Tacrolimus In Human Blood Research Articles

Application of volumetric absorptive microsampling device for quantification of tacrolimus in human blood as a model drug of high blood cell partition

Volumetric absorptive microsampling device (VAMS) was evaluated for bioanalysis of tacrolimus, which was used as a model drug with high blood cell partition. Aliquots of blood (ca. 10μL) with different hematocrits and fortified with tacrolimus were wicked up by VAMS and tacrolimus was extracted with a methanol-water mixture (1:1, v/v) via sonication. After chromatography on an AQUITY UPLC HSS T3 column (100×2.1 i.d., mm, 1.8μm), tacrolimus and the internal standard ascomycin, were detected in the positive ion mode with electrospray ionization by monitoring of transitions m/z 826.6→616.4 and m/z 814.6→604.0, respectively. An assay method to quantify tacrolimus from 1 to 250ng/mL in whole blood was qualified by ensuring that linearity, selectivity, intra- and inter-batch reproducibility, and stability were within the acceptance criteria. Consistent and high extraction recovery of tacrolimus was ensured from blood with low- (20%), mid- (45%), and high-hematocrit (65%) levels with minimal matrix effects. Apparent instability at ambient temperature or 4°C possibly due to reduced recovery suggests that tacrolimus in VAMS should be stored at −25°C until assay. Potential reduced recovery over time from VAMS should be taken into consideration in method optimization.

Validation of methods to study the distribution and protein binding of tacrolimus in human blood

Introduction: Tacrolimus is a macrolide immunosuppressant that has a narrow therapeutic index, displays considerable variability in response, and has the potential for serious drug interactions. Therapeutic drug monitoring and dose individualisation for tacrolimus is complicated but essential. Few studies have investigated the blood distribution and protein binding of tacrolimus and the results of these studies are conflicting. The aim of the present study is to establish and validate methods to investigate the distribution of tacrolimus in human blood. To conduct these studies at clinically relevant concentrations the use of 3H-dihydro-tacrolimus instead of tacrolimus was investigated. Methods: The use of radiolabelled tacrolimus was validated by conducting studies with a mixture of both labelled and unlabelled drug where tacrolimus was analysed by LC-MS/MS. The in vitro distribution of tacrolimus and 3H-dihydro-tacrolimus was investigated in blood collected from healthy subjects using Ficoll–Paque reagent and density gradient ultracentrifugation, respectively. The unbound fraction of tacrolimus in plasma was studied using equilibrium dialysis conducted at 37 °C. Results: In blood, tacrolimus was found to be mainly associated with erythrocytes (85.3±1.5%), followed by diluted plasma proteins (14.3±1.5%) and lymphocytes (0.46±0.10%). In plasma, tacrolimus was found to mainly be associated with the soluble protein fraction (61.2±2.5%), high-density lipoproteins (HDL, 28.1±5.4%), low-density lipoproteins (LDL, 7.8±1.6%), and very low-density lipoproteins (VLDL, 1.4±0.3%). The unbound fraction of tacrolimus was found to be only 1.2±0.12%. Statistical comparison indicated that there was no significant difference in the blood distribution and plasma protein binding of 3H-dihydro-tacrolimus when compared with tacrolimus. Discussion: These results have important implications for therapeutic drug monitoring of tacrolimus and subsequent studies of tacrolimus distribution in transplant recipients.

An HPLC/MS/MS assay for tacrolimus in patient blood samples Correlation with results of an ELISA assay

An HPLC/MS/MS assay for tacrolimus in whole blood using FR900520 as an internal standard was validated over the standard curve range of 0.100–10.040 ng ml −1. The calibration curve for tacrolimus in human blood gave a slope of 0.2481, an intercept of 0.007, and a correlation coefficient ( r) of 0.9996, with no interference noted from human blood, analyte, or internal standard stock solutions. Use of EDTA or heparin as the preservative in blood resulted in no significant differences. Samples were stable for at least the time required to assay the maximum number of samples that could be placed in the automated system. The limit of sensitivity of the assay was set at the concentration of the lowest nonzero standard tested, i.e., 0.100 ng ml −1. However, validation of the assay to a limit of 0.010 ng ml −1 is currently underway. The within-run and between-run precision and accuracy of the method were determined for four quality control samples. The highest CV was seen at 0.1 ng ml −1 (17.6% within-run and 15.9% between-run), with other CV < 5%. The recovery ranged 79.6–81.3% for tacrolimus over the range 0.3–8.0 ng ml −1 and was 63.10 ± 1.37% for FR900520. There was a linear correlation ( r 2 = 0.963) between assay results by HPLC/MS/MS and ELISA in whole blood from atopic dermatitis patients treated with topical tacrolimus ointment. The difference between the means ± S.D. determined by HPLC/MS/MS (1.22 ± 1.46 ng ml −1) and ELISA (1.12 ± 1.29 ng ml −1) was significant by a paired t-test ( P < 0.001). Similarly, there was a linear correlation ( r 2 = 0.841) between assay results by HPLC/MS/MS and IMx in whole blood from solid organ transplant patients treated with tacrolimus. The difference between the means was significantly higher ( P < 0.001) for the IMx (15.80 ± 8.37 ng ml −1) than the HPLC/MS/MS (13.42 ± 6.87 ng ml −1).