Determination method of 3-hydroxypropyl nortadalafil

Abstract

The invention provides a determination method of 3-hydroxypropyl nortadalafil, which comprises the following steps: weighing a 3-hydroxypropyl nortadalafil sample, adding a methanol solution, dissolving with methanol, fixing the volume, and preparing a standard stock solution; taking a proper amount of standard stock solution, diluting with a methanol-water solution, and preparing a standard working solution; transferring into a centrifugal tube for centrifugal treatment; filtering the supernate through a microfiltration membrane, and taking a subsequent filtrate; taking a proper amount of the standard working solution, injecting the standard working solution into a high performance liquid chromatography-tandem mass spectrometer, and recording a chromatogram and a mass spectrum; and sequentially injecting the series of standard working solutions into a high performance liquid chromatography-tandem mass spectrometer, and drawing a standard curve. According to the present invention, the blank of the ultra-high performance liquid chromatography-tandem mass spectrometry detection method for the 3-hydroxypropyl nortadalafil is filled, the detection basis is provided for ensuring the food safety law enforcement method, the drug abuse risk is avoided, and the food quality is ensured to meet the standard.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, in particular to a method for determining 3-hydroxypropyl nor-tadalafil. Background Art

[0002] The earliest synthesized sildenafil is a drug for treating male erectile dysfunction that was accidentally invented during the development of drugs for treating cardiovascular diseases. Nafil drugs are a class of drugs mainly used to treat male erectile dysfunction (ED), and they belong to phosphodiesterase type 5 inhibitors (PDE5 inhibitors). Nafil is classified as a drug and is not allowed to be added to food (including health foods). Due to the special efficacy of the drug, it is often illegally added to food (including health foods), mainly in foods such as biscuits, candies, wine, jelly and coffee, or health foods containing similar matrices.

[0003] The existing technology is unable to effectively detect this type of component, and the detection scheme for this type of substance is still blank. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for determining 3-hydroxypropyl nor-tadalafil to solve the problems raised in the above-mentioned background technology. The present invention fills the gap in the ultra-high performance liquid chromatography-tandem mass spectrometry detection method of 3-hydroxypropyl nor-tadalafil, provides a detection basis for ensuring food safety law enforcement legislation, avoids the risk of drug abuse, and ensures that food quality meets the standards.

[0005] In order to achieve the above object, the present invention is implemented by the following technical scheme: a method for determining 3-hydroxypropyl nor-tadalafil, comprising the following steps:

[0006] Step 1, accurately weigh a certain amount of 3-hydroxypropyl nor-tadalafil sample, add methanol solution, perform ultrasonic extraction on the standard stock solution, dissolve and dilute with methanol, and prepare the standard stock solution;

[0007] Step 2: Take an appropriate amount of standard stock solution, dilute it with methanol-water solution, and prepare standard working solution;

[0008] Step 3, transferring to a centrifuge tube for centrifugation;

[0009] Step 4: Filter the supernatant through a microporous membrane, take the filtrate, and dilute it with methanol-water solution to the linear range according to the actual concentration;

[0010] Step 5: Take an appropriate amount of standard working solution, inject it into the HPLC-MS / MS, record the chromatogram and mass spectrum, calculate the theoretical plate number, separation degree and tailing factor;

[0011] Step 6, drawing a standard curve with the concentration of 3-hydroxypropyl nortadalafil as the abscissa and the corresponding peak area as the ordinate;

[0012] Step 7: Calculate the concentration of 3-hydroxypropyl nor-tadalafil in the sample according to the standard curve.

[0013] Furthermore, the mixed standard working solution is measured according to the instrument reference conditions to obtain the chromatographic peak area of ​​the corresponding standard solution. Then, the concentration of the mixed standard working solution is used as the abscissa, and the peak area of ​​the chromatographic peak of the quantitative ion is used as the ordinate, and the standard curve is drawn through these data.

[0014] Furthermore, the standard curve is used for subsequent determination of the concentration of the corresponding component in the sample, and the corresponding concentration value is found on the standard curve according to the chromatographic peak area measured in the sample.

[0015] Furthermore, the content of 3-hydroxypropyl nor-tadalafil in the sample is calculated based on the specific concentration value of 3-hydroxypropyl nor-tadalafil in the sample, the dilution multiple during the sample processing, and the mass or volume parameters of the sample.

[0016] Further, the specific formula for calculating the content of 3-hydroxypropyl nortadalafil in a solid or semi-solid sample is as follows:

[0017]

[0018] Wherein, X is the content of 3-hydroxypropyl nortadalafil in the sample, in milligrams per kilogram (mg / kg);

[0019] c is the concentration of 3-hydroxypropyl nortadalafil in the sample solution obtained from the standard curve, in micrograms per liter (μg / L);

[0020] V is the sample volume, in milliliters (mL);

[0021] n is the sample dilution multiple;

[0022] m is the sample weight in grams (g).

[0023] Further, the specific formula for calculating the content of 3-hydroxypropyl nor-tadalafil in the liquid sample is as follows:

[0024]

[0025] Wherein, X is the content of 3-hydroxypropyl nortadalafil in the sample, in milligrams per liter (mg / L);

[0026] c is the concentration of 3-hydroxypropyl nortadalafil in the sample solution obtained from the standard curve, in micrograms per liter (μg / L);

[0027] V is the sample volume, in milliliters (mL);

[0028] n is the sample dilution multiple;

[0029] V0 is the volume of the sample aspirated, in milliliters (mL).

[0030] Furthermore, the mobile phases were 0.1% formic acid aqueous solution and acetonitrile.

[0031] Furthermore, by adjusting the mobile phase gradient and changing the chromatographic column, 3-hydroxypropyl nor-tadalafil was completely separated from the existing 2-hydroxypropyl nor-tadalafil.

[0032] Furthermore, in view of the differences in separation effects between chromatographic columns of different brands and specifications, a single label was used to measure their respective peak times.

[0033] Furthermore, during the sample determination process, an H+ is added to the liquid-mass ionization to form a molecular ion, i.e., a parent ion. The molecular ion with the addition of an H+ on the mass spectrum is calculated, and a full scan is performed into the mass spectrometer through single-label injection to obtain a full scan image. The parent ion is subjected to collision cracking with argon gas, and the generated fragment ions, i.e., daughter ions, are fully scanned to obtain a secondary mass spectrometry scan image.

[0034] Beneficial effects of the present invention:

[0035] 1. The determination method of 3-hydroxypropyl nor-tadalafil fills the long-standing blank area of ​​3-hydroxypropyl nor-tadalafil ultra-high performance liquid chromatography-tandem mass spectrometry detection. It has high sensitivity and can accurately capture extremely small amounts of 3-hydroxypropyl nor-tadalafil components in food samples, effectively eliminating the interference of complex components in the food matrix on the test results, ensuring the authenticity and reliability of each test data.

[0036] 2. The determination method of 3-hydroxypropyl nor-tadalafil provides technical support in the process of food safety law enforcement, enabling accurate investigation and evidence collection and rapid generation of test data when faced with foods suspected of illegally adding 3-hydroxypropyl nor-tadalafil, thus providing a quantitative basis for the formulation of more complete and practical food safety regulations.

[0037] 3. The determination method of 3-hydroxypropyl nor-tadalafil can avoid serious interactions when tadalafil-type drugs are used simultaneously with certain drugs (such as nitrate drugs), which may lead to a sharp drop in blood pressure. At the same time, this method can also be used to ensure that the ingredients of food meet their due quality and safety standards, so that consumers can buy food with confidence. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the molecular structure diagram of 3-hydroxypropyl nor-tadalafil;

[0039] Figure 2 This is a full scan image obtained by injecting a single label into the mass spectrometer for full scan;

[0040] Figure 3 The secondary mass spectrometry scan is obtained by performing a full scan on the generated fragment ions;

[0041] Figure 4 This is a spike recovery test diagram of 3-hydroxypropyl nortadalafil in an embodiment of the present invention;

[0042] Figure 5 This is a standard curve diagram provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0043] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0044] See also Figures 1 to 5 The present invention provides the following technical solutions: a method for determining 3-hydroxypropyl nor-tadalafil, which is based on ultra-high performance liquid chromatography-tandem mass spectrometry for detection. In this embodiment, in order to reduce duplication of work and improve detection effectiveness, the measured 3-hydroxypropyl nor-tadalafil and other nafil-like substances are subjected to only one pretreatment extraction. The chromatographic column is selected to have an ACQUITY UPLC HSS T3 (2.1×100mm, 1.8μm) separation effect that can meet the detection baseline separation requirements. The mobile phase is selected to have 0.1% formic acid aqueous solution and acetonitrile, which is convenient for configuration and replacement. The acetonitrile pump has low pressure, and the non-polar substances are rinsed more cleanly, which prolongs the service life of the chromatographic column. In terms of gradient, since 3-hydroxypropyl nor-tadalafil and the existing 2-hydroxypropyl nor-tadalafil are isomers, the parent ions and daughter ions of the two substances are the same when scanned on the mass spectrometer. The two substances can only be completely separated by adjusting the mobile phase gradient and changing the chromatographic column. Chromatographic columns of different brands and specifications have different separation effects, so a single label is required for each peak time.

[0045] The relative molecular mass of 3-hydroxypropyl nortadalafil is 433.46, and its molecular structure is as follows Figure 1 According to liquid-mass ionization, one H+ is usually required to form a molecular ion, i.e., the parent ion. It can be calculated that the molecular ion of adding one H+ on the mass spectrum is approximately 434. By injecting a single standard into the mass spectrometer for full scan, a full scan image is obtained, see Figure 2 , it can be seen that the first heavy molecular ion, i.e., the parent ion, is 434.1. Then, the parent ion is subjected to collision pyrolysis with argon gas, and the generated fragment ions, i.e., the daughter ions, are fully scanned to obtain a secondary mass spectrometry scan, as shown in FIG. Figure 3 , we can see that the product ion signals of 312, 135 and 169 are obvious, so these three are selected as the qualifier ions.

[0046] The above determination method fills the long-standing blank area of ​​3-hydroxypropyl nor-tadalafil ultra-high performance liquid chromatography-tandem mass spectrometry detection. It has high sensitivity and can accurately capture extremely small amounts of 3-hydroxypropyl nor-tadalafil in food samples, effectively eliminating the interference of complex components in the food matrix on the test results, ensuring the authenticity and reliability of each test data.

[0047] In this embodiment, the mixed standard working solution is measured according to the instrument reference conditions to obtain the chromatographic peak area of ​​the corresponding standard solution. Then, the concentration of the mixed standard working solution is used as the abscissa, and the peak area of ​​the chromatographic peak of the quantitative ion is used as the ordinate, and a standard curve is drawn through these data. The standard curve is used for subsequent determination of the concentration of the corresponding component in the sample, and the corresponding concentration value is found on the standard curve according to the chromatographic peak area measured in the sample.

[0048] The content value of 3-hydroxypropyl nor-tadalafil in the sample is calculated based on the specific concentration value of 3-hydroxypropyl nor-tadalafil in the sample, the dilution multiple during the sample processing, and the mass or volume parameters of the sample.

[0049] In this embodiment, the specific formula for calculating the content of 3-hydroxypropyl nortadalafil in a solid or semi-solid sample is as follows:

[0050]

[0051] Wherein, X is the content of 3-hydroxypropyl nortadalafil in the sample, in milligrams per kilogram (mg / kg);

[0052] c is the concentration of 3-hydroxypropyl nortadalafil in the sample solution obtained from the standard curve, in micrograms per liter (μg / L);

[0053] V is the sample volume, in milliliters (mL);

[0054] n is the sample dilution multiple;

[0055] m is the sample weight in grams (g).

[0056] In this embodiment, the specific formula for calculating the content of 3-hydroxypropyl nortadalafil in the liquid sample is as follows:

[0057]

[0058] Wherein, X is the content of 3-hydroxypropyl nortadalafil in the sample, in milligrams per liter (mg / L);

[0059] c is the concentration of 3-hydroxypropyl nortadalafil in the sample solution obtained from the standard curve, in micrograms per liter (μg / L);

[0060] V is the sample volume, in milliliters (mL);

[0061] n is the sample dilution multiple;

[0062] V0 is the volume of the sample sucked, in milliliters (mL)

[0063] This embodiment also provides the following specific steps:

[0064] 1. Accurately weigh 1g of sample and place it in a 50mL volumetric flask, add an appropriate amount of methanol, perform ultrasonic extraction for 15min, cool to room temperature, make up to volume with methanol, transfer to a 50mL centrifuge tube, centrifuge at 4000r / min for 5min, filter the supernatant through a microporous filter membrane, take the filtrate, and dilute it appropriately with methanol-water solution (1+1) to the linear range according to the actual concentration for later use.

[0065] 2. Instrument reference conditions

[0066] Chromatographic conditions: Column: ACQUITY UPLC HSS T3 (2.1×100 mm, 1.8 μm)

[0067] Injection volume: 2 μL, column temperature: 30 °C

[0068] Table 1: Mobile phase gradient change table

[0069] Gradient time / min 0.1% formic acid aqueous solution / % Acetonitrile / % Flow rate mL / min 0 90 10 0.3 4.5 10 90 0.3 6.1 10 90 0.3 6.1 90 10 0.3 9.5 90 10 0.3

[0070] Mass spectrometry conditions:

[0071] A. Ion source: electrospray ionization (ESI);

[0072] B. Detection method: multiple reaction monitoring (MRM);

[0073] C. Scanning mode: positive ion mode;

[0074] D. Detection tube voltage: 3.5kV;

[0075] E. Capillary temperature: 320℃;

[0076] F. Sheath gas pressure: 35psi;

[0077] G. Atomizing gas temperature: 300℃;

[0078] H. Auxiliary gas pressure: 15psi;

[0079] Table 2 Qualitative and quantitative ions and mass spectrometry analysis parameters of 3-hydroxypropyl nortadalafil

[0080]

[0081]

[0082] 3. The recovery rate of 3-hydroxypropyl nor-tadalafil is in the range of 71%-116%. See the recovery test chart for the spiked recovery. Figure 4 (4.52min is 3-hydroxypropyl nor-tadalafil, 4.62min is 2-hydroxypropyl nor-tadalafil), standard curve is shown in Figure 5 .

[0083] The basic principles and main features of the present invention and the advantages of the present invention are shown and described above. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention.

[0084] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for determining 3-hydroxypropyl nor-tadalafil, characterized in that: The following steps are involved: Step 1, accurately weigh a certain amount of 3-hydroxypropyl nor-tadalafil sample, add methanol solution, perform ultrasonic extraction on the standard stock solution, dissolve and dilute with methanol, and prepare the standard stock solution; Step 2: Take an appropriate amount of standard stock solution, dilute it with methanol-water solution, and prepare standard working solution; Step 3, transferring to a centrifuge tube for centrifugation; Step 4: Filter the supernatant through a microporous membrane, take the filtrate, and dilute it with methanol-water solution to the linear range according to the actual concentration; Step 5: Take an appropriate amount of standard working solution, inject it into the high performance liquid chromatography-tandem mass spectrometer, record the chromatogram and mass spectrum, and calculate the theoretical plate number, separation degree and tailing factor; Step 6, drawing a standard curve with the concentration of 3-hydroxypropyl nortadalafil as the abscissa and the corresponding peak area as the ordinate; Step 7: Calculate the concentration of 3-hydroxypropyl nor-tadalafil in the sample according to the standard curve.

2. The method for determining 3-hydroxypropyl nor-tadalafil according to claim 1, wherein: The mixed standard working solution is measured according to the instrument reference conditions to obtain the chromatographic peak area of ​​the corresponding standard solution. Then the concentration of the mixed standard working solution is used as the horizontal coordinate and the peak area of ​​the chromatographic peak of the quantitative ion is used as the vertical coordinate to draw the standard curve.

3. The method for determining 3-hydroxypropyl nor-tadalafil according to claim 2, wherein: The standard curve is used to subsequently determine the concentration of the corresponding component in the sample, and the corresponding concentration value is found on the standard curve according to the chromatographic peak area measured in the sample.

4. The method for determining 3-hydroxypropyl nor-tadalafil according to claim 1, characterized in that: The content value of 3-hydroxypropyl nor-tadalafil in the sample is calculated based on the specific concentration value of 3-hydroxypropyl nor-tadalafil in the sample, the dilution multiple during the sample processing, and the mass or volume parameters of the sample.

5. The method for determining 3-hydroxypropyl nor-tadalafil according to claim 4, characterized in that: The specific formula for calculating the content of 3-hydroxypropyl nor-tadalafil in solid or semi-solid samples is as follows: Wherein, X is the content of 3-hydroxypropyl nortadalafil in the sample, in milligrams per kilogram (mg / kg); c is the concentration of 3-hydroxypropyl nortadalafil in the sample solution obtained from the standard curve, in micrograms per liter (μg / L); V is the sample volume, in milliliters (mL); n is the sample dilution multiple; m is the sample weight in grams (g).

6. The method for determining 3-hydroxypropyl nor-tadalafil according to claim 1, characterized in that: The specific formula for calculating the content of 3-hydroxypropyl nor-tadalafil in the liquid sample is as follows: Wherein, X is the content of 3-hydroxypropyl nortadalafil in the sample, in milligrams per liter (mg / L); c is the concentration of 3-hydroxypropyl nortadalafil in the sample solution obtained from the standard curve, in micrograms per liter (μg / L); V is the sample volume, in milliliters (mL); n is the sample dilution multiple; V0 is the volume of the sample aspirated, in milliliters (mL).

7. The method for determining 3-hydroxypropyl nor-tadalafil according to claim 1, characterized in that: The mobile phases were 0.1% formic acid aqueous solution and acetonitrile.

8. The method for determining 3-hydroxypropyl nor-tadalafil according to claim 1, characterized in that: By adjusting the mobile phase gradient and changing the chromatographic column, 3-hydroxypropyl nor-tadalafil was completely separated from the existing 2-hydroxypropyl nor-tadalafil.

9. The method for determining 3-hydroxypropyl nor-tadalafil according to claim 1, characterized in that: In view of the differences in separation effects between chromatographic columns of different brands and specifications, a single standard is used to measure their respective peak times.

10. The method for determining 3-hydroxypropyl nor-tadalafil according to claim 1, characterized in that: During the sample determination process, the liquid-mass ionization carries an H+ to form a molecular ion, namely the parent ion, and the molecular ion with the added H+ on the mass spectrum is calculated. Through single-label injection, a full scan is performed into the mass spectrometer to obtain a full scan image. The parent ion is collision-crackered with argon gas, and the generated fragment ions, namely the daughter ions, are fully scanned to obtain a secondary mass spectrum scan image.

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