Restricted-access materials

Restricted-access materials (RAM) are biocompatible sample preparation supports that enable the direct injection of biological fluid into a chromatographic system. The technique was introduced in 1991 by Desilets et al., who also established the acronym RAM. Sorbents used in RAM represent a special class of materials that are able to fractionate a biological sample into a protein matrix and an analyte fraction, based on molecular weight cutoff. Macromolecules are excluded and interact only with the outer surface of the particle support, which is coated with hydrophilic groups. This minimizes the adsorption of matrix proteins. Applications of RAMs have been reviewed by several research groups. 

The basis of RAMs is the simultaneous size exclusion of macromolecules and extraction/enrichment of low-molecular-weight compounds into the interior phase via partition. The outer surface of the particles, which is in contact with biological matrix components such as proteins and nucleic acids, possesses a special chemistry to prevent adsorption of these molecules. Macromolecules can be excluded by a physical barrier, the pore diameter, or by a chemical diffusion barrier created by a protein (or polymer) network at the outer surface of the particle. RAMs can be classified according to the protein exclusion mechanism used into the following two groups RAM with a 

Oliveira and Cass described a method for the analysis of cephalosporin antibiotics in bovine milk using RAM columns for on-line sample clean-up. The system was composed of a RAM bovine serum albumin (BSA) phenyl column coupled to a C18 analytical column. Milk samples were directly injected after addition of 0.8 mM solution of tetrabutylammonium phosphate. The standard curve was linear over the range 0.100-2.50 tig/ml for five cephalosporin antibiotics (cefoperazone, cephacetril, cephalexin, cephapirin, and ceftiofur). The limits of quantification and detection reported were 0.100 and 0.050 ttg/ml, respectively. The method showed high intermediate precision [coefficient of variation percent (CV%) 2.37-2.63] and recovery (CV% 90.7-94.3) with adequate sensitivity for drug monitoring in bovine milk samples. 

Ding et al. described an automated on-line SPE-LC-MS/MS method for the determination of macrolide antibiotics, including erythromycin, roxithromycin, tylosin, and tilmicosin in environmental water samples. A Capcell Pak ME Ph-1 packed-column RAM was used as SPE column for the concentration of the analytes and clean-up of the sample. One millilitre of a water sample was injected into the conditioned SPE column, and the matrix was washed out with 3 ml high-purity water. By rotation of the switching valve (see Fig. 4.2), macrolides were eluted in the back-flush mode and transferred to the analytical column. The limits of detection and quantification obtained were 2-6 and 7-20 ng/1, respectively, which is suitable for trace analysis of macrolides. The intra- and inter-day precisions ranged within 2.9-12% and 3.3-8.9%, respectively. At the three fortification concentrations tested (20, 200, and 2000 ng/1), recoveries of macrolides ranged from 86.5% to 98.3%. 

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The first attempts employing two Cjg columns showed that the selectivity was not high enough, although this improved when the first column was substituted by a 5 p.m GFF n internal surface rcversed-phase material. This is known as a restricted-access-material (RAM) column which, since it restricts some compounds because of their size and includes rcversed-phase interaction and ionic exchange, is very useful for analysing herbicides in samples with high contents of humic and fulvic acids (54). 

Another solution to this particular problem is to use a restricted-access-material (RAM) column prior to the precolumn (54). 

FIGURE 9.1 Liquid chromatography workflow strategy options in proteomics. (a) bottom-up approach (b) top-down approach (c) selective sample cleanup directly combined with chromatographic separation (d) peptide capture with affinity restricted access material. 

Silica-based restricted access materials (RAM) have been developed for cleanup in bioanalysis, first for low molecular weight compounds in biofluids (Rbeida et al., 2005) and subsequently for biopolymers such as peptides (Wagner et al., 2002). A classification of different types of RAM has been given by Boos and Rudolphi (1997). Novel RAMs with strong cation-exchange functionality have been synthesized and implemented in the sample cleanup of biofluids. Racaityte et al. (2000) have shown that this type of RAM is highly suitable for the online extraction and analysis of... 

A wide variety of SPE materials and cartridges are commercially available for example, alkyl-diol silica-based restrictive access materials (RAMs) and a variety of silica- and polymer-based SPE materials of different binding abilities and capacities. Reversed-phase, size-exclusion, ion-exchange SPE, and turbulence flow methods will be discussed in this chapter related to real-world applications. 

Christiaens B. et al., 2004. Fully automated method for the liquid chromatographic-tandem mass spectrometric determination of cyproterone acetate in human plasma using restricted access material for online sample clean-up, J Chromatogr A 1056. 

Vintiloiu A. et al., 2005. Combining restricted access material (RAM) and turbulent flow for the rapid online extraction of the cyclooxygenase-2 inhibitor rofecoxib in plasma samples. J Chromatogr A 1082 150. 

Petrovic and coworkers [6] used PLE applying MeOH acetone (1 1) for the extraction of APEOs, APECs, APs and their halogenated derivatives from sediment samples. In combination with column-switching LC, using a restricted access material precolumn, hours of sample treatment were saved. 

High flow rate is generally the basis for the high-throughput efficiency of several Turbulent Flow Chromatography (TFC), Restricted Access Material (RAM), and monolithic columns methodologies. 

Ciogli, A. et al., A new chiral and restricted access material containing teicoplanin as selector (chiro-RAM-TE) for the HPLC determination of chiral drugs in biological matrices, presented at 16th Int. Symp. on Chirality, New York, July 11-14, 2004, 62. 

The sample homogenization with an MeCN THF mixture was used for the simultaneous determination of SMM, miloxacin, and oxolinic acid. The supernatant was filtered and injected directly into the ion-pair chromatographic system using a shielded hydrophobic phase. This method did not require time-consuming and complex extraction procedures moreover, the use of a restricted-access-material column prevented both column clogging and peak broadening throughout the analysis. On the other hand, no preconcentration of the sample affected the LOD... 

The simultaneous HPLC-UV determination of sulfamonomethoxine (SMM), miloxacin (MLX), and OXO in serum and muscle of cultured fish was developed (153). A sample of muscle was extracted with MeCN-THF (95 5) after centrifugation, the supernatant was injected into the HPLC system. A Hisep column, used in this study, is packed with restricted-access materials (RAMs) consisting of the polymeric hydrophilic/hydrophobic phase bound to silica gel. This column did not require time-consuming and complex extraction procedures. The RAM sorbent could also be applied in short precolumns, which are combined directly on-line with the HPLC equipment. This approach is much more convenient than that applied in the present paper. The guard column had to be changed very often in order to protect the analytical column in a sufficient way. The extraction recovery was 79.5%, RSD of 6.0%. 

Souverain, S., Rudaz, S., and Veuthey, J. L. (2004b). Restricted access materials and large particle supports for on-line sample preparation An attractive approach for biological fluids analysis. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 801 141-156. 

Body fluids, such as serum, contain several different carotenoids in low amounts. The crucial point in the isolation and analysis of these samples is the enrichment factor. Serum samples can be directly analysed with hyphenated extraction-sample enrichment-separation systems, such as on-line SPE-HPLC employing tailored stationary phases [29]. By using special restricted access materials (RAMs) for sample enrichment, the carotenoids are retarded on the pre-column while the protein binding is broken and the macromolecules are eluted. The preparation of artifacts is hindered, as the whole analysis steps take place under conditions of light- and oxygen-exclusion. The scheme of on-line SPE-HPLC is presented in Figure 5.2.2. 

Conversely, restricted access materials or restricted access media (RAM) retain small molecules while excluding macromolecules such as biological proteins in their presence (Figure 2.29). Small molecules are retained by sorption processes in the pores of the sorbent while the large molecules are excluded and elute at the interstitial volume of the sorbent. This separation leads to size-selective disposal of interfering macromolecular matrix constituents. 

Schlauch, M., Fulde, K., Erahm, A.W. Enantioselective determination of (R)- and (S)-sotalol in human plasma by on-line coupling of a restricted-access material precolumn to a cellobio-hydrolase I-based chiral stationary phase. J. Chromatogr. B 775, 197-207 (2002)... 

Restricted-access material (RAM) columns combine the size-exclusion of proteins by the hydrophilic outer surface of the packing and the simultaneous enrichment by SPE of analytes that interact with hydrophobic groups at the inner surface of the packing. These columns allow the direct injection of plasma samples without protein precipitation. Often, on-line RAM-LC-MS is described, following a procedure identical to on-line SPE-LC-MS (Ch. 1.5.4). The use of RAM columns has been reviewed by Souverain et al. [29]. 

Restricted-access materials (RAM, Ch. 1.5.6), as demonstrated for the analysis of triazines in water [81] and of azole pesticides in urine [82],... 

The use of on-line immrmoaffinity extraction in combination with coupled-column LC-MS-MS was demonstrated for the characterization of benzodiazepine libraries [34]. The benzodiazepine library was injected onto a Protein G colurtm loaded with benzodiazepine antibodies. The benzodiazepine-antibody complexes are stripped of the column at low pH and eluted to a restricted-access material in order to separate the benzodiazepines from the antibodies. In the last step, the compotmds interacting with the benzodiazepine antibodies are separated on a Cg-teversed-phase colunm and identified by MS-MS. 

Solid-phase extraction (SPE), which can be done off-hne or on-line by means of cartridges, cartridge columns, or extraction disks (Ch. 11.6.4), and eventually by using special packing materials, such as restricted access materials (RAM) or turbulent-flow chromatography (TFC) (Ch. 11.6.5). 

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