Sample preparation online methods

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. 

In our laboratories, a cycle time of 90 sec can be achieved with a dilution factor of 1 25 for a given sample concentration, allowing the purity and identity control of two and a half 384-well microtiter plates per day. The online dilution eliminated an external step in the workflow and reduced the risks of decomposition of samples in the solvent mixture (weakly acidic aqueous solvent) required for analysis. Mao et al.23 described an example in which parallel sample preparation reduced steps in the workflow. They described a 2-min cycle time for the analysis of nefazodone and its metabolites for pharmacokinetic studies. The cycle time included complete solid phase extraction of neat samples, chromatographic separation, and LC/MS/MS analysis. The method was fully validated and proved rugged for high-throughput analysis of more than 5000 human plasma samples. Many papers published about this topic describe different methods of sample preparation. Hyotylainen24 has written a recent review. 

Because the instability of the N-oxide metabolite, which was subjected to decomposition during sample preparation (solvent evaporation during offline SPE), online SPE LC/MS became the method of choice for the application. Hsieh et al. (2004) built a system with two TFC cartridges and one analytical column, and another system with two TFC cartridges and two analytical columns for GLP quantitative bioanalysis of drug candidates. A Turbo C18 (50 x 1.0 mm, 5 /.mi, Cohesive Technologies), an Xterra MS C18 (30 x 2.0 mm, 2.5 /mi), and a guard column were used. Protein precipitation preceded injection. The cycle times for the two systems were 0.8 and 0.4 min. 

Online SPE LC/MS/MS is commonly used for bioanalytical applications in the pharmaceutical industry. Column switching systems and TFC systems are easy to build and control. Sophisticated commercial systems and SPE cartridges are readily available. Compared to offline sample preparation, the online approach can save time and labor. However, the development of online SPE bioanalytical assays remains analyte-dependent. Generic methods can be applied to many analytes. For extremely hydrophobic, hydrophilic, and ionic analytes at normal pH range and analytes with a variety of hydrophobicity and pKa values, analyte-specific methods must be developed. An understanding of the chemistry of the analytes and SPE is critical. 

The preparation of oil samples for the determination of pesticides by GC requires the complete removal of fat. The online combination of GPC and the GC method allowed trace-level determination of 17 OPPs in olive oil samples. Sample preparation was negligible when a column packed with PLGel for GPC and UV detector was used (70). 

LC-MS/MS techniques moved the bottleneck for analyzing NCEs from sample analysis to sample preparation. Several methodologies have been developed to reduce sample preparation time. Online extraction methods are becoming more popular and have many advantages over offline techniques. For example, online sample preparation is easier to automate and minimizes recovery issues. 

Online coupling of supercritical fluid extraction and high-performance liquid chromatography considerably decreases sample preparation time and analysis time [175]. Dunkers [128] showed that by using dilute dichloromethane as a static modifier, 20-30 minute supercritical fluid extractions gave results comparable to those obtained by conventional four-hour sampling methods in soil extractions. 

Arce et al. [39] developed a flow injection analysis (F1A) system (Fig. 5.3) for online filtration of water samples prior to CE analysis. They also constructed a pump-driven unit for extraction and filtration of soil samples combined with CE in an online mode (automated sample transfer between pre-CE sample preparation step and the CE) [40]. The method was precise and four times faster than conventional methods of sample preparation with an off-line unit. Blood samples are centrifuged immediately to remove red blood cells and the serum is stored as discussed above. Sometimes, urine samples also contain precipitates which are removed by centrifuge. 

Sample preparation in NLC and NCE is the most important step in analysis due to the nano nature of these modalities. The sampling should be carried out in such a way as to avoid changes in the chemical composition of the sample. The quantitative values of species depend on the strategy adopted in sample preparation. Extraction recoveries may vary from one species to another and they should, consequently, be assessed independently for each compound as well as for the compounds together. Materials with an integral analyte, that is, bound to the matrix in the same way as the unknown, which is preferably labeled (radioactive labeling) would be necessary, which is called method validation. As discussed above few papers described off- and online sample preparation methods on microfluidic devices. Of course, online methods are superior due to lower risk of contamination and error of methods. Not much work been carried out on online nanosample preparation devices, which need more research. Briefly, to get maximum extraction of analytes, sample preparation should be handled very carefully. 

An alternative method involving coupled LC-GC has been proposed (Grob et al., 1992), which considerably reduces analysis time as the only sample preparation is dilution of the oil to a 20% solution in hexane. HPLC removes the large amounts of triglycerides and isolates the steroidal hydrocarbons from other interfering components such as alkanes before online transfer of the steradiene fraction to GC. 

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