CN117347546A - Detection method and application of azilsartan in Chinese patent medicine - Google Patents

Detection method and application of azilsartan in Chinese patent medicine Download PDF

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Publication number
CN117347546A
CN117347546A CN202310379552.7A CN202310379552A CN117347546A CN 117347546 A CN117347546 A CN 117347546A CN 202310379552 A CN202310379552 A CN 202310379552A CN 117347546 A CN117347546 A CN 117347546A
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China
Prior art keywords
azilsartan
chinese patent
ion mobility
detecting
medicines according
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CN202310379552.7A
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刘亚雄
何嘉雯
温家欣
赖宇红
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Guangdong Institute For Drug Control (guangdong Institute For Drug Quality
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Guangdong Institute For Drug Control (guangdong Institute For Drug Quality
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

The invention discloses a detection method and application of azilsartan in a Chinese patent medicine, comprising the following steps of: s1: preparing a sample solution; s2: preparing azilsartan standard solution; s3: the sample solution and azilsartan standard solution were determined using liquid chromatography-quadrupole-ion mobility-time-of-flight mass spectrometry, respectively. According to the method, the azilsartan in the Chinese patent medicine is measured by adopting a liquid chromatography-quadrupole rod-ion mobility-time-of-flight mass spectrometry (UPLC-Q-IMS-Qtof), and the Collision Cross Section (CCS) provided by IMS is utilized instead of the chromatographic retention time for auxiliary qualitative, so that the false negative caused by retention time drift due to complex matrix, high sensitivity to chromatographic conditions and other reasons in the traditional chromatographic retention time auxiliary qualitative process is avoided.

Description

Detection method and application of azilsartan in Chinese patent medicine
Technical Field
The invention relates to the technical field of medicine detection, in particular to a detection method and application of azilsartan in a Chinese patent medicine.
Background
Antihypertensive products have the problem of improper addition of chemicals.
Azilsartan has the chemical name 2-ethoxy-1- [ [2' - (4, 5-dihydro-5-oxo-1, 2, 4-oxadiazol-3-yl) biphenyl-4-yl ] methyl ] benzimidazole-7-carboxylic acid. The medicine is an antihypertensive medicine newly marketed in 2021, and is used for treating adult essential hypertension, belonging to chemical medicine management. The addition of azilsartan to the Chinese patent medicine is forbidden. The hypertension needs long-term stable treatment, and the addition of the antihypertensive product in the Chinese patent medicine cheats the patient with pure natural traditional Chinese medicine without Western medicine and other gimmick, interferes with normal medication, has great health risk on the patient, has bad behavioral properties and has to be hit seriously.
However, there is no report on the detection method of azilsartan in Chinese patent medicines and foods at home and abroad, the detection standard cannot be determined, the azilsartan is in the dilemma of being undetectable, and the supervision loopholes exist. Therefore, there is a need to establish a method for detecting azilsartan in Chinese patent medicines.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a method for detecting azilsartan in a Chinese patent medicine, which adopts liquid chromatography-quadrupole rod-ion mobility-time-of-flight mass spectrometry (UPLC-Q-IMS-Qtof) to measure the azilsartan in the Chinese patent medicine, and utilizes the Collision Cross Section (CCS) provided by IMS instead of chromatographic retention time to carry out auxiliary qualitative, thereby avoiding false negatives caused by retention time drift due to complex matrixes, high sensitivity to chromatographic conditions and other reasons in the traditional chromatographic retention time auxiliary qualitative process. The method is quick, sensitive and accurate, makes up the related technical blank, and provides technical support for the action of adding azilsartan into the striking antihypertensive products.
A detection method of azilsartan in a Chinese patent medicine is characterized by comprising the following steps: the method comprises the following steps:
s1: preparing a sample solution;
s2: preparing azilsartan standard solution;
s3: the sample solution and azilsartan standard solution were determined separately using liquid chromatography-quadrupole-ion mobility-time-of-flight mass spectrometry.
In some embodiments of the invention, the Trap gas flow rate of the ion mobility is 2mL/min, the helium bath gas flow rate is 180mL/min, and the IMS gas flow rate is 80mL/min.
In some embodiments of the invention, the ion mobility Trap DC bias is 45V and the imsdc bias is 3V.
In some embodiments of the invention, the ion mobility has a Trap wave velocity of 311m/s and a Trap wave height of 4V.
In some embodiments of the present invention, the ion mobility detection period has a wave velocity gradient of 1000m/s to 400m/s, the ion mobility detection period has a wave velocity of 1000m/s at the beginning of detection, and the ion mobility detection period has a wave velocity of 400m/s at the end of detection.
In some embodiments of the invention, the ion mobility has a Transfer wave speed of 220m/s and a Transfer height of 4V.
In some embodiments of the invention, the manual release time of the IMS Trap of ion mobility is 500s, the Trap height is 15V, and the extract height is 0V.
In some embodiments of the invention, the post-capture release IMS separation delay time for the ion mobility is 800 μs.
In some embodiments of the invention, the liquid chromatograph-quadrupole-ion mobility-time-of-flight mass spectrum has an input of 5 to 10uL.
In some embodiments of the invention, the time-of-flight mass spectrum has a capillary voltage of 2.5KV, a cone voltage of 40V, an ion source temperature of 120 ℃, a desolvation temperature of 450 ℃, a desolvation gas flow rate of 800L/h, a nebulizer pressure of 6.5Bar, and a cone gas flow rate of 50L/h.
In some embodiments of the invention, the detection method is applied to Chinese patent medicine dosage form products such as tablets, capsules, pills, granules, oral liquid, tea and the like.
Compared with the prior art, the invention has the beneficial effects that:
the application range of the detection method of the invention covers the common dosage forms of antihypertensive Chinese patent medicines, such as tablet, capsule, pill, granule, oral liquid, tea and other Chinese patent medicine dosage forms. The method has the advantages of wide application range, high detection speed and accurate result, can be used as a qualitative and quantitative method of azilsartan in a blood pressure reducing product, fills the blank of the detection field, and provides an effective means for striking the improper adding behavior of novel components.
Drawings
The database of figure 1 records TIC figures of azilsartan standard.
Figure 2 database records a graph of azilsartan standard drift time.
Figure 3 a TIC diagram of azilsartan in a real sample 1 of example 1.
Figure 4 a plot of azilsartan drift time in real sample 1 of example 1.
Figure 5 a TIC diagram of azilsartan in a real sample 2 of example 1.
Fig. 6 is a graph of azilsartan drift time in a real sample 2 of comparative example 1.
Figure 7 a TIC diagram of azilsartan in a real sample 3 of example 2.
Fig. 8 is a graph of azilsartan drift time in real sample 3 of comparative example 2.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
In the description of the present invention, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
In the following examples and comparative examples, the following instruments, reagents, and criteria for qualitative analysis were used, and standard solutions and sample solutions were prepared by the following methods:
instrument and reagent:
1. instrument: ACQUITY UPLC liquid phase System (Waters Co.); a Synapt G2-Si HDMS mass spectrometer (Waters Co.); a vortex mixer; an ultrasonic generator; a reciprocating oscillator; and (5) an ultrapure water machine.
2. Reagent: azilsartan standard; methanol, acetonitrile and formic acid are all chromatographic pure.
3. Preparation of the reagent:
methanol-acetonitrile mixed solution (1+1, volume ratio): 500mL of methanol and 500mL of acetonitrile are measured and mixed evenly.
0.1% formic acid solution (volume fraction): sucking 1mL of formic acid, diluting to 1000mL with water, uniformly mixing, and filtering by a 0.22 mu m aqueous phase microporous filter membrane for later use.
(II) standard stock solution:
preparing a standard solution: 0.01004g of azilsartan (content 98% of source leaf organism) is weighed respectively, and a standard stock solution with the mass concentration of 1mg/mL is prepared by using a methanol-acetonitrile mixed solution.
(III) preparation of a sample solution:
tablets, capsules, pills, granules, oral liquids and teas: 1g of a sample is weighed, placed in a 50mL volumetric flask, 40mL of a methanol-acetonitrile mixed solution is added, vortex is carried out for 1min, and ultrasonic extraction is carried out for 10min. Taking out, cooling, fixing volume to scale with methanol-acetonitrile mixed solution, shaking, passing through 0.22 μm microporous membrane, and testing.
(IV) qualitative screening:
judgment standard of qualitative analysis: after data acquisition is carried out on the sample, the sample is compared with a database established by using a standard substance, the relative deviation between the CCS value of the target compound and the database is measured to be within +/-2%, the mass deviation range is +/-5 ppm, and fragment ions are more than or equal to 2, namely the sample is detected.
The real sample 1 is a tablet, the real sample 2 is a capsule, and the real sample 3 is a pill.
Example 1
A detection method of azilsartan in a Chinese patent medicine comprises the following steps:
10 mu L of azilsartan real sample 1 sample solution is injected into a liquid chromatograph-mass spectrometer and measured according to the following chromatographic-mass spectrometry conditions.
Chromatographic conditions: ACQUITY UPLC@C18 chromatographic column100×2.1mm,1.8 μm), flow rate 0.2mL/min, column temperature 35 ℃,0.1% formic acid as mobile phase a,0.1% acetonitrile formate as mobile phase B, gradient elution according to the following procedure: 0 to 1.0min, A80%;1.0 to 15.0min, and 80 to 20 percent of A; 15.0 to 25.0min, and 20 to 0 percent of A; 25.1-30 min and A80%. The temperature of the sample was controlled at 10 ℃.
Mass spectrometry conditions: ion mobility-mass spectrometry (IMS-MS) analysis was performed on a hybrid quadrupole TWIMS-orthogonal acceleration time of flight (ToF) mass spectrometer (Synapt G2-Si HDMS, waters company) equipped with an electrospray ionization (ESI) interface. The ESI parameters were fixed as follows: capillary voltage (positive mode), 2.5KV; taper hole voltage 40V; the ion source temperature is 120 ℃, the desolventizing temperature is 450 ℃, and the desolventizing air flow rate is 800L/h; sprayer pressure 6.5Bar; the gas flow rate of the taper hole is 50L/h. The IMS parameters are as follows: trap gas flow rate 2mL/min, helium pool gas flow rate 180mL/min, IMS gas flow rate 90mL/min; the Trap DC bias and IMSDC bias are 45V and 3V, respectively; the speed and the height of the transfer wave are respectively 311m/s and 4V, and the speed and the height of the transfer wave are respectively fixed to 220m/s and 4V; the wave speed gradient change in the whole IMS period is from 1000m/s to 400m/s (from the beginning to the end); the manual release time of IMS Trap was 500 μs, the Trap height was 15V, the extract height was 0V, and the IMS separation delay time after trapping release was 800 μs.
Results: after data acquisition, the real sample 1 is compared with the database standard, and targets with mass deviation of + -5 ppm and fragment ions not less than 2 within + -2% of CCS of the database azilsartan standard appear in the real sample (figures 1 and 2).
Table 1. Azilsartan results in real samples 1,2 are aided qualitatively based on cross-sectional area of collision.
Through table 1, we can find that azilsartan is detected in the real sample 1, but because of the complex sample matrix, compared with the standard substance, the retention time of the target substance is drifted (> 2.5%), and the sample may be misjudged as false negative by the traditional retention time auxiliary qualitative means. While the mobility mass spectrum provides the cross-sectional area of impact of the compoundThe method has stronger reliability, and compared with a database standard, the method has the advantages that the deviation of the CCS value of the target object in the real positive sample is less than 2%, and the omission of the positive sample is avoided.
Example 2
Detection limit, quantitative limit and linear range relation test:
the standard stock solution was diluted with a tablet blank matrix solution to prepare a series of standard solutions having mass concentrations of 2, 5, 10, 25, 50ng/mL, respectively, and the mass concentration (x) was linearly regressed with the peak area (y). The azilsartan regression equation is y=2069.7x+15062, the correlation coefficient r 2 0.9936. The matrix such as capsules, pills, granules, oral liquid, tea and the like is inspected by the same method, and the correlation coefficient is more than 0.99 within the range of 2 ng/mL-100 ng/mL. Considering the state of a comprehensive instrument, the detection limit and the quantitative limit of the azilsartan method are both set to be 0.1mg/kg (namely, the target mass concentration is 2 ng/mL), so that the requirement of the test can be met.
Comparative example 1
The comparative example provides a detection method of azilsartan in a Chinese patent medicine, which is different from the embodiment 1 in chromatographic conditions, and comprises the following specific processes:
gradient elution was performed as follows: 0 to 1.0min, A80%;1.0 to 20.0min, and 80 to 20 percent of A; 15.0 to 30.0min, and 20 to 0 percent of A; 35.1-40 min and A80%. The temperature of the sample was controlled at 10 ℃.
Table 2. The results of azilsartan in the real sample 3 were determined based on the cross-sectional area of collision.
To further verify the reliability of the IMS-provided cross-sectional collision area CCS value to assist in the characterization of azilsartan, the target was caused to peak late by changing chromatographic conditions. The results show that azilsartan detection based on CCS value assisted qualitative determination shows excellent reliability even if the target substance is late-peaked (rt=20.06).
Comparative example 2
The comparative example provides a detection method of azilsartan in a Chinese patent medicine, which is different from the embodiment 1 in chromatographic conditions, and comprises the following specific processes:
gradient elution was performed as follows: 0 to 1.0min, A50%; 1.0 to 8.0min, 50 to 90 percent of A; 8.1 to 10.0min, and 90 to 0 percent of A; 10.1-15 min and A80%. The temperature of the sample was controlled at 10 ℃.
Table 2. The results of azilsartan in the real sample 3 were determined based on the cross-sectional area of collision.
In order to further examine the reliability of the collision cross-sectional area CCS value provided by the IMS, which assists in the characterization of azilsartan, the target is caused to peak in advance by changing the chromatographic conditions. The results show that even though the target advanced the peak (rt=1.42), the azilsartan detection based on CCS value assisted qualitative still shows excellent reliability on the premise of complex sample matrix, although resulting in more co-flow peaks.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. A detection method of azilsartan in a Chinese patent medicine is characterized by comprising the following steps: the method comprises the following steps:
s1: preparing a sample solution;
s2: preparing azilsartan standard solution;
s3: the sample solution and azilsartan standard solution were determined separately using liquid chromatography-quadrupole-ion mobility-time-of-flight mass spectrometry.
2. The method for detecting azilsartan in Chinese patent medicines according to claim 1, which is characterized in that: the Trap gas flow rate of the ion mobility is 2mL/min, the helium pool gas flow rate is 180mL/min, and the IMS gas flow rate is 80mL/min.
3. The method for detecting azilsartan in Chinese patent medicines according to claim 1, which is characterized in that: the Trap DC bias for the ion mobility is 45V and the IMSDC bias is 3V.
4. The method for detecting azilsartan in Chinese patent medicines according to claim 1, which is characterized in that: the Trap wave speed of the ion mobility is 311m/s, and the Trap wave height is 4V.
5. The method for detecting azilsartan in Chinese patent medicines according to claim 1, which is characterized in that: the wave speed gradient in the ion mobility detection period is 1000 m/s-400 m/s, the wave speed of the ion mobility is 1000m/s at the beginning of detection, and the wave speed of the ion mobility is 400m/s at the end of detection.
6. The method for detecting azilsartan in Chinese patent medicines according to claim 1, which is characterized in that: the Transfer wave speed of the ion mobility is 220m/s, and the Transfer height is 4V.
7. The method for detecting azilsartan in Chinese patent medicines according to claim 1, which is characterized in that: the manual release time of the IMS Trap with the ion mobility is 500s, the Trap height is 15V, and the extract height is 0V.
8. The method for detecting azilsartan in Chinese patent medicines according to claim 1, which is characterized in that: the IMS separation delay time after the trapping release of the ion mobility was 800 mus.
9. The method for detecting azilsartan in Chinese patent medicines according to claim 1, which is characterized in that: the sample injection amount of the liquid chromatography-quadrupole-ion mobility-time-of-flight mass spectrum is 5-10uL.
10. The method for detecting azilsartan in Chinese patent medicines according to any one of claims 1 to 9, characterized in that: the detection method is applied to Chinese patent medicine dosage forms such as tablets, capsules, pills, granules, oral liquid, tea and the like.
CN202310379552.7A 2023-04-10 2023-04-10 Detection method and application of azilsartan in Chinese patent medicine Pending CN117347546A (en)

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