Determination method of vardenafil impurity 31

Abstract

The invention discloses a determination method of a vardenafil impurity 31. Relates to the technical field of food detection. The determination method of the vardenafil impurity 31 comprises the following steps: respectively preparing a to-be-determined sample solution and a standard curve solution of the vardenafil impurity 31; and detecting the to-be-detected sample solution and the standard curve solution by adopting an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry method. According to the present invention, the vardenafil impurity 31 detection method is established for the first time, the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is adopted to determine the vardenafil impurity 31, and the method has advantages of simple sample pretreatment mode, fast detection speed, strong specificity, high sensitivity and good repeatability, and is suitable for the rapid detection requirement.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and in particular to a method for determining vardenafil impurity 31. Background Art

[0002] Among many health foods, products with the functions of improving immunity, assisting sleep, and nourishing the kidney and strengthening yang are very popular among people. Therefore, it is not uncommon to see cases of illegal addition of nafil-like substances and their derivatives in health foods, Chinese patent medicines and ordinary foods that explicitly or implicitly claim the above functions. Nafil-like substances are a class of inhibitors of type 5 phosphodiesterase, including sildenafil, tadalafil, vardenafil, etc., which can be used to treat male erectile dysfunction and are prescription drugs that must be used under the guidance of a doctor.

[0003] Instead, various derivatives are produced by modifying the structural core of the non-substance. These derivatives are numerous and difficult to detect. The addition method has changed from large-scale addition to small-scale addition, or the addition of a certain drug derivative, which further increases the difficulty of detection and analysis.

[0004] Among them, vardenafil impurity 31 is a new type of vardenafil derivative, which is obtained by replacing the ethoxy group on the benzene ring of vardenafil with a propoxy group and the ethyl group on the piperazine ring with an ethyl alcohol group. This structurally modified substance is more concealed, and there are no reports on its detection methods at home and abroad, and there are no relevant detection standards available. At the same time, the conventional detection methods applicable to vardenafil cannot specifically detect vardenafil impurity 31, which makes this impurity easily missed in routine inspections. This not only brings challenges to the fight against illegal additives, but also poses a potential threat to consumers' health.

[0005] Based on this, it is urgent to establish a detection method for vardenafil impurity 31 that is simple to operate, highly specific and sensitive, so as to provide technical support for illegal additive detection and provide strong protection for food safety. Summary of the invention

[0006] The object of the present invention is to provide a method for detecting vardenafil impurity 31 which has simple pretreatment operation, strong specificity and high sensitivity.

[0007] A first aspect of the present invention is:

[0008] Provided is a method for determining impurity 31 of vardenafil.

[0009] A second aspect of the present invention is:

[0010] Application of the determination method of the vardenafil impurity 31.

[0011] Specifically, the technical solution adopted according to the first aspect of the present invention is:

[0012] A method for determining vardenafil impurity 31 comprises the following steps:

[0013] preparing a vardenafil impurity 31 sample solution and a standard curve solution;

[0014] Ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry was used to detect the sample solution and the standard curve solution;

[0015] Wherein, the organic phase in the mobile phase system of ultra-high performance liquid chromatography is acetonitrile and / or methanol, and the aqueous phase is formic acid aqueous solution;

[0016] Wherein, the chromatographic column is a C18 chromatographic column;

[0017] Wherein, in the gradient elution program, at 7 to 8 minutes, the volume ratio of the organic phase in the mobile phase system is 100%.

[0018] According to the embodiments of the present invention, one of the technical solutions has at least one of the following advantages or beneficial effects:

[0019] The present invention establishes a method for detecting vardenafil impurity 31 for the first time. The method uses ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry to determine vardenafil impurity 31. The method can perform qualitative and quantitative analysis of vardenafil impurity 31 in various types of food such as candy, beverages, tablets, capsules, oral liquids, etc. within 10 minutes. The sample pretreatment method is simple, the detection speed is fast, the specificity is strong, the sensitivity is high, and the repeatability is good. It is suitable for the needs of rapid detection; at the same time, the method fills the gap in the detection method of vardenafil impurity 31 and provides a scientific and accurate detection method for food safety.

[0020] Among them, the organic phase in the mobile phase system of ultra-high performance liquid chromatography is acetonitrile and / or methanol, and the aqueous phase is formic acid aqueous solution, which can ensure the complete detection of vardenafil impurity 31, and the target peak shape is good and the response value is high.

[0021] Among them, the chromatographic column is a C18 chromatographic column, which can ensure the complete detection of the vardenafil impurity 31.

[0022] Among them, in the gradient elution program, at 7 to 8 minutes, the volume ratio of the organic phase in the mobile phase system is 100%, which can ensure that the vardenafil impurity 31 is completely flushed out.

[0023] When the method of the present invention is used to detect the vardenafil impurity 31, the vardenafil impurity 31 has a good linear relationship in the range of 0.2 to 100 μg / L, and the correlation coefficient (r 2) is greater than 0.99, the detection limit (S / N≥3) is 0.01 mg / kg, and the quantification limit (S / N≥10) is 0.02 mg / kg. The average recovery rate is between 96.84% and 110.77% at three concentration levels of low (0.02 mg / kg), medium (0.04 mg / kg), and high (0.2 mg / kg), and the relative standard deviation (RSD) is between 2.18% and 6.17%. The method of the present invention has the advantages of simplicity, high efficiency, good accuracy, and strong anti-interference ability, and is suitable for accurate qualitative and quantitative analysis of illegally added vardenafil impurity 31 in food.

[0024] According to one embodiment of the present invention, the aqueous phase is a 0.1-0.15% formic acid aqueous solution.

[0025] According to one embodiment of the present invention, the aqueous phase is a 0.1-0.12% formic acid aqueous solution.

[0026] According to one embodiment of the present invention, the aqueous phase is a 0.1% formic acid aqueous solution.

[0027] According to one embodiment of the present invention, the initial water phase volume ratio in the mobile phase system is less than or equal to 95%. Because the C18 used in the present invention is a hydrophobic chromatographic column, when the water phase ratio is greater than 95%, the chromatographic column may collapse, affecting the separation performance, so the initial water phase volume ratio in the mobile phase system of the present invention is less than or equal to 95%.

[0028] According to one embodiment of the present invention, the volume ratio of the initial organic phase to the aqueous phase in the mobile phase system is 5-10:95-90. Under this volume ratio, the peak time of the vardenafil impurity 31 can be avoided to be too early or too late.

[0029] According to one embodiment of the present invention, the volume ratio of the initial organic phase to the aqueous phase in the mobile phase system is 5:95. Under this preferred volume ratio, the peak time of the vardenafil impurity 31 can be ensured to be optimal.

[0030] According to one embodiment of the present invention, the gradient elution procedure is as follows: 0-7 min, 95%-0% aqueous phase; 7-8 min, 0% aqueous phase; 8-8.1 min, 0%-95% aqueous phase; 8.1-10 min, 95% aqueous phase. Only by using the gradient elution procedure of the present invention can the accurate detection of vardenafil impurity 31 be guaranteed.

[0031] According to one embodiment of the present invention, the gradient elution program is as shown in Table 1:

[0032] Table 1

[0033]

[0034] According to one embodiment of the present invention, the conditions of the ultra-high performance liquid chromatography also include a flow rate of 0.2-0.3 mL / min.

[0035] According to one embodiment of the present invention, the conditions of the ultra-high performance liquid chromatography also include a flow rate, and the flow rate is 0.25-0.3 mL / min.

[0036] According to one embodiment of the present invention, the conditions of the ultra-high performance liquid chromatography also include a flow rate, and the flow rate is 0.3 mL / min.

[0037] According to one embodiment of the present invention, the conditions of the ultra-high performance liquid chromatography also include column temperature, and the column temperature is 20-30°C.

[0038] According to one embodiment of the present invention, the conditions of the ultra-high performance liquid chromatography also include column temperature, and the column temperature is 25-30°C.

[0039] According to one embodiment of the present invention, the conditions of the ultra-high performance liquid chromatography also include a column temperature, and the column temperature is 30°C.

[0040] According to one embodiment of the present invention, the ultra-high performance liquid chromatography conditions further include an injection volume, and the injection volume is 1.5-2 μL.

[0041] According to one embodiment of the present invention, the ultra-high performance liquid chromatography conditions further include an injection volume, and the injection volume is 1.8-2 μL.

[0042] According to one embodiment of the present invention, the ultra-high performance liquid chromatography conditions further include an injection volume, and the injection volume is 2 μL.

[0043] According to one embodiment of the present invention, the C18 chromatographic column comprises Waters BEH C18, 2.1×50 mm, 1.7 μm. As the most preferred C18 chromatographic column, Waters BEH C18 has a better separation effect in the method of the present invention, and the retention time of vardenafil impurity 31 is appropriate, and the peak shape is good and free of interference.

[0044] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the capillary voltage is 1.50-3.50 kV. Within this voltage range, the detection of the vardenafil impurity 31 can be guaranteed.

[0045] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the capillary voltage is 1.50 kV, 2.00 kV, 2.50 kV, 3.00 kV or 3.50 kV.

[0046] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the capillary voltage is preferably 2.50 kV, at which time the response value of vardenafil impurity 31 is the largest.

[0047] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the cone voltage is 0-20 V. Within this voltage range, the detection of the vardenafil impurity 31 can be guaranteed.

[0048] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the cone voltage is 0V, 5V, 10V, 15V or 20V.

[0049] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the cone voltage is preferably 10V, at which time the response value of the vardenafil impurity 31 is the largest.

[0050] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the daughter ion collision voltage is 10-50V. For the parent ion 519.24 of vardenafil impurity 31, the present invention limits the daughter ion collision voltage to 10-50V to ensure the appearance of the daughter ion of vardenafil impurity 31. Specifically, when the collision voltage is 10V, the parent ion begins to break and daughter ions appear, but the abundance is very low; as the voltage increases, the daughter ion abundance increases, reaching a peak at a collision voltage of 30V, and then as the voltage increases, the daughter ion abundance decreases.

[0051] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the product ion collision voltage is 10V, 20V, 30V, 40V or 50V.

[0052] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the product ion collision voltage is preferably 30V.

[0053] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the mass spectrometry conditions also include: ion source ESI source; positive ion mode; scanning mode: multiple reaction monitoring; ion source temperature: 100-150°C; desolvation gas temperature: 250-300°C; desolvation flow rate: 600-800L / Hr; cone gas flow rate: 100-150L / Hr; nebulizer pressure: 6.0-7.0bar.

[0054] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the ion source temperature is 150°C.

[0055] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the desolvation temperature is 300°C.

[0056] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the desolvation flow rate is 800 L / Hr.

[0057] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the cone gas flow rate is 150 L / Hr.

[0058] According to one embodiment of the present invention, in the triple quadrupole mass spectrometry, the nebulizer gas pressure is 7.0 bar.

[0059] According to one embodiment of the present invention, the method of the present invention further includes the following steps: mixing and grinding the sample to be tested, adding an extracting solution for ultrasonic extraction, centrifuging, taking the supernatant and filtering the membrane to obtain the sample solution to be tested.

[0060] According to one embodiment of the present invention, in the method of the present invention, when the test sample is solid or semi-solid, the sample is first ground into powder, and an appropriate sample is accurately weighed for preparation; when the test sample is liquid, an appropriate sample is accurately measured for preparation; when the test sample is an oil matrix (soft capsule), the sample is first mixed in an appropriate amount, and an appropriate sample is accurately weighed for preparation.

[0061] According to one embodiment of the present invention, in the method of the present invention, the extraction method of the sample to be tested is: when the test sample is a solid, semi-solid, or liquid sample, methanol is used as the extracting solution and ultrasonic extraction is performed; when the test sample is an oil matrix (soft capsule), ethyl acetate is first added, shaken to disperse it, and then methanol is added for ultrasonic extraction.

[0062] According to one embodiment of the present invention, in the method of the present invention, the time for ultrasonic extraction with methanol is 15-30 minutes, preferably 15 minutes.

[0063] According to one embodiment of the present invention, in the method of the present invention, the sample to be tested is added with an extracting solution for ultrasonic extraction and centrifugation, and the filtrate can be diluted before testing and appropriately diluted with a methanol-water solution (1:1) to a linear range.

[0064] According to one embodiment of the present invention, the preparation method of the standard stock solution of vardenafil impurity 31 comprises the following steps: accurately weighing the vardenafil impurity 31 reference substance, placing it in a volumetric bottle, adding methanol to dissolve and make up the volume, and shaking well to obtain a 100 mg / L standard stock solution.

[0065] According to one embodiment of the present invention, a method for preparing a standard intermediate working solution of vardenafil impurity 31 comprises the following steps: accurately measuring the standard stock solution, diluting it with a methanol-water solution (1:1), and shaking it well to prepare a 1 mg / L standard intermediate working solution.

[0066] According to one embodiment of the present invention, a method for preparing a standard curve solution of vardenafil impurity 31 comprises the following steps: accurately measuring an appropriate amount of the standard intermediate working solution, diluting it with a methanol aqueous solution (1:1), and shaking it well to prepare a standard curve solution having concentrations of 0.2 μg / L, 0.5 μg / L, 1.0 μg / L, 10 μg / L, 50 μg / L, and 100 μg / L, respectively.

[0067] The second aspect of the present invention also relates to the application of the method in detecting vardenafil impurity 31 in food. It includes the method for determining vardenafil impurity 31 as described in the embodiment of the first aspect above. Since the application adopts all the technical solutions of the method for determining vardenafil impurity 31 above, it has at least all the beneficial effects brought by the technical solutions of the above embodiments.

[0068] According to one embodiment of the present invention, the food includes protein powder, oyster powder, biscuits, candy and coffee; the health care products include tablet-type health care products and capsule-type health care products.

[0069] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0071] Figure 1 It is the chromatogram of the vardenafil impurity 31 standard solution in Example 1.

[0072] Figure 2 It is the mass spectrum of the vardenafil impurity 31 standard solution in Example 1 and Examples 3-6.

[0073] Figure 3 It is the mass spectrum of the vardenafil impurity 31 standard solution in Example 1 and Examples 7-10.

[0074] Figure 4 It is the mass spectrum of the vardenafil impurity 31 standard solution in Example 1 and Examples 11-14.

[0075] Figure 5 It is the chromatogram of the standard solution of vardenafil impurity 31 in Comparative Example 5.

[0076] Figure 6 It is the chromatogram of the standard solution of vardenafil impurity 31 in Comparative Example 6.

[0077] Figure 7 It is the chromatogram of the standard solution of vardenafil impurity 31 in Comparative Example 7.

[0078] Figure 8 This is the test chart for optimizing the elution program in Example 1.

[0079] Fig. 9 It is the chromatogram of the standard solution of vardenafil impurity 31 in Comparative Example 8.

[0080] Fig.10 This is a specificity test diagram of the method in Example 1. DETAILED DESCRIPTION

[0081] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0082] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0083] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of the present invention.

[0084] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0085] Sources of reagents and instruments used in the embodiments of the present invention and the comparative examples: vardenafil impurity 31 (purity 95.7%, CAS No.: 1339092-18-0, Shanghai Anpu Cuishi Standard Technology Service Co., Ltd.); acetonitrile and methanol are chromatographically pure (Merck, Germany); the water used in the experiment is Milli-Q ultrapure water; ethyl acetate (chromatographically pure, Thermo Fisher Scientific, USA); formic acid (mass spectrometry pure, Thermo Fisher Scientific, USA); instruments and equipment used: Xevo TQ-Smicro ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometer (Waters, USA); chromatographic workstation is Masslynx V 4.2; XPR226DR / AC electronic balance (Mettler, USA); PS-G60A CNC ultrasonic cleaner (Dongguan Jiekang Ultrasonic Equipment Co., Ltd.); 3-18KS high-speed desktop refrigerated centrifuge (Sigma, USA); Waters ACQUITY UPLC BEH C18 (2.1×50mm, 1.7μm) chromatographic column.

[0086] The pretreatment of the samples in the embodiments of the present invention and the comparative examples comprises the following steps:

[0087] Solid or semi-solid samples: Take an appropriate amount of solid samples (protein powder, oyster powder, biscuits, candy, coffee and health foods containing the same matrix as the above, tablets, capsules and other dosage forms) and mix them evenly, grind them into powder, or take an appropriate amount of semi-solid sample (jelly) and mix them evenly, accurately weigh 1g (accurate to 0.001g) and place it in a 50mL volumetric flask, add an appropriate amount of methanol, ultrasonically extract for 15min, cool to room temperature, make up to volume with methanol, transfer to a 50mL centrifuge tube, centrifuge at 4000r / min for 5min, filter the supernatant through a microporous membrane, take the filtrate, and dilute it appropriately with methanol-water solution (1:1) to the linear range according to the actual concentration, and prepare three replicates in parallel.

[0088] Liquid samples: Take an appropriate amount of sample (beverage, wine), shake well, accurately pipette 1 mL into a 50 mL volumetric flask, add an appropriate amount of methanol, perform ultrasonic extraction for 15 min, cool to room temperature, make up to volume with methanol, filter through a microporous filter membrane, take the filtrate, dilute it appropriately with methanol-water solution (1:1) to the linear range according to the actual concentration, and prepare three replicates in parallel.

[0089] Oil matrix (soft capsule) sample: Take an appropriate amount of sample (soft capsule) and mix well. Accurately weigh 1g (accurate to 0.001g) and place it in a 50mL volumetric flask. Add 5mL of ethyl acetate and shake to disperse it. Add an appropriate amount of methanol and perform ultrasonic extraction for 15min. Cool to room temperature, make up to volume with methanol, transfer to a 50mL centrifuge tube, centrifuge at 4000r / min for 5min, filter the supernatant through a microporous filter membrane, take the filtrate, dilute it appropriately with methanol-water solution (1:1) to the linear range according to the actual concentration, and prepare three replicates in parallel.

[0090] The standard solutions in the examples and comparative examples of the present invention are prepared as follows:

[0091] Standard stock solution: Accurately weigh 1 mg of vardenafil impurity 31 reference substance, place it in a 10 mL volumetric flask, add methanol to dissolve and make up to volume, shake well to obtain a 100 mg / L standard stock solution.

[0092] Standard intermediate working solution: Accurately measure 0.1 mL of the standard stock solution, dilute it to 10 mL with methanol-water solution (1:1), shake well, and prepare a 1 mg / L standard intermediate working solution.

[0093] Standard curve solution: Accurately measure an appropriate amount of the standard intermediate working solution, dilute it with methanol-water solution (1:1), shake well, and use it as the standard curve solution. The concentrations are 0.2μg / L, 0.5μg / L, 1.0μg / L, 10μg / L, 50μg / L, and 100μg / L, respectively.

[0094] In the embodiments and comparative examples, the mass spectrometry conditions are: ion source ESI source; positive ion mode; scanning mode: multiple reaction monitoring; ion source temperature: 150°C; desolvation gas temperature: 300°C; desolvation flow rate: 800L / Hr; cone gas flow rate: 150L / Hr; nebulizer pressure: 7.0bar.

[0095] Example 1

[0096] A method for determining vardenafil impurity 31 comprises the following steps:

[0097] Preparation of the sample solution to be tested: accurately sample, place in a volumetric flask, add methanol, extract at 240W power for 15 minutes, cool to room temperature, make up to volume with methanol, centrifuge at 4000r / min for 5 minutes, take the supernatant and filter through a 0.22μm organic phase microporous filter membrane, and take the filtrate to obtain the sample solution to be tested;

[0098] Preparation of standard curve solution: prepare a standard working solution with a concentration of 1 mg / L using vardenafil impurity 31 standard, and dilute the standard working solution to prepare standard curve solutions with concentrations of 0.2 μg / L, 0.5 μg / L, 1.0 μg / L, 10 μg / L, 50 μg / L, and 100 μg / L;

[0099] Ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry was used to detect the sample solution and the standard curve solution;

[0100] The mobile phase system of the ultra-high performance liquid chromatography is acetonitrile-0.1% formic acid aqueous solution with a volume ratio of 5:95;

[0101] The gradient elution program used is shown in Table 1:

[0102] Table 1

[0103]

[0104] In the gradient elution program, the flow rate was 0.3 mL / min, the column temperature was 30 °C, and the injection volume was 2 μL.

[0105] Among them, the chromatographic column was Waters BEH C18 (2.1×50 mm, 1.7 μm);

[0106] Among them, in the triple quadrupole mass spectrometry, the capillary voltage is 2.50kV; the cone voltage is 10V; and the product ion collision voltage is 30V.

[0107] Among them, the mass spectrometry ion parameters are shown in Table 2:

[0108] Table 2

[0109]

[0110] * indicates quantitative ion

[0111] Example 2

[0112] The difference between Example 2 and Example 1 is that the mobile phase system of the ultra-high performance liquid chromatography in Example 2 is methanol-0.1% formic acid aqueous solution.

[0113] Compared with Example 1, when the organic phase is selected as methanol or acetonitrile, the response value of vardenafil impurity 31 is basically the same.

[0114] Since acetonitrile has a strong elution ability and the sample matrix is ​​complex and difficult to elute, acetonitrile is selected as the organic phase in Example 1 to achieve the best optimization.

[0115] Example 3

[0116] The difference between Example 3 and Example 1 is that in the triple quadrupole mass spectrometry of Example 3, the capillary voltage is 1.50 kV.

[0117] Example 4

[0118] The difference between Example 4 and Example 1 is that in the triple quadrupole mass spectrometry of Example 4, the capillary voltage is 2.00 kV.

[0119] Example 5

[0120] The difference between Example 5 and Example 1 is that in the triple quadrupole mass spectrometry of Example 5, the capillary voltage is 3.00 kV.

[0121] Example 6

[0122] The difference between Example 6 and Example 1 is that in the triple quadrupole mass spectrometry of Example 6, the capillary voltage is 3.50 kV.

[0123] Example 7

[0124] The difference between Example 7 and Example 1 is that in the triple quadrupole mass spectrometry of Example 7, the cone voltage is 0V.

[0125] Example 8

[0126] The difference between Example 8 and Example 1 is that in the triple quadrupole mass spectrometry of Example 8, the cone voltage is 5V.

[0127] Example 9

[0128] The difference between Example 9 and Example 1 is that in the triple quadrupole mass spectrometry of Example 9, the cone voltage is 15V.

[0129] Example 10

[0130] The difference between Example 10 and Example 1 is that in the triple quadrupole mass spectrometry of Example 10, the cone voltage is 20V.

[0131] Embodiment 11

[0132] The difference between Example 11 and Example 1 is that in the triple quadrupole mass spectrometry of Example 11, the daughter ion collision voltage is 10V.

[0133] Example 12

[0134] The difference between Example 12 and Example 1 is that in the triple quadrupole mass spectrometry of Example 12, the daughter ion collision voltage is 20V.

[0135] Embodiment 13

[0136] The difference between Example 13 and Example 1 is that in the triple quadrupole mass spectrometry of Example 13, the daughter ion collision voltage is 40V.

[0137] Embodiment 14

[0138] The difference between Example 14 and Example 1 is that in the triple quadrupole mass spectrometry of Example 14, the daughter ion collision voltage is 50V.

[0139] Comparative Example 1

[0140] The difference between Comparative Example 1 and Example 1 is that in Comparative Example 1, the mobile phase system of ultra-high performance liquid chromatography is acetonitrile-water.

[0141] Compared with Example 1, in Comparative Example 1, since the aqueous phase in Comparative Example 1 is pure water, the peak shape of the target vardenafil impurity 31 is poor, the response value is low, and detection cannot be achieved.

[0142] Comparative Example 2

[0143] The difference between Comparative Example 2 and Example 1 is that in Comparative Example 2, the mobile phase system of ultra-high performance liquid chromatography is methanol-water.

[0144] Compared with Example 1, since the aqueous phase in Comparative Example 2 is pure water, the peak shape of the target vardenafil impurity 31 is poor, the response value is low, and detection cannot be achieved.

[0145] Comparative Example 3

[0146] The difference between Comparative Example 3 and Example 1 is that in Comparative Example 3, the mobile phase system of ultra-high performance liquid chromatography is acetonitrile-5mmol / L ammonium acetate.

[0147] Compared with Example 1, since the aqueous phase in Comparative Example 3 is ammonium acetate, the peak of the target vardenafil impurity 31 is tailing, which affects the mass spectrometry quantification.

[0148] Comparative Example 4

[0149] The difference between Comparative Example 4 and Example 1 is that in Comparative Example 4, the mobile phase system of ultra-high performance liquid chromatography is methanol-5mmol / L ammonium acetate.

[0150] Compared with Example 1, since the aqueous phase in Comparative Example 4 is ammonium acetate, the peak of the target vardenafil impurity 31 is tailing, which affects the mass spectrometry quantification.

[0151] Comparative Example 5

[0152] The difference between Comparative Example 5 and Example 1 is that in Comparative Example 5, the volume ratio of acetonitrile-0.1% formic acid aqueous solution is 10:90.

[0153] Comparative Example 6

[0154] The difference between Comparative Example 6 and Example 1 is that in Comparative Example 6, at 7-8 min of the gradient elution program, the volume ratio of the 0.1% formic acid aqueous solution is not 0%, but 15%, that is, the acetonitrile increase ratio is set at 85%.

[0155] Comparative Example 7

[0156] The difference between Comparative Example 7 and Example 1 is that in Comparative Example 7, at 7-8 min of the gradient elution program, the volume ratio of the 0.1% formic acid aqueous solution is not 0%, but 5%, that is, the acetonitrile rising ratio is set at 95%.

[0157] Comparative Example 8

[0158] The difference between Comparative Example 8 and Example 1 is that in Comparative Example 8, the chromatographic column is Waters HSS T3, 2.1×50 mm, 1.7 μm.

[0159] Performance Testing:

[0160] The determination method of vardenafil impurity 31 in Example 1 was used to determine the 10 μg / L vardenafil impurity 31 standard solution for detection and analysis to obtain qualitative and quantitative ion pair chromatograms. The results are as follows: Figure 1 As shown, under the detection conditions, the chromatographic peak of vardenafil impurity 31 is sharp and symmetrical, with an elution time of about 2.67 min, and there is no interference peak near the elution time of the qualitative and quantitative ion pairs.

[0161] The difference between Example 1 and Examples 3-6 is that the capillary voltage is different. The capillary voltage of Example 1 is 2.50 kV, and the capillary voltages of Examples 3-6 are 1.50 kV, 2.00 kV, 3.00 kV, and 3.50 kV, respectively. Figure 2 The effect of capillary voltage on the detection of vardenafil impurity 31 is shown. It can be seen from the figure that when the cone voltage is 1.50 kV, the response value of vardenafil impurity 31 is 9.45e 3 At 2.00 kV, the response value of vardenafil impurity 31 was 5.03 e 4 At 2.50 kV, the response value of vardenafil impurity 31 was 8.83 e 5 ; At 3.00 kV, the response value of vardenafil impurity 31 was 6.42 e 5 ; At 3.50 kV, the response value of vardenafil impurity 31 was 5.81e 5 The results showed that as the capillary voltage increased, the response value of the parent ion of vardenafil impurity 31 gradually increased. At 2.50 kV, the response value reached the highest. Then, as the capillary voltage increased, the response value decreased. Therefore, a capillary voltage of 2.50 kV was the most preferred.

[0162] The difference between Example 1 and Examples 7-10 is that the cone hole voltage is different, wherein the cone hole voltage of Example 1 is 10V, and the cone hole voltages of Examples 7-10 are 0V, 5V, 15V, and 20V. Figure 3 The effect of cone voltage on the detection of vardenafil impurity 31 is shown. From the figure, it can be seen that when the cone voltage of the mass spectrometer is 0V, the response value of vardenafil impurity 31 is 2.42e 3 At 5V, the response value of vardenafil impurity 31 was 3.17e 4 At 10V, the response value of vardenafil impurity 31 was 1.37e 5 At 15V, the response value of vardenafil impurity 31 was 9.83e 4At 20V, the response value of vardenafil impurity 31 is 8.10e 4 The results showed that as the cone voltage increased, the response value of the parent ion of vardenafil impurity 31 gradually increased. At 10V, the response value reached the highest. Then, as the cone voltage increased, the response value decreased. Therefore, a cone voltage of 10V was the most preferred.

[0163] The difference between Example 1 and Examples 11-14 is that the ion collision voltage is different, wherein the ion collision voltage of Example 1 is 30V, and the ion collision voltages of Examples 11-14 are 10V, 20V, 40V, and 50V, respectively. Figure 4 The effect of ion collision voltage on the detection of vardenafil impurity 31 is shown. It can be seen from the figure that when the ion collision voltage is 10V, the response value of vardenafil impurity 31 is 347; when the ion collision voltage is 20V, the response value of vardenafil impurity 31 is 6.18e 3 At 30V, the response value of vardenafil impurity 31 was 4.13e 5 ; At 40V, the response value of vardenafil impurity 31 was 1.99e 5 ; At 50V, the response value of vardenafil impurity 31 is 2.76e 5 . The results showed that when the collision voltage was 10V, the parent ion began to break and daughter ions appeared, but the abundance was very low; with the increase of voltage, the abundance of daughter ions increased, reaching a peak at a collision voltage of 30V, and then with the increase of voltage, the abundance of daughter ions decreased, so the collision voltage of 30V was the most preferred. It should be noted that when the collision voltage is less than 10V, the parent ion will not be fragmented; when the collision voltage is greater than 50V, the parent ion is completely fragmented into extremely small ions, and the target daughter ions cannot be found, resulting in the inability to detect the vardenafil impurity 31.

[0164] The method of Comparative Example 5 was used to determine the vardenafil impurity 31. Figure 5 As shown, since the volume ratio of acetonitrile-0.1% formic acid aqueous solution is 10:90, the retention time of vardenafil impurity 31 is 1.21 min, and the peak is too early, which is not conducive to quantitative analysis.

[0165] The method of Comparative Example 6 was used to determine the vardenafil impurity 31. The results are as follows: Figure 6 As shown, it can be known that from 0 to 7 minutes, when the proportion of acetonitrile rises to 85%, the impurities cannot be completely flushed out, resulting in a large impurity peak at the end of the sample chromatogram, affecting the accuracy of sample detection.

[0166] The method of Comparative Example 7 was used to determine the vardenafil impurity 31. The results are as follows: Figure 7As shown, it can be known that from 0 to 7 minutes, when the proportion of acetonitrile rises to 95%, the impurities cannot be completely flushed out, resulting in a large impurity peak at the end of the sample chromatogram, affecting the accuracy of sample detection.

[0167] The method of Example 1 was used to determine the vardenafil impurity 31. The results are as follows: Figure 8 As shown, it can be known that when the proportion of acetonitrile rises to 100% during 0-7 minutes and the elution is continued for 1 minute, the sample impurities can be completely eluted.

[0168] The method of Comparative Example 8 was used to measure the vardenafil impurity 31. The measurement results are as follows: Fig. 9 As shown, it can be known that when the HSS T3 column is used, the peak shape of vardenafil impurity 31 is relatively broad, which affects the quantitative analysis.

[0169] The linear range, correlation coefficient, detection limit and quantification limit of the method of the present invention were tested:

[0170] The standard curve solution of vardenafil impurity 31 in Example 1 was measured according to the mass spectrometry detection conditions in Example 1, and the standard curve was drawn with the mass concentration of vardenafil impurity 31 as the horizontal axis and the peak area corresponding to its quantitative ion pair as the vertical axis, and the regression equation and its correlation coefficient were calculated. The results showed that the correlation coefficient (r 2 ) is greater than 0.99, and the linear relationship is good. The detection limit (LOD, S / N=3) and quantification limit (LOQ, S / N=10) of vardenafil impurity 31 were investigated by blank matrix spike method. The detection limit of vardenafil impurity 31 was 0.01 mg / kg, and the quantification limit was 0.02 mg / kg (see Table 3). The detection limit and quantification limit of the present invention are lower than BJS201710 "Detection of 75 Illegally Added Chemical Drugs in Health Foods", which can meet the qualitative and quantitative detection of extremely low content of vardenafil impurity 31 in actual samples.

[0171] Table 3 Linear equation and correlation coefficient (r 2 ), LOD and LOQ

[0172]

[0173] The specificity of the method of the present invention was tested:

[0174] The present invention selects 5 kinds of food blank matrices (candy, substitute tea, beverage, tablet, soft capsule) for spike addition (the amount of vardenafil impurity 31 added is 0.05 mg / kg), and adopts the method of Example 1 for pretreatment and detection analysis to investigate whether each sample matrix and reagent interferes with the measured component. The results show that there are no other impurity peaks in the chromatogram of each matrix sample, indicating that the method has strong specificity (see Fig.10 ).

[0175] The matrix effect of the method of the present invention was tested:

[0176] Five kinds of food blank matrices (candy, substitute tea, beverage, tablet, soft capsule) were selected, and blank matrix solutions were obtained according to the sample pretreatment method of Example 1. The 5 kinds of blank matrix solutions and methanol were used to prepare the standard solution of vardenafil impurity 31 with a concentration of 10 μg / L, and the detection conditions optimized in Example 1 were measured, and the peak areas were recorded 3 times in parallel, and the average values ​​were taken to calculate the different matrix effect values. The results are shown in Table 4. The results show that except for the medium matrix effect of the soft capsule matrix, the other matrices are weak matrix effects, so considering comprehensively, the non-matrix standard curve can be used to quantitatively analyze the target compound.

[0177] Table 4 Matrix effect of vardenafil impurity 31

[0178]

[0179] The recovery and precision of the method of the present invention were tested:

[0180] Three spike levels (0.02, 0.04, 0.2 mg / kg) were added to blank samples (candy, substitute tea, beverage, tablet, soft capsule), and each concentration level was measured 6 times in parallel. The recovery and precision of the method were calculated, and the results are shown in Table 5. From the values ​​in the table, it can be seen that the average recovery of vardenafil impurity 31 in different sample matrices is between 96.84% and 110.77%, and the relative standard deviation (RSD) is between 2.18% and 6.17%, indicating that the method of the present invention has good accuracy and precision.

[0181] Table 5 Average recovery and precision of vardenafil impurity 31 spiked in different sample matrices (n=6)

[0182]

[0183] The method of the present invention is used to test actual samples:

[0184] The detection method established by the present invention was used to detect 29 batches of commercially available samples, including candies, substitute teas, beverages, tablets, and soft capsules. The results showed that among the 29 batches of samples, 5 brands of candies, 1 brand of substitute teas, and 3 brands of beverages were found to contain vardenafil impurity 31. The amount of vardenafil impurity 31 added to solid and semi-solid samples was between 31.01 and 14559.70 μg / kg, and the amount of vardenafil impurity 31 added to liquid samples was between 2903.31 and 641577.30 μg / L. The specific measurement results are shown in Table 6. It can be seen from Table 6 that the detection method provided by the present invention can detect whether vardenafil impurity 31 is added to food, and can also accurately detect the specific content of vardenafil impurity 31, which fully meets the detection requirements of vardenafil impurity 31 in food.

[0185] Table 6 Detection results of vardenafil impurity 31 in 29 samples

[0186]

[0187]

[0188]

[0189] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for determining vardenafil impurity 31, characterized in that: The following steps are involved: preparing a vardenafil impurity 31 sample solution and a standard curve solution; Ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry was used to detect the sample solution and the standard curve solution; Wherein, the organic phase in the mobile phase system of ultra-high performance liquid chromatography is acetonitrile and / or methanol, and the aqueous phase is formic acid aqueous solution; Wherein, the chromatographic column is a C18 chromatographic column; Wherein, in the gradient elution program, at 7 to 8 minutes, the volume ratio of the organic phase in the mobile phase system is 100%.

2. The measuring method according to claim 1, characterized in that The initial water phase volume proportion in the mobile phase system is less than or equal to 95%.

3. The measuring method according to claim 1, characterized in that The volume ratio of the initial organic phase to the aqueous phase in the mobile phase system is 5-10:95-90.

4. The measuring method according to claim 1, characterized in that The gradient elution program is as follows: 0-7 min, 95%-0% aqueous phase; 7-8 min, 0% aqueous phase; 8-8.1 min, 0%-95% aqueous phase; 8.1-10 min, 95% aqueous phase.

5. The measuring method according to claim 1, characterized in that The C18 chromatographic column includes Waters BEH C18, 2.1×50 mm, 1.7 μm.

6. The measuring method according to claim 1, characterized in that In the triple quadrupole mass spectrometry, the capillary voltage is 1.50-3.50 kV.

7. The measuring method according to claim 1, characterized in that In the triple quadrupole mass spectrometry, the cone voltage is 0-20V.

8. The measuring method according to claim 1, characterized in that In the triple quadrupole mass spectrometry, the product ion collision voltage is 10-50V.

9. Use of the method according to any one of claims 1 to 8 in detecting vardenafil impurity 31 in food or health products.

10. The use according to claim 9, characterized in that: The food includes protein powder, oyster powder, biscuits, candy and coffee; the health care products include tablet health care products and capsule health care products.

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