Document 4Jg4pqeDKB1XmgmJa70O52z4Q

AKA _ m -o r 9 000130 Aknas- zvof s T itle: Bioanalytical Method Validation for the Quantitation o f Perfluorooctanoic A dd in Human Serum A uthors: David J. Anderson1; Kimberly L. Norwood1; Stephen Lowes1; Mary A. Kaiser2; Barbara S. Larsen3 Institutes: `Advion BioSciences, Inc., Ithaca, New York; 2Dupont Experimental Station, Wilmington, Delaware Novel Aspect: A rigorous bioanalytical method validation o f a representative, ubiquitous perfluorinated compound. In tro d u c tio n Perfluorinated surfactants have received increased attention in the environmental and human health communities due to their ubiquitous presence, as well as their environmental persistence and possibly long half-lives in the human bloodstream. Advances in analytical methodology have facilitated the detection o f these compounds at background levels in the low parts-per-billion range in human blood plasma and serum. The analytical uncertainty associated with these measurements is open to scientific criticism, and the errors are often unacceptable when compared to typical bioanalytical acceptance criteria. Application o f the bioanalytical approach to method validation w ill be presented for the quantitation o fperfluorooctanoic acid (PFOA) in human serum for two LC/MS/MS methods. M ethods The bioanalytical methods both utilized ion-pairing, liquid-liquid extraction followed by gradient elution liquid chromatography and tandem mass spectrometry. Sbimadzu LC 10AD pumps, a PE200 autosampler, and ionspray SRM MS/MS (positive ion) on a PE Sciex API 3000 were used in both methods. The target ranges o f quantitation for the two methods were l ng/mL to 100 ng/mL raid 0.05 pg/m Lto 10 pg/mL. A structural analog, 9H-hexadecafluorononaaoic acid, was used as the internal standard (IS). Due to the presence o f PFOA in control human serum, the standard curves for the lower-level method were prepared in rabbit serum. Quality control (QC) samples were assayed in both human and rabbit serum to validate the use of rabbit serum standards for the quantitation o f PFOA in human serum. Background subtraction was used to correct for the presence o f PFOA in the control serum in both methods. In addition, extensive measures were taken to eliminate potential sources o f PFOA from the extraction and the chromatographic system. Prelim inary D ata Higher-Level Method; The assay demonstrated a lower lim it o f quantitation o f 50 ng/mL based upon a 25 pL aliquot o f human serum. The calibration curves were fitted with a 1/x* weighted quadratic regression, and the coefficients o f determination ( f ) were > 0.9950. The intra- and inter-assay accuracy (RE, -12.3 to 9.00%) and precision (CV, 2.07 to 7.70%), based upon the QC samples, were within the acceptance criteria ( 15%). The extraction recoveries o f PFOA and file IS were 81.8 and 90.9%, respectively. Lower-Level Method; The assay demonstrated a Iow a lim it o fquantitation o f 1 ng/mL based upon a 50 pL aliquot o f human serum. The calibration curves were fitted with a 1/x* weighted quadratic regression, and the coefficients o f determination (r1) were > 0.9929. The intra- and 000131 inter-assay accuracy (RE, -0.567 to 5,57%) and precision (CV, 1.77 to 4.66%), based upon the human serum QC samples, were within the acceptance criteria ( 15%). Keywords: Ionspray, MS/MS, Liquid Chromatography; Quantitative Analysis; Selected Reaction Monitoring (Revision: 31 January 2003)l l i 000132