Online sample cleanup

Rbeida, O., Christiaens, B., Hubert, Ph., Lubda, D., Boos, K.-S., Crommen, J., Chiap, P. (2005). Integrated online sample cleanup using cation exchange restricted access sorbent for the LC determination of atropine in human plasma coupled to UV detection. J. Pharm. Biomed. Anal. 36/5, 947-954. 

Achiral-chiral multidimensional chromatography remains one of the best ways to separate chiral analytes from interfering matrix components or other compounds. The flexibility offered by different operation modes, stationary and mobile phases, and configurations allows analysis methods to be tailored to the analytical problem. By offering possible configurations for both online sample cleanup and concentration, achiral/chiral LC/LC reduces manual sample preparation. The ability to be coupled to... 

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. 

The typical scheme for an online sample cleanup procedure incorporating two pumps and a six-port switching valve is shown in Figure 2. Pump B is used to deliver the mobile phase and pump A is used to deliver the wash solvent, which is usually water or buffer, though a small percentage of organic solvent is sometimes added. In position 1, the wash solution... 

Pre- and guard columns can be used for online sample cleanup in conjunction with LC, which helps maintain sample integrity while protecting expensive and fragile stationary phases. Samples may also be preconcentrated prior to elution onto separation column. 

Spectroscopic, luminescence, turbidimetric, and electrochemical methods of detection have been combined with SIA for the successful determination of amino acids, sugars, and trace elements in matrices such as meats, vegetables, breads, wines, juices, and milks. Many of these methodologies required sample pretreatment and whilst most performed this in an offline manner there have been some reports of online sample cleanup. Microwave assisted digestion was performed in-line for the determination of phosphorous, calcium, magnesium, and iron in slurried foodstuffs, wine, milk, and soft drinks whilst gaseous diffusion allowed interference removal for the determination of urea in milk. 

Restricted access materials (Figure 9.8) are mostly used with online sample cleanup and enrichment of protein-rich biological samples. The crude samples, even as complex as serum or plasma, can be injected without any pretreatment. The commercially available RAM cartridges are reusable. The sorbents (alkyl-diol sUica ADS) are hydrophilic on the outside, while the surface in the pores is modified with hydrophobic groups (C4, C8, or C18). 

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... 

Using mentioned extraction/deproteinization procedures, the obtained aqueous or organic extracts often represent very dilute solutions of the analyte(s). These extracts may also contain coextractives that, if not efficiently separated prior to analysis of the final extract, will increase the background noise of the detector making it impossible to determine the analyte(s) at the trace residue levels likely to occur in the analyzed samples. Hence, to reduce potential interferences and concentrate the analyte(s), the primary sample extracts are often subjected to some kind of additional sample cleanup such as liquid-liquid partitioning, solid-phase extraction, or online trace enrichment and liquid chromatography. In many instances, more than one of these cleanup procedures may be applied in combination to allow higher purification of the analyte(s). 

To reduce coextractives in the primary sample extract and concentrate the analyte(s), various types of sample cleanup procedures can be applied. They include conventional liquid-liquid partitioning, solid-phase extraction, matrix solid-phase dispersion, and online dialysis and subsequent trace enrichment (Table 29.5). In many applications, more than one of these procedures is applied in combination to decrease the background noise of the detector, thus making it possible to quantify trace level residue concentrations. 

Owing to its high specificity and sample cleanup efficiency, immunoaffinity chromatography has also received widespread acceptance for the determination of -agonists in biological matrices (470, 471, 473, 475, 479, 487, 490). The potential of online immunoaffinity extraction for the multiresidue determination of -agonists in bovine urine was recently demonstrated, using an automated... 

Sample cleanup with an NP-HPLC column has been shown to be an efficient, robust way to separate triglycerides from organochlorine compounds for analysis in a wide range of fatty samples, such as milk, pork fat, animal feed, and cod liver (67). Complete fat-OCP separation is obtained in a small fraction volume. The method showed average recoveries of 80-110% in the concentration range of 1-510 /zg/kg, with relative standard deviations of less than 10%. The limits of detection ranged from 0.5 to 50 /ug/kg. The process can be monitored online with a UV detector. 

The major difficulty in analyzing OPPs in fatty samples has to do with the wide polarity range for both pesticides and lipids present in the matrix. Normal-phase HPLC is an adequate technique for cleaning up this type of sample using silica gel and modifiers with different polarity. In fact, an automated sample-cleanup system based on normal-phase HPLC using a silica gel column has been reported efficiently to clean up and fractionate chlorpyriphos, chlorpyriphos methyl, and their metabolites in molluscs. The system presents several advantages The procedure is fully automated, from the injection of the extract to the collection of fractions, which are injected directly into the GC system, and a diode array detector (DAD) allows online monitoring of the elution of lipids (68). 

The general methodology for online cleanup and concentration is the same as that used for isocratic focusing when assaying for known compounds, but sample cleanup and transfer steps are repeated as many times as necessary to concentrate the sample. Once the concentration is complete, elution from the analytical column occurs. LC-MS responses of a single standard injection to one that has been concentrated 10 times (10 injections) are compared in Fig. 10.12. Note that the response remains linear as long as the trapping analytical column is not over loaded. 

Figure 10.13. Online cleanup and concentration method (a) sample cleanup (b) sample transfer (c) sample elution, clean turbulent-flow column, and fill loop (6) sample elution, equilibrate turbulent-flow column (Herman, 2005). 

Different reversed phase [195,239,240], mixed mode (ion exchange and reversed phase) SPE cartridges [173,218] and online SPE column [193, 238] have been also reported for samples preparation and extraction. Some of these assays combined both PP and SPE in order to achieve an extensive sample cleanup [193, 195, 238-240], Likewise SPE, LLE provides cleaner plasma extracts than PP. Nevertheless, LLE procedure does not always provide satisfactory results with regard to extraction recovery and selectivity, especially with polar analytes and particularly in the case of multicomponent analysis such as in drug-metabolism studies, where analytes polarity varies widely. This issue was addressed by Zweigenbaum J and Henion J [235] and extraction solvent optimization, using isoamyl alcohol, to achieve acceptable extraction selectivity and recovery for polar analytes has been discussed. 

Mitulovic etal., 2003). An additional benefit of online HPLC/ESI-MS is that sample cleanup, analyte concentration and separation are accomplished in an automated process. However, polypeptides of high hydrophobicity are often retained on reversed-phase columns using a standard gradient and will therefore not be transferred into the MS device for mass analysis. Additionally, very hydrophobic peptides and proteins (e.g., membrane proteins) tend to precipitate in the small glass capillaries during the electrospray process, which leads to the breakup of the spray. 

Marchi et al. [64] reported on the utility of various sample cleanup procedures for reducing the matrix effects that are caused by various plasma constituents. They found that the best sample preparation procedure was to use PPT followed by an online SPE system. The authors also stated that with this sample preparation procedure, atmospheric pressure photoionization (APPI) was the least affected by matrix effects, followed by atmospheric pressure chemical ionization (APCI) and then electrospray ionization (ESI). 

SPE on ODS or Tenax can be used for sample cleanup in online extraction-analysis systems. Cleanup of extracts in polar solvents is generally performed using... 

Figure 2 Instrument arrangement for online solid-phase extraction with column switching. Pa and Pb, pumps Ci, extraction column for the online SPE C2, analytical column Vi and V2, injection and switching valves position 1, switching valve is positioned for online SPE cleanup and preconcentration position 2, switching valve is positioned to transport sample to the analytical column.

Today, there is strong interest in the development of online sample treatment techniques that allow the handling of untreated biological samples. Thus, in online SPE-LC, deproteination of plasma and serum is required before extraction, especially if the same cartridge is used for repeated analysis. For this purpose, restricted-access materials (RAMs) have been developed, which combine size-exclusion and reversed phase mechanisms, allowing extraction and cleanup of samples in the same step. RAMs have became quite popular for the direct injection of biological fluids, since they prevent the access of matrix components (e.g., proteins) while retaining the analytes in the interior of the sorbent. 

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