Method for simultaneously determining sildenafil impurity 2 and sildenafil impurity 9 by adopting ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a method for simultaneously determining a sildenafil impurity 2 and a sildenafil impurity 9 by adopting ultra-high performance liquid chromatography-tandem mass spectrometry. Relates to the technical field of detection. The method for simultaneously determining the sildenafil impurity 2 and the sildenafil impurity 9 comprises the following steps: taking a sample to be detected, adding a solution containing dichloromethane and / or acetonitrile, and carrying out solid-liquid separation to obtain a sample solution to be detected; detecting the sample solution to be detected by adopting a liquid chromatography-tandem mass spectrometry method; in the detection process, liquid chromatography conditions are as follows: a mobile phase A is an acidic solution; the mobile phase B is acetonitrile; a separation medium in the chromatographic column is octadecyl silane bonded silica gel. The invention develops a UPLC-MS / MS detection method based on multiple reaction monitoring (MRM). By optimizing a pretreatment reagent and detection conditions, the specific simultaneous determination and identification of the sildenafil impurity 2 and the sildenafil impurity 9 in a complex matrix are realized.

Description

Technical Field

[0001] The present invention relates to the field of inspection and detection technology, and in particular to a method for simultaneously determining sildenafil impurity 2 and sildenafil impurity 9 by ultra-high performance liquid chromatography tandem mass spectrometry. Background Art

[0002] With the significant increase in the incidence of erectile dysfunction (ED), the market for anti-ED drugs continues to expand. PDE-5 inhibitors, represented by sildenafil, have been widely used as effective treatments for male ED. However, some foods, health supplements, and traditional Chinese medicines labeled as "kidney-tonifying and yang-strengthening" are systematically adulterated with PDE-5 inhibitors and their structural modifications that have not been approved by the drug regulatory authorities. These compounds, which have not undergone rigorous safety evaluations, may pose a serious threat to consumers' health and lives.

[0003] The current supplementary inspection method of "Determination of Nafil-like Substances in Food" (BJS201805) has effectively solved the detection problem of the second-generation derivatives by establishing an LC-MS / MS multiple reaction monitoring database of 90 nafil-like derivatives. However, with the development of molecular building block technology and computer-aided drug design, illegal additives have entered the fourth generation. The fourth generation uses modification strategies such as the introduction of halogen substituents (such as flunafil) and cyclic amino side chains (such as cyclohexylaminosildenafil) into the parent nucleus to enable new derivatives to have characteristics such as fast metabolism and low ionization efficiency, thereby circumventing detection. Due to the novelty of their structure, these illegal additives are often outside the coverage of existing detection standards, forming a regulatory blind spot.

[0004] Sildenafil impurity 9 and sildenafil impurity 2, as fourth-generation illegal additives, both undergo key structural modifications to the sildenafil nucleus to evade detection using existing technologies: first, the N-methylpiperazine group at the C4 position of the pyrimidinone ring is deleted; second, a sulfonyl chloride group (-SO2Cl) is introduced to modulate electronic effects. These modifications reduce the polarity of the molecule, significantly increasing the difficulty of detection and reducing both accuracy and sensitivity.

[0005] Sildenafil impurities 2 and 9 may pose potential toxicological risks, particularly to the cardiovascular system. They may cause excessive vascular dilation, leading to symptoms such as hypotension, dizziness, and palpitations, and in severe cases, may even trigger a heart attack or stroke. Furthermore, these two impurities may affect the nervous system, manifesting as headaches and dizziness. Long-term exposure may also impair nerve function, affecting cognitive ability, memory, and emotional stability. Regarding the reproductive system, they may interfere with normal reproductive endocrine function, leading to decreased sperm quality and quantity, and thus infertility. Furthermore, these two impurities may also have toxic effects on the liver and kidneys, increasing the burden on them. Long-term exposure may lead to diseases such as hepatitis and nephritis.

[0006] Furthermore, on March 16, 2012, the "List of Substances That May Be Illegally Added to Health Foods (First Batch)" explicitly stipulated that sildenafil and other pharmaceutical ingredients must not be detected in health foods. As unapproved derivatives, sildenafil impurities 2 and 9 are also strictly prohibited from being added to health foods, foods, or medicines. If these impurities are illegally added, they would violate relevant regulations and pose a serious threat to consumer health. Currently, existing detection standards do not include mass spectrometry databases for these two impurities, and relevant literature lacks reports on their detection methods. This makes the need to detect sildenafil impurities 2 and 9 particularly urgent. Therefore, establishing reliable detection methods is crucial to ensuring effective enforcement of regulations and protecting consumer health. Summary of the Invention

[0007] The purpose of the present invention is to perform simultaneous qualitative and quantitative detection of sildenafil impurity 2 and sildenafil impurity 9 in food and health products.

[0008] A first aspect of the present invention is:

[0009] Provided is a method for simultaneously determining sildenafil impurity 2 and sildenafil impurity 9.

[0010] The second aspect of the present invention is:

[0011] Application of the method for simultaneously determining sildenafil impurity 2 and sildenafil impurity 9.

[0012] Specifically, the technical solution adopted according to the first aspect of the present invention is:

[0013] A method for simultaneously determining sildenafil impurity 2 and sildenafil impurity 9 comprises the following steps:

[0014] Take the sample to be tested, add a solution containing dichloromethane and / or acetonitrile, and perform solid-liquid separation to obtain a solution of the sample to be tested;

[0015] The sample solution is detected by liquid chromatography-tandem mass spectrometry;

[0016] During the detection process, the liquid chromatography conditions include:

[0017] Mobile phase A is an acidic solution;

[0018] Mobile phase B was acetonitrile;

[0019] The separation medium in the chromatographic column is octadecylsilane bonded silica gel;

[0020] The gradient elution program was:

[0021] Table 1

[0022] Time min Mobile phase A% Mobile phase B% 0.0 65-95 5-35 7.0 0 100 9.0 0 100 9.1 65-95 5-35 10.0 65-95 5-35

[0023] According to the embodiments of the present invention, one of the technical solutions has at least one of the following advantages or beneficial effects:

[0024] The present invention develops a UPLC-MS / MS detection method based on multiple reaction monitoring (MRM). By optimizing the pretreatment reagents and detection conditions, the specific simultaneous determination and identification of sildenafil impurity 2 and sildenafil impurity 9 in complex matrices was achieved. Method validation showed that in matrices covering common dosage forms such as beverages, candies, tablets, and capsules, the method sensitivity (LOQ 0.05 mg / kg), precision (RSD < 2.5%) and recovery (90.5% to 115.0%) all met the trace detection requirements. After systematic methodology verification, this technology successfully solved the problem of missed detection of the fourth-generation illegal additives due to their rapid metabolism and difficulty in ionization, and provided a fast, accurate and effective technical method for the systematic screening of new PDE-5 inhibitor structural analogs.

[0025] Specifically:

[0026] First, sildenafil impurity 2 and sildenafil impurity 9 contain sulfonyl chloride groups (-SO2Cl), which are highly active functional groups that are easily hydrolyzed with water in common solvent systems to generate corresponding sulfonic acid (-SO2OH), resulting in changes in the structure of the compound, affecting the specificity and accuracy of detection. Therefore, the present invention uses a solution containing dichloromethane and / or acetonitrile to pre-treat the sample to be tested. Dichloromethane has moderate polarity (dielectric constant is about 8.9) and can better dissolve the compound containing the sulfonyl chloride group, while the polarity of acetonitrile is higher (dielectric constant is about 37.5), which can provide a certain solvation effect and stabilize the electronic structure of the sulfonyl chloride group. The use of dichloromethane and / or acetonitrile significantly reduces the chance of contact between the sulfonyl chloride group and water molecules during the pre-treatment sample extraction process, thereby suppressing the occurrence of hydrolysis reaction. In addition, the chemical inertness of dichloromethane and / or acetonitrile systems also plays an important role in the stability of the sulfonyl chloride group.

[0027] Secondly, sildenafil impurity 2 and sildenafil impurity 9 easily react with the commonly used mobile phase methanol, so the present invention uses acetonitrile as mobile phase B; because the sulfonyl chloride groups of sildenafil impurity 2 and sildenafil impurity 9 will hydrolyze in an alkaline environment, therefore, the mobile phase A of the present invention uses an acidic solution to improve the separation and stability of sildenafil impurity 2 and sildenafil impurity 9;

[0028] Finally, the present invention also improves the sensitivity of sildenafil impurity 2 and sildenafil impurity 9 through a specific gradient elution program and chromatographic column, further promoting the specific simultaneous determination and identification of sildenafil impurity 2 and sildenafil impurity 9 in a complex matrix.

[0029] According to one embodiment of the present invention, the structural formulas of the sildenafil impurity 2 and the sildenafil impurity 9 are:

[0030]

[0031] According to one embodiment of the present invention, the solution containing dichloromethane and / or acetonitrile is a solution containing dichloromethane and acetonitrile.

[0032] According to one embodiment of the present invention, the method for simultaneously determining sildenafil impurity 2 and sildenafil impurity 9 comprises the following steps: taking a sample to be tested, adding a solution containing dichloromethane and acetonitrile, and performing solid-liquid separation to obtain a sample solution to be tested. The combination of dichloromethane and acetonitrile has a synergistic effect of solvent polarity (dichloromethane has moderate polarity, and acetonitrile has high polarity), which significantly reduces the chance of contact between the sulfonyl chloride group and water molecules during the extraction process, thereby inhibiting the occurrence of the hydrolysis reaction.

[0033] Compared to methanol, the combination of dichloromethane and acetonitrile is less likely to chemically react with the sulfonyl chloride groups of sildenafil impurities 2 and 9 under conventional extraction conditions, thus avoiding the problem of solvent-induced byproduct formation. This choice of solvent system not only improves the extraction efficiency and purity of the target compounds, but also ensures the structural integrity of the compounds, providing a more reliable sample basis for subsequent analytical testing.

[0034] The dichloromethane-acetonitrile binary solvent system, through the synergistic effect of solvent polarity and chemical inertness, inhibits the hydrolysis of the sulfonyl chloride group during the extraction process while also circumventing the formation of methyl ester byproducts caused by methanol solvation. This effectively addresses the stability and purity issues of the sulfonyl chloride group during the extraction process, a major innovation and advantage of the present method. The establishment of this pretreatment technology provides key technical support for the trace detection of illegal PDE5 inhibitor additives in health foods, demonstrating significant advantages in addressing the challenges of detecting illegal additions of novel derivatives.

[0035] According to one embodiment of the present invention, in the solution containing dichloromethane and acetonitrile, the volume ratio of dichloromethane to acetonitrile is 1:4-5. Preferably, the volume ratio of dichloromethane to acetonitrile is 1:4.

[0036] According to one embodiment of the present invention, the mobile phase A is an aqueous formic acid solution.

[0037] According to one embodiment of the present invention, the mobile phase A is a formic acid aqueous solution with a mass concentration of 0.1%.

[0038] According to one embodiment of the present invention, the model of the chromatographic column is Waters ACQUITY UPLC BEHC18.

[0039] According to one embodiment of the present invention, the dimensions of the Waters ACQUITY UPLC BEH C18 chromatographic column are: 50 mm×2.1 mm, 1.7 μm.

[0040] According to one embodiment of the present invention, the liquid chromatography conditions further include:

[0041] Flow rate: 0.3-0.4 mL / min;

[0042] and / or, the column temperature is 30-35°C;

[0043] and / or, the sample tray temperature is 10-15°C;

[0044] And / or, the injection volume is 1-2 μL.

[0045] According to one embodiment of the present invention, the gradient elution program is selected from one of Table 2, Table 3 or Table 4:

[0046] Table 2

[0047]

[0048]

[0049] Table 3

[0050] Time min Mobile phase A% Mobile phase B% 0.0 80 20 7.0 0 100 9.0 0 100 9.1 80 20 10.0 80 20

[0051] Table 4

[0052] Time min Mobile phase A% Mobile phase B% 0.0 65 35 7.0 0 100 9.0 0 100 9.1 65 35 10.0 65 35

[0053] According to one embodiment of the present invention, during the detection process, the mass spectrometry conditions include: the ion source is an electrospray ion source.

[0054] According to one embodiment of the present invention, during the detection process, the mass spectrometry conditions include: the ionization mode is a positive mode.

[0055] According to one embodiment of the present invention, during the detection process, the mass spectrometry conditions include: a capillary voltage of 1500-1600V.

[0056] According to one embodiment of the present invention, during the detection process, the mass spectrometry conditions include: an ion source temperature of 150-160°C.

[0057] According to one embodiment of the present invention, during the detection process, the mass spectrometry conditions include: the desolvation temperature is 300-310°C.

[0058] According to one embodiment of the present invention, during the detection process, the mass spectrometry conditions include: a desolvation flow rate of 650-660 L / Hr.

[0059] According to one embodiment of the present invention, during the detection process, the mass spectrometry conditions include: a cone gas flow rate of 150-160 L / Hr.

[0060] According to one embodiment of the present invention, during the detection process, the mass spectrometry conditions include: the cone voltage is 0-20V.

[0061] According to one embodiment of the present invention, during the detection process, the mass spectrometry conditions include: the nebulizer gas pressure is 7.0-8.0 bar.

[0062] According to one embodiment of the present invention, during the detection process, the mass spectrometry conditions include: the scanning mode is multiple reaction monitoring (MRM).

[0063] According to one embodiment of the present invention, during the detection process, the mass spectrometry conditions include: the qualitative and quantitative LC-MS / MS analysis parameters of Sildenafil Impurity 2 and Sildenafil Impurity 9 are shown in Table 5 below:

[0064] Table 5

[0065]

[0066] * indicates quantification ion.

[0067] According to one embodiment of the present invention, the method of taking a sample to be tested, adding a solution containing dichloromethane and / or acetonitrile, and performing solid-liquid separation to obtain a sample solution to be tested specifically includes the following steps: for solid, semi-solid, and liquid samples, accurately weighing 1-1.2 g or 1-1.2 mL of the sample into a volumetric flask, adding 10-12 mL of dichloromethane-acetonitrile (1:4), ultrasonicating, taking out and cooling, and adjusting the volume with dichloromethane-acetonitrile (1:4), and transferring the sample to a centrifuge tube; centrifuging at 4000-4100 r / min, filtering the supernatant through a 0.22-0.25 μm filter membrane, taking the filtrate, and diluting it with dichloromethane-acetonitrile (1:4) according to the actual concentration to the linear range, and preparing three replicates in parallel.

[0068] According to one embodiment of the present invention, when the sample to be tested is a fat matrix (soft capsule) sample, the following method is used for pretreatment: first, the sample is crushed and thoroughly mixed, 1-1.2 g is accurately weighed, placed in a volumetric flask, 5-6 mL of ethyl acetate is added, and then 10-12 mL of dichloromethane-acetonitrile (1:4) is added, ultrasonicated, taken out and cooled, and the volume is fixed with dichloromethane-acetonitrile (1:4), and transferred to a centrifuge tube; centrifuged at 4000-4100 r / min, the supernatant is filtered through a 0.22-0.25 μm filter membrane, and the filtrate is taken. It is diluted with dichloromethane-acetonitrile (1:4) according to the actual concentration to the linear range, and three parallel preparations are made.

[0069] According to one embodiment of the present invention, the sample to be tested includes foods having kidney-tonifying and yang-strengthening effects, such as functional foods and health foods.

[0070] According to one embodiment of the present invention, the liquid chromatography-tandem mass spectrometry method is used to detect the sample solution to be tested, and the process also includes the preparation of a standard, which includes the following steps:

[0071] Prepare standard stock solution: Accurately weigh sildenafil impurity 2 and sildenafil impurity 9 reference substances, place in a volumetric flask, add dichloromethane to dissolve, then add acetonitrile to the volume, shake well, and obtain;

[0072] Prepare standard working solution: accurately measure the standard stock solution and dilute it with acetonitrile to prepare a mixed standard working solution;

[0073] Prepare standard curve solutions: Accurately measure the standard intermediate working solution, dilute it with acetonitrile, and shake well to prepare the standard curve solutions with concentrations of 1.0 μg / L, 2.0 μg / L, 5.0 μg / L, 10 μg / L, 50 μg / L, and 100 μg / L, respectively.

[0074] According to one embodiment of the present invention, the concentration of the standard stock solution is 100-120 μg / mL.

[0075] According to one embodiment of the present invention, the concentration of the standard working solution is 1-2 μg / mL.

[0076] According to one embodiment of the present invention, the concentration of the mixed standard working solution is 1-2 μg / mL.

[0077] Specifically, the technical solution adopted according to the second aspect of the present invention is:

[0078] A method for detecting a PDE-5 inhibitor comprises the method for simultaneously determining sildenafil impurity 2 and sildenafil impurity 9.

[0079] Another aspect of the present invention relates to the use of the method for simultaneously determining sildenafil impurity 2 and sildenafil impurity 9 in food testing. This method includes the determination method described in the embodiment of the first aspect. Because this application utilizes all the technical solutions of the above-described determination method, it at least has all the beneficial effects brought about by the technical solutions of the above-described embodiment.

[0080] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0082] Figure 1 The chromatograms are those of Example 1-3 and Comparative Example 1-2.

[0083] Figure 2 It is the chromatogram of Example 1 and Comparative Examples 3-4.

[0084] Figure 3 The chromatograms are those of Example 1 and Example 4-5.

[0085] Figure 4 The chromatograms are shown in Figures 1 and 6-9.

[0086] Figure 5 10-13 are chromatograms of Examples 10-13.

[0087] Figure 6 This is a collision energy optimization test diagram for sildenafil impurities 2 and 9 in Example 14.

[0088] Figure 7 The qualitative and quantitative ion spectra of sildenafil impurities 2 and 9 in Example 14 are shown.

[0089] Figure 8 The specificity test charts for 5 kinds of food blank matrices. DETAILED DESCRIPTION

[0090] The terms "preferred," "more preferred," and the like, used herein refer to embodiments of the present invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are unsuitable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0091] When a numerical range is disclosed herein, the range is considered 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 a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0092] 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 embodiments described are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of the present invention.

[0093] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0094] Sources of reagents and instruments used in the Examples and Comparative Examples:

[0095] Sildenafil impurity 2 (purity 99.4%, CAS number 139756-22-2, Shenzhen Hengfeng Wanda Pharmaceutical Technology Co., Ltd.);

[0096] Sildenafil impurity 9 (purity 96.3%, CAS number 139756-31-3, Shenzhen Hengfeng Wanda Pharmaceutical Technology Co., Ltd.);

[0097] Acetonitrile was chromatographically pure and purchased from Merck, Germany;

[0098] The experimental water was Milli-Q ultrapure water;

[0099] Dichloromethane, ethyl acetate (chromatographic grade), and formic acid (mass spectrometry grade) were from Thermo Fisher Scientific, USA;

[0100] Experimental samples: Xiongqi candy, oyster peptide compressed candy, ginseng oyster deer penis compressed candy, oyster essence compressed candy, peptide coffee fruit, deer ginseng candy, ginseng deer blood sugar, oyster ginseng candy, Lubao sea ginseng peptide compressed candy, Chang Huanyu ginseng candy, deer penis candy, Zhanlikang compressed candy, Longba coffee flavored solid beverage, coffee powder, oyster peptide coffee solid beverage, Cistanche deserticola oyster herbal coffee solid beverage, golden wolfberry solid beverage, Youjiukang oligopeptide solid beverage, ginseng wolfberry plant beverage, ginseng oyster peptide plant beverage, Hungry Deer No. 7 beverage, ginseng Cordyceps militaris and Polygonatum tablets, ginseng maca oyster tablets, deer blood oyster ginseng tablets, Lubao Cordyceps Essence Tablets, Miao Erguo ginseng oyster tablets, deer blood oyster tablets, propolis soft capsules are all samples sent by the manufacturer for inspection.

[0101] Instruments and equipment include: Xevo TQ-Smicro ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometer from Waters, USA;

[0102] XPR226DR / AC electronic balance from Mettler, USA;

[0103] PS-G60A CNC ultrasonic cleaner from Dongguan Jiekang Ultrasonic Equipment Co., Ltd.;

[0104] 3-18KS high-speed desktop refrigerated centrifuge from Sigma, USA;

[0105] and a Waters ACQUITY UPLC BEH C18 column (specifications: 2.1 mm × 50 mm, particle size: 1.7 μm).

[0106] In the Examples and Comparative Examples, the preparation of the standard stock solution (100 μg / mL) comprises the following steps:

[0107] Accurately weigh 1 mg of sildenafil impurity 2 and sildenafil impurity 9 reference substances, place them in a 10 mL volumetric flask, add 2 mL of dichloromethane to dissolve them, then add acetonitrile to the volume, shake well, and obtain the products.

[0108] In the Examples and Comparative Examples, the preparation of the standard working solution (1 μg / mL) comprises the following steps:

[0109] Accurately measure 0.1 mL of the standard stock solution (100 μg / mL), dilute it to 10 mL with acetonitrile, and shake well to prepare a 1 μg / mL mixed standard working solution.

[0110] In the Examples and Comparative Examples, the preparation of the standard curve solution comprises the following steps:

[0111] Accurately measure an appropriate amount of the standard intermediate working solution (1 μg / mL), dilute it with acetonitrile, and shake well to prepare the standard curve solution with concentrations of 1.0 μg / L, 2.0 μg / L, 5.0 μg / L, 10 μg / L, 50 μg / L, and 100 μg / L, respectively.

[0112] Example 1

[0113] A method for simultaneously determining sildenafil impurity 2 and sildenafil impurity 9 comprises the following steps:

[0114] Accurately weigh 1 g of the sample to be tested into a 50 mL volumetric flask, add 10 mL of dichloromethane-acetonitrile (volume ratio of 1:4), sonicate for 15 min, remove and cool, dilute to the mark with dichloromethane-acetonitrile, transfer the sample to a centrifuge tube and centrifuge at 4000 r / min for 5 min, take the supernatant and pass it through a 0.22 μm filter membrane, take the filtrate, and dilute it with dichloromethane-acetonitrile to the linear range. Prepare three replicates in parallel.

[0115] The sample solution is detected by liquid chromatography-tandem mass spectrometry;

[0116] During the detection process, the liquid chromatography conditions include:

[0117] Mobile phase A: 0.1% formic acid in water;

[0118] Mobile phase B: acetonitrile;

[0119] Chromatographic column: Waters ACQUITY UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm);

[0120] The gradient system program used is:

[0121] Table 3

[0122] Time (min) Mobile phase A (%) Mobile phase B (%) 0.0 80 20 7.0 0 100 9.0 0 100 9.1 80 20 10.0 80 20

[0123] Flow rate: 0.3 mL / min;

[0124] Column temperature: 30°C;

[0125] Sample tray temperature: 10°C;

[0126] Injection volume: 1 μL;

[0127] During the detection process, the mass spectrometry conditions include:

[0128] The ion source was electrospray ionization (ESI);

[0129] Ionization mode: positive mode;

[0130] Capillary voltage: 1500V;

[0131] Ion source temperature: 150°C;

[0132] Desolvation temperature: 300°C;

[0133] Desolventization flow rate: 650L / Hr;

[0134] Cone hole air flow rate: 150L / Hr;

[0135] Sprayer air pressure: 7.0 bar;

[0136] Cone voltage 20V;

[0137] Scanning mode: ion monitoring (SIR) mode;

[0138] The qualitative and quantitative LC-MS / MS analysis parameters for Sildenafil Impurity 2 and Sildenafil Impurity 9 are shown in Table 5 below:

[0139] Table 5

[0140]

[0141]

[0142] * indicates quantification ion

[0143] Example 2

[0144] The difference between Example 2 and Example 1 is that in Example 2, the sample to be tested is extracted with a dichloromethane solution.

[0145] Example 3

[0146] The difference between Example 3 and Example 1 is that in Example 3, the sample to be tested is extracted with acetonitrile.

[0147] Example 4

[0148] The difference between Example 4 and Example 1 is that the gradient system program adopted in Example 4 is different.

[0149] Table 2

[0150] Time (min) Mobile phase A (%) Mobile phase B (%) 0.0 95 5 7.0 0 100 9.0 0 100 9.1 95 5 10.0 95 5

[0151] Example 5

[0152] The difference between Example 5 and Example 1 is that the gradient system program adopted in Example 5 is different.

[0153] Table 4

[0154] Time (min) Mobile phase A (%) Mobile phase B (%) 0.0 65 35 7.0 0 100 9.0 0 100 9.1 65 35 10.0 65 35

[0155] Example 6

[0156] The difference between Example 6 and Example 1 is that the mass spectrometry conditions used in Example 6 are different. Among them, the mass spectrometry conditions of Example 6 include:

[0157] Capillary voltage: 2000V;

[0158] Ion source temperature: 150°C;

[0159] Desolvation temperature: 300°C;

[0160] Desolventization flow rate: 650L / Hr;

[0161] Cone hole air flow rate: 150L / Hr;

[0162] Sprayer air pressure: 7.0 bar;

[0163] Cone voltage 20V.

[0164] Example 7

[0165] The difference between Example 7 and Example 1 is that the mass spectrometry conditions used in Example 7 are different. Among them, the mass spectrometry conditions of Example 7 include:

[0166] Capillary voltage: 2500V;

[0167] Ion source temperature: 150°C;

[0168] Desolvation temperature: 300°C;

[0169] Desolventization flow rate: 650L / Hr;

[0170] Cone hole air flow rate: 150L / Hr;

[0171] Sprayer air pressure: 7.0 bar;

[0172] Cone voltage 20V.

[0173] Example 8

[0174] The difference between Example 8 and Example 1 is that the mass spectrometry conditions used in Example 8 are different. The mass spectrometry conditions in Example 8 include:

[0175] Capillary voltage: 3000V;

[0176] Ion source temperature: 150°C;

[0177] Desolvation temperature: 300°C;

[0178] Desolventization flow rate: 650L / Hr;

[0179] Cone hole air flow rate: 150L / Hr;

[0180] Sprayer air pressure: 7.0 bar;

[0181] Cone voltage 20V.

[0182] Example 9

[0183] The difference between Example 9 and Example 1 is that the mass spectrometry conditions used in Example 9 are different. The mass spectrometry conditions in Example 9 include:

[0184] Capillary voltage: 3500V;

[0185] Ion source temperature: 150°C;

[0186] Desolvation temperature: 300°C;

[0187] Desolventization flow rate: 650L / Hr;

[0188] Cone hole air flow rate: 150L / Hr;

[0189] Sprayer air pressure: 7.0 bar;

[0190] Cone voltage 20V.

[0191] Example 10

[0192] The difference between Example 10 and Example 1 is that the mass spectrometry conditions used in Example 10 are different. The mass spectrometry conditions in Example 10 include:

[0193] Capillary voltage: 1500V;

[0194] Ion source temperature: 150°C;

[0195] Desolvation temperature: 300°C;

[0196] Desolventization flow rate: 650L / Hr;

[0197] Cone hole air flow rate: 150L / Hr;

[0198] Sprayer air pressure: 7.0 bar;

[0199] Cone voltage 0V.

[0200] Example 11

[0201] The difference between Example 11 and Example 1 is that the mass spectrometry conditions used in Example 11 are different. The mass spectrometry conditions in Example 11 include:

[0202] Capillary voltage: 1500V;

[0203] Ion source temperature: 150°C;

[0204] Desolvation temperature: 300°C;

[0205] Desolventization flow rate: 650L / Hr;

[0206] Cone hole air flow rate: 150L / Hr;

[0207] Sprayer air pressure: 7.0 bar;

[0208] Cone voltage 5V.

[0209] Example 12

[0210] The difference between Example 12 and Example 1 is that the mass spectrometry conditions used in Example 12 are different. The mass spectrometry conditions in Example 12 include:

[0211] Capillary voltage: 1500V;

[0212] Ion source temperature: 150°C;

[0213] Desolvation temperature: 300°C;

[0214] Desolventization flow rate: 650L / Hr;

[0215] Cone hole air flow rate: 150L / Hr;

[0216] Sprayer air pressure: 7.0 bar;

[0217] Cone voltage 10V.

[0218] Example 13

[0219] The difference between Example 13 and Example 1 is that the mass spectrometry conditions used in Example 13 are different. The mass spectrometry conditions in Example 13 include:

[0220] Capillary voltage: 1500V;

[0221] Ion source temperature: 150°C;

[0222] Desolvation temperature: 300°C;

[0223] Desolventization flow rate: 650L / Hr;

[0224] Cone hole air flow rate: 150L / Hr;

[0225] Sprayer air pressure: 7.0 bar;

[0226] Cone voltage 15V.

[0227] Example 14

[0228] Example 14 Based on Example 1, the daughter ion collision energy of sildenafil impurities 2 and 9 was further optimized.

[0229] Specifically:

[0230] The parent ions of sildenafil impurity 2 and sildenafil impurity 9 were subjected to secondary mass spectrometry full scan (MS2Scan) to obtain fragment ion information, and the two daughter ions with the best response were selected as characteristic fragment ions, among which the daughter ion with the highest response value was used as the quantitative ion.

[0231] Finally, the collision energy was optimized in the multiple reaction monitoring (MRM) mode, and the collision energy with the best response of the two product ions was determined to be 30 V for the sildenafil impurity 2 product ion 283.3, 20 V for the sildenafil impurity 2 product ion 311.2, 30 V for the sildenafil impurity 9 product ion 297.2, and 20 V for the sildenafil impurity 9 product ion 369.2. Figure 6 .

[0232] The optimal ion conditions for Sildenafil Impurity 2 and Sildenafil Impurity 9 were obtained by optimization (Table 6). The qualitative and quantitative ion spectra of Sildenafil Impurity 2 and Sildenafil Impurity 9 are shown in Table 6. Figure 7 .

[0233] Table 6

[0234]

[0235] Comparative Example 1

[0236] The difference between Comparative Example 1 and Example 1 is that in Comparative Example 1, the sample to be tested is extracted with water.

[0237] Comparative Example 2

[0238] The difference between Comparative Example 2 and Example 1 is that in Comparative Example 2, the sample to be tested is extracted with methanol.

[0239] Comparative Example 3

[0240] The difference between Comparative Example 3 and Example 1 is that in Comparative Example 3, the mobile phase A is water.

[0241] Comparative Example 4

[0242] The difference between Comparative Example 4 and Example 1 is that in Comparative Example 4, mobile phase A is 10 mM ammonium formate.

[0243] Performance testing:

[0244] 1. Example 1-3 and Comparative Example 1-2 used different extraction solvents, and the recoveries of sildenafil impurity 2 and sildenafil impurity 9 in Example 1-3 and Comparative Example 1-2 were tested. The test results are shown in Table 7.

[0245] Table 7

[0246]

[0247] It can be seen from Table 7 that when the aqueous phase of Comparative Example 1 was used to extract the test sample, the sulfonyl chloride groups of sildenafil impurities 2 and 9 underwent significant hydrolysis reaction in the polar medium ( Figure 1 ), the recovery rate dropped to 6.3-6.6%;

[0248] In the methanol system of Comparative Example 2, sulfonyl chloride reacts with methanol to generate the corresponding sulfonate product. This reaction will cause the peak of the original compound to become smaller, making it difficult to accurately quantify. In addition, the generated methyl ester peak may overlap with other component peaks, further interfering with quantitative analysis ( Figure 1 ), and at the same time, the recovery rate dropped to 69.3-69.5% due to the partial conversion of the original compound into derivatives;

[0249] Example 3 Acetonitrile is used as the extraction solvent to maintain the chemical stability of the target compound ( Figure 1 ), but the extraction efficiency of the target compound was only 84.4-86.9%.

[0250] Although the single dichloromethane extraction in Example 2 can effectively maintain the stability of the compound (degradation products <10%, Figure 1 ), but the extract was turbid and the recovery rate was 90.5-91.5%;

[0251] In Example 1, when dichloromethane-acetonitrile (1:4, v / v) was used as the extraction solvent, the recovery rate was increased to 98.1%, the solution was clear, and the matrix effect index was optimized to 0.8-1.2, achieving a balance between chemical stability and extraction efficiency.

[0252] 2. In Example 1 and Comparative Examples 3-4, different mobile phases A were used. The liquid chromatography of Example 1 and Comparative Examples 3-4 was as follows: Figure 2 As shown. Figure 2It is known that comparative example 4 adopts 10mM ammonium formate-acetonitrile system, and the sensitivity of sildenafil impurity 2 and sildenafil impurity 9 is significantly reduced, and it is speculated that its sulfonyl chloride group is hydrolyzed under alkaline environment; comparative example 3 adopts water-acetonitrile system, and the sensitivity of sildenafil impurity 2 and sildenafil impurity 9 is improved relative to comparative example 4, and sildenafil impurity 2 and sildenafil impurity 9 separation degree increase, but considering that sildenafil impurity 2 and sildenafil impurity 9 are more stable under acidic conditions, it is considered to add a suitable concentration of formic acid in the aqueous phase (i.e., obtain the mobile phase A of Example 1). At the same time, in an acidic environment, the applicability of other acids such as acetic acid and trifluoroacetic acid (TFA) is also considered, but it is found that although acetic acid and TFA can also provide an acidic environment, in actual operation, the 0.1% formic acid aqueous solution used in the mobile phase A of Example 1 is more outstanding in improving peak shape and improving separation efficiency. Therefore, 0.1% formic acid aqueous solution-acetonitrile system is the optimal mobile phase system.

[0253] 3. In Example 1 and Examples 4-5, different gradient elution procedures were used, and the corresponding chromatograms are shown in FIG. Figure 3 As shown. Figure 3 It is known that Examples 1 and 4-5 can all achieve the separation of sildenafil impurity 2 and sildenafil impurity 9. The sensitivity of sildenafil impurity 2 and sildenafil impurity 9 corresponding to Example 4 is slightly reduced; the separation of sildenafil impurity 2 and sildenafil impurity 9 corresponding to Example 5 is relatively poor; the sensitivity and separation of sildenafil impurity 2 and sildenafil impurity 9 corresponding to Example 1 are good.

[0254] 4. In Examples 1 and 6-13, different mass spectrometry parameters were used, and the corresponding chromatograms are shown in FIG. Figure 4-5 As shown. Figure 4 It is known that in the extracted ion chromatograms corresponding to Examples 1 to 6-9, when Method 1 is used, the response values ​​of Sildenafil Impurity 2 and Sildenafil Impurity 9 reach the highest. Figure 5 The extracted ion current chromatograms corresponding to Examples 1 to 10-13 are shown in FIG. Figure 5 It is known that the sensitivity of sildenafil impurity 2 reaches the highest in Example 13, and the sensitivity of sildenafil impurity 9 reaches the highest in Example 1. Therefore, the mass spectrometry parameters corresponding to Example 13 and Example 1 are respectively used as the optimal conditions for sildenafil impurity 2 and sildenafil impurity 9.

[0255] 5. Testing the specificity of the method of Example 1

[0256] The present invention selected five food blank matrices (candy, tea substitute, beverage, tablet, soft capsule) for spike addition and used the method of Example 1 for detection and analysis to investigate whether each sample matrix and reagent interfered with the measured components. The results showed that there were no other impurity peaks in the chromatograms of each matrix sample, indicating that the method has strong specificity (see Figure 8 ).

[0257] 6. Test for Matrix Effects

[0258] Five food blank matrices (candy, tea substitute, beverage, tablet, and soft capsule) were selected and pretreated with dichloromethane-acetonitrile according to the method of Example 1 to obtain blank matrix solutions. Sildenafil impurity 2 and sildenafil impurity 9 standard solutions were diluted (1:1) with the five blank matrix solutions (concentration after dilution was 10 μg / L). The assay was performed according to the detection conditions of Example 1, with three replicates. Peak areas were recorded and averaged to calculate different matrix effect values. The results are shown in Table 8. The results showed that all five food blank matrices showed weak matrix effects. Therefore, considering all factors, a non-matrix standard curve can be used for quantitative analysis of the target compounds.

[0259] Table 8

[0260] Compound Matrix candy Substitute tea drinks tablet soft capsules Sildenafil Impurity 2 1.2 0.8 0.8 0.8 0.9 Sildenafil Impurity 9 1.2 0.9 0.9 0.9 1.1

[0261] 7. Test the linear range, correlation coefficient, detection limit and quantification limit

[0262] The standard curve solutions of sildenafil impurity 2 and sildenafil impurity 9 were measured according to the mass spectrometry detection conditions optimized in Example 1. The standard curve was drawn with the mass concentration of sildenafil impurity 2 and sildenafil impurity 9 as the abscissa and the peak area corresponding to the quantitative ion pair as the ordinate, and the regression equation and its correlation coefficient were calculated. The test results are shown in Table 9.

[0263] The results in Table 9 show that the correlation coefficients (R2) of sildenafil impurity 2 and sildenafil impurity 9 are greater than 0.99 in the concentration range of 1.0 μg / L to 100 μg / L, and the linear relationship is good. The detection limit (LOD, S / N=3) and quantification limit (LOQ, S / N=10) of sildenafil impurity 2 and sildenafil impurity 9 were investigated by the blank matrix spike method. The experimental results showed that the detection limit of sildenafil impurity 2 and sildenafil impurity 9 was 0.025 mg / kg and the quantification limit was 0.05 mg / kg. It can be seen from Table 9 that the detection limit and quantification limit of the present invention are lower than BJS 201710 "Detection of 75 Illegally Added Chemical Drugs in Health Foods", which can meet the qualitative and quantitative detection of extremely low levels of sildenafil impurity 2 and sildenafil impurity 9 in actual samples.

[0264] Table 9

[0265]

[0266] 8. Test the recovery and precision

[0267] Three spike levels (0.05 mg / kg, 0.5 mg / kg, 5.0 mg / kg) were added to blank samples (candy, substitute tea, beverages, tablets, soft capsules). Each concentration level was measured three times in parallel. The recovery and precision of the method were calculated. The results are shown in Table 10.

[0268] From the values ​​in Table 10, it can be seen that the average recoveries of sildenafil impurity 2 and sildenafil impurity 9 in different sample matrices are between 96.84% and 110.77%, and the relative standard deviations (RSDs) are between 2.18% and 6.17%, indicating that the method of the present invention has good accuracy and precision.

[0269] Table 10

[0270]

[0271]

[0272] 9. Test actual samples

[0273] The method of Example 1 of the present invention was used to test 29 batches of commercially available samples. The matrices included candies, tea substitutes, beverages, tablets, and soft capsules. The results showed that among the 29 batches of samples, 6 brands of candies, 6 brands of tea substitutes, and 1 brand of beverages were found to contain sildenafil impurities 2 and 9. The addition amounts of sildenafil impurities 2 and 9 in solid and semisolid samples were between 27.3 and 60.8 μg / kg, and the addition amounts of sildenafil impurities 2 and 9 in liquid samples were 31.3 μg / L. The specific measurement results are shown in Table 11.

[0274] Table 11

[0275]

[0276]

[0277] The above are merely 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 simultaneously determining sildenafil impurity 2 and sildenafil impurity 9, characterized in that: The following steps are involved: Take the sample to be tested, add a solution containing dichloromethane and / or acetonitrile, and perform solid-liquid separation to obtain a solution of the sample to be tested; The sample solution is detected by liquid chromatography-tandem mass spectrometry; During the detection process, the liquid chromatography conditions include: Mobile phase A is an acidic solution; Mobile phase B was acetonitrile; The separation medium in the chromatographic column is octadecylsilane bonded silica gel; The gradient elution program was: Table 1 2. The method according to claim 1, wherein: The solution containing dichloromethane and / or acetonitrile is a solution containing dichloromethane and acetonitrile.

3. The method according to claim 2, wherein: In the solution containing dichloromethane and acetonitrile, the volume ratio of dichloromethane to acetonitrile is 1:4-5.

4. The method according to claim 1, wherein: The mobile phase A is a formic acid aqueous solution.

5. The method according to claim 1, wherein: The model of the chromatographic column is Waters ACQUITYUPLC BEH C18.

6. The method according to claim 1, wherein: The liquid chromatography conditions also include: Flow rate: 0.3-0.4 mL / min; and / or, the column temperature is 30-35°C; and / or, the sample tray temperature is 10-15°C; And / or, the injection volume is 1-2 μL.

7. The method according to claim 1, wherein: The gradient elution program is selected from one of Table 2, Table 3 or Table 4: Table 2 Table 3 Table 4 8. The method according to claim 1, wherein: During the detection process, the mass spectrometry conditions include: the qualitative and quantitative LC-MS / MS analysis parameters of Sildenafil Impurity 2 and Sildenafil Impurity 9 are shown in Table 5 below: Table 5 * indicates quantification ion.

9. The method according to claim 1, wherein: The mass spectrometry conditions include: cone voltage is 0-20V.

10. Use of the method for simultaneously determining sildenafil impurity 2 and sildenafil impurity 9 according to any one of claims 1 to 9 in food testing.

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