Restricted-access media, RAM

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. 

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... 

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. 

Two recently introduced direct injection techniques have been developed to deal with the special problems posed by biological samples. These techniques involve the use of restricted access media (RAM) [11,12] and turbulent flow chromatography [13] and are described later. 

Another technique that allows for the direct injection of plasma or serum online with the chromatographic system uses an analytical coliunn containing particles referred to as restricted-access media (RAM) traditional laminar flow liquid chromatography is employed. These RAM particles are designed to prevent or restrict large macromolecules from accessing the inner adsorption sites of the bonded phase. Commercially available RAM columns, all... 

Many different sample preparation techniques are available to the drug discovery scientist. Off-line sample preparation procedures include protein precipitation, filtration, dilution followed by injection, liquid-liquid extraction (LLE), and solid-phase extraction (SPE). Typically, these procedures are performed in an automated, high-throughput mode that features a 96-well plate format. Online sample preparation procedures include SPE and turbulent flow chromatography (TFC) with conventional chromatographic media or restricted access media (RAM). These online approaches are often simple and easy to automate. 

Hollow fibers have proved to be useful to separate labeled hapten from the complex labeled hapten-Ab after a competitive on-line HPLC-immunochromato-graphic detection method [121]. First, hapten eluting from the HPLC column was made to react with Ab. Then, labeled hapten was added to react with the excess of Ab. Separation of free labeled hapten from labeled hapten-Ab was performed using a hollow-fiber module. The cross-flow membrane of this module allowed separation based on size, as opposed to a restricted-access media (RAM) column in which size and hydrophobicity account for separation [122]. No restriction on polarity and size of the label and no need for regeneration of the separation device arc characteristic of the use of hollow fibers. Although the method has been used for analysis of haptens, it could be used to analyze proteins as it has been shown that proteins of 40kDa can be separated from the corresponding protein-Ab complex if the appropriate hollow fiber is chosen. 

FIGURE 11.1 A generalized flow scheme that indicates the fundamental elements of LC-MS-based bioanalysis. Abbreviations LLE = liquid-liquid extraction SPE = solid-phase extraction RAM = restricted-access media TEC = turbulent flow liquid chromatography API = atmospheric-pressure ionization APCI = atmospheric-pressure chemical ionization ESI = electrospray ionization SQMS = single-quadrupole mass spectrometry TQMS = triple-quadrupole mass spectrometry TOF = time-of-flight Q-TOF = quadrupole TOF. (Reprinted from Ackermann et al. [4], with permission from John Wiley Sons, Inc.)... 

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