Method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisavodine and indapamide in soft capsule

Abstract

The invention discloses a method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodine and indapamide in a soft capsule, which comprises the following steps: preparing 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL mixed standard working curve solutions; separating capsule shells and contents of the soft capsule samples, and fully and uniformly mixing to obtain capsule contents; weighing capsule contents, adding acetonitrile, extracting, centrifuging, taking supernate, diluting with acetonitrile to a constant volume, and uniformly shaking to obtain an extracting solution; transferring the extracting solution into a purifying column, and collecting a purifying solution to prepare a sample solution; and firstly determining the standard working curve solution by using a chromatographic instrument, and then determining the sample solution to obtain a sample concentration value. The method comprises the following steps: uniformly mixing a sample in a vortex manner, carrying out centrifugal treatment, purifying the centrifuged supernatant, and determining; a first sieve plate, a PSA purification layer, a second sieve plate, a silica purification layer, a microporous filter membrane and a third sieve plate are arranged in the adopted purification column, so that a better purification effect is achieved, and the problem of matrix interference is effectively solved.

Description

Technical Field

[0001] The invention belongs to the technical field of detection methods, and particularly relates to a method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyl and indapamide in soft capsules. Background Art

[0002] The basic operation of the current national standard "Determination of Sibutramine and Other Compounds in Food" (BJS201701) for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyl, and indapamide is: the sample is first extracted and then measured by liquid chromatography-mass spectrometry. This method will have matrix interference if the sample is directly measured without purification, resulting in inaccurate measurement results.

[0003] At present, the main methods for dealing with matrix interference are: increasing the dilution degree and preparing a matrix standard curve, but both methods have disadvantages. If the content of the component to be tested in the sample is very small, increasing the dilution degree may cause the concentration on the machine to be too low to reach the detection limit, resulting in false negative consequences. The preparation of a matrix standard curve requires a negative sample with a matrix that is basically the same as the matrix of the sample to be tested, but most of the samples to be tested are now mixed with a variety of special components, and it is difficult to find a negative sample with a basically identical matrix. Summary of the invention

[0004] The invention provides a method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyne and indapamide in soft capsules. The method solves the problem of matrix interference by purification and is suitable for soft capsule samples with small contents of hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyne and indapamide, and does not require the preparation of a matrix spike curve.

[0005] The invention provides a method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyl and indapamide in soft capsules to solve the above technical problems.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyne and indapamide in soft capsules comprises: preparing a standard curve solution: weighing standard substances of hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyne and indapamide, diluting and fixing the solution with acetonitrile to obtain mixed standard working curve solutions with concentrations of 2, 5, 10, 20, 50 and 100 ng / mL respectively; preparing a sample solution: separating a capsule shell from the contents of a soft capsule sample, and fully mixing to obtain the capsule contents; weighing the capsule contents, adding acetonitrile, extracting, centrifuging, taking a supernatant, diluting and fixing the solution with acetonitrile, and shaking to obtain an extract; transferring the extract into a purification column, and collecting the purified solution to obtain the extract; and determining: using a chromatographic instrument to first determine the standard working curve solution, and then determining the sample solution to obtain the sample concentration value.

[0008] Furthermore, the extraction in the sample solution preparation is vortex extraction, and the time is 10-25 minutes.

[0009] Furthermore, the centrifugal treatment speed in the sample solution preparation is 2000-5000 r / min, and the time is 3-7 min.

[0010] Furthermore, a filler is provided in the purification column in the sample solution preparation, and the fillers are arranged from top to bottom: a first sieve plate, a PSA purification layer, a second sieve plate, a silica purification layer, a microporous filter membrane, and a third sieve plate.

[0011] Furthermore, the amount of the PSA purification layer in the filler is 400-600 mg, and the amount of the silica purification layer is 450-600 mg.

[0012] Furthermore, the pore size of the first sieve plate is 30-50 μm, the pore size of the second sieve plate is 20-30 μm, and the pore size of the third sieve plate is 10-15 μm.

[0013] Furthermore, during the determination, mobile phase A was acetonitrile containing 0.1% formic acid, and mobile phase B was 10 mmol / L ammonium acetate solution containing 0.1% formic acid.

[0014] Furthermore, during the determination, the mobile phase gradient was: mobile phase A: 5%, 5%, 98%, 98%, 5%, 5%; mobile phase B: 95%, 95%, 2%, 2%, 95%, 95%; mobile phase time: 0 min, 5 min, 15 min, 25 min, 27 min, 32 min.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The method of the present invention firstly vortexes and mixes the sample, then centrifuges it, and then purifies the supernatant of the centrifuge before measuring it; the purification column used is provided with a first sieve plate, a PSA purification layer, a second sieve plate, a silica purification layer, a microporous filter membrane, and a third sieve plate, has a good purification effect, and effectively solves the problem of matrix interference.

[0017] It can be seen from the data in the examples that the method of the present invention is compared with the national standard "Determination of Sibutramine and Other Compounds in Food" (BJS201701), and the average results and relative errors of hydrochlorothiazide, caffeine, and phenolphthalein are several times or dozens of times different, and the data of the two are significantly different; from the test results of the detection limit and the quantification limit, it can be seen that the detection limit results of the present invention are less than 0.86 μg / kg, and the quantification limit is less than 2.9 μg / kg. Compared with the national standard, the present invention has higher sensitivity; when the repeatability and recovery rate are measured, the recovery rate is between 85-110%, indicating good accuracy and reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the mass spectrum of hydrochlorothiazide.

[0019] Figure 2 This is the mass spectrum of caffeine.

[0020] Figure 3 This is the mass spectrum of phenolphthalein.

[0021] Figure 4 This is the mass spectrum of fenofibrate.

[0022] Figure 5 This is the mass spectrum of bisacodyl.

[0023] Figure 6 This is the mass spectrum of indapamide. DETAILED DESCRIPTION

[0024] In order to facilitate a better understanding of the present invention, the following is an explanation in conjunction with the accompanying drawings and embodiments. These embodiments belong to the protection scope of the present invention, but do not limit the protection scope of the present invention.

[0025] Example 1

[0026] A method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyne and indapamide in soft capsules comprises: preparing a standard curve solution: weighing standard substances of hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyne and indapamide, diluting and fixing the solution with acetonitrile to obtain mixed standard working curve solutions with concentrations of 2, 5, 10, 20, 50 and 100 ng / mL respectively; preparing a sample solution: separating a capsule shell from the contents of a soft capsule sample, and fully mixing to obtain the capsule contents; weighing the capsule contents, adding acetonitrile, extracting, centrifuging, taking a supernatant, diluting and fixing the solution with acetonitrile, and shaking to obtain an extract; transferring the extract into a purification column, and collecting the purified solution to obtain the extract; and determining: using a chromatographic instrument to first determine the standard working curve solution, and then determining the sample solution to obtain the sample concentration value.

[0027] The extraction in the sample solution preparation was vortex extraction, and the time was 10 min.

[0028] The centrifugal speed in the sample solution preparation was 2000 r / min and the time was 3 min.

[0029] The purification column in the sample solution preparation is provided with fillers, and the fillers are arranged from top to bottom: a first sieve plate, a PSA purification layer, a second sieve plate, a silica purification layer, a microporous filter membrane, and a third sieve plate.

[0030] The amount of PSA purification layer in the filler is 400 mg, and the amount of silica purification layer is 450 mg.

[0031] The pore size of the first sieve plate is 30 μm, the pore size of the second sieve plate is 20 μm, and the pore size of the third sieve plate is 10 μm.

[0032] During the determination, mobile phase A was acetonitrile containing 0.1% formic acid, and mobile phase B was 10 mmol / L ammonium acetate solution containing 0.1% formic acid.

[0033] During the determination, the mobile phase gradient was: mobile phase A: 5%, 5%, 98%, 98%, 5%, 5%; mobile phase B: 95%, 95%, 2%, 2%, 95%, 95%; mobile phase time: 0 min, 5 min, 15 min, 25 min, 27 min, 32 min.

[0034] Example 2

[0035] A method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyne and indapamide in soft capsules comprises: preparing a standard curve solution: weighing standard substances of hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyne and indapamide, diluting and fixing the solution with acetonitrile to obtain mixed standard working curve solutions with concentrations of 2, 5, 10, 20, 50 and 100 ng / mL respectively; preparing a sample solution: separating a capsule shell from the contents of a soft capsule sample, and fully mixing to obtain the capsule contents; weighing the capsule contents, adding acetonitrile, extracting, centrifuging, taking a supernatant, diluting and fixing the solution with acetonitrile, and shaking to obtain an extract; transferring the extract into a purification column, and collecting the purified solution to obtain the extract; and determining: using a chromatographic instrument to first determine the standard working curve solution, and then determining the sample solution to obtain the sample concentration value.

[0036] The extraction in the sample solution preparation was vortex extraction, and the time was 25 minutes.

[0037] The centrifugal speed in the sample solution preparation was 5000 r / min and the time was 7 min.

[0038] The purification column in the sample solution preparation is provided with fillers, and the fillers are arranged from top to bottom: a first sieve plate, a PSA purification layer, a second sieve plate, a silica purification layer, a microporous filter membrane, and a third sieve plate.

[0039] The amount of PSA purification layer in the filler is 600 mg, and the amount of silica purification layer is 600 mg.

[0040] The pore size of the first sieve plate is 50 μm, the pore size of the second sieve plate is 30 μm, and the pore size of the third sieve plate is 15 μm.

[0041] During the determination, mobile phase A was acetonitrile containing 0.1% formic acid, and mobile phase B was 10 mmol / L ammonium acetate solution containing 0.1% formic acid.

[0042] During the determination, the mobile phase gradient was: mobile phase A: 5%, 5%, 98%, 98%, 5%, 5%; mobile phase B: 95%, 95%, 2%, 2%, 95%, 95%; mobile phase time: 0 min, 5 min, 15 min, 25 min, 27 min, 32 min.

[0043] Example 3

[0044] A method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyne and indapamide in soft capsules comprises: preparing a standard curve solution: weighing standard substances of hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyne and indapamide, diluting and fixing the solution with acetonitrile to obtain mixed standard working curve solutions with concentrations of 2, 5, 10, 20, 50 and 100 ng / mL respectively; preparing a sample solution: separating a capsule shell from the contents of a soft capsule sample, and fully mixing to obtain the capsule contents; weighing the capsule contents, adding acetonitrile, extracting, centrifuging, taking a supernatant, diluting and fixing the solution with acetonitrile, and shaking to obtain an extract; transferring the extract into a purification column, and collecting the purified solution to obtain the extract; and determining: using a chromatographic instrument to first determine the standard working curve solution, and then determining the sample solution to obtain the sample concentration value.

[0045] The extraction in the sample solution preparation was vortex extraction, and the time was 25 minutes.

[0046] The centrifugal speed in the sample solution preparation was 3000 r / min and the time was 4 min.

[0047] The purification column in the sample solution preparation is provided with fillers, and the fillers are arranged from top to bottom: a first sieve plate, a PSA purification layer, a second sieve plate, a silica purification layer, a microporous filter membrane, and a third sieve plate.

[0048] The amount of PSA purification layer in the filler is 550 mg, and the amount of silica purification layer is 550 mg.

[0049] The pore size of the first sieve plate is 40 μm, the pore size of the second sieve plate is 25 μm, and the pore size of the third sieve plate is 10 μm.

[0050] During the determination, mobile phase A was acetonitrile containing 0.1% formic acid, and mobile phase B was 10 mmol / L ammonium acetate solution containing 0.1% formic acid.

[0051] During the determination, the mobile phase gradient was: mobile phase A: 5%, 5%, 98%, 98%, 5%, 5%; mobile phase B: 95%, 95%, 2%, 2%, 95%, 95%; mobile phase time: 0 min, 5 min, 15 min, 25 min, 27 min, 32 min.

[0052] Example 4

[0053] A method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyne and indapamide in soft capsules comprises: preparing a standard curve solution: weighing standard substances of hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyne and indapamide, diluting and fixing the solution with acetonitrile to obtain mixed standard working curve solutions with concentrations of 2, 5, 10, 20, 50 and 100 ng / mL respectively; preparing a sample solution: separating a capsule shell from the contents of a soft capsule sample, and fully mixing to obtain the capsule contents; weighing the capsule contents, adding acetonitrile, extracting, centrifuging, taking a supernatant, diluting and fixing the solution with acetonitrile, and shaking to obtain an extract; transferring the extract into a purification column, and collecting the purified solution to obtain the extract; and determining: using a chromatographic instrument to first determine the standard working curve solution, and then determining the sample solution to obtain the sample concentration value.

[0054] The extraction in the sample solution preparation was vortex extraction, and the time was 20 minutes.

[0055] The centrifugal speed in the sample solution preparation was 4000 r / min and the time was 5 min.

[0056] The purification column in the sample solution preparation is provided with fillers, and the fillers are arranged from top to bottom: a first sieve plate, a PSA purification layer, a second sieve plate, a silica purification layer, a microporous filter membrane, and a third sieve plate.

[0057] The amount of PSA purification layer in the filler is 500 mg, and the amount of silica purification layer is 500 mg.

[0058] The pore size of the first sieve plate is 30 μm, the pore size of the second sieve plate is 20 μm, and the pore size of the third sieve plate is 10 μm.

[0059] During the determination, mobile phase A was acetonitrile containing 0.1% formic acid, and mobile phase B was 10 mmol / L ammonium acetate solution containing 0.1% formic acid.

[0060] During the determination, the mobile phase gradient was: mobile phase A: 5%, 5%, 98%, 98%, 5%, 5%; mobile phase B: 95%, 95%, 2%, 2%, 95%, 95%; mobile phase time: 0 min, 5 min, 15 min, 25 min, 27 min, 32 min.

[0061] The determination was performed using an ultra-high performance liquid phase-tandem mass spectrometer, and the specific determination operation was as follows:

[0062] The standard working curve solution was injected into a triple quadrupole ultra-high performance liquid phase-tandem mass spectrometer; the chromatographic determination conditions were as follows: chromatographic column ACQUITY BEH C18 2.1×100mm, 1.7μm; mobile phase A was acetonitrile containing 0.1% formic acid, and mobile phase B was 10mmol / L ammonium acetate solution containing 0.1% formic acid; column temperature: 30℃; flow rate: 0.3mL / min; injection volume: 2μL; mobile phase gradient and time were as follows: mobile phase gradient was: mobile phase A: 5%, 5%, 98%, 98%, 5%, 5%, mobile phase B: 95%, 95%, 2%, 2%, 95%, 95%, mobile phase time: 0min, 5min, 15min, 25min, 27min, 32min; mass spectrometry conditions were as follows: ion source: electrospray ion source (ES), multiple reaction monitoring (MRM); positive and negative ion simultaneous scanning mode; capillary voltage (Capillary) 2.5kV; ion source temperature (Source Temperature) 150℃; desolvation temperature (Desolvation Temperature) 500℃; Cone Gas Flow 150L / Hr; Desolvation Gas Flow 1000L / Hr; Collision Gas Flow 0.13mL / min

[0063] Among them, the ion pair, cone voltage, and collision voltage conditions are as follows:

[0064] ①Hydrochlorothiazide: negative ion mode; parent ion 296.0, quantitative ion 269.1, cone voltage 60.0V, collision energy 16.0V; parent ion 296.0, daughter ion 205.1, cone voltage 60.0V, collision energy 22.0V;

[0065] ② Caffeine: positive ion mode; parent ion 195.1, quantitative ion 138.0, cone voltage 40.0V, collision energy 19.0V; parent ion 195.1, daughter ion 110.1, cone voltage 40.0V, collision energy 21.0V;

[0066] ③ Phenolphthalein: positive ion mode; parent ion 319.1, quantitative ion 225.0, cone voltage 23.0V, collision energy 25.0V; parent ion 319.1, daughter ion 141.0, cone voltage 23.0V, collision energy 49.0V;

[0067] ④ Fenofibrate: positive ion mode; parent ion 361.1, quantitative ion 223.0, cone voltage 25.0V, collision energy 18.0V; parent ion 361.1, daughter ion 138.9, cone voltage 25.0V, collision energy 30.0V;

[0068] ⑤ Bisacodyl: positive ion mode; parent ion 362.1, quantitative ion 184.0, cone voltage 25.0V, collision energy 28.0V; parent ion 362.1, daughter ion 226.0, cone voltage 25.0V, collision energy 17.0V;

[0069] ⑥ Indapamide: positive ion mode; parent ion 366.0, quantitative ion 132.0, cone voltage 34.0V, collision energy 16.0V; parent ion 366.0, daughter ion 91.0, cone voltage 34.0V, collision energy 44.0V;

[0070] test

[0071] The method of Example 4 was used to conduct the following test.

[0072] 1. Accuracy determination

[0073] Take 1g of blank soft capsule sample, add 0.25mL of 1ug / mL mixed standard solution, and the addition level is 250ng / g. According to the method of the present invention and the national standard BJS201701 "Determination of Sibutramine and other compounds in food", three parallel experiments were performed. The results and relative errors are shown in Table 1.

[0074] Table 1 Accuracy test results

[0075]

[0076] From the data in Table 1, it can be seen that the relative error ranges of the method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyne and indapamide are all less than 6.9%, while the relative errors of the national standard method for determining hydrochlorothiazide, caffeine and phenolphthalein are 29.9%, 18.8% and 19.0%, respectively; in general, compared with the national standard method, the method of the present invention has a smaller relative error and higher accuracy.

[0077] 2. Determination of detection limit and quantification limit

[0078] The standard solution was added to the blank capsule matrix, and the detection limit of the target object was determined at a signal-to-noise ratio of 3 times, and the quantification limit of the target object was determined at a signal-to-noise ratio of 10 times. The method of the present invention was used for determination, and the results are shown in Table 2.

[0079] Table 2 Results of detection limit and quantification limit test

[0080]

[0081] From the data in Table 2, it can be seen that the detection limits of hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyl and indapamide determined by the method of the present invention are all less than 0.86 μg / kg, and the quantification limits are all less than 2.9 μg / kg. The results of the detection limit and the quantification limit are much smaller than the national standard "Determination of Sibutramine and Other Compounds in Food" (BJS201701), indicating that the method of the present invention has high sensitivity.

[0082] 3. Determination of Repeatability and Recovery

[0083] Three kinds of blank soft capsules available on the market were taken and measured by the method of the present invention. The repeatability and recovery rate were measured in three levels and three parallel tests. The results are shown in the following table.

[0084] Table 3 Repeatability and recovery test structure

[0085]

[0086] From the data in Table 3, it can be seen that the recovery rate of the method of the present invention is in the range of 85%-110%, and the accuracy is good.

[0087] Figure 1-Figure 6 These are the ion mass spectra of hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyl, and indapamide.

[0088] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the statement "including an element defined by ... does not exclude the existence of other identical elements in the process, method, article or device including the element".

[0089] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyl and indapamide in soft capsules, characterized in that: include: Preparation of standard curve solution: weigh the standard substances of hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyl, and indapamide, dilute to volume with acetonitrile to obtain mixed standard working curve solutions with concentrations of 2, 5, 10, 20, 50, and 100 ng / mL, respectively; Preparation of sample solution: Separate the capsule shell and contents of the soft capsule sample, and mix thoroughly to obtain the capsule contents; weigh the capsule contents, add acetonitrile, extract, centrifuge, take the supernatant and dilute to volume with acetonitrile, and shake well to obtain the extract; The extract is transferred into a purification column, and the purified liquid is collected to obtain; Determination: Use a chromatographic instrument to first measure the standard working curve solution, then measure the sample solution to obtain the sample concentration value.

2. The method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyl and indapamide in soft capsules according to claim 1, characterized in that: The extraction in the sample solution preparation is vortex extraction, and the time is 10-25min.

3. A method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyl and indapamide in soft capsules according to claim 2, characterized in that: The centrifugal speed in the preparation of the sample solution is 2000-5000 r / min and the time is 3-7 min.

4. The method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyl and indapamide in soft capsules according to claim 1, characterized in that: The purification column in the sample solution preparation is provided with fillers, and the fillers are arranged from top to bottom: a first sieve plate, a PSA purification layer, a second sieve plate, a silica purification layer, a microporous filter membrane, and a third sieve plate.

5. A method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyl and indapamide in soft capsules according to claim 4, characterized in that: The amount of PSA purification layer in the filler is 400-600 mg, and the amount of silica purification layer is 450-600 mg.

6. A method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyl and indapamide in soft capsules according to claim 5, characterized in that: The pore size of the first sieve plate is 30-50 μm, the pore size of the second sieve plate is 20-30 μm, and the pore size of the third sieve plate is 10-15 μm.

7. A method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyl and indapamide in soft capsules according to any one of claims 1 to 4, characterized in that: During the determination, mobile phase A was acetonitrile containing 0.1% formic acid, and mobile phase B was 10 mmol / L ammonium acetate solution containing 0.1% formic acid.

8. The method for determining hydrochlorothiazide, caffeine, phenolphthalein, fenofibrate, bisacodyl and indapamide in soft capsules according to claim 5, characterized in that: During the determination, the mobile phase gradient was: Mobile phase A: 5%, 5%, 98%, 98%, 5%, 5%, Mobile phase B: 95%, 95%, 2%, 2%, 95%, 95%, Mobile phase time: 0min, 5min, 15min, 25min, 27min, 32min.

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