A High-Performance Liquid Chromatography-Mass Spectrometry Method Using a Novel Atmospheric Pressure Chemical Ionization Approach for the Rapid Simultaneous Measurement of Tacrolimus and Cyclosporin in Whole Blood

Abstract

Concentration monitoring and dose individualization is required to optimize either tacrolimus or cyclosporin therapy. In this study, the validation of a simple, rapid high-performance liquid chromatography-tandem mass spectrometry method for the simultaneous measurement of tacrolimus and cyclosporin in whole blood is reported. Blood samples (100 microL) were prepared by protein precipitation with zinc sulphate followed by acetonitrile (containing the internal standards ascomycin and cyclosporin D). The chromatographic run time was 1.5 minutes per sample. Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in negative ionization mode (tacrolimus: m/z 802.5 --> 560.6, cyclosporin: m/z 1200.8 --> 1088.4). The assay had an analytical range of 1.0 to 30 mug/L (r > 0.998, n = 6) for tacrolimus and 25 to 2000 microg/L (r > 0.999, n = 6) for cyclosporin. Tacrolimus inter- and intraday inaccuracy and precision [coefficient of variation (CV)] using quality control samples (2.5, 12.5, 25 microg/L) was less than +/-10.0% and CV less than 5.0%, respectively (n = 5). Similarly, cyclosporin inter- and intraday inaccuracy and precision using quality control samples (70, 400, 1500 microg/L) was less than +/-2.0% and CV less than 5.0%, respectively (n = 5). The lower limit of quantification for tacrolimus was 1.0 mug/L and cyclosporin 25 microg/L. The assay had an absolute mean recovery of 86.7% for tacrolimus and 89.0% for cyclosporin (n = 15). Intersubject variability, as a measure of potential matrix effects on results, was less than 6.0% CV for both analytes (n = 15). Extracted samples were stable for at least 20 hours. Results obtained from external proficiency testing samples measured by this method compared with the mean of all liquid chromatography-tandem mass spectrometry methods used by scheme participants revealed a strong correlation and good agreement for tacrolimus (r = 0.993, mean bias = -10.3%, n = 19) and cyclosporin (r = 0.996, mean bias = 3.0%, n = 20). In conclusion, this is the first reported high-throughput method that uses negative atmospheric pressure chemical ionization for the simultaneous measurement of tacrolimus and cyclosporin in whole blood.

Quantification of Tacrolimus and Three Demethylated Metabolites in Human Whole Blood Using LC–ESI–MS/MS

“…[13][14][15][16] Since 2008, at least 7 methods for TAC analysis have been published, all using LC-MS/MS, indicating that this is the method of choice at present. 13,[17][18][19][20][21][22] Most of the above-mentioned methods use whole blood as matrix, because about 90% of the TAC binds to cellular components of blood and thus needs to be extracted from the cellular components first. 5 One previous article has been published on the measurement of TAC and its metabolites in human blood.…”

Therapeutic drug monitoring and LC–MS/MS

“…Some methods perform direct injection of the supernatant from the protein crash [57,61,62], whilst others use on line SPE to further purify the sample [63][64][65]. Analytical sensitivity is not an issue with these drugs and all methods can be operated on modern entry level instruments which generally have sufficient scan speeds to support multiplexed assays.…”

“…Sodium in particular can cause sensitivity problems in routine methods using ammonium adduct formation because of preferential adduct formation, the contamination can arise from solvents or source contamination with sample matrix. It has been reported that APCI sources operating in negative ion mode suffer less with ion suppression than ESI in positive ion mode for the analysis of immunosuppressant drugs [57], but the most popular methods are still those using fragmentation of ammonium adducts in electrospray positive ion mode. The first reported methods using LC-MS/MS were derived from HPLC-UV methods with long and cumbersome sample clean up steps such as off line SPE and LLE.…”

Strategies for Developing Sensitive and Automated LC-MS/MS Assays of a Pharmaceutical Compound and Its Metabolite from Whole Blood Matrix

“…Sample preparation techniques for whole blood assays are as diverse as those for plasma assays [3,4,5,6,7,8,9,10,11]. Although simple sample preparation techniques like protein precipitation (PPT) have been used in the whole blood analysis, whole blood analysis typically calls for more labor-intensive treatment such as liquid-liquid extraction (LLE) or solid-phase extraction (SPE).…”