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Day 2: TP32
LC/MS/MS Quantitation of Polypeptides, Proteins, and Antisense Nucleotides

Yongdong Zhu, Larry Duan, Daniel Meyer, Sherwin Jiang, Zamas Lam, and Ben Chien
Quest Pharmaceuticals Services, L.L.C., 3 Innovation Way, Suite 240, Delaware Technology Park, Newark, Delaware, 19711

This presentation describes the use of LC/MS/MS for the quantitation of large biomolecules (such as polypeptide, antisense nucleotide). Our experience has, so far, indicated that biomolecules of molecular weight over 5000 amu can be quantitated with the same methodology as small "drug-like" organic molecule to give similar sensitivity, selectivity, specificity, robustness and ruggedness. Using insulin analogues and a synthetic 14mer antisense nucleotide (molecular weight 4,500 amu), we will discuss calibration linearity, precision, accuracy, recovery, matrix effect, dilution effect, and specificity in the context of supporting a clinical trial. Using a "5-in-1" approach, we have developed and validated method for simultaneous quantitation of human insulin, its analogue, and their catabolites in plasma (molecular weight from 4500 to 6000 amu). The quantitation range for all five analytes was easily achievable from 1 ­ 500 ng/mL. The intraday precision and accuracy are listed below:

Analyte	6-Point Calibration Linearity (r2)	Intraday Precision n=5 @ 3conc (%cv)	Intraday Accuracy n=5 @ 3conc (%Diff)
Human Insulin	0.9960	3.3 - 5.6	0.1 - 9.7
Insulin Catabolite 1	0.9953	2.3 - 9.6	0.1 - 6.1
Insulin Analogue	0.9982	5.1 - 8.2	4.9 - 8.6
Insulin Analogue Catabolite 1	0.9921	2.3 - 3.8	-2.9 - 11.5
Insulin Analogue Catabolite 2	0.9991	2.9 - 3.7	-1.4 - 14.0

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