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Transplant patients receiving the immunosuppressive drug cyclosporin A require regular monitoring to maintain levels within a narrow therapeutic range. A stable, accurate and reproducible high-performance liquid chromatographic method for analysis of cyclosporin A in whole blood has been developed using the Varian Advanced Automated Sample Processor. Starting with 200 microliters of blood, absolute recovery of both cyclosporin A and the internal standard was 81% with a detection limit of 12.5 ng/ml. The assay is perfectly linear over the range 0-1000 ng/ml (r2 = 1.0). At a concentration of 250 ng/ml, the coefficient of variation, both between samples and between assays, is 1.87%. Chromatographic cycle time is 10.2 min per sample. Up to eighty samples can be processed by one person in a working day, with final results within 16 h.

A highly sensitive and semi-automated high-performance liquid chromatographic method, utilizing acetonitrile protein precipitation and column-switching, is described for the determination of cyclosporine A in whole blood. Following a rapid manual acetonitrile treatment of the blood samples, the supernatant is loaded automatically onto a 5-micron high-speed protein separation column without any further clean-up operations. The fraction containing cyclosporine A is switched to a 3-micron C18 reversed-phase high-speed column by a microprocessor-controlled column-switching unit for final separation and detection by absorption at 214 nm. Minimal sample handling and efficient separation resulted in a high recovery (75 +/- 3%) of cyclosporine A from blood and a detection limit as low as 2 micrograms/l with a highly reproducible and linear response up to 2500 micrograms/1 using 0.5 ml of sample. A separation cycle including regeneration of the first column is finished in 15 min, and this system was used continuously for ca. 1000 blood samples from heart, liver, kidney, pancreas and bone marrow recipients without change in separation parameters or material replacement. The method described allows accurate and very fast daily routine monitoring of cyclosporine A in large numbers of blood samples from transplant recipients.

A sensitive, semi-automated high-performance liquid chromatographic method utilizing column switching is described for the determination of cyclosporine in plasma and blood. This method involves a short and improved manual protein precipitation of the sample followed by an automated clean-up of the supernatant. After automatic loading of the clean supernatant onto an LC-8 column for initial separation, the segment containing cyclosporine is loaded (automatically) onto an LC-18 column for final separation and quantitation. Cyclosporine is detected by its ultraviolet absorption at 202 nm. The rate of analysis was four samples per h running 24 h per day (ca. 100 samples per day). The method is sensitive enough to measure with confidence cyclosporine concentrations of 8 microgram/l in plasma and 20 microgram/l in blood with a linear response up to 2500 microgram/l using only 0.5 ml of sample. No internal standard is required. The method was applied continuously (24 h per day) to approximately 1000 samples without deterioration in method parameters.

We describe a rapid, precise, cost-effective, and accurate isocratic liquid chromatographic (LC) procedure for determining cyclosporine in whole blood. The cyclosporine is extracted from 0.5 ml of whole blood together with 200 micrograms of cyclosporin D, added per liter as internal standard, by using an Advanced Automated Sample Processing (AASP) unit. The on-line solid-phase extraction is performed on an octasilane sorbent cartridge which is interfaced with a Perkin-Elmer 83 X 4.6 mm I.D. cartridge column, packed with 3-micron octadecyl packing. The column is eluted with a mobile phase containing acetonitrile-water (13:7) at a flow-rate of 1.0 ml/min at a column temperature of 70 degrees C. The column effluent is monitored at 210 nm. The absolute recovery of cyclosporine exceeded 87% and the linearity extended up to 2000 micrograms/l. Within-run and day-to-day coefficients of variation were less than 8%. The correlation between AASP-LC and manual Bond-Elut extraction-LC method was excellent (r = 0.97).

A simple reliable liquid chromatographic method for assay of cyclosporin A in serum or urine is described. Samples were cleaned up on a solid-phase extraction system (cyanopropyl column). The system involved a reversed-phase C18 Ultrasphere column maintained at 72 degrees C and an acetonitrile linear gradient (65 to 95%) in 0.14% triethylammonium phosphate. Liquid chromatographic analysis of radioimmunoassay standards shows that some samples contain a contaminant peak. Comparison of cyclosporin A levels obtained by radioimmunoassay and high-performance liquid chromatography in clinical investigations show that the former values are generally, but not always, higher than the latter, and that cyclosporin A is very differently metabolized depending on the patient, disease and treatment.

The large inter-patient variability in cyclosporin pharmacokinetics coupled with the agent's narrow therapeutic index with adverse effects resulting from supratherapeutic levels, necessitates individualization of drug dosage and therapeutic monitoring of cyclosporin blood levels. The performance of a liquid chromatographic method for the measurement of cyclosporin was evaluated and the results obtained by this method and by a specific radioimmunoassay were correlated. The method described is sensitive, selective, reproducible and easier to perform than other chromatographic methods. It is suitable for the daily measurement of cyclosporin in batches of up to 40 samples and the results correlate well with another chromatographic method and with the specific radioimmunoassay.

A high-performance liquid chromatographic procedure using cyclosporin D as internal standard for the routine measurement of cyclosporin A and four of its metabolites is described. Whole-blood samples were purified on refillable solid-phase glass extraction columns. The chromatographic method includes a gradient elution using acetonitrile and water (pH 3.0) as eluents and an RP-8 analytical column. More than 1000 samples have already been analysed without any loss. The inter-assay variation was 6.3% and the intra-assay variation 4.9%. A linear correlation was found over a range of 0-3000 ng cyclosporin A per ml whole blood. The detection limit was 20 ng and the recovery was found to be 80-90%. Metabolites 1, 17, 18 and 21 could be characterized.

An automated isocratic high-performance liquid chromatographic (HPLC) method is described for the determination of cyclosporin A and G in human serum. This method involves the use of an automated solid-liquid extraction procedure following rapid protein precipitation with acetonitrile. The use of a disposable C8 extraction cartridge allows a good recovery of cyclosporine (87%) from serum and a detection limit of 20 ng/ml with good reproducibility using 0.5 ml of sample. This method can also be adapted to whole blood measurements. The choice of a 3-micron cyano analytical column and of the mobile phase hexane-isopropanol (85:15) permitted a low column temperature (50 degrees C), a low flow-rate (0.6 ml/min) and a short run time (14 min). This method allows the accurate and fast routine monitoring of cyclosporine by HPLC, which is particularly important in hepatic transplantations.

High-performance liquid chromatography was used to determine cyclosporin A (CsA) concentrations in the serum of kidney transplant patients by rapid-flow fractionation (RFF) followed by silica gel normal-phase high-performance liquid chromatography (HPLC). The extraction of CsA from serum was achieved by RFF using a short diatomaceous earth column eluted with diethyl ether-n-hexane (50:50, v/v). The recovery was more than 80% at concentrations of 50-150 micrograms/l. The concentration of this compound was determined by HPLC using a conventional silica gel column with 3.3 M ammonia solution-ethanol-n-hexane (0.31:10.69:89, v/v) as eluent. Concentration calibration was made on the basis of the peak-height ratio of CsA to CsD as the internal standard. The coefficient of variation of this assay was less than 6.5% and the results were used for the therapeutic drug monitoring of CsA administered to kidney transplant patients. Measurements of the CsA concentrations in 160 serum specimens were also made by conventional radioimmunoassay (RIA) using commercial kits. The data obtained by RIA were on average 2.5 times those obtained by HPLC. Higher values in RIA were observed characteristically with patients with severe disfunction resulting from CsA hepatotoxicity. From the results, it appeared that HPLC rather than RIA provides more precise and reliable values for the concentration of this drug.

A fully automated high-performance liquid chromatographic column-switching system is presented for the determination of cyclosporin A in whole blood. After blood proteins were precipitated with acetonitrile, the supernatant was automatically loaded on to a cyanopropyl column for initial separation, and then the fraction containing cyclosporin A was loaded on to a trimethylsilica column for final separation and quantitation. Cyclosporin A was detected by ultraviolet absorption at 205 nm. The minimum detectable concentration of cyclosporin A was 5 ng/ml in 100 microliter of blood. The coefficient of variation of the method was 1.755, 1.748 and 0.655% in whole blood when spiked at the 170, 425 and 850 ng/ml levels, respectively. One assay was completed in 15 min.

Cyclosporine (CSA) is biotransformed to many metabolites which may contribute to its immunosuppressive and nephrotoxic activity. We report a rapid and sensitive, automated column-switching high-performance liquid chromatographic (HPLC) method for measuring CSA-M17 in whole blood; the method also separates CSA-M1. CSA metabolite standards were isolated by a preparative-scale HPLC method. Samples were prepared by protein precipitation with acetonitrile followed by dilution with water. CSA-M17 was initially separated on a C8 column; final separation was on a C18 column. The inter-day relative standard deviation at 50 ng/ml was 8% (n = 3). Limit of detection was 20 ng/ml.