Detection method for sialic acid in proteinic drug
A technology of sialic acid and protein, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve problems such as poor reproducibility, shortened column life, and long time
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Embodiment 1
[0149] Embodiment 1 The effect and specificity investigation of hydrophilic chromatographic column separation
[0150] Take 500 μg of protein sample, add water to 25 μl, then add 25 μl of 4M acetic acid and mix well, react at 80°C for 2 hours, then add 50 μl of labeling reagent that has been aliquoted, and react for 2 hours at 50°C in the dark, after the reaction, add 400 μl of acetonitrile, mix well After centrifugation at 13000r / min for 5 minutes, the supernatant was taken for chromatographic analysis.
[0151] The chromatographic parameters are: chromatographic column Waters Glycan BEH Amide; flow rate 0.4ml / min; detection wavelength ex=373nm, em=448nm; running time 12 minutes; injection volume 5μl. The sample tray temperature was set at 4°C. The eluent was a gradient of acetonitrile / water (85 / 15). The elution gradient of chromatographic analysis is shown in Table 6.
[0152] Table 6 The elution gradient of BEH amide
[0153] time flow rate ACN(%) h 2 O...
Embodiment 2
[0156] Example 2 Sample injection precision experiment
[0157] Repeat Example 1 to prepare 1 nmol of sialic acid standard substance, inject 6 times continuously, and investigate the stability of peak area and retention time.
[0158] The experimental results show that the retention time of six consecutive experiments is basically the same.
[0159] The results of the injection precision experiment are shown in Table 7.
[0160] Table 7 Injection precision results
[0161] frequency Nana RT Nana area NGNA RT NGNA area 1 3.00 396213 3.70 410972 2 3.00 390613 3.70 407435 3 3.00 393292 3.71 410560 4 3.00 391216 3.70 406978 5 3.00 386582 3.70 402065 6 3.00 389568 3.70 404223 average value 3.00 391247 3.70 407039 RSD(%) 0.0 0.8 0.1 0.9
[0162] The experimental results show that the peak areas and relative retention times of NANA and NGNA injected six times in a row are very stable (RSDs are n...
Embodiment 3
[0164] Embodiment 3 sialic acid standard substance linear inspection
[0165] Sialic acid standard handling
[0166] Take 1 μmol NANA and NGNA that have been aliquoted, take 10 μl each in the same centrifuge tube, add 980 μl ultrapure water and mix to obtain a 1 μmol / ml mixed standard sample as standard stock 1, take 100 μl stock 1 and add 900 μmol ultrapure water to obtain 0.1 μmol The mixed standard sample of NANA and NGNA / ml is used as standard stock 2, take 100 μl of stock 2 and add 900 μl ultrapure water to obtain 0.01 µmol / ml of NANA and NGNA mixed standard sample as standard stock 3, prepare different moles according to Table 8 Amount of standard solution, acid hydrolyzed at 80°C for 2 hours, after the reaction, add 50 μl of labeled reagent that has been dispensed, and react in the dark at 50°C for 2 hours, then add 400 μl of acetonitrile to terminate the reaction, centrifuge at 13000r / min for 5 minutes to take the supernatant chromatogram analyze.
[0167] Table 8 St...
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