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The benefits of supported liquid extraction (SLE) in sample cleanup and, in particular, the use of a synthetic SLE sorbent are discussed.
Sample cleanup is an important step in the analytical process, but many laboratories are under pressure to reduce analysis time. Liquid–liquid extraction (LLE) has historically been the technique of choice because of its relative simplicity, but supported liquid extraction (SLE) has recently gained in popularity in high-throughput laboratories. Although SLE alleviates emulsions and other LLE-associated challenges, it is dependent on diatomaceous earth packing. Diatomaceous earth is a naturally occurring product mined from the earth, and as a result of this natural variation between materials can occur. To counter these issues, a synthetic SLE sorbent can be used as an alternative.
Sample cleanup is an important step in the analytical process because it can greatly impact downstream analysis. Proper sample cleanup can extend instrumentation lifetime, increase method sensitivity, and remove contaminants that may interfere with analysis. Liquid–liquid extraction (LLE) has historically been a popular sample cleanup technique because it is relatively simple. However, LLE does have inherent challenges, the most common of which is the formation of emulsions. LLE relies on the mixing of non-miscible solvents, resulting in the formation of emulsions or droplets between the liquid phases that may trap analytes of interest and result in analyte loss. Furthermore, emulsions and incomplete analyte collection can make LLE difficult to automate. For these reasons many laboratories have adopted supported liquid extraction (SLE), which allows LLE automation.
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SLE relies on diatomaceous earth as the sorbent in liquid extractions. Samples are diluted in a buffered aqueous solvent and loaded onto the diatomaceous earth sorbent, allowing the aqueous solvent to soak into the sorbent like a sponge. A water-immiscible solvent is then applied to the diatomaceous earth. Target analytes partition into the water-immiscible solvent ready to be eluted from the sorbent while interferences such as proteins, phospholipids, and salts are retained in the aqueous phase that is left behind in the sorbent. While not as clean as more targeted techniques such as solid-phase extraction (SPE), SLE is simple, requires little method development, and can be automated therefore making it attractive to laboratories that are looking to reduce sample preparation time.
Figure 1: SEM (1000× zoom) of (a) salt water vs. (b) fresh water diatomaceous earth.
Although SLE provides many benefits to laboratories, some improvements to the technique can be made. Relying on diatomaceous earth can pose downstream challenges including lot-to-lot inconsistency and availability. Diatomaceous earth used in SLE products is mined from the ground and like all natural products, the composition of diatomaceous earth will vary, producing lot-to-lot inconsistencies such as a change in water holding capacity when used for liquid separation purposes (Figure 1). Also, as in all natural products, there is a finite amount of material available which could cause potential availability issues.
Figure 2: Measurement of phospholipids remaining in plasma samples after cleanup with three different extraction solvents on traditional diatomaceous earth SLE.
Despite the challenges associated with traditional SLE using diatomaceous earth, laboratories are willing to overlook the inconsistencies and availability issues because the technique drastically shortens analysis time. As SLE becomes ever more popular as the preferred cleanup technique, a need to alleviate the inconsistencies and availability has grown. To address these challenges, a synthetic SLE sorbent can be used.
Figure 3: Measurement of phospholipids remaining in plasma samples after cleanup with three different extraction solvents on a synthetic SLE sorbent.
A comparison between a synthetic SLE sorbent (Novum Simplified Liquid Extraction [SLE], Phenomenex) and a traditional diatomaceous earth sorbent was performed. The first comparison tested the cleanliness of plasma samples after being processed by the two SLE sorbents using several extraction solvents. To measure the cleanliness of the samples, the concentration of phospholipids that remained in the eluents after cleanup by SLE was measured. Both SLE sorbents were able to remove >99% of phospholipids using method tert-butyl ether (MTBE); however, the diatomaceous earth SLE displayed >10% breakthrough of phosphotidylcholines using dichloromethane (DCM) as the extraction solvent and >20% breakthrough of phosphotidylcholines using ethyl acetate (EtOAc) (Figure 2) (Lyso 1: 1-Palmitoyl-2-OH-sn-glycero-phosphocholine [m/z 496–184]; Lyso 2: 1-Oleoyl-2-OH-sn-glycero-phosphocholine [m/z522–184]; PC 1: 1-Palmitoyl-2-Oleoyl-sn-glycero-phosphocholine [m/z 761–184]; PC 2: 1-Stearoyl-2-Lindoleoyl-sn-glycerol-phosphocholine [m/z 787–184]; PC 4: 1-Oleoyl-2-Lindoleoyl-sn-glycerol-phosphocholine [m/z 784–184]). The synthetic SLE sorbent displayed >99.9% removal of phospholipids using all three extraction solvents (Figure 3).
Figure 4: Lot-to-lot cleanup of traditional diatomaceous earth SLE.
The ability to remove phospholipids was studied further as we looked at the performance of different lots of SLE sorbents. Extractions using EtOAc were performed on the diatomaceous earth sorbent as well as the synthetic SLE sorbent. The diatomaceous earth sorbent showed a large variability in the cleanup ability from lot-to-lot (Figure 4). This is thought to be a result of differences in the sorbent characteristics. Diatomaceous earth is a naturally occurring mined substance made of fossilized diatoms, and the media characteristics can vary from mine to mine. However, the synthetic SLE sorbent resulted in >99.9% removal of phospholipids from lot-to-lot (Figure 5). Because the sorbent is a synthetic lab-manufactured media, the water-holding characteristics of the sorbent (and therefore the ability to retain phospholipids) can be stringently controlled resulting in consistent results from lot-to-lot.
Figure 5: Lot-to-lot cleanup of synthetic SLE sorbent.
After it was confirmed that the synthetic SLE sorbent provided cleaner samples as compared to the traditional diatomaceous earth SLE product, analyte recovery was tested. A suite of seven corticosteroids was extracted from plasma using EtOAc on the synthetic SLE sorbent. Recoveries of all seven corticosteroids were consistently higher than 70% across three separate lots (Figure 6), indicating that the extraction method and sorbent provide robust and reproducible results.
Figure 6: Recovery of seven corticosteroids from plasma across three different synthetic SLE sorbent lots.
In today's laboratory, consistency and time are of the utmost importance. Sample preparation consumes a large portion of the time dedicated to analysis and simplifying this step can lead to significant time savings; however, it is also important to ensure that cleanliness is not compromised to the point at which it negatively affects downstream analysis. While SLE has helped laboratories reduce their sample preparation time without significantly compromising cleanliness, diatomaceous earth has proven to be inconsistent in terms of cleanup ability. A synthetic SLE sorbent can remedy these problems by providing consistent cleanup and recoveries while also cutting down on the amount of method development time required. In addition to improved results and time-savings, a synthetic sorbent can be used like traditional SLE and can be automated, making it easy to incorporate into existing methods.
Erica Pike is the sample preparation brand manager at Phenomenex. She received her bachelors degree in biochemistry and molecular biology and her masters degree in biotechnology from Boston University, Boston, USA.