4. FULLY AUTOMATED PRETREATMENT OF BIOLOGICAL
4.2. Fully Automated Pretreatment Systems Coupled On-
The chief restrictions arising from the combination of a CFS and a gas chromato- graph are due to the different aggregation states of the carrier and the mobile phase, and also to the pressure difference. The interface must be designed to cause minimal reversible changes in the gas chromatograph in such a way that it can be used in the conventional injection mode, using an injected volume of only a few microliters and adapting the split ratio to it.
The simplest way of directly coupling a home-made CFS to a GC–MS instrument is by using a high-pressure injection valve [32,43]. A schematic dia- gram of the interface is shown in Figure 6. The outgoing organic stream from
262 Valca´rcel et al.
Figure 6 Scheme of a CFS for the preconcentration/determination of abuse drugs in human serum and urine. IV⫽injection valve; W⫽waste. See text for details.
the CFS fills the 4.5àl loop of the injection valve (inner volume 2.5àl), con- structed from polytetrafluoroethylene (PTFE). A single helium input through the injection valve at a flow rate of 0.7 ml/min allows the organic plug to be trans- ferred to the gas chromatograph as the valve is switched, which shuts the carrier inlet to the instrument. A stainless steel tube (70 mm⫻0.3 mm ID) furnished with an injection needle was fitted to the carrier outlet and the needle was inserted into the septum of the injection port [43]. The interface unit was kept at room temperature and no instrumental modifications were required. Various drugs of toxicological interest (viz., methamphetamine, alphaprodine, lidocaine, metha- done, cocaine, and codeine) were determined in human urine and serum by using this on-line configuration. Dilute samples (equivalent to 1 ml of urine and serum) were continuously aspirated into a CFS similar to those previously described;
drugs were adsorbed onto a C18 column packed with approximately 40 mg of sorbent and then eluted with CHCl3; under these conditions, the column can be
Drug Determinations in Biological Fluids 263
reused for at least three months. The analytical features of the method are summa- rized in Table 2. When using this on-line configuration, the split ratio of the instrument must be increased at least fourfold in order to avoid saturation of the stationary phase.
Finally, an interface similar to that previously described was developed for the direct coupling of a commercially available automated sample preparation with extraction columns (ASPEC) unit and large-volume PTV–GC–MS [44].
The interface between the ASPEC system and the programmed temperature va- porization (PTV) injector was made from the standard Rheodyne injection valve of the ASPEC unit; however, the eluate was transferred in a discrete manner.
Minor instrumental modifications were needed for the coupling; an injected vol- ume of 100àl was chosen and two gas chromatographs (on-line connected) were used. Although the proposed system was not applied to drug analyses—only to determine caffeine spiked to human urine—it will probably be used for the determination of drugs. A 4-ml sample was passed through a 200-mg C18cartridge previously conditioned with methanol and water. After retention, the cartridge was cleaned up withn-hexane and caffeine was finally eluted with 4 ml of CHCl3. The cartridge cannot be reused. No analytical features were given other than the reproducibility of the large injected volume (100 àl), which was found to be 6.9%
Although the examples clearly show the potential of the method for han- dling sample volumes in the ml region, one must admit that on-line coupled SPE–GC–MS has not yet become such a routine application as on-line SPE–
LC, mainly because of the incompatibility of the chromatographic system with aqueous phases (the final injected volume must contain absolutely no traces of water if deactivation and deterioration of the chromatographic column are to be avoided). This problem can be especially serious when using polar organic sol- vents (methanol, acetonitrile, etc.) as eluents for the retained compounds.
5. CONCLUSIONS
The high potential of GC–MS for identifying and quantifying drugs and their metabolites in biological fluids depends critically on preliminary analytical opera- tions, which are the source of major errors, both systematic and random, that strongly affect the quality of the results upon which decisions must be based.
Matrix problems in biological fluid analysis are the most serious ‘‘bottleneck’’
for the corresponding analytical processes. Any improvement in sample prepara- tion also improves the quality of the analytical information derived. This chapter demonstrated the potential of automated systems coupled to GC–MS for such a purpose and the state of the art and prospects in this respect.
264 Valca´rcel et al.
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Gas Chromatography–Mass Spectrometry Analysis of Anesthetics and Metabolites Using Multidimensional
Detection Strategies