Detection method of polyamine substances in blood plasma
A measurement method and plasma technology, applied in the field of medicine, can solve the problems of low content in the body and the incomplete establishment of polyamine detection methods, and achieve the effects of good repeatability, reduced side reactions, and easy operation
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Embodiment 1
[0037] (1) Blood sample pretreatment
[0038] a. Take 250 μL of plasma sample and add 5% HClO 4 125 μL, vortexed and centrifuged at high speed.
[0039] b. Take the supernatant after treatment in step a, add 2 mol mL -1 400 μL of NaOH, 125 μL of a mixed solution of benzoyl chloride and acetonitrile with a mass concentration of 4% benzoyl chloride, vortex to dissolve and mix well, 30 o C. Ultrasonic reaction for 30 min, adding 1 mL of saturated sodium chloride solution to terminate the reaction, extracting the derivatized polyamine with 2 mL of ether, drying under air flow, reconstitution with 50 μL of methanol, centrifuging at 15000 r.m.p. for 3 min, and taking 10 μL The supernatant was analyzed by LC-MS.
[0040] (2) Liquid phase separation: the chromatographic column is Kromasil C 18 Column (250 mm×4.6 mm, 5 μm); mobile phase A: methanol, B: water; gradient elution: 0-14 min: 55 % A→74 % A. Flow rate is 1.0 mL ? min -1 , the column temperature is 35 o C, inject 10...
Embodiment 2
[0047] (1) Blood sample pretreatment
[0048] a. Take 250 μL of plasma sample, add mass concentration of 9% HClO 4 125 μL, vortexed and centrifuged at high speed.
[0049] b. Take the supernatant after treatment in step a, add 0.5 mol mL -1 400 μL of NaOH, 125 μL of a mixed solution of benzoyl chloride and acetonitrile with a mass concentration of 4% benzoyl chloride, vortex to dissolve and mix well, 30 o C. Ultrasonic reaction for 30 min, adding 1 mL of saturated sodium chloride solution to terminate the reaction, extracting the derivatized polyamine with 2 mL of ether, drying under air flow, reconstitution with 50 μL of methanol, centrifuging at 15000 r.m.p. for 3 min, and taking 10 μL The supernatant was analyzed by LC-MS.
[0050] (2) Liquid phase separation: the chromatographic column is Kromasil C 18 Column (250 mm×4.6 mm, 5 μm); mobile phase A: methanol, B: water; gradient elution: 0-14 min: 55 % A→74 % A. Flow rate is 1.0 mL ? min -1 , the column temperature ...
Embodiment 3
[0057] (1) Blood sample pretreatment
[0058] a. Take 250 μL of plasma sample, add 150 μL of 1% acidic methanol, vortex to mix, and centrifuge at high speed.
[0059] b. Take the supernatant after treatment in step a, add 2 mol mL -1 NaOH 400 μL, 125 μL benzoyl chloride mass concentration of 1% mixed solution of benzoyl chloride and acetonitrile, vortex to dissolve and mix well, 30 o C. Ultrasonic reaction for 30 min, adding 1 mL of saturated sodium chloride solution to terminate the reaction, extracting the derivatized polyamine with 2 mL of ether, drying under air flow, reconstitution with 50 μL of methanol, centrifuging at 15000 r.m.p. for 3 min, and taking 10 μL The supernatant was analyzed by LC-MS.
[0060] (2) Liquid phase separation: the chromatographic column is Kromasil C 18 Column (250 mm×4.6 mm, 5 μm); mobile phase A: methanol, B: water; gradient elution: 0-14 min: 55 % A→74 % A. Flow rate is 1.0 mL ? min -1 , the column temperature is 35 o C, inject 10 μL...
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