High-Throughput, Simplified Protein Precipitation: A Novel 96-Well Format for Precipitation and Filtration

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Kimvan Tran, Diane M. Diehl, Brian P. Murphy [Waters Corporation] Tim Schultz, Wilf Wixwat, Jeff Kane, Emily Berlin, Kevin Seeley [Pall Life Sciences, Ann Arbor, MI]
EAS, Somerset, New Jersey, November 17-20
Content Type:
Oasis HLB 5 mg µElution 96-well Plate
Basic Analytes: Propranolol; Trimethoprim; Pipenzolate; Risperidone; Terfenadine; Methoxyverapamil; Reserpine; Ketoconazole Acidic Analytes: Fumaric acid; Malic acid; Etidronic acid; Clodronic acid; Niflumic acid; Canrenoic acid; Cholic acid; Raffinose
Protein precipitation is a simple sample preparation method, but can be long and tedious, and is difficult to automate. There several commercially available protein precipitation filter plates, but they suffer from poor performance. Depending on the procedure and the plate, plasma often leaks into the filter before a vacuum is applied, leading to a cloudy filtrate. Other plates suffer from a high percentage of blocked wells. To address these performance issues, we developed a new 96-well protein precipitation and filtration plate that offers a dripless technology with a gradient of purpose-selected filtration media, together with a vented closure to allow for rapid ‘in-well’ sample preparation. We ran a series of ion suppression and extraction studies to ensure the cleanliness of the plate materials. We validated the plate performance for a variety of polar and non-polar acids, bases and neutrals spiked into porcine and rat plasmas. We used both manual and automated methods to evaluate the plate and compared the results to traditional centrifugation methods and other commercially available products. In our studies, we did not observe liquid flow until vacuum is applied. Additionally, less than 5% of the wells plug. The time for filtration is less than 1 minute, and the time to process an entire plate of 96 samples is approximately 20 minutes. There is no observable cross talk between the wells. The eluents are free of observable particulates and offer a cleaner eluent than the traditional centrifugation method. The plate works equally well in a manual or automated process. The plates are highly reproducible and linear, as well as result in greater than 95% recoveries for the analytes examined. This plate out-performs current commercially available products.

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