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Serum diaminopimelic acid detection kit based on liquid chromatography-mass spectrometry, detection method and application

A technology of diaminopimelic acid and detection kit, which is applied in the directions of measuring devices, instruments, scientific instruments, etc., can solve the problems of limited disease indication effect, large fluctuation range, unstable content of diaminopimelic acid, etc. The effect of good guidance, improved sensitivity, improved accuracy and stability

Active Publication Date: 2021-09-03
FIRST AFFILIATED HOSPITAL OF DALIAN MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the detection method of diaminopimelic acid in blood. Because the content of diaminopimelic acid in urine is unstable and affected by various factors (such as urine collection time, water intake, etc.), the fluctuation range is large. Therefore, the indication of disease is limited

Method used

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  • Serum diaminopimelic acid detection kit based on liquid chromatography-mass spectrometry, detection method and application
  • Serum diaminopimelic acid detection kit based on liquid chromatography-mass spectrometry, detection method and application
  • Serum diaminopimelic acid detection kit based on liquid chromatography-mass spectrometry, detection method and application

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Effect test

Embodiment 1

[0034] The present invention utilizes an isotope internal standard and a derivatization reagent, first conducts chromatographic separation through a reversed-phase column, and then conducts quantitative analysis using a triple quadrupole mass spectrometer, thereby more accurately quantifying the content of diaminopimelic acid in serum (plasma) samples .

[0035] The composition of the kit of the present invention: extraction reagent {diaminopimelic acid standard (100 mg), diaminopimelic acid isotope internal standard (deuterated lysine (Lys-d4)}; derivatization reagent {borate Buffer (PH8~9), 6-aminoquinoline-N-succinimide methyl ester (AQC)}.

[0036] The DAP standard is used to draw the standard curve: use 0.1M HCL to dissolve the DAP standard, and then use 50% acetonitrile / water for serial dilution. The dilution concentration includes: 230, 115, 23, 11.5, 2.3, 1.15, 0.23, 0.115, 0.023, 0.0115, 0.0023, 0 (ng / mL), draw the standard curve with the standard concentration as th...

Embodiment 2

[0047] The detection method was used to accurately quantify the serum DAP content of rats with severe acute pancreatitis.

[0048] In this experiment, 28 male SD rats were divided into 4 groups: sham operation group (SO group); severe acute pancreatitis model group (SAP); Qingyi granule treatment group (QYKL); neomycin pretreatment group (Neo) . Waters UPLC-TQXS liquid mass spectrometry system was used to quantitatively analyze the serum DAP content of rats in each group.

[0049] Serum pretreatment and detection methods are the same as in Example 1.

[0050] Test results: Compared with the sham operation (SO) group, the serum DAP level of SAP rats was significantly increased (p Figure 5 shown. It suggested that the serum DAP level of rats with severe pancreatitis was significantly increased.

Embodiment 3

[0052] The detection method was used to accurately quantify the serum DAP content of patients with acute pancreatitis of different severity and healthy subjects. A total of 121 serum samples were collected from patients with acute pancreatitis and healthy subjects, including 12 healthy subjects; 81 patients with mild acute pancreatitis (MAP); 13 patients with moderate to severe acute pancreatitis (MSAP); 15 patients with inflammatory disease (SAP). The detection method was used to determine the DAP content of the serum samples of subjects in each group. The results showed that the serum DAP content of SAP patients was significantly higher than that of other groups (p Figure 6 A). It has good efficacy for distinguishing MSAP and SAP, AUC is 0.731 (95% CI: 0.535~0.926), cut-off value is 1.27, sensitivity and specificity are 86.7% and 61.5% respectively ( Figure 6 B).

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Abstract

The invention discloses a serum diaminopimelic acid detection kit based on liquid chromatography-mass spectrometry, a detection method and application, and belongs to the technical field of biological detection. The detection kit comprises an extraction reagent and a derivatization reagent; the extraction reagent comprises a DAP standard substance and a DAP isotope internal standard substance, and the derivatization reagent comprises a borate buffer solution and 6-aminoquinoline-N-succinimide methyl ester (AQC). Through research and development of the kit, the content of DAP in serum / plasma can be relatively rapidly and accurately quantified, and the repeatability is good. And the detection accuracy and stability can be improved by using the isotope internal standard. And the detection sensitivity can be improved by utilizing derivatization.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a detection kit, a detection method and an application of serum diaminopimelic acid based on liquid chromatography-mass spectrometry. Background technique [0002] Diaminopimelic acid (DAP) belongs to the peptidoglycan component of bacterial cell wall, widely exists in most Gram-negative bacteria and some Gram-positive bacteria. Because it is widely distributed in bacteria, diaminopimelic acid is considered to be a highly specific bacterial taxonomic marker. However, due to the low content, detection is limited and accurate measurement is more difficult. At present, there have been studies on the detection of diaminopimelic acid in urine, which has been derivatized with o-phthalaldehyde (OPA) derivative reagents, but the derivative products are unstable. However, there is no report on the detection method of diaminopimelic acid in blood. Because the con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/72
CPCG01N30/02G01N30/06G01N30/72G01N2030/067
Inventor 尚东尹沛源刘建均焦巨英潘晨
Owner FIRST AFFILIATED HOSPITAL OF DALIAN MEDICAL UNIV
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