A kind of pretreatment method of free oxytocin in biological fluid sample

A technology for biological fluids and oxytocin, applied in the field of analytical chemistry, can solve the problems of long time-consuming, complicated maintenance and methods, and low oxytocin retention rate

Active Publication Date: 2020-08-11
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, SPE processing steps are cumbersome and time-consuming
Although online SPE sample pretreatment simplifies the entire processing process and reduces manual steps, online operation requires special equipment, its maintenance and method settings are complicated, and often results in long chromatographic run times. Therefore, laboratory and clinical samples rarely used in analysis
In addition, the strong hydrophilicity of free oxytocin will cause serious loss in SPE media
Ultrafiltration centrifugation is easy to operate, but the retention rate of oxytocin with a molecular weight of only 1007.2Da is low, and the sample loss is serious

Method used

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  • A kind of pretreatment method of free oxytocin in biological fluid sample
  • A kind of pretreatment method of free oxytocin in biological fluid sample
  • A kind of pretreatment method of free oxytocin in biological fluid sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1 Plasma free oxytocin pretreatment

[0090] (1) Plasma collection

[0091] Human whole blood was collected with a commercial vacuum blood collection tube (BDTM P800, Becton, Dickinson and Company, USA), centrifuged at 3000rpm for 10min, and the supernatant was taken to obtain plasma, which could be directly pretreated or stored in a refrigerator at -80°C.

[0092] (2) Protein denaturation

[0093] Take 600 μL of human plasma in step (1), put it in a 10ml centrifuge tube, add an internal standard of oxytocin isotope (Pro[13C5,15N]oxytocin, Invitrogen Trading Co., Ltd, Shanghai, China) aqueous solution (final concentration 200pg / mL), add 240 μL 5v% phosphoric acid aqueous solution, and vortex for 30s.

[0094] (3) Oxytocin extraction and impurity removal

[0095] After the plasma sample is processed in step (2), add 4 mL of dipotassium hydrogen phosphate aqueous solution (4M), vortex for 30 seconds, add 4 mL of isopropanol, shake fully 10 times, vortex for 30...

Embodiment 2

[0100] Example 2 Plasma free oxytocin pretreatment

[0101] The pretreatment method of plasma free oxytocin in this example is similar to Example 1. in:

[0102]In step (2) protein denaturation, "240 μL 5v% phosphoric acid aqueous solution" was replaced with "120 μL 4wt% sodium dodecylsulfonate aqueous solution";

[0103] In the step (3) of oxytocin extraction and impurity removal, the concentration of dipotassium hydrogen phosphate aqueous solution is 0.7M;

[0104] In step (4) concentration and secondary desalination, "1ml isopropanol" is replaced with "0.6ml isopropanol";

[0105] In step (5) oxytocin collection, "50 μL of 15% acetonitrile (15% by volume, water as solvent)" was replaced by "30 μL of 30% acetonitrile (30% by volume, water as solvent)".

[0106] The remaining operations and conditions are the same as in Example 1, and the supernatant finally obtained is the desired oxytocin-enriched fraction.

Embodiment 3

[0107] Example 3 Plasma free oxytocin pretreatment

[0108] (1) Plasma collection

[0109] The whole blood of SD rats was collected with a commercial vacuum blood collection tube (containing NaEDTA), centrifuged at 3000rpm for 10min, and the supernatant was obtained to obtain plasma, which could be directly pretreated or stored in a refrigerator at -80°C.

[0110] (2) Protein denaturation

[0111] Take 300 μL of the rat plasma in step (1), put it in a 10ml centrifuge tube, add an internal standard of oxytocin isotope (Pro[13C5,15N]oxytocin, Invitrogen Trading Co., Ltd, Shanghai, China) aqueous solution (final concentration 200pg / mL), add 120 μL 5v% phosphoric acid aqueous solution, and vortex for 30s.

[0112] (3) Oxytocin extraction and impurity removal

[0113] After the plasma sample is processed in step (2), add 4 mL of dipotassium hydrogen phosphate aqueous solution (4M), vortex for 30 seconds, add 4 mL of isopropanol, shake fully 10 times, vortex for 30 seconds, and ...

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Abstract

The present application discloses a method for the pretreatment of free oxytocin in a biological fluid sample, which at least includes: (1) subjecting the biological fluid sample to oxytocin desorption to obtain system I; (2) subjecting the system I in step (1) to Selective enrichment of oxytocin and removal of impurities to obtain System II; (3) Concentrating and secondary desalting of System II in step (2) to obtain System III; (4) Collection of oxytocin. The method described in this application can simply and efficiently enrich free oxytocin from biological fluids, remove high-abundance proteins, inorganic salts, small hydrophilic molecules, and lipids and other interfering substances for nanoLC‑MS analysis and quantification of biological fluids Free oxytocin in the sample.

Description

technical field [0001] The application relates to a pretreatment method for free oxytocin in biological fluid samples, which belongs to the field of analytical chemistry. Background technique [0002] Oxytocin is a neuropeptide that may be associated with complex emotional and social behaviors such as social cognition, stress buffering, aggression and trust. Oxytocin, which exists in the free state in biological fluids, is closely related to highly specific individual conditions such as age, sex, and drug abuse history as a neuromodulator. Accurate determination of oxytocin is important for understanding the role of this peptide in various neurophysiological processes and behaviors. The key to clinical diagnosis is more valuable. The accurate analysis of free oxytocin in biological fluids is facing great challenges. The composition of biological fluids, especially plasma, is complex, which is not compatible with high-efficiency analysis techniques (such as liquid mass analy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/06
CPCG01N30/06G01N2030/062
Inventor 张晓哲刘丹刘欣欣程孟春赵楠
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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