Ultra high performance liquid chromatography–tandem mass spectrometry method for cyclosporine a quantification in biological samples and lipid nanosystems

Abstract

Cyclosporine A (CyA) is an immunosuppressant cyclic undecapeptide used for the prevention of organ transplant rejection and in the treatment of several autoimmune disorders. An ultra high performance liquid chromatography-tandem mass spectrometry method (UHPLC-MS/MS) to quantify CyA in lipid nanosystems and mouse biological matrices (whole blood, kidneys, lungs, spleen, liver, heart, brain, stomach and intestine) was developed and fully validated.Chromatographic separation was performed on an Acquity UPLC ® BEH C18 column with a gradient elution consisting of methanol and 2 mM ammonium acetate aqueous solution containing 0.1% formic acid at a flow rate of 0.6 mL/min. Amiodarone was used as internal standard (IS). Retention times of IS and CyA were 0.69 min and 1.09 min, respectively. Mass spectrometer operated in electrospray ionization positive mode (ESI+) and multiple reaction monitoring (MRM) transitions were detected, m/z 1220.69→1203.7 for CyA and m/z 646→58 for IS. The extraction method from biological samples consisted of a simple protein precipitation with 10% trichloroacetic acid aqueous solution and acetonitrile and 5 µL of supernatant were directly injected into the UHPLC-MS/MS system. Linearity was observed between 0.001 µg/mL-2.5 µg/mL (r ≥ 0.99) in all matrices. The precision expressed in coefficient of variation (CV) was below 11.44% and accuracy in bias ranged from −12.78% to 7.99% including methanol and biological matrices. Recovery in all cases was above 70.54% and some matrix effect was observed. CyA was found to be stable in post-extraction whole blood and liver homogenate samples exposed for 6 h at room temperature and 72 h at 4 o C. The present method was successfully applied for quality control of lipid nanocarriers as well as in vivo studies in BALB/c mice.

Development and validation of UHPLC method for the determination of cyclosporine A in biological samples

“…Therefore, much more interest has been addressed to chromatographic methods. The use of LC coupled with UV detection, mass spectrometry or tandem mass spectrometry has allowed a significant increase of method selectivity to be achieved (Kabra et al, 1985;Guada et al, 2013;Tszyrsznic et al, 2013).…”

“…Therefore, some interference may occur with analyte quantification. At present, LC/MS and LC/MS/MS are more selective methods for the quantification of immunosuppressive drugs (Guada et al, 2013;Tszyrsznic et al, 2013). Despite the clear advantages of MS detection, our UHPLC method is still very attractive because of the short run time (in comparison with HPLC methods), wide linearity range, applicability to all ocular tissues and fluids and the fact that the MS technique is not widely available for many laboratories and routine drug level control because of the high cost of the equipment, reagents and analyses.…”

“…In recent years more specific and sensitive chromatographic methods have been developed to determine the low concentrations of CsA in biological tissues and fluids. Currently described techniques include liquid chromatography-mass spectrometry (LC/MS) [limit of quantitation (LOQ) = 50 ng/mL; Kanduru et al, 2010], LC-tandem mass spectrometry (LC/MS/MS; LOQ = 15 ng/mL; Karapirli et al, 2012), turbulent flow chromatography coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS; LOQ = 1 ng/mL; Mora et al, 2008), ultra-performance liquid chromatography interfaced to electrospray ionization mass spectrometry (UPLC/ESI-MS; Onoune et al, 2010), UPLC tandem MS/MS (LOQ in the range 1-25 ng/mL; Di Guada et al, 2013;Tszyrsznic et al, 2013) and ultra-high pressure liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/MS/MS; LOQ = 2 ng/mL; Rodriguez-Aller et al, 2012). However, in many laboratories mass spectrometry is unavailable, mainly because of its high cost.…”

Lipid nanoparticles for cyclosporine A administration: development, characterization, and in vitro evaluation of their immunosuppression activity

“…10 Briefly, 500 μL of chloroform were added to 5 mg of LN and vortexed for 30 seconds, then 1.5 mL of methanol were added to the mixture and vortexed for 1 minute. After centrifuging at 15,000× g for 10 minutes, the supernatant was diluted with methanol (1:10) and a 2 μL aliquot was injected into the UHPLC system for drug analysis.…”

“…Chemical stability of the formulations was studied by quantifying CsA in the dried powder over the same period of time using the UHPLC-MS/MS. 10 In vitro biological activity of csa lN cell culture Jurkat cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) cultured in suspension at a concentration between 1×10 5 and 1×10 6 viable cells/mL in Roswell Park Memorial Institute 1640 cell culture medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO 2 . Cells were subcultured every 3-4 days depending on cell density to an initial concentration of 2×10 5 viable cells/mL.…”