Direct injection column switching with

Haginaka, J. Wakai, J. Yasuda, H. Kimura, Y. Determination of anticonvulsant drugs and methyl xanthine derivatives in serum by liquid chromatography with direct injection column-switching method using a new internal-surface reversed-phase silica support as a precolumn. J.Chromatogr, 1990, 529, 455-461... 

Cass et al. [71] described a direct injection HPLC method, with column-switching, for the determination of omeprazole enantiomers in human plasma. A restricted access media of bovine serum albumin octyl column has been used in the first dimension for separation of the analyte from the biological matrix. The omeprazole enantiomers were eluted from the restricted access media column onto an amylose tris (3,5-dimethylphenylcarbamate) chiral column by the use of a columnswitching valve and the enantioseparation was performed using acetonitrile-water (60 40) as eluent. The analytes were detected by their UV absorbance at 302 nm. The validated method was applied to the analysis of the plasma samples obtained from 10 Brazilian volunteers who received a 40-mg oral dose of racemic omeprazole and was able to quantify the enantiomers of omeprazole in the clinical samples analyzed. 

Sample preparation Filter (0.5 p,m) serum, inject 200 p.L directly onto column A with mobile phase A, run with mobile phase A for 1.5 min then change to mobile phase B over 0.1 min, wash column A with mobile phase B for 10.5 min, backflush column A onto column B with mobUe phase C for 7.5 min then switch column B out of circuit, elute column B with mobile phase C and monitor the eluant, re-equilibrate column A with mobile phase A for at least 5 min. 

To determine ions at mid pg/1 to mg/1 (ppb to ppm) levels with IC, a sample size of 10 to 50/pi is sufficient. To determine ions at lower levels, then a preconcentration or trace enrichment technique has t3rpically to be utilized [20]. With this method, the analytes of interest are preconcentrated on another column in order to "strip" ions from a measured sample volume. This process concentrates the desired species resulting in lower detection limits. However, preconcentration has several disadvantages, compared with a direct method, additional hardware is required. A concentrator column is used to preconcentrate the ions of interest, a sample pump is needed for loading sample, an additional valve is often required for switching the concentrator column in and out-of line with the analytical column and extra time is required for the preconcentration step. It was of interest to explore the development of a high-volume direct-injection IC method that would facilitate trace ion determinations without a separate preconcentration step. This would represent a significantly simpler and more reliable means of trace analysis. 

An on-line MCAC-HPLC method was developed for OTC, TC, CTC, DMC residues in both liver and kidney samples. The drugs were extracted with succinate buffer, and the extract was diluted with EDTA-pentanesulphonic acid buffer. Diluted extract was then purified on C8 or XAD-2 SPE cartridges previously activated with MeOH, water, and PSA-containing phosphate buffer. The TCs were eluted with MeOH, and the eluate was then injected onto an Anagel-TSK-Chelate-SPW column that had been preloaded with copper(II) sulphate. The TCs were eluted directly onto the analytical column in a column-switching system after washing with both water and MeOH (29). 

HPLC with column switching and mass spectrometry was applied to the online determination and resolution of the enantiomers of donepezil HC1 in plasma [38]. This system employs two avidin columns and fast atom bombardment-mass spectrometry (FAB-MS). A plasma sample was injected directly into an avidin trapping column (10 mm x 4.0 mm i.d.). The plasma protein was washed out from the trapping column immediately while donepezil HC1 was retained. After the column-switching procedure, donepezil HC1 was separated enantioselectivity in an avidin analytical column. The separated donepezil HC1 enantiomers were specifically detected by FAB-MS without interference from metabolites of donepezil HC1 and plasma constituents. The limit of quantification for each enantiomer of donepezil HC1 in plasma was 1.0 ng/ml and the intra-and inter-assay RSDs for the method were less than 5.2%. The assay was validated for enantioselective pharmacokinetic studies in the dog. 

Good reproducibility has been reported for capillary supercritical fluid chromatography using a direct injection method without a split restrictor. This method (Fig. 1.2(b)) utilises a rapidly rotating internal-loop injector (Valeo Inst. Switzerland) which remains in-line with the column for only a short period of time. This then gives a reproducible method of injecting a small fraction of the loop into the column. For this method to be reproducible the valve must be able to switch very rapidly to put a small slug of sample into the column. To attain this a method called timed-split injection was developed (Lee Scientific). For timed split to operate it is essential that helium is used to... 

White, D.R., et al. 1991. Reverse phase HPLC/EC determination of folate in citrus juice by direct injection with column switching. J. Agr. Food Chem. 39 714-717. 

Determination of these compounds is carried out frequently in biological fluids. Analysis in urine requires a previous filtration to remove cells and other particulate matter then, the samples are diluted and directly injected onto the column. With cerebrospinal fluid, the samples are obtained by lumbar puncture each ahquot is centrifuged and decanted before analysis. Often in plasma or semm, some form of protein removal is needed because the presence of these compounds in injected samples can cause modifications of the column and bias in chromatographic results. Protein removal can be performed by various methods such as protein precipitation, ultrafiltra-tion, centrifugation, liquid-phase or solid-phase extraction, and column-switching techniques. 

---