CN115876926A - Method for detecting BOC butyric acid in water and algae culture medium by using HPLC-MS/MS - Google Patents
Method for detecting BOC butyric acid in water and algae culture medium by using HPLC-MS/MS Download PDFInfo
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Abstract
The invention discloses a method for detecting BOC butyric acid in water and algae culture medium by using HPLC-MS/MS, which adopts an LC30AD-AB6500PLUS liquid chromatography mass spectrometer and an MSD detector, uses a Hilic PLUS RRHD, a chromatographic column of 50mm multiplied by 2.1mm,1.8 mu m and uses UP water and methanol as flowing phase to detect the BOC butyric acid. The linear correlation coefficient of BOC butyric acid in aerated water and algae culture medium is 0.9996, and RSD in aerated water R.T. 0.60% of RSD Area RSD in algal Medium at 4.05% R.T. 0.43%, RSD Area 1.71%, LOD in aerated water 0.761 μ g/L; LOQ is 2.54 mug/L, LOD in algae culture medium is 0.902 mug/L; LOQ of 3.01 mu g/LThe average adding recovery rate in the aerated water is 94.9-100.3%, and the average adding recovery rate in the algae culture medium is 93.8-97.9%. The method has the advantages of strong specificity, good repeatability, simple operation, low cost, good separation effect, high sensitivity and high accuracy.
Description
Technical Field
The invention relates to a method for detecting BOC butyric acid in water and algae culture medium by using HPLC-MS/MS, belonging to the field of compound analysis and detection.
Background
BOC butyric acid, chemical name: (beta R) -beta- [ [ (1, 1-dimethylethoxy) carbonyl]Amino group]-2,4, 5-trifluorophenylbutyric acid of formula: c 15 H 18 F 3 NO 4 CAS number: 486460-00-8, the structural formula is shown below.
BOC butyrate is mainly used as an intermediate of sitagliptin, the first dipeptidyl peptidase-IV (DDP-4) inhibitor developed by the american merck company for the treatment of type ii diabetes, which can improve the control of blood glucose in type ii diabetics by increasing the level of active incretin, without inhibiting DPP-8 or DPP-9 enzymes closely related to DDP-4 enzymes at therapeutic concentrations. The characteristics of strong effect, high selectivity and small side effect of sitagliptin enable the sales volume of sitagliptin to grow explosively, and the global sales volume reaches more than 70 billion dollars at present. The BOC butyric acid serving as an important intermediate for synthesizing sitagliptin has great market potential, so that the quality control method of the BOC butyric acid is deeply researched, and the BOC butyric acid has important significance for production and application of the BOC butyric acid.
The only reported detection method of BOC butyric acid is high performance liquid chromatography.
[ Enterprise Standard ] Q/YONTA 1169-2019 discloses a high performance liquid chromatography detection method of BOC butyric acid, and specifically discloses (1) a standard solution: 20mg of a reference substance is precisely weighed, placed in a 50mL volumetric flask, dissolved by adding 5-10 mL of 50% acetonitrile, diluted to the scale with a diluent, and shaken up (0.4 mg/mL). (2) chromatographic conditions: mobile phase A: measuring 1mL of perchloric acid, putting the perchloric acid into a 1000mL volumetric flask containing 500mL of water, shaking up, diluting the perchloric acid to a scale with water, shaking up, filtering, and ultrasonically degassing; mobile phase B: acetonitrile; sample introduction amount: 5uL; wavelength: 210nm; flow rate: 1.0mL/min; column temperature; gradient elution at 25 ℃. The method has the advantages of complex operation, long instrument running time, poor separation effect and low sensitivity in the practical application process.
HPLC-MS/MS is widely applied to analytical research with good separation effect and high sensitivity, and no relevant report for detecting BOC butyric acid by using HPLC-MS/MS technology is found at present.
Disclosure of Invention
The invention aims to solve the problems of complex operation, long instrument running time, poor separation effect and low sensitivity in the prior art, and provides a method for detecting BOC butyric acid in water and algae culture medium by using HPLC-MS/MS.
The method comprises the following steps: (1) preparation of test solution: precisely weighing 0.2002g and 1.40141g of BOC butyric acid samples in 10mL and 100mL volumetric flasks respectively, dissolving with methanol, metering to a certain volume, shaking up, and preparing to obtain BOC butyric acid sample stock solutions with the concentrations of 20g/L and 14 g/L; sucking a proper amount of sample stock solution, and diluting with aerated water or an algae culture medium to obtain a test solution to be tested; (2) preparation of standard working solution: precisely weighing 0.01003g of BOC butyric acid standard, placing the BOC butyric acid standard in a 100mL volumetric flask, dissolving the BOC butyric acid standard with methanol, fixing the volume to scale, shaking up, preparing a BOC butyric acid standard stock solution with the concentration of 100.2mg/L, sucking a proper amount of the BOC butyric acid standard stock solution, diluting the BOC butyric acid standard stock solution with aerated water or a algae culture medium into a standard working solution with the concentration of the BOC butyric acid of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L and 1.00mg/L, and waiting for HPLC-MS/MS determination; (3) measuring and calculating: and setting the operation conditions of an HPLC-MS/MS instrument, respectively carrying out HPLC-MS/MS measurement on the test solution and the standard solution after the instrument is stabilized, and calculating the content of BOC butyric acid according to an external standard method.
In the HPLC-MS/MS, the HPLC operating conditions are as follows: a chromatographic column: hilic Plus RRHD,50mm × 2.1mm,1.8 μm; sample injection amount: 0.5-1.5 mu L; mobile phase: a is UP water, B is methanol, and the volume ratio of the mobile phase A to the mobile phase B is 10; column temperature: 35 to 45 ℃; flow rate: 0.1-0.5 mL/min.
Preferably, the sample injection amount is 0.8 to 1.2 μ L, and the flow rate is 0.2 to 0.4mL/min.
More preferably, the amount of the sample is 1.0. Mu.L.
More preferably, the flow rate is 0.3mL/min.
Preferably, the column temperature is 38 to 42 ℃.
More preferably, the column temperature is 40 ℃.
In the HPLC-MS/MS, the operation conditions of MSD are as follows:
an ion source: turbo V ion source (ESI mode); ion source parameters: air curtain air (CUR), 45psi; collision gas (CAD), 12; ionization voltage (IS), -4500V; temperature (TEM): 500 ℃; mist spray (GS 1): 50psi; auxiliary heating gas (GS 2): 50psi; and (3) monitoring mode: and (4) MRM.
The MRM ion acquisition parameters are as follows:
* To quantify the ions.
The LOD of BOC butyric acid in aerated water is 0.761 mug/L and the LOQ is 2.54 mug/L; LOD in the algal medium was 0.902. Mu.g/L, and LOQ was 3.01. Mu.g/L.
The invention has the beneficial effects that:
the invention overcomes the high detection limit of the prior high performance liquid chromatography, and the LOD of the invention in aerated water is 0.761 mug/L, and the LOQ is 2.54 mug/L; the LOD in the algae culture medium is 0.902 mu g/L, the LOQ is 3.01 mu g/L, and the sensitivity is high; the running time of the instrument is short, the peak of BOC butyric acid can be generated within 0.41min, the separation effect is good, and the specificity is strong; the BOC butyric acid has good linear relation in the range of 0.100 mg/L-1.00 mg/L, the linear equation in aerated water is y = 4.71X 107X + 4.57X 105, the linear correlation coefficient is 0.9996, the linear equation in the algae culture medium is y = 4.87X 107X-5.64X 105, and the linear correlation coefficient is 0.9996; the analysis method provided by the invention can respectively meet the concentration determination requirements of an daphnia acute activity inhibition test, an algae growth inhibition test and a fish acute toxicity test. The detection method has the advantages of strong specificity, good repeatability, simple and convenient operation, low cost, good separation effect, high sensitivity and high accuracy, can well detect the content of the BOC butyric acid in the aerated water and the algae culture medium, and provides a reference basis for quality control in the production and application process of the BOC butyric acid.
Drawings
FIG. 1 is a linear fitting curve diagram of BOC butyric acid in aerated water;
FIG. 2 is a graph of a linear fit of BOC butyric acid in algal medium.
Detailed Description
The invention will be further elucidated by means of specific embodiments, without being limited thereto, in conjunction with the accompanying drawings. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Reagents and instruments used in the following examples include, but are not limited to:
1. reagents and solvents
(1) Methanol: HPLC, shanghai an spectral experimental science & technology ltd., Y7011440;
(2) UP water: resistivity, 18.2M Ω cm;
(3) Aeration water: self-made in a laboratory;
(4) Algae culture medium: self-made in a laboratory;
(5) And (3) BOC butyric acid.
2. Main instrument equipment
(1) Liquid chromatography mass spectrometer: shimadzu LC30AD-AB6500 PLUS;
(2) An electronic balance: mettler-tollido (china) ltd, XSE205DU;
(3) Constant temperature culture shaker, ZWY-240.
Example 1:
(1) Preparing a test solution: respectively and precisely weighing 0.2002g and 1.40141g of BOC butyric acid samples in 10mL volumetric flasks and 100mL volumetric flasks, dissolving with methanol, fixing the volume to a scale, shaking up, and preparing to obtain BOC butyric acid sample stock solutions with the concentrations of 20g/L and 14 g/L; absorbing a proper amount of sample stock solution, and diluting with aerated water to obtain a to-be-tested sample solution;
(2) Preparation of standard working solutions: precisely weighing 0.01003g of BOC butyric acid standard, placing the BOC butyric acid standard in a 100mL volumetric flask, dissolving the BOC butyric acid standard with methanol, fixing the volume to scale, shaking up, preparing a BOC butyric acid standard stock solution with the concentration of 100.2mg/L, sucking a proper amount of the BOC butyric acid standard stock solution, diluting the BOC butyric acid standard stock solution with aerated water into a standard working solution with the concentration of BOC butyric acid of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L and 1.00mg/L, and waiting for HPLC-MS/MS determination;
(3) Measurement and calculation: and setting the operation conditions of an HPLC-MS/MS instrument, respectively carrying out HPLC-MS/MS measurement on the test solution and the standard solution after the instrument is stabilized, and calculating the content of BOC butyric acid according to an external standard method.
The operating conditions for the HPLC were: the chromatographic column is Hilic Plus RRHD,50mm multiplied by 2.1mm,1.8 mu m; the sample injection amount is 1.0 mu L; the mobile phase A is UP water, the mobile phase B is methanol, and the volume ratio of the mobile phase A to the mobile phase B is 10; the column temperature was 40 ℃; the flow rate was 0.3mL/min.
The operating conditions for MSD were:
an ion source: turbo V ion source (ESI mode);
ion source parameters: air curtain air (CUR), 45psi; collision gas (CAD), 12; ionization voltage (IS), -4500V; temperature (TEM): 500 ℃; mist spray (GS 1): 50psi; auxiliary heating gas (GS 2): 50psi;
a monitoring mode: and (4) MRM.
The MRM ion acquisition parameters are as follows:
* To quantify the ions.
Example 2:
(1) Preparing a test solution: precisely weighing 0.2002g and 1.40141g of BOC butyric acid samples in 10mL and 100mL volumetric flasks respectively, dissolving with methanol, metering to a certain volume, shaking up, and preparing to obtain BOC butyric acid sample stock solutions with the concentrations of 20g/L and 14 g/L; absorbing a proper amount of sample stock solution, and diluting with aerated water to obtain a to-be-tested sample solution;
(2) Preparation of standard working solutions: precisely weighing 0.01003g of BOC butyric acid standard substance, placing the BOC butyric acid standard substance in a 100mL volumetric flask, dissolving the BOC butyric acid standard substance with methanol, fixing the volume to scale, shaking up the BOC butyric acid standard substance to prepare a BOC butyric acid standard stock solution with the concentration of 100.2mg/L, sucking a proper amount of BOC butyric acid standard stock solution, diluting the BOC butyric acid standard stock solution with aerated water to prepare a standard working solution with the concentration of the BOC butyric acid of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L and 1.00mg/L, and waiting for HPLC-MS/MS measurement;
(3) Measurement and calculation: and setting the operation conditions of an HPLC-MS/MS instrument, respectively carrying out HPLC-MS/MS measurement on the test solution and the standard solution after the instrument is stabilized, and calculating the content of BOC butyric acid according to an external standard method.
The operating conditions for the HPLC were: the chromatographic column is Hilic Plus RRHD,50mm multiplied by 2.1mm,1.8 μm; the sample injection amount is 0.5 mu L; the mobile phase A is UP water, the mobile phase B is methanol, and the volume ratio of the mobile phase A to the mobile phase B is 10; the column temperature was 35 ℃; the flow rate was 0.1mL/min.
The operating conditions for MSD were:
an ion source: turbo V ion source (ESI mode);
ion source parameters: air curtain air (CUR), 45psi; collision gas (CAD), 12; ionization voltage (IS), -4500V; temperature (TEM): 500 ℃; mist spray (GS 1): 50psi; auxiliary heating gas (GS 2): 50psi;
and (3) monitoring mode: and (4) MRM.
The MRM ion acquisition parameters are as follows:
* To quantify the ions.
Example 3:
(1) Preparation of a test solution: respectively and precisely weighing 0.2002g and 1.40141g of BOC butyric acid samples in 10mL volumetric flasks and 100mL volumetric flasks, dissolving with methanol, fixing the volume to a scale, shaking up, and preparing to obtain BOC butyric acid sample stock solutions with the concentrations of 20g/L and 14 g/L; absorbing a proper amount of sample stock solution, and diluting with aerated water to obtain a to-be-tested sample solution;
(2) Preparation of standard working solutions: precisely weighing 0.01003g of BOC butyric acid standard, placing the BOC butyric acid standard in a 100mL volumetric flask, dissolving the BOC butyric acid standard with methanol, fixing the volume to scale, shaking up, preparing a BOC butyric acid standard stock solution with the concentration of 100.2mg/L, sucking a proper amount of the BOC butyric acid standard stock solution, diluting the BOC butyric acid standard stock solution with aerated water into a standard working solution with the concentration of BOC butyric acid of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L and 1.00mg/L, and waiting for HPLC-MS/MS determination;
(3) Measurement and calculation: and setting the operation conditions of an HPLC-MS/MS instrument, respectively carrying out HPLC-MS/MS measurement on the test solution and the standard solution after the instrument is stabilized, and calculating the content of BOC butyric acid according to an external standard method.
The operating conditions for the HPLC were: the chromatographic column is Hilic Plus RRHD,50mm multiplied by 2.1mm,1.8 μm; the sample injection amount is 0.8 mu L; the mobile phase A is UP water, the mobile phase B is methanol, and the volume ratio of the mobile phase A to the mobile phase B is 10; the column temperature was 38 ℃; the flow rate was 0.2mL/min.
The operating conditions for MSD were:
an ion source: turbo V ion source (ESI mode);
ion source parameters: air curtain air (CUR), 45psi; collision gas (CAD), 12; ionization voltage (IS), -4500V; temperature (TEM): 500 ℃; mist spray (GS 1): 50psi; auxiliary heating gas (GS 2): 50psi;
and (3) monitoring mode: and (4) MRM.
The MRM ion acquisition parameters were:
* To quantify the ions.
Example 4:
(1) Preparation of a test solution: respectively and precisely weighing 0.2002g and 1.40141g of BOC butyric acid samples in 10mL volumetric flasks and 100mL volumetric flasks, dissolving with methanol, fixing the volume to a scale, shaking up, and preparing to obtain BOC butyric acid sample stock solutions with the concentrations of 20g/L and 14 g/L; absorbing a proper amount of sample stock solution, and diluting with aerated water to obtain a to-be-tested sample solution;
(2) Preparation of standard working solutions: precisely weighing 0.01003g of BOC butyric acid standard, placing the BOC butyric acid standard in a 100mL volumetric flask, dissolving the BOC butyric acid standard with methanol, fixing the volume to scale, shaking up, preparing a BOC butyric acid standard stock solution with the concentration of 100.2mg/L, sucking a proper amount of the BOC butyric acid standard stock solution, diluting the BOC butyric acid standard stock solution with aerated water into a standard working solution with the concentration of BOC butyric acid of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L and 1.00mg/L, and waiting for HPLC-MS/MS determination;
(3) Measurement and calculation: and setting the operation conditions of an HPLC-MS/MS instrument, respectively carrying out HPLC-MS/MS measurement on the test solution and the standard solution after the instrument is stabilized, and calculating the content of BOC butyric acid according to an external standard method.
The operating conditions for the HPLC were: the chromatographic column is Hilic Plus RRHD,50mm multiplied by 2.1mm,1.8 mu m; the sample injection amount is 1.2 mu L; the mobile phase A is UP water, the mobile phase B is methanol, and the volume ratio of the mobile phase A to the mobile phase B is 10; the column temperature was 42 ℃; the flow rate was 0.4mL/min.
The operating conditions for MSD were:
an ion source: turbo V ion source (ESI mode);
ion source parameters: air curtain air (CUR), 45psi; collision gas (CAD), 12; ionization voltage (IS), -4500V; temperature (TEM): 500 ℃; mist spray (GS 1): 50psi; auxiliary heating gas (GS 2): 50psi;
and (3) monitoring mode: and (4) MRM.
The MRM ion acquisition parameters are as follows:
* To quantify the ions.
Example 5:
(1) Preparation of a test solution: respectively and precisely weighing 0.2002g and 1.40141g of BOC butyric acid samples in 10mL volumetric flasks and 100mL volumetric flasks, dissolving with methanol, fixing the volume to a scale, shaking up, and preparing to obtain BOC butyric acid sample stock solutions with the concentrations of 20g/L and 14 g/L; absorbing a proper amount of sample stock solution, and diluting with aerated water to obtain a to-be-tested sample solution;
(2) Preparation of standard working solutions: precisely weighing 0.01003g of BOC butyric acid standard, placing the BOC butyric acid standard in a 100mL volumetric flask, dissolving the BOC butyric acid standard with methanol, fixing the volume to scale, shaking up, preparing a BOC butyric acid standard stock solution with the concentration of 100.2mg/L, sucking a proper amount of the BOC butyric acid standard stock solution, diluting the BOC butyric acid standard stock solution with aerated water into a standard working solution with the concentration of BOC butyric acid of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L and 1.00mg/L, and waiting for HPLC-MS/MS determination;
(3) Measurement and calculation: and setting the operation conditions of an HPLC-MS/MS instrument, respectively carrying out HPLC-MS/MS measurement on the test solution and the standard solution after the instrument is stabilized, and calculating the content of BOC butyric acid according to an external standard method.
The operating conditions for HPLC were: the chromatographic column is Hilic Plus RRHD,50mm multiplied by 2.1mm,1.8 μm; the sample injection amount is 1.5 mu L; the mobile phase A is UP water, the mobile phase B is methanol, and the volume ratio of the mobile phase A to the mobile phase B is 10; the column temperature was 45 ℃; the flow rate was 0.5mL/min.
The operating conditions for MSD were:
an ion source: turbo V ion source (ESI mode);
ion source parameters: air curtain air (CUR), 45psi; collision gas (CAD), 12; ionization voltage (IS), -4500V; temperature (TEM): 500 ℃; mist spray (GS 1): 50psi; auxiliary heating gas (GS 2): 50psi;
and (3) monitoring mode: and (4) MRM.
The MRM ion acquisition parameters were:
* To quantify the ions.
Example 6:
(1) Preparation of a test solution: respectively and precisely weighing 0.2002g and 1.40141g of BOC butyric acid samples in 10mL volumetric flasks and 100mL volumetric flasks, dissolving with methanol, fixing the volume to a scale, shaking up, and preparing to obtain BOC butyric acid sample stock solutions with the concentrations of 20g/L and 14 g/L; sucking a proper amount of sample stock solution, and diluting with an algae culture medium to obtain a test sample solution to be tested;
(2) Preparation of standard working solutions: precisely weighing 0.01003g of BOC butyric acid standard, placing the BOC butyric acid standard in a 100mL volumetric flask, dissolving the BOC butyric acid standard with methanol, fixing the volume to scale, shaking up, preparing a BOC butyric acid standard stock solution with the concentration of 100.2mg/L, sucking a proper amount of the BOC butyric acid standard stock solution, diluting the BOC butyric acid standard stock solution with a algae culture medium into a standard working solution with the concentration of BOC butyric acid of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L and 1.00mg/L, and waiting for HPLC-MS/MS determination;
(3) Measurement and calculation: and setting the operation conditions of an HPLC-MS/MS instrument, respectively carrying out HPLC-MS/MS measurement on the test solution and the standard solution after the instrument is stabilized, and calculating the content of BOC butyric acid according to an external standard method.
The operating conditions for the HPLC were: the chromatographic column is Hilic Plus RRHD,50mm multiplied by 2.1mm,1.8 μm; the sample injection amount is 1.0 mu L; the mobile phase A is UP water, the mobile phase B is methanol, and the volume ratio of the mobile phase A to the mobile phase B is 10; the column temperature is 40 ℃; the flow rate was 0.3mL/min.
The operating conditions for MSD were:
an ion source: turbo V ion source (ESI mode);
ion source parameters: air curtain air (CUR), 45psi; collision gas (CAD), 12; ionization voltage (IS), -4500V; temperature (TEM): 500 ℃; mist spray (GS 1): 50psi; auxiliary heating gas (GS 2): 50psi;
a monitoring mode: and (4) MRM.
The MRM ion acquisition parameters were:
* To quantify the ions.
Example 7:
(1) Preparation of a test solution: respectively and precisely weighing 0.2002g and 1.40141g of BOC butyric acid samples in 10mL volumetric flasks and 100mL volumetric flasks, dissolving with methanol, fixing the volume to a scale, shaking up, and preparing to obtain BOC butyric acid sample stock solutions with the concentrations of 20g/L and 14 g/L; sucking a proper amount of sample stock solution, and diluting with an algae culture medium to obtain a test sample solution to be tested;
(2) Preparation of standard working solutions: precisely weighing 0.01003g of BOC butyric acid standard substance, placing the BOC butyric acid standard substance in a 100mL volumetric flask, dissolving the BOC butyric acid standard substance with methanol, fixing the volume to scale, shaking up the BOC butyric acid standard substance to prepare a BOC butyric acid standard stock solution with the concentration of 100.2mg/L, sucking a proper amount of BOC butyric acid standard stock solution, diluting the BOC butyric acid standard stock solution with an alga culture medium to prepare a standard working solution with the concentration of the BOC butyric acid of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L and 1.00mg/L, and waiting for HPLC-MS/MS measurement;
(3) Measurement and calculation: and setting the operation conditions of an HPLC-MS/MS instrument, respectively carrying out HPLC-MS/MS measurement on the test solution and the standard solution after the instrument is stabilized, and calculating the content of BOC butyric acid according to an external standard method.
The operating conditions for the HPLC were: the chromatographic column is Hilic Plus RRHD,50mm multiplied by 2.1mm,1.8 μm; the sample injection amount is 0.5 mu L; the mobile phase A is UP water, the mobile phase B is methanol, and the volume ratio of the mobile phase A to the mobile phase B is 10; the column temperature is 35 ℃; the flow rate was 0.1mL/min.
The operating conditions for MSD were:
an ion source: turbo V ion source (ESI mode);
ion source parameters: air curtain air (CUR), 45psi; collision gas (CAD), 12; ionization voltage (IS), -4500V; temperature (TEM): 500 ℃; mist spray (GS 1): 50psi; auxiliary heating gas (GS 2): 50psi;
and (3) monitoring mode: and (4) MRM.
The MRM ion acquisition parameters are as follows:
* To quantify the ions.
Example 8:
(1) Preparing a test solution: respectively and precisely weighing 0.2002g and 1.40141g of BOC butyric acid samples in 10mL volumetric flasks and 100mL volumetric flasks, dissolving with methanol, fixing the volume to a scale, shaking up, and preparing to obtain BOC butyric acid sample stock solutions with the concentrations of 20g/L and 14 g/L; sucking a proper amount of sample stock solution, and diluting with an algae culture medium to obtain a test sample solution to be tested;
(2) Preparation of standard working solutions: precisely weighing 0.01003g of BOC butyric acid standard substance, placing the BOC butyric acid standard substance in a 100mL volumetric flask, dissolving the BOC butyric acid standard substance with methanol, fixing the volume to scale, shaking up the BOC butyric acid standard substance to prepare a BOC butyric acid standard stock solution with the concentration of 100.2mg/L, sucking a proper amount of BOC butyric acid standard stock solution, diluting the BOC butyric acid standard stock solution with an alga culture medium to prepare a standard working solution with the concentration of the BOC butyric acid of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L and 1.00mg/L, and waiting for HPLC-MS/MS measurement;
(3) Measurement and calculation: and setting the operation conditions of an HPLC-MS/MS instrument, respectively carrying out HPLC-MS/MS measurement on the test solution and the standard solution after the instrument is stabilized, and calculating the content of BOC butyric acid according to an external standard method.
The operating conditions for the HPLC were: the chromatographic column is Hilic Plus RRHD,50mm multiplied by 2.1mm,1.8 μm; the sample injection amount is 0.8 mu L; the mobile phase A is UP water, the mobile phase B is methanol, and the volume ratio of the mobile phase A to the mobile phase B is 10; the column temperature was 38 ℃; the flow rate was 0.2mL/min.
The operating conditions for MSD were:
an ion source: turbo V ion source (ESI mode);
ion source parameters: air curtain air (CUR), 45psi; collision gas (CAD), 12; ionization voltage (IS), -4500V; temperature (TEM): 500 ℃; mist spray (GS 1): 50psi; auxiliary heating gas (GS 2): 50psi;
and (3) monitoring mode: and (4) MRM.
The MRM ion acquisition parameters are as follows:
* To quantify the ions.
Example 9:
(1) Preparation of a test solution: respectively and precisely weighing 0.2002g and 1.40141g of BOC butyric acid samples in 10mL volumetric flasks and 100mL volumetric flasks, dissolving with methanol, fixing the volume to a scale, shaking up, and preparing to obtain BOC butyric acid sample stock solutions with the concentrations of 20g/L and 14 g/L; sucking a proper amount of sample stock solution, and diluting with an algae culture medium to obtain a test sample solution to be tested;
(2) Preparation of standard working solutions: precisely weighing 0.01003g of BOC butyric acid standard, placing the BOC butyric acid standard in a 100mL volumetric flask, dissolving the BOC butyric acid standard with methanol, fixing the volume to scale, shaking up, preparing a BOC butyric acid standard stock solution with the concentration of 100.2mg/L, sucking a proper amount of the BOC butyric acid standard stock solution, diluting the BOC butyric acid standard stock solution with a algae culture medium into a standard working solution with the concentration of BOC butyric acid of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L and 1.00mg/L, and waiting for HPLC-MS/MS determination;
(3) Measurement and calculation: and setting the operation conditions of an HPLC-MS/MS instrument, respectively carrying out HPLC-MS/MS measurement on the test solution and the standard solution after the instrument is stabilized, and calculating the content of BOC butyric acid according to an external standard method.
The operating conditions for the HPLC were: the chromatographic column is Hilic Plus RRHD,50mm multiplied by 2.1mm,1.8 μm; the sample injection amount is 1.2 mu L; the mobile phase A is UP water, the mobile phase B is methanol, and the volume ratio of the mobile phase A to the mobile phase B is 10; the column temperature was 42 ℃; the flow rate was 0.4mL/min.
The operating conditions for MSD were:
an ion source: turbo V ion source (ESI mode);
ion source parameters: air curtain air (CUR), 45psi; collision gas (CAD), 12; ionization voltage (IS), -4500V; temperature (TEM): 500 ℃; mist spray (GS 1): 50psi; auxiliary heating gas (GS 2): 50psi;
and (3) monitoring mode: and (4) MRM.
The MRM ion acquisition parameters are as follows:
* To quantify the ions.
Example 10:
(1) Preparation of a test solution: respectively and precisely weighing 0.2002g and 1.40141g of BOC butyric acid samples in 10mL volumetric flasks and 100mL volumetric flasks, dissolving with methanol, fixing the volume to a scale, shaking up, and preparing to obtain BOC butyric acid sample stock solutions with the concentrations of 20g/L and 14 g/L; sucking a proper amount of sample stock solution, and diluting with an algae culture medium to obtain a test sample solution to be tested;
(2) Preparation of standard working solutions: precisely weighing 0.01003g of BOC butyric acid standard, placing the BOC butyric acid standard in a 100mL volumetric flask, dissolving the BOC butyric acid standard with methanol, fixing the volume to scale, shaking up, preparing a BOC butyric acid standard stock solution with the concentration of 100.2mg/L, sucking a proper amount of the BOC butyric acid standard stock solution, diluting the BOC butyric acid standard stock solution with a algae culture medium into a standard working solution with the concentration of BOC butyric acid of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L and 1.00mg/L, and waiting for HPLC-MS/MS determination;
(3) Measurement and calculation: and setting the operation conditions of an HPLC-MS/MS instrument, respectively carrying out HPLC-MS/MS measurement on the test solution and the standard solution after the instrument is stabilized, and calculating the content of BOC butyric acid according to an external standard method.
The operating conditions for the HPLC were: the chromatographic column is Hilic Plus RRHD,50mm multiplied by 2.1mm,1.8 mu m; the sample injection amount is 1.5 mu L; the mobile phase A is UP water, the mobile phase B is methanol, and the volume ratio of the mobile phase A to the mobile phase B is 10; the column temperature was 45 ℃; the flow rate was 0.5mL/min.
The operating conditions for MSD were:
an ion source: turbo V ion source (ESI mode);
ion source parameters: air curtain air (CUR), 45psi; collision gas (CAD), 12; ionization voltage (IS), -4500V; temperature (TEM): 500 ℃; mist spray (GS 1): 50psi; auxiliary heating gas (GS 2): 50psi;
and (3) monitoring mode: and (4) MRM.
The MRM ion acquisition parameters are as follows:
* To quantify the ions.
To further demonstrate the feasibility of the present invention, the inventors conducted the following methodological investigations.
1. Linear test
In examples 1 and 6, the standard working solutions having BOC butyric acid concentrations of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L and 1.00mg/L were precisely aspirated and measured by the method of the present invention, and the results are shown in Table 1. And (3) drawing a correction curve by taking the concentration of the BOC butyric acid as an abscissa and the peak area of the BOC butyric acid as an ordinate, wherein when the concentration of the BOC butyric acid is in a range of 0.100 mg/L-1.00 mg/L, a linear relation is formed between y and x. The equation of BOC butyric acid in aerated water is obtained by linear regression of the curve and is y = 4.71X 10 7 x+4.57×10 5 The linear correlation coefficient of the curve is 0.9996, and a linear fitting curve is shown in figure 1; the equation of BOC butyric acid in the algal culture medium is y = 4.87X 10 7 x-5.64×10 5 The linear correlation coefficient of the curve is 0.9996, and the linear fitting curve is shown in fig. 2.
Table 1: results of the Linear test
2. Specificity test
The detection method of the invention is used for measuring the aeration water blank sample, the aeration water test sample solution, the algae culture medium blank sample and the algae culture medium test sample solution, and comparing the chromatograms of the blank sample and the test sample solution. The retention time of the BOC butyric acid is 0.4 minute, a blank sample has no interference peak at the time point, the qualitative and quantitative determination of the BOC butyric acid can be realized through a qualitative and quantitative ion pair, and the interference phenomenon of other impurities does not exist, so that the result shows that the method has good specificity on the BOC butyric acid.
3. Repeatability test
The precision of the analytical method of the present invention was determined by continuously feeding 6 times the standard working solutions of 0.400mg/L in example 1 and example 6, respectively, wherein RSD of aerated water R.T. 0.60% of RSD Area 4.05%, precision test data are shown in Table 2. RSD of algae culture medium R.T. 0.43%, RSD Area 1.71%, and the precision measurement data are shown in Table 3. The result shows that the analysis precision of the same sample can meet the requirement by the analysis method.
Table 2: precision test result-aerated water
Table 3: results of precision test-Medium
4. Detection limit and quantification limit
The method is used for preparing and obtaining BOC butyric acid standard working solution with the concentration of 0.504mg/L of BOC butyric acid in aerated water and algae culture medium respectively, the chromatographic conditions are determined according to the invention, and the obtained atlas is analyzed, under the concentration, the average signal-to-noise ratio (S/N) =2002.3 of the target peak in aerated water and the average signal-to-noise ratio (S/N) =1699.8 of the target peak in algae culture medium. According to LOD =3 Xanalysis concentration/(S/N) and LOQ =10 Xanalysis concentration/(S/N), the LOD of the BOC butyric acid in the aerated water is 0.761 mu g/L and the LOQ is 2.54 mu g/L; the LOD of BOC butyric acid in the algae culture medium is 0.902 mu g/L, and the LOQ is 3.01 mu g/L. The detailed results are shown in tables 4 and 5.
Table 4: test results of detection limit and quantitative limit-aerated water
Table 5: test results of detection limit and quantitative limit-culture Medium
5. Recovery test
5.1 aerated Water recovery test
And (3) sucking 1.00mL of BOC butyric acid sample stock solution with the concentration of 14g/L in example 1, placing the BOC butyric acid sample stock solution in a 100mL volumetric flask, adding aerated water to a constant volume to scale, and uniformly mixing to obtain an aerated water high-concentration recovery test sample with the concentration of 140 mg/L. 0.36mL of BOC butyric acid sample stock solution with the concentration of 14g/L in example 1 was added to a 200mL volumetric flask, the volume was fixed to the scale with aerated water, and the mixture was mixed to obtain a low-concentration recovery test sample with the concentration of 25.2 mg/L. 5 parts of each concentration sample are prepared in parallel, the high concentration recovery rate sample is diluted by 200 times by using aerated water, the low concentration recovery rate sample is diluted by 50 times by using aerated water, and the low concentration recovery rate sample passes through a 0.45 mu m filter membrane and then is tested according to the operation conditions of the instrument. The recovery rate was calculated according to the formula (1), and the recovery rate Relative Standard Deviation (RSD) was calculated according to the formulas (2) and (3).
Taking 1.00mL of BOC butyric acid sample stock solution with the concentration of 20g/L in example 1 into a 100mL volumetric flask, adding aerated water to a constant volume to a scale, and uniformly mixing to obtain aerated water high-concentration recovery rate test samples with the concentration of 200mg/L, wherein 5 parts of each concentration sample are prepared in parallel. The high concentration recovery sample was diluted 250 times with aerated water and tested according to the operating conditions of the apparatus of the present invention. The recovery rate was calculated according to the formula (1), and the recovery rate Relative Standard Deviation (RSD) was calculated according to the formulas (2) and (3).
The analysis results are shown in Table 6, the average addition recovery rates of the 3 BOC butyric acids with different concentrations in the aerated water are respectively 94.9%, 100.3% and 99.4%, and the relative standard deviations of the recovery rates are respectively 3.64%, 1.70% and 1.01%.
In the formula:
r-recovery,%;
cd is the actually measured concentration of a target substance, mg/L;
ca-target Physics addition concentration, mg/L.
In the formula:
s-standard deviation;
xi-recovery,%, from the ith measurement;
x-average of recovery,%;
n-the number of recovery rates involved in the calculation.
In the formula:
RSD-relative standard deviation,%.
Table 6: test result of recovery rate in aerated water
5.2 recovery test in algal Medium
Taking 0.79mL of BOC butyric acid sample stock solution with the concentration of 14g/L in example 1, putting the BOC butyric acid sample stock solution into a 100mL volumetric flask, adding the algae culture medium to a constant volume to a scale, and uniformly mixing to obtain the algae culture medium high-concentration recovery test sample with the concentration of 110.6 mg/L. 0.09mL of BOC butyric acid sample stock solution with the concentration of 14g/L in example 1 was added to a 500mL volumetric flask, the volume was fixed to the scale with an algal culture medium, and the mixture was mixed to obtain low-concentration recovery test samples with the concentration of 2.52mg/L, and 5 parts of each concentration sample were prepared in parallel. The sample with high concentration recovery rate is diluted by 200 times by using the algae culture medium, the sample with low concentration recovery rate is diluted by 5 times by using the algae culture medium, and the sample passes through a 0.45 mu m filter membrane to be tested according to the operation conditions of the instrument. The recovery rate was calculated according to the formula (1), and the recovery rate Relative Standard Deviation (RSD) was calculated according to the formulas (2) and (3).
And (3) putting 1.00mL of BOC butyric acid sample stock solution with the concentration of 20g/L in the example 1 into a 100mL volumetric flask, adding an algae culture medium to a constant volume to a scale, and uniformly mixing to obtain an algae culture medium high-concentration recovery rate test sample with the concentration of 200mg/L, wherein 5 parts of each concentration sample are prepared in parallel. The high concentration recovery sample was diluted 250 times with algae culture medium and tested according to the instrument operating conditions of the present invention. The recovery rate was calculated according to the formula (1), and the recovery rate Relative Standard Deviation (RSD) was calculated according to the formulas (2) and (3).
The analysis results are shown in Table 7, the average addition recovery rate of 3 different concentrations of BOC butyric acid in the algae culture medium is 97.4%, 97.9% and 93.8%, and the relative standard deviation recovery rate is 3.49%, 4.95% and 4.12%.
Table 7: recovery in algal culture medium
6. Test for stability of test object
Weighing 1.02g of BOC butyric acid into a 250mL conical flask, adding an algae culture medium to a constant volume to a scale, oscillating for 24h in a constant temperature culture oscillator at 25 ℃, filtering a 0.45 mu m filter membrane to remove insoluble substances to obtain a BOC butyric acid saturated algae culture medium stock solution for later use, weighing 1.01g of BOC butyric acid into a 500mL conical flask, adding aeration water to a constant volume to a scale, oscillating for 24h in a constant temperature culture oscillator at 25 ℃, filtering a 0.45 mu m filter membrane to remove insoluble substances to obtain a BOC butyric acid saturated aeration water stock solution for later use, respectively diluting the saturated aeration water and the algae culture medium stock solution by 2000 times with methanol, filtering the solution by the 0.45 mu m filter membrane, measuring the concentration of a sample in the solution according to the operation conditions of the instrument, and parallelly measuring each saturated solution for 3 times.
Taking the average value of the results of the 3 times of measurement as the concentration of the saturated stock solution, and measuring to obtain the concentration of the BOC butyric acid saturated aerated water stock solution of 890mg/L and the concentration of the BOC butyric acid saturated algae culture medium stock solution of 288mg/L.
6.1 daphnia acute activity inhibition test stability
And adding 7.00mL of saturated aerated water stock solution into 100.0mL of aerated water, and uniformly mixing to obtain a stable sample solution for the daphnia acute activity inhibition test. Placing the solution under the daphnia test condition, and sampling at intervals; after the stability test solution is diluted by 100 times and filtered through a 0.45 mu m filter membrane, the concentration is measured according to the operation conditions of the instrument. The stability of the target in the sample solution in aerated water was calculated according to the formula (4), and the results are shown in Table 8. The result shows that the test object can be maintained at more than 80% of the initial concentration within 48h under the static method environment of the daphnia acute activity inhibition test.
Stability,% = (C) nh /C 0h )×100％...........(4)
Wherein, C0h: is the actual concentration of the target at 0 hour;
cn h: is the measured concentration of the target at n hours.
Table 8: stability result of daphnia acute activity inhibition test
6.2 stability of acute toxicity test for Fish
And adding 200mL of saturated aerated water stock solution into 2800mL of aerated water, and uniformly mixing to obtain a stable sample solution for the fish acute toxicity test. Sampling at intervals while the solution is placed under fish test conditions; after the stability test solution is diluted by 100 times and passes through a 0.45 mu m filter membrane, the concentration is measured according to the operating conditions of the instrument. The stability of the target in the sample solution in aerated water was calculated according to the formula (4), and the results are shown in Table 9. The result shows that the tested object can be maintained to be more than 80% of the initial concentration within 96h under the static method environment of the fish acute toxicity test.
Table 9: stability results of acute toxicity test of fish
6.3 algal production inhibition test stability
And adding 100.0mL of aeration water into 20.00mL of saturated algae culture medium stock solution, and uniformly mixing to obtain a stable sample solution for the algae growth inhibition test. Sampling at intervals while the solution is subjected to algae testing conditions; after the stability test solution is diluted by 100 times and passes through a 0.45 mu m filter membrane, the concentration is measured according to the operating conditions of the instrument. The stability of the target substance in the sample solution in the algal culture medium was calculated according to the formula (4), and the results are shown in Table 10. The results show that the sample can be maintained at 80% or more of the initial concentration within 72 hours under the algae growth inhibition test environment.
Table 10: parallel design of stability of algae production inhibition test
The stability test results are as follows: the analysis method provided by the invention can respectively meet the concentration determination requirements of an daphnia acute activity inhibition test, an alga growth inhibition test and a fish acute toxicity test.
While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for detecting BOC butyric acid in water and algae culture medium by using HPLC-MS/MS, which is characterized by comprising the following steps:
(1) Preparing a test solution: respectively and precisely weighing 0.2002g and 1.40141g of BOC butyric acid samples in 10mL volumetric flasks and 100mL volumetric flasks, dissolving with methanol, fixing the volume to a scale, shaking up, and preparing to obtain BOC butyric acid sample stock solutions with the concentrations of 20g/L and 14 g/L; sucking a proper amount of sample stock solution, and diluting with aerated water or an algae culture medium to obtain a test solution to be tested;
(2) Preparation of standard working solutions: precisely weighing 0.01003g of BOC butyric acid standard, placing the BOC butyric acid standard in a 100mL volumetric flask, dissolving the BOC butyric acid standard with methanol, fixing the volume to scale, shaking up, preparing a BOC butyric acid standard stock solution with the concentration of 100.2mg/L, sucking a proper amount of the BOC butyric acid standard stock solution, diluting the BOC butyric acid standard stock solution with aerated water or a algae culture medium into a standard working solution with the concentration of the BOC butyric acid of 0.100mg/L, 0.200mg/L, 0.400mg/L, 0.600mg/L, 0.800mg/L and 1.00mg/L, and waiting for HPLC-MS/MS determination;
(3) Measurement and calculation: and setting the operation conditions of an HPLC-MS/MS instrument, respectively carrying out HPLC-MS/MS measurement on the test solution and the standard solution after the instrument is stabilized, and calculating the content of BOC butyric acid according to an external standard method.
2. The method of claim 1, wherein the BOC butyric acid in water and algal culture medium is detected by HPLC-MS/MS,
the operating conditions for the HPLC were:
a chromatographic column: hilicPlusRRHD,50mm × 2.1mm,1.8 μm;
sample introduction amount: 0.5-1.5 mu L;
mobile phase: a is UP water, B is methanol, and the volume ratio of the mobile phase A to the mobile phase B is 10;
column temperature: 35 to 45 ℃;
flow rate: 0.1-0.5 mL/min.
3. The method for detecting BOC butyric acid in water and algal culture medium by HPLC-MS/MS of claim 2, wherein the sample volume is 0.8-1.2 μ L, and the flow rate is 0.2-0.4 mL/min.
4. The method for detecting BOC butyric acid in water and algal culture medium by HPLC-MS/MS of claim 3, wherein the sample amount is 1.0 μ L.
5. The method for detecting BOC butyric acid in water and algal culture medium by HPLC-MS/MS of claim 3, wherein said flow rate is 0.3mL/min.
6. The method for detecting BOC butyric acid in water and algal culture medium by HPLC-MS/MS of claim 2, wherein the column temperature is 38-42 ℃.
7. The method for detecting BOC butyric acid in water and algal culture medium by HPLC-MS/MS of claim 6, wherein the column temperature is 40 ℃.
8. The method of claim 1, wherein the BOC butyric acid in water and algal culture medium is detected by HPLC-MS/MS,
the operating conditions for MSD were:
an ion source: a TurboV ion source (ESI mode);
ion source parameters: air curtain air (CUR), 45psi; collision gas (CAD), 12; ionization voltage (IS), -4500V; temperature (TEM): 500 ℃; mist spray (GS 1): 50psi; auxiliary heating gas (GS 2): 50psi;
and (3) monitoring mode: and (4) MRM.
9. The method for detecting BOC butyric acid in water and algal culture medium by HPLC-MS/MS of claim 8, wherein the MRM ion collection parameters are: the compound name is BOC butyric acid, parent ion 332.1, daughter ion quantification ion 170.9, DP is-48V, EC is-17V, and polarity is negative; the parent ion 332.1, the daughter ion 257.8, DP-48V, EC-17V, and polarity negative.
10. The method for detecting BOC butyric acid in water and algal culture medium by HPLC-MS/MS according to any one of claims 1 to 9, wherein LOD of BOC butyric acid in aerated water is 0.761 μ g/L and LOQ is 2.54 μ g/L; LOD in the algal medium was 0.902. Mu.g/L, and LOQ was 3.01. Mu.g/L.
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