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]
AAPS, Salt Lake City Utah, October 26-30
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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
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Pharmaceutical chemists require fast and efficient bioanalytical procedures. Sample preparation is often the rate limiting step and much research and product development has been devoted to increasing the speed of sample preparation. In recent years, sample preparation in the 96-well format has risen in popularity. However, protein precipitation has not been as successfully developed in this format. Many of the commercially available protein precipitation filter plates 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, a gradient of purpose-selected filtration media, together with a vented closure to allow a rapid ‘in-well’ sample preparation. The 96-well plate was designed with 1 mL reservoirs where each well contains a stack of chemically resistant and low extractible membrane filters. The selection of filtration media and the design of the flow path was customized for the protein precipitation purification solvents in that the wells do not empty until vacuum is applied. This allows the precipitation reaction to occur in the filtration plate without the need of a transfer step. 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. We examined the reproducibility, linearity and precision of the calibration curves generated as well as the recoveries of quality control samples. 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 outperforms current commercially available products. The new 96-well protein precipitation and filtration plate allows for simple and fast protein precipitation. This is the first commercially available protein precipitation plate that offers reproducible results with high recoveries that is easy to use.

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