Sample processing plasma

Esterbauer et al. (1991) have demonstrated that /3-carotene becomes an effective antioxidant after the depletion of vitamin E. Our studies of LDL isolated from matched rheumatoid serum and synovial fluid demonstrate a depletion of /8-carotene (Section 2.2.2.2). Oncley et al. (1952) stated that the progressive changes in the absorption spectra of LDL were correlated with the autooxidation of constituent fatty acids, the auto-oxidation being the most likely cause of carotenoid degradation. The observation that /3-carotene levels in synovial fluid LDL are lower than those of matched plasma LDL (Section 2.2.2) is interesting in that /3-carotene functions as the most effective antioxidant under conditions of low fOi (Burton and Traber, 1990). As discussed above (Section 2.1.3), the rheumatoid joint is both hypoxic and acidotic. We have also found that the concentration of vitamin E is markedly diminished in synovial fluid from inflamed joints when compared to matched plasma samples (Fairburn etal., 1992). This difference could not be accounted for by the lower concentrations of lipids and lipoproteins within synovial fluid. The low levels of both vitamin E and /3-carotene in rheumatoid synovial fluid are consistent with the consumption of lipid-soluble antioxidants within the arthritic joint due to their role in terminating the process of lipid peroxidation (Fairburn et al., 1992). 

It can readily be seen from this example that the contributions of the extrapolated areas to the total areas are relatively more important for the higher order moments. In this example, the contributions are 28, 61 and 72% for AUC, AUMC and AUSC, respectively. Because of this effect, the applicability of the statistical moment theory is somewhat limited by the precision with which plasma concentrations can be observed. The method also requires a careful design of the sampling process, such that both the peak and the downslope of the curve are sufficiently covered. 

There are a number of solutions that have been proposed to address the limitations on throughput in PK assays, including cassette dosing [37], where typically five compounds are dosed in a mixture, pooling of plasma samples from multiple animals receiving a specific dose, or the cassette-accelerated rapid rat screen where the processing of samples is streamlined [38]. 

In addition to online filters and precolumns, a simple protein precipitation step often precedes online SPE LC/MS/MS to prolong cartridge life. Protein precipitation can also reduce analytical interference and shorten chromatographic separation time. Since an internal standard (IS) solution is often added to plasma samples and centrifugation is used to remove possible particles before loading into the autosampler, protein precipitation does not add labor to the process. 

When using PFT with a neutral selector, it is quite difficult to avoid any entrance of the chiral selector into the ionization source, particularly at a high pH, where EOF is important. The use of BGE at low pH and/or coated capillary to minimize EOF is therefore mandatory. However, the coaxial sheath gas, which generally assists the ionization process, leads to an aspirating phenomenon of the chiral selector in the MS direction. Javerfalk et al. were the first to apply PFT with a neutral methyl-/i-CD for the separation of racemic bupivacaine and ropivacaine with a polyacrylamide-coated capillary and an acidic pH buffer (pH 3). Cherkaoui et al. employed another neutral CD (HP-/1-CD) with a PVA-coated capillary for the analysis of amphetamines and their derivatives. To prevent a detrimental aspiration effect, analyses were carried out without nebulization pressure. Numerous other studies presented excellent results such as the enantioselective separation of adrenoreceptor antagonist drugs using tandem mass spectrometry (MS/MS) the separation of clenbuterol enantiomers after solid-phase extraction (SPE) of plasma samples or the use of CD dual system for the simultaneous chiral determination of amphetamine, methamphetamine, dimethamphetamine, and p-hydroxymethamphetamine in urine. 

A fully automated on-line SPE-HPLC-MS-MS method was developed and validated for the direct analysis of 14 antidepressants and their metabolites in plasma by de Castro et al. [75]. After direct injection of 50pL of plasma without prior sample pre-treatment, gradient RP separation was completed in 14 min, with a sample throughput of 3h. LOQs were estimated to be at 10 ng/ mL. Analytes proved to be stable during sample process with the exception of clomipramine and norclomipramine. 

A characteristic of the plasma etching process which is generally observed is that the etch rate of a sample decreases as the area of the sample exposed to the plasma increases This dependence of etch rate on batch size is referred to as the loading effect and an example is shown in Fig. 3.8. The reason for the loading effect is simply that the etching process consumes a significant fraction of the... 

Liquid chromatographic methods based on ultraviolet and/or electrochemical detection have also been developed (622-625). In the earliest of these methods (623), tissues were extracted with mcthanol/water (1 1), and the evaporated residue was taken up in dichloromethanc. The extract was then injected on a Kieselgel Merckosorb SI-60 liquid chromatographic column, and eluted with dichloromethane/ethanol/water. Monitoring at 280 nm allowed 5 ppb thiouracils to be readily detected in the tissue samples. In the latest method (625), cattle plasma samples were extracted with etliyl acetate in presence of ethylenediamine-tetraacetate (EDTA). The addition of ED I A could significantly improve the efficiency of the extraction process. Remarkable improvement of the ethyl acetate... 

Using a Symbiosis system, Alnouti et al. (2005) and Li et al. (2005) developed online SPE-LC-MS/MS methods for analysis of rat plasma without any prior sample processing. Direct plasma injection resulted in accuracy of 88-111 % and 41 -108% with and without on-line SPE, respectively. The precision was improved from 3-81% without SPE to 0.5-14% with SPE. Furthermore, Alnouti et al. (2005) demonstrated that the cost of quantitative bioanalysis can be reduced by reusing the on-line SPE cartridges up to 20 times without loss of accuracy, precision or analyte recovery. 

Without MDF processing, the BPC for the LC-MS data showed no distinct indication of metabolite peaks in any of the plasma samples. After MDF processing, the majority of endogenous interference ions were removed. As a result, the BPC of the plasma samples displayed predominantly metabolite peaks. For example, Fig. 6.6a shows the BPC of a plasma sample without MDF filtering (1 50 dilution of HLM with human plasma, the equivalent of a 1.0-mL plasma injection). Using Fig. 6.6a... 

Figure 6.6. Metabolite profiles of omeprazole in human plasma (a) base peak ion chromatogram of unprocessed data and (b) base peak ion chromatogram of MDF-processed data exhibiting all metabolite peaks present and some endogenous peaks. High-resolution LC-MS data were obtained for a human plasma sample spiked with omeprazole metabolites generated by microsomal incubation (the equivalent of a 1.0-mL plasma injection). A MDF ivas set at 50 mDa around the apparent mass defect of the omeprazole ion.

In contrast with the previous method, Gao et al. [35] reported a GC-MS method which did not require prior derivatization for the analysis of rocuronium bromide and 17-desacetylrocuronium in biological samples. To prevent hydrolysis of rocuronium, solutions containing 1 M NaH2P04 was added to plasma samples, which were then stored at 20 °C prior to analysis. Similar with the previously described method, the internal standard was 3-desacetylvecuronium. The extraction process employed here also involved liquid-liquid ion pair extraction with saturated... 

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