MSACL 2023 Abstract

INTRODUCTION: Many cancer patients who have received bone marrow transplantation (BMT) often have complications with graft versus host disease (GVHD). The donor immune cells that attack malignant tumors also attack other body parts which can lead to liver complications, digestive issues, and skin rashes. The first line treatment for acute GVHD includes the glucocorticosteroid, prednisone (PRED). PRED can have serious side effects for BMT patients whose immune system is already compromised from the transplant procedure. These side effects include susceptibility to infection, muscle atrophy and elevated blood sugar levels which can initiate diabetes and hypertension. PRED is potentially dangerous in high does, especially over long periods of time. When patients experience symptoms that can last months to years this can become problematic. PRED dosing for the treatment of acute GVHD has been set on the drug’s use for other medical conditions which may not be optimal for acute GVHD. It is necessary to have a sensitive and specific assay to quantify PRED routinely in the clinical laboratory to ensure optimal dosage is determined and standardized for treatment of acute GVHD.

OBJECTIVES: The objective was to develop a rapid and simple assay for the simultaneous measurement of PRED and prednisolone (PRDL) in serum by LC-MS/MS. This assay will be used to support clinical trials and pharmacokinetic studies to better define optimal dosing to improve patient outcomes with acute GVHD.

METHODS: PRED and PRDL were isolated from serum samples after protein precipitation with methanol containing internal standards (IS). PRED-D8 was the IS for PRED while PRDL-D8 was the IS for PRDL. Following centrifugation, the supernatant was injected into the turbulent flow liquid chromatography system followed by electrospray ionization tandem mass spectrometry (TFLC-MS/MS). Chromatographic separation was performed using a Thermo Scientific TLX-2 Transcend HPLC system interfaced to a SCIEX 6500+ mass spectrometer operated in positive ion ESI mode. MRM transitions were as follows: PRED 359.1>147.1 and 359.1>171.1 m/z and PRDL 361.2>147.1 and 361.2>128.1 m/z. The results were quantified using a six-point calibration curve. Assay accuracy was determined through recovery studies. Within-day imprecision was evaluated by analyzing 10 specimens in a single day, and between-day imprecision was evaluated by analyzing samples in triplicate over 20 days.

RESULTS: The LOQs of PRED & PRDL were 1 ng/mL; the CVs were <20% over 20 days. For both compounds the calibration curves were linear over the analytical measurement range (AMR) with correlation coefficients (R2) ≥0.999. Dilutions were validated providing a clinical reportable range (CRR) of 1 to 10,000 ng/mL. Within-day and between-day imprecision (CVs) at concentrations spanning the AMR were less than 5% for all analytes. PRED and PRDL were sufficiently stable under all relevant analytical conditions. No significant matrix effects were observed during the method validation. Recoveries ranged from 96-102% for three controls (250, 500, and 750 ng/mL) spanning the AMR for both compounds.

CONCLUSION: We have developed an accurate and sensitive TFLC-MS/MS method for the simultaneous quantification of PRED and PRDL in human serum. The method has been fully validated for imprecision, accuracy, linearity, recovery, carryover, specificity, and matrix effects. This is the first reported method to measure both compounds simultaneously using TurboFlow technology. Due to the assay’s performance, it will be used to support clinical trials and pharmacokinetic studies for patients with acute GVHD.