A NMR quantitative method for determining sucrose content in vaccines

Abstract

The present invention discloses a nuclear magnetic resonance quantitative method for determining the sucrose content in a vaccine. The method uses a water peak suppression pulse based on noesygppr1d pulse adjustment to determine the sample, obtains the quantitative peaks of potassium hydrogen phthalate and sucrose after phase adjustment and baseline correction of the spectrum, and integrates the quantitative peaks to obtain the quantitative peak integral area I of sucrose and potassium hydrogen phthalate. x ,I std , the sucrose content in the vaccine was calculated according to the formula. This method is accurate, precise and reliable. It can eliminate the interference of water peak without long-term freeze-drying, and solves the influence of pulse irradiation on quantitative results in the pre-saturation stage when general solvent peak suppression technology is used for quantitative determination. The accuracy and precision of this method are better than those of unoptimized methods.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and more specifically to a nuclear magnetic resonance quantitative method for determining sucrose content in vaccines. Background Art

[0002] Sucrose is a non-reducing disaccharide formed by the condensation of glucose and fructose through isomer hydroxyl groups. It plays a critical role in biological vaccine products, especially vaccines that need to be stored at low temperatures, and is a common stabilizer. Proteins can generally be stored for a long time in a hydrophobic environment. Adding certain stabilizers can reduce the polarity of protein solutions to improve their stability. The quality control of sucrose is of great significance for studying the quality, potency and stability of vaccines, the osmotic pressure of liquid vaccines, and the storage conditions of freeze-dried or liquid vaccines.

[0003] Since sucrose has no UV absorption, the most commonly used HPLC-UV in the laboratory cannot analyze it. The existing liquid phase method is cumbersome, time-consuming, and requires a special sugar analysis column. The current mainstream quantitative method for sucrose in vaccines is HPLC-RID, but the RID detector is not suitable for gradient elution, which undoubtedly limits the promotion and application of this method in drug analysis. Commonly used sugar analysis columns such as amino columns often require that the proportion of water phase in the mobile phase should not exceed 30%. The limitation from the mobile phase often further prolongs the analysis time.

[0004] The most significant advantage of quantitative NMR over HPLC is that it is convenient, fast, and highly automated. During analysis, there is no need for complex sample pretreatment, nor is there a need for long column equilibration, column flushing, and other steps. However, because the water peak will mask the signal peak near it, the interference of water must be eliminated when using quantitative NMR technology to quantify sucrose in vaccines. There are two commonly used methods: freeze-drying and water peak suppression. The freeze-drying method is cumbersome and time-consuming; the pulse used in the water peak suppression method will pre-saturate the water peak signal within the D1 time. If the pre-saturation time is too long, it will affect the integral area of ​​the signal near the water peak, resulting in inaccurate quantitative results. On the other hand, the continuous exposure of the sample to radio waves during the D1 time will cause the sample to heat up, resulting in an increase in T1, which will also affect the accuracy of the quantitative results.

[0005] Therefore, there is a need to provide a more effective method for quantitatively determining the sucrose content in vaccines. Summary of the invention

[0006] The purpose of the present invention is to provide a quantitative nuclear magnetic resonance hydrogen spectroscopy method with water peak suppression technology to achieve accurate quantification of sucrose in vaccines, thereby solving the pain points that the freeze-drying method and liquid phase method are cumbersome and time-consuming, and the quantitative results of the solvent peak suppression method are inaccurate.

[0007] The above technical objectives of the present invention are achieved through the following technical solutions.

[0008] The present invention provides a nuclear magnetic resonance quantitative method for determining the sucrose content in a vaccine, comprising the following steps:

[0009] S1: Measure a certain amount of vaccine liquid and dilute it with heavy water as the test solution;

[0010] S2: Take the internal standard potassium hydrogen phthalate, add 3-(trimethylsilyl) sodium deuterated propionate (TSP) as the internal reference for calibration, and dissolve it in heavy water as the internal standard solution;

[0011] S3: Mix the test solution and the internal standard solution, add them into the NMR tube, and place the NMR tube in a superconducting NMR spectrometer;

[0012] S4: Set the superconducting NMR spectrometer test parameters and optimize O1 using a pre-saturated single pulse experiment;

[0013] S5: Set the test parameters of the superconducting NMR spectrometer, use O1 optimized in step S4 to perform a water peak suppression test, use noesygppr1d.mod to measure the hydrogen nuclear magnetic resonance spectrum of the test sample, obtain the quantitative peaks of potassium hydrogen phthalate and sucrose, integrate the quantitative peaks after phase adjustment and baseline correction of the spectrum, obtain the respective integrated areas, and calculate the sucrose content in the vaccine according to the fitting formula.

[0014] The present invention is further configured as follows: in step S1, the solution is diluted 5-20 times with heavy water, preferably 10 times. Also preferably, step S1 includes measuring 1 ml of a quantitative vaccine liquid, diluting it with heavy water and fixing the volume to 10 ml as a test solution.

[0015] The present invention is further configured as follows: in step S2, the ratio of potassium hydrogen phthalate to heavy water is 3 ml of heavy water is used to dissolve every 32-38 mg of potassium hydrogen phthalate; preferably, 3 ml of heavy water is used to dissolve every 35 mg of potassium hydrogen phthalate.

[0016] The present invention is further configured as follows: the amount of 3-(trimethylsilyl) sodium deuterated propionate (TSP) used in step S2 is generally not more than one thousandth of the mass of heavy water.

[0017] The present invention is further configured as follows: the volume ratio of the test solution to the internal standard solution is 1-5:1, preferably 2-3:1, and more preferably 2.5:1.

[0018] The present invention is further configured as follows: the experimental parameters of the pre-saturation single pulse experiment in step S4 are D1=4s, the number of empty scans DS=0, the number of scans NS=1, and the gain RG=4. The FID signal of the experiment is collected, and the phase of the signal on the right side of the water peak is adjusted to be positive after Fourier transformation to observe whether the water peak is a dispersion peak. If so, the O1 value is appropriately adjusted until the two sides of the water peak are symmetrical and the baseline is flat. It is also preferred that O1P is set to 4.695ppm in this experiment, and the setting of O1P is directly related to the effect of water peak suppression.

[0019] The present invention is further configured as follows: in step S5, noesygppr1d.mod is optimized by noesygppr1d, a blank line is inserted before the 33rd line "d12 pl9:f1" of the noesygppr1d pulse file, and a waiting delay time of 21-29s is written, preferably, 23-38s is written, and more preferably, 26s is written.

[0020] The present invention is further configured as follows: in step S5, 1 H NMR parameters are as follows: the experimental template is PROF_1H, the pulse is noesygppr1d.mod, preferably, D1=4s is set, and the pre-saturation power in the edprosol table is 25Hz.

[0021] The present invention is further configured as follows: in step S5, the obtained one-dimensional NMR spectrum is subjected to phase adjustment and baseline correction, and the chemical shift δ is selected. H 7.529 (2H, q) as the quantitative peak of potassium hydrogen phthalate, select δ H 5.418 (1H, d) was used as the quantitative peak of sucrose. After integration, the sucrose content in the vaccine was calculated according to the following formula: x :

[0022]

[0023] Among them, C x is the content of sucrose in the vaccine; I x ,I std are the quantitative peak integrated areas of sucrose and potassium hydrogen phthalate, respectively; N std 、N x are the proton numbers of the quantitative peaks of potassium hydrogen phthalate and sucrose, respectively; M x 、M std are the relative molecular masses of sucrose and potassium hydrogen phthalate respectively; P std is the purity of the selected potassium hydrogen phthalate; m std V is the mass of potassium hydrogen phthalate in the final test sample; x is the volume of the test solution in the final test sample; n is the dilution multiple of the vaccine liquid.

[0024] In summary, the present invention has the following beneficial effects: the patented method optimizes the water peak suppression experimental pulse, reduces the influence of the presaturation signal on the sucrose integral area during the D1 time, and compared with the unoptimized pulse, the method has good accuracy and high precision; compared with the mainstream liquid phase method, the method is simple and fast, and only takes 10 minutes to complete the determination of the sucrose content in the vaccine. The present invention uses an optimized one-dimensional nuclear magnetic resonance hydrogen spectrum method using water peak suppression technology to achieve the quantification of sucrose in the vaccine. The method is simple, fast, specific, precise, accurate, and linear. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic diagram of modification of the noesygppr1d pulse in an embodiment of the present invention;

[0026] Figure 2 is a schematic diagram of a noesygppr1d pulse in an embodiment of the present invention;

[0027] Figure 3 This is a schematic diagram of a noesygppr1d.mod pulse in an embodiment of the present invention;

[0028] Figure 4 is a typical hydrogen nuclear magnetic resonance spectrum of noesygppr1d.mod in an embodiment of the present invention;

[0029] Figure 5 This is the linear range test result in the embodiment of the present invention;

[0030] Figure 6 This is the linear range test result in the comparative example of the present invention. DETAILED DESCRIPTION

[0031] The following will further explain the present invention in detail with reference to the accompanying drawings. The examples given are for better description of the invention content, and the invention content is not limited to the examples. Non-essential improvements and adjustments to the implementation scheme according to the invention content still belong to the scope of the invention.

[0032] The experimental methods in the following examples are all conventional methods unless otherwise specified. If no specific techniques or conditions are specified in the examples, they are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0033] Example 1: Quantification of sucrose in vaccines by optimized one-dimensional proton nuclear magnetic resonance spectroscopy using water peak suppression technology

[0034] Preparation of test solution: Measure 1 ml of the quantitative vaccine liquid, dilute it with heavy water and make up to 10 ml as the test solution.

[0035] Preparation of internal standard solution: Take 35.00 mg of internal standard potassium hydrogen phthalate, accurately weigh it, put it in an EP tube, add an appropriate amount of TSP as an internal reference, add 3 ml of heavy water to dissolve it, and shake it to prepare the internal standard solution.

[0036] Preparation of test samples: Use a pipette to draw 500 μl of the test solution and 200 μl of the internal standard solution, place them in an EP tube, mix them well, and then draw 600 μl into a 5 mm NMR tube, and place the NMR tube in the automatic sampler.

[0037] To ensure the accuracy of the results, set O1P = 4.695ppm.

[0038] To ensure the accuracy of the results, set D1 = 4s and use the noesygppr1d.mod pulse measurement. The pulse is optimized by noesygppr1d. The optimization content is as follows Figure 1 As shown, a blank line is inserted before the 33rd line "d12 pl9:f1" of the noesygppr1d pulse file, and a waiting delay time of 26s is written to obtain the noesygppr1d.mod pulse. The longitudinal relaxation time T1 is measured by the inversion recovery method, and the maximum relaxation time T1 = 4.824s is detected. Among them, the schematic diagram of the noesygppr1d pulse is shown as follows Figure 2 As shown, the schematic diagram of the noesygppr1d.mod pulse is as follows Figure 3 shown.

[0039] related 1 The H NMR parameters were set as follows: the experimental template was PROF_1H, the pulse was noesygppr1d.mod, the number of sampling points TD = 65536, the number of empty scans TD = 4, the number of scans NS = 16, the spectrum width SW = 11904.762 Hz, the center frequency O1 = 3900.84 Hz, the gain value RG = 32, the dwell time DW = 42.000 μs, the line width factor LB = 0.3 Hz, the probe temperature was 298.0 K, and the pre-saturation power in the edprosol table was 25 Hz.

[0040] The phase adjustment and baseline correction of the obtained one-dimensional NMR spectrum were performed, and the chemical shift δ H 7.529 (2H, q) as the quantitative peak of potassium hydrogen phthalate, select δ H 5.418 (1H, d) was used as the quantitative peak of sucrose. After integration, the sucrose content in the vaccine was calculated according to the following formula: x :

[0041]

[0042] Among them, C xis the content of sucrose in the vaccine; I x ,I std are the quantitative peak integrated areas of sucrose and potassium hydrogen phthalate, respectively; N x 、N std are the proton numbers of the quantitative peaks of sucrose and potassium hydrogen phthalate, respectively; M x 、M std are the relative molecular masses of sucrose and potassium hydrogen phthalate respectively; P std is the purity of the selected potassium hydrogen phthalate; m std V is the mass of potassium hydrogen phthalate in the final test sample; x is the volume of the test solution in the final test sample; n is the dilution multiple of the vaccine liquid.

[0043] like Figure 4 As shown, it is a typical H NMR spectrum of the method for quantitatively analyzing the sucrose content in the vaccine by H NMR spectrum using potassium hydrogen phthalate as an internal standard.

[0044] Specificity test:

[0045] According to the sample preparation method, prepare a sample for testing, and place the NMR tube in the autosampler. Select the Bruker standard experimental template PROF_1H, select the optimized experimental pulse noesygppr1d.mod, and set the parameters as described above. The injection experiment obtains the one-dimensional hydrogen spectrum of the sample. All peaks on the spectrum can be attributed, and no other peaks interfere with the determination of sucrose content.

[0046] Precision test:

[0047] According to the test sample preparation method, prepare a test sample and place the NMR tube in the automatic sampler. Set the experimental parameters according to the setting method of the relevant parameters described above, and conduct 6 consecutive tests; according to the NMR spectrum processing and sucrose content calculation method described above, process the NMR spectrum and calculate the sucrose content in the vaccine, and calculate the average value and RSD. The results are shown in Table 1:

[0048] Table 1 Precision test results

[0049]

[0050] Repeatability and sample recovery test:

[0051] The test samples were prepared according to the sucrose content of 120%, 100%, and 80% in the vaccine, and 3 copies were prepared in parallel for each concentration. The experimental parameters were set according to the setting method of the relevant parameters described above, and the sample recovery test was carried out. The nuclear magnetic resonance spectrum was processed and the sucrose content in the vaccine was calculated according to the nuclear magnetic resonance spectrum processing and sucrose content calculation method described above. The sample addition amount and the determination results are shown in Table 2:

[0052] Table 2 Repeatability and sample recovery test results

[0053]

[0054]

[0055] Linear:

[0056] Take different masses (0.5000-20.0000 mg) of internal standard potassium hydrogen phthalate standard, accurately weigh them, place them in EP tubes respectively, add appropriate amount of test solution to dissolve, vortex to mix, and then pipette 600 μl into 5 mm NMR tube, and place the NMR tube in the automatic sampler. Set the experimental parameters according to the setting method of the relevant parameters described above, and conduct a linear test. Process the NMR spectrum according to the NMR spectrum processing and sucrose content calculation method described above, and use the mass ratio of the analyte sucrose to the internal standard as the horizontal coordinate, and the peak area ratio of the analyte quantitative peak to the internal standard quantitative peak as the vertical coordinate to conduct a linear regression. The linear test results are as follows: Figure 5 As shown. The linear regression equation is y = 0.0254x-0.0046, where R 2 =0.9999. The results show that when the peak area ratio of the analyte quantitative peak to the internal standard quantitative peak is between 7.6103 and 0.2506:1, the linearity is good.

[0057] Comparative Example 1: Quantification of Sucrose in Vaccines Using One-Dimensional H-NMR Spectroscopy Using Unoptimized Water Peak Suppression

[0058] The test samples in the above-described embodiment were selected and placed in the automatic sample injector of the nuclear magnetic resonance spectrometer, and D1 was set to 30s, the experimental pulse was noesygppr1d, and the other parameters were set in the same way. Specificity, precision, accuracy, sample recovery rate, and linearity experiments were performed, and each tube of the test sample was subjected to the experiment in the comparative example immediately after the experiment in the embodiment was completed to minimize the influence of other influencing factors on the quantitative results.

[0059] Specificity test:

[0060] Each peak on the spectrum can be attributed, and no other peaks interfere with the determination of sucrose content.

[0061] Precision test:

[0062] According to the method of NMR spectrum processing and sucrose content calculation described above, the NMR spectrum was processed and the sucrose content in the vaccine was calculated, and the average value and RSD were calculated. The results are shown in Table 3:

[0063] Table 3 Precision test results

[0064]

[0065]

[0066] Repeatability and sample recovery test:

[0067] According to the method of NMR spectrum processing and sucrose content calculation described above, the NMR spectrum was processed and the sucrose content in the vaccine was calculated. The sample addition amount and the measurement results are shown in Table 2:

[0068] Table 4 Repeatability and sample recovery test results

[0069]

[0070] Linear:

[0071] The NMR spectrum was processed according to the method of NMR spectrum processing and sucrose content calculation described above, and linear regression was performed with the mass ratio of sucrose to the internal standard as the abscissa and the peak area ratio of the quantitative peak area of ​​the analyte to the quantitative peak area of ​​the internal standard as the ordinate. The linear test results are shown in Figure 6 As shown. The linear regression equation is y = 0.0254x + 0.0436, where R 2 =0.9992. The results show that when the peak area ratio of the analyte quantitative peak to the internal standard quantitative peak is between 7.6103 and 0.2502:1, the linearity is good.

[0072] The present invention adopts the optimized water peak suppressed nuclear magnetic resonance hydrogen spectroscopy and the unoptimized nuclear magnetic resonance hydrogen spectroscopy to determine the sucrose content in the vaccine. In the methodological verification part of the embodiment, the precision test RSD is 0.470%; the sample recovery rate is between 99% and 101%; the repeatability RSD is less than 1%; the regression equation is y=0.0254x-0.0046, where R 2 =0.9999, when the peak area ratio of the quantitative peak of the analyte to the quantitative peak of the internal standard is between 7.6103 and 0.2506:1, the linearity is good. In some experiments of the comparative example, the precision test RSD = 1.161%; the sample recovery rate is between 97% and 100%; the repeatability RSD is less than 1.5%, and some are greater than 1%; the regression equation is y = 0.0254x + 0.0436, where R 2=0.9992, and the linearity was good when the peak area ratio of the quantitative peak of the analyte to the quantitative peak of the internal standard was between 7.6103 and 0.2502:1. The results showed that the precision, accuracy, repeatability and linearity of the optimized water peak suppression experiment in determining the sucrose content in the vaccine were better than those of the method before optimization.

[0073] This specific embodiment is only used as an explanation of the invention and is not a limitation of the present invention. After reading this specification, those skilled in the art can make non-creative modifications to this embodiment as needed, but as long as it is within the scope of the claims of the present invention, it will be protected by the patent law.

Claims

1. A nuclear magnetic resonance quantitative method for determining sucrose content in vaccines, comprising the following steps: S1: Measure a certain amount of vaccine liquid and dilute it with heavy water as the test solution; S2: Take the internal standard potassium hydrogen phthalate, add 3-(trimethylsilyl) sodium deuterated propionate as the internal reference for calibration, and dissolve it in heavy water as the internal standard solution; S3: Mix the test solution and the internal standard solution, add them into the NMR tube, and place the NMR tube in a superconducting NMR spectrometer; S4: Set the superconducting NMR spectrometer test parameters, use the pre-saturation single pulse experiment to optimize O1, and after optimization, O1P is set to 4.695ppm; S5: Set the test parameters of the superconducting NMR spectrometer, use O1 optimized in step S4 to perform a water peak suppression test, use noesygppr1d.mod to measure the nuclear magnetic resonance hydrogen spectrum of the test sample, obtain the quantitative peaks of potassium hydrogen phthalate and sucrose, integrate the quantitative peaks after phase adjustment and baseline correction of the spectrum, obtain the respective integral areas, and calculate the sucrose content in the vaccine according to the fitting formula; the noesygppr1d.mod is optimized by noesygppr1d, insert a blank line before the 33rd line "d12 pl9:f1" of the noesygppr1d pulse file, and write the waiting delay time 21-29s.

2. The method according to claim 1, characterized in that In step S1, heavy water is used for dilution 5-20 times.

3. The method according to claim 2, characterized in that In step S1, the mixture was diluted 10 times with heavy water.

4. The method according to claim 1, characterized in that: The ratio of potassium hydrogen phthalate to heavy water in step S2 is 3 ml of heavy water for every 32-38 mg of potassium hydrogen phthalate.

5. The method according to claim 4, characterized in that Dissolve 35 mg of potassium hydrogen phthalate in 3 ml of heavy water.

6. The method according to claim 1, characterized in that The volume ratio of the test solution to the internal standard solution is 1-5:

1.

7. The method according to claim 6, characterized in that The volume ratio of the test solution to the internal standard solution is 2-3:

1.

8. The method according to claim 6, characterized in that The volume ratio of the test solution to the internal standard solution was 2.5:

1.

9. The method according to claim 1, characterized in that: In step S5, a waiting delay time of 23-28s is written.

10. The method according to claim 9, characterized in that In step S5, a waiting delay time of 26 s is written.

11. The method according to claim 1, characterized in that In step S5, 1 The H NMR parameters were as follows: the experimental template was PROF_1H and the pulse was noesygppr1d.mod.

12. The method according to claim 11, characterized in that In step S5, 1 The H NMR parameters were as follows: D1 = 4 s, and the presaturation power in the edprosol table was set to 25 Hz.

13. The method according to claim 1, characterized in that In step S5, the phase adjustment and baseline correction of the obtained one-dimensional NMR spectrum are performed, and the chemical shift δ H 7.529 (2H, q) as the quantitative peak of potassium hydrogen phthalate, select δ H 5.418 (1H, d) was used as the quantitative peak of sucrose. After integration, the sucrose content in the vaccine was calculated according to the following formula: C x : C x = ； in, C x is the sucrose content in the vaccine; I x , I std are the quantitative peak integrated areas of sucrose and potassium hydrogen phthalate, respectively; N std , N x are the proton numbers of the quantitative peaks of potassium hydrogen phthalate and sucrose, respectively; M x , M std are the relative molecular masses of sucrose and potassium hydrogen phthalate respectively; is the purity of the selected potassium hydrogen phthalate; is the mass of potassium hydrogen phthalate in the final test sample; is the volume of the test solution in the final test sample; It is the dilution multiple of the vaccine liquid.

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