Method for detecting new sildenafil compound in health food

Abstract

The present application relates to the technical field of health product ingredient detection, in particular to a detection method of a new type of sildenafil compound in health food, comprising the following steps: qualitative and quantitative detection by HPLC-MS / MS; the chromatographic conditions include: a chromatographic column, a C18 column; a column temperature, 40 DEG C; a mobile phase A, 0.1% formic acid aqueous solution; a mobile phase B, acetonitrile; a flow rate, 0.5 mL / min; a gradient elution program, 0~2.0 min, 10%B~95%B; 2.0~5.0 min, 95%B; 5.0~5.1 min, 95%B~5%B; 5.1~8.0 min, 5%B; a sample injection amount, 5 µL; the mass spectrometry conditions include: an ionization mode, an electrospray ionization source, a positive ion mode; an electrospray voltage, 5500 V; an ion source temperature, 550 DEG C; an atomization gas pressure, 345 kPa; an auxiliary gas pressure, 414 kPa; a curtain gas pressure, 276 kPa; a declustering voltage, 100 V; a collision energy, 45 V; a quantitative ion pair, 449 / 243; a qualitative ion pair, 449 / 259; the residence time of each ion pair, 250 ms; the new type of sildenafil compound contains a methyl piperazine group. The detection method of the present application is simple in operation, short in time consumption, high in sensitivity and good in stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of health product ingredient detection, and more particularly to a detection method of a new type of sildenafil compound in health food. BACKGROUND

[0002] In recent years, with the improvement of people's living standards and the improvement of health awareness, the use of health food has increased, and more and more people hope to adjust and improve their physical condition by taking health food. Some of these products have the effect of enhancing sexual function. Although many of these products are marked as containing natural ingredients, it is often found that sildenafil, tadalafil and vardenafil, which are phosphodiesterase 5 (PDE-5) inhibitors for treating male sexual dysfunction, are illegally added. With the increasing regulation of health food by the state, some unscrupulous manufacturers add new types of sildenafil drugs to avoid regulation. The purity, additive amount and impurity content of illegally added new types of sildenafil drugs are not subject to clear specifications, and long-term consumption of such health food by consumers can cause more harm to their health, and even endanger their lives. The inventors found a new type of unknown structure of sildenafil compound in the "Oyster Angelica Powder" sample submitted for inspection. The prior art does not have a related report on the detection method of the compound, so there is an urgent need for a method that can quickly detect the compound to fill the gap in this field. SUMMARY

[0003] The present application provides a detection method of a new type of sildenafil drug in health food to solve the above technical problems.

[0004] To achieve the above purpose, the present application realizes the following technical solutions:

[0005] A detection method of a new type of sildenafil compound in health food, comprising the following steps:

[0006] HPLC-MS / MS is used for qualitative and quantitative detection;

[0007] The chromatographic conditions include the following:

[0008] Chromatographic column: C18 column; column temperature: 40℃; mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile; flow rate: 0.5mL / min; gradient elution program: 0~2.0 min, 10%B~95%B; 2.0~5.0 min, 95%B; 5.0~5.1 min, 95%B~5%B; 5.1~8.0 min, 5%B; injection volume: 5 µL;

[0009] The mass spectrometry conditions include the following:

[0010] Ionization mode: electrospray ionization source, positive ion mode; electrospray voltage: 5500V; ion source temperature: 550 DEG C; atomization gas pressure: 345 kPa; auxiliary gas pressure: 414 kPa; gas curtain pressure: 276 kPa; de-clustering voltage: 100 V; collision energy: 45 V; quantitative ion pair: 449 / 243, qualitative ion pair: 449 / 259; each pair of ion residence time: 250 ms;

[0011] The novel PDE5 inhibitor compound contains a methyl piperazine group.

[0012] Further, the inventors identified the target as 5,6-Diethyl-2-[5-[(4-methyl-1-piperazinyl)sulfonyl]-2-propoxyphenyl]-4(3H)-pyrimidinone (no generic name, temporarily referred to as compound X) by means of liquid chromatography-quadrupole / time-of-flight mass spectrometer (HPLC-QTof), self-developed molecular structure identification software, purification technology, nuclear magnetic resonance instrument (NRM), etc., and the structural formula of the compound X is shown as formula (I):

[0013]

[0014] Formula (I).

[0015] In the preferred solution, the preparation method of the sample solution to be tested comprises: weighing 0.5 g of the sample to be tested, placing it in a 50 mL plastic centrifuge tube, adding 10 mL of methanol, ultrasonicating for 15 min, centrifuging at 4000 r / min for 5 min, taking an appropriate amount of supernatant to pass through a membrane, diluting 200000 times, and obtaining the sample solution to be tested.

[0016] In the preferred solution, the preparation method of the standard sample comprises: placing the sample to be tested into a chicken heart bottle, adding 100 ml of acetonitrile for ultrasonic extraction for 30 min, filtering, and concentrating the filtrate under reduced pressure to dryness to obtain a crude product; purifying the crude product through a chromatography column, collecting the fraction, and obtaining the target after purification, i.e., the standard sample.

[0017] In the preferred solution, the standard solution of different concentrations is prepared by using the control sample, and a working curve is drawn by taking the peak area as the mass concentration of each component in the standard solution.

[0018] Advantages of the present application

[0019] The application establishes a qualitative and quantitative detection method of liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS) by taking the purified target as a control. After the sample is ultrasonically extracted by methanol, centrifuged and filtered, an Eclipse Plus C18 chromatographic column (150 mm*3.0 mm, 3.5 µm) is used for separation, gradient elution is carried out by taking acetonitrile and 0.1% formic acid aqueous solution as the mobile phase, liquid chromatography-triple quadrupole mass spectrometry is used in the positive ion mode, and the external standard method is used for quantitative detection. The results show that the linear relationship of compound X is good in the range of 0.5-50 µg / L, the correlation coefficient is greater than 0.99995, the detection limit (LOQ) is 0.005 mg / kg, the quantitative limit (LOQ) is 0.01 mg / kg, the average recovery of the blank sample at three different levels is 99.1%-112.2%, and the relative standard deviation (RSD) is 2.6%-3.7% (n=6). n The method is simple in operation, short in time consumption, high in sensitivity, good in stability, and suitable for qualitative and quantitative detection of compound X in health food. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the HPLC-QTof primary total ion chromatogram (TIC) of oyster white peony powder sample;

[0021] Figure 2 is the HPLC-QTof secondary fragment mass spectrum of oyster white peony powder sample;

[0022] Figure 3 is the fragmentation mechanism analysis of the characteristic secondary fragment ion of sildenafil;

[0023] Figure 4 is the hydrogen spectrum of compound X;

[0024] Figure 5 is the carbon spectrum of compound X;

[0025] Figure 6 is the structural formula of compound X;

[0026] Figure 7 is the chromatogram of compound X standard solution (5 ng / mL); Figure 7 A is a quantitative ion pair, Figure 7 B is a qualitative ion pair;

[0027] Figure 8 is the chromatogram of compound X standard sample (0.01 mg / kg); Figure 8 A is a quantitative ion pair, Figure 8 B is a qualitative ion pair. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative efforts should fall within the protection scope of the present application.

[0029] In the present application, the scientific and technical terms used herein have meanings commonly understood by those skilled in the art, unless otherwise specified. The experimental methods in the embodiments are conventional methods, unless otherwise specified. The specific conditions not specified in the embodiments are carried out according to conventional conditions or manufacturer's recommended conditions. The reagents or instruments used, if not specified, are conventional products available on the market.

[0030] Recently, a batch of oyster Angelica dahurica powder samples were sent to our laboratory by a municipal public security bureau. The sample is a commercial sample and has been sold nationwide. The sending unit suspects that the sample may contain illegally added drugs. Several laboratories were sent to detect PDE5 inhibitors, but none was detected. When screening and detecting the sample, our laboratory found a new PDE5 inhibitor compound X with an unknown structure.

[0031] Our laboratory used the self-developed AI software for PDE5 inhibitors to identify the molecular structure of the compound, accurately identified the compound, and purified and identified the sample by nuclear magnetic resonance (NMR). The identification results are completely consistent with the self-developed software identification results. After consulting the literature of the identified compound, compound X is a PDE5 inhibitor synthesized by the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences for the first time, and no literature report on the detection method of compound X has been found. In order to protect people's health, it is urgent to establish a detection method for compound X. Since compound X cannot be purchased as a commercial standard or control, our laboratory purified the sample, and the purified target was used as a control to establish a qualitative and quantitative detection method for new PDE5 inhibitors in health food by liquid chromatography-triple quadrupole mass spectrometry, filling the gap in this field.

[0032] I. Experimental Section

[0033] (I) Instruments, reagents and materials

[0034] TripleTOF 5600+ liquid chromatography-quadrupole / time-of-flight mass spectrometer (AB Sciex, USA); QTRAP4000 liquid chromatography-triple quadrupole mass spectrometer (AB Sciex, USA); AVANCE NEO 500MHz nuclear magnetic resonance instrument (Bruker, USA); MSA225S-1CE-DA analytical balance (Sartorius, Germany, 0.01 mg sensitivity); JY3002 analytical balance (Shanghai Precision Scientific Instrument Co., Ltd., 0.01 g sensitivity); PRO VF-T ultrapure water system (Sartorius, Germany); Hermle Z323K centrifuge (Hermle, Germany); pipettes (10-100 µL, 20-200 µL, 100-1000 µL, Eppendorf, Germany).

[0035] Acetonitrile and methanol (chromatographically pure) were purchased from Shanghai Anpu Experiment Technology Co., Ltd.; formic acid (chromatographically pure) was purchased from Beijing Dikeyama Technology Co., Ltd.

[0036] The sample for submission was a solid oyster white peony powder beverage sample (produced by a biotechnology Co., Ltd. in Liaoning Province).

[0037] Sildenafil (molecular formula: C 22 H 30 N6O4S, CAS No.: 139755-83-2) reference substance was purchased from Shanghai Anpu Experiment Technology Co., Ltd., with a purity of 99.6%.

[0038] Compound X (molecular formula: C 22 H 32 N4O4S, CAS No.: 1229017-26-8) reference substance was prepared by column chromatography in the laboratory, about 40 mg, with an NMR quantitative purity of 99.1%.

[0039] (II) Preparation of standard solution

[0040] Accurately weigh 10 mg (accurate to 0.1 mg) of each of the column-purified compound X reference substance and the sildenafil reference substance, and transfer them into 10 mL volumetric flasks, respectively. Both are diluted to the mark with acetonitrile to prepare standard stock solutions with a mass concentration of 1000 mg / L, which are stored at 4°C in the dark.

[0041] Take an appropriate amount of the above standard stock solutions, respectively, and place them in 100 mL volumetric flasks. Both are diluted to the mark with acetonitrile to prepare standard intermediate solutions with a mass concentration of 1000 µg / L, which are stored at 4°C in the dark.

[0042] (III) Sample pretreatment

[0043] Weigh 0.5 g of the sample (accurate to 0.01 g), add 10 mL of methanol into a 50 mL plastic centrifuge tube, ultrasonic for 15 min, centrifuge at 4000 r / min for 5 min, take an appropriate amount of supernatant to pass through the membrane, dilute 2000 times for HPLC-QTof screening identification, and dilute 200000 times for HPLC-MS / MS qualitative and quantitative detection.

[0044] (IV) Analysis conditions

[0045] 1. HPLC-QTof detection

[0046] (1) Chromatographic conditions

[0047] Chromatographic column: ZORBAX Eclipse Plus C18 column (150 mm x 3.0 mm, 3.5 μm); column temperature: 40℃; mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile; flow rate: 0.5 mL / min. Gradient elution program: 0~3.0 min, 5%B~90%B; 3.0~10.0 min, 90%B; 10.0~10.1 min, 90%B~5%B; 10.1~12.0 min, 5%B. Injection volume: 10 μL.

[0048] (2) Mass spectrometry conditions

[0049] Ionization mode: electrospray ionization (ESI) source, positive ion mode; mass spectrometry correction method: sodium formate solution external standard correction; correction period: sodium formate correction solution and other test solutions are inserted for sampling (every time a standard solution or sample is inserted, a sodium formate solution is first inserted for correction of mass number before mass spectrometry); acquisition mode: primary and secondary acquisition at the same time (TOF MS-IDA-TOF MS / MS); primary and secondary scanning range: m / z 50~650, secondary in high sensitivity mode; electrospray voltage (IS): 5500 V; ion source temperature (TEM): 550℃; atomization gas pressure (GS1): 345 kPa; auxiliary gas pressure (GS2): 414 kPa; curtain gas pressure (CUR): 276 kPa; de-clustering voltage (DP): 30 V; collision energy (CE): 30±10 V.

[0050] 2. HPLC-MS / MS detection

[0051] (1) Chromatographic conditions

[0052] Chromatographic column: ZORBAX Eclipse Plus C18 column (100 mm x 2.1 mm, 3.5 μm); column temperature: 40 °C; mobile phase A was 0.1% formic acid in water, mobile phase B was acetonitrile; flow rate: 0.5 mL / min. Gradient elution program: 0~2.0 min, 10%B~95%B; 2.0~5.0 min, 95%B; 5.0~5.1 min, 95%B~5%B; 5.1~8.0 min, 5%B. Injection volume: 5 μL.

[0053] (2) Mass spectrometry conditions

[0054] Ionization mode: electrospray ionization (ESI) source, positive ion mode; electrospray voltage (IS): 5500 V; ion source temperature (TEM): 550 °C; nebulization gas pressure (GS1): 345 kPa; auxiliary gas pressure (GS2): 414 kPa; curtain gas pressure (CUR): 276 kPa; de-clustering voltage (DP): 100 V; collision energy (CE): 45 V. Quantitative ion pair: 449 / 243, qualitative ion pair: 449 / 259; each pair of ion residence time (Dwell time): 250 ms.

[0055] II. Results and discussion

[0056] (1) Liquid chromatography-time-of-flight mass spectrometry detection and molecular structure identification

[0057] According to the conditions in "Section 4, Part I, Section 1" and BJS 202405 Table A.1, the oyster white peony powder sample was subjected to target screening of 95 kinds of sildenafil compounds, and no above-mentioned 95 kinds of sildenafil compounds were detected. From the HPLC-QTof total ion chromatogram (TIC), it can be seen that there is a obvious chromatographic peak at retention time 3.535 min, with accurate molecular weight 449.22166, see Figure 1 ; the secondary fragment mass spectrum is shown in Figure 2 . The m / z 58, 99, 100 ions contained in the secondary fragments are completely consistent with sildenafil. The secondary fragment ion mass spectrum of sildenafil was analyzed, and it was speculated that the above fragment ions were from the cleavage of the methyl piperazine group, and the fragmentation mechanism analysis is shown in Figure 3 . Therefore, it can be determined that the compound is a sildenafil compound, and must contain a methyl piperazine group. The molecular formula C 22 H 32 N4O4S is speculated by the instrument masterview qualitative software. Input into the Chemspider website to find the possible compounds of the molecular formula is 1662, which cannot accurately identify the molecular structure formula of the target.

[0058] The structure of the target was identified by using the self-developed molecular structure identification software. The identification results were ranked according to the relative scores, as shown in Table 1. The molecular formula of the top 1-4 ranked compounds was consistent with the molecular formula identified above, so the compound ranked 5 was excluded. The compound ranked 4 was also excluded because it did not contain a methyl piperazine group. The top three compounds belonged to isomers and all contained a methyl piperazine group. Based on the different substituents on the pyrimidine ring and the secondary fragments, it was speculated that the compound ranked first in Table 1 (compound X) had the highest probability.

[0059] Table 1 Identification results of self-developed software

[0060]

[0061] (II) Purification of the compound

[0062] According to the HPLC-QTof detection and self-developed molecular structure identification software in “Part (I)”, it has been identified with high probability that the target is compound X, but in order to ensure the accuracy of the identification results, NMR structure identification is needed. NMR structure identification requires high purity of the compound, so the laboratory needs to purify the target compound using the test sample for NMR structure identification. At the same time, the purified target also provides a standard for establishing a qualitative and quantitative detection method of liquid chromatography-triple quadrupole mass spectrometry.

[0063] The test sample sent by the customer was combined and weighed, then placed in a chicken heart bottle, 100 ml of acetonitrile was added and ultrasonic extraction was performed for 30 min. The filtrate was concentrated to dryness at 45°C under reduced pressure to obtain a crude product. After purification by silica gel chromatography column, the fractions were collected to obtain about 50 mg of purified target control, and the purity was 99.1% by NMR detection.

[0064] (III) NMR structure identification

[0065] The target after purification in “Part (II)” was detected by NMR. The hydrogen spectrum and carbon spectrum of the compound are shown in Figure 4 and Figure 5 , and the data is shown in Table 2. The identification result of the compound is 5,6-Diethyl-2-[5-[(4-methyl-1-piperazinyl)sulfonyl]-2-propoxyphenyl]-4(3H)-pyrimidinone, and the structural formula is shown in Figure 6 .

[0066] Table 2 NMR data

[0067]

[0068] (IV) Investigation of liquid chromatography-triple quadrupole mass spectrometry detection method

[0069] Methodology investigation was carried out using purified compound X as the control.

[0070] 1. Linear range

[0071] The compound X standard intermediate solution (first part (ii)) was diluted with acetonitrile to a mass concentration of 0.5, 1, 5, 25 and 50 μg / L standard working solution, and detected under the conditions of "first part (iv) section 2" to draw a working curve of peak area versus mass concentration of each component in the standard solution. Compound X showed a linear relationship in the range of 0.5-50 μg / L, the linear equation was y = 4856.98812 x, and the correlation coefficient (R2) was 0.99995. Representative spectrum was shown in r Figure 7 Figure 7 A was the quantitative ion pair, Figure 7 B was the qualitative ion pair.

[0072] 2. Limit of detection, recovery, precision and limit of quantification

[0073] The limit of detection (LOD) of the method (referring to the detection method provided in "first part (iv) section 2", the same below) was determined according to the signal-to-noise ratio (S / N) ≥ 3, all of which were 0.005 mg / kg. According to the European document SANTE / 11813 / 2017, the limit of quantification (LOQ) was the lowest spiked level in which the recovery and RSD in the spiked recovery test met the performance requirements (recovery of 70%-120% and RSD ≤ 20%). S / N

[0074] The experiment selected blank weight loss capsule samples for 0.01, 0.02 and 0.10 mg / kg 3 levels of spiked recovery test, 6 parallel samples for each level (n = 6), and detected according to the method. The spiked recovery and precision data were shown in Table 3, and the representative spectrum was shown in n Figure 8 Figure 8 A was the quantitative ion pair, Figure 8 B was the qualitative ion pair. The results showed that the recovery of compound X was 99.1%-112.2%, and the relative standard deviation (RSD) was 2.6%-3.7%, indicating that the method had high accuracy and good repeatability, and was suitable for the detection of health food.

[0075] As can be seen from the above, the minimum spiked level of the method was 0.01 mg / kg, the average recovery and RSD of compound X in the blank matrix met the requirements (see Table 3), and the signal-to-noise ratio (S / N) was > 10, so the limit of quantification (LOQ) of the method was determined to be 0.01 mg / kg. S / N

[0076] ​​​​​​Table 3 Compound X 3 spiking level recovery and relative standard deviation n =6)

[0077]

[0078] (Four) content of the sample to be tested

[0079] Three bags of samples to be tested were weighed, sample pretreatment was carried out according to "Part I (Three)", the sample to be tested was obtained, and detection was carried out according to the conditions of "Part I (Four) Section 2", the average content of the sample to be tested was 26.3 mg / g, as shown in Table 4. According to the product label, each box of sample is 2 bags of product, and the net weight of each bag of sample is 1g (1-2 bags are recommended each time), that is, the content of compound X in each bag of sample is 26.3 mg. The specification of sildenafil medicine is 25 mg, 50 mg, 100 mg, etc. The specification of the sample to be tested is similar to the minimum specification of sildenafil.

[0080] Table 4 Determination results of the content of compound X in the sample to be tested

[0081]

[0082] III. Conclusion

[0083] A new sildenafil compound with inhibitory activity on PDE5 is found in health food. The compound is identified as 5,6-Diethyl-2-[5-[(4-methyl-1-piperazinyl)sulfonyl]-2-propoxyphenyl]-4(3H)-pyrimidinone by LC-QTof, laboratory self-developed software, chromatographic column purification, NMR and other means. The compound has not been approved as an active pharmaceutical ingredient at home and abroad, and this paper first reports the illegal addition of the compound in health food. No generic name of the compound has been found, and the inventors name the compound as "diethyl pyrimidine sildenafil" to facilitate its monitoring. Since commercial diethyl pyrimidine sildenafil reference cannot be purchased, the diethyl pyrimidine sildenafil obtained after sample purification is used as a reference to establish a liquid chromatography-triple quadrupole mass spectrometry qualitative and quantitative detection method for new sildenafil compounds in oyster white peony powder, filling the gap in this field, providing technical support for market supervision, public security, customs and other relevant departments, and having good application value. The research idea can provide a reference for the identification of other illegal additions of unknown substances.

[0084] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any skilled person in the art can make equivalent substitutions or changes within the scope of the technical disclosure of the present application according to the technical solution and inventive concept of the present application, which should be covered within the protection scope of the present application.

Claims

1. A method for detecting natriuretic compounds in health food products, characterized in that, Comprising the following steps: Qualitative and quantitative detection is performed by HPLC-MS / MS; The chromatographic conditions include the following: Chromatographic column: C18 column; column temperature: 40 DEG C; mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile; flow rate: 0.5 mL / min; gradient elution program: 0~2.0 min, 10%B~95%B; 2.0~5.0 min, 95%B; 5.0~5.1 min, 95%B~5%B; 5.1~8.0 min, 5%B; injection volume: 5 µL; The mass spectrometry conditions include the following: Ionization mode: electrospray ionization source, positive ion mode; electrospray voltage: 5500V; ion source temperature: 550 DEG C; atomization gas pressure: 345 kPa; auxiliary gas pressure: 414 kPa; curtain gas pressure: 276 kPa; declustering voltage: 100 V; collision energy: 45V; quantitative ion pair: 449 / 243, qualitative ion pair: 449 / 259; each pair of ion residence time: 250 ms; The PDE5 inhibitor compound contains a methylpiperazine group; The PDE5 inhibitor compound is compound X, and the structural formula of the compound X is shown in formula (I): Formula (I).

2. The method according to claim 1, wherein the PDE5 inhibitor is selected from the group consisting of sildenafil, vardenafil, tadalafil, udenafil, avanafil, and lodenafil. The preparation method of the sample solution to be tested includes: weighing 0.5 g of the sample to be tested, placing it in a 50 mL plastic centrifuge tube, adding 10 mL of methanol, ultrasonicating for 15 min, centrifuging at 4000 r / min for 5 min, taking an appropriate amount of supernatant to pass through a membrane, diluting 200000 times, and obtaining a sample solution to be tested.

3. The method according to claim 1, wherein the PDE5 inhibitor is selected from the group consisting of sildenafil, vardenafil, tadalafil, udenafil, avanafil, and lodenafil. The preparation method of the reference substance includes: placing the sample to be tested in a chicken heart bottle, adding 100 ml of acetonitrile, ultrasonicating for 30 min, filtering, and concentrating the filtrate at 45 DEG C under reduced pressure to dryness to obtain a crude product; the crude product is purified by a silica gel chromatographic column, and the fractions are collected to obtain the purified target substance, which is the reference substance.

4. The method according to claim 3, wherein the PDE5 inhibitor is selected from the group consisting of sildenafil, vardenafil, tadalafil, udenafil, avanafil, and lodenafil. The reference substance is used to configure standard solutions of different concentrations, and a working curve is drawn by taking the peak area as the mass concentration of each component in the standard solution.

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