DOI: 10.6084/m9.figshare.28623083
title: Bioanalytical method validation to quantify ketorolac in human vitreous and aqueous via surrogate matrix of human plasma

description: Intracameral phenylephrine 1.0%/ketorolac 0.3% (OMIDRIA<sup>®</sup>) is used during cataract surgery to prevent intraoperative miosis and reduce postoperative pain. Although studied in beagles, no human data exist showing the duration ketorolac remains in the eye postoperatively. A clinical trial measuring ketorolac concentrations in aqueous/vitreous samples necessitated the development of a validation process for acquiring these measurements. Due to limited human aqueous/vitreous humor sample availability, a bioanalytical method was developed and validated to quantify ketorolac levels using human plasma as a surrogate matrix. The developed process involves extracting ketorolac and its internal standard (ketorolac-<i>d</i>5) 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 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. 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. Cataract surgery often causes inflammation, managed using NSAIDs and steroids. Intraoperative anti-inflammatories like phenylephrine 1.0%/ketorolac 0.3% are increasingly used to reduce reliance on topical drops.No human data exists for ketorolac concentrations in aqueous and vitreous humors after intracameral administration, necessitating a sensitive analytical method for future clinical studies. Cataract surgery often causes inflammation, managed using NSAIDs and steroids. Intraoperative anti-inflammatories like phenylephrine 1.0%/ketorolac 0.3% are increasingly used to reduce reliance on topical drops. No human data exists for ketorolac concentrations in aqueous and vitreous humors after intracameral administration, necessitating a sensitive analytical method for future clinical studies. Develop and validate an LC-MS/MS method to quantify ketorolac in rare ocular matrices (aqueous and vitreous humors) using plasma as a surrogate matrix, ensuring compliance with ICH M10 guidelines. Develop and validate an LC-MS/MS method to quantify ketorolac in rare ocular matrices (aqueous and vitreous humors) using plasma as a surrogate matrix, ensuring compliance with ICH M10 guidelines. Calibration standards and QC samples were prepared using human plasma and validated over a concentration range of 2.5 to 5000 ng/mL.The method employed protein precipitation for sample preparation and multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer.Validation parameters included accuracy, precision, recovery, stability, carryover, dilution integrity, and selectivity/specificity. Calibration standards and QC samples were prepared using human plasma and validated over a concentration range of 2.5 to 5000 ng/mL. The method employed protein precipitation for sample preparation and multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. Validation parameters included accuracy, precision, recovery, stability, carryover, dilution integrity, and selectivity/specificity. A nine-point calibration curve in plasma demonstrated excellent linearity (R<sup>2</sup> &gt; 0.99) and sensitivity, with an LLOQ of 2.5 ng/mL.Accuracy and precision were within ±15%, meeting all ICH M10 criteria for bioanalytical methods.The method showed &gt;80% recovery and stability under bench-top, freeze-thaw, long-term storage, and autosampler conditions.Validation confirmed that human plasma can effectively serve as a surrogate matrix for ketorolac quantification in aqueous and vitreous humors. A nine-point calibration curve in plasma demonstrated excellent linearity (R<sup>2</sup> &gt; 0.99) and sensitivity, with an LLOQ of 2.5 ng/mL. Accuracy and precision were within ±15%, meeting all ICH M10 criteria for bioanalytical methods. The method showed &gt;80% recovery and stability under bench-top, freeze-thaw, long-term storage, and autosampler conditions. Validation confirmed that human plasma can effectively serve as a surrogate matrix for ketorolac quantification in aqueous and vitreous humors. The method enables accurate pharmacokinetic studies for ketorolac, reducing the need for extensive use of scarce ocular tissues.A clinical study is underway to evaluate ketorolac levels in patients undergoing cataract surgery, with the findings expected to inform future therapeutic strategies.The validated LC-MS/MS method is robust, sensitive, and suitable for quantifying ketorolac in aqueous and vitreous humors, addressing the challenges posed by rare ocular matrices. The method enables accurate pharmacokinetic studies for ketorolac, reducing the need for extensive use of scarce ocular tissues. A clinical study is underway to evaluate ketorolac levels in patients undergoing cataract surgery, with the findings expected to inform future therapeutic strategies. The validated LC-MS/MS method is robust, sensitive, and suitable for quantifying ketorolac in aqueous and vitreous humors, addressing the challenges posed by rare ocular matrices.