Inpatients and outpatients who were treated with tacrolimus (Prograf Capsules or Graceptor Capsules; Astellas Pharma US) or cyclosporin A (Neoral; Novartis Pharma) at the Department of Nephrology and Department of Rheumatology, Kanazawa University Hospital, from May 2018 to July 2019 were recruited with informed consent. The present study was performed after receiving approval from the Medical Ethics Committee of Kanazawa University (protocol no. 2017 − 195).

The CLAM-LC-MS/MS system consists of a CLAM-2000 CL automated sample pretreatment device, CBM-20A CL system controller, CTO-20AC CL column oven, SIL-30AC CL autosampler, two LC-30AD CL pumps, FCV-20AH2 CL valve, DGU-20A5R CL degasser and LCMS-8050 CL (Shimadzu Corporation, Kyoto, Japan), all of which are approved for use as medical equipment. A DOSIMMUNE trap column (trap column) and a DOSIMMUNE analytical column (Alsachim, France) were used in the LC system. Commercial DOSIMMUNE kits (Alsachim, France) including calibrators (6 concentrations), 4 QC samples, and stable isotope-labeled internal standards (ISs; [13C2, 2H4]-everolimus, [13 C, 2H3]-sirolimus, [13 C, 2H4]-tacrolimus, and [2H12]-cyclosporin A) for everolimus, sirolimus, tacrolimus and cyclosporin A were used respectively. The validation and acceptable ranges for QC samples are shown in supplemental Table 1.

A chemiluminescence immunoassay (CLIA) method was applied to measure tacrolimus using the Architect system (ARCHITECT i1000SR, Abbott, Illinois, U.S.A) with an ARCHITECT Tacrolimus Reagent Kit, ARCHITECT Tacrolimus Calibrators, ARCHITECT Tacrolimus Whole Blood Precipitation Reagent, and Multichem WBT (Technopath Clinical Diagnostics, Ireland). An affinity column-mediated immune assay (ACMIA) method was applied to measure cyclosporin A, using Dimension® Xpand Plus (Siemens, Germany) with Dimension Systems CSA Assay, Dimension CSA Calibrator, and MORE RAP/Tac/CsA Control (More Diagnostics, CA. USA).

Whole blood collected from patients was divided into two tubes containing EDTA-Na. One was stored at -30˚C for CLAM-LC-MS/MS measurement and the other was stored at 4˚C for immunoassay. For application to the CLAM-LC-MS/MS system, the frozen whole blood samples (95 µL) were thawed at room temperature for hemolysis and placed in the CLAM unit. The CLAM unit was programmed to perform pretreatment, including sample extraction and protein precipitation followed by filtration and sample collection. First, 20 µL of 75% 2-propanol was dispensed onto the filter to activate the dedicated hydrophobic filter. Next, 20 µL of whole blood sample, 150 µL of extraction buffer, and 12.5 µL of ISs were dispensed into a dedicated vial and stirred for 60 s. The samples were vacuum-filtered (approximately 50 to 60 kPa) for 60 s in a dedicated filter consisting of a polytetrafluoroethylene membrane with a pore size of 0.45 μm and collected in a collection vial. Finally, the filtrate was automatically transported to the HPLC unit for LC-MS/MS analysis. The MS/MS conditions are shown in Table 1.

For measuring tacrolimus with CLIA, sample preparation was conducted according to the manufacturer’s protocol. Briefly, Whole Blood Precipitation Reagent was added into the same volume (200 µL) of whole blood samples, QCs, or calibrators. After vortexing and centrifugation, the supernatant was immediately transferred to the Architect system. To measure cyclosporine A with ACMIA, whole blood samples (250 µL) were directly placed in the carousel of the Dimension system.

Regular maintenance of the CLAM-LC-MS/MS system was performed every 6 months as follows. The flow path of LC, lens system of the mass spectrometer, and sample probe of CLAM were cleaned, and the capillary, desolvation line, and PEEK tube were replaced. Technical support was promptly available if required at other times.

Statistical analyses were performed with MedCalc statistical software for Windows (Ostend, Belgium). Data collected by different assay methods were compared by means of Passing and Bablok regression analysis [23] and calculation of Spearman correlation coefficients. The Bland-Altman approach was used to compare immunological assay and CLAM-LC-MS/MS assay results by plotting the relative differences [24].　A p-value of 0.05 was considered statistically significant.