Method for developing L-phenylalanine solution standard substance based on solid state national primary standard substance

Abstract

The invention belongs to the technical field of metering, and particularly relates to a method for developing an L-phenylalanine solution standard substance based on a solid state national first-grade standard substance. On one hand, the fixed value is accurate and reliable, on the other hand, the uniformity and the stability of the L-phenylalanine solution standard substance are accurately and reliably analyzed, and on the other hand, the uncertainty of the L-phenylalanine solution standard substance is comprehensively evaluated; in addition, in the process of analyzing the uniformity and the stability of the L-phenylalanine solution standard substance, test parameters of an ultra-high performance liquid chromatography-ultraviolet absorption method are screened, and a method for analyzing the accuracy and the precision is established; therefore, the technical problem of lack of the L-phenylalanine solution standard substance in the prior art is solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of metrology, and particularly relates to a method for developing L-phenylalanine solution standard substance based on a solid national primary standard substance. BACKGROUND

[0002] L-phenylalanine is one of the essential amino acids for the human body, but the human body cannot synthesize it by itself and must obtain it from food. L-phenylalanine is widely present in meat, beans and their products, nuts, etc. L-phenylalanine has a variety of important functions in the human body, such as promoting dopamine synthesis, enhancing immunity, regulating blood sugar, reducing blood pressure, inhibiting cholesterol synthesis, promoting protein synthesis, regulating the nervous system, etc. In addition, L-phenylalanine is widely used in the food industry and the medical field.

[0003] The commonly used detection methods for phenylalanine at present include chemical analysis method (potentiometric titration method, fluorescence spectrophotometry), biochemical method (phenylalanine deaminase test, phenylalanine dehydrogenase method), chromatographic analysis method (high performance liquid chromatography, liquid chromatography-mass spectrometry), Guthrie bacterial inhibition method and fluorescence biosensor. The sensitivities of various methods are inconsistent, which may lead to large differences in analysis results. Therefore, it is necessary to use relevant standard substances to realize the uniformity of the value.

[0004] However, at present, some domestic enterprises producing L-phenylalanine solution standard substance may face the risk of supply interruption for some concentrations of L-phenylalanine solution standard substance, which is difficult to meet the needs of actual calibration work and customer production verification, and it is urgent to develop L-phenylalanine solution standard substance based on the national primary standard substance GBW09235 (China Institute of Metrology, purity 99.8%, U=0.4%, k=2). SUMMARY

[0005] Therefore, the application provides a method for developing L-phenylalanine solution standard substance based on a solid national primary standard substance, which is used to solve the technical problem of lack of L-phenylalanine solution standard substance in the prior art.

[0006] The first aspect of the application provides a method for developing L-phenylalanine solution standard substance based on a solid national primary standard substance, which comprises the following steps:

[0007] The step of selecting preparation raw materials: selecting the national primary standard substance GBW09235 and purified water as preparation raw materials;

[0008] The step of preparing L-phenylalanine solution standard substance: based on the value model shown in formula 1 and the buoyancy correction, the weighing mass of the national primary standard substance GBW09235 is obtained, which is prepared into L-phenylalanine solution standard substance according to the weight capacity method and subjected to value verification;

[0009] Steps for analyzing homogeneity and stability: After screening the test parameters, ultra-high performance liquid chromatography-ultraviolet absorption method was used to analyze the homogeneity and stability of L-phenylalanine solution standard material;

[0010] The steps for obtaining the uncertainty are as follows: Based on the uncertainty arising from the preparation steps of the L-phenylalanine solution standard, the uncertainty arising from the homogeneity and stability of the L-phenylalanine solution standard, and Equations 2 and 3, the relative expanded uncertainty U is calculated and obtained. rel ;

[0011] Formula 1;

[0012] Formula 2;

[0013] Formula 3;

[0014] In Equation 1, C L-苯丙氨酸 Prepare L-phenylalanine solution standard substances at concentrations of 135.2 μg / mL, 600.0 μg / mL, or 999.9 μg / mL; m L-苯丙氨酸 The vacuum mass is specified in national Class I standard GBW09235, and the unit is mg; q L-苯丙氨酸 The purity value is the national first-class standard GBW09235, in units of %; V is the final volume of the volumetric flask, in units of mL.

[0015] In Equation 2, u rel，定值 The uncertainty arising from the preparation steps of the L-phenylalanine solution standard, u rel，bb Uncertainty arising from the homogeneity of the L-phenylalanine solution standard substance, u rel，Its Uncertainty arising from the stability of L-phenylalanine solution standard;

[0016] In Equation 3, k is the inclusion factor of the national first-class standard GBW09235.

[0017] Preferably, in the step of selecting raw materials for preparation, the national first-class standard GBW09235 is qualitatively analyzed in advance using mass spectrometry and / or ultraviolet spectroscopy.

[0018] The purified water is purified water that has undergone reverse osmosis, ion exchange, and quartz distillation in sequence.

[0019] Preferably, the step of preparing L-phenylalanine solution standard material includes the following process for verifying the value: using different testers, following the same steps for preparing L-phenylalanine solution standard material, preparing one or more solutions with the same target value, measuring the two solutions using a validated analytical method, and comparing the difference between the measured value and the prepared value to determine whether there was an error in the preparation process.

[0020] The t-test for the consistency of the average value was used to determine whether the preparation process was correct. When all t values ​​were less than 2.228, the preparation process was deemed to be without error.

[0021] Preferably, in the steps of analyzing uniformity and stability, the process of screening test parameters includes: based on at least one of effective plate number, symmetry factor, elution time, absorption intensity, and response intensity, screening the test parameters for ultra-high performance liquid chromatography-ultraviolet absorption spectrometry as follows: detector: UV detector; chromatographic column: ZORBAX Eclipse Plus C18 2.1*50mm 1.8μm, PN959757-902; mobile phase: acetonitrile / water = 5:95; mobile phase flow rate: 0.1mL / min; injection volume: 5μL; column temperature: 30℃; detection wavelength: 210nm.

[0022] Preferably, in the step of analyzing uniformity and stability, the standard curve established by ultra-high performance liquid chromatography-ultraviolet absorption method uses L-phenylalanine solutions with concentration gradients of 50.07 μg / mL, 149.70 μg / mL, 250.88 μg / mL, 750.41 μg / mL, 998.65 μg / mL, and 1245.16 μg / mL.

[0023] Preferably, in the step of obtaining the uncertainty, the process of obtaining the uncertainty arising from the preparation step of the L-phenylalanine solution standard substance includes:

[0024] Based on the uncertainties arising from the purity of national primary standard GBW09235, the uncertainties arising from the weighing mass of national primary standard GBW09235, the uncertainties arising from the preparation volume of national primary standard GBW09235, and Equation 4, the uncertainty u arising from the preparation steps of the L-phenylalanine solution standard substance is calculated. rel，定值 ;

[0025] Equation 4;

[0026] In Equation 4, u rel(p) Uncertainty arising from the purity of national first-class standard GBW09235, u rel(m) The uncertainty in the weighing mass of the national first-class standard GBW09235, u rel(V) Uncertainty arising from the preparation volume of the national first-class standard GBW09235.

[0027] Preferably, the process for obtaining the uncertainty arising from the purity of the national first-class standard GBW09235 includes: calculating the uncertainty u arising from the purity of the national first-class standard GBW09235 based on Equation 5. rel(p) ;

[0028] The process of obtaining the uncertainty of the weighing mass of the national first-class standard GBW09235 includes: calculating the uncertainty u of the weighing mass of the national first-class standard GBW09235 based on equations 6 and 7. rel(m) ;

[0029] The process of obtaining the uncertainty in the preparation volume of national primary standard GBW09235 includes: calculating the uncertainty u in the preparation volume of national primary standard GBW09235 based on equations 8 and 9. rel(v) ;

[0030] Formula 5;

[0031] Formula 6;

[0032] Formula 7;

[0033] Formula 8;

[0034] Equation 9;

[0035] In equations 6 and 7, u m1 The uncertainty arising from the weighing of a balance for the national first-class standard GBW09235, u m2 The uncertainty arising from repeated weighing of the national first-class standard GBW09235, u m3 Uncertainty arising from the buoyancy correction of the weighing mass for the national first-class standard GBW09235;

[0036] In Equations 8 and 9, uV1 represents the uncertainty caused by the volume of the volumetric flask used in the preparation process, and uV2 represents the uncertainty caused by temperature fluctuations during the preparation process.

[0037] Preferably, the process of obtaining the uncertainty of the balance generated by the weighing mass of the national first-class standard GBW09235 includes: calculating the uncertainty u generated by the balance generated by the weighing mass of the national first-class standard GBW09235 based on Equation 10. m1 ;

[0038] The process of obtaining the uncertainty arising from repeated weighing of the national first-class standard GBW09235 includes: calculating the uncertainty u arising from repeated weighing of the national first-class standard GBW09235 based on Equation 11. m2 ;

[0039] The process of obtaining the uncertainty arising from the buoyancy correction of the weighing mass of the national first-class standard GBW09235 includes: calculating the uncertainty u arising from the buoyancy correction of the weighing mass of the national first-class standard GBW09235 based on Equation 12. m3 ;

[0040] Formula 10;

[0041] Formula 11;

[0042] Equation 12;

[0043] In Equations 10 and 11, s is the absolute value of the maximum permissible error of the balance for weighing the mass of the national first-class standard GBW09235, s is the standard deviation of the weighing indication error of the balance for repeated weighing of the mass of the national first-class standard GBW09235, and x is the value of the number of times the balance is used to repeatedly weigh the mass of the national first-class standard GBW09235.

[0044] In Equation 12, ρ k For the air density u(ρ) during buoyancy correction for the weighing mass of the national first-class standard GBW09235, k ρ is the standard uncertainty introduced by the air density. w For the national first-class standard GBW09235, the density of L-phenylalanine when weighing and buoyancy correction is given, u(ρ) w ) represents the standard uncertainty introduced by the density of L-phenylalanine, ρ F For the weighing mass buoyancy correction of the national first-class standard GBW09235, the nominal density of the calibration weights, u(ρ) F The standard uncertainty is introduced by the nominal density of the calibration weights.

[0045] Preferably, the process of obtaining the uncertainty of the volumetric flask volume used in the preparation process includes: calculating and obtaining the uncertainty uV1 of the volumetric flask volume used in the preparation process based on Equation 13;

[0046] The process of obtaining the uncertainty caused by temperature fluctuations during the preparation process includes: calculating and obtaining the uncertainty uV2 caused by temperature fluctuations during the preparation process based on Equation 14;

[0047] Equation 13;

[0048] Equation 14;

[0049] In Equations 13 and 14, This represents the absolute value of the maximum permissible tolerance of the volumetric flasks used in the preparation process. This represents the numerical value of the volumetric expansion coefficient of water. This is the absolute value of the maximum permissible deviation of temperature fluctuation during the preparation process.

[0050] Preferably, the process of obtaining the uncertainty arising from the homogeneity of the L-phenylalanine solution standard material includes: calculating the uncertainty u arising from the homogeneity of the L-phenylalanine solution standard material based on Equations 15 and 16. rel，bb ;

[0051] Formula 15;

[0052] Formula 16;

[0053] In Equation 15, M between For the mean square between groups, M within The mean square value is within the group, and n0 is the number of repetitions of the homogeneity analysis of L-phenylalanine solution standard by ultra-high performance liquid chromatography-ultraviolet absorption spectrometry.

[0054] Preferably, the process of obtaining the uncertainty arising from the stability of the L-phenylalanine solution standard material includes: calculating the uncertainty u arising from the stability of the L-phenylalanine solution standard material based on Equations 17 and 18. rel，Its ;

[0055] Equation 17;

[0056] Formula 18;

[0057] In Equation 17, β1 is the rate of change of the mass concentration of L-phenylalanine solution standard in methanol with time, s(β1) is the slope of the regression line of β1, and t is the total time for stability analysis of L-phenylalanine solution standard in methanol by ultra-high performance liquid chromatography-ultraviolet absorption method, in integer months.

[0058] Compared with existing technologies, the method for preparing L-phenylalanine solution standard material based on solid national primary standard material provided in this application has at least the following beneficial effects:

[0059] 1. The method provided in this application for developing L-phenylalanine solution standard material based on solid national primary standard material has the following advantages: Firstly, the determination of values ​​is accurate and reliable. Solid national primary standard material GBW09235 is selected as the raw material, and its value is traceable to the International System of Units (SI) unit kg. The value of solid national primary standard material GBW09235 is transferred to L-phenylalanine solution standard material and the value is verified by gravimetric method. Secondly, the homogeneity and stability of L-phenylalanine solution standard material are accurately and reliably analyzed. Thirdly, the uncertainty of L-phenylalanine solution standard material is comprehensively evaluated. Thus, L-phenylalanine solution standard material has been successfully developed based on solid national primary standard material. At the same time, it provides L-phenylalanine solution standard material with low, medium and high concentrations to meet different detection needs.

[0060] 2. The method for developing L-phenylalanine solution standard material based on solid national primary standard material provided in this application analyzes the homogeneity and stability of L-phenylalanine solution standard material. Optimal ultra-high performance liquid chromatography-ultraviolet absorption spectrometry (UHPLC-UV) test parameters were obtained by screening at least one of the following conditions: effective plate number, symmetry factor, elution time, absorption intensity, and response intensity. Reliable standard curves were established based on L-phenylalanine solutions with concentration gradients of 50.07 μg / mL, 149.70 μg / mL, 250.88 μg / mL, 750.41 μg / mL, 998.65 μg / mL, and 1245.16 μg / mL. Accuracy and precision analyses were performed, enabling accurate and reliable analysis of the homogeneity and stability of low, medium, and high concentrations of L-phenylalanine solution standard material.

[0061] 3. The method for preparing L-phenylalanine solution standard material based on solid national primary standard material provided in this application comprehensively considers the uncertainties introduced in the preparation process, uniformity and stability of the L-phenylalanine solution standard material during the evaluation of uncertainty. It combines the results of Equations 2 to 18 to make the analysis of uncertainty of solution standard material more accurate and comprehensive. Attached Figure Description

[0062] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0063] Figure 1 This is the mass spectrum of the raw material GBW09235 used in Example 1 of this application;

[0064] Figure 2 The ultraviolet spectrum of raw material GBW09235 used in Example 1 of this application;

[0065] Figure 3 This is a schematic diagram illustrating the traceability of L-phenylalanine solution standard material in methanol.

[0066] Figure 4 The results of detecting L-phenylalanine solution standard substance by ultra-high performance liquid chromatography-ultraviolet absorption method after screening test parameters are shown in the figure.

[0067] Figure 5 A standard curve was established using L-phenylalanine solutions with concentration gradients of 50.07 μmol / L, 149.70 μmol / L, 250.88 μmol / L, 750.41 μmol / L, 998.65 μmol / L, and 1245.16 μmol / L.

[0068] Figure 6 Long-term stability trend of L-phenylalanine solution standard (135.2 μg / mL);

[0069] Figure 7 Long-term stability trend of L-phenylalanine solution standard (600.0 μg / mL);

[0070] Figure 8 Long-term stability trend of L-phenylalanine solution standard (999.9 μg / mL);

[0071] Figure 9 Short-term stability trend of L-phenylalanine solution standard (135.2 μg / mL);

[0072] Figure 10 Short-term stability trend of L-phenylalanine solution standard (600.0 μg / mL);

[0073] Figure 11 Short-term stability trend of L-phenylalanine solution standard (999.9 μg / mL). Detailed Implementation

[0074] This application provides a method for developing L-phenylalanine solution standard material based on solid national primary standard material, which is used to solve the technical problem of the lack of L-phenylalanine solution standard material in the prior art.

[0075] The technical solutions of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0076] Example 1

[0077] This embodiment provides a method for developing L-phenylalanine solution standard material based on solid national primary standard material. The method includes the steps of selecting raw materials, preparing L-phenylalanine solution standard material, packaging L-phenylalanine solution standard material, screening test parameters, analyzing homogeneity and stability, and obtaining uncertainty.

[0078] 1.1 The steps for selecting and preparing raw materials include:

[0079] Raw material selection: The national first-class standard GBW09235 (China National Institute of Metrology, purity 99.8%, U=0.4%, k=2) and purified water that has been successively purified by reverse osmosis, ion exchange and quartz distillation are selected as raw materials for preparation.

[0080] Qualitative analysis of raw materials: Mass spectrometry analysis was performed using an Agilent 1260-6470 ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer equipped with an ESI ion impact source. Its sensitivity was <3.5 ppt (positive ion mode), minimum residence time was 1 ms, signal-to-noise ratio was >5000:1, and single-charge mass range was 5-3000. Ultraviolet analysis was performed using an ultra-high performance liquid chromatography system with a 1260 Infinity DAD detector (Agilent, 1290 Infinity). The results are as follows: Figures 1-2 As shown, the raw material used is L-phenylalanine.

[0081] 1.2 The steps for preparing L-phenylalanine solution standard material include:

[0082] Volumetric flasks (Grade A) of 25mL, 100mL, 250mL, 500mL, and 2000mL; 5mL...

[0083] 10mL and 25mL graduated pipettes (Grade A); medicine spoons; glassware such as beakers and weighing dishes; electronic balance (CPA225D (Max: 220g / 100g; d: 0.1mg / 0.01mg; Sartorius, Germany); vertical pressure steam sterilizer (YXQ-LS-75G, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory); and 2mL cryovials as instruments and glassware for preparation.

[0084] In a clean bench (SW-CJ-1F, Suzhou Antai Air Technology Co., Ltd.), accurately weigh 44.73 mg of GBW09235 L-phenylalanine using an electronic balance according to the gravimetric method (room temperature 20±2℃, humidity 55±5%RH). Transfer the solution to a beaker, dissolve and rinse the beaker three times with purified water, and transfer the solution to a 2000 mL volumetric flask. Finally, add purified water to the mark, tighten the cap, invert and shake thoroughly to prepare a 135.2 μmol / L L-phenylalanine solution standard. Accurately weigh 49.62 mg of GBW09235 L-phenylalanine using an electronic balance and prepare a 500 mL volumetric flask. Prepare a 600.0 μmol / L L-phenylalanine solution standard using the same gravimetric method. Accurately weigh GBW09235... Prepare 41.35 mg of L-phenylalanine and a 250 mL volumetric flask to prepare a 1000 μmol / L L-phenylalanine standard solution using the same gravimetric method.

[0085] L-phenylalanine standard solutions of 135.2 μmol / L, 600.0 μmol / L, and 1000 μmol / L were aliquoted into 2 mL cryovials, labeled with sample concentration, sample number, and aliquot date, and stored at -80°C.

[0086] In this preparation (determination) step, the gravimetric method is used, traceable to the national primary standard reference GBW09235 (e.g. Figure 3 As shown); simultaneously, according to the fixed value model shown in Equation 1 and buoyancy correction, the accurate weighing mass displayed by the electronic balance is obtained, and the buoyancy correction process is carried out with reference to Appendix C of "GB10738-2007 General Rules for Determination of Content of Working Standard Reagents: Weighing Titration Method"; according to

[0087] According to the formula Combining the constant value model shown in Equation 1, the weighing mass of GBW09235 is calculated, where m is the mass of GBW09235. k The measured mass displayed on the electronic balance (44.73 mg, 49.62 mg, or 41.35 mg), m z The vacuum mass of the object being weighed (the vacuum mass of the national first-class standard GBW09235, 44.759 mg, 49.652 mg, 41.377 mg), ρ k The density of air at the time of weighing (20℃, 55%RH, 1.2×10⁻⁶) is given. -3 g / cm 3 ), ρ w The density of the substance being weighed (L-phenylalanine is 1.5 g / cm³) 3 ), ρ F The density of the weights on the electronic balance is 8.0 g / cm³.3 ); and in accordance with the provisions of JJF 1343-2022 "Assignment and Homogeneity and Stability Assessment of Standard Reference Materials", the values ​​of three L-phenylalanine solution standard reference materials were verified. The verification results are shown in Table 1, and all values ​​are less than t. （0.05,10） =2.228, which meets the requirements for quantity verification.

[0088] Table 1: Measurement Verification Results

[0089]

[0090] 1.3 The steps for screening test parameters include:

[0091] Test parameters for ultra-high performance liquid chromatography-ultraviolet absorption spectrometry (UHPLC-UV absorption spectrometry) were screened based on at least one of the following: effective plate number, symmetry factor, elution time, absorption intensity, and response intensity. The liquid chromatograph used was an Agilent 1260infinity HPLC-1260 infinity DAD. The screened test parameters included column, mobile phase, mobile phase ratio, mobile phase flow rate, injection volume, column temperature, and wavelength.

[0092] The column selection process included: simultaneously using ZORBAX Eclipse Plus C18 2.1*50mm 1.8μm, PN959757-902 (column 1), ZORBAX SB-C18 2.1*100mm 1.8μm, PN858700-902 (column 2), and ZORBAX Hilic Plus C18 2.1*100mm... A 1.8 μm PN959758-901 (column 3) was used to detect purified water-soluble blank and L-phenylalanine solution standard. The test conditions were: mobile phase acetonitrile / water = 5 / 95 (V / V), flow rate 0.10 mL / min, column temperature 30℃, and UV wavelength 210 nm. The results are shown in Table 2. It can be seen that column 3 can effectively separate L-phenylalanine and purified water-soluble blank, and has a high effective plate number and response intensity, while having a small symmetry factor and suitable peak time.

[0093] Table 2: Results of L-phenylalanine solution standard substance detection by chromatographic column 1-3

[0094]

[0095] The screening process for the mobile phase included: based on the screening of the chromatographic column, acetonitrile / water = 5 / 95 (V / V) or methanol / water = 5 / 95 (V / V) were selected as the mobile phase, and L-phenylalanine solution standard was detected respectively. The results are shown in Table 3. It can be seen that when acetonitrile / water is used as the mobile phase, the effective plate number and response intensity are higher, while the symmetry factor is smaller, and the peak does not elute too quickly during detection.

[0096] Table 3: Results of L-phenylalanine solution standard reference for detection in acetonitrile / water or methanol / water

[0097]

[0098] The screening process for mobile phase ratios included: based on the screening of acetonitrile / water mobile phases, acetonitrile:water ratios of 10:90, 5:95, or 2:98 were selected for the detection of L-phenylalanine solution standard material, and the results are shown in Table 4. It can be seen that the acetonitrile / water ratio of 2:98 resulted in a longer peak time and a lower response intensity, while the acetonitrile / water ratio of 10:90 resulted in a too fast peak time and a lower effective plate number. Therefore, the acetonitrile / water ratio of 5:95 was selected for the detection of L-phenylalanine solution standard material.

[0099] Table 4: Results of L-phenylalanine solution standard reference with different acetonitrile / water ratios

[0100]

[0101] The screening process for mobile phase flow rate included: based on the initial screening using acetonitrile / water at a ratio of 5:95, mobile phase flow rates of 0.05 mL / min, 0.1 mL / min, 0.15 mL / min, and 0.20 mL / min were selected for the detection of L-phenylalanine solution standard. The results are shown in Table 5. It can be seen that at flow rates of 0.05 mL / min and 0.1 mL / min, the acetonitrile / water mobile phase exhibits higher response intensity and effective plate number, but lower symmetry factor. However, at a flow rate of 0.05 mL / min, the peak elution time is too long. Therefore, a mobile phase flow rate of 0.05 mL / min for acetonitrile / water was selected for the detection of L-phenylalanine solution standard.

[0102] Table 5: Results of L-phenylalanine solution standard substance detection at different mobile phase flow rates

[0103]

[0104] The injection volume screening process included: based on the mobile phase screening, L-phenylalanine solution standard substances of 2 μL, 3 μL, 5 μL, 6 μL or 8 μL were selected as injection volumes for detection. The detection results are shown in Table 6. It can be seen that the response intensity is low when the injection volume is 2 μL or 3 μL, while the symmetry factor is small when the injection volume is 6 μL or 8 μL. Considering all factors, 5 μL was selected as the injection volume.

[0105] Table 6: Results of L-phenylalanine solution standard substance detection under different injection volumes

[0106]

[0107] The column temperature screening process included: based on the injection volume screening, the L-phenylalanine solution standard was tested at column temperatures of 20℃, 25℃, 30℃, and 35℃. The test results are shown in Table 7. It can be seen that good test results can be obtained at column temperatures of 20℃ to 35℃. Considering all factors, room temperature of 30℃ was chosen as the optimal temperature for testing the L-phenylalanine solution standard.

[0108] Table 7: Results of L-phenylalanine solution standard at different column temperatures

[0109]

[0110] Based on column temperature screening, 200nm, 205nm, 210nm, 220nm, 230nm, and 240nm were selected as excitation wavelengths for detection, and the detection results are shown in Table 8. It can be seen that L-phenylalanine has good absorption intensity for excitation wavelengths of 200nm, 205nm, and 210nm. Considering the effective number of plates, 210nm was selected as the excitation wavelength.

[0111] Table 8: Results of L-phenylalanine solution standard at different excitation wavelengths

[0112]

[0113] The optimal test parameters for ultra-high performance liquid chromatography-ultraviolet absorption spectrometry (UHPLC-UV absorption spectrometry) were determined as follows: Detector: UV detector; Column: ZORBAX Eclipse Plus C18 2.1*50mm 1.8μm, PN959757-902; Mobile phase: acetonitrile / water = 5:95; Mobile phase flow rate: 0.1mL / min; Injection volume: 5μL; Column temperature: 30℃; Detection wavelength: 210nm. The results of detecting methyl-L-phenylalanine solution standard using UHPLC-UV absorption spectrometry under these selected parameters are as follows: Figure 4As shown, its comprehensive performance, including effective plate number, symmetry factor, elution time, absorption intensity, and response intensity, is excellent and meets the analytical requirements of L-phenylalanine solution standard material.

[0114] 1.4 Steps for analyzing homogeneity and stability:

[0115] Establishment of the standard curve: L-phenylalanine solutions with concentration gradients of 50.07 μmol / L, 149.70 μmol / L, 250.88 μmol / L, 750.41 μmol / L, 998.65 μmol / L, and 1245.16 μmol / L were prepared using the national primary standard GBW09235 as the raw material. Ultra-high performance liquid chromatography-ultraviolet absorption spectrometry (UHPLC-UV) was used for testing after screening the parameters. Each concentration was injected three times. The average measured value was plotted on the x-axis, and the prepared values ​​were plotted on the y-axis to show the relationship between concentration and detection signal. Figure 5 As shown in Table 9, the linear relationship is good, and it can be used to test the homogeneity and stability of the L-phenylalanine solution standard material prepared in this embodiment.

[0116] Table 9: Preparation and test values ​​of L-phenylalanine solution standard (unit: μg / mL)

[0117]

[0118] Accuracy Analysis: The accuracy of the GBW(E)100672 L-phenylalanine solution standard was analyzed by liquid chromatography. The test was repeated three times, and the peak area was measured by the liquid chromatograph. The concentration was read from the standard curve mentioned above. The results are shown in Table 10. It can be seen that the average value of the test results of the GBW(E)100672 phenylalanine solution standard is within the range of the set value of GBW(E)100672.

[0119] Table 10: Accuracy Analysis Results

[0120]

[0121] Precision analysis: Three concentrations of L-phenylalanine solution standard substances were selected for liquid chromatography precision analysis. The liquid chromatography was repeated 6 times. The results are shown in Table 11. It can be seen that the relative standard deviation (RSD) is 0.11%, which shows good precision.

[0122] Table 11: Precision analysis results (unit: μg / mL)

[0123]

[0124] Meanwhile, substituting the signal-to-noise ratios of 3 and 10 times into the standard curve reveals that the detection limit for L-phenylalanine is 0.17 μg / mL, and the quantitation limit is 0.28 μg / mL. Therefore, the test method provided in this embodiment, after screening test parameters, establishing a standard curve for L-phenylalanine based on concentration gradients of 50.07 μmol / L, 149.70 μmol / L, 250.88 μmol / L, 750.41 μmol / L, 998.65 μmol / L, and 1245.16 μmol / L, and performing accuracy and precision analysis, is suitable for analyzing the homogeneity and stability of the L-phenylalanine solution standard material prepared in this embodiment, and the test method is reliable.

[0125] Uniformity analysis:

[0126] At (20±0.3)℃, the concentration of the L-phenylalanine solution standard prepared in step 1.2 was measured by the absorption peak area in the chromatogram of ultra-high performance liquid chromatography-ultraviolet absorption (using the test parameters screened above and the established reliable standard curve). Before use, the frozen tube solution was shaken evenly and used immediately after opening. For simple homogeneity experiments or randomized block designs, in order to obtain an acceptable estimate of the inter-unit variance for uncertainty assessment, the degrees of freedom should be ≥9, i.e., at least 10 units should be selected. Therefore, using the randbetween function in Excel software, 11 random numbers were randomly generated for each level of standard. 11 units of candidates for each level were selected according to the random numbers. The candidate of each unit was tested three times, with a sample volume of 2 μL each time. In order to eliminate the error caused by instrument drift during the test, the samples were analyzed for homogeneity three times in the order of 1→11, 11→1, alternating between odd and even numbers.

[0127] The measurement data from three repeated tests were used as data for homogeneity analysis, and variance analysis was performed on them. All instruments and measuring tools used were first calibrated to ensure they were in normal working order and that the traceability of the measurements was guaranteed. Analysis of variance (ANOVA) is the most commonly used method for statistically testing homogeneity. It determines whether there are systematic differences between group measurements by comparing between-group variance and within-group variance. If the ratio is less than the critical value of the statistical test, the sample is considered homogeneous. The results of the homogeneity analysis are shown in Table 12-14. The formulas used in the homogeneity analysis include statistical model formula A, between-group mean square (between-group mean square) calculation formula B, within-group mean square (residual mean square) calculation formula C, mean of the i-th unit calculation formula D, and overall mean of all observations calculation formula E. It can be seen that the variance analysis results for the L-phenylalanine solution standard substance are all F < F<0.05. 0.05 Since there is no significant difference between the data groups, it can be considered that the L-phenylalanine solution standard material prepared in step 1.2 of this embodiment has good homogeneity.

[0128] Table 12: Results of homogeneity analysis of L-phenylalanine solution standard (concentration 135.2 μg / mL)

[0129]

[0130] Table 13: Results of homogeneity analysis of L-phenylalanine solution standard (concentration 600.0 μg / mL)

[0131]

[0132] Table 14: Results of homogeneity analysis of L-phenylalanine solution standard (concentration 999.9 μg / mL)

[0133]

[0134] Equation A, where x ij This represents the j-th observation in the i-th unit.

[0135] μ represents the total number of all possible outcomes (observation result x). ij Assuming the (true) mean is derived from it; δ i εi represents the effect of unit i on the result, i.e., the (true) deviation of unit i from μ; εij represents the random error of the j-th observation of the i-th unit, also known as the residual term;

[0136] Formula B;

[0137] Formula C;

[0138] Formula D;

[0139] Formula E.

[0140] Stability analysis:

[0141] The stability of a standard reference material refers to how a defined characteristic quantity changes over time.

[0142] Stability is affected by physical, chemical, and storage conditions, requiring long-term periodic testing using highly precise analytical methods to determine the relative stability period. The stability of a standard substance includes long-term stability and short-term stability. Long-term stability refers to the stability of the characteristic quantity of the standard substance under specified storage conditions, while short-term stability refers to the stability of the standard substance during transportation under transportation conditions. In this embodiment, following the principle of denser testing at the beginning and sparser testing at the end, the developed L-phenylalanine solution standard substance was subjected to a long-term stability test for 8 months at -20°C and a short-term stability test under simulated transportation conditions for 7 days.

[0143] Long-term stability was assessed by examining the changes in characteristic properties of L-phenylalanine solution standard material after storage at -80℃ for 0, 1, 2, 3, 4, 5, 6, 7, and 8 months. The tests were conducted using ultra-high performance liquid chromatography-ultraviolet absorption spectrometry (UHPLC-UV absorption spectrometry) with the selected test parameters and established reliable standard curve. Three samples were randomly selected at each time point. After the samples were removed from storage and equilibrated for at least 30 minutes, their concentrations were measured. Each sample was measured three times, and the average value at each time point was taken as the long-term stability assessment result. Regression analysis was used to evaluate the long-term stability. The experimental results of long-term stability are shown in Tables 15-17. Figures 6-8 As shown, it can be seen that it has excellent long-term stability.

[0144] Short-term stability was studied to examine the effect of transportation conditions on the stability of the standard substance. This embodiment simulated transportation conditions at 40±2℃ and 4±2℃. Six groups of samples were randomly selected, with three samples in each group. The L-phenylalanine solution standard substance candidate was removed from storage conditions at -20℃ on days 0, 2, 4, 5, 6, and 7 and placed under simulated transportation conditions. Seven days later, all samples were removed (the storage times under transportation conditions were 7, 5, 3, 2, 1, and 0 days, respectively). After the samples were allowed to equilibrate for at least 30 minutes, their concentrations were measured. The experimental results of short-term stability are shown in Tables 21-29. Figures 9-11 As shown, regression analysis was used to determine the impact of simulation conditions on its stability, and it can be seen that the short-term stability is excellent.

[0145] The regression analysis used a linear regression model, which is formula F. The slope (β1) was calculated using formula G, and the slope (β0) was calculated using formula H. The standard deviation of the slope (β1), s(β1), was calculated using formula I. The standard deviation (s) of each point on the straight line in the standard deviation s(β1) was calculated using formula J. After calculating the standard deviation of the slope, s(β1), the t-test was used to determine the significant difference between β1 and 0, and the t-statistic was calculated using formula K. It can be seen that the L-phenylalanine solution standard material prepared in this application has good long-term and short-term stability.

[0146] Equation F, where X is time, Y is the characteristic value of the standard substance candidate, and ε is the random error term, which is usually assumed to be a normal distribution with a mean of 0;

[0147] Equation G, where X i For the i-th time point, Y i For the i-th

[0148] Observations at time points, The average value across all time points. The average of all observations;

[0149] Formula H;

[0150] Formula I;

[0151] Equation J, where n is the number of tests;

[0152] Formula K;

[0153] Table 15: Long-term stability of L-phenylalanine solution standard (concentration 135.2 μg / mL)

[0154]

[0155] Table 16: Long-term stability of L-phenylalanine solution standard (concentration 600.0 μg / mL)

[0156]

[0157] Table 17: Long-term stability of L-phenylalanine solution standard (concentration 999.9 μg / mL)

[0158]

[0159] Table 18: Regression analysis of experimental data for L-phenylalanine solution standard material (concentration 135.2 μg / mL)

[0160]

[0161] Table 19: Regression Analysis of Experimental Data for L-Phenylalanine Solution Standard Material (Concentration 600.0 μg / mL)

[0162]

[0163] Table 20: Regression analysis of experimental data for L-phenylalanine solution standard material (concentration 999.9 μg / mL)

[0164]

[0165] Table 21: Short-term stability of L-phenylalanine solution standard at 40±2℃ (concentration 135.2 μg / mL)

[0166]

[0167] Table 22: Short-term stability of L-phenylalanine solution standard at 4±2℃ (concentration 135.2 μg / mL)

[0168]

[0169] Table 23: Short-term stability of L-phenylalanine solution standard at 40±2℃ (concentration 600.0 μg / mL)

[0170]

[0171] Table 24: Short-term stability of L-phenylalanine solution standard at 4±2℃ (concentration 600.0 μg / mL)

[0172]

[0173] Table 25: Short-term stability of L-phenylalanine solution standard at 40±2℃ (concentration 999.9 μg / mL)

[0174]

[0175] Table 26: Short-term stability of L-phenylalanine solution standard at 4±2℃ (concentration 999.9 μg / mL)

[0176]

[0177] Table 27: Regression analysis of experimental data for L-phenylalanine solution standard material (concentration 135.2 μg / mL)

[0178]

[0179] Table 28: Regression Analysis of Experimental Data for L-Phenylalanine Solution Standard Material (Concentration 600.0 μg / mL)

[0180]

[0181] Table 29: Regression analysis of experimental data for L-phenylalanine solution standard material (concentration 999.9 μg / mL)

[0182]

[0183] 1.5 The steps for obtaining uncertainty include:

[0184] Uncertainty introduced by the preparation steps: Uncertainty (u) introduced by the weighing mass according to the national primary standard GBW09235 was obtained. rel(m) Uncertainty introduced by preparation volume (u) rel(v) ) and the uncertainty introduced by purity (u rel(p) )analyze.

[0185] The process of obtaining the uncertainty introduced by weighing mass includes: the uncertainty u generated by the balance for weighing mass according to the national first-level standard GBW09235. m1 Based on Equation 10, since the balance used is GBW09235 and the maximum permissible error is ±0.5mg, the standard weight should be calculated twice: once with an empty pan and once with gross weight. Because each weighing is an independent observation, the standard uncertainty u is... m1 The value is 0.409 mg; the uncertainty u arising from repeated weighings m1 Based on Equation 11, the E2 grade 50mg weight was repeatedly weighed 10 times as the basis for evaluating the repeatability of the balance. The weights of the 10 weighings were (49.98, 50.01, 50.02, 50.04, 50.00, 50.02, 50.02, 50.03, 50.00, 50.02, in mg), with an average value of 50.01mg and a standard deviation of 0.017mg. Therefore, the standard uncertainty u m2 The uncertainty u arising from buoyancy correction is 0.0054 mg. m3 Based on Equation 12, where the laboratory temperature during weighing was 20±2℃, the air density ρ is known from the Langevin Chemical Handbook. k It is 0.0012 g / cm³ 3 The standard uncertainty u(ρ) introduced by air density k The value is 0.00005 g / cm³. 3 L-phenylalanine density ρ w It is 1.5 ± 0.10 g / cm³ 3 The nominal density ρ of the calibration weights F It is 8.00±0.02 g / cm³ 3 Treating it as a rectangular distribution, the standard uncertainty u(ρ) introduced by the L-phenylalanine density w The value is 0.057 g / cm³. 3 The standard uncertainty u(ρ) introduced by the nominal density of the weight F The value is 0.012 g / cm³. 3 Therefore, the standard uncertainty u m3The value is 0.0408 mg. Subsequently, based on Equations 6 and 7, the uncertainty u caused by the weighing mass is calculated. m and u rel(m) .

[0186] The process of obtaining the uncertainty introduced by the preparation volume includes: calculating the uncertainty uV1 caused by the volume of the volumetric flask used in the preparation process and the uncertainty uV2 caused by temperature fluctuation based on Equations 13 and 14; the 2000mL single-mark volumetric flask used in the preparation process is of Grade A qualified, with a given capacity tolerance of ±0.60mL on the calibration certificate, while the 500mL single-mark volumetric flask used in the preparation process has a given capacity tolerance of ±0.25mL on the calibration certificate, and the 250mL calibration certificate has a given capacity tolerance of ±0.15mL. Simultaneously, the temperature fluctuation is ±2℃, and the coefficient of water volume expansion is 2.1×10⁻⁶. -4 ℃ -1 Thus, the uV1 values ​​can be calculated to be 0.3464 mL (135.2 μg / mL), 0.1443 mL (600.0 μg / mL), and 0.0866 mL (999.9 μg / mL), respectively.

[0187] uV2 were 0.4850 mL (135.2 μg / mL), 0.1212 mL (600.0 μg / mL), and 0.0606 mL (999.9 μg / mL), respectively; then, based on Equations 8 and 9, the uncertainty u arising from the preparation volume was calculated. V and u rel(V) .

[0188] The process of obtaining the uncertainty introduced by purity includes: Since GBW09235 (National Institute of Metrology, China, purity 99.8%, U=0.4%, k=2), combined with Equation 5, the uncertainty u caused by the purity of GBW09235 is calculated. rel(p) The value is 0.200%; subsequently, the uncertainty u introduced by the preparation step is calculated based on Equation 4. rel，定值 .

[0189] Uncertainties introduced by homogeneity and stability:

[0190] The uncertainty introduced by the homogeneity of the prepared L-phenylalanine solution standard is based on the inter-group mean square M shown in Table 12-14. between and within-group mean square M within The number of tests n0 (3 times) and equations 15 and 16 are used to calculate u. bb and u rel，bb .

[0191] The uncertainty introduced by the stability of the prepared L-phenylalanine solution standard is calculated based on s(β1) shown in Table 18-20 and Equations 17 and 18.Its and u rel，Its .

[0192] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for developing L-phenylalanine solution standard substance based on solid state national primary standard substance, characterized in that, The method comprises the following steps: Select GBW09235 and purified water as preparation raw materials; Based on the fixed value model shown in formula 1 and the buoyancy correction, the weighing mass of GBW09235 is obtained, and the L-phenylalanine solution standard material is prepared according to the weight capacity method and the value verification is carried out; After screening the test parameters, the uniformity and stability of the L-phenylalanine solution standard material are analyzed by using the ultra-high performance liquid chromatography-ultraviolet absorption method; The uncertainty due to the preparation procedure of the L-phenylalanine solution certified reference material, the uncertainty due to the homogeneity and stability of the L-phenylalanine solution certified reference material, the relative expanded uncertainty U calculated from equation 2 and equation 3 rel ; Formula 1; Formula 2; Formula 3; In formula 1, C L-苯丙氨酸 is the concentration of the L-phenylalanine solution standard substance preparation; m L-苯丙氨酸 is the vacuum mass of GBW09235; q L-苯丙氨酸 is the purity value of GBW09235; V is the constant volume of the volumetric flask; In formula 2, u rel，定值 The uncertainty, u, produced by the preparation procedure of the L-phenylalanine solution standard material rel，bb The uncertainty, u, produced by the homogeneity of the L-phenylalanine solution standard material rel，Its The uncertainty, u, produced by the stability of the L-phenylalanine solution standard material.

2. The method for developing L-phenylalanine solution standard substance based on solid state national primary standard substance according to claim 1, characterized in that, The national primary standard GBW09235 is pre-qualitatively analyzed by using mass spectrometry and / or ultraviolet spectroscopy; The purified water is purified water treated by reverse osmosis, ion exchange and quartz distillation in sequence.

3. The method for developing L-phenylalanine solution standard substance based on solid state national primary standard substance according to claim 1, characterized in that, The process of screening the test parameters comprises: based on at least one of the effective plate number, the symmetry factor, the peak time, the absorption intensity and the response intensity, the test parameters of the ultra-high performance liquid chromatography-ultraviolet absorption method are screened: detector: UV detector; chromatographic column: ZORBAX Eclipse Plus C18 2.1*50 mm 1.8 μm, P.N. 959757-902; mobile phase: acetonitrile / water=5:95; mobile phase flow rate 0.1 mL / min; injection volume: 5 μL; column temperature: 30 DEG C; detection wavelength: 210 nm.

4. The method for developing L-phenylalanine solution standard substance based on solid state national primary standard substance according to claim 1, characterized in that, The standard curve of the ultra-high performance liquid chromatography-ultraviolet absorption method uses L-phenylalanine solutions with concentration gradients of 50.07 μg / mL, 149.70 μg / mL, 250.88 μg / mL, 750.41 μg / mL, 998.65 μg / mL and 1245.16 μg / mL.

5. The method for developing L-phenylalanine solution standard substance based on solid state national primary standard substance according to claim 1, characterized in that, The process of obtaining the uncertainty generated in the preparation step of the L-phenylalanine solution standard material comprises: The uncertainty of the preparation step of the L-phenylalanine solution standard material u is calculated based on the uncertainty of the purity of GBW09235, the uncertainty of the weighing mass of GBW09235, the uncertainty of the preparation volume of GBW09235, and formula 4. rel，定值 ; Equation 4; In formula 4, u rel(p) , u rel(m) , u rel(V) are the uncertainty of the purity of GBW09235, the uncertainty of the mass taken, and the uncertainty of the volume prepared, respectively.

6. The method for developing L-phenylalanine solution standard substance based on solid state national primary standard substance according to claim 5, characterized in that, The uncertainty, u, resulting from the calculation to obtain the purity of GBW09235 based on Equation 5 rel(p) ; The uncertainty u resulting from the mass taken to obtain GBW09235 is calculated based on formula 6 and formula 7 rel(m) The uncertainty u resulting from the volume taken to obtain GBW09235 is calculated based on formula 8 and formula 9 rel(v) ; Formula 5; Formula 6; Formula 7; Formula 8; Equation 9; u m1 u m2 u m3 are the uncertainty from the balance used to weigh the mass, the uncertainty from the repeated weighing of the mass, and the uncertainty from the buoyancy correction of the mass, respectively. In formula 8 and formula 9, uV1 and uV2 are the uncertainties generated by the volume of the volumetric flask used in the preparation process and the temperature fluctuation, respectively.

7. The method for developing L-phenylalanine solution standard substance based on solid state national primary standard substance according to claim 6, characterized in that, The uncertainty u generated by the balance for the mass taken of GBW09235 is calculated based on equation 10 m1 ; The uncertainty u of the repeated mass measurement of GBW09235 is calculated based on formula 11 m2 ; The uncertainty u of the mass of GBW09235 taken from the buoyancy correction is calculated based on formula 12 m3 ; Formula 10; Formula 11; Equation 12; In formula 10 and formula 11, s, x are respectively absolute value of maximum permissible error of the balance for weighing the mass, standard deviation of the balance weighing indication error, and the number of times of weighing the mass by the balance. In formula 12, p k is the air density at the buoyancy correction to the mass of GBW09235, u(p k ) is the standard uncertainty introduced by the air density, p w is the L-phenylalanine density at the buoyancy correction to the mass of GBW09235, u(p w ) is the standard uncertainty introduced by the L-phenylalanine density, p F is the nominal density of the calibration weight at the buoyancy correction to the mass of GBW09235, u(p F ) is the standard uncertainty introduced by the nominal density of the calibration weight.

8. The method for developing L-phenylalanine solution standard substance based on solid state national primary standard substance according to claim 6, characterized in that, The uncertainty uV1 generated by the volume of the volumetric flask used in the preparation process is calculated based on formula 13; The uncertainty uV2 generated by the temperature fluctuation in the preparation process is calculated based on formula 14; Equation 13; Formula 14; in formula 13 and formula 14, is the absolute value of the maximum allowable deviation of the volume of the volumetric flask used in the preparation process, is the numerical value of the water volume expansion coefficient, is the numerical value of the absolute value of the maximum allowable deviation of the temperature fluctuation in the preparation process.

9. The method for developing L-phenylalanine solution standard substance based on solid state national primary standard substance according to claim 1, characterized in that, Based on the formula 15 and formula 16, the uncertainty u of the L-phenylalanine solution standard material uniformity is calculated rel，bb ; Formula 15; Formula 16; In formula 15, M between is the mean square between groups, M within is the mean square within groups, n0is the number of repetitions of the uniformity analysis of the L-phenylalanine solution standard material by ultra-high performance liquid chromatography-ultraviolet absorption method.

10. The method for developing L-phenylalanine solution standard substance based on solid state national primary standard substance according to claim 1, characterized in that, Based on the formula 17 and formula 18, the uncertainty u generated by the stability of L-phenylalanine solution standard substance is calculated rel，Its ; Formula 17; Formula 18; In formula 17, β1 is the rate of change of the mass concentration of the L-phenylalanine solution standard material in methanol with time, s(β1) is the regression straight line slope of β1, and t is the numerical value of the total time of the stability analysis of the L-phenylalanine solution standard material in methanol by the ultra-high performance liquid chromatography-ultraviolet absorption method.

Images