Objective: To establish a method for the determination of hypoxanthine and
inosine content in Geosaurus, and to determine the constituents of Geosaurus of five origins,
so as to explore a new and efficient analytical research method for the study of extraction
process and quality analysis of Geosaurus 's constituents. Methods: A high performance liquid
chromatographic (HPLC) method was established for the determination of hypoxanthine and
inosine. The separation was carried out on an Agilent extend C18 column, 4.6 mm×250 mm,
particle size: 5 μm, with the mobile phase of methanol -0.2% phosphoric acid, injection volume of
20 μL, wavelength of 254 nm, column temperature of 30 ℃ , and gradient elution, to establish the
standard curves of hypoxanthine and inosine and carry out the methodology of precision, stability,reproducibility, and sample recovery. The methodology of precision test, stability test, repeatability
test and sample recovery test was investigated. Results: The standard curve of hypoxanthine: y
= 78.819x + 245.92, R2 = 0.999 9, with good linearity in the range of 10.08~161.28 μg·mL-1; the
standard curve of inosine: y = 56.566x + 68.333, R2 = 0.999 5, with good linearity in the range
of 20.08~321.28 μg·mL-1; the standard curve of inosine: y = 56.566x + 68.333, R2 = 0.999 5,
with good linearity in the range of 20.08~321.28 μg·mL-1. The linear relationship was good in
the range of 20.08~321.28 μg·mL-1. The average recovery of hypoxanthine was 95.43% with
the RSD value of 1.52% (n=6); the average recovery of inosine was 99.86% with the RSD value
of 2.36% (n=6); and the contents of hypoxanthine in the five types of Geosaurus of Guangdong,
Henan, Hainan, Shanghai and Guangxi were 0.533 5, 0.346 8, 0.687 4, 0.687 4, 0.566x + 68.333,
R2= 0.999 5, with good linear relationship in the range of 20.08~321.28 μg·mL-1. The contents
of hypoxanthine in Geosaurus from Guangdong, Henan, Hainan, Shanghai and Guangxi were
0.533 5, 0.346 8, 0.687 4, 0.569 4, 0.794 5 (mg·g-1), and the contents of inosine were 1.562 5,
0.567 4, 1.126 7, 0.843 6, 1.284 9 (mg·g-1). Conclusion: The established HPLC method of high
performance liquid chromatography (HPLC) has a good gradient elution effect, good experimental
accuracy, good precision, good stability and high reproducibility, which is an efficient analytical
determination.
