Method for detecting content of pyrroloquinoline quinine and / or pyrroloquinoline quinine salt and application

Abstract

The invention discloses a method for detecting the content of pyrroloquinoline quinine and / or pyrroloquinoline quinine salt and application. The provided method for detecting the content of pyrroloquinoline quinine and / or pyrroloquinoline quinine salt comprises the following steps: uniformly mixing a sample to be detected with a disodium hydrogen phosphate aqueous solution to prepare a solution to be detected, the pH of the solution to be detected being neutral; the to-be-detected solution is subjected to high performance liquid chromatography detection, and the conditions of high performance liquid chromatography detection are as follows: a disodium hydrogen phosphate aqueous solution is taken as a mobile phase A, acetonitrile is taken as a mobile phase B, and isocratic elution is carried out. In the provided method for detecting the content of pyrroloquinoline quinone, the pH value of the solution powder to be detected is controlled to be neutral, and the pyrroloquinoline quinone in the sample to be detected can be rapidly, accurately and conveniently detected by adopting a disodium hydrogen phosphate aqueous solution as a mobile phase A and combining conditions such as isocratic elution.

Description

Technical Field

[0001] This application belongs to the field of analytical detection technology and relates to methods and applications for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts. Background Technology

[0002] Pyrroloquinoline quinone (PQQ) is an aromatic ortho-quinone compound, usually existing as its disodium salt. It is readily soluble in water, exhibits good thermal stability, and is pH sensitive. Under weakly acidic to neutral conditions, it readily dissociates into anions and can form adducts with various compounds, including water, alcohols, and amines. PQQ is widely distributed in natural foods such as natto, parsley, green tea, and kiwifruit. It is a natural oxidoreductase coenzyme that participates in biocatalytic reactions, possessing functions such as scavenging free radicals, reducing oxidative damage, and promoting cell growth.

[0003] Pyrroloquinoline quinone, as a novel and potent antioxidant and cellular energy metabolism enhancer, is increasingly widely used in the field of beauty and anti-aging, and is extensively added to various beauty products, such as health supplements and functional food powders. Accurate detection of pyrroloquinoline quinone content is of great significance for improving product quality control, supporting efficacy verification, and optimizing production processes.

[0004] While existing methods for the quantitative detection of pyrroloquinoline quinones (PQQ) and their salts each have their advantages in specificity and sensitivity, they generally suffer from drawbacks such as easy column contamination, high equipment costs, cumbersome methods, slow analysis speed, insufficient reliability of pretreatment, and lack of methods for specific matrices. These limitations restrict the large-scale application of PQQ-related products in quality inspection, health foods, and cosmetics. Therefore, there is an urgent need to develop a robust method that does not require ion-pairing reagents, shortens analysis time, simplifies pretreatment, avoids strong acid mobile phases, and is based on conventional high-performance liquid chromatography combined with a universal detector (such as UV), to improve the practicality, economy, and repeatability of detection. Summary of the Invention

[0005] Therefore, it is necessary to provide a method and application for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts.

[0006] In some embodiments, a method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts is provided, comprising the following steps:

[0007] The sample to be tested is mixed with an aqueous solution of disodium hydrogen phosphate to prepare a test solution, the pH of which is neutral.

[0008] The test solution is subjected to high-performance liquid chromatography (HPLC) detection, and the HPLC detection conditions include:

[0009] Isocratic elution was performed using an aqueous solution of disodium hydrogen phosphate as mobile phase A and acetonitrile as mobile phase B.

[0010] In some embodiments, in the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt, the pH value of the test solution is 6.8 to 7.2.

[0011] In some embodiments, the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt includes high performance liquid chromatography (HPLC) detection conditions comprising: during isocratic elution, a volume ratio of mobile phase A to mobile phase B of 98:2.

[0012] In some embodiments, the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts, wherein the high-performance liquid chromatography (HPLC) detection conditions satisfy one or more of the following conditions:

[0013] (1) The detection wavelength is 248nm~250nm;

[0014] (2) The flow rate is 0.8 mL / min to 1.0 mL / min;

[0015] (3) The injection volume is 5 μL to 20 μL;

[0016] (4) The column temperature is 25℃~35℃;

[0017] (5) The chromatographic column packing material is C18 alkylsilane bonded silica gel;

[0018] (6) The concentration of the sodium hydrogen phosphate aqueous solution is 10 mmol / L to 50 mmol / L;

[0019] (7) The pH value of the sodium hydrogen phosphate aqueous solution is 8.8~9.2.

[0020] In some embodiments, the method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts involves mixing the sample to be tested with an aqueous solution of disodium hydrogen phosphate and then subjecting it to ultrasonic treatment to prepare the test solution.

[0021] In some embodiments, the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts satisfies one or more of the following conditions:

[0022] (1) The ultrasonic treatment time is 2 min to 15 min;

[0023] (2) The power of ultrasonic treatment is 80W~200W;

[0024] (3) The frequency of ultrasonic treatment is 40kHz~50kHz;

[0025] (4) Perform ultrasonic treatment at a temperature of 25°C or less.

[0026] In some embodiments, the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts satisfies one or both of the following conditions:

[0027] (1) The sample to be tested is a dietary supplement and / or functional food;

[0028] (2) The sample to be tested is a powder.

[0029] In some embodiments, the method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt further includes the following steps:

[0030] Take the standard of pyrroloquinoline quinone and prepare standard solutions of different concentrations;

[0031] The standard solutions of different concentrations were subjected to high performance liquid chromatography (HPLC) for detection, and a standard curve was constructed based on the peak area and concentration of the standard solution in the detection results.

[0032] The detection results of the high performance liquid chromatography of the test solution are substituted into the standard curve to calculate the content of pyrroloquinoline quinone in the test solution.

[0033] In some embodiments, the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts involves preparing a stock solution by dissolving a standard of the pyrroloquinoline quinone in an aqueous sodium hydroxide solution, and then diluting the stock solution with an aqueous acetonitrile solution to prepare standard solutions of different concentrations. In some embodiments, the concentration of the aqueous sodium hydroxide solution is 10 mM to 15 mM.

[0034] In some embodiments, the method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts is provided for use in the quality control of dietary supplements and / or functional foods.

[0035] The provided method for detecting pyrroloquinoline quinone content controls the pH of the test solution powder to neutral, uses disodium hydrogen phosphate aqueous solution as mobile phase A, and combines isocratic elution and other conditions to rapidly, accurately, and conveniently detect pyrroloquinoline quinone in the test sample. This method eliminates the need for ion-pairing reagents, shortens analysis time, simplifies pretreatment, avoids strong acid mobile phases, and is a robust method based on conventional high-performance liquid chromatography combined with a universal detector (such as UV), improving the practicality, economy, and repeatability of the detection. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0037] Figure 1 This is a full-wavelength scan result of the pyrroloquinoline quinone standard solution;

[0038] Figure 2 Optimize the chromatogram for isocratic elution of pyrroloquinoline quinone;

[0039] Figure 3 Solutions for dissolving pyrroloquinoline quinone standards in different solvents;

[0040] Figure 4 Peak areas of pyrroloquinoline quinone dissolved in different solvents;

[0041] Figure 5 The full-wavelength UV absorption scan results of PQQ dissolved in four solvents: ultrapure water, methanol-water (1:4), sodium hydroxide (10mM), and acetonitrile-water (1:3);

[0042] Figure 6 The peak areas of pyrroloquinoline quinone under different pH conditions;

[0043] Figure 7 The liquid chromatogram is for pyrroloquinoline quinone standards;

[0044] Figure 8 The above are the liquid chromatograms of samples A, B, and C from Example 2. Detailed Implementation

[0045] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0046] It should be noted that when a component is said to be "fixed to" another component, it can be directly attached to the other component or there may be an intervening component. When a component is said to be "connected to" another component, it can be directly connected to the other component or there may be an intervening component.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0048] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0049] The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and "a combination of A and B."

[0050] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0051] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0052] In this application, the term "suitable" as used in "suitable combination", "suitable method", "any suitable method", etc., refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0053] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0054] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0055] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0056] In this invention, the terms "first aspect," "second aspect," "third aspect," and "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," and "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0057] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0058] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0059] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0060] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0061] In this application, "room temperature" generally refers to 5℃~30℃, and more preferably 25±5℃.

[0062] In this application, "methanol-water (1:4)" in the context refers to an aqueous methanol solution prepared with a volume ratio of methanol to water of 1:4; "acetonitrile-water (1:3)" refers to an aqueous acetonitrile solution prepared with a volume ratio of acetonitrile to water of 1:3.

[0063] In existing technologies, quantitative detection methods for pyrroloquinoline quinones (PQQ) and their salts generally face a prominent contradiction between the need for high performance and practicality and economy. The essence of this contradiction is not the performance target itself, but rather the side effects of the technical means employed to achieve these targets. First, most chromatographic methods (such as various HPLC-UV / FLD, LC-MS / MS, and UPLC-MS / MS methods) generally rely on ion-pairing reagents such as tetrabutylammonium bromide and dibutylammonium acetate to improve the retention of the highly polar PQQ. For example, a liquid chromatography-tandem mass spectrometry method for the detection of pyrroloquinoline quinones in plasma (CN113030343A; Pan et al., 2025; Sun et al., 2022; Kato et al., 2018; Fukuda et al., 2017) is mentioned. While these reagents are effective, they are easily and irreversibly adsorbed into the chromatographic column and system flow path, leading to a rapid decline in column efficiency, increased risk of system contamination, and potential interference with mass spectrometry detection, severely affecting the long-term stability of the method and column life.

[0064] Secondly, high-sensitivity technical approaches are often accompanied by high equipment investment and complex operations. For example, pressurized capillary electrochromatography (pCEC) (Yang Jiewei 2021) and LC-MS / MS systems (Yang et al., 2015) are not only expensive to purchase and maintain, but also have demanding technical requirements for operators, making them difficult to popularize in routine quality inspection laboratories. On the other hand, GC-MS / MS-based methods require cumbersome derivatization steps. Although there were successful cases of silanization in the early days, most attempts failed due to the special physical properties of PQQ (Mostafa et al., 2025).

[0065] Furthermore, existing methods have significant shortcomings in terms of analytical efficiency and simplicity: some methods have excessively long analysis cycles, generally exceeding 20 minutes, such as the patented detection time of 40 minutes (a method for simultaneous detection of β-NMN and PQQ in health foods or cosmetics, CN118883781A), or require multi-step gradient elution to balance the time consumption; to cope with complex matrices (such as food and plasma), many methods have to adopt time-consuming and labor-intensive sample pretreatment steps such as solid-phase extraction, which reduces throughput and increases the risk of operational errors (Dai Kun et al. 2025); at the same time, the mobile phase of some methods requires precise preparation of buffer salts and pH control, which further increases the difficulty of method development and daily maintenance (a method for the detection of disodium pyrroloquinoline quinone in milk beverages, CN117471005A). For example, Fukuda et al. (2017) used a mixture of Tris-HNO3 buffer (pH 8.8, 50 mM) containing tetrabutylammonium bromide (4.0 mM) and acetonitrile (7:3) for the detection of PQQ by chemiluminescence immunoassay (HPLC-CL); Wei Jingyuan et al. (2015) used methanol-water (with the pH adjusted to 1.0 by trifluoroacetic acid) for gradient elution to detect PQQ. This strong acid condition will accelerate the dissolution of conventional silica matrix reversed-phase chromatography columns (such as C18) and the hydrolysis of the bonded phase, leading to problems such as irreversible decrease in column efficiency and retention time drift. Using acid-resistant chromatography columns will increase costs.

[0066] Meanwhile, in the more fundamental dissolution stage, the selection of solvents lacks systematic research. Although it is known that alkaline solutions (such as sodium hydroxide) can be used for dissolution, most methods still use conventional solvents such as methanol, acetonitrile, or water, without fully considering the solubility characteristics, stability, and potential speciation issues of PQQ in different solvents. This can easily lead to inaccurate standard solution concentrations and poor linearity of calibration curves, thus introducing quantitative errors. More importantly, current research on the detection of PQQ mainly focuses on fermentation broth, blood, or beverages. For cosmetic powders containing multiple interfering substances such as proteins, sugars, vitamins, or natural extracts, and with complex compositions, there is still a lack of specific detection methods.

[0067] In summary, while existing technologies each have their advantages in specificity and sensitivity, they generally suffer from drawbacks such as easy column contamination, high equipment costs, cumbersome methods, slow analysis speed, insufficient pretreatment reliability, and a lack of methods for specific matrices. These limitations restrict the large-scale application of PQQ-related products in quality inspection, health food, and cosmetics. Therefore, there is an urgent need to develop a robust method that requires no ion-pairing reagents, shortens analysis time, simplifies pretreatment, avoids strong acid mobile phases, and is based on conventional high-performance liquid chromatography combined with a universal detector (such as UV), in order to improve the practicality, economy, and repeatability of detection.

[0068] Therefore, this application is filed.

[0069] In some embodiments, a method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts is provided, comprising the following steps:

[0070] The sample to be tested was mixed with an aqueous solution of disodium hydrogen phosphate to prepare a test solution with a neutral pH.

[0071] The conditions for high-performance liquid chromatography (HPLC) detection of the test solution include:

[0072] Isocratic elution was performed using an aqueous solution of disodium hydrogen phosphate as mobile phase A and acetonitrile as mobile phase B.

[0073] The provided method for detecting pyrroloquinoline quinone content involves simple sample preparation, eliminating the need for complex and costly purification processes, and enabling rapid and accurate detection.

[0074] Compared with gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and liquid chromatography-fluorescence derivatization methods for the determination of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt content, the method provided in this application is more versatile, lower in cost, and simpler and easier to operate.

[0075] The provided detection method uses disodium hydrogen phosphate aqueous solution for elution, which does not require the addition of ion-pairing reagents to increase the retention time of the analyte. Compared with the current detection methods that use complex salt solutions and organic phase ratios for gradient elution, the baseline is more stable, the required time is shorter, and the modification effect of ion-pairing reagents on the chromatographic column is avoided, thus avoiding a reduction in the column life. It also eliminates the need for complex and costly purification treatments, achieving rapid and accurate detection.

[0076] Maintaining the test solution in a neutral state reduces the likelihood of PQQ complexing with other compounds under acidic conditions, thus improving detection accuracy. The provided detection method is time-efficient and highly effective.

[0077] In some embodiments, in the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt, the pH value of the test solution is 6.8 to 7.2. For example, the pH value of the test solution can be 6.8, 6.9, 7.0, 7.1, 7.2, etc., or it can be a range composed of any two of the aforementioned values.

[0078] In some embodiments, the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt includes high performance liquid chromatography (HPLC) detection conditions including a volume ratio of mobile phase A to mobile phase B of 98:2 during isocratic elution.

[0079] In some embodiments, the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt includes high-performance liquid chromatography (HPLC) detection conditions of a detection wavelength of 248 nm to 250 nm.

[0080] In some embodiments, the high-performance liquid chromatography (HPLC) detection conditions provided for the method of detecting pyrroloquinoline quinone and / or pyrroloquinoline quinone salts include a flow rate of 0.8 mL / min to 1.0 mL / min.

[0081] In some embodiments, the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt includes high performance liquid chromatography (HPLC) detection conditions including an injection volume of 5 μL to 20 μL.

[0082] In some embodiments, the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt includes high-performance liquid chromatography (HPLC) detection conditions including a column temperature of 25°C to 35°C.

[0083] In some embodiments, the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts includes high-performance liquid chromatography (HPLC) detection conditions comprising: the column packing material being C18 alkylsilane-bonded silica gel.

[0084] In some embodiments, the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt includes high performance liquid chromatography (HPLC) detection conditions including: the concentration of disodium hydrogen phosphate aqueous solution is 10 mmol / L to 50 mmol / L, for example, it can be 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, etc., or it can be a range composed of any two of the aforementioned values.

[0085] In some embodiments, the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt includes high performance liquid chromatography (HPLC) detection conditions including: a pH value of 8.8 to 9.2 for an aqueous solution of disodium hydrogen phosphate, for example, the pH value of mobile phase A can be 8.8, 8.9, 9, 9.1, 9.2, etc., or a range of any two of the aforementioned values.

[0086] In some embodiments, the method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts involves mixing the sample to be tested with an aqueous solution of disodium hydrogen phosphate and then subjecting it to ultrasonic treatment to prepare the test solution.

[0087] In some embodiments, the ultrasonic treatment time in the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt is 2 min to 15 min.

[0088] In some embodiments, the ultrasonic treatment power is 80W to 200W in the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt.

[0089] In some embodiments, the ultrasonic treatment frequency is 40 kHz to 50 kHz in the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt.

[0090] In some embodiments, the method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts is provided by ultrasonic treatment at a temperature of less than or equal to 25°C, or optionally, ultrasonic treatment is performed at a temperature of 0°C to 25°C.

[0091] In some embodiments, the method provided for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts uses a dietary supplement and / or functional food as the test sample. For example, the test sample may be one or more of GREEN EVERS Ergothioneine 5-in-1 Skin Rejuvenation Anti-Aging Tablets, FineNutri NAD+ Cell Revitalizing Gold Bottle, feelwell Ergothioneine-27267, and biovoz Ergothioneine Capsules.

[0092] In some embodiments, the sample to be tested is a powder in the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt.

[0093] In some embodiments, the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt further includes the following steps:

[0094] Take the standard of pyrroloquinoline quinone and prepare standard solutions of different concentrations;

[0095] High-performance liquid chromatography was used to detect standard solutions of different concentrations, and a standard curve was constructed based on the peak area and concentration of the standard solution in the detection results.

[0096] Substitute the results of the high-performance liquid chromatography (HPLC) detection of the test solution into the standard curve to calculate the content of pyrroloquinoline quinone in the test solution.

[0097] In some embodiments, the provided method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts involves preparing a stock solution by dissolving a standard of pyrroloquinoline quinone in an aqueous sodium hydroxide solution, and then diluting the stock solution with an aqueous acetonitrile solution to prepare standard solutions of different concentrations. In some embodiments, the concentration of the aqueous sodium hydroxide solution is 10 mM to 15 mM.

[0098] First, sodium hydroxide is used to dissolve the pyrroloquinoline quinone in a reversible state to stabilize it. Then, acetonitrile aqueous solution is used for dissolution, which has the best peak area and high dissolution efficiency. Methanol-water and ultrapure water do not dissolve completely and require long-term ultrasonic treatment. However, the increase in temperature affects the accuracy of the standard.

[0099] In some embodiments, methods for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salts are provided for use in the quality control of dietary supplements and / or functional foods.

[0100] The following are specific embodiments. They are intended to provide a more detailed description of this application to help those skilled in the art and researchers better understand it. The technical conditions described do not constitute any limitation on this application. Any modifications made within the scope of the claims of this application are protected by the claims.

[0101] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods not specifying particular conditions in the examples were performed under conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0102] The reagent sources used in the examples are as follows: Disodium hydrogen phosphate, analytical grade, purchased from Aladdin Shanghai; Acetonitrile, chromatographic grade, purchased from Aladdin Shanghai; PQQ standard (CAS: 72909-34-3, purity ≥98%), purchased from Aladdin Shanghai; Filter membrane (0.45μm) and filter (0.22μm) purchased from Jinteng Experimental Equipment Co., Ltd.; Capsules purchased from Biovoz, product model Biovoz Ergothioneine Capsules, with a PQQ content of 5mg / capsule.

[0103] Example 1

[0104] This embodiment establishes a method for detecting the PQQ content in powder.

[0105] 1. Optimization of chromatographic conditions

[0106] 1.1 Detection Wavelength

[0107] The standard was dissolved in acetonitrile and water at a volume ratio of 1:3, and a full-wavelength scan was performed. The results are as follows: Figure 1 As shown, according to Figure 1 The full-wavelength scan results show that PQQ has strong ultraviolet absorption at 248nm and 331nm. Since the absorption peak response value is higher at 248nm, 250nm was selected as the detection wavelength.

[0108] 1.2 Optimization of the mobile phase

[0109] The standard was dissolved in acetonitrile and water at a volume ratio of 1:3. 50 mM sodium dihydrogen phosphate (pH=5.0) and disodium hydrogen phosphate (pH=8.9) were used as mobile phase A, and acetonitrile as mobile phase B. The flow rate was 1.0 mL / min. Gradient elution was performed: 0–6 min, 0–20%; 6.01–20 min, 0%. The solution was then analyzed.

[0110] Testing revealed that 50mM sodium dihydrogen phosphate could not elute PQQ, and no peak was observed. Therefore, disodium hydrogen phosphate with pH=8.9 was selected as the mobile phase A.

[0111] 1.3 Optimization of the elution gradient

[0112] The optimized mobile phase detection results showed that the baseline drifted in the later stage of gradient elution. Therefore, isocratic elution optimization was performed. The standard was dissolved in acetonitrile and water at a volume ratio of 1:3, and elution was carried out under the following conditions to select a gradient with a suitable peak time:

[0113] (1) 100% mobile phase A, isocratic elution, flow rate of 0.8 mL / min;

[0114] (2) 100% mobile phase A, isocratic elution, flow rate 0.9 mL / min;

[0115] (3) The volume ratio of mobile phase A to mobile phase B is 98:2, isocratic elution is performed, and the flow rate is 1.0 mL / min;

[0116] (4) The volume ratio of mobile phase A to mobile phase B is 98:2, isocratic elution is performed, and the flow rate is 0.8 mL / min.

[0117] Test results as follows Figure 2 As shown, Figure 2 In the diagram, black represents the chromatographic peak of condition (1), pink represents the chromatographic peak of condition (2), blue represents the chromatographic peak of condition (3), and brown represents the chromatographic peak of condition (4).

[0118] from Figure 2 As can be seen, when eluting with 100% mobile phase A salt solution, the chromatographic peak is slightly broad. Then, by increasing the organic phase ratio to 2% and optimizing the flow rate, when the flow rate is 0.8 mL / min, the chromatographic peak is narrow, tall, and symmetrical, and the response value is improved. The PQQ peak elution time is 3.98 min. Therefore, the mobile phase A / B = 98:2, isocratic elution, and flow rate = 0.8 mL / min are selected as the isocratic elution conditions.

[0119] In summary, the optimized HPLC chromatographic conditions were determined as follows: High-performance liquid chromatograph (Shimadzu LC-20AT, Japan); column: InterSustain AQ-C18 250 × 4.6 mm, 5 μm; column temperature: 30℃; mobile phase A: 50 mM disodium hydrogen phosphate solution, mobile phase B: acetonitrile; flow rate: 0.8 mL / min; injection volume: 10 μL; isocratic elution: A:B = 98:2; detection wavelength: 250 nm.

[0120] 2. Selection of solvents for standard products

[0121] (1) When PQQ standard is dissolved in pure methanol, the solution is turbid. Therefore, a new solvent needs to be found.

[0122] (2) PQQ standard was dissolved in tap water, ultrapure water, methanol-water (1:4), sodium hydroxide (10mM), and acetonitrile-water (1:3) respectively to prepare stock solutions, with a concentration of 2.6 mg / mL for each solution. The results are as follows: Figure 3 As shown, except for the fourth solution in the picture, which is yellowish-brown when dissolved in sodium hydroxide, the other four solvents produce different shades of reddish-brown. Furthermore, the solutions dissolved in tap water, ultrapure water, and methanol-water (1:4) showed black precipitate, indicating incomplete dissolution; even after repeated vortexing, precipitate remained. In contrast, the solutions dissolved quickly in sodium hydroxide (10mM) and acetonitrile-water (1:3), and were clear and transparent without any precipitate.

[0123] (3) Dilute the above five stock solutions to 260 μg / ml with the corresponding solvents, and perform detection under these conditions. Calculate the peak area. Chromatographic conditions: High performance liquid chromatograph (Shimadzu LC-20AT, Japan); Column: InterSustain AQ-C18 250 × 4.6 mm, 5 μm; Column temperature: 30℃; Mobile phase A: 50 mM disodium hydrogen phosphate solution; Mobile phase B: acetonitrile; Flow rate: 0.8 mL / min; Injection volume: 10 μL. Isocratic elution: A:B = 98:2. Detection wavelength: 250 nm.

[0124] Figure 4 The results are shown in the high-performance liquid chromatography (HPLC) diagram, where A) is the chromatogram and B) is the peak area diagram. Figure 4 The results showed that the PQQ chromatographic peak area was the highest when dissolved in sodium hydroxide (10mM), indicating the best dissolution effect. Figure 5 The full-wavelength UV absorption scan results of PQQ dissolved in four solvents: ultrapure water, methanol-water (1:4), sodium hydroxide (10mM), and acetonitrile-water (1:3).

[0125] (4) In order to verify that the abnormal color of the PQQ stock solution does not affect the solution concentration, the ultraviolet scanning spectra of the five diluted solutions were compared. The results showed that the maximum absorption wavelength did not change, but there was a red shift or blue shift, which were 331 nm and 248 nm respectively. This indicates that the PQQ did not undergo any changes in the conjugated system or produce other metabolites. It was initially judged that although the stock solution color changed to yellowish-brown, the low concentration after dilution could reversibly change to the same light pink as other diluted solutions, which did not affect the accuracy of the solution concentration.

[0126] (5) Therefore, all subsequent standards are diluted with sodium hydroxide (10mM) and acetonitrile water (1:3) because the organic solvent mixture can ensure the stability of the solution and prevent degradation.

[0127] 3. Calculation and Expression of Test Results for the Samples to be Tested

[0128] The formula for calculating the content of active substances in the sample to be tested is as follows:

[0129] ;

[0130] In the formula:

[0131] X - Percentage of PQQ in the sample to be tested (g / 100g).

[0132] C - Calculate the concentration of PQQ in the sample (μg / mL) from the standard curve;

[0133] V - The final volume of the sample to be tested (mL);

[0134] F - Dilution factor;

[0135] M - Sample volume (mg) to be tested.

[0136] 4. Sample pretreatment optimization

[0137] Accurately weigh 100 mg of the capsule contents powder into a 15 mL centrifuge tube, dissolve it in pure water to the mark, and measure the pH to 5.85. Then, adjust the pH to 7.0, 7.82, and 8.4 with 1 M sodium hydroxide. Take a sample before adjusting the pH, pass it through a membrane, and perform HPLC analysis under the conditions determined above.

[0138] Test results as follows Figure 6 As shown, from Figure 6 It can be seen that pH has a significant impact on the extraction of PQQ from the sample, with the best extraction effect achieved at pH 7.0. Based on the selected mobile phase A, disodium hydrogen phosphate aqueous solution was chosen as the solvent to dissolve the sample, ensuring the pH was maintained at neutral.

[0139] Therefore, PQQ extraction was subsequently performed using a 50mM disodium hydrogen phosphate aqueous solution. Approximately 300mg of the capsule contents powder was placed in a 50mL volumetric flask, diluted to volume, and the pH was kept neutral. The mixture was sonicated for 10min, with ice added during the sonication process to prevent overheating. After 10min of sonication, the supernatant was aspirated with a syringe, filtered through a 0.22μm aqueous filter, and then passed through a membrane for analysis.

[0140] 5. Standard Curve

[0141] Preparation of standard solutions: Accurately weigh an appropriate amount of PQQ standard and place it in a 10 mL volumetric flask. Add sodium hydroxide (10 mM) solution to the mark, shake well, and prepare a stock solution of 3.2 mg / mL. Then dilute with acetonitrile water (1:3) to prepare a series of standard solutions: 20, 40, 80, 160, 320, and 640 μg / mL. All solutions should be prepared fresh and used immediately.

[0142] The prepared series of standard working solutions were injected and analyzed under the optimized HPLC chromatographic conditions described above. A series of standard curves were plotted with concentration as the abscissa and peak area (mAU) response value as the ordinate, and the linear regression equation of the standard curves was obtained.

[0143] Limit of detection (LOQ): the concentration at a signal-to-noise ratio of 3:1; Limit of quantitation (LOD): the concentration at a signal-to-noise ratio of 10:1.

[0144] The liquid chromatogram of standard PQQ is as follows: Figure 7 As shown. From Figure 7 The peak elution time is 3.985 min. Table 1 shows the linear equation, correlation coefficient, and detection limit. The detection results shown in Table 1 indicate that the linearity is good and meets the requirements of the chromatographic methodology.

[0145] Table 1. Linear equations, correlation coefficients, and detection limits of the analytes.

[0146]

[0147] 6. Repeatability Experiments

[0148] HPLC chromatographic conditions were as follows: High-performance liquid chromatograph (Shimadzu LC-20AT, Japan); column: InterSustain AQ-C18 250 × 4.6 mm, 5 μm; column temperature: 30℃; mobile phase A: 50 mM disodium hydrogen phosphate solution, mobile phase B: acetonitrile; flow rate: 0.8 mL / min; injection volume: 10 μL. Isocratic elution: A:B = 98:2. Detection wavelength: 250 nm.

[0149] Accurately weigh an appropriate amount of PQQ standard and place it in a 10 mL volumetric flask. Add sodium hydroxide (10 mM) solution to the mark, shake well, and prepare a 3.2 mg / mL stock solution. Then dilute with acetonitrile-water (1:3) to prepare a 160 μg / mL PQQ standard solution. Perform HPLC analysis under the optimized conditions described above, repeating the process 6 times. Calculate the relative standard deviation based on the peak area. The results are shown in Table 2. As can be seen from the repeatability test results in Table 2, the RSD of repeatability is 2.62%, and the relative standard deviation is less than 3%, meeting the methodological requirements.

[0150] 7. Precision Experiment

[0151] HPLC chromatographic conditions were as follows: High-performance liquid chromatograph (Shimadzu LC-20AT, Japan); column: InterSustain AQ-C18 250 × 4.6 mm, 5 μm; column temperature: 30℃; mobile phase A: 50 mM disodium hydrogen phosphate solution, mobile phase B: acetonitrile; flow rate: 0.8 mL / min; injection volume: 10 μL. Isocratic elution: A:B = 98:2. Detection wavelength: 250 nm.

[0152] Six portions of the capsule contents powder were accurately weighed and extracted using a 50 mM disodium hydrogen phosphate aqueous solution. Approximately 300 mg of the capsule contents powder was placed in a 50 mL volumetric flask, and the volume was brought to a final volume. The pH was adjusted with hydrochloric acid or sodium hydroxide to maintain neutrality. The mixture was sonicated for 10 min, with ice added during the sonication process to prevent overheating. After 10 min of sonication, the supernatant was aspirated using a syringe, filtered through a 0.22 μm aqueous filter, and then passed through a membrane for analysis. Analysis was performed for three consecutive days. The relative standard deviation (RSD) was calculated based on the peak area to assess intra-day and inter-day precision. The results are shown in Table 2. As can be seen from the intra-day and inter-day precision test results in Table 2, the RSDs for both intra-day and inter-day precision are less than 5%, meeting the methodological requirements.

[0153] 8. Stability test

[0154] HPLC chromatographic conditions were as follows: High-performance liquid chromatograph (Shimadzu LC-20AT, Japan); column: InterSustain AQ-C18 250 × 4.6 mm, 5 μm; column temperature: 30℃; mobile phase A: 50 mM disodium hydrogen phosphate solution, mobile phase B: acetonitrile; flow rate: 0.8 mL / min; injection volume: 10 μL. Isocratic elution: A:B = 98:2. Detection wavelength: 250 nm.

[0155] Six portions of the capsule contents powder were accurately weighed and extracted with 50 mM disodium hydrogen phosphate aqueous solution using PQQ extraction. Approximately 300 mg of the capsule contents powder was placed in a 50 mL volumetric flask, and the volume was brought to a final volume. The pH was adjusted with hydrochloric acid or sodium hydroxide to maintain neutrality. The mixture was sonicated for 10 min, with ice added during the sonication process to prevent overheating. After 10 min of sonication, the supernatant was aspirated using a syringe, filtered through a 0.22 μm aqueous filter, and then passed through a membrane for analysis. Measurements were taken every 4 hours, for a total of 3 measurements. The relative standard deviation (RSD) was calculated based on the peak area. The results are shown in Table 2. The stability test results in Table 2 show that the RSDs were all less than 5%, meeting the methodological requirements.

[0156] Table 2. Methodological validation of the analytes (repeatability, intra-day and inter-day precision)

[0157]

[0158] 9. Spike Recovery Experiment

[0159] Nine aliquots of the capsule contents powder were accurately weighed and extracted with PQQ using a 50 mM disodium hydrogen phosphate aqueous solution. Approximately 300 mg of the capsule contents powder was placed in a 50 mL volumetric flask, and a known concentration of PQQ standard solution was precisely added to achieve the predetermined low (100 μg / mL), medium (200 μg / mL), and high (300 μg / mL) concentrations. The solution was brought to volume with a 50 mM disodium hydrogen phosphate aqueous solution, and the pH was adjusted to neutral using hydrochloric acid or sodium hydroxide. The mixture was sonicated for 10 min, with ice added during sonication to prevent overheating. After 10 min of sonication, the supernatant was aspirated using a syringe, filtered through a 0.22 μm aqueous filter, and then passed through a membrane for analysis. The spiked recovery rate and relative standard deviation were calculated based on the peak area to assess the accuracy of the method. Three replicates were performed for each concentration.

[0160] The calculation formula is as follows:

[0161] ;

[0162] In the formula:

[0163] C1 - Calculate the PQQ concentration (μg / mL) of the spiked sample from the standard curve.

[0164] C2 - Calculate the PQQ concentration (μg / mL) of the unspiked sample from the standard curve;

[0165] C3-PQQ spiked concentration (μg / mL).

[0166] The test results are shown in Table 3. As can be seen from the results in Table 3, the spiked recoveries were 98-103%, and the RSDs were all less than 5%, meeting the methodological requirements.

[0167] Table 3. Methodological Validation of Analytes (Spiked Recovery)

[0168]

[0169] Example 2

[0170] The content of PQQ powder capsules was determined using three representative and complex types of capsules.

[0171] 1. Test Sample

[0172] Sample A: GREEN EVERS, Ergothioneine Five-in-One Skin Beautifying and Anti-Aging Tablets, PQQ label indicates content of 10mg / tablet;

[0173] Sample B: FineNutri, FineNutri nad+ gold bottle, PQQ labeled content is 20mg / capsule;

[0174] Sample C: feelwell, ergothioneine-27267, PQQ labeled content is 20mg / capsule.

[0175] 2. Detection Method

[0176] Preparation of standard solutions: Accurately weigh an appropriate amount of PQQ standard and place it in a 10 mL volumetric flask. Add sodium hydroxide (10 mM) solution to the mark, shake well, and prepare a stock solution of 3.2 mg / mL. Then dilute with acetonitrile water (1:3) to prepare a series of standard solutions: 20, 40, 80, 160, 320, and 640 μg / mL. All solutions should be prepared fresh and used immediately.

[0177] HPLC chromatographic conditions were as follows: High-performance liquid chromatograph (Shimadzu LC-20AT, Japan); column: InterSustain AQ-C18 250 × 4.6 mm, 5 μm; column temperature: 30℃; mobile phase A: 50 mM disodium hydrogen phosphate solution, mobile phase B: acetonitrile; flow rate: 0.8 mL / min; injection volume: 10 μL. Isocratic elution: A:B = 98:2. Detection wavelength: 250 nm.

[0178] Specific sample processing methods:

[0179] Sample content detection: Accurately weigh product A into a 100mL volumetric flask, and place product B and product C into separate 250mL volumetric flasks. Dilute to volume with 50mM disodium hydrogen phosphate aqueous solution. Adjust the pH with hydrochloric acid or sodium hydroxide to maintain neutrality. Sonicate for 10 minutes, adding ice during sonication to prevent overheating. After 10 minutes of sonication, use a syringe to aspirate the supernatant, filter through a 0.22μm aqueous filter, and then perform chromatographic analysis. Perform three replicates for each sample. Calculate the content based on the chromatographic peak area.

[0180] Spiked recovery test: Accurately weigh product A into a 100 mL volumetric flask, and place product B and product C into separate 250 mL volumetric flasks. Precisely add a known concentration of PQQ standard solution to each flask to achieve a standard concentration of 100 μg / mL. Dilute to volume with 50 mM disodium hydrogen phosphate aqueous solution, ensuring the pH remains neutral. Sonicate for 10 min, adding ice during sonication to prevent overheating. After 10 min of sonication, aspirate the supernatant with a syringe, filter through a 0.22 μm aqueous filter, and then perform sample analysis using a membrane analyzer. Perform three replicates for each sample.

[0181] The calculation formula is as follows:

[0182] ;

[0183] In the formula:

[0184] C1 - Calculate the PQQ concentration (μg / mL) of the spiked sample from the standard curve.

[0185] C2 - Calculate the PQQ concentration (μg / mL) of the unspiked sample from the standard curve;

[0186] C3-PQQ spiked concentration (μg / mL).

[0187] 3. Test Results

[0188] Test results as follows Figure 8 As shown in Table 4.

[0189] Table 4. Content of PQQ in three special powder formulations

[0190]

[0191] Table 4 shows that the provided detection method can accurately detect the content of PQQ. Figure 8 It is evident that the provided detection method can separate PQQ from impurities such as natural extracts, achieving accurate detection.

[0192] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0193] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt, characterized in that, Includes the following steps: The sample to be tested is mixed with an aqueous solution of disodium hydrogen phosphate to prepare a test solution, the pH of which is neutral. The test solution is subjected to high-performance liquid chromatography (HPLC) detection, and the HPLC detection conditions include: Isocratic elution was performed using an aqueous solution of disodium hydrogen phosphate as mobile phase A and acetonitrile as mobile phase B.

2. The method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt according to claim 1, characterized in that, The pH value of the solution to be tested is 6.8~7.

2.

3. The method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt according to claim 1, characterized in that, The conditions for high performance liquid chromatography detection include: during isocratic elution, the volume ratio of mobile phase A to mobile phase B is 98:

2.

4. The method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt according to any one of claims 1 to 3, characterized in that, The conditions for the high-performance liquid chromatography detection meet one or more of the following conditions: (1) The detection wavelength is 248nm~250nm; (2) The flow rate is 0.8 mL / min to 1.0 mL / min; (3) The injection volume is 5 μL to 20 μL; (4) The column temperature is 25℃~35℃; (5) The chromatographic column packing material is C18 alkylsilane bonded silica gel; (6) The concentration of the sodium hydrogen phosphate aqueous solution is 10 mmol / L to 50 mmol / L; (7) The pH value of the sodium hydrogen phosphate aqueous solution is 8.8~9.

2.

5. The method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt according to any one of claims 1 to 3, characterized in that, The sample to be tested is mixed with an aqueous solution of disodium hydrogen phosphate and then subjected to ultrasonic treatment to prepare the test solution.

6. The method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt according to claim 5, characterized in that, One or more of the following conditions must be met: (1) The ultrasonic treatment time is 2 min to 15 min; (2) The power of ultrasonic treatment is 80W~200W; (3) The frequency of ultrasonic treatment is 40kHz~50kHz; (4) Perform ultrasonic treatment at a temperature of 25°C or less.

7. The method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt according to any one of claims 1 to 3, characterized in that, One or both of the following conditions must be met: (1) The sample to be tested is a dietary supplement and / or functional food; (2) The sample to be tested is a powder.

8. The method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt according to any one of claims 1 to 3, characterized in that, The method further includes the following steps: Take the standard of pyrroloquinoline quinone and prepare standard solutions of different concentrations; The standard solutions of different concentrations were subjected to high performance liquid chromatography (HPLC) for detection, and a standard curve was constructed based on the peak area and concentration of the standard solution in the detection results. Substitute the detection results of the high performance liquid chromatography of the test solution into the standard curve to calculate the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt in the test solution.

9. The method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt according to claim 8, characterized in that, A stock solution was prepared by dissolving the standard of pyrroloquinoline quinone in an aqueous sodium hydroxide solution, and then the stock solution was diluted with an aqueous acetonitrile solution to prepare standard solutions of different concentrations.

10. The application of the method for detecting the content of pyrroloquinoline quinone and / or pyrroloquinoline quinone salt according to any one of claims 1 to 9 in the quality control of dietary supplements and / or functional foods.

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