Pharmaceutical
A Simplified Approach to Bioanalytical Sample Preparation
he complexity of biological samples can pose challenges to down- stream analysis in the pharmaceutical industry. Bioanalytical and drug metabolism and pharmacokinetics (DMPK) groups often work with plasma or serum samples and are under pressure to keep cost per sample low while keeping throughput high. For this reason, sample preparation is often compromised because it is the most time- consuming step in the analytical process.
T
A simple protein precipitation step has become a popular sample prepara- tion technique in the pharmaceutical environment because it is rapid, requires little method development and is cost-effective. However, pro- tein precipitation only removes proteins from samples, leaving behind cumbersome phospholipids that can negatively affect analysis and column lifetime (Figures 1 and 2). Another popular technique, traditional liquid–liquid extraction (LLE), removes proteins, phospholipids and salts. However, this process can be difficult to automate, which limits through- put capabilities. To alleviate these challenges, newer techniques such as supported or simplified liquid extraction (SLE) are gaining popularity.
Traditional SLE relies on diatomaceous earth to provide a solid support for loading aqueous samples. The samples are allowed to soak into the sorbent, creating a large surface area. Target analytes are then eluted from
Table 1 – SLE protocol
1. Dilute sample at least 1:1 with water or buffer, ensuring that target analytes are in a nonionized state.
2. Load onto the SLE sorbent. Wait 5 min, allowing the sample to completely soak into the sorbent.
3. Apply elution solvent and elute via gravity. Apply vacuum for 10–60 sec to complete elution.
the sorbent by applying a solvent, typically a water-immiscible organic solvent such as ethyl acetate (EtOAc), methyl tert-butyl ether (MTBE) or dichloromethane (DCM). Target analytes partition into the elution solvent as it passes through the solid support, leaving behind interferences such as proteins, phospholipids and salts. The solvent can be eluted via gravity and collected using a plate or tube (Table 1). While this process is simple, requires minimal method development and is easily automated, the dia- tomaceous earth used in traditional SLE products can pose challenges such as inconsistencies and availability issues, which are inherent with all naturally occurring materials.
Synthetic simplified liquid extraction sorbent A novel, synthetic SLE sorbent was developed to overcome the challenges
associated with diatomaceous earth. Novum Simplified Liquid Extraction (Phenomenex, Torrance, Calif.) is manufactured in the laboratory to ensure adequate supplies of material and undergoes strict quality-control tests to make sure that results are consistent and reliable. The synthetic sorbent can be used in the same manner as diatomaceous earth SLE, following the simple load and elute procedure depicted in Table 1.
Figure 1 – Phospholipids in protein precipitated plasma.
To verify that the new, synthetic SLE sorbent provided adequate clean- liness, phospholipid depletion studies were performed using plasma samples and DCM as an extraction solvent and the presence of five major phospholipid classes were measured in the extracted samples. The syn- thetic SLE sorbent removed >99% of all five phospholipid classes, while the diatomaceous earth SLE sorbent allowed >10% breakthrough of phosphotidyl cholines (Figure 3).
To assess the effect of phospholipid build up, repetitive 20 µL injections of diclofenac in protein precipitated plamsa were made.
Figure 2 – HPLC/UHPLC column lifetime study after 250 injections of protein precipitated plasma.
After determining that the synthetic SLE sorbent provided a clean sample, an analyte recovery study was performed. Nine nonsteroidal anti- inflammatory drugs (NSAIDs) were extracted from plasma using the synthetic SLE sorbent and DCM as an extraction solvent. Recovery of the NSAID compounds was >75%, the exception being salicylic acid at <60%. In an effort to boost recoveries, a second extraction using an optimized elution solvent of 10% ethyl acetate in DCM was performed. The optimized elution solvent improved recovery for salicylic acid to >90% without significantly altering recoveries of the other eight NSAID compounds (Figure 4).
AMERICAN LABORATORY • 20 • MARCH 2015
by Erica Pike
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