Comparative study on the determination of the anti-neoplastic drug teniposide in plasma using micellar liquid chromatography and surfactant-mediated plasma clean-up.

The potential of micellar liquid chromatography and of an on-line surfactant-mediated sample cleanup, which involves column-switching prior to conventional reversed-phase high-performance liquid chromatography, has been evaluated for the determination of the anti-neoplastic drug teniposide in plasma by using electrochemical detection. A major advantage of surfactant-mediated techniques is that they allow fully automated processing of plasma samples, because protein precipitation is prevented by the addition of the surfactant sodium dodecylsulphate. With the automated column-switching technique, a degree of sample enrichment and of selectivity can be attained, which is similar to that for the conventional procedure which, however, involves a labour-intensive off-line isolation of teniposide, using liquid-liquid extraction prior to chromatography. An inherent drawback of automated micellar liquid chromatography is that no sample clean-up or preconcentration can be carried out, which results in only a moderate detection limit and selectivity. The linearity, reproducibility and recovery of the surfactant-mediated techniques are similar to those of the conventional procedure. Based on the presented results, it was concluded that the surfactant-mediated column-switching technique is a highly attractive sample enrichment technique with respect to simplicity, speed and cost.

Determination of penicillin in pharmaceutical formulations by flow injection analysis using an optimised immobilised penicillinase reactor and iodometric detection.

An automated assay for the determination of penicillin in formulations suitable for use in pharmaceutical quality control is presented. The method is based on the classical iodometric penicillin assay which is incorporated in a flow injection analysis (FIA) system. The required hydrolysis is performed on-line by using an immobilised penicillinase reactor. Packed-bed and single-bead-string enzyme reactors are compared. It turns out that a packed-bed penicillinase reactor (10 cm x 1.5 mm i.d.) provides complete hydrolysis within short residence time, while only little back-pressure is generated. This enzyme reactor is stable for at least 9 months. Enzymatic hydrolysis of the beta-lactam ring results in the formation of the corresponding penicilloic acid, which consumes iodine. The iodine consumption is determined colorimetrically by measuring the decrease of the absorbance of the blue coloured iodine/starch complex. The optimum reactor length and flow rate for the colourimetrical detection reaction are determined. The optimised method is applied to the assay of penicillin in formulations and the results are compared with the "true" results obtained with a reference method: a mercurimetric titration. The reliability of the flow injection method is evaluated quantitatively by determining the maximum total error (MTE). The reliability is shown to be highest when measuring at a 0.3-mM level. Eight formulations including capsules, tablets and injectables containing penicillin G, amoxicillin or flucloxacillin are assayed. The MTE does not exceed the 6% level and the most probable MTE is between 1.5 and 3.5%.

Automated reversed-phase chromatographic analysis of etoposide and teniposide in plasma by using on-line surfactant-mediated sample clean-up and column-switching.

An automated high-performance liquid chromatographic method for the plasma assay of two neutral drugs, etoposide and teniposide, involving direct plasma injection is presented. The problematic nature of protein precipitation has been circumvented by adding the anionic surfactant sodium dodecyl sulphate to the plasma at a final concentration of 38 mM. Plasma samples are loaded on to a clean-up column with an aqueous mobile phase with which the analyte(s) is (are) retained, whereas the solubilized plasma proteins are flushed to waste. Next, the retained compounds are eluted from the clean-up column on to the analytical column by using the chromatographic mobile phase with a higher elution capacity. The column-switching technique is used to achieve an automated assay. At least 10 ml of plasma, representing 100 repeated injections of 100 microliters or five repeated injections of 2 ml, can pass through the clean-up column without increasing the back-pressure. The recovery increased considerably from 10-30% to 90-95% on adding surfactant to the plasma samples prior to the analysis. The relative standard deviation of the proposed clean-up procedure is 3.5% (n = 6) for both drugs measured at the 2 micrograms/ml level without using an internal standard. The limit of determination with 100-microliters injections is 0.10-0.15 microgram/ml for ultraviolet detection and is seven times lower with electrochemical detection. Teniposide was determined in patients' plasma and the results agreed well with those obtained by the conventional procedure involving manual liquid-liquid extraction prior to chromatographic analysis.

Determination of glutathione in biological material by flow-injection analysis using an enzymatic recycling reaction.

A sensitive and specific assay for glutathione using a recycling reaction followed by spectrophotometric detection in a flow-injection analysis system is presented. The proposed method provides specific amplification of the response to glutathione by combined use of the enzyme GSSG reductase and the chromogenic reagent 5,5'-dithiobis(2-nitrobenzoic acid). Both oxidized (GSSG) and reduced (GSH) glutathione are detected, so that GSSG must be determined separately after alkylation of the GSH with N-ethylmaleimide. The sensitivity is controlled by the number of times the cycle occurs and therefore by the residence time of the sample in the reactor. This time depends on the reactor length and the flow rate. The influence of residence time, temperature, and enzyme concentration on the response has been studied and the optimum reaction conditions have been selected. The sample throughput is as high as 30 h(-1) and the detection limit is 1 pmol GSH at a signal-to-noise ratio of 3. The method has been evaluated by the quantification of GSH and GSSG in isolated hepatocytes. A high correlation between the new flow-injection analysis method and the original spectrophotometric batch assay has been found (slope = 1.039, intercept = 0.6, n = 216, r = 0.977). The main advantages of the proposed method are high sample throughout, high sensitivity, and good reproducibility.