| BackgroundSignificant challenge to keep balance between immunosuppression and rejection remains in the field of treatment on post-transplantation. Common used immunosuppressants include cytotoxics (anti-metabolism and alkylation agents), antibiotics, corticosteroids and biological agents. Tacrolimus, one of antibiotic immunosuppressants, was approved for treatment of liver or kidney transplantation recipients. It shows extraordinary potent immunosuppressive properties with50-100fold higher potency than cyclosporine A in vitro. It has been used as rescue therapy for cyclosporine A-and corticosteroid-resistant rejection. However, tacrolimus has lower therapeutic index, a narrow therapeutic window associated with their nephrotoxicity and neurotoxicity. It could be influenced by some factors in the aspects of adsorption and metabolism, showing as pharmacokinetic intra-and inter-variation. Therapeutic drug monitoring (TDM) for tacrolimus is therefore mandatory. Although trough concentration in whole blood has usually served as indicator for dose titration in case of overdose toxicity or underdose rejection, it is not the best option for TDM. Trough concentration of tacrolimus shows good correlation with side effects such as nephrotoxicity, but not so good with drug effect. Limited sampling strategies developed based on population pharmacokinetics (PPK) has been applied through sampling2-4whole blood specimens during dose-interval for detecting drug concentrations, which have been used for estimation of area under curve (AUC) with the help of PPK software. The value of AUC has been supposed to reflect drug exposure more accurately and consequently show good correlation with drug effect. However, multiple sampling in a drug-interval would increase physiological burden of patients, particularly in special groups of population (such as children and the elderly), and prolong lenghth of stay for outpatients. Otherwise, transplantation recipients have kinds of original disease, special physiologic and pathogenic alternations, and drug co-administration. A facilitated, accurate and specific assay with low-volume sample required was demanded. The dried blood spots (DBS) sample collection techniques shows significant advantages over conventional blood collection, including longer lifespan of samples with reduced need for refrigeration, less invasive, more cost effective, easy shipment and storage, and reduction of infection risks by deactivation of potential pathogens on the filter paper. LC-MS technique is characteristic of high sensitivity and specificity. Thus, we set up a sensitive method for determination of tacrolimus on DBS with high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Moreover, the assay was strictly tested and validated.MethodA6mm diameter of disk was punched out from the center of dried blood spots into a2mL polypropylene centrifuge tube, followed by addition of10%methanol-water solution (10:90, v/v) contained with internal standard (IS). After30min in ultrasonic bath,1mL of methyl tert-butyl ether was added for liquid-liquid extraction (LLE). Extraction solution was transferred to a clean tube for evaporation to dryness under nitrogen stream in a heating block at45℃. The residue was reconstituted with50μL of MeOH:water (50:50, v/v) solution and10μL of the supernatant was injected into the LC-MS/MS system for analysis. Chromatographic separation was performed on an XBridge Phenyl column with an isocratic elution of mobile phase compromised of MeOH:water (95:5, v/v) containing2mmol/L ammonium acetate and0.1%formic acid (v/v) at a flow rate of0.3mL/min. The column was kept at50℃in a column oven. Mass spectrometric detection was performed on a triple quadrupole tandem mass spectrometer API3000instrument equipped with and Turbo Ionspray source and operated in positive ionization mode. The ion transitions were monitored on m/z821.5/768.4for TAC and809.4/756.3for IS under multiple reaction monitoring (MRM) mode.Method validation was conducted in terms of linearity, specificity, sensitivity, accuracy, precision, matrix effect, extraction recovery, stability, chromatographic effect of filter paper, effect of volume and hematocrit (HCT) of whole blood.Established method (DBS-LC-MS/MS) was applied to detect50clinical specimens from50patients, which concentration data was compared with those obtained from Microparticle Enzyme-linked Immunoassay (MEIA) and whole blood-LC-MS/MS (WB-LC-MS/MS). Statistical software was used for analyzing and plotting.ResultsWith the chromatographic and mass spectrometric condition described above, tacrolimus and IS exhibited good chromatography with baseline resolution at retention time of1.67min. Calibration range was1-80ng/mL for tacrolimus with correlation coefficient (r) of0.998. Lower limit of quantification and limit of detection was1ng/mL and0.25ng/mL respectively. Intra-assay, inter-assay imprecision and biases were all less than15%for DBS. The recovery of tacrolimus and IS was73.2～79.5%and71.7～77.9%respectively. Matrix effect was not detected. Tacrolimus on DBS was stable for at least10days at room temperature and at4℃, for at least24h at50℃, and at least35d at-20°℃. Chromatographic effect of filter paper was not detected. Volume of blood (15-50uL) and hematocrit of blood (range from23.2%to48.6%) did not show significant influence on detection of tacrolimus concentration by DBS-LC-MS/MS.Tacrolimus concentrations measured by DBS-LC-MS/MS, WB-LC-MS/MS and MEIA were compared. Passing-Bablok regression analysis showed that DBS-LC-MS/MS has good identity with WB-LC-MS/MS, while identity between DBS-LC-MS/MS and MEIA or between WB-LC-MS/MS and MEIA was poor. Bland-Altman plot and Folded empirical cumulative distribution plot (Mountain plot) showed that tacrolimus concentrations detected by DBS-LC-MS/MS or WB-LC-MS/MS were lower apparently than by MEIA.95%of difference between tacrolimus concentrations determined by DBS-LC-MS/MS versus those determined by MEIA was in the range of1.125～9.975ng/mL.95%of difference between tacrolimus concentration determined by DBS-LC-MS/MS versus those determined by WB-LC-MS/MS was range from-1.125ng/mL to2.25ng/mLConclusionsA sensitive and specific LC-MS/MS method for quantitative analysis of tacrolimus from DBS samples was successfully developed, with good performance in strictly validation including chromatographic effect of filter paper, effects of volume and HCT of whole blood applied on filter paper. It would facilitate clinical therapeutic drug monitoring of tacrolimus. For the difference exists between established method and MEIA, which has been applying generally in clinics, application of DBS-LC-MS/MS assay for determination of tacrolimus still needs further clinical validation. |
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