Abstract: SA-PO571
Quantification of Oxalate in Human Plasma by Novel Liquid Chromatography-Tandem Mass Spectrometry: Method Development, Validation, and Application in Lumasiran Clinical Trials
Session Information
- Genetic Diseases: Diagnosis
November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1102 Genetic Diseases of the Kidneys: Non-Cystic
Authors
- Clausen, Valerie, Alnylam Pharmaceuticals Inc, Cambridge, Massachusetts, United States
- Cao, Karen H., Alnylam Pharmaceuticals Inc, Cambridge, Massachusetts, United States
- Gansner, John M., Alnylam Pharmaceuticals Inc, Cambridge, Massachusetts, United States
- Robbie, Gabriel, Alnylam Pharmaceuticals Inc, Cambridge, Massachusetts, United States
- Wu, Jing-Tao, Alnylam Pharmaceuticals Inc, Cambridge, Massachusetts, United States
Background
Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive genetic disease characterized by hepatic oxalate overproduction. Oxalate is excreted primarily by the kidneys, where it can cause kidney stones and/or nephrocalcinosis, leading to progressive kidney damage. PH1 is characterized by increases in both urinary and plasma oxalate (POx). In PH1 patients with compromised kidney function, POx is monitored. However, measuring POx is challenging due to its intrinsic chemical property and nonenzymatic conversion of ascorbate to oxalate in vitro. We present the development and validation of a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to determine oxalate concentration in human K2EDTA plasma.
Methods
A validated LC-MS/MS assay capable of measuring oxalate in 100 µL of K2EDTA plasma was developed. Samples were spiked with internal standard (13C2-labeled oxalic acid), acidified, and extracted by protein precipitation prior to analysis using anion exchange high-performance liquid chromatography with electrospray ionization MS/MS detection. The method was assessed for linearity, sensitivity, accuracy, precision, selectivity, hemolyzed plasma, lipemic plasma, interference, recovery, matrix effect, and stability. The validated LC-MS/MS assay was used to quantify POx in the lumasiran clinical trials.
Results
The LC-MS/MS assay was developed and validated successfully with a quantitation range of 0.500-50.0 µg/mL (5.55-555 µmol/L). The validation met acceptance criteria of 15% (20% at the lower limit of quantitation) for accuracy, precision, and other parameters tested. Oxalate was shown to be stable in K2EDTA human plasma for 125 days and for 5 freeze/thaw cycles at −20°C and −70°C. Analysis of POx levels in 75 healthy adults indicated the normal range to be 1.71-12.11 µmol/L.
Conclusion
A novel LC-MS/MS assay was developed and validated successfully and in accordance with regulatory guidelines. The required sample volume was only 100 µL of K2EDTA plasma, which is especially favorable in the pediatric population, and there is no need to acidify blood before processing. The assay accurately determines POx levels, which were used as an efficacy endpoint in the clinical development of lumasiran.
Funding
- Commercial Support –