Restricted access column

Fig. 5.14 Principle of label-free ligand binding MS assays. Protein (P) molecules react with the test ligand (L) to form a protein-ligand complex (PL). Unbound compounds are separated from PL by passage through a restricted-access column. Subsequently, PL is dissociated at low pH, and active ligands L are detected by LC-ESI-MS.

Fig. 5.17 Demonstration of MS-based bioassay functionality using a plant extract. MS instrument Ion-trap mass spectrometer (LCQ Deca, Thermo Electron), (a) MS analysis of pure extract by direct injection onto restricted-access column 2 in the absence of affinity protein, (b) Analysis of the same natural extract spiked with digoxin using the label-free MS assay method as shown in Fig. 5.15.

In other bioanalytical applications, the emphasis lies more on overcoming cross-reactivity. For example, the heart glycoside digoxin is cross-reactive with several of its metabolites as well as with plasma constituents, thus hampering approaches solely based on immunoassays. By treating plasma samples with sohd-phase extraction on a restricted access column, coupled online to reversed-phase LC/immunodetection, digoxin and two of its cross-reactive metabolites were analyzed in patients treated with the heart glycoside. 

When operating restricted access columns, some basic rules should be followed. It is strongly recommended that the analysis be performed within the linear range of the adsorption isotherm of the sorbent in question in order to avoid the over-... 

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). 

E. A. Hogendoorn, E. Dijkman, B. Baumann, C. Hidalgo, J. V. Sancho and E. Hernandez, Strategies in using analytical restricted access media columns for the removal of humic acid interferences in the trace analysis of acidic herbicides in water... 

Restricted access phases are another approach to exploiting the differences in characteristics of analytes. Large analytes are excluded from an internal surface on which an adsorptive stationary phase is present. A herbicide analysis for Metsulfuron methyl, Bentazone, Bromoxynil, methylchlorophenoxy acid, and Mecoprop in the presence of humic acid was performed on restricted access reversed phase media.52 The cytostatic compound epirubicin and its metabolites were separated from plasma using a Pinkerton GFF II column.53 Gradient separations of polymers on reversed phase and on normal phase represent an alternative to gel permeation chromatography. Polyesters of noncrystalline materials were separated on a variety of such phases.54... 

Direct injection of pretreated biological samples (also called online sample cleanup) greatly simplified sample preparation for LC/MS/MS analysis. The normal process involves sample aliquot steps, internal standard addition, and centrifugation. Compared to traditional off-line LLE and SPE sample preparation procedures, online methods are easier and faster. Two types of online SPE columns are commercially available. One is the restricted access media (RAM) column. The other is the turbulent flow chromatography (TFC) column. 

Viehauer S. et al., 1995. Evaluation and routine application of the novel restricted-access precolumn packing material Alkyl-Diol Silica Coupled-column high-performance liquid chromatographic analysis of the photoreactive drug 8-methoxypsoralen in plasma. J Chromatogr B 666 315. 

Many other types of solid phase adsorbents, including those based on conventional and specialty materials like restricted access media (RAM), can increase analysis speed and improve assay performance. These types of materials, also known as internal reversed-phase packings, are especially useful for assaying target compounds in biological samples such as serum and plasma. They are chemically modified porous silicas that have hydrophilic external surfaces and restricted-access hydrophobic internal surfaces. The ratio of interior to external surface areas is large. Macromolecules such as proteins cannot enter the pores of the RAM (they are excluded from the hydrophobic internal surface) and they elute quickly through the column. However, the smaller analyte molecules that can enter the pores are retained via interactions with the hydrophobic bonded phase within... 

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. 

Gustavson et al. (2000) developed a convenient and novel solid phase extraction (SPE) method for the removal of methyl oleate from SPMD dialysates containing PAHs. A small SPE column (1 g or 0.5 g) containing a dual-zone silica (normal phase)-based restricted-access sorbent (Diazem, Midland, MI, USA) is used for the separation. The capacity of this sorbent to remove methyl oleate is about 1.8% (lipid/sorbent wt wt ). The PAHs are eluted with 19 mL of hexane and methylene chloride (97 3 VV ) and recoveries of all PAHs are typically >72%. 

On the basis of the separation mechanism, restricted-access media can be classified into physical or chemical diffusion barrier types. The limited accessibility of the former type is due to the pore structure of the support that represents physical diffusion barriers for macromolecular compounds. The restricted access of the latter type is due to covalently or adsorptively bonded synthetic or natural polymers that cover the support surface, preventing macromolecules from being adsorbed on or denatured by the column packing material. 

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%. 

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