Method for preparing coenzyme Q10 standard substance and evaluating quality of coenzyme Q10 standard substance

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for preparing a coenzyme Q10 standard substance and evaluating the quality of the coenzyme Q10 standard substance. The method provided by the invention comprises the following steps: S01, refining a commercially available coenzyme Q10 sample under a first liquid chromatography condition to obtain a purified solution; s02, freeze-drying and storing the refined purified liquid; s03, carrying out purity analysis on the purified coenzyme Q10 liquid under a second liquid chromatography condition; and S04, carrying out moisture control on the coenzyme Q10 standard sample and carrying out quality detection on the coenzyme Q10 standard sample. The method system provided by the invention provides implementation conditions for meeting the requirements of analysis detection, quality control management and standard substance development in the fields of related foods, medicines and health care products and ensuring the accuracy, comparability and traceability of detection results.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and particularly relates to a method for preparing a coenzyme Q 10 standard and its quality evaluation. Background Art

[0002] A reference material is a material with sufficiently uniform and stable specified properties that has been determined to meet the intended use of the measurement process. It is a physical sample with uniform quality whose properties are compared with those of the test sample, and has a purity suitable for the intended use. A reference material is an essential reference standard for confirming product quality, and has characteristics such as high uniformity, accurate quantity value, and good stability.

[0003] Coenzyme Q 10 is an unsaturated nonameric isoprenoid primary alcohol, belonging to sesquarterpenenol, and has a unique chain-like isolated polyene structure. Coenzyme Q 10 has the functions of nourishing the myocardium and improving myocardial metabolism, and at the same time has the functions of promoting oxidative phosphorylation reaction and protecting the integrity of the biological membrane structure. It is a raw material for a variety of health foods. However, there is currently no reference material for this compound in China, and the quality of the reagents or reference substances provided on the market varies, and the standards are inconsistent. There is an urgent need for a coenzyme Q 10 reference material to meet the needs of the analysis and detection and quality control of coenzyme Q 10 and its related products.

[0004] In summary, it is necessary to improve the existing technology. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing a coenzyme Q 10 standard and its quality evaluation, which partially solves or alleviates the above deficiencies in the existing technology. The present invention specifically adopts the following technical solutions.

[0006] A method for preparing a coenzyme Q 10 standard and its quality evaluation, the method comprising the following steps: S01: Refine the commercially available coenzyme Q 10 sample under the first liquid chromatography condition, the first liquid chromatography condition being gradient elution, specifically as follows: The chromatographic column is a C18 chromatographic column; the mobile phase is methanol; The mobile phase program is: 0 - 30 min, 90% - 10% methanol; 30 - 35 min, 10% methanol; 35.1 - 45 min, 10% - 90% methanol; Collect the eluted fractions, set the collection time as t1 - t2; the t1 - t2 is 15 - 18 min, and the fraction is the coenzyme Q 10 purified solution; S02: Freeze-dry and store the purified coenzyme Q solution. 10 S03: Analyze the purity of the purified coenzyme Q solution under the second liquid chromatography conditions. The second liquid chromatography conditions are isocratic elution, and the purity of the eluate within the range of t1'-t2' of the detection elution time is detected. A sample with a purity greater than 99% is determined to be a coenzyme Q standard sample. The duration of t1'-t2' is less than the duration of t1-t2. 10 10 S04: Detect the moisture content of the obtained coenzyme Q standard sample. The water content of the coenzyme Q standard sample should not be greater than 0.1%. 10 10 For a coenzyme Q standard sample with a water content greater than 0.1%, perform vacuum low-temperature static drying treatment until the water content is lower than 0.1%. 10 S05: Perform the first-level quality inspection on the coenzyme Q standard sample. The indicators of the first-level quality inspection include one or more of thin-layer chromatography purity analysis, HPLC purity analysis, peak purity analysis, moisture detection, ash detection, solvent residue detection, and / or structure confirmation. 10 S06: Perform the second-level quality inspection on the coenzyme Q standard sample. The indicators of the second-level quality inspection include one or more of uniformity detection, stability detection, and / or value determination analysis. 10

[0007] Furthermore, the chromatographic column for the second liquid chromatography conditions is a C18 column; the mobile phase is a mixed solution of methanol and ethanol, and the volume ratio of methanol to ethanol in the mixed solution is 25:75; t1'-t2' is 10 - 11 min.

[0008] Furthermore, the column temperature for the first liquid chromatography conditions and the second liquid chromatography conditions is 35°C; the detection wavelength is 275 nm; the flow rate is 1.0 mL / min.

[0009] As a preference, the chromatographic columns for the first liquid chromatography conditions and the second liquid chromatography conditions are ZORBAX SB-C 18 chromatographic columns.

[0010] In some other embodiments, the chromatographic columns for the first liquid chromatography conditions and the second liquid chromatography conditions are Supersil ODS2 chromatographic columns.

[0011] Furthermore, the vacuum low-temperature static drying treatment is to 10The sample is placed in a vacuum desiccator containing a desiccant, and the vacuum desiccator is placed at -20°C for a drying time of not less than 30 days; the desiccant is a mixture of anhydrous calcium chloride and silica gel; the silica gel is used to indicate the change in moisture.

[0012] In some embodiments, when the silica gel absorbs water, its color changes from blue to pink.

[0013] Furthermore, the mass ratio of the anhydrous calcium chloride to the silica gel is 2 - 5:1.

[0014] In some preferred embodiments, when the mass ratio of the anhydrous calcium chloride to the silica gel is 5:1, the drying effect is the best.

[0015] Furthermore, in the moisture detection of S05, the coenzyme Q 10 The water content of the standard sample should be less than 0.1%; For the coenzyme Q 10 standard sample with a water content greater than 0.1%, it is processed according to the low-temperature static drying method described in S04.

[0016] The low-temperature static drying method provided by the present invention is a mild and effective drying method, which can protect coenzyme Q 10 from being damaged or lost to the greatest extent, and at the same time uses a color-changing indicator to indicate the degree of drying, with good drying effect and visual observation.

[0017] Furthermore, in the ash content detection of S05, the coenzyme Q 10 The ash content of the standard sample should be less than 0.07%; The coenzyme Q 10 standard sample with an ash content greater than 0.07% is purified by an ion exchange column.

[0018] Furthermore, in the solvent residue detection of S05, the coenzyme Q 10 No methanol and ethanol should be detected in the standard sample; For the coenzyme Q 10 standard sample detected with methanol and ethanol, it is processed according to the first liquid chromatography conditions described in S01.

[0019] Furthermore, the stability detection described in S06 includes short-term stability detection, solution stability detection and long-term stability detection, and the detection temperature condition is 25°C; the stability detection is carried out for purity detection according to the second liquid chromatography conditions described in S03, and the quality index for stability investigation is the purity of the coenzyme Q 10 standard sample detected by the second liquid chromatography conditions, and the purity should be not less than 99%; The short-term stability investigation uses a 14-day detection cycle; the long-term stability investigation uses an 18-month (calculated as 30 days) detection cycle; The solution stability investigation involves dissolving the coenzyme Q 10 standard sample in the mobile phase under the second liquid chromatography condition and performing a 48-hour purity detection; For the short-term stability detection, a straight line is used as the first empirical model, and the short-term stability of the coenzyme Q 10 standard sample is predicted by whether the change in the slope is significant. The slope calculation formula for the first empirical model is as follows: , where =99.55%, =6.25; For the long-term stability detection, a straight line is used as the second empirical model, and the long-term stability of the coenzyme Q 10 standard sample is predicted by whether the change in the slope is significant. The slope calculation formula for the second empirical model is as follows: ; where =99.76%, =6; where n is the total number of measurements; i is the measurement number; a slope less than 1% represents a non-significant change.

[0020] Furthermore, in the first empirical model and the second empirical model, if the slope is not significant, it is determined that the sample is stable and can be stored at 25°C.

[0021] The shelf life of the coenzyme Q 10 standard sample is determined through the long-term stability investigation.

[0022] In some preferred embodiments, in the thin-layer chromatography purity analysis of S05, a mixed solution of petroleum ether and acetone is used as the developing agent, and the volume ratio of petroleum ether to acetone is 93:7; iodine is used as the developer, and the developing method is iodine vapor fumigation; Five spotting amounts of 20 μg, 40 μg, 60 μg, 80 μg, and 100 μg are designed; No impurity spots should appear at each spotting amount.

[0023] Beneficial technical effects: (1) The present invention provides a method system for the preparation and quality evaluation of a set of coenzyme Q 10 standard products, providing implementation conditions for meeting the requirements of analysis and detection, quality control management, and development of standard products in related food, drug, and health product fields, as well as for ensuring the accuracy, comparability, and traceability of detection results.

[0024] (2)The method system for the preparation and quality evaluation of the coenzyme Q 10 standards provided by the present invention is very complete, involving multiple preparation and detection method steps. These method steps do not achieve independently, but cooperate with each other before and after to form a method system. For example, the method of the present invention provides two sets of chromatographic conditions, one is gradient elution and the other is isocratic elution. The peak time range of the target substance under the gradient elution condition is longer and can be used for the collection of fractions; the peak time range of the target substance under the isocratic elution condition is short and is used for the quality detection of standard samples, which can improve efficiency and save costs. After refining the coenzyme Q 10 standard sample with the first set of chromatographic conditions, the second set of chromatographic conditions is subsequently used for purity detection and stability detection, and the detection results are accurate, reliable and efficient. Another example is that the present invention proposes a vacuum low-temperature static drying method to control moisture. In the later quality detection of standard samples, this method is still used to control the moisture of the water-absorbed standard samples.

[0025] (3)The method of the present invention also verifies other detection items, such as thin-layer chromatography purity analysis, selection of chromatographic columns, etc., which can improve the efficiency and accuracy of the entire method system. In addition, the present invention also uses a straight line as an empirical model to detect the stability of the coenzyme Q 10 standard sample, making the stability investigation of the coenzyme Q 10 standard sample more scientific and objective.

[0026] (4)The quality evaluation method system provided by the present invention includes multiple detection items and reference to the standard sample structure. Therefore, the method system of the present invention also provides multiple method options and references for detection units and scientific research institutions at different levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts do not necessarily draw according to the actual proportion. Obviously, the following-described drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without creative efforts.

[0028] Figure 1 For the chromatogram of separation and purity analysis of the coenzyme Q 10 sample in one of the embodiments of the present invention; Figure 2 For the thin-layer chromatogram with petroleum ether: acetone (93:7, v / v) as the developing agent in one of the embodiments of the present invention; Figure 3The thin layer chromatogram with petroleum ether: ethyl acetate (90:10, v / v) as the developing agent in one embodiment of the present invention; Figure 4 The thin layer chromatogram with n-hexane: ethyl acetate (85:15, v / v) as the developing agent in one embodiment of the present invention; Figure 5 The HPLC chromatogram of coenzyme Q 10 standard sample analyzed using different chromatographic columns in one embodiment of the present invention; Figure 6 The purity chromatogram of coenzyme Q 10 in one embodiment of the present invention; Figure 7 The three-dimensional full-spectrum scan chromatogram of coenzyme Q 10 in one embodiment of the present invention; Figure 8 The TGA curve of water content determination of coenzyme Q 10 in one embodiment of the present invention; Figure 9 The TGA curve of ash content determination of coenzyme Q 10 in one embodiment of the present invention; Figure 10 The ultraviolet spectrum of coenzyme Q 10 standard sample in one embodiment of the present invention; Figure 11 The infrared spectrum of coenzyme Q 10 standard sample in one embodiment of the present invention; Figure 12 The mass spectrum of coenzyme Q 10 standard sample in negative ion mode in one embodiment of the present invention; Figure 13 The solution stability test results of coenzyme Q 10 standard sample in one embodiment of the present invention; Figure 14 The results of three parallel samples measured at the 0th month and the 18th month of the stability test of coenzyme Q 10 standard sample in one embodiment of the present invention; Figure 15 The trend line of the 0 - 18-month stability test of coenzyme Q 10 standard sample in one embodiment of the present invention; Detailed implementation manners

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0030] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0031] As used herein, "a plurality of" means two or more, that is, it includes two, three, four, five, etc.

[0032] As used in this specification, the term "about" typically represents + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0033] In this specification, certain embodiments may be disclosed in a format within a certain range. It should be understood that this description of "within a certain range" is only for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as the individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0034] Example 1 This example provides a method for the preparation and quality evaluation of a coenzyme Q 10 standard sample.

[0035] 1. Coenzyme Q 10 Standard sample preparation and purity determination 1.1 Main equipment and instruments: Dalian Elite iChrom W5100 high performance liquid chromatograph, freeze dryer (Christ Company, Germany) Materials and reagents: Methanol and ethanol for high performance liquid chromatography are of chromatographic purity, water is ultrapure water, and the remaining reagents are of analytical purity.

[0036] 1.2 Method steps Weigh an appropriate amount of the purchased coenzyme Q 10 sample, dissolve it with the mobile phase, and then refine it under the preparative liquid chromatography conditions as follows: Chromatographic column: Agilent ZORBAX SB-C 18 chromatographic column (4.6 mm × 250 mm, 5 μm); Mobile phase elution program: 0 - 30 min, 90% - 10% methanol; 30 - 35 min, 10% methanol; 35.1 - 45 min, 10% - 90% methanol. Column temperature: 35°C; Detection wavelength: 275 nm; Flow rate: 1.0 mL / min; Elution time: 45 min, collect the fractions at 15 - 18 min, see Figure 1 (a).

[0037] Freeze-dry and store the collected fractions.

[0038] Analyze the purity of the enriched fractions.

[0039] Chromatographic column: Agilent ZORBAX SB-C 18 chromatographic column (4.6 mm × 250 mm, 5 μm); Mobile phase: methanol / ethanol = 25 / 75 (v / v); Injection volume: 10 μL; Column temperature: 35°C; Detection wavelength: 275 nm; Flow rate: 1.0 mL / min; Elution time: 15 min.

[0040] The measured chromatogram is shown in Figure 1 (b). As can be seen from the figure, there are no obvious impurity peaks. After deducting the solvent peak, the purity of the prepared coenzyme Q 10 standard sample is determined, and the result is 99.57%, which can meet the requirements.

[0041] 2. Moisture determination Coenzyme Q 10 The standard sample is prone to absorbing water. After preliminary determination, its moisture content > 0.5%, which does not meet the requirements. The moisture content of the coenzyme Q 10 standard sample should be controlled below 0.1%.

[0042] Dry the coenzyme Q 10 sample, and the method steps are as follows: Place the coenzyme Q 10 sample in a vacuum dryer containing anhydrous calcium chloride and silica gel (mass ratio 5:1), and place the vacuum dryer in a -20°C refrigerator. For this coenzyme Q 10The sample was subjected to low-temperature static drying for 1 month. After 1 month, the moisture content of the sample was detected, and the result showed 0.12%. The vacuum dryer containing the sample was continuously placed in a -20°C refrigerator for low-temperature static drying for 1 month. The moisture content of the sample was detected again. When the moisture content remained unchanged in two consecutive determinations, it was coenzyme Q 10 The final content of the sample moisture, the result was about 0.084%, meeting the requirements.

[0043] Quality inspection was carried out on the coenzyme Q10 standard sample prepared above.

[0044] 3. Purity analysis The purity of the prepared sample was examined by thin-layer chromatography and high-performance liquid chromatography (HPLC).

[0045] 3.1 Instruments and equipment Dalian Elite iChrom W5100 high-performance liquid chromatograph; Shimadzu LC-20AT type high-performance liquid chromatograph: SPD-M20A diode array detector (PDA), SIL-20A automatic sampler, DGU-20As online degasser, CTO-10ASvp column oven and Shimadzu LC-solution workstation; PerkinElmer TGA 8000 thermogravimetric analyzer; Shimadzu Nexis GC-2030 gas chromatograph.

[0046] For thin-layer chromatography, petroleum ether, acetone, ethyl acetate, and n-hexane were of chromatographic purity, for high-performance liquid chromatography, methanol and ethanol were of chromatographic purity, water was ultrapure water, and the rest of the reagents were of analytical purity.

[0047] 3.2 Thin-layer chromatography purity analysis Three developing systems were used for detection. The thin-layer plate was silica gel G plate. Five sample application amounts were designed, namely 20 μg (spot 1), 40 μg (spot 2), 60 μg (spot 3), 80 μg (spot 4), and 100 μg (spot 5).

[0048] Preparation of the test solution: Precisely weigh 20 mg of the sample, dissolve it in 100 mL of anhydrous ethanol, shake well, filter, and take the subsequent filtrate to obtain a 0.20 mg / mL reference solution.

[0049] (1) System I Thin-layer plate: Merck HPTLC silica gel 60 prefabricated plate Developing agent: Petroleum ether: Acetone (93:7, v / v) Developer: I2 Developing method: Fumigation with iodine vapor Rf value: 0.31 Conclusion: No impurity spots were observed at each quantity. See Figure 2 .

[0050] (2) System II TLC plate: Pre-coated HPTLC silica gel 60 plate from Merck Developing solvent: Petroleum ether: Ethyl acetate (90:10, v / v) Developer: I2 Development method: Fumigation with iodine vapor Rf value: 0.51 Conclusion: No impurity spots were observed at each quantity. See Figure 3 .

[0051] (3) System III TLC plate: Pre-coated HPTLC silica gel 60 plate from Merck Developing solvent: n-Hexane: Ethyl acetate (85:15, v / v) Developer: I2 Development method: Fumigation with iodine vapor Rf value: 0.60 Conclusion: No impurity spots were observed at each quantity. See Figure 4 .

[0052] From the high-performance thin-layer chromatograms obtained with 3 different developing solvents, it can be seen that for the coenzyme Q 10 sample, in the spotting amount range of 20 - 100 μg, only one obvious colored spot was observed, and no impurity spots were seen, indicating that the purity of this sample is relatively high. However, overall in terms of thin-layer chromatography, the best coloring effect was obtained when using petroleum ether: acetone (93:7, v / v) as the developing solvent.

[0053] 3.3 HPLC purity analysis Different chromatographic column conditions Using the iChrom W5100 high-performance liquid chromatograph from Dalian Elite, the liquid chromatography conditions are as follows: Chromatographic column: Elite Supersil ODS2 chromatographic column (4.6 mm × 250 mm, 5 μm) Agilent ZORBAX SB-C 18 chromatographic column (4.6 mm × 250 mm, 5 μm) Mobile phase: Methanol / ethanol = 25 / 75 (v / v), both methanol and ethanol are chromatographic grade reagents; Injection volume: 10 μL; Column temperature: 35 °C; Detection wavelength: 275 nm; Flow rate: 1.0 mL / min; Elution time: 35 min.

[0054] Weigh 10.0 mg of the sample, a total of 6 portions, add mobile phase, ultrasonically dissolve for 10 min, and make up the volume to 10 mL in a brown volumetric flask, filter through a 0.22 μm filter membrane to obtain a 1.0 mg / mL sample solution. Precisely pipette 10 μL for injection, and use chromatographic columns of different brands and models to measure coenzyme Q 10 standard samples, and their chromatograms are shown in Figure 5 (a) and Figure 5 (b). Among them, Figure 5 (a) is the HPLC chromatogram of coenzyme Q 18 standard sample measured using an Agilent ZORBAX SB-C 10 chromatographic column; Figure 5 (b) is the HPLC chromatogram of coenzyme Q 10 standard sample measured using an Elite Supersil ODS2 chromatographic column.

[0055] The results show that the retention time of coenzyme Q 10 measured under the Agilent chromatographic column is about 10 min, and the retention time of coenzyme Q 10 under the Elite chromatographic column is about 15 min. Considering the time cost of HPLC determination, it is recommended to use an Agilent ZORBAX SB-C 18 chromatographic column. At the same time, the purity of coenzyme Q 10 standard sample was determined under the conditions of the two chromatographic columns respectively, and the purities of coenzyme Q 10 were 99.53% and 99.55% respectively, meeting the requirements.

[0056] 3.4 Peak purity analysis 3.4.1 Impurity detection The purity test of the chromatographic peak of coenzyme Q 10 was carried out by HPLC. No impurities were detected by HPLC, the peak purity index was 1.000000, and the single-point threshold: 0.999762, indicating that the chromatographic peak at 10.845 min has no impurities and is a single substance. The results are shown in Figure 6 .

[0057] 3.4.2 Three-dimensional full-spectrum scan Using the isocratic elution condition experimental conditions in 3.2.2, HPLC-DAD determination was carried out on coenzyme Q 10 to obtain the three-dimensional full-spectrum scan (200 - 800 nm) of the coenzyme Q 10 sample. The results show that no obvious impurity peaks were detected, and coenzyme Q10 The purity value is 99.54%, meeting the requirements. The results are shown in Figure 7 .

[0058] 3.5 Moisture determination The moisture content of the standard sample was determined using a thermogravimetric analyzer TGA 8000 under an air atmosphere. Heating rate: 30 °C / min, set temperature: rising from 40 °C to 800 °C, holding time 5 min, parallel determination 3 times. The measured moisture value of coenzyme Q 10 is 0.084%. The results are shown in Table 1, and the TGA curve is shown in Figure 8 .

[0059] Table 1 Determination results of coenzyme Q 10 moisture content 3.6 Ash determination The ash content of the standard sample was determined using a thermogravimetric analyzer TGA 8000 under an air atmosphere. Heating rate: 30 °C / min, set temperature: rising from 40 °C to 800 °C, holding time 5 min, parallel determination 3 times. The measured ash value of coenzyme Q 10 is 0.062%. The results are shown in Table 2, and the TGA curve is as shown in Figure 9 .

[0060] Table 2 Determination results of coenzyme Q 10 ash content 3.7 Solvent residue detection In this experiment, headspace injection gas chromatography was used, with n-propanol as the internal standard. The residual amounts of methanol and ethanol solvents in this sample were detected according to the internal standard single-point calibration method. The experimental conditions were as follows: Instrument: Shimadzu Nexis GC-2030, headspace injection; Chromatographic column: SHIMADZU Rtx-624 (30.0 m * 0.32 mm * 1.80 μm); Front inlet temperature: 200 °C; Detector temperature: 220 °C; Injection volume: 1 mL; Split ratio: 10:1; Chromatographic column flow rate: 2.0 mL / min; Temperature program: Hold at 40 °C for 5 min, rise to 180 °C at a rate of 15 °C / min, and hold for 2 min; Internal standard: n-propanol.

[0061] The results showed that no methanol and ethanol solvent residues were detected in this sample.

[0062] 4. Structure confirmation In this laboratory, ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, and nuclear magnetic resonance were used to confirm the structure of the prepared solanesol standard sample. In practice, any of the above methods can be used to confirm the structure of the sample to be detected.

[0063] 4.1 Ultraviolet Spectroscopy Detection For the coenzyme Q 10 standard sample, ultraviolet spectroscopy scanning was carried out, and its maximum absorption value was λ = 275 nm, as shown in Figure 10 .

[0064] 4.2 Infrared Spectroscopy Detection For the coenzyme Q 10 standard sample, infrared spectroscopy detection was carried out using the KBr tablet pressing method. The coenzyme Q 10 standard sample has -CH3, -CH2-, -CH3O-, C=C, C=O, as shown in Figure 11 .

[0065] 4.3 Mass Spectrometry Analysis For the coenzyme Q 10 standard sample, mass spectrometry analysis was carried out. The exact mass number of the coenzyme Q 10 minus hydrogen ion measured in the negative ion scanning mode of the mass spectrometry ESI source was 862.6835, and the speculated molecular formula was C 59 H 90 O4, as shown in Figure 12 .

[0066] 4.4 Nuclear Magnetic Resonance Analysis For the coenzyme Q 10 standard sample, nuclear magnetic resonance analysis was carried out. The sample to be detected was dissolved in deuterated chloroform (CDCl3), and tetramethylsilane (TMS) was used as the internal standard. The 1 H, 13 C, COSY, HSQC, and HMBC spectra were measured respectively, and the data were assigned. Compared with the literature reports, they were basically consistent, as shown in Table 3.

[0067] Table 3 Assignment Table of Nuclear Magnetic Resonance Data of Coenzyme Q 10 Standard Sample Example 2 This example provides another set of quality detection of coenzyme Q 10 standard sample with higher requirements. The indicators of the quality detection include homogeneity detection, stability detection, and value determination analysis.

[0068] First, about 10.0 g of coenzyme Q prepared 10The reference standard samples are aliquoted into 2-mL brown sample bottles. The aliquoted samples are placed in a vacuum desiccator containing anhydrous calcium chloride and silica gel for color change, evacuated, and then stored in a refrigerator at -20 °C.

[0069] 1. Homogeneity test 1.1 Test method Analytical instrument: Dalian Elite iChrom W5100 high-performance liquid chromatograph. Analytical conditions: Agilent ZORBAX SB-C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm); mobile phase: 25% methanol + 75% absolute ethanol; flow rate: 1.0 mL / min; column temperature: 35 °C; running time: 15 min; injection volume: 10 μL; detection wavelength: 275 nm. The purity of the reference standard sample is determined by HPLC area normalization method.

[0070] Weigh 10.0 mg of the sample, dissolve it by ultrasonic treatment with the mobile phase for 10 min, and make up the volume to 10 mL in a volumetric flask. Filter through a 0.22-μm filter membrane to obtain a 1.0-mg / mL sample solution.

[0071] Under the above liquid chromatography conditions, first measure the chromatographic peaks of the solvent used, and then measure the chromatographic peaks of the sample solution. Quantitative analysis is carried out on the sample chromatographic peaks after subtracting the solvent chromatographic peaks by area normalization method.

[0072] 1.2 Data acquisition The method of random order repeated measurement is adopted. Randomly select 12 bottles of samples from the aliquoted samples. Weigh 3 portions of 1 mg of the sample from each bottle according to three procedures respectively. Dissolve each portion of the sample with 1 mL of the mobile phase and perform HPLC analysis. Read the purity value of the sample as the area percentage.

[0073] 1.3 Data processing 1.3.1 Trend analysis Take the average value of the content in each bottle as a function of the bottle serial number to study the trend in sample preparation and the logical relationship in the aliquoting process. The results show that the preparation trend of the sample is stable, the aliquoting is uniform, and the data is stable.

[0074] 1.3.2 Variance analysis Using F test to determine whether the homogeneity data of coenzyme Q 10 reference standard sample conforms to the normal distribution. The results are shown in Table 4.

[0075] Table 4 Coenzyme Q 10 Analysis of variance table for homogeneity study As can be seen from the above table, with υ1 (i.e., between groups) = 11 and υ2 (i.e., within groups) = 24, referring to the F critical value table, we get F 0.05 (11, 24) = 2.22. Since F = MS 瓶间 / MS 瓶内 = 0.4848 < F 0.05 (11, 24), so this sample is homogeneous. Therefore, the minimum sampling amount of this sample is determined to be 1.0 mg.

[0076] Since MS 瓶间 < MS 瓶内 , thus .

[0077] 2. Stability detection Stability investigation analysis conditions: Shimadzu LC - 20AT type high - performance liquid chromatograph, Agilent ZORBAX SB - C 18 chromatographic column (4.6 mm × 250 mm, 5 μm); mobile phase: 25% methanol + 75% absolute ethanol; flow rate: 1.0 mL / min; column temperature: 35 °C; running time: 15 min; injection volume: 10 μL; detection wavelength: 275 nm. The purity of the standard sample was determined by HPLC area normalization method.

[0078] 2.1 Short - term stability Considering that the melting point temperature of coenzyme Q 10 is 48.7 - 49.7 °C and the stability of the sample cannot be investigated at a higher temperature, so in this experiment, the short - term stability of coenzyme Q 10 standard sample at room temperature (25 °C) was investigated. Three bottles of samples were randomly selected from the aliquoted samples and placed in an environment of 25 °C. In this study, with a period of 14 days, the coenzyme Q 10 standard sample was tested on the 1st, 3rd, 7th, and 14th days respectively. The purity of each test sample was the average value of two determinations.

[0079] Table 5 Test results of short - term stability Using a straight line as the first empirical model, observe whether there is a significant change in the slope value to predict the stability change of the standard sample. The slope can be calculated by the following formula.

[0080] , where = 99.55%, = 6.25, The intercept is calculated by the following formula: The standard deviation of the points on the straight line can be calculated by the following formula: ; The uncertainty related to the slope is calculated by the following formula: .

[0081] Where n = total number of measurements; i = measurement number; a slope less than 1% indicates insignificant change.

[0082] The degrees of freedom are n -2 and p = 0.95 (95% confidence level) distribution t The factor is equal to 4.30. Since , the slope is insignificant, and thus the instability of the sample is not observed. It shows that coenzyme Q 10 The standard sample can be stored short-term at 25 °C.

[0083] 2.2. Investigation of solution stability The stability of coenzyme Q 10 standard sample solution at 25 °C was investigated, and the mobile phase was selected as the storage solvent.

[0084] The results are shown in Figure 13 , and coenzyme Q 10 has good solution stability during storage in the mobile phase within 48 h.

[0085] 2.3. Investigation of long-term stability This sample was taken, simulated the commercial packaging, and placed at -20 °C for 1.5 years. At 0, 1, 2, 3, 6, 12, and 18 months after packaging the samples, 3 samples were taken each time, and their purities were determined by high performance liquid chromatography. The HPLC stability determination spectra at 0 month and 18 months are shown in Figure 14 and Figure 15 .

[0086] So far, 18 months have been measured. The results are shown in Table 6. Trend analysis was performed using the average content per bottle as a function of the bottle number to study the trend of the sample during storage.

[0087] Table 6 Results of coenzyme Q 10 stability test Using the linear model as the second empirical model, t test was used to analyze the data obtained for stability. The calculation process is as follows: Slope: ; In the formula: = 99.76%, = 6; The intercept is calculated by the following formula: ; The standard deviation of the points on the straight line can be calculated by the following formula: ; Take its square root s = 0.06554%, and the uncertainty related to the slope is calculated by the following formula: .

[0088] n = total number of measurements; i = number of measurements; a slope less than 1% represents insignificant change.

[0089] The degrees of freedom are n - 2 and P = 0.95 (95% confidence level) of the t factor is equal to 2.57, ; Therefore, the slope is insignificant and no instability is observed.

[0090] .

[0091] Coenzyme Q 10 No obvious instability was observed in the standard sample within 18 months. The validity period of the coenzyme Q 10 standard sample is tentatively set at 18 months, and a longer stability period will be determined according to continuous test results.

[0092] 3. Characterization Characterization refers to the determination of the characteristic values or characteristic attributes of the standard sample, which is part of the production process that generates a standard sample certificate or corresponding documents. The standard sample characterization is generally completed by technically valid and rigorously recognized methods. This standard sample is characterized by collaborative tests in multiple laboratories in accordance with the requirements of GB / T15000.3 - 2008 standard. Select laboratories that are recognized by the state or department, have qualifications, or are national standard sample characterization laboratories inspected by the Natural Products Standard Sample Working Group, or laboratories with long - term experience in natural products for sample characterization.

[0093] It should be noted that in this article, the term "including", "comprising" or any other variant thereof is intended to cover non - exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "including an..." does not exclude the existence of additional identical elements in the process, method, article or device including that element.

[0094] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit of the present invention and the scope protected by the claims. All of these are within the protection scope of the present invention.

Claims

1. A coenzyme Q 10 The method for preparing and evaluating the quality of a standard sample is characterized in that: The method comprises the following steps: S01: Using the first liquid chromatography condition to analyze the commercially available coenzyme Q 10 The sample is purified, and the first liquid chromatography condition is gradient elution, which is as follows: The chromatographic column was C18 column; the mobile phase was methanol; The mobile phase program was: 0-30 min, 90%-10% methanol; 30-35 min, 10% methanol; 35.1-45 min, 10%-90% methanol; Collect the eluted fractions, set the collection time to t1-t2; the t1-t2 is 15-18min, and the fraction is coenzyme Q 10 Purified solution; S02: The coenzyme Q 10 The purified solution was freeze-dried and stored; S03: Using the second liquid chromatography condition to 10 The purified solution was subjected to purity analysis. The second liquid chromatography condition was isocratic elution. The purity of the eluate within the elution time range of t1'-t2' was detected. Samples with a purity greater than 99% were determined to be coenzyme Q 10 Standard sample; the duration of t1'-t2' is less than the duration of t1-t2; S04: The obtained coenzyme Q 10 The standard sample was tested for moisture content, and the coenzyme Q 10 The water content of the standard sample should not be greater than 0.1%; For coenzyme Q with water content greater than 0.1% 10 The standard sample was vacuum dried at low temperature until the water content was less than 0.1%; S05: The coenzyme Q 10 The standard sample is subjected to a first-level quality test, wherein the indicators of the first-level quality test include one or more of thin layer chromatography purity analysis, HPLC purity analysis, peak purity analysis, moisture detection, ash detection, solvent residue detection and / or structure confirmation; S06: The coenzyme Q 10 The standard sample undergoes a second-level quality inspection, and the indicators of the second-level quality inspection include one or more of uniformity inspection, stability inspection and / or fixed value analysis.

2. The method according to claim 1, characterized in that The chromatographic column of the second liquid chromatography condition is a C18 column; the mobile phase is a mixed solution of methanol and ethanol, and the volume ratio of methanol to ethanol in the mixed solution is 25:75; and the t1'-t2' is 10-11 min.

3. The method according to claim 1, characterized in that The column temperature of the first liquid chromatography condition and the second liquid chromatography condition is 35° C.; the detection wavelength is 275 nm; and the flow rate is 1.0 mL / min.

4. The method according to claim 1 or claim 2, characterized in that The chromatographic columns for the first liquid chromatography condition and the second liquid chromatography condition are ZORBAX SB-C 18 Chromatographic column.

5. The method according to claim 1, characterized in that The vacuum low temperature static drying process is to 10 The sample is placed in a vacuum dryer filled with a desiccant, and the vacuum dryer is placed at -20°C for a drying time of not less than 30 days; the desiccant is a mixture of anhydrous calcium chloride and color-changing silica gel; the color-changing silica gel is used to indicate changes in moisture.

6. The method according to claim 5, characterized in that The mass ratio of the anhydrous calcium chloride to the color-changing silica gel is 2-5:

1.

7. The method according to claim 1, characterized in that In the moisture detection described in S05, the coenzyme Q 10 The water content of the standard sample should be less than 0.1%; For coenzyme Q with water content greater than 0.1% 10 The standard sample was processed according to the low temperature static drying method described in S04.

8. The method according to claim 1, characterized in that In the ash content detection described in S05, the coenzyme Q 10 The ash content of the standard sample should be less than 0.07%; Coenzyme Q 10 The standard samples were purified using ion exchange columns.

9. The method according to claim 1, characterized in that In the solvent residue detection described in S05, the coenzyme Q 10 Methanol and ethanol should not be detected in the standard samples; Coenzyme Q for the detection of methanol and ethanol 10 The standard sample is processed according to the first liquid chromatography condition described in S01.

10. The method according to claim 1, characterized in that The stability test described in S06 includes short-term stability test, solution stability test and long-term stability test, and the test temperature condition is 25°C; the quality index of the stability test is the coenzyme Q detected by the second liquid chromatography condition. 10 Purity of standard samples; The short-term stability test uses the first empirical model to predict the coenzyme Q by whether the change in slope is significant. 10 The short-term stability of the standard sample, the slope calculation formula of the first empirical model is as follows: ,in =99.55%, =6.25; The long-term stability test uses the second empirical model to predict the coenzyme Q by whether the change in slope is significant. 10 The long-term stability of the standard sample, the slope calculation formula of the second empirical model is as follows: ;in =99.76%, =6; Wherein, n=total number of measurements; i=number of measurements; a slope less than 1% indicates an insignificant change.

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