(W0930-06-36) ICH M10 Bioanalytical Method Validation for Quantitation of Ketorolac in Human Vitreous and Aqueous Humor using Human Plasma as a Surrogate Matrix

Purpose: A planned clinical trial required accurate measurement of ketorolac levels in human vitreous and aqueous humor samples. Due to limited sample availability to develop and validate a test to measure ketorolac, a bioanalytical method was developed and validated for the quantitation of ketorolac levels using human plasma as a surrogate matrix.

Methods: The developed process involves extracting ketorolac and its internal standard (ketorolac-d₅) from plasma as a surrogate for aqueous and vitreous humor using a protein precipitation sample preparation technique, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was assessed per ICH M10 Bioanalytical Guidelines for selectivity/specificity, matrix effect, linearity/range, accuracy/precision, carryover, dilution integrity, and stability. A recovery assessment was also made.

Results: The validated method can be successfully applied for quantitation of ketorolac over a concentration range of 2.5 ng/mL to 5000 ng/mL. The method met the acceptance criteria with respect to selectivity, specificity, precision, accuracy, linearity, dilution integrity, and stability.

Conclusion: The validated method can use plasma as a surrogate matrix for quantitation of ketorolac in aqueous and vitreous humor, thereby eliminating the need to procure human vitreous and aqueous samples for validation prior to initiation of a clinical trial.

Acknowledgements: This study was funded by Rayner Surgical, Inc. The authors would like to acknowledge Lions World Vision Institute for providing aqueous and vitreous humor.

Sample Preparation Workflow

2-Day Calibration Curve Accuracy Results

Accuracy and Precision of Ketorolac in Plasma (Surrogate Matrix), Aqueous, and Vitreous Humor