Screening and quantitative determination method for illegally added novel vardenafil structural analogue in food

Abstract

The invention provides a screening and quantitative determination method for illegally added novel vardenafil structural analogues in food, and relates to the technical field of medicinal chemistry. The O-propyl hydroxyl vardenafil is qualitatively and quantitatively detected by adopting ultra-high performance liquid chromatography in series with triple quadrupole mass spectrometry. An unknown vardenafil analogue is found when an ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry screening technology is adopted to screen illegal addition of anti-fatigue food, analysis is carried out according to a cracking law, and a target compound is obtained through separation and purification of preparative liquid chromatography. The molecular weight and structural fragments of the compound are obtained by using an ultra-high performance liquid chromatography-tandem mass spectrometry technology, and the structure of the compound is finally identified. A brand-new and effective screening and quantifying method is provided for the novel vardenafil derivative lacking a screening and detecting method in the food center.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a method for screening and quantitatively determining a novel vardenafil structural analogue illegally added into food. Background Art

[0002] In recent years, with the rapid growth of market demand, unscrupulous merchants have frequently illegally added phosphodiesterase 5 inhibitors (PDE-5) and their derivatives such as sildenafil, tadalafil, and vardenafil to health foods that tonify the kidneys and strengthen yang, regulate immunity, or resist fatigue in order to make huge profits. PDE-5 inhibitors are drugs that inhibit the activity of phosphodiesterases. They are mainly used in clinical practice to treat male erectile dysfunction (ED) and other related diseases. Vardenafil (English name: Vardenafil) is a PDE-5 inhibitor that was launched in 2003. It has the characteristics of small dosage, rapid onset, and high dose efficiency. It quickly became a classic prescription drug for the first-line treatment of ED.

[0003] When screening for illegal additives in anti-fatigue foods, an unknown vardenafil derivative was found. This derivative has the same parent nucleus as the approved vardenafil, with little structural difference. Therefore, it is necessary to establish a screening and quantitative determination method for this substance. Summary of the invention

[0004] The purpose of the present invention is to provide a method for screening and quantitatively determining novel vardenafil structural analogues illegally added to food.

[0005] The inventors used ultra-high performance liquid chromatography-diode array detector combined analysis to find a compound with a similar structure to vardenafil in the health wine, and determined the precise molecular weight and molecular formula of the new vardenafil derivative by high-resolution mass spectrometry; using the precise molecular weight of high-resolution mass spectrometry, combined with the mass spectrometry fragmentation rules of vardenafil and hydroxyvardenafil, the inventors speculated that the new derivative may be a vardenafil impurity 31 in which an ethyl group on the piperazine ring of vardenafil is added with a hydroxyl group, and a methyl group is added with a phenoxyethyl group. The derivative is confirmed to be vardenafil impurity 31 (CAS: 1339092-18-0) by ultra-high performance liquid chromatography-quadrupole electrostatic field orbital ion trap high-resolution mass spectrometry, which can be called O-propyl hydroxyvardenafil from a structural point of view.

[0006] In view of the fact that there are few research reports on this substance at home and abroad, and the basic data are seriously missing, there is a lack of objective detection data and results as support in the supervision process. This paper establishes a detection method for the illegal addition of chemical substance O-propyl hydroxyvardenafil in foods such as health wine and health foods by high performance liquid chromatography-tandem mass spectrometry. Due to the presence of hydroxyl groups in the structure, the acid-resistant HSS T3 column was selected to improve the separation efficiency, and acetonitrile-0.1% formic acid aqueous solution was selected as the mobile phase to ensure the separation effect and stability. The matrix effect of health wine was investigated, and the MRM mode was selected to enhance the ion selectivity, anti-interference ability and specificity. This method is simple, fast and accurate, and can accurately determine O-propyl hydroxyvardenafil in food. It provides a new and effective screening and quantitative method for new vardenafil derivatives that lack screening and detection methods, and provides technical support for food and drug safety supervision.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] The present invention provides a method for screening and quantitatively determining a novel vardenafil structural analogue illegally added to food, comprising the following steps:

[0009] Ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry was used for qualitative and quantitative detection, wherein the liquid chromatography conditions were as follows: chromatographic column: HSS T3 chromatographic column, flow rate: 300 μL / min, column temperature: 30°C, injection volume: 2 μL, mobile phase: A was 0.1% formic acid aqueous solution, B was acetonitrile, and the gradient elution program was as follows: 0-1.5 min, 90% A-50% A; 1.5-4.5 min, 50% A-15% A; 4.5-5 min, 15% A; 5-5.1 min, 15% A-90% A; 5.1-7 min, 90% A;

[0010] The mass spectrometry conditions were as follows: ion source: ESI, scanning mode: multiple reaction monitoring; interface temperature: 300°C; solvent temperature: 526°C; DL temperature: 250°C; heating block temperature: 400°C; nebulizing gas flow rate: 3 L / min; heating gas flow rate: 10 L / min; drying gas flow rate: 10 L / min;

[0011] The novel vardenafil structural analog is O-propyl hydroxyvardenafil.

[0012] In some embodiments, the multiple reaction monitoring conditions are: select ion pairs with mass-to-charge ratios of 519.3>299.1 and 519.3>284.1 as qualitative ion pairs, and select ion pairs with mass-to-charge ratios of 519.3>299.1 as quantitative ion pairs; wherein the Q1 deviation voltage, collision voltage CE and Q3 deviation voltage corresponding to the ion pair 519.3>299.1 are -22V, -42V, and -20V, respectively, and the Q1 deviation voltage, collision voltage CE and Q3 deviation voltage corresponding to the ion pair 519.3>284.1 are -24V, -48V, and -28V, respectively, and the retention time is 2.52min.

[0013] In some embodiments, the specific determination steps include: taking O-propyl hydroxyvardenafil standard substance, preparing a series of standard working solutions of different concentrations, injecting and drawing a standard curve to obtain a linear regression equation, then injecting the sample solution to be tested, and calculating the content of O-propyl hydroxyvardenafil therein by the linear regression equation.

[0014] In some embodiments, the sample solution is obtained by the following method:

[0015] Take 1g or 1mL of food sample in a 50mL volumetric flask, add 40mL of methanol, sonicate for 30 minutes, cool to room temperature, and dilute to the mark with methanol to obtain the extraction solution;

[0016] Purification: Activate the solid phase extraction column with 5 mL of water and 10 mL of methanol in turn. The filler composition of the solid phase extraction column is: 1 g PSA, 0.75 g GCB, 0.25 g C18; accurately pipette 5 mL of the extraction sample solution into the solid phase extraction column, elute with 20 mL of methanol, collect all the effluent in a 50 mL centrifuge tube, concentrate to 1 mL with nitrogen blowing (temperature controlled below 40°C), and pass through a 0.22 μm organic phase microporous filter membrane to obtain the sample solution.

[0017] Technical effects:

[0018] Conventional liquid chromatography-tandem mass spectrometry requires the presence of standard substances for qualitative and quantitative detection. This experiment used high-resolution mass spectrometry to screen out a new chemical substance outside the conventional monitoring range through material analysis in the absence of standard substances.

[0019] The present invention provides a new and effective screening and quantitative method for novel vardenafil derivatives which are lacking of screening and detection methods in food centers.

[0020] The present invention has been described in detail above, but the above embodiments are only illustrative in nature and are not intended to limit the present invention. In addition, this article is not limited by any theory described in the above prior art or invention content or the following examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is a comparison of the characteristic absorption spectra of the unknown derivative and vardenafil.

[0022] Figure 2 This is the first-order mass spectra of the unknown derivative.

[0023] Figure 3 This is the high-resolution mass spectrum of O-propyl hydroxyvardenafil. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with examples. It should be noted that the following examples are provided for illustrative purposes only and do not constitute a limitation on the scope of protection claimed for the present invention.

[0025] Unless otherwise specified, the raw materials, reagents, methods, etc. used in the examples are conventional raw materials, reagents, methods in the art.

[0026] Example

[0027] When inspecting an imported health wine that claims to have aphrodisiac function, we detected a new vardenafil analog using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap tandem mass spectrometry (UPLC-Q-Exactive Orbitrap / MS, abbreviated as UPLC-QExactive) and liquid chromatography-coupled diode array detector (HPLC-PDA). The screening and quantification method of the vardenafil structural analogue includes the following steps:

[0028] 1.1 Discovery of unknown derivatives

[0029] Take 1.00 mL of the sample and add 10 mL of methanol. Ultrasonicate for 30 min. Take an appropriate amount of the test solution and filter it with a 0.22 μm organic filter membrane to use as the HPLC-PDA test solution.

[0030] HPLC instrument conditions: Waters e2695 / 2998 liquid chromatograph, PDA diode array detector: scanning range: 210-400 nm; chromatographic column: Agilent 5HC-C18(2) column (4.6×250 mm, 5 μm); column temperature: 25°C; mobile phase: v (0.1％甲酸水溶液) :v (甲醇) =60:40, isocratic elution, running time: 30min; flow rate: 1mL / min; injection volume: 2μL.

[0031] A chromatographic peak was detected in the liquid chromatogram, and its absorption spectrum was compared with the spectrum of vardenafil. The two were very similar ( Figure 1), both have the characteristic UV absorption spectrum of vardenafil at 230nm, but the retention time is inconsistent. The retention time of vardenafil is 5.391min, and the retention time of the positive sample is 7.903min, which is speculated to be a vardenafil derivative.

[0032] 1.2 Structural inference of unknown derivatives

[0033] Take the above samples and inject them into UPLC-Q Exactive. The UPLC-Q Exactive instrument conditions are as follows:

[0034] (1) Chromatographic conditions: Chromatographic column: Hypersil GOLD C18 column (2.1×100 mm, 1.9 μm); column temperature: 35°C; mobile phase: 0.1% formic acid aqueous solution (containing 10 mmol ammonium acetate) (phase A)-acetonitrile (phase B); gradient elution mode: 0-1 min, 90% A; 1-6 min, 90%-10% A; 6-8 min, 10% A; 8-9 min, 10%-90% A; 9-12 min, 90% A. Flow rate: 0.3 mL / min, injection volume: 2 μL.

[0035] (2) Mass spectrometry conditions: ion source: HESI-II; spray voltage: 3800 V (positive ion mode); vaporization temperature: 350 °C; sheath gas pressure (N2): 35 arb; auxiliary gas pressure (N2): 10 arb; ion transfer tube temperature: 320 °C; mass range: m / z 80-1200; Full MS resolution: 70000; dd-MS 2 Resolution: 17500; (N)CE: 20,40,60.

[0036] During routine screening of samples by high-resolution mass spectrometry, the samples did not contain any of the 90 nafil-like chemicals in the established method (BJS201805), but a quasi-molecular ion peak was found: 519.23962 ([M+H] + ) compounds ( Figure 2 ), and its molecular formula is inferred to be: C 24 H 34 N6O5S, the fragment ions of m / z 99.09206, m / z 151.08563, m / z 284.12717, and m / z 299.11407 in its secondary spectrum are typical fragments of vardenafil. Compared with the quasi-molecular ion peak of vardenafil 489.22192 ([M+H] + ), the mass-to-charge ratio of the quasi-molecular ions of the two differs by 30.00177amu, indicating that the compound has the core structure of vardenafil, but the substituents are slightly different. Compared with the quasi-molecular ion peak of hydroxyvardenafil 505.22277 ([M+H]+ ), the mass-to-charge ratio of the new derivative is 14.01685 amu more, and the fragment ion peak m / z 326.17310 in the new derivative is 14.01492 amu more than the m / z 312.15818 of vardenafil, which is speculated to be the replacement of the H of the phenoxyethyl group of vardenafil by -CH3. The new derivative also has a fragment ion peak of m / z 501.22565, which is 18.01397 amu more than the quasi-molecular ion peak of the new derivative, which is speculated to be hydroxyl substitution.

[0037] Combining the mass spectrometry fragmentation rules of vardenafil and hydroxyvardenafil, and using the precise mass spectrometry molecular weight of the substance, it is speculated that the new derivative may be an ethyl group added to the piperazine ring of vardenafil with a hydroxyl group, and a methyl group added to the phenoxyethyl group to form a phenoxypropyl group. Structurally, it can be called O-propyl hydroxyvardenafil, and the molecular structure is shown in Formula I. According to the fragmentation rule, its fragmentation pathway is shown below.

[0038]

[0039] 1.3 Confirmation of standard substances

[0040] After inferring that its structure is O-propyl hydroxyvardenafil, it was suspected to be vardenafil impurity 31 (CAS: 1339092-18-0) according to the structural formula query, and relevant standard substances were purchased from the market for confirmation. UPLC-QExactive was used to determine the 500μg / mL vardenafil impurity 31 (i.e., O-propyl hydroxyvardenafil) standard solution in the same method as the sample. The primary and secondary mass spectra are shown in the following figure. Figure 3 As shown. Figure 2 and Figure 3 It can be seen that the chromatographic retention time, primary and secondary spectra and high-resolution mass spectra of the standard substance O-propylhydroxyvardenafil are completely consistent with the new derivative in the sample.

[0041] 2 Quantitative detection

[0042] Since this compound is not covered by the current national, industry, and local standards, nor is it included in the detection methods promulgated by relevant departments, except for the structural formula and a small amount of reports on physical and chemical properties such as theoretical density and boiling point, the rest of the reports at home and abroad are almost zero. Therefore, it is necessary to establish a quantitative determination method for this substance and determine the content of O-propylhydroxyvardenafil in the sample to fill the gap in the detection method and provide technical support for food and drug safety supervision.

[0043] 2.1 Sample preparation

[0044] Take 1g or 1mL of food sample in a 50mL volumetric flask, add 40mL of methanol, sonicate for 30 minutes, cool to room temperature, and dilute to the mark with methanol to obtain the extraction solution;

[0045] Purification: Activate the solid phase extraction column with 5 mL of water and 10 mL of methanol in turn. The filler composition of the solid phase extraction column is: 1 g PSA, 0.75 g GCB, 0.25 g C18; accurately pipette 5 mL of the extraction sample solution into the solid phase extraction column, elute with 20 mL of methanol, collect all the effluent in a 50 mL centrifuge tube, concentrate to 1 mL with nitrogen blowing (temperature controlled below 40°C), and pass through a 0.22 μm organic phase microporous filter membrane to obtain the sample solution.

[0046] According to the actual concentration, it can be appropriately diluted with methanol-water solution (volume ratio 1:1) to within the linear range. Take a blank sample that is basically the same or similar to the sample matrix and prepare it in the same way as the corresponding sample to obtain a blank matrix solution. When injecting the sample for determination, the test compound should not be detected.

[0047] O-propyl hydroxyvardenafil is a new synthetic compound, and there are few reports on it at home and abroad. The substance has good solubility in methanol and acetonitrile after testing. This method uses cheap methanol as a solvent. The extraction effects of four extraction methods, namely ultrasonic extraction for 5min, 10min, 15min and 30min, on two matrix spiked samples (concentration 1mg / kg) of health wine and compressed candy were investigated. The results showed that the extraction rate was basically stable after ultrasonic extraction for 15min, and the effect of extending the ultrasonic time on the extraction rate was not significant. Therefore, ultrasonic extraction of samples was used for 15min. Due to the complexity of food matrix, solid phase extraction technology was used to purify it in order to eliminate matrix interference. The homemade solid phase extraction column (filler: 1g PSA, 0.75g GCB, 0.25gC18) can eliminate interferences such as organic acids, pigments and oils, among which PSA can remove pigment and metal ion interference, and GCB can remove pigment interference. For soft capsule samples with high oil content, C18 effectively removes oil. The experiment examined the ratio of each filler, and ultimately the optimal ratio was PSA:GCB:C18=1:0.75:0.25.

[0048] 2.2 Liquid chromatography-tandem mass spectrometry instrument conditions

[0049] Shimadzu LCMS-8050 ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (with electrospray ion source) was used. Liquid chromatography conditions: chromatographic column: ZORBAX SB-Aq RRHD (2.1×100mm, 1.8μm), flow rate: 300μL / min, column temperature: 30℃, injection volume: 2μL, mobile phase: A is 0.1% formic acid aqueous solution, B is methanol, gradient elution mode: 0-1.5min, 90%A-50%A; 1.5-4.5min, 50%A-15%A; 4.5-5min, 15%A; 5-5.1min, 15%A-90%A; 5.1-7min, 90%A. When there are co-eluting components that affect the detection of target compounds, the mobile phase ratio can be appropriately adjusted, or the organic phase can be replaced with methanol to make the chromatographic peak retention time later and separate it from the interfering components as much as possible.

[0050] Mass spectrometry conditions: Shimadzu LCMS-8050 liquid chromatograph-mass spectrometer, electrospray ion source (ESI source); scanning mode: multiple reaction monitoring (MRM); interface temperature: 300°C; solvent temperature: 526°C; DL temperature: 250°C; heating block temperature: 400°C; nebulizing gas flow rate: 3 L / min; heating gas flow rate: 10 L / min; drying gas flow rate: 10 L / min; qualitative and quantitative ion pairs and mass spectrometry analysis parameters are shown in Table 1.

[0051] Table 1 Qualitative and quantitative ion pairs and mass spectrometry analysis parameters of the compounds

[0052]

[0053] 2.2.1 Optimization of liquid phase conditions

[0054] The experiment investigated the commonly used organic mobile phases methanol and acetonitrile for LC-MS. The results showed that under the same conditions, the elution time of acetonitrile was 2.52min and the elution time of methanol was 4.00min. Both could achieve effective separation, but acetonitrile had a faster elution time and a better peak shape. Volatile formic acid was used to adjust the acidity of the aqueous mobile phase to improve the ionization efficiency, and the formic acid concentration was selected to be a lower 0.1%. The mobile phase composition was determined to be 0.1% formic acid aqueous solution-acetonitrile. O-propyl hydroxyvardenafil can be effectively separated under the gradient elution conditions of various brands of C18 columns, but HSS T3 has a better peak shape and higher column efficiency, so HSS T3 column was selected as the analytical column.

[0055] 2.2.1 Optimization of mass spectrometry conditions

[0056] O-propyl hydroxyvardenafil can produce peaks in both positive and negative ion modes, but the positive ion mode is obviously one order of magnitude higher than the negative ion mode, and has better stability. Therefore, this method selects the positive ion mode with high peak response and strong stability. The experiment investigated the responses of 5 major ion pairs: 519.2<99.0, 519.3<151.1, 519.2<283.1, 519.2<284.1 and 519.2<299.1. The results showed that the three groups of ions 519.3<151.1, 519.2<284.1 and 519.2<299.1 had better responses, good peak shapes, no bifurcation, and less interference. 519.2<283.1 had a poor response and could not even be collected on Shimadzu 8050. Although 519.2<99.0 has a better response than 519.30<284.1, it overlaps with the characteristic ion pair 519.2<99.0 of propoxyphenyl hydroxyhomoserine. The two are too close, which affects the ion collection efficiency. After comprehensive consideration, the experiment selected the ion with the highest ion abundance ratio and no interference as the quantitative ion, and took another ion with higher ion abundance and low baseline noise as the qualitative ion pair.

[0057] 2.3 Qualitative and quantitative determination

[0058] The O-propyl hydroxyvardenafil standard was dissolved and diluted with methanol in a 50 mL volumetric flask to a concentration of 200 μg / mL. The standard stock solution was diluted with blank matrix to prepare a series of 1 ng / mL to 50 ng / mL standard working solutions.

[0059] The standard working solution is measured according to the instrument conditions to obtain the chromatographic peak area of ​​the corresponding standard solution. When a chromatographic peak with the same retention time as the chromatographic peak of the standard solution is detected in the sample, and the relative abundance of the qualitative ion pair selected in the sample chromatogram is consistent with the relative abundance of the ions of the standard solution of equivalent concentration, it can be determined that the corresponding compound is detected in the sample. Draw a standard curve with the concentration of the standard working solution as the horizontal axis and the peak area of ​​the chromatographic peak as the vertical axis.

[0060] In the linear range of 1ng / mL to 50ng / mL, the linear relationship between the methanol solvent preparation curve y=22588.72x+3675.231 (r=0.9987) and the blank matrix matching curve y=20464.66x-142.5542 (r=0.9996) is good and can be used for quantitative detection. The slope ratio of the two is 90%, >85%, indicating that the matrix effect of the method in health wine is small, and both methanol and blank matrix solutions can be used to prepare standard curve solutions.

[0061] 2.4 Recovery and precision

[0062] Take 1.00mL of blank matrix sample and place it in a 50mL polypropylene stoppered centrifuge tube. Prepare 1μg / mL standard intermediate solution with methanol as solvent. Accurately pipette 0.1, 0.2, and 1.0mL of 1μg / mL standard intermediate solution and add it to the blank matrix sample. The rest is prepared in the same way as the sample, and the final volume is 50mL. Each level is measured in parallel 6 times and the average value is taken. The results show that the average recovery rates of health wine at 3 different levels are 99.8%, 102.3%, and 104.2%, respectively, and the RSDs are 4.0%, 1.9%, and 4.4%, respectively. It shows that the test results obtained using this method are accurate, reliable, and highly reproducible.

[0063] 2.5 Detection limit

[0064] 1.00mL of blank matrix sample was placed in a 50mL polypropylene stoppered centrifuge tube, and a certain amount of standard solution was added respectively. The samples were prepared in the same way as the samples and then injected. The detection limit was calculated based on a 3-fold signal-to-noise ratio, and the quantification limit was calculated based on a 10-fold signal-to-noise ratio. The measurements were repeated 3 times and the average values ​​were taken. The results showed that the detection limit of O-propyl hydroxyvardenafil was 4μg / mL, and the quantification limit was 10μg / mL.

[0065] 2.6 Actual sample testing

[0066] The above positive samples were tested according to this method, and the results were repeated 3 times to take the average value. The results showed that O-propyl hydroxyvardenafil was detected in this batch of products, with a content of 620.6 mg / L. The results of testing the other 25 batches of commercial health wines that claimed to have aphrodisiac effects showed that O-propyl hydroxyvardenafil was detected in 1 batch, with a content of 34.06 mg / L.

[0067] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents, without departing from the spirit and essence of the claims of the present invention; and these modifications or replacements are still within the scope defined by the claims of the present invention.

Claims

1. A method for screening and quantitative determination of novel vardenafil structural analogs illegally added to food, characterized in that: The following steps are involved: Ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry was used for qualitative and quantitative detection, wherein the liquid chromatography conditions included: chromatographic column: HSS T3 chromatographic column, mobile phase: A was 0.1% formic acid aqueous solution, B was acetonitrile, gradient elution program was: 0-1.5 min, 90% A-50% A; 1.5-4.5 min, 50% A-15% A; 4.5-5 min, 15% A; 5-5.1 min, 15% A-90% A; 5.1-7 min, 90% A; The mass spectrometry conditions included: ion source: ESI, scanning mode: multiple reaction monitoring; interface temperature: 300°C; solvent temperature: 526°C; DL temperature: 250°C; heating block temperature: 400°C; nebulizing gas flow: 3 L / min; heating gas flow: 10 L / min; drying gas flow: 10 L / min; The novel vardenafil structural analog is O-propyl hydroxyvardenafil.

2. The method according to claim 1, characterized in that Liquid chromatography conditions also include: flow rate: 300 μL / min, column temperature: 25-30°C, injection volume: 2 μL.

3. The method according to claim 1, characterized in that The multiple reaction monitoring conditions were as follows: ion pairs with mass-to-charge ratios of 519.3>299.1 and 519.3>284.1 were selected as qualitative ion pairs, and ion pairs with mass-to-charge ratios of 519.3>299.1 were selected as quantitative ion pairs; the Q1 deviation voltage, collision voltage CE and Q3 deviation voltage corresponding to the ion pair 519.3>299.1 were -22 V, -42 V and -20 V, respectively, and the Q1 deviation voltage, collision voltage CE and Q3 deviation voltage corresponding to the ion pair 519.3>284.1 were -24 V, -48 V and -28 V, respectively, and the retention times were all 2.52 min.

4. The method according to claim 1, characterized in that: The specific determination steps include: taking O-propyl hydroxyvardenafil standard substance, preparing a series of standard working solutions with different concentrations, injecting and drawing a standard curve to obtain a linear regression equation, then injecting the sample solution to be tested, and calculating the content of O-propyl hydroxyvardenafil therein by the linear regression equation.

5. The method according to claim 4, characterized in that The sample solution is obtained by the following method: Take 1g or 1mL of food sample in a 50mL volumetric flask, add 40mL of methanol, sonicate for 30 minutes, cool to room temperature, and dilute to the mark with methanol to obtain the extraction solution; Purification: Activate the solid phase extraction column with 5 mL of water and 10 mL of methanol in turn. The filler composition of the solid phase extraction column is: 1 g PSA, 0.75 g GCB, 0.25 g C18; accurately pipette 5 mL of the extraction sample solution into the solid phase extraction column, elute with 20 mL of methanol, collect all the effluent in a 50 mL centrifuge tube, concentrate to 1 mL with nitrogen blowing, and pass through a 0.22 μm organic phase microporous filter membrane to obtain the sample solution.

6. The method according to claim 5, characterized in that Food samples include liquid beverages or alcoholic beverages.

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