Method for measuring content of sialic acid in serum
A technology of sialic acid and free sialic acid, applied in the field of detection, can solve the problems of complicated operation and difficult ionization, and achieve the effect of simple operation, small matrix effect and good reproducibility
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Embodiment 1
[0078] Embodiment one, measure total sialic acid content in serum
[0079] The present embodiment provides the method for determining the total sialic acid content in serum, which specifically includes the following steps:
[0080] 1) Mix 10 μL human serum with 90 μL ultrapure water;
[0081] 2) Add 100 μL of 1mol / L trifluoroacetic acid, mix well, place in an oven at 80°C for acid hydrolysis for 1 hour, after cooling, centrifuge at 1500 g for 5 minutes, take the supernatant, aliquot, 20 μL per tube, vacuum at room temperature Dry, add 98 μL ultrapure water, 2 μL internal standard;
[0082] 3) Add 100 μL of GP reagent (1 mg / mL, dissolved in water: acetic acid: methanol = 6:3:1, v / v / v) to obtain the derivative product;
[0083] 4) Take 20 μL of the derivative product and dry it under vacuum at 60° C., then reconstitute with 1 mL of methanol, and load 1 μL of liquid mass.
[0084] Chromatographic column: flow rate 0.3mL / min; running time 10 minutes; injection volume 1μL. The ...
Embodiment 2
[0085] Embodiment two, measure free sialic acid content in serum
[0086] The present embodiment provides the method for measuring free sialic acid content in serum, specifically comprises the following steps:
[0087] 1) Take 40 μL of human serum and add 8 μL of internal standard substance (5000ng / mL) to mix well, then add 144 μL of acetonitrile, mix well, centrifuge at 15000g for 10 minutes, take the supernatant, aliquot each tube with a volume of 4.8 μL, and vacuum dry at room temperature , add 95.2 μL ultrapure water;
[0088] 2) Add 100 μL of GP reagent (1 mg / mL, dissolved in water: acetic acid: methanol = 6:3:1, v / v / v) 5000 g, centrifuge for 5 minutes, take out 20 μL, vacuum-dry at 60 ° C, and then use After reconstitution in 100 μL of methanol, add 1 μL of LC / MS.
[0089] Chromatographic column: flow rate 0.3mL / min; running time 10 minutes; injection volume 1μL. The sample tray temperature was 4°C. Elution gradient: 0-4min, 5%-30%B, 4-6min, 30%-95%B, 6-8min, keep 95...
Embodiment 3
[0091] Embodiment three, method verification
[0092] The present embodiment uses sialic acid standard solution to verify the assay method, which specifically includes the following steps:
[0093] 1) Take 5 μL of sialic acid standard solution (10 μg / ml) and mix with 93 μL of ultrapure water, then add 2 μL of internal standard and mix evenly to prepare a low-concentration spiked solution;
[0094] 2) Take 5 μL of sialic acid standard solution (100 μg / ml) and mix with 93 μL of ultrapure water, then add 2 μL of internal standard, and mix evenly to prepare a medium-concentration spiked solution;
[0095] 3) Mix 10 μL of sialic acid standard solution (100 μg / ml) with 88 μL of ultrapure water, then add 2 μL of internal standard, and mix evenly to prepare a high-concentration spiked solution;
[0096] 4) Then add 100 μL of GP solution, mix evenly, centrifuge at 5000 g for 5 min, take out 20 μL, dry in vacuum at 60°C, and reconstitute with 1 mL of methanol. LC-MS injection 1 μL.
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