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Recently, the development of cyclic peptide drugs has accelerated. To develop an analytical method to determine the physicochemical properties of these lipophilic drug candidates, we investigated the separation mechanism of cyclosporine congeners A, B, C, and D using HPLC with a column packed with 2-μm nonporous octadecylsilyl silica particles at high temperature. The four congeners were eluted with good repeatability in terms of retention time, peak area, and theoretical plate number. A difference of one amino acid in the eleven amino-acid sequence of the cyclosporine congeners was able to be recognized by our system within 4 min by isocratic elution, and the resolution was greater than 1.68. The calculated logP values of these congeners were well correlated with the retention factors with a correlation coefficient of 0.991. We could elucidate the separation mechanism of cyclosporine congeners on the high-temperature HPLC system. These results show that this method using HPLC on a column packed with 2-μm nonporous octadecylsilyl silica particles can be used for studying the lipophilicity of cyclosporine congeners., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The extensive use of routine monitoring of cyclosporin A (INN, ciclosporin) whole blood levels of patients undergoing such therapy has resulted in a wide variety of chromatographic conditions for analysing this drug. The aim of this study was to evaluate the importance of essential parameters in the chromatographic determination of cyclosporin A and its main metabolites, AM1, AM9 and AM4N. A D-optimal design was used to evaluate the effect of type and amount of organic modifier, temperature, flow rate, pH and gradient steepness. The optimal chromatographic conditions were determined by multi-linear regression. In the final chromatographic method separation of the compounds was carried out on a reversed phase C(8) column maintained at 80 degrees C. The mobile phase consisted of a linear gradient with two mobile phases containing acetonitrile and water. The flow rate was set at 0.8 ml/min. UV detection was carried out at 214 nm. Validation of the analytical method showed linearity over the range 25-1000 ng/ml (r>0.997). The detection limits of cyclosporin A, AM1, AM9 and AM4N were 1.3 pmol on column. The within-day and between-day relative standard deviations were <15% for cyclosporin A at all concentrations and for the metabolites at 250 and 1000 ng/ml, and <21% for the metabolites at limit of quantification (25 ng/ml).

The suitability of the United States Pharmacopeia (USP) assay method for analysing stressed samples of cyclosporine oral solution USP was evaluated for stability samples by analyzing cyclosporine oral solution after acid, alkali, hydrogen peroxide, heat and light treatment. Some of the degradants generated during stress testing, as well as dihydrocyclosporine A, which is a known degradant of cyclosporine A, were not adequately resolved from the cyclosporine peak and mobile phase adjustments did not improve the resolution. In addition, isocyclosporine A, another known degradant of cyclosporine, could not be quantitated as it was eluting too early with the system peaks. Therefore, a binary gradient, reverse phase, stability indicating, HPLC method for the assay of cyclosporine in cyclosporine oral solution USP has been developed and validated. Analysis of degraded samples showed that the cyclosporine A eluted as a spectrally pure peak resolved from its degradation products.

A significant overestimation of cyclosporin A (CsA) by a radioimmunoassay using 125I-labeled monoclonal antibody (125I-RIA), compared to the reference HPLC method, has been reported for a limited number of samples from transplant patients. However, the extent of the discrepancy, with respect to bioavailability parameters, has not been examined for the case of the oral administration of a single dose CsA to healthy subjects where a number of factors which might be involved in this overestimation (e.g. under steady state condition and a significant accumulation of CsA metabolites) would be absent. Therefore, the objective of this study was to assess the effect of potential difference manifested by the two analytical procedures, 125I-RIA and HPLC, on the bioavailability analysis of CsA. An oral CsA formulation was administered to 22 healthy male subjects and the blood samples were analysed by both 125I-RIA and HPLC. Significant discrepancies in the estimated CsA concentrations by the two methods (paired t-test, P < 0.001) were found. The difference (bias) increased with increasing concentrations of blood CsA (P < 0.001). However, despite the bias in CsA estimations, the AUC and Cmax, obtained by 125I-RIA and HPLC methods showed only small differences (i.e. 2% for AUC and 7% for Cmax). Thus, our results suggest that the bias of the 125I-RIA vis-a-vis the HPLC method in the estimation of CsA blood levels may not, in practice, affect the bioavailability analysis (e.g. bioequivalence study) of CsA in a situation where a single dose CsA is orally administered to healthy subjects.

Competitive immunoassays have been developed for the immunosuppressant cyclosporin A and the anti-asthmatic drug theophylline utilizing identical controlled pore glass-protein A microcolumns and flow injection techniques. For cyclosporin A the assay was based on a monoclonal antibody with fluorescence detection whilst for theophylline sheep antiserum and electrochemical detection was used.