Tacrolimus (FK 506) measurement by immunoassays in clinical samples of organ transplant patients often lacks a specific reference method. A method combining liquid chromatography (LC) with tandem mass spectrometry (MS/MS) was developed to quantify tacrolimus in whole blood. Liquid-liquid extraction was performed on 1 ml of sample before narrow-bore LC/MS/MS analysis. Ascomycin was used as an internal standard. The standard curve was composed of seven points ranging from 1 to 50 micrograms/l (average r2 = 0.9999). Limits of detection and quantitation were 0.25 and 0.75 microgram/l, respectively. Imprecision was < 5% across the therapeutic range. Tacrolimus recovery averaged 62%. The most abundant metabolites detected in clinical samples were 13-O- and 15-O-demethyl tacrolimus. This method was used in a comparison study with a microparticle enzyme immunoassay (MEIA): MEIA = 1.03 LC/MS/MS -0.084 (microgram/l), (Sy/x = 1.43), r2 = 0.933. With its high sensitivity and specificity, this LC/MS/MS method presents a good reference method for immunoassay evaluation as well as a valuable tool for metabolism studies.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.