LC-MS grade methanol was purchased from J.T Baker (Phillipsburg, NJ, USA). Formic acid and ammonium formate were from Sigma–Aldrich (St. Louis, MO,US). Water was prepared in-house using a Millipore Milli-Q Advantage A10 purification system (Darmstadt, Germany). Liquid MPA and MPAG were purchased from Cerilliant corporation (Round Rock, Texas, USA). MPA-d3, MPAG-d3 and AcMPAG-d3 were from Toronto Research Chemicals (Toronto, Canada). The structure of MAP, MPAG and AcMPAG are shown in Fig. 1 . The Chromsystems 6 PLUS1 multilevel calibrator mycophenolic acid/glucuronide in plasma/serum calibration kit (Munich, Germany) was used to evaluate accuracy. For immunoassays, the mycophenolic acid Flex reagent cartridge, calibrators, and controls were all purchased from Siemens Healthcare Diagnostics (Frimley, UK).
Mpa d3
Manufactured by LGC
Sourced in Canada
The MPA-d3 is a laboratory instrument used for the analysis and quantification of specific compounds. It utilizes direct mass spectrometry detection to provide precise and reliable measurements. The core function of the MPA-d3 is to facilitate accurate analytical testing within a laboratory setting.
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2 protocols using mpa d3
1
Quantitative Determination of Mycophenolic Acid and Metabolites
2
Immunosuppressant Drug Quantification in Renal Tissue
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All drug concentrations were determined 4 hours after last dosing, when animals were sacrificed following renal clearance function measurements. For TAC and SRL measurements, whole blood samples were collected in heparinized tubes. For MPA, the active metabolite of MMF, heparin plasma was prepared following standard centrifugation procedures.
Flash-frozen renal tissue (100 to 200 mg) was mortared in liquid nitrogen and homogenized in 2 mL KH2PO4 buffer (pH 7.4). For protein precipitation, 800 µL methanol and 0.2 mmol/L ZnSO4 (80/20, v/v) were added to 200 µL of tissue homogenate or blood sample. Ascomycin (20 ng/mL; Sigma-Aldrich, St. Louis, MO) was added as internal standard for SRL and TAC [34] (link), and MPA-d3 (Toronto Research Chemicals, Toronto, CA) served as internal standard for quantitation of MPA. All immunosuppressant drugs were quantified using a validated HPLC-MS assay [34] (link), [35] (link).
Flash-frozen renal tissue (100 to 200 mg) was mortared in liquid nitrogen and homogenized in 2 mL KH2PO4 buffer (pH 7.4). For protein precipitation, 800 µL methanol and 0.2 mmol/L ZnSO4 (80/20, v/v) were added to 200 µL of tissue homogenate or blood sample. Ascomycin (20 ng/mL; Sigma-Aldrich, St. Louis, MO) was added as internal standard for SRL and TAC [34] (link), and MPA-d3 (Toronto Research Chemicals, Toronto, CA) served as internal standard for quantitation of MPA. All immunosuppressant drugs were quantified using a validated HPLC-MS assay [34] (link), [35] (link).
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