Method for detecting creatinine in creatine product

Abstract

The invention belongs to the technical field of analytical chemistry, and discloses a method for detecting creatinine in a creatine product, which comprises the following steps: (1) precisely weighing a sample, adding a buffer solution, carrying out vortex oscillation dissolution, volume metering, shaking up, centrifuging, and passing through a water phase filter membrane to obtain a creatine test solution; (2) a creatine test solution is taken, a liquid chromatograph is adopted for liquid phase detection, and the liquid chromatography conditions are as follows: an SCX chromatographic column is adopted, an ammonium dihydrogen phosphate solution is taken as a mobile phase for isocratic elution, an ultraviolet detector is adopted, and the content of creatinine is obtained according to a liquid phase detection result. According to the method, the situation that the detection result of the creatinine in the creatine product is virtually high can be remarkably reduced, accurate quantification of the creatinine byproduct creatinine is achieved, and a more perfect data basis is provided for quality control of the creatine product.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, and more specifically relates to a method for detecting creatinine in creatine products. Background Technology

[0002] Creatinine is the main degradation product of creatine, formed through dehydration and cyclization under acidic conditions or during long-term storage. In the pharmaceutical and health supplement industries, creatine is widely used due to its effects on improving athletic performance and treating neuromuscular diseases. Currently, there are clear standards for the quality control of creatine raw materials. For example, the United States Pharmacopeia (USP) explicitly requires that the creatinine content in creatine raw materials be controlled below 1000 ppm. This standard aims to ensure the initial purity and stability of the raw materials, controlling the introduction of major impurities at the source.

[0003] However, existing quality standards have a significant technical gap: they only control raw materials, without specifying clear requirements and testing standards for the creatinine content in the final product. This deficiency leads to a series of technical and regulatory problems, such as the risk of secondary degradation during production, stability risks during storage, and potential risks to efficacy and safety. Furthermore, during testing, some creatine products that dissolve in water and are acidic, such as creatine gummies, will have their sample solutions acidic if pretreated with purified water using conventional methods, which could increase the rate of creatine degradation to creatinine.

[0004] Therefore, there is an urgent need in this field for a solution to accurately monitor the creatinine content in creatine products. This requires developing an efficient, sensitive method for detecting creatinine in complex finished product matrices (such as gummies, tablets containing multiple excipients, powders, or capsules), overcoming the shortcomings of existing quality standards, and achieving quality monitoring throughout the entire lifecycle of the finished product from production to storage. This will ensure that the content and purity of the active ingredient in the final product consistently meet design specifications. This is not only a key technological means to control product quality and ensure product efficacy and safety, but also a necessary innovation to enhance brand reputation, meet potential future regulatory requirements, and drive the advancement of industry quality standards.

[0005] There are few existing literature reports on methods for determining creatinine content in food, which cannot be readily adapted to increasingly complex and diverse product matrices. For example, invention patent CN110596269A uses purified water to dissolve and extract creatine powder samples, and the operation steps are relatively simple. However, creatinine is a degradation product of creatine, and the degradation conditions are low pH, high temperature, and water solvent. Most products require water dissolution during creatine content detection, which leads to a continuous increase in creatinine in the test sample solution, failing to accurately reflect the true creatinine content in the product.

[0006] Therefore, how to provide a method for detecting creatinine in creatine products to eliminate the possibility of falsely high creatinine test results is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the present invention provides a method for detecting creatinine in creatine products.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for detecting creatinine in a creatine product, comprising the following steps:

[0010] (1) Accurately weigh the sample, add buffer solution, vortex to dissolve, make up to volume, shake well, centrifuge, and filter through an aqueous phase membrane to obtain the creatine test solution.

[0011] (2) Take the creatine test solution and perform liquid chromatography detection using a liquid chromatograph. The liquid chromatography conditions are: use an SCX column, use ammonium dihydrogen phosphate solution as the mobile phase for isocratic elution, use an ultraviolet detector, and obtain the creatinine content based on the liquid chromatography detection results.

[0012] Preferably, the buffer salt of the buffer solution in step (1) is selected from at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

[0013] Preferably, the concentration of the buffer solution in step (1) is 0.05 mol / L to 0.1 mol / L.

[0014] Preferably, the pH value of the buffer solution in step (1) is 7-9.

[0015] More preferably, the buffer salt in step (1) is potassium dihydrogen phosphate with a concentration of 0.05 mol / L, and the pH is adjusted to 7.5-8.5 using 5M sodium hydroxide solution.

[0016] Preferably, the vortex oscillation time in step (1) is 5 min to 10 min.

[0017] Preferably, the centrifugation in step (1) is refrigerated centrifugation, with a temperature of 4-10℃ and a rotation speed of 4000-10000 r / min.

[0018] Preferably, the creatine test solution described in step (1) is subjected to liquid phase detection within 2-5 minutes after completion.

[0019] The beneficial effects of the above technical solution are: creatine is easily converted into creatinine in an aqueous environment, so the detection can be performed within 2-5 minutes.

[0020] Preferably, the SCX column in step (2) has the following specifications: ChromCore SCX, 250mm × 4.0mm, 5μm.

[0021] Preferably, the molar concentration of the ammonium dihydrogen phosphate solution in step (2) is 0.15 mol / L-0.25 mol / L, and the pH value is adjusted to 4.0 using phosphoric acid.

[0022] Preferably, the wavelength of the detector in step (2) is 220nm-225nm.

[0023] Preferably, the flow rate of the mobile phase for liquid phase detection in step (2) is 0.6 mL / min to 1.2 mL / min;

[0024] Preferably, the column temperature for liquid phase detection in step (2) is 25-35℃, the running time is 12-30 min, and the injection volume is 5-20 μL.

[0025] As can be seen from the above technical solution, compared with the prior art, the present invention provides a method for detecting creatinine in creatine products, which has the following beneficial effects:

[0026] (1) Creatine readily undergoes an intramolecular condensation reaction in aqueous solution to form a stable cyclic structure called creatinine. This is a spontaneous and irreversible process. This hydrolysis reaction is sensitive to hydrogen ion concentration, and the pH of pure water is easily affected by environmental factors (such as the decrease in pH when dissolving gummies), leading to changes in the reaction rate. This invention uses a buffer solution instead of pure water, which can provide a stable and suitable pH chemical environment, effectively inhibiting the non-enzymatic conversion of creatine to creatinine and significantly improving its stability in aqueous solution.

[0027] (2) During the centrifugation process after sample preparation, high-speed centrifugation generates heat, causing the sample temperature to rise. This invention utilizes refrigerated centrifugation, which significantly reduces the sample temperature, thereby slowing down or "freezing" the conversion of creatine to creatinine. This invention can significantly reduce the occurrence of falsely high creatinine test results in creatine products, achieving accurate quantification of creatine byproducts, and providing more comprehensive data for the quality control of creatine products. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0029] Figure 1This is the standard curve for creatinine standard solution.

[0030] Figure 2 This is a graph showing the linear relationship between peak height and concentration.

[0031] Figure 3 The HPLC chromatogram is for a negative solution.

[0032] Figure 4 This is the HPLC chromatogram of the creatinine reference solution.

[0033] Figure 5 The results show the stability of sample a in Experiment 2 after treatment with different solvents.

[0034] Figure 6 The results show the stability of sample b in Experiment 2 after treatment with different solvents. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Experiment 1

[0037] I. Instruments and Reagents

[0038] 1. Instruments

[0039] 1.1 High performance liquid chromatograph equipped with ultraviolet detector.

[0040] 1.2 Analytical balance: sensitivity of 1 mg and 0.1 mg.

[0041] 1.3 Ultrasonic cleaner.

[0042] 1.4 Centrifuge.

[0043] 2. Reagents

[0044] 2.1 Ammonium dihydrogen phosphate: Superior grade.

[0045] 2.2 Potassium dihydrogen phosphate: Superior grade.

[0046] 2.3 Glacial acetic acid: Superior grade.

[0047] 3. Reagent preparation

[0048] 3.1 pH=8 buffer solution: Take 6.8g of potassium dihydrogen phosphate, add 800ml of water, adjust the pH to 8.0 with 5M sodium hydroxide solution, make up to 1000ml of water, refrigerate, and use the next day.

[0049] 3.2 Mobile phase (v / v): Dissolve 23 g of ammonium dihydrogen phosphate in 1 L of water, and adjust the pH to 4.0 with phosphoric acid. (Isocratic)

[0050] 4. Standards and standard solutions

[0051] 4.1 Standard: Creatinine: Purity ≥ 98.0%.

[0052] 4.2 Preparation of Standard Solutions

[0053] 4.2.1 Weigh 10 mg (accurate to 0.01 mg) of creatinine standard into a 100 mL volumetric flask, add water, shake to disperse evenly, and then dilute to the mark with water. Quantitatively transfer 1 mL and dilute to 10 mL with water. The concentration of the creatinine standard solution is 0.01 mg / mL.

[0054] 5. Analysis Steps

[0055] 5.1 Preparation of sample solution

[0056] Weigh 25-30 creatine gummies, freeze-dry them in liquid nitrogen, and grind them into powder. Accurately weigh 500 mg of the sample powder and place it in a 50 mL volumetric flask. Add 25 mL of pH=8 buffer solution, vortex for 10 min to dissolve the sample, add water to make up to 50 mL, shake well, and centrifuge at 10000 r / min and 4℃ for 2 min. Take the supernatant and filter it through an aqueous phase filter membrane to obtain the creatine test solution. Perform liquid chromatography analysis within 2-5 min after completion.

[0057] 5.2 Instrument Reference Conditions

[0058] 5.2.1 Chromatographic column: Nano-Spectro ChromCore SCX, 250mm×4.0mm, 5μm column.

[0059] 5.2.2 Column temperature: 25℃.

[0060] 5.2.3 Detection wavelength: 225nm.

[0061] 5.2.4 Mobile phase: Dissolve 23g of ammonium dihydrogen phosphate in 1L of water and adjust the pH to 4.0 with phosphoric acid. Perform isocratic elution.

[0062] 5.2.5 Flow rate: 1.0 mL / min.

[0063] 5.2.6 Injection volume: 5 μL; Run time: 12 min.

[0064] 5.3 Sample Determination

[0065] The standard solution in 4.2.1 and the sample solution in 5.1 were determined according to the above chromatographic conditions. The HPLC chromatograms are shown below. Figure 1 .

[0066] 6. Result Calculation

[0067] The creatinine content in the sample is calculated using the following formula:

[0068]

[0069] In the formula:

[0070] X—The creatinine content in the sample, expressed in grams per 100 grams (g / 100g).

[0071] c — the concentration of creatinine in the sample solution being tested, expressed in milligrams per liter (mg / mL).

[0072] V—The final volume of the sample solution to be tested, in milliliters (mL);

[0073] m—Sample mass, expressed in grams (mg).

[0074] 7. Methodological Validation

[0075] 7.1 Linear Range

[0076] 7.1.1 Creatinine standard solution (2.5 mg / mL): Weigh 251.74 mg (accurate to 0.01 mg) of creatinine standard into a 100 mL volumetric flask, add an appropriate amount of water, dissolve it by sonication, and dilute to the mark with water.

[0077] 7.1.2 Pipette 0 mL, 0.05 mL, 0.1 mL, 1.0 mL, 5.0 mL, and 10.0 mL of the above standard solution into 25 mL volumetric flasks, and dilute to the mark with water. Prepare standard curves with creatinine concentrations of 0.000 mg / mL, 0.005 mg / mL, 0.010 mg / mL, 0.100 mg / mL, 0.500 mg / mL, and 1.000 mg / mL.

[0078] The instrument was tested according to the conditions outlined in [5.2. Instrument Reference Conditions]. The correlation between the obtained response values ​​and the concentration fitting data was analyzed. (To meet the requirements for subsequent quantitative analysis using the single-point external standard method, the standard curve fitting was forced to pass through the origin in this verification). The R² value of the standard curve was used as the linearity evaluation within the concentration range of the curve. The results are shown in Table 1, and the standard curve is shown in [Table 1]. Figure 1 .

[0079] Table 1 Linear Range

[0080] Plot a standard working curve with concentration on the x-axis and peak area on the y-axis, as shown below. Figure 1 As shown, the results indicate that the correlation of creatinine is 1.000. Therefore, the method for determining creatinine exhibits good linearity between concentrations of 0000 mg / mL and 1.0070 mg / mL, which meets the requirements of GB / T27404-2008 "Laboratory Quality Control Standard" (GB / T27404-2008 requires a correlation ≥0.99).

[0081] 7.2 Limit of Detection and Limit of Quantification

[0082] 7.2.1 Limit of Detection

[0083] According to GB / T 5009.1-2003, the minimum response value of the instrument is S=3N (N is the instrument noise level). [5.2. Instrument Reference Conditions] Use the lowest concentration standard solution, dilute it until the signal-to-noise ratio meets the requirements, and confirm the minimum response value S.

[0084] The peak height response value S (peak height) for a concentration with a signal-to-noise ratio of 3 times was obtained from the experiment as 0.01422.

[0085] A linear equation was established using the peak height measurements of the 7.1 standard series and the creatinine concentration. The slope of the regression equation for the standard curve is b. Data are shown in Table 2, and the linear relationship graph is shown below. Figure 2 .

[0086] Table 2. Linear equation of peak height-concentration

[0087] The instrument detection limit was calculated from the above data. The instrument detection limit (mg / mL) = S / b, and the result is 1.80 × 10⁻⁶. -5 mg / mL. Therefore, when the sample size is 0.5g and the volume is 50mL, the detection limit of creatinine calculated by the method is 1.80mg / kg.

[0088] 7.2.2 Limit of Quantification

[0089] According to GB / T 5009.1-2003, the minimum response value of the instrument is S=10N (N is the instrument noise level). [6.2. Instrument Reference Conditions] Use the lowest concentration standard solution, dilute it until the signal-to-noise ratio meets the requirements, and confirm the minimum response value S.

[0090] The peak height response value S (peak height) for a concentration with a signal-to-noise ratio of 10 times was obtained from the experiment as 0.02478.

[0091] Creatinine:

[0092] The instrument's limit of quantitation (LOQ) was calculated from the above data. The LQ (mg / mL) = S / b, and the result is 3.14 × 10⁻⁶. -5 mg / mL. Therefore, when the sample size is 0.5g and the volume is 50mL, the limit of quantification for creatinine calculated by the method is 3.14mg / kg.

[0093] Conclusion: Meets acceptance criteria (limit of detection ≤ 3.0 mg / kg; limit of quantitation ≤ 10.0 mg / kg).

[0094] 7.3. Specificity

[0095] Take a negative sample and prepare it according to the sample processing methods in [5.1 Sample Preparation] and [5.2 Instrument Reference Conditions], and then perform the test. Detect whether there is interference in the negative sample. The test results are shown in Table 3 below. The HPLC chromatogram of the negative solution is shown below. Figure 3 As shown. The HPLC chromatogram of the creatinine reference solution is shown below. Figure 4 As shown.

[0096] Table 3 Specificity

[0097] Conclusion: Based on Figure 3-4 It can be seen that the negative sample did not have an absorption peak at the retention time of the creatinine peak in the reference solution, indicating that the sample substrate did not interfere with the measurement results.

[0098] 7.4 Precision

[0099] 7.4.1 Repeatability

[0100] Take 5g of negative sample and accurately weigh 18 well-mixed samples, dividing them into 3 groups of 6 samples each. Add 0.2mL of creatinine standard solution (2.5mg / mL) to each group in group 1 (lv-1), 2.0mL to each group in group 2 (lv-2), and 5.0mL to each group in group 3 (lv-3). Prepare the samples according to the sample processing methods in [5.1 Sample Preparation] and [5.2 Instrument Reference Conditions]. Calculate the RSD of the sample content for each group. The test results are shown in Table 4.

[0101] Table 4 Repeatability

[0102] Calculation formula: Creatinine content (g / 100g) = Measured concentration (mg / mL) × Volume (mL) × Dilution factor / Sample amount (mg) × 100.

[0103] Conclusion: Meets the acceptance criteria (RSD≤2.7%).

[0104] 7.5 Accuracy

[0105] Using the sample test results (measured concentration) under section 7.4 (Precision), calculate the measured spiking amount and compare it with the theoretical spiking amount to obtain the recovery rate. The accuracy of the method is evaluated using the recovery rate results for each sample group. The test results are shown in Table 5.

[0106] Table 5 Accuracy

[0107] Conclusion: Meets acceptance criteria (recovery rate 95%-105%).

[0108] 8. Conclusion

[0109] Conclusion: After testing for detection limit, linear range, specificity, precision, and accuracy, the results met the acceptance criteria. This method is suitable for determining the creatinine content in products containing creatine.

[0110] Experiment 2 Optimization of Sample Extraction Solvent Selection

[0111] Two commercially available creatine powder samples, a and b, were taken. Sample a contained 5g of creatine per 9g, and sample b contained 5g of creatine per 7g. Creatinine content was determined using potassium dihydrogen phosphate buffer (pH=8) and purified water, respectively, as sample extraction solvents. Specific data were compared below. Figure 5 and Figure 6 As shown in the figure, the creatinine content of the sample solution using pure water as a solvent increases significantly with prolonged preparation time, and is significantly higher than that of the sample solution using potassium dihydrogen phosphate buffer as a solvent. This indicates that using a buffer solution instead of pure water as the sample extraction solvent can significantly slow down the conversion rate of creatine to creatinine.

[0112] Further optimization of the sample extraction solvent of this invention was achieved through the following comparative methods:

[0113] Method 1: Use potassium dihydrogen phosphate as the sample extraction solvent, adjust the pH to 8.0 with sodium hydroxide solution, and perform other treatments as in Experiment 1;

[0114] Method 2: Use sodium dihydrogen phosphate-disodium hydrogen phosphate buffer as the sample extraction solvent, adjust the pH to 8.0 with sodium hydroxide solution, and perform other treatments as in Experiment 1;

[0115] Method 3: Use potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer as the sample extraction solvent, adjust the pH to 8.0 with sodium hydroxide solution, and perform other treatments as in Experiment 1;

[0116] Method 4: Use water as the sample extraction solvent, adjust the pH to 8.0 with sodium hydroxide solution, and perform other treatments as in Experiment 1.

[0117] The test results show that:

[0118] Taking creatine gummies as an example, the results of creatinine content determination were as follows: Method 4 > Method 3 > Method 2 > Method 1; Method 1 yielded the lowest content, and the result was set as 1; Method 2's result was 1.1 of the pretreatment method of this invention; Method 3's result was 1.2 of the pretreatment method of this invention; Method 4's result was 10.5 of the pretreatment method of this invention; the data obtained by Methods 1, 2, and 3 were all significantly lower than the result of Method 4. Therefore, buffer solutions prepared using potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate can all be used as extraction solvents for creatine samples.

[0119] Experiment 3 Optimization of pH value for sample extraction solvent

[0120] The pH value of the sample extraction solvent in this invention was selected using the following method for comparison:

[0121] Method 1: Use potassium dihydrogen phosphate as the sample extraction solvent, adjust the pH to 7.0 with sodium hydroxide solution, and perform other treatments as in Experiment 1;

[0122] Method 2: Use potassium dihydrogen phosphate as the sample extraction solvent, adjust the pH to 9.0 with sodium hydroxide solution, and perform other treatments as in Experiment 1;

[0123] Method 3: Use potassium dihydrogen phosphate as the sample extraction solvent, adjust the pH to 4.0 with sodium hydroxide solution, and perform other treatments as in Experiment 1;

[0124] Method 4: Use potassium dihydrogen phosphate as the sample extraction solvent, adjust the pH to 10.0 with sodium hydroxide solution, and perform other treatments as in Experiment 1.

[0125] The test results show that:

[0126] Taking creatine gummies as an example, the results of creatinine content determination are as follows: Method 3 > Method 4 > Method 1 > Method 2; Method 2 yielded the lowest content, and the result is set as 1; the result of Method 1 corresponds to 1.2 of the pretreatment method of this invention; the result of Method 3 corresponds to 4.5 of the pretreatment method of this invention; and the result of Method 4 corresponds to 2.1 of the pretreatment method of this invention. Therefore, the preferred pH value of the sample extraction solvent is 7-9.

[0127] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting creatinine in a creatine product, characterized in that, Includes the following steps: (1) Accurately weigh the sample, add buffer solution, vortex to dissolve, make up to volume, shake well, centrifuge, and filter through an aqueous phase membrane to obtain the creatine test solution. (2) Take the creatine test solution and perform liquid chromatography detection using a liquid chromatograph. The liquid chromatography conditions are: use an SCX column, use ammonium dihydrogen phosphate solution as the mobile phase for isocratic elution, use an ultraviolet detector, and obtain the creatinine content based on the liquid chromatography detection results.

2. The method for detecting creatinine in a creatine product according to claim 1, characterized in that, The buffer salt of the buffer solution in step (1) is selected from at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

3. The method for detecting creatinine in a creatine product according to claim 1, characterized in that, The concentration of the buffer solution in step (1) is 0.05 mol / L-0.1 mol / L.

4. The method for detecting creatinine in a creatine product according to claim 1, characterized in that, The pH value of the buffer solution in step (1) is 7-9.

5. The method for detecting creatinine in a creatine product according to claim 1, characterized in that, The vortex oscillation time in step (1) is 5 min to 10 min.

6. The method for detecting creatinine in a creatine product according to claim 1, characterized in that, The centrifugation in step (1) is refrigerated centrifugation, with a temperature of 4-10℃ and a rotation speed of 4000-10000r / min.

7. The method for detecting creatinine in a creatine product according to claim 1, characterized in that, The molar concentration of the ammonium dihydrogen phosphate solution in step (2) is 0.15 mol / L-0.25 mol / L, and the pH value is adjusted to 4.0 using phosphoric acid.

8. The method for detecting creatinine in a creatine product according to claim 1, characterized in that, The wavelength of the detector in step (2) is 220nm-225nm.

9. The method for detecting creatinine in a creatine product according to claim 1, characterized in that, The flow rate of the mobile phase for liquid phase detection in step (2) is 0.6 mL / min to 1.2 mL / min.

10. The method for detecting creatinine in a creatine product according to claim 1, characterized in that, In step (2), the column temperature for liquid phase detection is 25-35℃, the running time is 12-30 min, and the injection volume is 5-20 μL.

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