A Specific Method for the Measurement of Cyclosporin A in Human Whole Blood by Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Therapeutic monitoring of the immunosuppressant cyclosporin A (CsA) is routinely performed by immunoassays to make individual dosage adjustments for patients after organ transplantation. High-performance liquid chromatography with ultraviolet detection (HPLC-UV) has been used as the reference method. However, HPLC-UV methods frequently suffer from chromatographic interferences that affect accuracy and reproducibility. A sensitive, specific HPLC-tandem mass spectrometry (HPLC/MS/MS) method for the quantitation of CsA has been developed. One hundred microliters CsA whole blood sample containing cyclosporin C (CsC) as the internal standard was extracted with ethyl ether. High-performance liquid chromatography separation was accomplished on an RP-C18 narrow-bore column at 50 degrees C with a linear gradient elution followed by on-line ion-spray ionization MS/MS analysis. The standard curve was established in the range of 10 to 1000 microg/l (r = 0.9989, n = 8). Limits of detection and quantitation were 1 microg/l and 5 microg/l, respectively. Imprecision was <4% across three control levels. Cyclosporine A recovery averaged 88%. Six metabolites: AM1, AM9, AM4N, Am1c, AM1a, and AM19 were identified with this method. AM1, AM9, and AM1c were further differentiated with a modification to the MS/MS conditions. This method was used in a comparison study with an HPLC-UV method: HPLC = 1.055 LC/MS/MS + 7.05 (microg/l), (Sy/x = 25.7), r2 = 0.982. With its high degree of sensitivity and specificity, this LC/MS/MS method offers a valuable reference method for immunoassay evaluation and a tool for metabolite investigation.

Quantitative bioanalysis of peptides by liquid chromatography coupled to (tandem) mass spectrometry

“…Several MRM analyses measure the [M + NH 4 ] + ion as the parent ion with the molecular ion as product ion after collision-induced dissociation (CID) [90,100,121,123,[125][126][127]131,132], as no stable abundant fragment of cyclosporin A could be measured. However, MRM analysis with the [M + H] + ion as parent ion has also been reported, measuring an undefined product ion with m/z 425.4 [86,124].…”

Evaluation of a cyano stationary phase for the determination of tacrolimus, sirolimus and cyclosporin A in whole blood by high-performance liquid chromatography–tandem mass spectrometry

“…Most liquid chromatography-mass spectrometry (LC-MS) methods for the analysis of tacrolimus, sirolimus and/or cyclosporin A in blood employ a C 8 or C 18 silica stationary phase [1,2,4,6,7,23,24]. These methods are highly robust and have found wide range applicability in hospital labs around the world.…”

Improvement of cyclosporin A determination in whole blood by reversed‐phase high‐performance liquid chromatography

“…Chromatographic separation of cyclosporin A in blood samples has been well documented by several publications (Sharma et al, 2002;Chimalakonda et al, 2002;Brozmanova et al, 2000;Zeng et al, 1999;Simpson et al, 1998), in which a relatively high temperature, e.g. 70°C, was recommended to improve the peak shape.…”

“…One method is to increase the column temperature that was actually applied to the cyclosporin A determination by many authors (Min et al, 2000;Bowers and Mathews, 1985;Kahn et al, 1986;Giesbrecht et al, 1989;Sharma et al, 2002;Chimalakonda et al, 2002;Brozmanova et al, 2000;Zeng et al, 1999;Simpson et al, 1998). The capacity factor (k) was efficiently decreased at relatively higher working temperatures, leading to short retention times and sharp peak shapes as shown in Fig.…”