A Simplified Approach to Bioanalytical Sample Preparation

The complexity of biological samples can pose challenges to downstream 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.

A simple protein precipitation step has become a popular sample preparation technique in the pharmaceutical environment because it is rapid, requires little method development and is cost-effective. However, protein 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 throughput capabilities. To alleviate these challenges, newer techniques such as supported or simplified liquid extraction (SLE) are gaining popularity.

Figure 1 – Phospholipids in protein precipitated plasma.
Figure 2 – HPLC/UHPLC column lifetime study after 250 injections of protein precipitated plasma.

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 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 diatomaceous earth used in traditional SLE products can pose challenges such as inconsistencies and availability issues, which are inherent with all naturally occurring materials.

Table 1 – SLE protocol

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.

To verify that the new, synthetic SLE sorbent provided adequate cleanliness, 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 synthetic SLE sorbent removed >99% of all five phospholipid classes, while the diatomaceous earth SLE sorbent allowed >10% breakthrough of phosphotidyl cholines (Figure 3).

Figure 3 – Synthetic SLE versus diatomaceous earth SLE using DCM as an extraction solvent.
Lyso 1: 1-Palmitoyl-2-OH-sn-glycero-phosphocholine (m/z 496–184).
Lyso 2: 1-Oleoyl-2-OH-sn-glycero-phosphocholine (m/z 522–184).
PC 1: 1-Palmitoyl-2-oleoyl-sn-glycero-phosphocholine (m/z 761–184).
PC 2: 1-Stearoyl-2-lindoleoyl-sn-glycero-phosphocholine (m/z 787–184).
PC 4: 1-Oleoyl-2-lindoleoyl-sn-glycero-phosphocholine (m/z 784–184).

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).

Figure 4 – Recovery of nine NSAID compounds using synthetic SLE under DCM and EtOAc in DCM extraction conditions.


While a reduction in the amount of time spent performing sample preparation can improve total analysis time, analysts must be careful that downstream results and column lifetime are not negatively affected as a result of the sample preparation process. Rapid techniques such as protein precipitation are often adopted; however, this technique simply removes proteins and does not remove phospholipids, which can cause a huge decrease in HPLC/ UHPLC (ultrahigh-performance liquid chromatography) column lifetime. As column lifetime decreases, consumable costs and the amount of time spent changing columns will increase, effectively canceling out the time-saving benefits of the quick protein precipitation.

An acceptable compromise to protein precipitation, LLE removes proteins, phospholipids and salts from samples and thus improves downstream results and column lifetime, but can be a cumbersome process and is difficult to automate. SLE is gaining popularity because it requires minimal method development and optimization time, follows a simple procedure and is easily automated. Further improvements to the SLE process have resulted in a synthetic SLE sorbent that alleviates the reliance on a natural resource as well as the inherent challenges associated with the diatomaceous earth sorbent.

Erica Pike is the sample preparation brand manager at Phenomenex, 411 Madrid Ave., Torrance, Calif. 90501, U.S.A.; tel.: 310-212-0555; e-mail: [email protected]; phenomenex.com.