues to become more critical.

Bioanalytical scientists are often asked to detect drug levels in the parts-per-trillion to parts-per-quadrillion range. Because solid phase extraction (SPE) technology is based on chromatographic separation, analysts can develop robust methods that offer high analyte recoveries. More importantly, SPE offers the selectivity necessary to specifi cally target the retention and elution of analytes of interest in the presence of complicated biological matrix components.

Even with SPE technology’s advantages, many analysts do have reservations for using the technology. The most common disadvantage is the wide perception that SPE is overly complex. The wide selection of phase chemistries coupled with the large number of potential reagents/ solvents used for each step of the SPE process makes method development and troubleshooting a daunting and time consuming task. In effect, many researchers find it diffi cult to develop rugged SPE methods that meet their analytical objectives.

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96-well SPE MD (Method Development) Plate – BAN, for extracting basic, acidic, and neutral compounds (BAN)

To address this widespread concern, we have developed a 96-well SPE platform to ease the method development process. Our new 96-well SPE MD (Method Development) Plate – BAN contains a selection of 8 different SPE chemistries commonly used in the extraction of basic, acidic, and/or neutral compounds from biological fl uids (Figure 1). The mix of phase chemistries contained within this 96-well SPE plate allows researchers to screen for the phase(s) that offer the best analyte recovery, selectivity, and reproducibility when using the generic methods described in Table 1.

Figure 1 Phase Chemistry Template for 96-well SPE MD Plate-BAN, 25 mg/well (577522-U)

Table 1 Recommended Generic Methods for 96-well SPE MD Plate-BAN

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The Benefits of Evaluating Multiple Phase Chemistries

Most method developers often focus their method development efforts on popular reversed-phase chemistries such as C18 and hydrophilic polymer phases. Such phases often offer good retention of a broad range of analytes and can typically yield high recoveries under generic methodology. However, because of this broad affinity, matrix interferences can often co-retain and elute with analytes of interest.

For example, in this application, we compare the extraction of 2 μg/mL amphetamine and methyl amphetamine spiked in human urine on both a standard C18 SPE and Discovery DSC-MCAX using the generic protocols described in Table 1 (reversed-phase protocol for C18 and cation-exchange protocol for MCAX) followed by subsequent LC-UV analysis (Figure 2). The MCAX phase offered ~100% absolute recovery, whereas the C18 phase offered 79 and 48% recovery for the compounds tested. Also note that the C18 background was 20 times greater than the MCAX phase.

Figure 2 DSC-MCAX SPE vs. C18 SPE for the Extraction of Amphetamine and Methylamphetamine in Urine

In this second application, four corticosteroids (0.5 and 1.0 μg/mL) were extracted from urine on both Discovery DSC-CN and standard C18 (100 mg/well) using the reversed-phase procedure described in Table 1 followed by LC-UV analysis. Note that the background level on C18 was so high, HPLC system failure occurred early in the run (Figure 3). DSC-CN also offered excellent recovery for the cortocosteroids tested (Figure 4).

Figure 3 Background Comparison of Blank Urine Sample

Figure 4 Recovery and Example Chromatogram for DSC-CN Extraction of Corticosteroids

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Conclusion

In pharmaceutical bioanalysis, sample prep selectivity and recovery is of vital importance when achieving very low limits of quantitation in diffi cult sample matrices. Supelco’s new 96-well SPE MD Plate-BAN offers a convenient format for researchers to screen an array of SPE phase chemistries during SPE method development. Once one or more phase chemistries are selected, further method optimization can be conducted to offer maximum assay selectivity, recovery, and accuracy/precision. In this report, we demonstrate the importance of evaluating multiple phase chemistries during method development.

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