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A method for determining the concentration of endogenous amino acids asparagine and glutamine by liquid chromatography-mass spectrometry

A technology of asparagine and glutamine, which is applied in the field of medical testing, can solve problems such as inability to accurately reflect the dynamic changes of asparagine and glutamine, and the inability to obtain blank plasma without endogenous amino acids, reaching the analysis cycle Short, low cost, simple operation effect

Active Publication Date: 2015-08-26
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

None of these methods can obtain blank plasma without endogenous amino acids to accurately measure the concentration of asparagine and glutamine in the body, especially it cannot accurately reflect the dynamic changes of low-concentration asparagine and glutamine in the patient's body. Evaluate the efficacy of asparaginase in vivo
[0005] At present, there are no literature reports at home and abroad on how to deal with other endogenous amino acids to obtain a blank matrix for the preparation of a standard curve, so as to accurately determine the concentration of endogenous amino acids asparagine and glutamine in plasma (serum) samples

Method used

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  • A method for determining the concentration of endogenous amino acids asparagine and glutamine by liquid chromatography-mass spectrometry
  • A method for determining the concentration of endogenous amino acids asparagine and glutamine by liquid chromatography-mass spectrometry
  • A method for determining the concentration of endogenous amino acids asparagine and glutamine by liquid chromatography-mass spectrometry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Investigation of the extraction rate of amino acids in different proportions of methanol-water or in different proportions of acetonitrile-water solution

[0047] Serum samples were naturally thawed at room temperature (20°C), and 200 μL was added to 800 μL methanol-water or acetonitrile-water solution in different proportions, vortexed for 15 seconds, and then centrifuged at 11,000 rpm for 7 minutes. Transfer the supernatant to the vial of the autosampler, and wait for the sample injection at 20°C in the autosampler (the pretreatment process of each blood sample is completed within 3 hours).

[0048] Table 1 Different ratios of methanol-water or different ratios of acetonitrile-water solution on the extraction rate of amino acids

[0049]

[0050]

[0051] It can be seen from Table 1 that 60-70% methanol or 60-70% acetonitrile cannot ensure that the amino acid is fully extracted, and the concentration of high-concentration glutamine pure methanol or ac...

Embodiment 2

[0052] Embodiment 2: Investigate and analyze asparaginase potency and substrate elimination time

[0053] After the serum sample was naturally thawed at room temperature (20°C), 10IU, 20IU, 80IU, 150IU and 200IU asparaginase were added in parallel to each 15ml, and after standing for 24h, 16h, 8h, 4h and 2h respectively, 200μL was added to 20μL methanol - Mix 20 μL of water (80:20, V / V) solution. Add 780 μL of acetonitrile-water (80:20) solution, vortex for 15 seconds and centrifuge at 11000 rpm for 7 minutes. After transferring the supernatant, transfer it to the vial of the autosampler, and wait for the sample injection at 20°C in the autosampler (Note: the pretreatment process of each blood sample is completed within 3 hours).

[0054] Analysis of the relationship between asparaginase titer and substrate elimination time figure 1 As shown, there is a proportional relationship between the time required for asparaginase to catalyze the substrates asparagine and glutamine an...

Embodiment 3

[0055] Example 3: Investigate the effect of trypsin under different pH

[0056] Add 200IU asparaginase to healthy human plasma or serum samples and mix well, then immediately add a small amount of concentrated H 3 PO 4 After adjusting the pH to 2, 3, 4, 5, 6 and 7 respectively, add 200 IU of trypsin and mix well. After standing for 30 minutes, take 200 μL and add 20 μL internal standard solution and mix well. Add 780 μL of acetonitrile-water (80:20) solution, and other operations are consistent with Example 1.

[0057] Table 2: Comparison of the effect of trypsin at different pH

[0058]

[0059]

[0060] It can be seen from Table 2 that after plasma is acidified to pH 3-5 by phosphoric acid or acidification, trypsin can eliminate asparaginase, and when pH5, trypsin autolyzes and cannot inactivate asparaginase .

[0061] Table 3: 72h stability test of endogenous asparagine and glutamine after acidification with hydrochloric acid or phosphoric acid to pH 3-5 and addin...

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Abstract

The invention belongs to the medical examination field, and aims to provide a method for accurately determining the concentrations of endogenous amino acid asparagines and glutamine in a blood plasma or serum sample. The method can be used for accurately, simply and rapidly evaluating the in-vivo medicine effect of a leukemia patient treatment medicine asparaginase. The method adopts an LC / MS spectrometer to determine the concentrations of endogenous amino acid asparagines and glutamine in the blood plasma or serum sample. The method comprises the following steps: processing a blank matrix, preprocessing the blood plasma or serum sample to be detected, and separating and analyzing the blood plasma or serum sample to be detected.

Description

technical field [0001] The invention belongs to the field of medical examination, and in particular relates to a method for measuring the concentration of endogenous amino acids asparagine and glutamine in vivo by liquid mass spectrometry. Background technique [0002] Asparaginase (L-asp) has been used in the treatment of acute lymphoblastic leukemia (ALL) for more than 30 years. Studies from various countries have reported that adding L-asp to the ALL induction regimen can improve the complete remission rate (CR) and cure rate, especially in acute lymphoblastic leukemia (ALL). The 5-year event-free survival rate in the treatment of B lymphocytic leukemia can reach 76-86% (reference: 1. Pui, C.H., Carroll, W.L., Meshinchi, S., & Arceci, R.J. Biology, risk stratification, and therapy of pediatric acute leukemias: An update.Journal of Clinical Oncology,2011;29(5):551-565.2. Chinese Society of Clinical Oncology (CSCO), Chinese Society of Hematology (CHS), Chinese Society of Pe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
Inventor 王卓张文静高申万思慧许孟雪丁雪鹰何金凤丁楠
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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