Geoffrey Rule, Ph.D.- Principal Scientist, MilliporeSigma, Bellefonte, PA- A business of Merck KGaA, Darmstadt, Germany; Hugh Cramer- Scientist,MilliporeSigma, Bellefonte, PA- A business of Merck KGaA, Darmstadt, Germany
Many scientists doing bioanalysis in the pharmaceutical industry use protein precipitation to clean up samples prior to analysis of small molecule drugs by LC-MS/MS. While this technique removes proteins quickly and inexpensively, it does not address the issue of ion suppression caused by phospholipids, which are present in biological sample matrices such as serum, plasma, and whole blood. During chromatography, coelution of phospholipids with the analyte of interest results in ion suppression and thus a decrease of mass spec signal that can cause variability and impact accuracy in quantitation. If the phospholipids do not immediately coelute with the analyte of interest, they can accumulate on the analytical column and elute later, unpredictably, during downstream analyses.
Ballistic gradients and small particles amplify the issue
Advances in LC-MS technology have allowed analysts to decrease LC run times by using ballistic HPLC gradients and columns with particles sizes of 2 μm or less. However, ballistic gradients often do not purge the column well enough of phospholipids that remain after typical protein precipitation protocols, and HPLC columns with small particles are generally more prone to clogging than ones with larger particles. Because contaminant phospholipids are often highly retained on the analytical column, they can take a prolonged period to elute. With the shorter run times however, phospholipids can accumulate on the column unless the analyst also adds a long column washing step. This added step can decrease laboratory throughput.
Traditional SPE versus chemical filtration
One approach to overcome the problem is to use traditional solid phase extraction (SPE). These methods are often based on a hydrophobic retention mechanism to separate the phospholipids from the sample’s analyte of interest. This mechanism, however, leads to problems if the analyte is also hydrophobic. Such compounds are removed along with the hydrophobic phospholipids, which decreases analyte recovery and makes results inaccurate. These methods also often require time-consuming and analyte-dependent method development while still only removing nominal amounts of phospholipids. Remaining phospholipids can still accumulate on the analytical column and thus impact future analyses, add to column replacement costs, and increase instrument downtime. This problem led to the development of a new approach to phospholipid removal. Unlike with traditional SPE, where the analyte is retained on the sorbent through a washing step, the new approach utilizes a type of chemical filtration that selectively removes undesired phospholipids while allowing analytes to pass through unretained. This method is performed from the same high organic solvent composition, typically acetonitrile, that is used to precipitate the proteins. A variety of products designed specifically for the removal of both proteins and phospholipids have become commercially available. The stationary phase is typically packed into syringe-shaped cartridges, 96-well microtiter plates, flat disks, or pipette tip microextraction devices for small sample volumes. Most of these products use standardized, simple, and fast procedures requiring little method development.
How it works
A technology introduced in recent years offers a means of removing phospholipids from a high organic solvent “protein crash” while allowing analytes to pass through, in essence a kind of chemical filtration. HybridSPE®- Phospholipid combines the simple, standardized methodology of traditional protein precipitation in an SPE format for simultaneous removal of proteins and phospholipids from biological samples. Unlike phospholipid removal products that use a hydrophobic retention mechanism, the technology is based on zirconia (ZrO₂) coated onto the silica stationary phase. The zirconium (Zr) atoms act as a Lewis acid (electron acceptor), having empty d orbitals, while the phosphate moieties of phospholipids act as a strong Lewis base (electron donor). This mechansim provides for strong interaction between the phospholipids and the Zr atoms. The technology is capable of separating phospholipids from even highly hydrophobic analytes.
A diverse list of challenging analytes
Recently, we have placed the same HybridSPE® sorbent we use in common SPE formats into a Dispersive Pipette Extraction (DPX) tip format. The HybridSPE®-DPX tips are available in a variety of tip types for use with manual, semi-automated or fully automated liquid handlers. In this case, the sorbent is allowed to disperse and mix freely within the sample solution (Figure 1). Using it, applications have been developed for analysis of a range of challenging analytes. These analytes include clenbuterol, warfarin, verapamil, steroid hormones, omeprazole, digoxin, as well as antineoplastic, antidepressant, antiarrhythmic and immunosuppressant drugs. A further example is segesterone acetate (Nestorone®), a synthetic progestin for female and male contraception. The compound was previously measured in serum by radioimmunoassay but had non-specific interferences which led to erroneous, false-positive levels being reported in men. HybridSPE® DPX Tips allowed a sensitive LC-MS/MS assay for segesterone acetate in human serum to be developed and validated for use in clinical and research studies.
To find out more about HybridSPE®-Phospholipid to remove phospholipids from biological samples, visit sigmaaldrich.com/hybridSPE
See applications in the MilliporeSigma chromatogram database for which HybridSPE® technology was successfully used: sigmaaldrich.com/hybridspeProtocol
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