The LCGC Blog: Restricted-Access Media for Biomonitoring Applications: A Solution That Deserves More Attention

Our research group works on a wide variety of analytical chemistry topics. This is what makes my job so interesting. We are regularly approached with a problem that needs to be solved, and often it is something that we have not thought about previously. The joy comes in evaluating the needs of a particular analysis. We figure out the best course of action, design and carry out the experiments, and in the course of this work, think about what we can do to make our work better, faster, and cheaper. As such, we are always operating on a learning curve, which is a good thing. I have much less interest in performing “routine” analyses in my own laboratory, unless they are ones that we have made routine through our own efforts.

In my mind, one of the most challenging topics in small molecule research is targeted ultratrace (sub parts-per-billion) quantitative analysis from biological fluids (that is, biomonitoring). Biological fluids comprise a wide variety of compounds of varying sizes, physical character, and abundance. In our laboratory, we have worked on plasma, serum, saliva, urine, seminal fluid, and cerebrospinal fluid. Our analytical technique of choice is liquid chromatography–mass spectrometry (LC–MS), but as anyone knows, the sample preparation is as critical to successful analysis as any other part of the workflow. To achieve the most sensitive and reproducible analysis, analytes should be segregated from proteins, lipids, and salts, which can foul separation hardware and reduce signal intensity. In the spirit of avoiding time-consuming and resource-intensive traditional off-line sample preparation techniques, such as liquid–liquid extraction, solid-phase extraction, or protein precipitation, we have placed significant effort in the use of restricted-access media (RAM) to achieve efficient and reproducible on-line sample preparation.

The term RAM refers to specialized chromatographic phases that combine a size-exclusion facet of separation with some of other retention mechanism (1,2). In our lab, we currently use internal surface reversed phase (ISRP) RAM. In these media, the inner pores (~120 Å) of a silica gel particle are functionalized with typical reversed-phase chemical moieties (such as C18, C8, or C4). The outer surface of the particles is hydrophilic (consisting of a material such as methyl cellulose). Thus, when a complex sample is injected onto the phase, large molecules that cannot access the pores are not retained, whereas small molecules are. With biological fluids, proteins and salts can be washed from the ISRP RAM so that they do not complicate the analysis when the analytes in the trap column are subsequently eluted onto the analytical column.

Surprisingly, although RAM have been around for a long time (since about 1985 (3)), it has not been highly used in the mainstream by researchers. Could it be a coincidence that approximately 20 years passed since the first reports of RAM (also presumably, the filing of patents) and the significant offerings of these materials by major commercial manufacturers?

In our lab, we have worked primarily on developing methods for ultratrace analysis of endocrine disruptors and estrogens by LC–MS-MS. Our first work with the ISRP RAM was to develop a method for determination of bisphenol A (BPA, a common polymer additive and endocrine disruptor) in human saliva (4). We performed dansyl chloride derivatization of BPA to achieve detection limits in the low parts-per-trillion concentration range (I will talk specifically about the merits and limits of derivatization for LC–MS in my next blog post). We also demonstrated the capability of ISRP RAM for multiple injection loading as a preconcentration strategy. Of course, in comparison to other biological fluids, saliva is pretty simple; it does not have a lot of proteins and lipids. In more complex matrices, such as plasma, even the high abundance of proteins is not a large problem, as long as the analyte of interest does not bind strongly to them. However, lipids remain a big challenge because they too will be retained by the reversed-phase media in RAM. We are now working on the determination of dansyl-derivatized estrogens in plasma and cerebrospinal fluid, and we find that the highly hydrophobic nature of the compounds causes them to be retained to a similar degree as high abundance phospholipids. Thus, when the trapped compounds are eluted to the analytical column, the presence of lipids in the electrospray droplets induces significant ion suppression relative to matrix-free analysis. It is a problem that we are currently working to solve through the variation of loading conditions and trapping phases.

Overall, the advantages of RAM still outweigh potential limitations. In laboratory tests, we have found that RAM provide higher recovery than other off-line sample preparation techniques, such as solid-phase extraction. The workflow is also less time-consuming than liquid–liquid extraction. The fact that RAM offer the ability to perform all sample preparation on-line is a major advantage. Although lipid interferences remain a problem when highly hydrophobic analytes are to be determined, RAM have been demonstrated to be effective in the analysis of a wide variety of analytes present in a wide variety of matrices. And, to address some of the problems with coeluted interferences, materials that provide alternative internal surface retention mechanisms are available.

I think that as more researchers demonstrate the promise of RAM, more manufacturers will provide products to enable their routine use in the analytical laboratory. Given the many advantages of RAM, that would be a welcome development.

References

(1) N.M. Cassiano, V.V. Lima, R.V. Oliveira, A.C. de Pietro, and Q.B. Cass, Anal. Bioanal. Chem.384, 1462–1469 (2006).

(2) P. Sadilek, D. Satinsky, and P. Solich, Trends Anal. Chem. 26, 375–384 (2007).

(3) I.H. Hagestam and T.C. Pinkerton, Anal. Chem. 57, 1757–1763 (1985).

(4) S.H. Yang, A.A. Morgan, H.P. Nguyen, H. Moore, B.J. Figard, and K.A. Schug,. Environ. Toxicol. Chem. 30(6) 1243–1251 (2011).

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