Development and Validation of the New Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Unbound Tacrolimus in the Plasma Ultrafiltrate of Transplant Recipients

Abstract

(1) Background: Only unbound tacrolimus particles are considered to be active and capable of crossing cellular membranes. Thus, the free-drug concentration might be better associated with clinical effects than the total drug concentration used for dosage adjustment. We propose a new, fully validated online liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for unbound tacrolimus concentration measurement. (2) Methods: The determination of the unbound tacrolimus concentration in plasma ultrafiltrate was performed with the Nexera LC system with LCMS-8050 triple quadrupole MS using ascomycin as an internal standard. Chromatographic separation was made using a HypurityC18 analytical column. MS/MS with electrospray ionization and positive-ion multiple-reaction monitoring was used. The unbound tacrolimus level was determined in 36 patients after solid organ transplantation (n = 140). (3) Results: A lower limit of quantification 0.1 pg/mL was achieved, and the assay was linear between 0.1 and 20 pg/mL (R2 = 0.991). No carry-over was detected. The within-run and between-run accuracies ranged between 97.8–109.7% and 98.3–107.1%, while the greatest imprecision was 10.6% and 10.7%, respectively. Free tacrolimus in patients’ plasma ultrafiltrate varied between 0.06 and 18.25 pg/mL (median: 0.98 pg/mL). (4) Conclusions: The proposed method can be easily implemented. The significance of the unbound tacrolimus concentration needs to be investigated. This may facilitate the individualization and optimization of immunosuppressive treatment.

Therapeutic Drug Monitoring of Tacrolimus Based on Volumetric Absorptive Microsampling Technique (VAMS) in Renal Transplant Pediatric Recipients—LC-MS/MS Method Development, Hematocrit Effect Evaluation, and Clinical Application

“…The results of the working-solution stability examination were reported in a previous study by Bodnar-Broniarczyk et al [ 16 , 17 ]. For TAC LC-MS/MS assays in our laboratory, we used the same working solution as the reference standards and ITSD, for which the long-term stability tests are described above.…”

In-situ preparation of norepinephrine-functionalized silver nanoparticles and application for colorimetric detection of tacrolimus in plasma samples

“…A comparison between the performance of the developed method and some of the recent analytical approaches for Tac determination is made in Table 1 . The proposed platform quantifies Tac in a suitable linear range, which is comparable with chromatography and MS-based determinations [ 9 , 11 , 15 , 47 ]. Although chromatographic methods detect lower concentration of Tac in comparison to the developed method, these methods require extensive training, a time-consuming process, and high-cost sophisticated equipment, making them an inappropriate platform for rapid and real-time routine detection.…”

“… Technique Matrix LOD (ng/mL) Dynamic range (ng/mL) Ref. Coupling of solid-phase microextraction to mass spectrometry Whole blood 0.3 1–50 [ 47 ] UHPLC Pharmaceutical Formulation – 1.0 × 10 5 –3.0 × 10 5 [ 15 ] LC/MS Plasma 0.01 0.01–2 [ 11 ] LC-MS/MS Whole blood – 1–40 [ 12 ] LC-MS/MS Whole blood – 0.5–60 [ 13 ] UPLC-MS/MS Whole blood – 50–5000 [ 16 ] HPLC-MS/MS Whole blood – 2.25–42.9 [ 50 ] Millifluidic microwave Whole blood 0.00012 10–500 [ 51 ] Microwave Blood 0.032 – [ 52 ] Immunoassay Blood ...…”

“…So far, numerous analytical methods have been used for the quantification of Tac, such as liquid chromatography tandem mass spectrometry (LC-MS/MS) [ [10] , [11] , [12] , [13] ], ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) [ [14] , [15] , [16] ], UHPLC [ 17 ], enzyme-linked immunosorbent assay (ELISA) [ 18 ], enzyme multiplied immunoassay technique (EMIT) [ 19 ], automated microparticle enzyme immunoassay (MEIA) [ 20 ], electro-chemiluminescence immunoassay (ECLIA) [ 21 ], fluorescent aptasensor [ 22 ], and electrochemical immunosensor [ 9 ]. Some of these methods require extensive training, a high degree of technical ability, a time-consuming process, and high-cost sophisticated equipment, which are the main drawbacks for rapid and real-time routine quantifications [ 23 ].…”

Importance of Pharmacogenetics and Drug–Drug Interactions in a Kidney Transplanted Patient

“…Moreover, 99% of TAC is bound to albumin and x1-acidclycoprotein in plasma, whereas <1% of it is freely distributed [ 3 ]. Although unbound TAC is the pharmacologically active part of the drug, the measurement of this free fraction involves technical difficulties [ 4 ]. Recently, new methods have been developed for the measurement of free levels in plasma, but at the moment there is not any standardized target plasma concentration, and their implications must be investigated before being carried out in clinical practice [ 5 ].…”