Method for improving methionine enkephalin blood sample detection sensitivity

Abstract

The invention discloses a method for improving methionine enkephalin blood sample detection sensitivity, which comprises the following steps: (1) adding a blood sample containing methionine enkephalin into normal saline for dilution, and adding pure acetonitrile protein for precipitation and centrifugation; (2) taking supernate, adding an adsorbent, and centrifuging to adsorb methionine enkephalin; the adsorbent is methyl cellulose or chitosan; (3) after adsorption is completed, discarding an upper-layer solution, then adding an eluent, and centrifuging to elute methionine enkephalin from the adsorbent; the eluent is methanol or a PBS (Phosphate Buffer Solution); and taking the supernate in the step (3), and carrying out LC-MS / MS detection. According to the method, the sensitivity of methionine enkephalin plasma sample detection can be improved, the lower quantification limit reaches 0.8 ng / mL, the linearity is good in the range of 1.0-4000.0 ng / mL, and meanwhile, the extraction and recovery rate of methionine enkephalin can be improved.

Description

Technical Field

[0001] The invention relates to the field of polypeptide biological sample analysis, and in particular to a method for improving the detection sensitivity of methionine enkephalin rat blood samples. Background Art

[0002] Peptide drugs have become one of the hot spots in new drug research today because of their clear targets and good drugability. In recent years, clinical research in the field of peptides has grown significantly. As of 2020, more than 80 peptide drugs have been launched, more than 150 peptides are in the clinical research stage, and 400-600 peptide compounds are in the preclinical research stage. However, compared with ordinary drugs, the detection of biological samples of peptide drugs has problems such as low response, nonspecific adsorption, endogenous interference, and poor stability. Therefore, the development of technical methods for the analysis of biological samples of peptide drugs is also continuing to develop.

[0003] Methionine enkephalin is an endogenous opioid pentapeptide, also known as opioid growth factor due to its growth regulatory properties. Methionine enkephalin can regulate immune function and provide potential therapeutic activity for immune-related diseases and cancer. Although its anti-tumor evaluation is in its preliminary stage, there are still many problems to be solved. However, based on its potential advantages such as safety, immunomodulation and potential anti-cancer activity. Therefore, studies believe that methionine enkephalin will have the potential to be used as part of the adjuvant therapy for human cancer treatment, and researchers are increasingly studying its anti-tumor efficacy. However, there are few reports on the detection methods of methionine enkephalin. According to known literature reports, due to the regulation of enzymes, the concentration of methionine enkephalin in plasma is very low, making its quantitative analysis difficult. Therefore, exploring the bioanalysis method of methionine enkephalin drugs and improving the sensitivity of blood sample detection can provide a useful reference for the in vivo detection of such drugs. Summary of the invention

[0004] The present invention provides a method for improving the sensitivity of methionine enkephalin rat blood sample detection. The methionine enkephalin is first pre-treated, and after the methionine enkephalin is enriched, the treated rat plasma sample is detected by LC-MS / MS and the experimental data is analyzed. Compared with the prior art, the present invention improves the sensitivity of methionine enkephalin rat blood sample LC-MS / MS detection, effectively improves the problem of low recovery rate of methionine enkephalin pre-treatment, and enhances its detection response.

[0005] The technical solution of the present invention is as follows:

[0006] A method for improving the sensitivity of methionine enkephalin blood sample detection comprises the following steps:

[0007] (1) After diluting the blood sample containing methionine enkephalin with physiological saline, pure acetonitrile was added to the protein precipitate and centrifuged;

[0008] (2) adding an adsorbent to the supernatant and centrifuging to adsorb the methionine enkephalin; the adsorbent is methylcellulose or chitosan; the mass volume ratio of the adsorbent to the supernatant is 5 to 50 mg / 900 μL;

[0009] (3) After the adsorption is completed, the upper layer solution is discarded, and then an eluent is added and centrifuged to elute the methionine enkephalin from the adsorbent; the eluent is methanol or PBS solution;

[0010] (4) Take the supernatant of step (3) and perform LC-MS / MS detection.

[0011] Preferably, the mass volume ratio of the adsorbent to the supernatant is 10-20 mg / 900 μL.

[0012] Preferably, the volume ratio of the blood sample to physiological saline is 1:(0.5-2.0).

[0013] Preferably, the volume ratio of the pure acetonitrile to the blood sample is (2-4):1.

[0014] Preferably, the mass spectrometry conditions for the LC-MS / MS detection include: the ionization mode of the AB Sciex 4500 mass spectrometer is positive ionization, the electrospray ion source (ESI), and the monitoring mode is the multiple reaction monitoring (MRM) mode; the quantitative ion pair m / z of methionine enkephalin is 120.1 / 573.9; the curtain gas (Curtain Gas) is 40psi, the ionization voltage (IonSprayVoltage) is 5500V, the auxiliary heating gas temperature (TEM) is 500°C, the collision gas (Collision Gas) is 9unit, the collision energy (Collision Energy) is 71eV, the declustering potential (Declustering Potential) is 56V, the entrance voltage (Entrance Potential) is 10eV, the collision cell exit voltage (Collision Cell Exit Potential) is 6V, and the dwell time (Dwell time) is 100msec.

[0015] Preferably, the liquid chromatography conditions for LC-MS / MS detection include: using Hypersil GOLD TMChromatographic separation was performed on a C18 column (3 μm 100×2.1 mm) at a flow rate of 0.6 mL / min and a column temperature of 40°C. Mobile phase A was 0.1% FA in water, mobile phase B was 0.1% FA in acetonitrile, and the gradient elution program was as follows: 0.00-1.50 min, 10% B; 1.50-2.00 min, 10-90% B; 2.00-3.00 min, 90% B; 3.00-3.50 min, 90-10% B; 3.50-4.00, 10% B, maintained at 10% B until the end of the run (4.00 min). The injection volume was set to 10.0 μL.

[0016] The present invention compares the protein precipitation method and the commercial WAX solid phase extraction column pretreatment of methionine enkephalin rat plasma samples, and compares the sensitivity, extraction recovery rate, precision, accuracy and other indicators based on the LC-MS / MS detection data to prove the advantages of the pretreatment method for methionine enkephalin rat blood sample analysis. The specific advantages are as follows:

[0017] (1) Compared with the protein precipitation method, the present invention can improve the sensitivity of methionine enkephalin plasma sample detection and reduce the quantitative limit of methionine enkephalin detection, the quantitative limit of which reaches 0.8 ng / mL, and has good linearity in the range of 1.0-4000.0 ng / mL, while effectively improving the extraction recovery rate.

[0018] (2) Compared with the WAX ​​solid phase extraction column, the precision of the WAX ​​column is poor when pre-treating the methionine enkephalin plasma sample. Compared with the WAX ​​column, the present invention not only has a higher detection response, but also has good precision and accuracy. At the same time, the commercial WAX solid phase extraction column is expensive and has a high cost of use. The application of methylcellulose dispersed micro solid phase extraction can reduce the detection cost and improve efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for describing the embodiments of the present invention are briefly introduced below.

[0020] Figure 1 It is a flow chart of the specific steps of the detection method of the present invention.

[0021] Figure 2 is a chromatogram of LC-MS / MS detection after pre-treatment of methylcellulose dispersive micro-solid phase extraction (a) and protein precipitation (b), WAX solid phase extraction column (c), sodium citrate dispersive micro-solid phase extraction (d), and graphene oxide dispersive micro-solid phase extraction (e) at the same concentration.

[0022] Figure 3 This is a graph showing the effect of the amount of methylcellulose adsorbent used on the detection sensitivity in Example 1.

[0023] Figure 4 This is a graph showing the effect of eluent type on detection sensitivity in Example 1.

[0024] Figure 5 This is a graph showing the effect of the amount of chitosan adsorbent used on the detection sensitivity in Example 2.

[0025] Figure 6 This is a diagram showing the effect of eluent type on detection sensitivity in Example 2.

[0026] Figure 7 This is the chromatogram of LC-MS / MS detection after chitosan dispersion micro solid phase extraction pretreatment. DETAILED DESCRIPTION

[0027] The specific implementation steps of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. The specific experimental parameters involved in the present invention, the technical operations of the dispersed micro-solid phase extraction of methionine enkephalin using methylcellulose and the LC-MS / MS detection steps, all belong to the scope of protection of the present invention.

[0028] Example 1

[0029] The specific implementation steps of the present invention for improving the sensitivity of methionine enkephalin blood sample detection based on methylcellulose dispersed micro-solid phase extraction can be referred to Figure 1 , which shows a specific step flow chart of the technical method, and the specific steps are as follows:

[0030] Step 1. Take 20 μL of methionine enkephalin working solution, add 180 μL of rat plasma in a 1.5 mL EP tube to prepare 200 μL of methionine enkephalin plasma matrix solution. Add 200 μL of saline to dilute the plasma matrix solution 1:1, vortex for 10 seconds, add 600 μL of pure acetonitrile solution (1:3) for extraction, vortex for 60 seconds, centrifuge at 15000 rpm for 5 minutes, and take 900 μL of supernatant.

[0031] Step 2: Add the supernatant in the above step into an EP tube containing 20 mg of methylcellulose powder, vortex for 60 seconds, centrifuge at 15,000 rpm for 5 minutes, and discard the supernatant.

[0032] It should be noted that the amount of adsorbent methylcellulose mentioned in step 2 is 20 mg, which is the optimal amount determined by experimentally comparing the response values ​​of methionine enkephalin biological samples detected by LC-MS / MS after extraction pre-treatment at different amounts (including 5 mg, 10 mg, 20 mg, 30 mg, 50 mg, and 75 mg).

[0033] Step 3: Add 50 μL of PBS as the eluent, vortex for 60 seconds, centrifuge at 15,000 rpm for 5 minutes, aspirate 60 μL of the supernatant into an injection bottle, and use LC-MS / MS to detect and analyze the treated plasma samples.

[0034] It should be noted that the eluent mentioned in step 3 is PBS, which is the optimal amount determined by experimentally comparing the response values ​​of LC-MS / MS detection of methionine enkephalin biological samples after elution with different eluents (including PBS, methanol, acetonitrile, and 75% acetonitrile). The experiment found that the volume of the eluent had little effect on the enrichment of methionine enkephalin, so the volume of the eluent was set to 50 μL.

[0035] Step 4: LC-MS / MS detection:

[0036] The mass spectrometry conditions included: AB Sciex 4500 mass spectrometer ionization mode was positive ionization, electrospray ionization source (ESI), monitoring mode was multiple reaction monitoring (MRM) mode; the quantitative ion pair of methionine enkephalin was m / z 120.1 / 573.9; the curtain gas (Curtain Gas) was 40 psi, and the ionization voltage was

[0037] (IonSpray Voltage) is 5500V, the auxiliary heating gas temperature (TEM) is 500℃, and the collision gas

[0038] (Collision Gas) is 9unit, Collision Energy is 71eV, Declustering Potential is 56V, Entrance Potential is 10eV, Collision Cell Exit Potential is 6V, and Dwell time is 100msec.

[0039] The HPLC conditions included: using Hypersil GOLD TM Chromatographic separation was performed on a C18 column (3 μm 100×2.1 mm) with a flow rate of 0.6 mL / min and a column temperature of 40°C. Mobile phase A was 0.1% FA in water, mobile phase B was 0.1% FA in acetonitrile, and the gradient elution program was as follows: 0.00-1.50 min, 10% B; 1.50-2.00 min, 10-90% B; 2.00-3.00 min, 90% B; 3.00-3.50 min, 90-10% B;

[0040] 3.50-4.00, 10% B, hold at 10% B until the end of the run (4.00 min). The injection volume was set to 10.0 μL.

[0041] Comparative Example 1 (Pretreatment before protein precipitation)

[0042] Step (1) is the same as in Example 1.

[0043] The supernatant obtained in step (1) was directly subjected to LC-MS / MS detection, and the detection conditions were the same as those in Example 1.

[0044] Comparative Example 2 (WAX solid phase extraction column pretreatment)

[0045] A WAX solid phase extraction column was used for pretreatment, and then LC-MS / MS detection was performed. The detection conditions were the same as those in Example 1.

[0046] The advantages of the present invention for methionine enkephalin rat blood sample analysis are specifically reflected by examining the quantitative lower limit, sensitivity, extraction recovery rate, precision and accuracy of LC-MS / MS detection data. The specific implementation steps and method advantages will be described in conjunction with Figures 2, 3, 4 and 5. The specific steps for examining and comparing the relevant indicators are as follows:

[0047] Step 1, taking 20 μL of methionine enkephalin working solution with a concentration of 1000 ng / mL, adding 180 μL of rat plasma to prepare a methionine enkephalin plasma matrix solution with a concentration of 100 ng / mL, respectively using the methylcellulose dispersed micro solid phase extraction pretreatment, protein precipitation pretreatment, and WAX solid phase extraction column pretreatment of the present invention, and analyzing the response values ​​of samples treated by different methods by LC-MS / MS detection. FIG2 is the chromatogram response of the three methods. It can be seen that the method of the present invention has the highest detection response after pretreatment of the methionine enkephalin plasma matrix solution, indicating that its enrichment effect on methionine enkephalin is significantly better than the other two methods.

[0048] It should be noted that, since the reproducibility and precision of the WAX ​​solid phase extraction column were found to be poor when the experiment was compared, only a preliminary comparison was made between it and the method of the present invention.

[0049] Step 2, using the methylcellulose dispersed micro-solid phase extraction method of the present invention to pre-treat the eight concentrations of methionine enkephalin rat plasma matrix solution set for linear investigation, 60 μL of supernatant is drawn into the injection bottle, and LC-MS / MS is used to detect and analyze the eight treated samples, investigate the linearity of the method, and calculate the quantitative lower limit according to the linear equation and baseline response. At the same time, it is compared with the quantitative lower limit obtained by the protein precipitation method detection and analysis. As shown in Table 1, the linear equation and quantitative lower limit obtained by analyzing the LC-MS / MS data after the pre-treatment of the method of the present invention and the protein precipitation method, the method of the present invention is combined with LC-MS / MS quantitative analysis of methionine enkephalin, and the linearity is good in the range of 1.0-4000.0ng / mL.

[0050] Table 1

[0051]

[0052] According to Table 1, the lower limit of quantification of methionine enkephalin after protein precipitation pretreatment is 4.00 ng / mL, while the lower limit of quantification of methionine enkephalin after methylcellulose dispersion micro solid phase extraction pretreatment is 0.8 ng / mL. The present invention can effectively improve the detection sensitivity of methionine enkephalin.

[0053] Step 3, set three concentrations of low, medium and high respectively, and each concentration has six parallel quality control points, and use the methylcellulose dispersion micro solid phase extraction method of the present invention to pre-treat six points of methionine enkephalin rat plasma matrix solution of low, medium and high concentrations, draw 60 μL of supernatant into the injection bottle, and use LC-MS / MS to detect and analyze the samples pre-treated with methylcellulose dispersion micro solid phase extraction and protein precipitation, and combine the above-obtained linear equation to investigate the precision and accuracy of low, medium and high concentration quality control samples. The results are shown in Table 2, and the methylcellulose dispersion micro solid phase extraction method involved in the present invention has good precision and accuracy in pre-treating methionine enkephalin.

[0054] Table 2

[0055]

[0056] It should be noted that the peak area obtained by LC-MS / MS detection of the low, medium and high concentration samples processed in step 3 is the peak area A of the methionine enkephalin pre-spiked sample mentioned in the subsequent steps.

[0057] Step 4: To calculate the extraction recovery, a methionine enkephalin post-spiking solution is prepared. The peak area of ​​the methionine enkephalin post-spiking solution detected by LC-MS / MS is B. The extraction recovery is calculated according to (A / B)×100%.

[0058] It should be noted that the specific operation steps for preparing the methionine enkephalin post-spiking solution and treatment in step 4 are as follows: Take 20 μL of solvent, add 180 μL of rat plasma in a 1.5 mL EP tube, and prepare 200 μL of blank plasma matrix solution. Add 200 μL of physiological saline to dilute the blank plasma matrix solution 1:1, vortex for 10 seconds, add 600 μL of pure acetonitrile solution (1:3) for extraction, vortex for 60 seconds, centrifuge at 15000 rpm for 5 minutes, take 900 μL of supernatant and add it to an EP tube containing 20 mg of methylcellulose powder, vortex for 60 seconds, centrifuge at 15000 rpm for 5 minutes, and discard the supernatant. Add 50 μL of eluent PBS, vortex for 60 seconds, centrifuge at 15000 rpm for 5 minutes, and aspirate the supernatant solution. Use the obtained solution to prepare the post-extraction spike solution samples (6 at low, medium, and high concentration levels).

[0059] The extraction recovery rate obtained by the detection and analysis after the pretreatment of methionine enkephalin in the present invention was compared with the data after the protein precipitation method. The relevant data are shown in Table 3.

[0060] Table 3

[0061]

[0062] According to Table 3, the average extraction recovery rates of plasma samples at low, medium and high concentration levels of methionine enkephalin extracted by the method of the present invention are 49.79%±5.05%, 63.61%±3.19% and 50.72%±1.41% respectively; the average extraction recovery rates of plasma samples at low, medium and high concentration levels of methionine enkephalin extracted by the protein precipitation method are 28.56%±5.11%, 22.18%±3.46% and 30.24%±0.81% respectively. The present invention uses methylcellulose to perform dispersed micro-solid phase extraction on methionine enkephalin, which can effectively improve the extraction recovery rate of methionine enkephalin and enhance the detection response of LC-MS / MS compared with the protein precipitation method, and can provide a useful reference for the subsequent quantitative analysis of methionine enkephalin.

[0063] Example 2

[0064] Dispersive micro-solid phase extraction using chitosan materials:

[0065] The optimal adsorption and elution conditions of chitosan were determined: the optimal adsorption dosage of chitosan was 10 mg, and the optimal elution agent was PBS solution.

[0066] Chromatogram comparison (100ng / mL), see Figure 7 .

[0067] The linear equation and lower limit of quantification (1.0-4000.0 ng / mL have good linearity) and related data are shown in Table 4.

[0068] Table 4

[0069]

[0070] The accuracy (relative recovery) and intra-batch precision are shown in Table 5.

[0071] Table 5

[0072]

[0073] The extraction recovery rate and related data are shown in Table 6.

[0074] Table 6

[0075]

[0076] Comparative Example 3

[0077] The difference between this embodiment and embodiment 1 is that the adsorbent is sodium citrate, see Figure 2d .

[0078] Comparative Example 4

[0079] The difference between this embodiment and embodiment 1 is that the adsorbent is graphene oxide (GO), see Figure 2e .

[0080] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for improving the sensitivity of methionine enkephalin blood sample detection, characterized in that: The following steps are involved: (1) After diluting the blood sample containing methionine enkephalin with physiological saline, pure acetonitrile was added to the protein precipitate and centrifuged; (2) adding an adsorbent to the supernatant and centrifuging to adsorb the methionine enkephalin; the adsorbent is methylcellulose or chitosan; the mass volume ratio of the adsorbent to the supernatant is 5 to 50 mg / 900 μL; (3) After the adsorption is completed, the upper layer solution is discarded, and then an eluent is added and centrifuged to elute the methionine enkephalin from the adsorbent; the eluent is methanol or PBS solution; (4) Take the supernatant of step (3) and perform LC-MS / MS detection.

2. The method according to claim 1, characterized in that The mass volume ratio of the adsorbent to the supernatant is 10-20 mg / 900 μL.

3. The method according to claim 2, characterized in that The volume ratio of the blood sample to physiological saline is 1:(0.5-2.0).

4. The method according to claim 3, characterized in that The volume ratio of the pure acetonitrile to the blood sample is (2-4):

1.

5. The method according to any one of claims 1 to 4, characterized in that: The mass spectrometry conditions for the LC-MS / MS detection include: the mass spectrometer ionization mode is positive ionization, the electrospray ion source, the monitoring mode is the multiple reaction detection mode; the quantitative ion pair m / z of methionine enkephalin is 120.1 / 573.9; the curtain gas is 40psi, the ionization voltage is 5500V, the auxiliary heating gas temperature is 500°C, the collision gas is 9unit, the collision energy is 71eV, the declustering voltage is 56V, the inlet voltage is 10eV, the collision cell outlet voltage is 6V, and the residence time is 100msec.

6. The method according to claim 5, characterized in that The liquid chromatography conditions for the LC-MS / MS detection include: using Hypersil GOLD TM Chromatographic separation was carried out on a C18 chromatographic column with a flow rate of 0.6 mL / min and a column temperature of 40°C; mobile phase A was an aqueous solution containing 0.1% FA, mobile phase B was acetonitrile containing 0.1% FA, and the gradient elution program was as follows: 0.00-1.50 min, 10% B; 1.50-2.00 min, 10-90% B; 2.00-3.00 min, 90% B; 3.00-3.50 min, 90-10% B; 3.50-4.00, 10% B, and maintained at 10% B until the end of the run.

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