Hybrid Stationary Phases: The Answer to All of Your Matrix Effect Problems?

Keywords::

• hybrid stationary phase
• matrix effects
• phospholipids

Matrix effects can result in proportional errors in quantitative measurements by MS. They are commonly caused by signal enhancement or suppression resulting from co-elution of matrix components that compete with the target analyte for ionization or contribute to their ionization efficiency [1,2]. These components are often undetected at the mass transitions used for analysis, but can significantly affect analyte sensitivity and reproducibility [3]. Matrix effects can be of particular concern due to variability in the concentrations of matrix effect-causing components from one matrix source to another [4]. Furthermore, accumulation of endogenous matrix components on an analytical column may occur, and over time these components may start to elute or bleed from the column, leading to a difference between the responses of early injected samples and late injected samples during an analytical run, or from run to run [5].

There are many endogenous and exogenous components that have been shown to demonstrate ion suppression or enhancement reactions resulting in matrix effects. Phospholipids have been indicated as the main class of endogenous components that result in matrix effects in biologic samples [6]. Endogenous lipid components other than phospholipids, such as cholesterols and triacylglycerols, may also result in major matrix effects. Optimization of the sample extraction procedure is one of the most important approaches for minimization of matrix effects [7]. Selection of the most appropriate extraction approach, which yields the lowest degree of matrix effects for the matrix in question is an important consideration in LC–MS/MS method development. It is reported that no single extraction procedure has been efficient in removing all of these different lipid components [8]. Zirconia-coated silica (hybrid stationary phases) is a SPE material produced by many manufacturers; and is available in either a 96-well plate format or as cartridges. It is a simple sample purification stationary-phase material for which highly selective phospholipid binding and protein depletion have been claimed by the manufacturers. This stationary phase is also claimed to leave most analyte compounds unbound. Precipitated proteins are removed physically by filtration, which can be carried out as a separate step or in the well plate. Phospholipids are removed by selective interaction of the phosphate moiety of the phospholipids with zirconia ions functionally bonded to the hybrid stationary phase (Lewis acid–base interaction).

A hybrid stationary phase procedure was also found to be efficient for removal of cholesterols and triacylglycerols. This may have been due to an interaction with the stationary phase combined with the limited solubility of cholesterols and triacylglycerols in acetonitrile [8]. This interaction of cholesterol and triglycerides with the stationary phase should be further investigated by using other organic solvents to ensure capability of the hybrid SPE technique to remove these lipids without affecting recoveries of various analytes.

Hybrid stationary phases have been shown to minimize matrix effects in plasma samples [9]. An online extraction procedure using a POROS-R1 perfusion column with a switching valve configuration has been used with a hybrid stationary phase to reduce matrix effects [10]. Since two columns were necessary, this implies that in some cases, direct injection of the hybrid stationary phase extracts into an analytical column is not sufficient to minimize matrix ionization effects. A hybrid stationary phase has also been applied for the extraction of sirolimus from whole blood samples in order to minimize matrix effects. A somewhat complicated procedure was used for whole blood, which consisted of three steps; protein precipitation to remove proteins; solid-phase (octadecylsilane [ODS]) extraction to remove water-soluble compounds such as salts; and finally passage of the extracts over the hybrid stationary phase column to selectively remove phospholipids (mainly lysophosphatidylcholine which was unretained by the solid-phase ODS). Under the proposed elution conditions a C18 stationary phase retained phosphatidylcholine (one of the main phospholipid components and one of the main causes of matrix effects in LC–MS/MS). The procedure successfully eliminated ion suppression from the whole blood matrix [11]; however, the hybrid stationary phase was only one of several steps used to eliminate the matrix effects, suggesting that there are other matrix effect-causing sample components that must be removed by other means. Hybrid stationary phases can be used as an extraction step to selectively remove phospholipids but should not be considered a comprehensive one step procedure to remove matrix effects. Another multiple SPE extraction protocol that consisted of a hydrophilic–lipophilic balanced cartridge, hybrid stationary phase and a mixed-mode strong cation exchange (MCX) cartridge, respectively, has been shown to be necessary to reduce matrix effects from urine [8]. Differences in matrix effect causing compounds in urine, plasma and whole blood have been reported previously [9–12], which would suggest that hybrid stationary-phase extraction may be an important complement to other sample-extraction protocols, but that there may be some benefits and drawbacks to this technique when applied to different matrices. One should not consider hybrid stationary-phase benefits as described by the manufacturers to be absolute with regard to the many matrix effect-causing components in various matrices. Multiple step extraction procedures can also affect analyte recovery and method sensitivity. Nonphospholipid endogenous matrix components, such as inorganic water soluble components, other lipid components, fatty acids, concomitant drugs and metabolites, may require multiple extraction steps to ensure removal of matrix effect-causing components from sample extracts.

Exogenous compounds such as sodium citrate, phthalates, plasticizers and polyethylene glycol may also lead to ion suppression and these compounds are un-retained on the hybrid stationary phase materials [13]. Further extraction steps may be needed to ensure the absence of ionization matrix effects, depending on the type of sample and other factors, such as the type of anticoagulant used (lithium/heparin is known to cause matrix effects); additives such as surfactants and bovine serum albumin; and finally, the type of containers and seals used to store and extract samples.

In most literature that study the effectiveness of hybrid stationary phases for removal of matrix effects, hybrid stationary-phase extracts are compared with protein precipitation alone. Protein precipitation alone is known to yield a high degree of matrix effects in LC–MS/MS and it is more appropriate to compare hybrid stationary phases to more powerful purification techniques, such as SPE and supported liquid extraction (SLE). These have been shown to produce cleaner extracts than protein precipitation alone and SLE alone when compared with hybrid stationary phase alone, SLE alone demonstrated <0.007% of the phosphatidylcholine levels that were found in the protein precipitation extracts, while the hybrid stationary phase procedure produced approximately 0.02% [4]. This suggests the two phases were comparable, or even perhaps that the SLE extract was more effective in removing phospholipids.

The hybrid stationary-phase approach has been reported to merge the simplicity of protein precipitation-only procedures with the selectivity of SPE. High affinity towards phospholipids has been claimed while remaining nonselective towards a wide range of basic, neutral and acidic compounds. Lack of selectivity toward these analytes, however, could pose another potential problem in the use of hybrid stationary phases. Use of various SPE stationary phases other than hybrid stationary phases with optimization of washing and elution conditions have been shown to improve selectivity for target compounds, and LLE at different pHs is an effective way to purify basic, neutral and acidic compounds with regard to various unwanted sample components. The lack of selectivity of hybrid stationary phases with regard to various unwanted sample components limits their ability to remove interfering compounds and explains why hybrid SPE is often used in conjunction with other purification procedures.

The limited amount of sample volume that can be applied to some hybrid stationary phases (maximum 300 µl with phospholipid breakthrough potentially occurring with larger volumes) may be considered a problem in cases where larger volumes are needed for sensitivity enhancement. Larger sample volumes (up to 2 ml) can be used in SPE 96-well plates and single cartridges.

Analysis of large molecules through quantification of peptides from digests of proteins is a fast-growing endeavor in pharmaceutical and diagnostic sciences [14]. It remains to be seen whether or not this approach may encounter difficulties with the use of hybrid stationary phase for sample purification. However, low recovery of large molecules may be observed due to their retention with precipitated protein, which is a necessary step with the hybrid stationary phase approach.

Conclusion

Hybrid stationary phases may be a simple effective tool for selective removal of phospholipids from a variety of samples. It may be very useful as part of a matrix effects assessment protocol. However, use of hybrid stationary phase alone may not be sufficient, especially when different matrices such as whole blood and urine are evaluated. The costs of hybrid stationary phases are comparable to other SPE phases, although they should not be considered a full matrix effect solution and additional phases may be needed to achieve desired selectivity. Hybrid stationary phases should not be considered suitable for all applications, and potential drawbacks such as limited sample volume, low recovery and the potential need for multistep extraction procedures should be considered.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.