Performance of an Ultra Low Elution Volume 96-Well Plate

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Claude Mallet, Ziling Lu, Jeff Mazzeo, Uwe Neue
HPLC 2002; Montreal; 3-4 June 2002
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Achieving both high throughput and low detection limits (LOQ''s) is now the challenge of in pharmaceutical analysis. Since the mid 90''s, additional emphasis has been directed on sample preparation for sub ng/mL levels and extraction speed. Classical extraction techniques, like liquid-liquid extraction (LLE) or protein precipitation (PPT), are not a suitable formats for high throughput or good clean-up. For example, LLE gives better recovery and clean-up but requires extensive manual labor and is not easily automated. PPT, on the other hand, is fast and can be automated, but the extract is not as clean as LLE or SPE techniques (potential loss of signal due to ion suppression). As more drug candidates show increased levels of potency, less dosage is required, hence the need for lower LOQ''s in order to define the pharmacokinetic profile. During pre-clinical trials of a drug candidate, sample volume is relatively low, most commonly in the 50 mL range. Clearly, using solid phase extraction (SPE) in a 96-well plate format (1,2) can keep up with these new demands for low sensitivity using less sample volume. With the assistance of a robotic liquid handler, a 96-well plate can be prepared in less than a minute per sample (3). The performance of a new 96-well ultra low elution volume plate for LC/MS/MS applications will be summarized. Each well in the plate contains 2 mg of a high capacity solid phase extraction polymer (Oasis HLB) (4) that has the capability of extracting analytes from several hundred microliters of plasma. A novel tip design permits quantitative recovery of the extracted analytes with as little as 50 microliters of elution solvent. Therefore, the plate provides as much as a 5-fold concentration of the analyte from plasma. The plate allows for simple dilution of the elution solution with water prior to LC/MS/MS analysis, avoiding an evaporation and reconstitution step. 1- Zweigenbaum, T., Heing, K., Steinborner, S., Wachst, T., Henion, J., Anal.Chem., 1999, 71: 2294 2- Show, W.Z., Jiang, X., Beato, B.D., Naidong, W., Rapid Commun. Mass Spectrom., 2001, 15: 466 3- Jemal, M., Huang, M., Mao, Y., Whigan, D., Powell, M., Rapid Commun. Mass Spectrom., 2001, 15: 994 4- Cheng, Y.F., Neue, D.U., Bean, L., J. Chromatogr. A, 1998, 828: 273

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