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Volume 4, Issue 2 American Journal of Ethnomedicine
ISSN 2348-9502
Natural Products Congress & World Pharma Congress 2017
October 16-18, 2017
3rd World Congress on
NATURAL PRODUCTS CHEMISTRY AND RESEARCH
&
12th WORLD PHARMA CONGRESS
October 16-18, 2017 Budapest, Hungary
Optimization and validation of a fluorescent kinetic analysis for the measurement of enzymatic
activity of plasma DPP4
Hyunyee Yoon1, 2, Yu Rim Seo1, Su Hee Cho1, Sung Sup Park1 and Moon Jung Song2
1Seoul National University Hospital, South Korea
2Korea University, South Korea
In the course of a clinical study developing evoglipitin (DA-1229) tartrate, a specific dipeptidyl peptidase 4 (DPP4) inhibitor
for the treatment of type 2 diabetes, an analytical method of fluorescent kinetic assay was optimized and verified to determine
an enzymatic activity of soluble DPP4 in human plasma using a spectrofluorometer. The validation was performed for the
parameters including the accuracy, the precision, the Limit of Detection (LOD), the linearity, the dynamic range, the short/
long-term stability, the freezing-thawing stability, the Km constant, the dilution effect, and the recovery efficiency. The plasma
DPP4 enzymatic activity (mU/min) was measured as the Initial Velocity (VO) of enzymatic reaction over time. After the
reaction, the deviation of the mean from the nominal value, the Coefficient of Variation (CV) within/between runs, and the
relative determinant constant (R2) were calculated. Accuracy and precision were within the deviation of the mean ≤15%, CV
≤15%, R2>0.99 except for LOD, where it did not exceed the deviation of the mean ≤20%, CV ≤15%, R2>0.95, respectively.
The linearity of VO and the dynamic range of DPP4 values were reliable in the range of 6.06x103–5.13 x 105 mU/min and
62.5-1,500 ng/mL, respectively. Plasma DPP4 was stable under the various temperatures and even after three cycles of the
freezing-thawing. The Km constant of plasma DPP4 was similar to that of the recombinant DPP4. Evoglipitin (DA-1229)
tartrate effectively inhibited the DPP4 enzymatic activity in a dose-dependent manner without the dilution effect of sample.
Due to the limited recovery efficiency of DPP4 in sample larger than 10 uL, the volume of sample was determined to be 10 uL
for reliable assays. The optimized and validated analysis method of the DPP4 activity was successfully set up and employed for
the measurement of the DPP4 activity in human plasma.
Biography
Hyunyee Yoon has expertise in developing the bio-analytic methods in the field of clinical studies. She has been operating the protein immunology core facility
to support a number of clinical researchers at Seoul National University Hospital and Biomedical Research Institute since 1999. The major assays that she has
worked on include protein quantification by an ELISA and a fluorescent multiplex bead array, protein qualification by a western blot and a Simple Western™ assays,
enzymatic kinetic assay using a multi-detection analyzer, and immunogenicity analysis using a Meso Scale Discovery immunoassay. She has accumulated in-depth
knowledge and experiences on various assays that are essential for the development and validation of the pharmacokinetic and pharmacodynamics analysis of
clinical researches. Her continuous effort to resolve the complicated study derives the precise and reliable results.
Hyunyee Yoon et al., American Journal of Ethnomedicine, 4:2
DOI: 10.21767/2348-9502-C1-003