Improved Clean Up and Recovery of Pharmaceutical Compounds From Plasma using Strata™-X Solid Phase Extraction (SPE) vs. Traditional Liquid-Liquid Extraction

Diclofenac is a slightly acidic (pK_a = 4.0) non-steroidal anti-inflammatory drug (NSAID) that has been used as a post operative pain reliever in adult and pediatric patients. The subsequent quantization of diclofenac from the small volumes of biological matrices, such as plasma, has been of significant concern. Therefore, this article explores two popular extraction methods, solid phase extraction (SPE) and liquid-liquid extraction (LLE), for the isolation of diclofenac from plasma, using a water matrix as the control.

Materials and Methods

The plasma pretreatment step was the same for SPE and LLE and was comprised of filtration through a gauze cloth. Afterwards, 500 μL of diclofenac, which was dissolved in 5% methanol, was added to 500 μL of plasma, and the solution mixture was then acidified with 600 μL of 1 M phosphoric acid.

Solid Phase Extraction

The pretreated plasma samples were further cleaned up and concentrated using SPE as shown in Figure 1.

Figure 1: SPE experimental information.

Liquid-Liquid Extraction

After pretreatment, 5 mL of hexane:IPA (95:5) was added to the pretreated solution, which was followed by 1 min of vortexing, and 10 min of centrifugation at 2000 rpm. Subsequently, 4 mL of the top organic layer was transferred to a clean glass centrifuge tube and then evaporated to dryness under a stream of nitrogen at 53 °C for 20 min.

LC–UV

Figure 2 shows a diclofenac spiked plasma sample (50 μg/mL) after extraction with Strata-X. Flurbiprofen (IS) was added post-extraction (after blow-down) at a concentration of 160 μg/mL.

Figure 2: A diclofenac spiked plasma sample (50 μg/mL) after extraction with Strata-X. Flurbiprofen.

Results and Discussion

Traditionally, liquid-liquid extraction (LLE) has been used as the standard clean up procedure for a variety of pharmaceutical samples. While performing this work, it was discovered that solid phase extraction provided many benefits over LLE including improved recoveries, time, and solvent savings, as well as more consistent results (Table I).

Table I: % absolute recovery for diclofenac

Conclusion

The Strata-X SPE sorbent utilizes the chemical bonding properties of a pyrrolidone ligand, which makes it a prime target for the retention of hydrophobic neutral compounds, while also retaining basic and acidic compounds under strong organic wash conditions. With the presence of the phenyl ring in the pyrrolidone ligand, pi-pi interactions are prevalent for compounds with aromatic structures, and acidic and basic compounds are able to bind to the nitrogen of the secondary amine and the carbonyl carbon, respectively.

LLE instead utilized two immiscible solvents that compete for interaction with the analyte of interest. While LLE has been a universal choice of extraction, SPE poses many advantages over LLE for the extraction of diclofenac, via the existence of multiple binding sites on the Strata-X sorbent. Consequently, this data shows that SPE provides greater absolute recovery of diclofenac when compared to LLE; and is less time-intensive, consumes less solvent than traditional LLE procedures, and provides better reproducibility, thereby demonstrating that the extraction method of choice for pharmaceuticals, such as diclofenac, is SPE.

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