Method for detecting tadalafil analogue

Abstract

The invention relates to the technical field of inspection and detection, in particular to a method for detecting tadalafil analogues. According to the method disclosed by the invention, the tadalafil impurity O, the EP impurity 1 and the impurity 82 in the sample are rapidly screened and quantitatively detected by adopting UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry). Extracting and purifying the sample by adopting a four-salt dispersion solid-phase extraction method; a C18 chromatographic column is adopted, 5-20 mM ammonium formate-acetonitrile is used as a mobile phase for gradient elution, and an MRM mode is combined for detection. A verification result of the method shows that the linear relation of target impurities is good, the quantitation limit of oral liquid is 0.02 mg / kg, the detection limit is as low as 0.007 mg / kg, the quantitation limit of a semi-solid preparation and a solid preparation is 0.05 mg / kg, the detection limit is as low as 0.02 mg / kg, and the average recovery rate reaches 80%-116% (RSDlt; 5.3%).

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of inspection and detection, and in particular to a method for detecting tadalafil analogues. BACKGROUND

[0002] In recent years, the phenomenon of illegally adding drug analogues in health food has been repeated, especially in products claiming to have the functions of "tonifying kidney and invigorating yang" and "anti-fatigue", unapproved chemical substances are often detected. The current detection standard (such as BJS202405, BJS201710) focuses on traditional tadalafil substances such as sildenafil, vardenafil and some tadalafil mother nucleus derivatives (such as desmethyl tadalafil), and the list of compounds is mainly based on the characteristics of illegal additives before 2018. However, in recent years, the illegal addition behavior presents a "structural avoidance" trend, and new modified impurities (such as tadalafil impurity O, tadalafil EP impurity 1, tadalafil impurity 82) escape supervision through directional modification of key pharmacophore groups, for example, tadalafil impurity O (CAS: 2169996-11-4, 3 ketone is replaced by methoxy carboxylate group, the polarity is significantly reduced, which causes the retention time of tadalafil impurity O on C18 chromatographic column to be prolonged); tadalafil EP impurity 1 (CAS: 2378614-29-8, indole ring is introduced into a spiro ring structure, molecular rigidity is enhanced, and the characteristic fragment ion is completely deviated from the original drug spectrum fragmentation path); tadalafil impurity 82 (CAS: 629652-67-1, 2-methyl is replaced by cyclohexyl, hydrophobicity is improved, and the risk of co-elution with fat-soluble substrates (such as maca extract) in the conventional acetonitrile-water elution system is increased).

[0003] The analogues of tadalafil (such as tadalafil impurity O, tadalafil EP impurity 1, tadalafil impurity 82, etc.) have similar structures to the original drug, and are often illegally added to enhance the effect of the product, but such substances may cause cardiovascular problems such as hypotension, arrhythmia, and even shock, heart attack or sudden cardiac death in severe cases; may also cause nervous system symptoms such as headache, dizziness, sensory reduction, insomnia and sleepiness; may cause gastrointestinal reactions such as indigestion, abdominal pain and diarrhea in the digestive system; the visual system may also be damaged, showing blurred vision, eye pain, etc., and severe cases may have permanent vision loss. In addition, it may also increase the burden on the liver and kidneys, leading to abnormal liver and kidney function. Such analogues are not approved for medicinal use, but due to their concealment, they have become a regulatory difficulty. At the same time, such impurities are often illegally added in trace amounts to complex substrates, and the existing standards and literature have not reported a method for simultaneously detecting the three substances. Therefore, developing a specific detection method for the above-mentioned three tadalafil impurities has important significance for filling the standard gap, blocking the technical loopholes of illegal addition, and protecting the health rights and interests of consumers. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes a method for detecting tadalafil analogs, filling the gap in the existing detection standard, and ensuring the qualitative and quantitative accuracy of the detection method by optimizing the pretreatment method of different sample matrices and method development.

[0005] The method for detecting tadalafil analogs in food according to the present application comprises the following steps:

[0006] Prepare a test sample solution and a standard solution, respectively, and detect the test sample solution and the standard solution by liquid chromatography-tandem mass spectrometry, and qualitatively and quantitatively analyze the detection results of the test sample solution according to the detection results of the standard solution;

[0007] The conditions of the liquid chromatography include:

[0008] The chromatographic column is octadecylsilane bonded silica gel filler,

[0009] The mobile phase A is 5-20 mM ammonium formate solution, and the mobile phase B is acetonitrile,

[0010] The gradient elution program is as follows: 0.0-7.0 min, 5%-100% B (the volume fraction of B phase increases from 5% to 100%); 7.0-9.0 min, 100% B (the volume fraction of B phase remains at 100%); 9.0-9.1 min, 100%-5% B (the volume fraction of B phase decreases from 100% to 5%); 9.1-10.0 min, 5% B (the volume fraction of B phase remains at 5%);

[0011] The conditions of the mass spectrometry include:

[0012] Capillary voltage: 2-4 KV, for example, 2000-3500 V, 2500-3500 V, 2000-3000 V,

[0013] Conical hole voltage: 5-30 V, for example, 5-25 V, 5-20 V, 5-15 V, 10-15 V,

[0014] Collision energy: 15-25 V, for example, 20-25 V, 15-20 V,

[0015] Collection mode: SIR (selected ion monitoring), MRM (multiple reaction monitoring).

[0016] In some embodiments of the present application, the preparation of the test sample solution comprises the following steps:

[0017] The non-liquid sample to be tested is contacted with an extraction agent to obtain an extract, and the extract is contacted with a purification agent to obtain a test sample solution; or, the liquid sample to be tested is mixed with methanol to obtain a test sample solution.

[0018] In some embodiments of the present application, the extraction agent comprises magnesium sulfate, sodium chloride, sodium citrate and disodium hydrogen citrate in a mass ratio of 5-10:1-4:1-4:1; preferably, magnesium sulfate, sodium chloride, sodium citrate and disodium hydrogen citrate in a mass ratio of 6-10:1-3:1-3:1; more preferably, magnesium sulfate, sodium chloride, sodium citrate and disodium hydrogen citrate in a mass ratio of 7-9:1-3:1-3:1; and further preferably, magnesium sulfate, sodium chloride, sodium citrate and disodium hydrogen citrate in a mass ratio of 8:2:2:1.

[0019] The purification agent comprises ethylenediamine-N-propylsilane (PSA), magnesium sulfate, graphitized carbon black (GCB) and C18 in a mass ratio of 5-10:15-30:1:10-20; preferably, ethylenediamine-N-propylsilane (PSA), magnesium sulfate, graphitized carbon black (GCB) and C18 in a mass ratio of 5-9:15-25:1:12-20; more preferably, ethylenediamine-N-propylsilane (PSA), magnesium sulfate, graphitized carbon black (GCB) and C18 in a mass ratio of 5-8:15-25:1:12-18; and further preferably, ethylenediamine-N-propylsilane (PSA), magnesium sulfate, graphitized carbon black (GCB) and C18 in a mass ratio of 6-7:20-25:1:12-15.

[0020] Ethylenediamine-N-propylsilane (PSA) can remove polar matrix interferents such as sugars, organic acids, etc. in the extract; magnesium sulfate (MgSO4) has the function of dehydrating and drying, and promotes the precipitation of non-polar interferents (such as fats); graphitized carbon black (GCB) can remove pigments and planar structured compounds, and improve the background signal of UPLC-MS / MS; C18 can adsorb non-polar matrices such as fats, oils, waxes, etc. through hydrophobic interaction, and reduce the pollution of the ion source of mass spectrometry. The specific ratio combination of the four kinds of fillers can achieve excellent purification effect on the sample.

[0021] In some embodiments of the present application, the non-liquid sample to be tested is mixed with methanol, and then contacted with an extraction agent to obtain an extract, and the extract is contacted with a purification agent to obtain a test sample solution.

[0022] Preferably, the mass-volume ratio of the non-liquid sample to be tested to methanol is 1g:5-20mL.

[0023] Preferably, the mass ratio of the non-liquid sample to be tested to the extraction agent is 1:4-10, for example, 1:5-8, 1:6-7.

[0024] Preferably, the volume-mass ratio of the extraction liquid to the purification agent is 1 mL: 250-400 mg, for example, 1 mL: 300-400 mg, 1 mL: 250-350 mg, 1 mL: 300-350 mg.

[0025] In some embodiments of the present application, the test liquid sample is mixed with methanol at a mass-volume ratio of 1 g: 10-30 mL to obtain a test sample solution.

[0026] In some embodiments of the present application, the standard sample is mixed with methanol to prepare the standard sample solution; wherein the concentration of the standard sample solution ranges from 0.1 ng / mL to 100000 ng / mL.

[0027] In some embodiments of the present application, the mobile phase A is a 5-15 mM ammonium formate solution.

[0028] In some embodiments of the present application, the chromatographic column is a Waters ACQUITY UPLC BEH C18 chromatographic column (50 mm x 2.1 mm, 1.7 μm).

[0029] In some embodiments of the present application, the conditions of the liquid chromatography further include that the flow rate is 0.1-0.5 mL / min, for example, 0.2-0.4 mL / min.

[0030] In some embodiments of the present application, the conditions of the liquid chromatography further include that the column temperature is 20-40℃, for example, 25-35℃.

[0031] In some embodiments of the present application, the conditions of the liquid chromatography further include that the injection volume is 1-5 μL, for example, 1-3 μL.

[0032] In some embodiments of the present application, the detection conditions of the mass spectrometry further include any one of (i)-(vii):

[0033] (i) an electrospray ionization (ESI) source,

[0034] (ii) a negative ion mode,

[0035] (iii) an ion source temperature of 120-180℃, for example, 120-150℃, 150-180℃,

[0036] (iv) a desolvation gas temperature of 250-350℃, for example, 300-350℃, 250-300℃,

[0037] (v) desolvation gas flow rate 500-750 L / hr, for example, 550-750 L / hr, 550-700 L / hr, 600-700 L / hr,

[0038] (vi) cone gas flow rate 120-180 L / hr, for example, 150-180 L / hr, 120-150 L / hr,

[0039] (vii) nebulizer gas pressure 6-7 bar.

[0040] In some embodiments of the present application, the tadalafil analogues include tadalafil impurity O, tadalafil EP impurity 1, and tadalafil impurity 82.

[0041] In some embodiments of the present application, the qualitative and quantitative ion pair parameters of tadalafil impurity O, tadalafil EP impurity 1, and tadalafil impurity 82 are as follows:

[0042]

[0043]

[0044] wherein * represents a quantitative ion.

[0045] In some embodiments of the present application, a standard solution is determined, so as to obtain the chromatographic peak area of the corresponding standard solution, and then a standard curve is plotted with the concentration of the standard solution as the abscissa and the chromatographic peak area of the quantitative ion as the ordinate; the measured peak area of each tadalafil analogue in the ion chromatogram of the test sample solution is substituted into the standard curve equation, so as to perform quantitative analysis.

[0046] In some embodiments of the present application, by using the method, the detection limit of a liquid sample is 0.007 mg / kg, and the quantification limit is 0.02 mg / kg; the detection limit of a non-liquid sample is 0.02 mg / kg, and the quantification limit is 0.05 mg / kg.

[0047] In some embodiments of the present application, the test sample includes a functional food, a health food, or a drug. Preferably, the functional food, the health food, or the drug can be a tablet, a powder, a capsule, a pill, a granule, a decoction, an oral liquid, or an injection.

[0048] According to some embodiments of the present application, at least the following beneficial effects are achieved:

[0049] The application provides a screening, qualitative and quantitative analysis method for three new tadalafil analogs (tadalafil impurity O, tadalafil EP impurity 1 and tadalafil impurity 82) in food. The method makes full use of the high separation capacity of liquid chromatography and the high specificity of mass spectrometric detection, effectively eliminates false positive results by combining secondary mass spectrometry technology, and ensures the accuracy and reliability of the detection results. Through the multiple reaction monitoring (MRM) mode, the retention time and ion relative abundance are used for qualitative and quantitative analysis, and the precise detection of the target compound is realized. The method has the advantages of simple pretreatment operation, strong specificity, high sensitivity, etc., and can be suitable for rapid detection of a large number of samples, and meets the daily determination needs of three new tadalafil analogs in food.

[0050] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent to those skilled in the art from the description, or can be learned by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0052] Figure 1 Qualitative and quantitative ion spectrum of tadalafil impurity O, tadalafil EP impurity 1 and tadalafil impurity 82.

[0053] Figure 2 Extraction mode optimization result graph.

[0054] Figure 3 Flow phase system optimization result graph.

[0055] Figure 4 Elution gradient optimization result graph.

[0056] Figure 5 Collision energy influence on secondary mass spectrum of tadalafil impurity O, tadalafil EP impurity 1 and tadalafil impurity 82. DETAILED DESCRIPTION

[0057] The concept and technical effects of the present application will be described below in combination with embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all embodiments, and other embodiments obtained by those skilled in the art based on the embodiments of the present application without creative labor are within the scope of protection of the present application.

[0058] When a numerical range is disclosed herein, the range is to be construed as a continuous range and include each and every value within the range. Further, where a range is provided, the range includes the minimum and maximum values of the range, as well as every integer within the range. Additionally, where multiple ranges are provided, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein are to be understood to encompass any and all sub-ranges of the same.

[0059] Unless otherwise specified, "about" as used herein means ±10% error, further ±5% error, and further ±2% error.

[0060] The reagent materials used in the present application are as follows: tadalafil impurity O (purity 95.32%, CAS No. 2169996-11-4, Shenzhen Hengfeng Wanda Pharmaceutical Technology Co., Ltd.); tadalafil impurity 82 (purity 96.43%, CAS No. 629652-67-1, Shenzhen Hengfeng Wanda Pharmaceutical Technology Co., Ltd.); tadalafil EP impurity 1 (purity 95.27%, CAS No. 2378614-29-8, Shenzhen Hengfeng Wanda Pharmaceutical Technology Co., Ltd.); acetonitrile (chromatographically pure, Merck, Germany); experimental water is Milli-Q ultrapure water; ammonium formate (mass spectrometry pure), magnesium sulfate (MgSO4), sodium chloride (NaCl), sodium citrate (Na3C6H5O7), sodium hydrogen citrate (Na2C6H6O7), ethylenediamine-N-propylsilane (PSA), graphitized carbon black (GCB), etc. are commercially available, for example, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; C18 (analytically pure, Guangzhou Boshimeng Scientific Instrument Co., Ltd.).

[0061] Experimental samples: Baihuwan (batch number: 20240302, Bozhou Lvzhiyuan Biotechnology Co., Ltd.), Anshen Buanao Pills (batch number: 2406218, Shanghai Hehuang Pharmaceutical Co., Ltd.), Guilu Erxian Gao (batch number: Z14020687, Hunan Qianjin Xielv Pharmaceutical Co., Ltd.), Guyuan Gao (batch number: 45505, Shandong Donga Runchengtang Ejiao Products Co., Ltd.), Renshen Qicao Plant Beverage (batch number: 20240513, Xuzhou Kanghui Century Food Co., Ltd.), Renshen Muli Pei Plant Beverage (batch number: 20240807, Xuzhou Kanghui Century Food Co., Ltd.), Heli No. 7 Beverage (batch number: 20240619, Xuzhou Kanghui Century Food Co., Ltd.), Renshen Ju Chongcao Huangjing Tablet (batch number: DJ20240816, Ganzhou Mengan Food Co., Ltd.), Renshen Macha Muli Tablet (batch number: BS20240812, Ganzhou Mengan Food Co., Ltd.), Lvxue Muli Renshen Tablet (batch number: VM2024112801, Ganzhou Mengan Food Co., Ltd.), Luba Chongcao Jing Tablet (batch number: DB20240823, Xuzhou Kanghui Century Food Co., Ltd.), Miao Er Guo Renshen Muli Tablet (batch number: 20240913, Xuzhou Kanghui Century Food Co., Ltd.), Lvxue Muli Tablet (batch number: LB20240819, Ganzhou Mengan Food Co., Ltd.), Propolis Soft Capsule (batch number: 2018050901, Fuzhou Fowan Biotechnology Engineering Co., Ltd.), Gegen Zhijiang Jiujiu Capsule (batch number: 20240907, Wuhan Maxin Food Co., Ltd.).

[0062] The instruments and equipment used in the present application include: Xevo TQ-Smicro ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometer of Waters Corporation of the United States; XPR226DR / AC electronic balance of Mettler Corporation of the United States; PS-G60A numerical control ultrasonic cleaner of Dongguan Jiekang Ultrasonic Equipment Co., Ltd.; 3-18KS high-speed table centrifuge of Sigma Corporation of the United States; and Waters ACQUITY UPLC BEH C18 chromatographic column (specification: 2.1 mm x 50 mm, particle size: 1.7 μm).

[0063] The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased on the market.

[0064] Example 1

[0065] The present embodiment provides a method for screening and quantifying tadalafil analogues in food.

[0066] 1. Preparation of solution

[0067] 1.1. Preparation of test solution

[0068] (1) Extraction of tablet, capsule, pill, decoction sample

[0069] Accurately weigh 1 g of tablet, capsule, pill, decoction into a 50 mL centrifuge tube, add 10 mL of methanol, vortex for 2 min, ultrasonic for 30 min, add 4 g of magnesium sulfate (MgSO4), 1 g of sodium chloride (NaCl), 1 g of sodium citrate (Na3C6H5O7), 0.5 g of sodium hydrogen citrate (Na2C6H6O7), shake vigorously for 5 min, centrifuge at 4000 r / min for 5 min, take the supernatant, which is the extract.

[0070] (2) Purification of tablet, capsule, pill, decoction sample

[0071] Take 1 mL of the above extract into a 2 mL centrifuge tube, accurately add 50 mg of PSA, 150 mg of MgSO4, 7.5 mg of GCB and 100 mg of C18, vortex for 2 min, centrifuge at 10000 r / min for 5 min, take the upper clear liquid, pass through a 0.22 μm nylon filter membrane, and obtain the test solution, which is ready for use.

[0072] (3) Extraction of liquid sample

[0073] Accurately weigh 0.5 g of liquid into a 10 mL volumetric flask, dilute to the mark with methanol, shake well, pass through a 0.22 μm nylon filter membrane, and determine on the machine.

[0074] 1.2, Preparation of standard stock solution

[0075] Accurately weigh 1 mg of tadalafil impurity O, tadalafil EP impurity 1, tadalafil impurity 82 standard into a 10 mL volumetric flask, dissolve and dilute to the mark with methanol, respectively, to prepare a standard stock solution of 100 μg / mL, and store at -18°C, away from light and sealed.

[0076] 1.3, Preparation of mixed standard solution

[0077] Take 1 mL of the standard stock solution obtained in step 1.2, place it in a 100 mL volumetric flask, dilute to the mark with methanol, and obtain a mixed standard solution with a concentration of 1 μg / mL, store at 4°C.

[0078] 1.4, Preparation of mixed standard working solution

[0079] Take 1 mL of the mixed standard solution obtained in step 1.3, place it in a 10 mL volumetric flask, dilute to the mark with methanol, and obtain a mixed standard working solution with a concentration of 100 ng / mL.

[0080] 1.5, Preparation of mixed standard curve solution

[0081] Accurately pipette 10 μL, 50 μL, 100 μL, 500 μL, 1000 μL of the mixed standard working solution obtained in step 1.4 into a 10 mL volumetric flask, respectively, and dilute to the mark with methanol to prepare mixed standard curve solutions with concentrations of 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL. Prepare immediately before use.

[0082] 1.6, Blank sample matrix solution of tablets, capsules, pills, decoction pieces, oral liquid

[0083] Take the Lubaoshengcao refined tablets (tablets), Gegenjiegongzhejue capsules (capsules), Baihu pills (pills), and Guyu decoction pieces (decoction pieces) that have been tested to be free of Dalafin impurity O, tadalafil EP impurity 1, and tadalafil impurity 82. Refer to the steps under the item “1.1, Preparation of the test solution” to prepare the blank sample matrix solutions of tablets, capsules, pills, and decoction pieces, respectively, and wait for use.

[0084] Weigh 0.5 g (accurate to 0.001 g) of the Renshenqicao plant beverage (oral liquid) that has been tested to be free of Dalafin impurity O, tadalafil EP impurity 1, and tadalafil impurity 82 into a 10 mL volumetric flask, dilute to the mark with methanol, shake well, and pass through a 0.22 μm filter to obtain the blank sample matrix solution of the oral liquid, and wait for use.

[0085] 2, Chromatographic and mass spectrometric detection conditions

[0086] Take each solution for sampling and perform ultra-high performance liquid chromatography-mass spectrometry detection, wherein,

[0087] (1) The detection conditions for ultra-high performance liquid chromatography are as follows:

[0088] Chromatographic column: Waters ACQUITY UPLC BEH C18 (50×2.1 mm, 1.7 μm);

[0089] Mobile phase A: 10 mM ammonium formate solution;

[0090] Mobile phase B: acetonitrile;

[0091] The gradient elution program is as follows: 0.0-7.0 min, 5%-100% B; 7.0-9.0 min, 100% B; 9.0-9.1 min, 100%-5% B; 9.1-10.0 min, 5% B (for column equilibration);

[0092] Flow rate: 0.3 mL / min;

[0093] Column temperature: 30°C;

[0094] Injection volume: 2 μL.

[0095] (2) The detection conditions of mass spectrometry are as follows:

[0096] Capillary voltage 2500V;

[0097] Ion source temperature 150℃;

[0098] Desolvation gas temperature 300℃;

[0099] Desolvation gas flow 650L / hr;

[0100] Conespray gas flow 150L / hr;

[0101] Spray gas pressure 7.0bar;

[0102] The scanning mode is multiple reaction monitoring (MRM);

[0103] The qualitative and quantitative ion pair parameters of tadalafil impurity O, tadalafil EP impurity 1 and tadalafil impurity 82 are shown in Table 1 below, and the total ion chromatogram of the three chemical drugs is shown in Figure 1 , in which the three impurities are arranged in the order of peak appearance from left to right as follows: 1, tadalafil EP impurity 1; 2, tadalafil impurity O; and 3, tadalafil impurity 82.

[0104] Table 1

[0105]

[0106] 3. Matrix effect test

[0107] The matrix refers to the components in the sample other than the analyte, and the phenomenon that the matrix affects the ionization of the target is the matrix effect (ME). Reducing the matrix effect is the key to achieving efficient analysis. The following steps are taken to analyze the matrix effect.

[0108] The "1.6, blank sample matrix liquid of tablets, capsules, pills, decocted pastes and oral liquids" or methanol is mixed with "1.4, mixed standard working solution preparation" according to the volume ratio of 1:1 to prepare a standard solution or a matrix-added standard solution with a concentration of 50μg / L. The machine is measured under the conditions of "2, chromatographic and mass spectrometric detection conditions", each sample is measured in parallel for 3 times, the peak areas of different impurities in the sample are recorded respectively, and the average value is calculated according to the following formula (1) to calculate the ME value of different impurities.

[0109] ME = B / A, formula (1);

[0110] Wherein, A is the average value of the peak area of the target impurity in the standard solution, and B is the average value of the peak area of the target impurity in the matrix-added standard solution.

[0111] When ME = 0.9-1.1 indicates that there is no matrix effect, 0.8

[0112] The matrix effect test results are shown in Table 2.

[0113] Table 2

[0114] Impurities Oral solution Tablets Capsules Pills Decoction Tadalafil impurity O 0.9 1.0 1.0 0.9 1.0 Tadalafil EP impurity 1 0.9 0.9 0.9 0.9 0.9 Tadalafil impurity 82 1.0 1.0 1.0 1.0 1.0

[0115] The results of Table 2 show that the ME values of the three illegally added chemical drugs, tadalafil impurity O, tadalafil EP impurity 1 and tadalafil impurity 82, in the five matrices of tablets, capsules, pills, decocting pastes and oral liquids are in the range of 0.9-1.0, indicating that there is no matrix effect.

[0116] 4. Linear range, correlation coefficient, detection limit and quantification limit test

[0117] Take "1.5, mixed standard curve solution", and determine it on the machine under the conditions of "2, chromatographic and mass spectrometric detection conditions". The standard curve is drawn with the target impurity concentration as the abscissa and the impurity quantitative ion pair peak area as the ordinate, and the regression equation and its correlation coefficient are calculated. See Table 3 for details.

[0118] Take "1.6, blank sample matrix solution of tablets, capsules, pills, decocting pastes and oral liquids", and add standard solution of a certain concentration. The concentration at a signal-to-noise ratio of 3 is taken as the detection limit, and the concentration at a signal-to-noise ratio of 10 is taken as the quantification limit. The detection results are shown in Table 3. The detection limit and quantification limit of the method of the present application are lower than those of SN / T 5357-2021 "Determination of multiple illegally added substances in exported health food by liquid chromatography-mass spectrometry / mass spectrometry", and can meet the qualitative and quantitative detection of tadalafil impurity O, tadalafil EP impurity 1 and tadalafil impurity 82 with extremely low content in actual samples.

[0119] Table 3

[0120]

[0121]

[0122] 5. Recovery rate and precision

[0123] The ginseng and gypenoside plant beverage (oral liquid) without positive components was used as the matrix, and 0.02 mg / kg, 0.2 mg / kg and 2.0 mg / kg were set as the three addition levels to analyze the recovery rate and precision. The deer and cordyceps essence tablets (tablets), kudzu vine and hovenia dulcis alcohol-reducing capsules (capsules), white tiger pills (pills) and solid essence paste (decocting paste) without positive components were used as the matrix, and 0.05 mg / kg, 0.1 mg / kg and 1.0 mg / kg were set as the three addition levels to analyze the recovery rate and precision. The control product was added in an appropriate amount according to the sample preparation method, and was determined on the machine under the conditions of “2, chromatographic and mass spectrometric detection conditions”, 6 times in parallel, and the recovery rate was calculated. The average recovery rate and precision of the three impurities in the liquid matrix with the addition of the standard are shown in Table 4, and the average recovery rate and precision of the three impurities in the non-liquid matrix with the addition of the standard are shown in Table 5.

[0124] Table 4

[0125]

[0126] Table 5

[0127]

[0128]

[0129] 6. Optimization of detection method

[0130] 6.1. Optimization of extraction method

[0131] Liquid food matrix is relatively simple, which can be directly extracted by methanol, and can efficiently enrich target compounds, and the operation is simple and the cost is low. However, semi-solid and solid food matrixes have complex structures, and often contain sugars, organic acids, lipids, pigments and other substances. After direct extraction, a large amount of impurities often accompanies, which will interfere with the subsequent detection process, increase the matrix effect, and reduce the accuracy and reliability of the detection results. In order to effectively reduce the influence of matrix effect on quantitative accuracy, the present application adopts the method of extracting the target compound after extracting the extraction agent combined with purification for semi-solid and solid food. By systematically comparing the adsorption performance of different proportions of purification fillers to the target, the filler combination with the lowest adsorption is selected to minimize the interference of impurities and ensure that the recovery rate of the target compound meets the range of 70% to 120% specified in the national standard GB 23200.113-2018.

[0132] Reference is made to the preparation of the test solution under “1.1, Preparation of test solution”, the following four fillers are adopted, and the machine is determined under the conditions of “2, Chromatographic and mass spectrometric detection conditions”,

[0133] A, 30 mg PSA+150 mg MgSO4+5 mg GCB+30 mg C18,

[0134] B. 50mg PSA+150mg MgSO4+5mg GCB+30mg C18,

[0135] C. 50mg PSA+150mg MgSO4+5mg GCB+50mg C18,

[0136] D. 50mg PSA+150mg MgSO4+7.5mg GCB+100mg C18.

[0137] The distribution of the effects of four combined packing materials on the recovery rate of three impurities in food is shown in the figure. Figure 2 Table 6.

[0138] Table 6

[0139]

[0140] according to Figure 2 The response values ​​of tadalafil EP impurity 1 in extraction methods A, B, C, and D were 1.25e 5 1.27e 5 1.57e 5 1.81e 5 The response values ​​of tadalafil impurity 82 in extraction methods A, B, C, and D were 4.89e. 5 5.23e 5 6.99e 5 8.19e 5 The response values ​​of tadalafil impurity O in the extraction methods of combinations A, B, C, and D were 2.80 eE. 5 2.85e 5 3.74e 5 4.41e 5 This indicates that combination D is the optimal extraction method, significantly improving the response value of the target compound.

[0141] The results in Table 6 show that using 50 mg PSA + 150 mg MgSO4 + 7.5 mg GCB + 100 mg C18 as the dispersive extraction purification packing material resulted in the best purification effect and recovery rate. The recovery rate of the target compound remained stable at 97.33%–109.02%, indicating that this method can effectively control the matrix effect, improve the accuracy and precision of quantitative analysis, and provide a reliable and efficient technical means for the detection of target compounds in health foods.

[0142] 6.2 Optimization of the mobile phase system

[0143] Reference "1.1, preparation of test solution" under the preparation of test solution, according to "2, chromatography and mass spectrometry detection conditions" under the conditions of machine measurement, the only difference is: take the following three kinds of mobile phase,

[0144] A: 10 mM ammonium formate (A) - acetonitrile (B) system,

[0145] B: 0.1% formic acid water (A) - acetonitrile (B) system,

[0146] C: water (A) - acetonitrile (B) system.

[0147] The influence of three kinds of mobile phase on the separation effect of three kinds of impurities in food is shown in Figure 3 . It is found that the separation degree of the sample is poor and the response is low when using 0.1% formic acid water (A) - acetonitrile (B) system; when using water (A) - acetonitrile (B) system, the response values of three target compounds are increased, but the separation degree is poor; when using 10 mM ammonium formate (A) - acetonitrile (B) system, the response value and separation degree are obviously improved. Therefore, considering the mobile phase, it is more reasonable to use 10 mM ammonium formate (A) - acetonitrile (B) system.

[0148] 6.3, elution gradient condition optimization

[0149] Reference "1.1, preparation of test solution" under the preparation of test solution, according to "2, chromatography and mass spectrometry detection conditions" under the conditions of machine measurement, the only difference is: take the three elution gradients in table 7.

[0150] Table 7

[0151]

[0152] Set the liquid flow into the waste liquid channel to avoid the interference of the front high polarity matrix and the rear high lipid impurity into the mass spectrometer, and produce pollution.

[0153] The influence of three kinds of elution gradient on the separation effect of three kinds of impurities in food is shown in Figure 4 . In liquid chromatography-mass spectrometry, if the peak appears too fast, it will lead to insufficient response time of the instrument, and the mass spectrum signal cannot be accurately captured and recorded, which seriously affects the accuracy and reliability of quantitative analysis, and the solvent peak will also interfere with the accuracy of quantification. If the peak appears too slowly, it will lead to peak broadening and baseline drift, reduce the analysis efficiency and the resolution and sensitivity of the mass spectrum peak, and make the measurement of peak area difficult. The retention time of the target compound is significantly changed by optimizing the elution gradient. The final method can accurately quantify the three target compounds with retention time precisely controlled in the interval of 2.5-5.0 min without reducing the analysis efficiency.

[0154] 6.4, mass spectrometry condition optimization

[0155] The single standard stock solution was diluted to 100 ng / mL with methanol, and the single standard solution was injected into the mass spectrometer in negative ion mode. The MS Scan full scan mode was used to obtain the molecular ion of the compound, and then the compound parent ion was determined. The capillary voltage and cone voltage were adjusted to maximize the response value of the parent ion. The daughter MS mode was selected, and the rearrangement reaction of the parent ion was carried out by nitrogen collision. Two ion fragments with high response value and less interference were selected as the qualitative and quantitative ion fragments of the compound. Finally, the collision voltage was optimized to maximize the response of the daughter ion fragments. See Table 8 for details.

[0156] Table 8

[0157]

[0158] Different capillary voltages and cone voltages significantly affect the response value of the target compound, and thus affect the sensitivity. Table 8 shows that the response value of tadalafil EP impurity 1, tadalafil impurity 82, and tadalafil impurity O is the highest at a capillary voltage of 2500 V. At the same time, the response value of tadalafil EP impurity 1 is the highest at a cone voltage of 20 V, tadalafil impurity 82 is the highest at a cone voltage of 10 V, and tadalafil impurity O is the highest at a cone voltage of 10 V.

[0159] By optimizing the capillary voltage and cone voltage, the response value of the parent ion is maximized, and the characteristic ion pairs m / z 434.24→262.13, m / z 434.24→317.44, m / z 404.14→278.06, m / z 404.14→386.08, m / z 456.28→262.09, and m / z 456.28→209.03 are optimized. Table 8 shows that the response value of each ion pair is the highest under the collision energy (20 V) condition, while the response value significantly decreases by about 2-20000 times under other parameters such as collision energy of 10 V, 30 V, 40 V, 50 V, and 60 V, which cannot meet the detection requirements (see Figure 5 ) The method has a detection limit (LOD) of 0.007 mg / kg and a quantification limit (LOQ) of 0.02 mg / kg, which is significantly better than the 0.1 mg / kg limit specified by the national standard.

[0160] The analysis method established by the present application is optimized by three technologies, which significantly improves the sensitivity of the method while ensuring the accuracy of the detection, and provides a reliable technical solution for the accurate quantification of trace target substances in complex matrices.

[0161] Example 2

[0162] The commercial samples (Shenpi Zhuangyang drink, tablets, capsules, pills, decoction) are detected by the method of the application, and the results show that among 14 batches of samples, 1 brand of pills, 2 brands of oral liquid, 1 brand of tablets and 1 brand of capsules detect tadalafil impurity O, tadalafil EP impurity 1 and tadalafil impurity 82; the addition amount of tadalafil impurity O, tadalafil EP impurity 1 and tadalafil impurity 82 in solid and semi-solid samples is between 0.01-0.53 mg / kg; the addition amount of tadalafil impurity O, tadalafil EP impurity 1 and tadalafil impurity 82 in liquid samples is between 0.51-0.64 mg / kg. The specific determination results are shown in Table 9.

[0163] Table 9

[0164]

[0165] In summary, the application provides a rapid screening and quantitative detection method for three new tadalafil analogues (tadalafil impurity O, EP impurity 1 and impurity 82) in food by using high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The sample is extracted and purified by QuEChERS method. A Waters ACQUITY UPLC BEH C18 column (50x2.1mm, 1.7μm) is used for separation and detection by gradient elution (mobile phase: 10mM ammonium formate-acetonitrile) combined with negative ion electrospray multi-reaction monitoring mode (MRM). The method verification results show that the target has a good linear relationship in the range of 1.0-100μg / L (R 2 >0.995), the limit of quantification of oral liquid is 0.02mg / kg, the detection limit is as low as 0.007mg / kg, the limit of quantification of semi-solid and solid preparations is 0.05mg / kg, the detection limit is as low as 0.02mg / kg, and the average recovery rate is 80%-116% (RSD<5.3%). The method has strong specificity and high sensitivity, and can provide efficient and reliable technical support for market supervision of related products.

[0166] The above describes the embodiments of the application in detail, but the application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the purpose of the application. In addition, the embodiments of the application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for detecting tadalafil analogues, characterized in that, Includes the following steps: A test solution and a standard solution were prepared separately. The test solution and the standard solution were then subjected to liquid chromatography-tandem mass spectrometry for detection. Based on the detection results of the standard solution, the detection results of the test solution were qualitatively and quantitatively analyzed. The conditions for the liquid chromatography include: Column: Octadecylsilane-bonded silica gel packing material. Mobile phase A: 5-20 mM ammonium formate solution; Mobile phase B: acetonitrile. The gradient elution program was as follows: 0.0–7.0 min, 5%–100% B; 7.0–9.0 min, 100% B; 9.0–9.1 min, 100%–5% B; 9.1–10.0 min, 5% B. The conditions for the mass spectrometry include: Capillary voltage: 2-4KV Tapered hole voltage: 5-30V Impact energy: 15-25V Acquisition modes: Select ion monitoring or multiple reaction monitoring.

2. The method according to claim 1, characterized in that, The preparation of the test solution includes the following steps: The non-liquid sample to be tested is contacted with the extractant to obtain an extract, and the extract is contacted with the purifying agent to obtain a test solution; or, the liquid sample to be tested is mixed with methanol to obtain a test solution.

3. The method according to claim 2, characterized in that, The extractant comprises magnesium sulfate, sodium chloride, sodium citrate, and disodium hydrogen citrate in a mass ratio of 5-10:1-4:1-4:

1. The purifying agent comprises ethylenediamine-N-propylsilane, magnesium sulfate, graphitized carbon black, and C18 in a mass ratio of 5-10:15-30:1:10-20.

4. The method according to claim 1, characterized in that, The standard solution is prepared by mixing the standard with methanol. The concentration range of the standard solution is 0.1-100000 ng / mL.

5. The method according to claim 1, characterized in that, The chromatographic column was a Waters ACQUITY UPLC BEHC18 column with dimensions of 50 mm × 2.1 mm and a diameter of 1.7 μm.

6. The method according to claim 1, characterized in that, The mass spectrometry conditions also include any one of the following (i)-(vii): (i) Electrospray ionization source (ii) Negative ion mode. (iii) Ion source temperature 120-180℃, (iv) Desolvation gas temperature: 250-350℃ (v) Desolvation gas flow rate 500-750 L / hr, (vi) Conical orifice airflow velocity 120-180 L / hr (vii) Sprayer pressure 6-7 bar.

7. The method according to claim 1, characterized in that, The tadalafil analogues include Tadalafil impurity O, Tadalafil EP impurity 1 and Tadalafil impurity 82; The qualitative and quantitative ion pair parameters for tadalafil impurity O, tadalafil EP impurity 1, and tadalafil impurity 82 are as follows: In this context, * indicates a quantitative ion.

8. The method according to claim 1, characterized in that, The chromatographic peak areas of the standard solutions are obtained by measuring the standard solution concentrations. Then, a standard curve is plotted with the concentrations of the standard solutions on the x-axis and the peak areas of the quantitative ions on the y-axis. The measured peak areas of each tadalafil analog in the ion chromatogram of the test solution are substituted into the standard curve equation for quantitative analysis.

9. The method according to claim 2, characterized in that, Using the method described above, the limit of detection for liquid samples is 0.007 mg / kg, and the limit of quantitation is 0.02 mg / kg; the limit of detection for non-liquid samples is 0.02 mg / kg, and the limit of quantitation is 0.05 mg / kg.

10. The method according to any one of claims 1-9, characterized in that, The test samples include functional foods, health foods, or pharmaceuticals.

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