From a 95% ethanolic extract of H. diffusa, four marker compounds (HD1~HD4) were isolated, which were relatively unique and exist in comparably high contents. The structures of marker compounds were identified as digitolutein (1), 2-hydroxy-3-methylanthraquinone (2), (E/Z)-6-O-p-coumaroyl scandoside methyl ester (4:1 mixture) (3), and (E/Z)-6-O-p-methoxycinnamoyl scandoside methyl ester (4:1 mixture) (4), respectively, on the basis of 13 C and 1 H-NMR analyses. The calibration curves of marker compounds showed high linearity, as their correlation coefficient (R 2 ) were in the range of 0.99910 .9999. In addition, the limit of detection (LOD) and the limit of quantification (LOQ) were 0.03~0.07 μg/ml and 0.099~0.231 μg/ml, respectively. The intra-day/inter-day precision and accuracy were 0.23~2.00%/0.25~1.16% and 94.60~108.44%/94.73-110.23%, respectively. The optimal HPLC conditions for the simultaneous quantification of HD1~HD4 were as follows: stationary phase; Merck Chromolith RP-18e (100×4.6 mm, 5 μm), column temp.; room temperature, UV detection at 280 nm, flow rate; 2.0 ml/min, injection volume; 10 μl, mobile phase; start with the mixture of 80% solvent A (H2O containing 0.5% acetic acid) and 20% solvent B (methanol containing 0.5% acetic acid) and gradually decrease solvent A to 40% in 9 min., then retain this condition to 18 min. Under the HPLC condition, the four marker compounds 1~4 were successfully separated without any interference of other constituents. The results obtained in this study are expected to be helpful for the development of nutraceutics and natural medicines and for the quality control of this plant.
Nucleotides play important roles in numerous intracellular biochemical processes
and are used in infant formulas and other dairy products. However, domestic
analytical methods for assessing nucleotide content in products have not yet
been established, and therefore, methods for determining nucleotide content are
urgently required. A rapid and simple analytical method for determining the
content of five types of nucleotides in dairy products was improved using solid
phase extraction clean-up and high-performance liquid chromatography with diode
array detector. The extraction solvent used in the AOAC method was not well
dissolved and was changed to hydrophilic EDTA-Na. In addition, the results
obtained using the isocratic elution method and a single wavelength were similar
to those obtained using the AOAC method, and the time taken for analysis was
shortened from 40 min to 25 min. The process of method validation revealed the
following parameters: accuracy (84.69%–102.72%), precision
(1.51%–6.82%), linearity (0.999), and limit of detection
(cytidine 5′-monophosphate, 0.09 mg/L; uridine 5′-monophosphate,
0.11 mg/L; adenosine 5′-monophosphate, 0.12 mg/L; guanosine
5′-monophosphate, 0.11 mg/L; and inosine 5′-monophosphate, 0.14
mg/L). The method was also used to determine the nucleotide concentration in 25
samples (infant formulas, 1.99–29.39 mg/100 g; and cow milk,
0.28–0.83 mg/100 g). The newly improved method was appropriate for
analyzing nucleotides in infant formulas and other dairy products faster when
compared to conventional methods.
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