Detection method and application of vardenafil impurity 30

Abstract

The invention discloses a detection method and application of vardenafil impurity 30, and relates to the technical field of pharmaceutical analysis. Comprising the following steps: ultrasonically extracting a sample by methanol, filtering, determining filtrate by an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer (electrospray ionization source, MRM mode), and quantifying by an external standard method by taking 503.3-326.2 and 503.3-299.1 as qualitative ion pairs and 503.3-151.1 as quantitative ion pairs. The method is suitable for determining the vardenafil impurity 30 in foods such as drinks, jellies, protein powder, biscuits, candies, wines, coffee and the like (including health-care foods, tablets, capsules, soft capsules and other dosage forms which are the same as the matrix).

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and more specifically to a method and application for detecting vardenafil impurity 30. Background Technology

[0002] Erectile dysfunction (ED) is one of the most common diseases, resulting in a large market for anti-ED drugs. The illegal addition of anti-ED drugs to food (including health foods) is widespread, with sildenafil being the most common culprit. To evade detection, criminals are turning to adding novel derivatives of sildenafil-like drugs.

[0003] To effectively combat the illegal addition of new types of substances by criminals, the State Administration for Market Regulation (SAMR) has successively issued national standards for the illegal addition of sildenafil-like substances to food (including health food) in recent years. In 2018, SAMR released a supplementary testing method for "Determination of Sildenafil-like Substances in Food" (BJS 201805), which detects 90 kinds of sildenafil-like substances in food (including health food). However, the authorities are constantly evolving, and new types of sildenafil-like substances continue to be detected in literature. These include hydroxycarbazinavir, cis-tetrahydrocarbazinavir, N-cyclohexylnortadalafil, phenylpropylcarbazinavir, tadalafil impurity 37, and morphine sildenafil. On December 22, 2024, SAMR released "Determination of Sildenafil, Tadalafil, and Other Compounds in Food" (BJS 202405). In addition, there are literature reports on the use of ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry to detect 102 kinds of erectile dysfunction (ED) inhibitors in health food. However, a novel nalaviroid substance was recently detected in a blackberry tablet candy. After separation, purification, nuclear magnetic resonance spectroscopy, mass spectrometry, and spectral analysis, the novel nalaviroid substance was confirmed to be Vardenafil Impurity 30 (CAS No.: 2840532-32-1). This substance has no known presence in the food industry and is a novel nalaviroid substance that has not been monitored. The structure of this compound is modified based on the structure of the prescription drug Vardenafil and has the same pharmacodynamic group as Vardenafil, therefore it may have the same drug efficacy as Vardenafil.

[0004] Appendix 2 of document No. 74 of 2022 issued by the Market Supervision and Inspection Bureau (Expert Opinion on the Toxic and Harmful Effects of Phenylephrine-modified Carbadinafil, Lafil, and Their Derivatives) states that "...consuming food containing larfine, lafil, and their derivatives poses a risk of toxic side effects to the human body, affecting human health and even endangering life." Appendix A of the United States Pharmacopeia (USP) lists six qualitative detection methods for 64 sexually functional compounds, but none of them include vardenafil impurity 30. Furthermore, vardenafil impurity 30 lacks systematic and comprehensive pharmacological, toxicological, and related clinical studies. Inappropriate or prolonged use by consumers without their knowledge poses a significant risk to public food safety.

[0005] Therefore, in order to effectively combat the illegal addition of new types of valdenafil-like substances, safeguard people's food safety, and promote effective supervision by regulatory authorities, it is an urgent problem for those skilled in the art to provide a method for detecting valdenafil impurity 30 in food. Summary of the Invention

[0006] In view of this, the present invention provides a method for detecting vardenafil impurity 30 and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for detecting vardenafil impurity 30 includes the following steps: the sample is extracted with methanol by ultrasonication, filtered, and the filtrate is used for determination by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (electrospray ionization source, MRM mode). 503.3→326.2 and 503.3→299.1 are used as qualitative ion pairs, and 503.3→151.1 is used as quantitative ion pair. Quantification is performed using the external standard method.

[0008] Furthermore, when the sample is a solid sample or a semi-solid sample: Take an appropriate amount of solid sample, mix well, and grind finely; or take an appropriate amount of semi-solid sample, mix well; weigh 1 g of sample (accurate to 0.001 g) and place it in a 50 mL volumetric flask, add 25 mL of methanol, extract by ultrasonication at 1000 W for 15 min, cool to room temperature, make up to the mark with methanol, transfer to a 50 mL centrifuge tube, centrifuge at 6000 r / min for 5 min, filter the supernatant through a 0.22 μm organic microporous membrane, and keep the filtrate for later use.

[0009] Furthermore, the solid samples include: protein powder, biscuits, candy, coffee, and health foods containing the same matrix as described above, as well as tablet and capsule dosage forms; The semi-solid sample includes: jelly.

[0010] Furthermore, when the sample is a liquid sample: Take an appropriate amount of sample and shake well. Accurately measure 1 mL of sample and place it in a 50 mL volumetric flask. Add 25 mL of methanol and extract by ultrasonication at 1000W for 15 min. Cool to room temperature and dilute to the mark with methanol. Filter through a 0.22 μm organic microporous membrane and collect the filtrate for later use.

[0011] Furthermore, the liquid sample includes beverages and alcohol.

[0012] Furthermore, when the sample is an oil-based sample: Take an appropriate amount of sample and mix well. Weigh 1 g of sample (accurate to 0.001 g) and place it in a 50 mL volumetric flask. Add 5 mL of ethyl acetate, shake to disperse, add 25 mL of methanol, and extract by sonication for 15 min. Cool to room temperature, and make up to the mark with methanol. Transfer to a 50 mL centrifuge tube, centrifuge at 4000 r / min for 5 min, filter the supernatant through an organic microporous membrane, and keep the filtrate for later use.

[0013] Furthermore, the oil-based sample includes: soft capsules.

[0014] Furthermore, the chromatographic analysis conditions are as follows: a) Chromatographic column: WATERS ACQUITY-UPLC® BEH C18 column, 2.1 mm × 50 mm, 1.7 µm; b) Mobile phase: A: 0.1% formic acid aqueous solution, B: methanol; c) Column temperature: 35 ℃; d) Injection volume: 2 µL; e) Flow rate: 0.3 mL / min.

[0015] Furthermore, the mobile phase gradient elution is as follows: Gradient time: 0-1 min; Mobile phase A: 90%~50%; Mobile phase B: 10%~50%; Gradient time 1-2.5 min, mobile phase A: 50%~10%, mobile phase B: 50%~90%; Gradient time: 2.5-4.0 min; Mobile phase A: 10%; Mobile phase B: 90%; Gradient time 4.0-4.1 min, mobile phase A: 10%~90%, mobile phase B: 90%~10%; Gradient time 4.1-6.5 min, mobile phase A: 90%, mobile phase B: 10%.

[0016] Application of a method for detecting vardenafil impurity 30 in food testing. As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:The sample was extracted with methanol by ultrasound, filtered, and the filtrate was analyzed by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (electrospray ionization source, MRM mode). Qualitative ion pairs were 503.3→326.2 and 503.3→299.1, and quantitative ion pairs were 503.3→151.1. Quantification was performed using the external standard method. Vardenafil impurity 30 showed good linearity in the concentration range of 1 µg / L to 50 µg / L. The limit of detection (LOD) was 0.05 mg / kg and the limit of quantitation (LOQ) was 0.1 mg / kg in coffee, soft capsules, and capsule matrices. The LOD was 0.025 mg / kg and the LOQ was 0.05 mg / kg in jelly, protein powder, biscuits, candy, and tablet matrices. The LOD was 0.025 mg / L and the LOQ was 0.05 mg / L in beverages and alcoholic beverages. Three-level spiked tests were conducted on 10 food matrices. The average recoveries of vardenafil impurity 30 ranged from 92.09% to 115.15%, and the relative standard deviations (RSDs) ranged from 0.71% to 18.32%. The average recoveries in tablet matrix were 111.55%, 99.43%, and 106.37%, with RSDs of 7.27%, 5.49%, and 5.34%, respectively; in beverage matrix, the average recoveries were 105.93%, 111.82%, and 99.75%, with RSDs of 5.65%, 9.29%, and 8.74%, respectively; in confectionery matrix, the average recoveries were 106.77%, 107.97%, and 96.44%, with RSDs of 7.42%, 12.71%, and 2.85%, respectively; in coffee matrix, the average recoveries were 110.05%, 112.74%, and 106.98%, with RSDs of 6.96%, 4.11%, and 2.93%, respectively; and in soft capsule matrix, the average recoveries were 105.55%, 105.14%, and 110.21%, with RSDs of 7.11%, 4.97%, and 3.21%, respectively. The average recoveries in the jelly matrix were 106.74%, 95.90%, and 107.29%, with RSDs of 7.63%, 18.32%, and 3.69%, respectively; in the protein powder matrix, the average recoveries were 92.09%, 115.15%, and 97.21%, with RSDs of 17.05%, 2.26%, and 7.33%, respectively; in the wine matrix, the average recoveries were 109.00%, 110.51%, and 107.58%, with RSDs of 5.22%, 5.05%, and 3.24%, respectively; in the capsule matrix, the average recoveries were 111.15%, 112.75%, and 102.24%, with RSDs of 5.26%, 6.46%, and 0.71%, respectively; and in the biscuit matrix, the average recoveries were 105.24%, 112.24%, and 101.60%, with RSDs of 3.17%, 5.73%, and 1.39%, respectively.This invention is applicable to the determination of vardenafil impurity 30 in beverages, jellies, protein powders, biscuits, candies, alcoholic beverages, coffee and other foods (including health foods with the same matrix as above and dosage forms such as tablets, capsules, and soft capsules). Attached Figure Description

[0017] 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.

[0018] Figure 1. Chemical structural formulas of vardenafil and vardenafil impurity 30; Figure 2 Recovery rates of vardenafil impurity 30 in 10 food matrices under different extraction solvents; Figure 3 Recovery rate of vardenafil impurity 30 at different extraction times; Figure 4 Vardenafil impurity 30 and the mass spectrometric fragmentation pathway of vardenafil. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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. Example

[0020] 1.1 Raw materials Vardenafil Impurity 30 (Molecular Formula: C24H34N6O4S, CAS No.: 2840532-32-1), purity ≥ 98%, Shanghai Anpu Cuishi Standard Technical Service Co., Ltd. Acetonitrile, formic acid, methanol, ammonium acetate, ethyl acetate, chromatographic grade, Merck, Germany. 0.22 μm organic filter membrane, Tianjin Jinteng Experimental Equipment Co., Ltd. Laboratory water was ultrapure water. 50 mL centrifuge tubes, Corning Incorporated, USA. Waters ACQUITY UPLC® BEH C18 (50mm × 2.1mm, 1.7m), Waters Atlantis™ T3 (150mm × 2.1mm, 3μm), Waters Technology (Shanghai) Co., Ltd.

[0021] 1.2 Instruments and Equipment Mettler ME204 electronic balance, Mettler Incorporated, USA; KW-500TDB high-efficiency CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; MV-3000 multi-tube oscillator, Chengdu Laipu Technology Co., Ltd.; 4-16KS high-speed refrigerated centrifuge, SIGMA GmbH, Germany; TQS liquid chromatography-triple quadrupole mass spectrometer, Waters Technology (Shanghai) Co., Ltd.

[0022] 1.3 Test Methods 1.3.1 Requirements for Sample Preparation Weigh approximately 200 g of representative samples of jelly, protein powder, biscuits, candy, coffee, tablets, capsules (containing the contents), and soft capsules (containing the contents). Crush and mix thoroughly, then dry and store away from light for later use. Measure approximately 200 mL of representative beverage and alcohol samples and refrigerate for later use.

[0023] 1.3.2 Selection and optimization of sample pretreatment conditions 1.3.2.1 Investigation of the extraction solvent Vardenafil impurity 30 is readily soluble in organic reagents such as methanol, acetonitrile, and ethanol. The recovery rate of vardenafil impurity 30 was used as the evaluation index in this experiment. Spiked samples with a content of 0.5 mg / kg were prepared using a blank sample plus standard method (subsequent samples were prepared using this method). The extraction effects of methanol, acetonitrile, and ethanol on the target compound in 10 food matrices, including tablets, beverages, candies, coffee, soft capsules, jellies, protein powders, wines, capsules, and biscuits, were investigated. Triple parallel determinations were performed, and the results are shown below. Figure 2 The results showed that methanol was the most stable extraction solvent for the target compound in all 10 matrices, with recoveries ranging from 72% to 114%. Therefore, methanol was chosen as the extraction solvent. Furthermore, the study found that for oil-rich soft capsules, adding an appropriate amount of ethyl acetate to disperse the matrix before adding methanol for extraction can improve extraction efficiency.

[0024] 1.3.2.2 Optimization of Sample Extraction Time Extraction time is a crucial parameter. Too short an extraction time may fail to extract the target compound completely, while too long an extraction time may lead to the dissolution of more matrix components, resulting in a matrix effect on the target compound's response. Using spiked samples with a concentration of 0.5 mg / kg, the extraction efficiency of the target compound in 10 food matrices at different extraction times (10, 15, and 20 minutes) was investigated. Triple parallel determinations were performed, and the results are shown below. Figure 3Experiments showed that the extraction efficiency of the target compound gradually increased with increasing extraction time. At an extraction time of 15 minutes, the recovery rate of the target compound in 10 matrices reached its maximum, ranging from 72% to 114%. When the extraction time reached 20 minutes, the recovery rate of the target compound in soft capsules exceeded 120%, indicating a matrix enhancement effect. To obtain a high recovery rate in a shorter time and reduce the influence of the matrix effect, this invention selected an ultrasonic extraction time of 15 minutes.

[0025] 1.3.2.3 Examination of the number of sample extractions To ensure extraction efficiency, the recovery rate of the target compound extracted from 10 food matrices under different ultrasonic cycles was investigated using spiked samples with a content of 0.5 mg / kg. The results were obtained in three parallel determinations and are shown in Table 1.

[0026] Table 1 Recovery rates of extraction under different ultrasound cycles

[0027] The experimental results showed that in the first extraction, the target compound was almost completely extracted from 10 food matrices, with a recovery rate of over 72%; in the second extraction, the recovery rate was between 0.02% and 0.9%. Considering the recovery rate, detection efficiency, and experimental cost, the method was determined to be a single ultrasonic extraction.

[0028] 1.3.3 Determination of Pretreatment Methods for Different Types of Samples 1.3.3.1 Solid or semi-solid samples Take an appropriate amount of solid sample (protein powder, biscuits, candy, coffee, and health foods containing the same matrix as above, as well as tablets, capsules, and other dosage forms), mix well, and grind finely. Alternatively, take an appropriate amount of semi-solid sample (jelly), mix well, weigh 1 g of sample (accurate to 0.001 g), place it in a 50 mL volumetric flask, add an appropriate amount of methanol, extract ultrasonically for 15 min, cool to room temperature, dilute to the mark with methanol, transfer to a 50 mL centrifuge tube, centrifuge at 6000 r / min for 5 min, filter the supernatant through a 0.22 μm organic microporous membrane, and keep the filtrate for later use.

[0029] 1.3.3.2 Liquid Samples Take an appropriate amount of sample (beverage, wine) and shake well. Accurately measure 1 mL of sample and place it in a 50 mL volumetric flask. Add an appropriate amount of methanol and extract by ultrasonication for 15 min. Cool to room temperature and dilute to the mark with methanol. Filter through a 0.22 μm organic microporous membrane and collect the filtrate for later use.

[0030] 1.3.3.3 Oil-based (soft capsule) samples Take an appropriate amount of sample (soft capsules) and mix well. Weigh 1 g of sample (accurate to 0.001 g) and place it in a 50 mL volumetric flask. Add 5 mL of ethyl acetate, shake to disperse, add an appropriate amount of methanol, and extract by sonication for 15 min. Cool to room temperature, and make up to the mark with methanol. Transfer to a 50 mL centrifuge tube, centrifuge at 4000 r / min for 5 min, filter the supernatant through an organic microporous membrane, and keep the filtrate for later use.

[0031] 1.4 Selection and optimization of instrument measurement conditions 1.4.1 Optimization of mass spectrometry conditions Vardenafil impurity 30 is a derivative of vardenafil and possesses similar chemical properties. According to literature reports, vardenafil is primarily detected using a positive ion mode. Therefore, this study selected positive ion mode, injecting a 0.1 µg / mL standard solution of vardenafil impurity 30 into the mass spectrometer for primary mass spectrometry scanning, obtaining a stable molecular ion peak [M+H]+. After optimizing the declustering voltage, the daughter ions were scanned by changing the collision energy, yielding secondary mass spectrometry ions of vardenafil impurity 30. Ions with high signal responses at m / z 326.2, m / z 299.1, and m / z 151.1 were obtained. Considering the ion signal responses and literature reports that m / z 151 is a characteristic ion fragment of vardenafil-like substances, m / z 151.1 was selected as the quantitative ion.

[0032] from Figure 4 The mass spectrometry fragmentation pathway revealed that the molecular structure of vardenafil impurity 30 differs from that of vardenafil in that the ethoxy group attached to the benzene ring is modified with a propoxy group. Therefore, it can fragment ion m / z 326, which has a molecular weight 14 greater than that of the characteristic fragment ion m / z 312 of vardenafil. This fragmentation behavior is a typical feature of vardenafil impurity 30, so m / z 326 was selected as the qualitative ion. The signal at m / z 299 was also relatively high, so m / z 326.2 and m / z 299.1 were finally selected as the qualitative ions.

[0033] Table 2. Qualitative, quantitative ion and mass spectrometry parameters of compounds

[0034] Note: *Quantitative ions 1.4.2 Selection of Chromatographic Column According to existing standards and literature, the main chromatographic column used for separating natriuretic compounds is the reversed-phase C18 column, and the mobile phase is mainly a 0.1% formic acid aqueous solution-methanol system. To determine the optimal separation conditions, the research group compared two columns: Waters ACQUITYUPLC® BEH C18 (50 mm × 2.1 mm, 1.7 m) and Waters Atlantis™ T3 (150 mm × 2.1 mm, 3 μm). The results showed that the target compound could elute normally under both column conditions, and the response intensity was stable.

[0035] 1.4.3 Selection of Mobile Phase The effects of five mobile phase systems—water-acetonitrile, 0.1% formic acid-water-methanol, 0.1% formic acid-water-acetonitrile, 5 mmol / L ammonium acetate solution (containing 0.1% formic acid)-methanol, and 5 mmol / L ammonium acetate solution (containing 0.1% formic acid)-acetonitrile—on the peak shape and ion response of the target compound were investigated. The results showed that the target compound exhibited some peak tailing in the water-acetonitrile system, while the peak shape was symmetrical in the other four mobile phase systems. To improve ionization efficiency, stabilize ion strength, and reduce the introduction of salt ions, the mobile phase system was kept consistent with that used in BJS202405; therefore, 0.1% formic acid-water-methanol was selected as the mobile phase system for this detection method. The optimized chromatographic analysis conditions are as follows: a) Chromatographic column: WATERS ACQUITY-UPLC® BEH C18 column, 2.1 mm × 50 mm, 1.7 µm; b) Mobile phase: A: 0.1% formic acid aqueous solution, B: methanol, gradient elution table is shown in Table 3; c) Column temperature: 35 ℃; d) Injection volume: 2 µL; e) Flow rate: 0.3 mL / min; Table 3. Mobile phase gradient elution program

[0036] 1.5 Examining the linear range and matrix effect of the method Standard solutions with concentrations of 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 50 ng / mL were prepared using blank extraction solutions of 10 food matrices and methanol-water (1:1). Standard curves were plotted with the peak area of ​​the quantitative ion as the ordinate (Y) and the mass concentration as the abscissa (X, ng / mL). The linear regression equation for the standard solution prepared with methanol-water (1:1) was: Y = 7894.72x - 2.93697, with a correlation coefficient R² = 0.998. The standard curve equations, correlation coefficients, and matrix effects for the 10 food matrix blank extraction solutions are shown in Table 4.

[0037] The matrix effect (ME) is calculated using the formula ME = (k2 / k1 - 1) × 100%, where k1 is the slope of the standard solution curve and k2 is the slope of the matrix standard curve. A matrix effect of -20% to 20% indicates that it is within an acceptable range. Experiments have shown that coffee and capsules exhibit a certain matrix effect. This effect can be largely eliminated by diluting the blank extract 5 times with methanol-water (1:1) before using it to prepare the standard curve. Since the recommended dosage of vardenafil is generally between 5 mg and 20 mg, and vardenafil impurity 30 is a derivative of vardenafil that may have similar effects, unscrupulous merchants often illegally add higher doses to their products to achieve advertised efficacy. Therefore, positive samples usually require additional dilution during pretreatment to ensure the concentration of the test solution is within the range of the standard curve. Thus, a blank solvent can be used to prepare the standard curve for quantification of positive samples.

[0038] 1.6 Limits of Detection and Limits of Quantification of the Method Spiked samples were prepared using a blank matrix and the optimal pretreatment method. The samples were then analyzed. The limit of detection (LOD) was calculated using a signal-to-noise ratio (SNR) of 3, and the limit of quantitation (LOQ) was calculated using a SNR of 10. Six parallel determinations were performed, and the average value was taken. The results are shown in Table 4. The LOD of the target analyte was 0.025 mg / kg or 0.025 mg / mL in most matrices, and the LOQ was 0.050 mg / kg or 0.050 mg / mL. The LODs of capsules and coffee matrices were more complex and exhibited a higher matrix effect, resulting in slightly higher LODs of detection and LOQ.

[0039] Table 4. Linear regression equation, correlation coefficient, matrix effect, limit of detection and limit of quantitation for vardenafil impurity 30.

[0040] 1.7 Accuracy and precision (repeatability) of the method Weigh 1.000 g of blank sample into 50 mL volumetric flasks, add 3 levels of vardenafil impurity 30 standard solution to each flask, and prepare the remaining samples according to the optimal sample pretreatment method. Perform 6 parallel determinations. The results are shown in Table 5-14. The recoveries of the target analyte in 10 food matrices ranged from 70.20% to 120.24%, with relative standard deviations (RSD, n=6) ranging from 0.71% to 18.32%.

[0041] Table 5. Recovery and precision of vardenafil impurity 30 in tablet matrix

[0042] Table 6. Recovery and precision of vardenafil impurity 30 in beverage matrix

[0043] Table 7 Recovery and precision of vardenafil impurity 30 in confectionery matrix

[0044] Table 8 Recovery and precision of vardenafil impurity 30 in coffee matrix

[0045] Table 9 Recovery and precision of vardenafil impurity 30 in soft capsule matrix

[0046] Table 10 Recovery and precision of vardenafil impurity 30 in jelly matrix

[0047] Table 11 Recovery and precision of vardenafil impurity 30 in protein powder matrix

[0048] Table 12 Recovery and precision of vardenafil impurity 30 in the wine matrix

[0049] Table 13 Recovery and precision of vardenafil impurity 30 in capsule matrix

[0050] Table 14 Recovery and precision of vardenafil impurity 30 in biscuit matrix

[0051] 1.8 Stability (Stability of standard solution and stability of sample solution) 1.8.1 Stability of standard solutions The stability of the vardenafil impurity 30 standard stock solution (200 μg / mL) was investigated under room temperature, refrigeration (4℃), and freezing (-20℃) conditions for 3 months. The solution was diluted to 10 ng / mL every month for analysis. The experiment showed that after approximately 7 days at room temperature, the volume of solvent visibly decreased due to evaporation of the organic solvent, indicating that the standard stock solution should not be stored at room temperature for extended periods. Therefore, the study only measured the standard stock solution under refrigeration (4℃) and freezing (-20℃) conditions. The results are shown in Table 15. Under refrigeration (4℃), the detection concentration of vardenafil impurity 30 ranged from 10.05 ng / mL to 9.66 ng / mL, with an RSD of 1.83%; under freezing (-20℃), the detection concentration ranged from 10.11 ng / mL to 9.72 ng / mL, with an RSD of 1.67%. This indicates that the target standard solution is stable within 3 months.

[0052] Table 15 Stability of Vardenafil Impurity 30 under Two Storage Environments

[0053] 1.8.2 Stability of Sample Solution Spiked samples at 0.5 mg / kg were treated according to the planned sample pretreatment method to obtain the test solution. Positive samples, without the addition of standards, were treated according to the planned pretreatment method and diluted 25,000 times with methanol-water (1:1) to obtain the positive sample solution. Detection was performed at 0, 2, 12, 24, 36, and 48 hours, and the results are shown in Table 16. This indicates that the target analyte exhibits good stability in the matrix solution within 48 hours.

[0054] Table 16 Stability of Sample Solutions

[0055] 1.9 Specificity Ten blank matrices and spiked blank matrices were analyzed according to the sample pretreatment method determined in section “2.4” to investigate whether the substances present in the matrices interfered with the analyte. The results showed that the target compound was not detected in any of the ten blank matrices, but the target compound was detected in all the spiked blank matrices. The target compound did not interfere with the sample background.

[0056] 1.10 Sample Determination Vardenafil impurity 30 was detected in a batch of blackberry tablets. The contents of the samples tested according to the proposed method were 14970 mg / kg, 14070 mg / kg, 14100 mg / kg, 14860 mg / kg, 14530 mg / kg, and 14210 mg / kg, with an average value of 14460 mg / kg and an RSD of 2.72%. result The sample was extracted with methanol by ultrasonication, filtered, and the filtrate was used for analysis by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (electrospray ionization source, MRM mode). The qualitative ion pairs were 503.3→326.2 and 503.3→299.1, and the quantitative ion pair was 503.3→151.1. The external standard method was used for quantification.

[0057] Vardenafil impurity 30 exhibits good linearity in the concentration range of 1 µg / L to 50 µg / L. The limit of detection (LOD) is 0.05 mg / kg and the limit of quantitation (LOQ) is 0.1 mg / kg in coffee, soft capsules, and capsule matrices. The LOD is 0.025 mg / kg and the LOQ is 0.05 mg / kg in jelly, protein powder, biscuits, candy, and tablet matrices. The LOD is 0.025 mg / L and the LOQ is 0.05 mg / L in beverages and alcoholic beverages.

[0058] Three-level spiked tests were conducted on 10 food matrices. The average recoveries of vardenafil impurity 30 ranged from 92.09% to 115.15%, and the relative standard deviations (RSDs) ranged from 0.71% to 18.32%.

[0059] The average recoveries in the tablet matrix were 111.55%, 99.43%, and 106.37%, respectively, with RSDs of 7.27%, 5.49%, and 5.34%. The average recoveries in the beverage matrix were 105.93%, 111.82%, and 99.75%, with RSDs of 5.65%, 9.29%, and 8.74%, respectively. The average recoveries in the candy matrix were 106.77%, 107.97%, and 96.44%, with RSDs of 7.42%, 12.71%, and 2.85%, respectively; while the average recoveries in the coffee matrix were 110.05%, 112.74%, and 106.98%, with RSDs of 6.96%, 4.11%, and 2.93%, respectively. The average recoveries in the soft capsule matrix were 105.55%, 105.14%, and 110.21%, with RSDs of 7.11%, 4.97%, and 3.21%, respectively. The average recoveries in the jelly matrix were 106.74%, 95.90%, and 107.29%, with RSDs of 7.63%, 18.32%, and 3.69%, respectively. The average recoveries in the protein powder matrix were 92.09%, 115.15%, and 97.21%, with RSDs of 17.05%, 2.26%, and 7.33%, respectively. The average recoveries in the wine were 109.00%, 110.51%, and 107.58%, respectively, with RSDs of 5.22%, 5.05%, and 3.24%. The average recoveries in the capsule matrix were 111.15%, 112.75%, and 102.24%, with RSDs of 5.26%, 6.46%, and 0.71%, respectively. The average recoveries in the biscuit matrix were 105.24%, 112.24%, and 101.60%, with RSDs of 3.17%, 5.73%, and 1.39%, respectively.

[0060] This invention is applicable to the determination of vardenafil impurity 30 in beverages, jellies, protein powders, biscuits, candies, alcoholic beverages, coffee and other foods (including health foods with the same matrix as above and dosage forms such as tablets, capsules, and soft capsules).

[0061] 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 a vardenafil impurity 30, characterized by, Comprising the following steps: The sample is ultrasonically extracted with methanol, filtered, and the filtrate is determined by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry. The qualitative ion pairs are 503.3→326.2 and 503.3→299.1, and the quantitative ion pair is 503.3→151.

1. The external standard method is used for quantification.

2. The method of claim 1, wherein, When the sample is a solid sample or a semi-solid sample: An appropriate amount of the solid sample is mixed and finely ground, or an appropriate amount of the semi-solid sample is mixed. 1 g of the sample is weighed into a 50 mL volumetric flask, 25 mL of methanol is added, ultrasonic extraction is performed for 15 min at 1000 W, it is cooled to room temperature, and then it is diluted to the mark with methanol. It is transferred to a 50 mL centrifuge tube, centrifuged at 6000 r / min for 5 min, the supernatant is filtered through a 0.22 μm organic microporous filter membrane, and the filtrate is reserved.

3. The detection method according to claim 2, characterized in that, The solid sample includes: protein powder, biscuits, candies, coffee, and health foods and tablet and capsule formulations containing the same matrix as described above; The semi-solid sample includes: jelly.

4. The method of claim 1, wherein, When the sample is a liquid sample: An appropriate amount of the sample is shaken, 1 mL of the sample is accurately measured and placed in a 50 mL volumetric flask, 25 mL of methanol is added, ultrasonic extraction is performed for 15 min at 1000 W, it is cooled to room temperature, and then it is diluted to the mark with methanol. It is filtered through a 0.22 μm organic microporous filter membrane, and the filtrate is reserved.

5. The detection method according to claim 4, characterized in that, The liquid sample includes: beverages, wine.

6. The method of claim 1, wherein, When the sample is a fat matrix sample: An appropriate amount of the sample is mixed, 1 g of the sample is weighed into a 50 mL volumetric flask, 5 mL of ethyl acetate is added, it is shaken to disperse, 25 mL of methanol is added, ultrasonic extraction is performed for 15 min, it is cooled to room temperature, and then it is diluted to the mark with methanol. It is transferred to a 50 mL centrifuge tube, centrifuged at 4000 r / min for 5 min, the supernatant is filtered through an organic microporous filter membrane, and the filtrate is reserved.

7. The detection method according to claim 6, characterized in that, The fat matrix sample includes: soft capsules.

8. The method of claim 1, wherein, The chromatographic analysis conditions are as follows: a) Chromatographic column: WATERS ACQUITY-UPLC® BEH C18 chromatographic column, 2.1 mm×50 mm, 1.7 μm; b) Mobile phase: A: 0.1% formic acid aqueous solution, B: methanol; c) Column temperature: 35 ℃; d) Injection volume: 2 μL; e) Flow rate: 0.3 mL / min.

9. The detection method according to claim 8, characterized in that, The mobile phase is gradient eluted as follows: Gradient time 0-1 min, mobile phase A: 90%~50%, mobile phase B: 10%~50%; Gradient time 1-2.5 min, mobile phase A: 50%~10%, mobile phase B: 50%~90%; Gradient time 2.5-4.0 min, mobile phase A: 10%, mobile phase B: 90%; Gradient time 4.0-4.1 min, mobile phase A: 10%~90%, mobile phase B: 90%~10%; Gradient time 4.1-6.5 min, mobile phase A: 90%, mobile phase B: 10%.

10. Use of the method for detecting impurity 30 of vardenafil according to any one of claims 1-9 in food detection.

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