Method for detecting buthiazide in food

Abstract

The invention discloses a method for detecting buthiazide in food, which comprises the following steps: uniformly mixing a sample to be detected with methanol, ultrasonically extracting, fixing the volume with methanol, centrifuging, passing through a 0.2 mu m filter head to obtain a liquid to be detected, separating by a liquid chromatograph, and detecting in a multi-reaction ion monitoring mode by adopting a triple quadrupole tandem mass spectrometer. According to the method, the linear relation of the buthiazide is good in the range of 0-500 ng / mL, the correlation coefficient R2 is larger than 0.999, and the method is accurate, reliable, simple and rapid and can be used for rapidly detecting the buthiazide in food.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection, and specifically relates to a detection method for determining buthiazine in food by using liquid chromatography-tandem mass spectrometry. Background Art

[0002] Buthiazine, also known as isobuthiazine, is a moderate-acting diuretic that mainly acts on the urinary system. Buspirone is a commonly used drug that is mainly used to treat anxiety and depression. It belongs to the category of antianxiety drugs that improve mood and emotional state by regulating chemicals in the brain. The mechanism of action of buthiazine is different from other antianxiety drugs. It produces sedative and anti-anxiety effects by increasing the concentration of serotonin in the brain. Serotonin is a neurotransmitter that plays an important role in regulating mood and anxiety. Buthiazine binds to serotonin receptors, increases its activity, and thus relieves anxiety symptoms. The advantage of buthiazine is that it is relatively safe and does not cause serious side effects and dependence. Compared with benzodiazepines, buthiazine does not cause adverse reactions such as drowsiness, muscle relaxation, and memory loss. Therefore, it can be used for a long time without withdrawal reactions. However, buthiazine also has some side effects. Common side effects include dizziness, headache, nausea, nervousness, and muscle discomfort. These side effects are usually mild and will be alleviated or disappear as treatment progresses.

[0003] According to inquiries, there is currently no detection method for butiazide, and it has not been reported in standards or literature. Butiazide is a new type of drug. In view of this, it is urgent to study a method for detecting butiazide. The present invention fills the gap in the detection method of butiazide and provides technical support for the detection of butiazide in foods such as compressed candies. Summary of the invention

[0004] The present invention aims at the problems existing in the above-mentioned prior art and provides a method for detecting buthiazine in food. The method of the present invention is simple, rapid, has a low detection limit and low cost, and is suitable for rapid and accurate quantitative detection of buthiazine in food.

[0005] The instrument used in the present invention is a liquid chromatography-triple quadrupole tandem mass spectrometer LC-MS / MS, comprising a liquid chromatograph LC and a triple quadrupole tandem mass spectrometer MS / MS.

[0006] The method for detecting buthiazine in food of the present invention comprises the following steps:

[0007] Step 1: Determination of buthiazine retention time and monitoring ions

[0008] A standard substance of buthiazine was taken to prepare a standard solution with a concentration of 500 ng / mL. The standard solution was scanned for parent ions by a triple quadrupole tandem mass spectrometer to determine the monitoring ions and quantitative ion pairs of buthiazine. The standard solution was then subjected to LC-MS / MS detection to determine the retention time of buthiazine. The mass spectrum parameters of buthiazine of the present invention are shown in Table 1 below.

[0009]

[0010] Step 2: Preparation of the standard curve of buthiazine

[0011] A series of standard working solutions were prepared with buthiazine standard substances, and then 2 µL of the buthiazine series of standard working solutions of different concentrations were sampled for injection, and LC-MS / MS detection was performed. The peak area of ​​buthiazine was used to perform linear regression with its corresponding mass concentration to obtain the linear regression equation of buthiazine. The curve corresponding to the linear regression equation was the standard curve of buthiazine.

[0012] The linear regression equation and correlation coefficient R corresponding to the buthiazine 2 See Table 2, where Y in the equation is the peak area corresponding to buthiazine, and X is the mass concentration of buthiazine.

[0013]

[0014] Step 3: Pretreatment of the sample to be tested

[0015] Solid or semi-solid sample: Take a solid sample or a semi-solid sample and add methanol, extract it by ultrasonic, cool it to room temperature, make up to volume with methanol, centrifuge it, filter it through a 0.22μm microporous filter membrane, and obtain the test solution for injection.

[0016] Liquid sample: Take an appropriate amount of the sample and add methanol, perform ultrasonic extraction, cool to room temperature, make up to volume with methanol, filter through a microporous filter membrane to obtain the test liquid, and prepare for injection.

[0017] Step 4: Testing of the sample to be tested

[0018] Take 1 mL of the sample to be tested after pretreatment in step 3 for LC-MS / MS detection, and obtain the mass spectrum and chromatogram of the sample to be tested in multiple reaction monitoring mode. Perform qualitative analysis on the sample to be tested according to the retention time and the monitoring ions of buthiazine, and perform quantitative analysis on the sample to be tested according to the buthiazine standard curve.

[0019] The detection parameters of the present invention are set as follows:

[0020] The conditions of liquid chromatography LC are:

[0021] Chromatographic column: C 18 (3.0mm*100mm, 2.7μm), or equivalent performance;

[0022] Column temperature: 35°C;

[0023] Flow rate: 0.4 mL / min;

[0024] Injection volume: 2 μL;

[0025] Mobile phase: A: 0.1% formic acid aqueous solution; B: acetonitrile;

[0026] Gradient elution program:

[0027]

[0028] The conditions for triple quadrupole tandem mass spectrometry MS / MS were:

[0029] Ion source: electrospray ionization source (ESI+);

[0030] Scanning mode: positive ion scanning;

[0031] Detection method: multiple reaction monitoring mode (MRM);

[0032] Capillary voltage: 2000V;

[0033] Drying gas temperature: 300℃;

[0034] Drying gas flow: 5L / min;

[0035] Atomizing gas pressure: 241 kPa;

[0036] Sheath gas temperature: 250℃;

[0037] Sheath gas flow rate: 11L / min.

[0038] In the solid or semi-solid sample pretreatment step, the sample weight is 2 g (accurate to 0.001 g); the ultrasonic extraction time is 30 min; the volume of methanol is 20 mL; the centrifugal speed is 4000 r / min, and the time is 5 min.

[0039] In the liquid sample pretreatment step, the sample dosage is 2 mL; the ultrasonic extraction time is 30 min; and the volume of the methanol is 20 mL.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] 1. The present invention establishes a method for detecting buthiazine in food, which is the best method for detecting buthiazine. It can accurately conduct qualitative and quantitative analysis of buthiazine, and provide a scientific basis for the accurate determination and rapid detection of buthiazine in food.

[0042] 2. The liquid chromatography-triple quadrupole tandem mass spectrometer of the present invention is simple and fast to operate, accurate and reliable, and has good repeatability.

[0043] 3. The method of the present invention is simple, rapid, has a low detection limit and low cost, and is suitable for the rapid and accurate quantitative detection of buthiazine in a large number of foods, providing technical support for the detection of buthiazine in foods.

[0044] 4. The validation of the method of the present invention is combined for the first time with GB 5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for National Food Safety Standards", ensuring that the test data is scientific, accurate and reliable.

[0045] 5. The quantitative limit and detection limit in the present invention are formulated using innovative methods, which provides an effective technical support for method validation.

[0046] 6. The present invention adopts rapid scanning technology, which improves the response value of samples with the same concentration, greatly reduces the detection limit of the method, and realizes accurate analysis of trace components. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The TIC diagram and single ion current diagram of buthiazine standard solution. DETAILED DESCRIPTION

[0048] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0049] The present invention provides a method for detecting buthiazine in food, the instrument used is a liquid chromatography-triple quadrupole tandem mass spectrometer LC-MS / MS, including a liquid chromatograph LC and a triple quadrupole tandem mass spectrometer MS / MS, and the method is carried out according to the following steps:

[0050] Step 1: Determination of buthiazine retention time and monitoring ions

[0051] A standard substance of buthiazine was taken to prepare a standard solution with a concentration of 500 ng / mL, the standard solution was subjected to parent ion scanning by a triple quadrupole tandem mass spectrometer to determine the monitoring ion and quantitative ion pair of buthiazine, and then the standard solution was subjected to LC-MS / MS detection to determine the retention time of buthiazine;

[0052] The buthiazine standard solution was tested, and the ions that needed to be further analyzed were selected by full scan. The parent ion was obtained by parent ion scanning, and the characteristic daughter ions were selected for daughter ion scanning. Finally, the mass spectrum of buthiazine was obtained by multiple reaction monitoring mode.

[0053] Step 2: Preparation of the standard curve of buthiazine

[0054] The standard series working solution of buthiazine was prepared by taking the standard substance of buthiazine, and then 2 µL of the standard series working solution of buthiazine was sampled for LC-MS / MS detection. The peak area of ​​buthiazine was used to perform linear regression with its corresponding mass concentration to obtain the linear regression equation of buthiazine. The curve corresponding to the linear regression equation was the standard curve of buthiazine. The linear regression equation and correlation coefficient R corresponding to buthiazine were 2 See Table 2, linear correlation coefficient R 2 Greater than 0.999.

[0055] 3. Pretreatment of samples to be tested

[0056] Solid or semi-solid specimens

[0057] Take an appropriate amount of solid sample and mix it evenly, grind it into powder, or take an appropriate amount of semi-solid sample and mix it evenly, accurately weigh 2 g (accurate to 0.001 g) and place it in a 20 mL volumetric flask, add an appropriate amount of methanol, ultrasonically extract for 30 min, cool to room temperature, make up to volume with methanol, transfer to a 50 mL centrifuge tube, centrifuge at 4000 r / min for 5 min, filter the supernatant through a 0.22 μm microporous membrane, take the filtrate, dilute it appropriately with methanol according to the actual concentration to the linear range, and set aside.

[0058] Liquid sample

[0059] Take an appropriate amount of the sample and shake it evenly. Accurately pipette 2 mL into a 20 mL volumetric flask, add an appropriate amount of methanol, perform ultrasonic extraction for 30 min, cool to room temperature, make up to volume with methanol, filter through a 0.22 μm microporous filter membrane, take the filtrate, and appropriately dilute it with methanol to within the linear range according to the actual concentration for later use.

[0060] 4. Testing of samples to be tested

[0061] A certain amount of standard working solution was added to the sample determined to be free of buthiazine as the sample to be tested for pretreatment, and the sample to be tested was injected with an injection volume of 2 µL, and LC-MS / MS detection was performed to obtain a chromatogram and a mass spectrum of the sample to be tested.

[0062] The instrument conditions used were set as follows:

[0063] The conditions of the liquid chromatograph LC were set as follows:

[0064] Chromatographic column: C 18 (3.0mm*100mm, 2.7μm), or equivalent performance;

[0065] Column temperature: 35°C;

[0066] Flow rate: 0.4 mL / min;

[0067] Injection volume: 2 μL;

[0068] Mobile phase: A: 0.1% formic acid aqueous solution; B: acetonitrile;

[0069] Gradient elution program:

[0070]

[0071] The conditions for setting the triple quadrupole tandem mass spectrometer MS / MS are:

[0072] Ion source: electrospray ionization source (ESI+);

[0073] Scanning mode: positive ion scanning;

[0074] Detection method: multiple reaction monitoring mode (MRM);

[0075] Capillary voltage: 2000V;

[0076] Drying gas temperature: 300℃;

[0077] Drying gas flow: 5L / min;

[0078] Atomizing gas pressure: 241 kPa;

[0079] Sheath gas temperature: 250℃;

[0080] Sheath gas flow rate: 11L / min.

[0081] The sensitivity test of the method of the present invention is performed: the sensitivity is expressed by the detection limit and the quantification limit. According to 4.1.2.2.2 of GB5009.295-2023 "General Rules for Validation of Chemical Analysis Methods in National Food Safety Standards", at least 20 parallel samples of blank sample matrices are selected, and the target analytes with estimated detection limit concentrations are added for verification. Two representative blank matrices are selected for verification, and the results are shown in Tables 3 and 4. According to the determination of buthiazine by liquid chromatography-mass spectrometry / mass spectrometry: when the sample weight is 2g and the fixed volume is 20mL, the detection limit is 0.030mg / kg.

[0082]

[0083]

[0084] According to GB5009.295-2023 "General Rules for Validation of Chemical Analysis Methods in National Food Safety Standards" 4.1.3, for each representative sample matrix, a standard spiked sample at the quantitative limit concentration level is used for independent testing, and at least 6 parallel samples are tested. Two representative blank matrices were selected for validation, and the results are shown in Tables 5 and 6. According to the determination of buthiazine by liquid chromatography-mass spectrometry / mass spectrometry: when the sample weight is 2g and the fixed volume is 20mL, the detection limit is 0.10mg / kg.

[0085]

[0086]

[0087] The accuracy experiment of the method of the present invention is carried out: According to GB5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for Food Safety National Standards" 4.1.5.2.2, for each sample matrix, at least 3 concentrations should be added, and the recovery rate of the known amount of target analyte in the standard added sample is obtained. For each concentration, at least 6 parallel independent tests are required, and the concentration of the target analyte in each sample is calculated, and the average recovery rate of each concentration is calculated. Two representative blank matrices were selected for validation, and the results are shown in Tables 7-12. It can be concluded from Tables 7 and 8 that the recovery rates are 100.8% and 97.8%, respectively, which meet the requirements of GB5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for Food Safety National Standards" that the recovery rate is 70%-120% when the target analyte content is 0.01-0.1 mg / kg. The relative standard deviations of 2.7% and 3.4% are in line with the requirements of GB5009.295-2023 "General Principles for Validation of Chemical Analysis Methods for Food Safety National Standards" that the relative standard deviation is less than or equal to 15% when the target analyte content is 0.01-0.1 mg / kg. It can be concluded from Tables 9 and 10 that the recovery rates are 98.6% and 83.6% respectively, which are in line with the requirements of GB5009.295-2023 "General Principles for Validation of Chemical Analysis Methods for Food Safety National Standards" that the recovery rate is 80%-110% when the target analyte content is 0.1-1000 mg / kg. The relative standard deviations of 1.6% and 2.4% are in line with the requirements of GB5009.295-2023 "General Principles for Validation of Chemical Analysis Methods for Food Safety National Standards" that the relative standard deviation is less than or equal to 10% when the target analyte content is 0.1-10000 mg / kg. It can be concluded from Table 11 and Table 12 that the recoveries are 94.3% and 92.3% respectively, both of which meet the requirements of GB5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for Food Safety National Standards" that the recovery rate is 80%-110% when the target analyte content is 0.1-1000 mg / kg. The relative standard deviations are 2.9% and 0.9%, both of which meet the requirements of GB5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for Food Safety National Standards" that the relative standard deviation is less than or equal to 10% when the target analyte content is 0.1-10000 mg / kg.

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094] The method of the present invention was subjected to precision experiment:

[0095] Repeatability precision: Same as the above accuracy, the relative standard deviation of each concentration of different matrices meets the requirements of GB5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for National Food Safety Standards".

[0096] Intermediate precision: According to 4.1.6.3 of GB5009.295-2023 "General Principles for Validation of Chemical Analysis Methods for Food Safety National Standards", different personnel were used to perform validation at different times, and the intermediate precision of each concentration was calculated, as shown in Tables 13 to 16. It can be concluded from Table 16 that the intermediate precision meets the requirements of GB 5009.295-2023 "General Principles for Validation of Chemical Analysis Methods for Food Safety National Standards".

[0097]

[0098]

[0099]

[0100] From Table 6, Table 8, Table 12, Table 13 and Table 14, we can get the following table:

[0101]

[0102] The method of the present invention is subjected to a specific experiment: according to GB 5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for Food Safety National Standards", a certain concentration of substances that may interfere with the qualitative and / or quantitative properties of the target analyte is added to the representative sample, and then analyzed to check whether these interfering substances can be effectively identified. According to the liquid chromatography-mass spectrometry / mass spectrometry method for the determination of buthiazine, substances that may interfere with the qualitative and / or quantitative properties of the target analyte and the target analyte are added to the empty matrix for the validation test. The results are shown in Tables 7 to 12. The spike recovery rate and relative standard deviation meet the requirements of GB5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for Food Safety National Standards", so the liquid chromatography-mass spectrometry / mass spectrometry method for the determination of buthiazine can effectively identify these interfering substances.

[0103] The stability test of the method of the present invention was conducted: According to GB 5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for Food Safety National Standards" 4.1.7.2, different storage conditions and time intervals were selected according to the properties of the sample solution, and the target analytes in the sample solution were determined using the same analysis method and analysis conditions. The verification results are shown in Table 17. According to GB5009.295-2023 "General Rules for Validation of Chemical Analysis Methods for Food Safety National Standards", the absolute value of the difference between the two determination results meets the requirement of being less than or equal to 15%.

[0104]

[0105] The above examples demonstrate that the method of the present invention can accurately perform qualitative and quantitative detection of buthiazine in food.

Claims

1. A method for detecting buthiazine in food, characterized in that The steps include: Step 1: Determination of buthiazine retention time and monitoring ions A standard substance of buthiazine was taken to prepare a standard solution with a concentration of 500 ng / mL, the standard solution was subjected to parent ion scanning by a triple quadrupole tandem mass spectrometer to determine the monitoring ion and quantitative ion pair of buthiazine, and then the standard solution was subjected to LC-MS / MS detection to determine the retention time of buthiazine; Step 2: Preparation of the standard curve of buthiazine A series of standard working solutions are prepared with buthiazine standard substances, and then different concentrations of buthiazine series standard working solutions are sampled for LC-MS / MS detection, and a linear regression is performed with the peak area of ​​buthiazine and its corresponding mass concentration to obtain a linear regression equation of buthiazine. The curve corresponding to the linear regression equation is the standard curve of buthiazine. Step 3: Pretreatment of the sample to be tested Solid or semi-solid sample: Take a solid or semi-solid sample and add methanol, perform ultrasonic extraction, cool to room temperature, make up to volume with methanol, centrifuge, filter through a 0.22μm microporous filter membrane to obtain the test solution, and then inject it; Liquid sample: Take an appropriate amount of the sample and add methanol, perform ultrasonic extraction, cool to room temperature, make up to volume with methanol, filter through a microporous filter membrane to obtain the liquid to be tested, and then inject the sample; Step 4: Testing of the sample to be tested Take 1 mL of the sample to be tested after pretreatment in step 3 for LC-MS / MS detection, and obtain the mass spectrum and chromatogram of the sample to be tested in multiple reaction monitoring mode. Perform qualitative analysis on the sample to be tested according to the retention time and the monitoring ions of buthiazine, and perform quantitative analysis on the sample to be tested according to the buthiazine standard curve.

2. The detection method according to claim 1, characterized in that The mass spectrometry parameters for buthiazine are: 。 3. The detection method according to claim 1, characterized in that: In the solid or semi-solid sample pretreatment step, the sample weight is 2 g (accurate to 0.001 g); the ultrasonic extraction time is 30 min; the volume of methanol is 20 mL; the centrifugal speed is 4000 r / min, and the time is 5 min.

4. The detection method according to claim 1, characterized in that: In the liquid sample pretreatment step, the sample dosage is 2 mL; the ultrasonic extraction time is 30 min; and the volume of the methanol is 20 mL.

5. The detection method according to claim 1, characterized in that: The detection conditions of the liquid chromatograph LC were set as follows: Chromatographic column: C 18 (3.0mm*100mm, 2.7μm); Column temperature: 35°C; Flow rate: 0.4 mL / min; Injection volume: 2 μL; Mobile phase: A: 0.1% formic acid aqueous solution; B: acetonitrile; gradient elution.

6. The detection method according to claim 5, characterized in that The gradient elution program was set as: 。 7. The detection method according to claim 1, characterized in that: The detection conditions of triple quadrupole tandem mass spectrometer MS / MS were set as follows: Ion source: electrospray ion source ESI+; Scanning mode: positive ion scanning; Detection method: Multiple reaction monitoring mode MRM; Capillary voltage: 2000V; Drying gas temperature: 300℃; Drying gas flow: 5L / min; Atomizing gas pressure: 241 kPa; Sheath gas temperature: 250℃; Sheath gas flow rate: 11L / min.

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