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CN-122017073-A - Method and kit for detecting plasma renin mass by utilizing liquid chromatography tandem mass spectrometry

CN122017073ACN 122017073 ACN122017073 ACN 122017073ACN-122017073-A

Abstract

The invention relates to the technical field of liquid chromatography tandem mass spectrometry detection, and provides a method and a kit for detecting plasma renin mass by using liquid chromatography tandem mass spectrometry. The invention provides a mass spectrometry method for detecting the quality of plasma sample renin for the first time, and adopts a pretreatment method of immunomagnetic bead capture to enrich the sample, so that the detection sensitivity can be improved. The isotopes N15 and C13 are adopted to mark the renin specific peptide fragment as an internal standard, and the peak area ratio and the concentration of the renin specific peptide fragment and the internal standard peak area ratio are used for calibration. The method has good precision, repeatability and laboratory imprecision CV of less than 15 percent, and the method and the kit for detecting the quality of the plasma renin based on the liquid chromatography tandem mass spectrometry can be popularized and applied in clinical detection.

Inventors

  • YU SONGLIN
  • QIU LING
  • WANG DANCHEN
  • MA XIAOLI
  • LI XUEZHI

Assignees

  • 中国医学科学院北京协和医院

Dates

Publication Date
20260512
Application Date
20260226

Claims (9)

  1. 1. A method for detecting plasma renin mass by liquid chromatography tandem mass spectrometry, comprising the steps of: A. preparing renin standard substance solutions with different concentrations; B. Adding magnetic beads into a standard substance solution for extraction, incubating, firstly cleaning the magnetic beads by using a cleaning solution, eluting by using an eluent, then adding ammonia water, an isotope internal standard and a pancreatin solution into the collected eluent, incubating and enzyme cutting; C. replacing the standard substance solution in the step B with a plasma sample to be detected, and detecting by adopting the same method; D. according to the detection result of the plasma sample to be detected, comparing the standard curve to obtain the concentration of the renin mass in the plasma sample to be detected; The isotope internal standard is a renin specific polypeptide marked by N15 and C13, and comprises a quantitative internal standard and a qualitative internal standard, wherein the renin specific polypeptide corresponding to the quantitative internal standard is LIKTGVWQIQMKGVS, the K lysine is marked by N15 and C13, and the renin specific polypeptide corresponding to the qualitative internal standard is GRVTPIFDNIISQGVLKED, and the F phenylalanine is marked by C13 and N15.
  2. 2. The method of claim 1, wherein in step a, the standard solution is formulated with bovine serum.
  3. 3. The method according to claim 1, wherein the step B is characterized in that 40-200 mu L of magnetic bead solution is added into 100-600 mu L of standard substance solution for mixing, shaking and incubation are carried out at 37 ℃ for 30-90min, then 50-200 mu L of cleaning liquid is used for washing 1-5 times sequentially, 20-100 mu L of eluent is used for eluting, 2-6 mu L of 5-15% ammonia water, 2-20 mu L of mixed internal standard solution and 5-20 mu L of 10-50 ng/mu L of pancreatin solution are sequentially added into the collected eluent, shaking and incubation are carried out at 37 ℃ for 10-30h, and samples are collected for on-machine detection; Wherein, the mixed internal standard solution is 1-50 ng/mL of quantitative internal standard and 1-200ng/mL of qualitative internal standard, and the reagent for preparing the isotope internal standard solution is 10-100mM NH 4 HCO 3 solution.
  4. 4. The method according to claim 1, wherein the liquid chromatography column used in step B is ACQUITY UPLC HSS T3.1.8 μm,2.1 mm. Times.50 mm; The liquid chromatography detection condition is that the flow rate is 0.3mL/min, and the column temperature is 40 ℃; Gradient elution was performed according to the following procedure: ; Wherein, the mobile phase A is 0.1% v/v formic acid aqueous solution, and the mobile phase B is methanol.
  5. 5. The method of claim 1, wherein the mass spectrometry is performed in step B under conditions of a Waters Xevo TQ-S for mass spectrometry, an electrospray ionization source for positive mode acquisition, a capillary voltage of 2.5kv, an ion source temperature of 150 ℃, a desolvation temperature of 550 ℃, desolvation gas of 1100L/Hr, and a cone gas of 150L/Hr.
  6. 6. The method according to claim 1, wherein the characteristic peptide fragment for quantitative detection of renin is LIKTGVWQIQMKGVS, the corresponding ion pair is 545.79 → 833.43 or 546→833, the characteristic peptide fragment for qualitative detection of renin is GRVTPIFDNIISQGVLKED, and the corresponding ion pair is 822.47 → 722.41.
  7. 7. The method of claim 1, wherein the ion pair generated in the mass spectrum for quantitatively detecting the isotope internal standard is 549.79-841.44 or 550-841.44, and the ion pair for qualitatively detecting the isotope internal standard is 827.49-727.46.
  8. 8. The method of any one of claims 1-7, wherein the linear detection range of renin in the plasma sample is 0.01-4 ng/mL.
  9. 9. The kit for detecting the renin by liquid chromatography tandem mass spectrometry is characterized by comprising at least one of a renin standard solution, an isotope internal standard, magnetic beads, a cleaning solution and an eluent; The isotope internal standard is a renin specific polypeptide marked by N15 and C13, and comprises a quantitative internal standard and a qualitative internal standard, wherein the renin specific polypeptide corresponding to the quantitative internal standard is LIKTGVWQIQMKGVS, the K lysine is marked by N15 and C13, and the renin specific polypeptide corresponding to the qualitative internal standard is GRVTPIFDNIISQGVLKED, and the F phenylalanine is marked by C13 and N15.

Description

Method and kit for detecting plasma renin mass by utilizing liquid chromatography tandem mass spectrometry Technical Field The invention relates to the technical field of liquid chromatography tandem mass spectrometry detection, in particular to a method and a kit for detecting plasma renin mass by using liquid chromatography tandem mass spectrometry. Background Primary aldosteronism is a common cause of secondary hypertension, and refers to excessive secretion of aldosterone by the adrenal cortex, leading to in vivo sodium retention and potassium release, increased blood volume, and inhibited renin-angiotensin system activity. Clinically, hypertension is mainly manifested as hypokalemia. The diagnosis of the protoaldehyde type influences the selection of a treatment scheme, and therefore a clinician is required to judge the lesion type by means of imaging, and comprehensive judgment is carried out by combining biochemical indexes, imaging performance, bilateral adrenal vein blood sampling (AVS) results and the like. Whereas renin (Renin) is a core regulator of the renin-angiotensin system (Renin-Angiotensin System, RAS), direct measurement of renin is the most laboratory of choice, currently the primary immunological method, without mass spectrometry. The mutation among the immunization methods is less active, but compared with the mass spectrometry methodology principle, the problems of large mutation, cross reaction and the like still exist. Therefore, there is a need to develop a stable, efficient mass spectrometry method for performing renin mass measurement. Disclosure of Invention The invention aims to provide a method and a kit for detecting plasma renin mass by utilizing liquid chromatography-tandem mass spectrometry. To achieve the object of the present invention, in a first aspect, the present invention provides a method for detecting plasma renin mass by liquid chromatography tandem mass spectrometry, comprising the steps of: A. preparing renin standard substance solutions with different concentrations; B. adding magnetic beads into a standard substance solution for extraction, incubating, firstly cleaning the magnetic beads by using a cleaning solution, eluting by using an eluent, then adding ammonia water, an isotope internal standard and a pancreatin solution into the collected eluent, incubating for a period of time, and performing enzyme digestion; C. replacing the standard substance solution in the step B with a plasma sample to be detected, and detecting by adopting the same method; D. According to the detection result of the plasma sample to be detected, comparing the standard curve to obtain the concentration of renin in the plasma sample to be detected; Wherein the isotope internal standard is a renin specific polypeptide marked by N15 and C13, and comprises a quantitative internal standard and a qualitative internal standard, wherein the renin specific polypeptide corresponding to the quantitative internal standard is LIKTGVWQIQMK (N15, C13) GVS, wherein K lysine is marked (N15, C13), and the renin specific polypeptide corresponding to the qualitative internal standard is GRVTPIF (C13, N15) DNIISQGVLKED, wherein F phenylalanine is marked (C13, N15). Further, in step a, a standard solution is prepared with bovine serum. Further, the step B comprises the steps of adding 40-200 mu L of magnetic bead solution into 100-600 mu L of standard substance solution, mixing, vibrating and incubating at 37 ℃ for 30-90 min times, sequentially washing with 50-200 mu L of cleaning solution for 1-5 times, eluting with 20-100 mu L of eluent, sequentially adding 2-6 mu L of 5-15% ammonia water, 2-20 mu L of mixed internal standard solution and 5-20 mu L of 10-50 ng/mu L of pancreatin solution into the collected eluent, vibrating and incubating at 37 ℃ for 10-30 h, collecting samples and preparing for machine detection; wherein, the mixed internal standard solution is 1-50 ng/mL of quantitative internal standard and 1-200 ng/mL of qualitative internal standard, and the reagent for preparing the isotope internal standard solution is 10-100mM NH 4HCO3 solution. Preferably, step B is to add 100. Mu.L of magnetic bead solution to 400. Mu.L of standard solution, mix, incubate for 1h with shaking at 37 ℃, wash with 100. Mu.L of washing liquid sequentially for 3 times, elute with 50. Mu.L of eluent, then add 3.4. Mu.L of 10% ammonia water to the collected eluent, 10. Mu.L of mixed internal standard solution (25 ng/mL of quantitative internal standard and 125ng/mL of qualitative internal standard) and 15. Mu.L of 16 ng/mu.L of pancreatin solution sequentially, incubate with shaking at 37 ℃ for 24h, collect samples ready for on-machine detection, wherein the reagent for preparing isotope internal standard solution is 50mM NH 4HCO3 solution. Preferably, the liquid chromatography column used in step B is ACQUITY UPLC HSS T, 3.8 μm,2.1 mm. Times.50 mm. Preferably, the liquid chromatography detection conditions are a