A method for detecting BOC butyric acid in water and algae culture medium using HPLC-MS / MS

Abstract

The present invention discloses a method for detecting BOC butyric acid in water and algae culture medium using HPLC-MS / MS. The method uses an LC30AD-AB6500PLUS liquid chromatography-mass spectrometer and an MSD detector, a HilicPlusRRHD, 50mm×2.1mm, 1.8μm chromatographic column, and UP water and methanol as mobile phases to detect BOC butyric acid. The linear correlation coefficient of BOC butyric acid in aerated water and algae culture medium is 0.9996, the average addition recovery rate in aerated water is 94.9% to 100.3%, and the average addition recovery rate in algae culture medium is 93.8% to 97.9%, respectively. The method has strong specificity, good repeatability, simple operation, low cost, good separation effect, high sensitivity and accuracy.

Description

Technical Field

[0001] The invention relates to a method for detecting BOC butyric acid in water and algae culture medium by utilizing HPLC-MS / MS, and belongs to the field of compound analysis and detection. Background Art

[0002] BOC butyric acid, chemical name: (βR)-β-[[(1,1-dimethylethoxy)carbonyl]amino]-2,4,5-trifluorophenylbutyric acid, molecular formula: C 15 H 18 F3NO4, CAS number: 486460-00-8, the structural formula is shown below.

[0003]

[0004] BOC butyric acid is primarily used as an intermediate for sitagliptin, the first dipeptidyl peptidase-IV (DDP-4) inhibitor developed by Merck for the treatment of type 2 diabetes. Sitagliptin improves blood sugar control in patients with type 2 diabetes by increasing levels of active incretin. At therapeutic concentrations, it does not inhibit the closely related DPP-8 or DPP-9 enzymes. Sitagliptin's potent efficacy, high selectivity, and minimal side effects have led to explosive growth in sales, with global sales currently exceeding $7 billion. As a key intermediate in the synthesis of sitagliptin, BOC butyric acid holds enormous market potential. Therefore, in-depth research on its quality control methods is crucial for its production and application.

[0005] Currently, the only reported method for detecting BOC butyric acid is high performance liquid chromatography.

[0006] [Enterprise Standard] Q / YONTA 1169-2019 discloses a high-performance liquid chromatography detection method for BOC butyric acid, specifically disclosing (1) Standard solution: accurately weigh 20 mg of the reference substance, place it in a 50 mL volumetric flask, add 5-10 mL of 50% acetonitrile to dissolve it and dilute it to the scale with a diluent, and shake it evenly (0.4 mg / mL). (2) Chromatographic conditions: Mobile phase A: measure 1 mL of perchloric acid, place it in a 1000 mL volumetric flask containing 500 mL of water, shake it evenly, then dilute it to the scale with water, shake it evenly, filter it, and ultrasonically degas it; Mobile phase B: acetonitrile; Injection volume: 5 uL; Wavelength: 210 nm; Flow rate: 1.0 mL / min; Column temperature: 25 ° C, gradient elution. In actual application, this method is more complicated to operate, the instrument running time is long, the separation effect is poor, and the sensitivity is low.

[0007] HPLC-MS / MS is widely used in analytical research due to its good separation effect and high sensitivity. However, there are currently no reports on the detection of BOC butyric acid using HPLC-MS / MS technology. Summary of the Invention

[0008] The purpose of the present invention is to overcome the problems of complex operation, long instrument operation time, poor separation effect and low sensitivity in the prior art, and to provide a method for detecting BOC butyric acid in water and algae culture medium using HPLC-MS / MS.

[0009] The method comprises the following steps: (1) preparation of test solution: accurately weigh 0.2002 g and 1.40141 g of BOC butyric acid sample into 10 mL and 100 mL volumetric flasks respectively, dissolve with methanol and dilute to the mark, shake well, and prepare BOC butyric acid sample stock solutions with concentrations of 20 g / L and 14 g / L; take an appropriate amount of sample stock solution and dilute with aerated water or algae culture medium to obtain the test solution; (2) preparation of standard working solution: accurately weigh 0.01003 g of BOC butyric acid standard, place in a 100 mL volumetric flask, dissolve with methanol and dilute to the mark, shake well, and prepare a BOC butyric acid sample stock solution with a concentration of 10 g / L and 14 g / L; take an appropriate amount of sample stock solution and dilute with aerated water or algae culture medium to obtain the test solution; (2) preparation of standard working solution: accurately weigh 0.01003 g of BOC butyric acid standard, place in a 100 mL volumetric flask, dissolve with methanol and dilute to the mark, shake well, and prepare a BOC butyric acid sample stock solution with a concentration of 10 g / L and 14 g / L. 0.2 mg / L BOC butyric acid standard stock solution, take an appropriate amount of BOC butyric acid standard stock solution, dilute it with aerated water or algae culture medium to form standard working solutions with BOC butyric acid concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, and 1.00 mg / L, and wait for HPLC-MS / MS determination; (3) Determination and calculation: Set the HPLC-MS / MS instrument operating conditions, and after the instrument stabilizes, perform HPLC-MS / MS determination on the test solution and the standard solution, respectively, and calculate the BOC butyric acid content according to the external standard method.

[0010] In the HPLC-MS / MS, the HPLC operating conditions are as follows: chromatographic column: Hilic Plus RRHD, 50 mm × 2.1 mm, 1.8 μm; injection volume: 0.5 to 1.5 μL; mobile phase: A is UP water, B is methanol, and the volume ratio of mobile phases A to B is 10:90; column temperature: 35 to 45°C; flow rate: 0.1 to 0.5 mL / min.

[0011] Preferably, the injection volume is 0.8 to 1.2 μL, and the flow rate is 0.2 to 0.4 mL / min.

[0012] More preferably, the injection volume is 1.0 μL.

[0013] More preferably, the flow rate is 0.3 mL / min.

[0014] Preferably, the column temperature is 38-42°C.

[0015] More preferably, the column temperature is 40°C.

[0016] In the HPLC-MS / MS, the operating conditions of MSD are:

[0017] Ion source: Turbo V ion source (ESI mode); ion source parameters: curtain gas (CUR), 45 psi; collision gas (CAD), 12; ionization voltage (IS), -4500 V; temperature (TEM): 500°C; spray gas (GS1): 50 psi; auxiliary heating gas (GS2): 50 psi; monitoring mode: MRM.

[0018] The MRM ion acquisition parameters are:

[0019]

[0020]

[0021] * is the quantitative ion.

[0022] The LOD and LOQ of BOC butyrate in aerated water were 0.761 μg / L and 2.54 μg / L, respectively. The LOD and LOQ of BOC butyrate in algae culture medium were 0.902 μg / L and 3.01 μg / L, respectively.

[0023] The beneficial effects of the present invention are:

[0024] The present invention overcomes the high detection limit of conventional high performance liquid chromatography methods. The LOD of the present invention in aerated water is 0.761 μg / L, and the LOQ is 2.54 μg / L; the LOD of the present invention in algae culture medium is 0.902 μg / L, and the LOQ is 3.01 μg / L, with high sensitivity. The instrument has a short operating time, and a BOC butyric acid peak can be obtained in 0.41 minutes, resulting in good separation effect and strong specificity. The present invention has a good linear relationship for BOC butyric acid in the range of 0.100 mg / L to 1.00 mg / L, and the linear equation in aerated water is y=4.71×107x+4.57×105, with a linear correlation coefficient of 0.9996; the linear equation in algae culture medium is y=4.87×107x-5.64×105, with a linear correlation coefficient of 0.9996. The analytical method of the present invention can meet the concentration determination requirements of a Daphnia acute activity inhibition test, an algae growth inhibition test, and a fish acute toxicity test, respectively. The detection method of the present invention has strong specificity, good repeatability, simple operation, low cost, good separation effect, high sensitivity and accuracy, and can well detect the content of BOC butyric acid in aeration water and algae culture medium, providing a reference basis for quality control in the production and application process of BOC butyric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the linear fitting curve of BOC butyric acid in aerated water;

[0026] Figure 2 This is the linear fitting curve of BOC butyrate in algae culture medium. DETAILED DESCRIPTION

[0027] The present invention is further described below by way of specific examples and in conjunction with the accompanying drawings, but the present invention is not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0028] The reagents and instruments used in the following examples include but are not limited to:

[0029] 1. Reagents and solvents

[0030] (1) Methanol: HPLC, Shanghai Anpu Laboratory Technology Co., Ltd., Y7011440;

[0031] (2) UP water: resistivity, 18.2 MΩ×cm;

[0032] (3) Aerated water: homemade in the laboratory;

[0033] (4) Algae culture medium: homemade in the laboratory;

[0034] (5) BOC butyrate.

[0035] 2. Main instruments and equipment

[0036] (1) Liquid chromatography-mass spectrometry: Shimadzu LC30AD-AB 6500PLUS;

[0037] (2) Electronic balance: Mettler-Toledo (China) Co., Ltd., XSE205DU;

[0038] (3) Constant temperature culture oscillator, ZWY-240.

[0039] Example 1:

[0040] (1) Preparation of test solution: Accurately weigh 0.2002 g and 1.40141 g of BOC butyric acid sample into 10 mL and 100 mL volumetric flasks, respectively. Dissolve with methanol and dilute to the mark. Shake well to prepare BOC butyric acid sample stock solutions with concentrations of 20 g / L and 14 g / L. Draw an appropriate amount of the sample stock solution and dilute with aerated water to obtain the test solution.

[0041] (2) Preparation of standard working solution: Accurately weigh 0.01003 g of BOC butyric acid standard, place it in a 100 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well, and prepare a BOC butyric acid standard stock solution with a concentration of 100.2 mg / L. Pipette an appropriate amount of BOC butyric acid standard stock solution and dilute it with aerated water to prepare standard working solutions with BOC butyric acid concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, and 1.00 mg / L, for HPLC-MS / MS determination;

[0042] (3) Determination and calculation: Set the operating conditions of the HPLC-MS / MS instrument. After the instrument is stable, perform HPLC-MS / MS determination on the test solution and the standard solution, and calculate the content of BOC butyric acid by the external standard method.

[0043] The operating conditions of HPLC were as follows: Hilic Plus RRHD column, 50 mm × 2.1 mm, 1.8 μm; injection volume was 1.0 μL; mobile phase A was UP water, and mobile phase B was methanol, with a volume ratio of A to B of 10:90; column temperature was 40°C; and flow rate was 0.3 mL / min.

[0044] The operating conditions of the MSD are:

[0045] Ion source: Turbo V ion source (ESI mode);

[0046] Ion source parameters: curtain gas (CUR), 45 psi; collision gas (CAD), 12; ionization voltage (IS), -4500 V; temperature (TEM): 500 °C; spray gas (GS1): 50 psi; auxiliary heating gas (GS2): 50 psi;

[0047] Monitoring mode: MRM.

[0048] The MRM ion acquisition parameters are:

[0049]

[0050] * is the quantitative ion.

[0051] Example 2:

[0052] (1) Preparation of test solution: Accurately weigh 0.2002 g and 1.40141 g of BOC butyric acid sample into 10 mL and 100 mL volumetric flasks, respectively. Dissolve with methanol and dilute to the mark. Shake well to prepare BOC butyric acid sample stock solutions with concentrations of 20 g / L and 14 g / L. Draw an appropriate amount of the sample stock solution and dilute with aerated water to obtain the test solution.

[0053] (2) Preparation of standard working solution: Accurately weigh 0.01003 g of BOC butyric acid standard, place it in a 100 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well, and prepare a BOC butyric acid standard stock solution with a concentration of 100.2 mg / L. Pipette an appropriate amount of BOC butyric acid standard stock solution and dilute it with aerated water to prepare standard working solutions with BOC butyric acid concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, and 1.00 mg / L, for HPLC-MS / MS determination;

[0054] (3) Determination and calculation: Set the operating conditions of the HPLC-MS / MS instrument. After the instrument is stable, perform HPLC-MS / MS determination on the test solution and the standard solution, and calculate the content of BOC butyric acid by the external standard method.

[0055] The operating conditions of HPLC were as follows: Hilic Plus RRHD column, 50 mm × 2.1 mm, 1.8 μm; injection volume, 0.5 μL; mobile phase A, UP water, and B, methanol, with a volume ratio of A to B of 10:90; column temperature, 35°C; and flow rate, 0.1 mL / min.

[0056] The operating conditions of the MSD are:

[0057] Ion source: Turbo V ion source (ESI mode);

[0058] Ion source parameters: curtain gas (CUR), 45 psi; collision gas (CAD), 12; ionization voltage (IS), -4500 V; temperature (TEM): 500 °C; spray gas (GS1): 50 psi; auxiliary heating gas (GS2): 50 psi;

[0059] Monitoring mode: MRM.

[0060] The MRM ion acquisition parameters are:

[0061]

[0062] * is the quantitative ion.

[0063] Example 3:

[0064] (1) Preparation of test solution: Accurately weigh 0.2002 g and 1.40141 g of BOC butyric acid sample into 10 mL and 100 mL volumetric flasks, respectively. Dissolve with methanol and dilute to the mark. Shake well to prepare BOC butyric acid sample stock solutions with concentrations of 20 g / L and 14 g / L. Draw an appropriate amount of the sample stock solution and dilute with aerated water to obtain the test solution.

[0065] (2) Preparation of standard working solution: Accurately weigh 0.01003 g of BOC butyric acid standard, place it in a 100 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well, and prepare a BOC butyric acid standard stock solution with a concentration of 100.2 mg / L. Pipette an appropriate amount of BOC butyric acid standard stock solution and dilute it with aerated water to prepare standard working solutions with BOC butyric acid concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, and 1.00 mg / L, for HPLC-MS / MS determination;

[0066] (3) Determination and calculation: Set the operating conditions of the HPLC-MS / MS instrument. After the instrument is stable, perform HPLC-MS / MS determination on the test solution and the standard solution, and calculate the content of BOC butyric acid by the external standard method.

[0067] The operating conditions of HPLC were as follows: Hilic Plus RRHD column, 50 mm × 2.1 mm, 1.8 μm; injection volume was 0.8 μL; mobile phase A was UP water, and mobile phase B was methanol, with a volume ratio of A to B of 10:90; column temperature was 38°C; and flow rate was 0.2 mL / min.

[0068] The operating conditions of the MSD are:

[0069] Ion source: Turbo V ion source (ESI mode);

[0070] Ion source parameters: curtain gas (CUR), 45 psi; collision gas (CAD), 12; ionization voltage (IS), -4500 V; temperature (TEM): 500 °C; spray gas (GS1): 50 psi; auxiliary heating gas (GS2): 50 psi;

[0071] Monitoring mode: MRM.

[0072] The MRM ion acquisition parameters are:

[0073]

[0074] * is the quantitative ion.

[0075] Example 4:

[0076] (1) Preparation of test solution: Accurately weigh 0.2002 g and 1.40141 g of BOC butyric acid sample into 10 mL and 100 mL volumetric flasks, respectively. Dissolve with methanol and dilute to the mark. Shake well to prepare BOC butyric acid sample stock solutions with concentrations of 20 g / L and 14 g / L. Draw an appropriate amount of the sample stock solution and dilute with aerated water to obtain the test solution.

[0077] (2) Preparation of standard working solution: Accurately weigh 0.01003 g of BOC butyric acid standard, place it in a 100 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well, and prepare a BOC butyric acid standard stock solution with a concentration of 100.2 mg / L. Pipette an appropriate amount of BOC butyric acid standard stock solution and dilute it with aerated water to prepare standard working solutions with BOC butyric acid concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, and 1.00 mg / L, for HPLC-MS / MS determination;

[0078] (3) Determination and calculation: Set the operating conditions of the HPLC-MS / MS instrument. After the instrument is stable, perform HPLC-MS / MS determination on the test solution and the standard solution, and calculate the content of BOC butyric acid by the external standard method.

[0079] The operating conditions of HPLC were as follows: Hilic Plus RRHD column, 50 mm × 2.1 mm, 1.8 μm; injection volume was 1.2 μL; mobile phase A was UP water, and mobile phase B was methanol, with a volume ratio of A to B of 10:90; column temperature was 42°C; and flow rate was 0.4 mL / min.

[0080] The operating conditions of the MSD are:

[0081] Ion source: Turbo V ion source (ESI mode);

[0082] Ion source parameters: curtain gas (CUR), 45 psi; collision gas (CAD), 12; ionization voltage (IS), -4500 V; temperature (TEM): 500 °C; spray gas (GS1): 50 psi; auxiliary heating gas (GS2): 50 psi;

[0083] Monitoring mode: MRM.

[0084] The MRM ion acquisition parameters are:

[0085]

[0086] * is the quantitative ion.

[0087] Example 5:

[0088] (1) Preparation of test solution: Accurately weigh 0.2002 g and 1.40141 g of BOC butyric acid sample into 10 mL and 100 mL volumetric flasks, respectively. Dissolve with methanol and dilute to the mark. Shake well to prepare BOC butyric acid sample stock solutions with concentrations of 20 g / L and 14 g / L. Draw an appropriate amount of the sample stock solution and dilute with aerated water to obtain the test solution.

[0089] (2) Preparation of standard working solution: Accurately weigh 0.01003 g of BOC butyric acid standard, place it in a 100 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well, and prepare a BOC butyric acid standard stock solution with a concentration of 100.2 mg / L. Pipette an appropriate amount of BOC butyric acid standard stock solution and dilute it with aerated water to prepare standard working solutions with BOC butyric acid concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, and 1.00 mg / L, for HPLC-MS / MS determination;

[0090] (3) Determination and calculation: Set the operating conditions of the HPLC-MS / MS instrument. After the instrument is stable, perform HPLC-MS / MS determination on the test solution and the standard solution, and calculate the content of BOC butyric acid by the external standard method.

[0091] The operating conditions of HPLC were as follows: Hilic Plus RRHD column, 50 mm × 2.1 mm, 1.8 μm; injection volume was 1.5 μL; mobile phase A was UP water, and mobile phase B was methanol, with a volume ratio of A to B of 10:90; column temperature was 45°C; and flow rate was 0.5 mL / min.

[0092] The operating conditions of the MSD are:

[0093] Ion source: Turbo V ion source (ESI mode);

[0094] Ion source parameters: curtain gas (CUR), 45 psi; collision gas (CAD), 12; ionization voltage (IS), -4500 V; temperature (TEM): 500 °C; spray gas (GS1): 50 psi; auxiliary heating gas (GS2): 50 psi;

[0095] Monitoring mode: MRM.

[0096] The MRM ion acquisition parameters are:

[0097]

[0098] * is the quantitative ion.

[0099] Example 6:

[0100] (1) Preparation of test solution: Accurately weigh 0.2002 g and 1.40141 g of BOC butyric acid sample into 10 mL and 100 mL volumetric flasks, respectively. Dissolve with methanol and dilute to the mark. Shake well to prepare BOC butyric acid sample stock solutions with concentrations of 20 g / L and 14 g / L. Draw an appropriate amount of the sample stock solution and dilute with algae culture medium to obtain the test solution.

[0101] (2) Preparation of standard working solution: Accurately weigh 0.01003 g of BOC butyric acid standard, place it in a 100 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well, and prepare a BOC butyric acid standard stock solution with a concentration of 100.2 mg / L. Pipette an appropriate amount of BOC butyric acid standard stock solution and dilute it with algae culture medium to prepare standard working solutions with BOC butyric acid concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, and 1.00 mg / L, for HPLC-MS / MS determination;

[0102] (3) Determination and calculation: Set the operating conditions of the HPLC-MS / MS instrument. After the instrument is stable, perform HPLC-MS / MS determination on the test solution and the standard solution, and calculate the content of BOC butyric acid by the external standard method.

[0103] The operating conditions of HPLC were as follows: Hilic Plus RRHD column, 50 mm × 2.1 mm, 1.8 μm; injection volume was 1.0 μL; mobile phase A was UP water, and mobile phase B was methanol, with a volume ratio of A to B of 10:90; column temperature was 40°C; and flow rate was 0.3 mL / min.

[0104] The operating conditions of the MSD are:

[0105] Ion source: Turbo V ion source (ESI mode);

[0106] Ion source parameters: curtain gas (CUR), 45 psi; collision gas (CAD), 12; ionization voltage (IS), -4500 V; temperature (TEM): 500 °C; spray gas (GS1): 50 psi; auxiliary heating gas (GS2): 50 psi;

[0107] Monitoring mode: MRM.

[0108] The MRM ion acquisition parameters are:

[0109]

[0110] * is the quantitative ion.

[0111] Example 7:

[0112] (1) Preparation of test solution: Accurately weigh 0.2002 g and 1.40141 g of BOC butyric acid sample into 10 mL and 100 mL volumetric flasks, respectively. Dissolve with methanol and dilute to the mark. Shake well to prepare BOC butyric acid sample stock solutions with concentrations of 20 g / L and 14 g / L. Draw an appropriate amount of the sample stock solution and dilute with algae culture medium to obtain the test solution.

[0113] (2) Preparation of standard working solution: Accurately weigh 0.01003 g of BOC butyric acid standard, place it in a 100 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well, and prepare a BOC butyric acid standard stock solution with a concentration of 100.2 mg / L. Pipette an appropriate amount of BOC butyric acid standard stock solution and dilute it with algae culture medium to prepare standard working solutions with BOC butyric acid concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, and 1.00 mg / L, for HPLC-MS / MS determination;

[0114] (3) Determination and calculation: Set the operating conditions of the HPLC-MS / MS instrument. After the instrument is stable, perform HPLC-MS / MS determination on the test solution and the standard solution, and calculate the content of BOC butyric acid by the external standard method.

[0115] The operating conditions of HPLC were as follows: Hilic Plus RRHD column, 50 mm × 2.1 mm, 1.8 μm; injection volume, 0.5 μL; mobile phase A, UP water, and B, methanol, with a volume ratio of A to B of 10:90; column temperature, 35°C; and flow rate, 0.1 mL / min.

[0116] The operating conditions of the MSD are:

[0117] Ion source: Turbo V ion source (ESI mode);

[0118] Ion source parameters: curtain gas (CUR), 45 psi; collision gas (CAD), 12; ionization voltage (IS), -4500 V; temperature (TEM): 500 °C; spray gas (GS1): 50 psi; auxiliary heating gas (GS2): 50 psi;

[0119] Monitoring mode: MRM.

[0120] The MRM ion acquisition parameters are:

[0121]

[0122] * is the quantitative ion.

[0123] Example 8:

[0124] (1) Preparation of test solution: Accurately weigh 0.2002 g and 1.40141 g of BOC butyric acid sample into 10 mL and 100 mL volumetric flasks, respectively. Dissolve with methanol and dilute to the mark. Shake well to prepare BOC butyric acid sample stock solutions with concentrations of 20 g / L and 14 g / L. Draw an appropriate amount of the sample stock solution and dilute with algae culture medium to obtain the test solution.

[0125] (2) Preparation of standard working solution: Accurately weigh 0.01003 g of BOC butyric acid standard, place it in a 100 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well, and prepare a BOC butyric acid standard stock solution with a concentration of 100.2 mg / L. Pipette an appropriate amount of BOC butyric acid standard stock solution and dilute it with algae culture medium to prepare standard working solutions with BOC butyric acid concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, and 1.00 mg / L, for HPLC-MS / MS determination;

[0126] (3) Determination and calculation: Set the operating conditions of the HPLC-MS / MS instrument. After the instrument is stable, perform HPLC-MS / MS determination on the test solution and the standard solution, and calculate the content of BOC butyric acid by the external standard method.

[0127] The operating conditions of HPLC were as follows: Hilic Plus RRHD column, 50 mm × 2.1 mm, 1.8 μm; injection volume was 0.8 μL; mobile phase A was UP water, and mobile phase B was methanol, with a volume ratio of A to B of 10:90; column temperature was 38°C; and flow rate was 0.2 mL / min.

[0128] The operating conditions of the MSD are:

[0129] Ion source: Turbo V ion source (ESI mode);

[0130] Ion source parameters: curtain gas (CUR), 45 psi; collision gas (CAD), 12; ionization voltage (IS), -4500 V; temperature (TEM): 500 °C; spray gas (GS1): 50 psi; auxiliary heating gas (GS2): 50 psi;

[0131] Monitoring mode: MRM.

[0132] The MRM ion acquisition parameters are:

[0133]

[0134] * is the quantitative ion.

[0135] Example 9:

[0136] (1) Preparation of test solution: Accurately weigh 0.2002 g and 1.40141 g of BOC butyric acid sample into 10 mL and 100 mL volumetric flasks, respectively. Dissolve with methanol and dilute to the mark. Shake well to prepare BOC butyric acid sample stock solutions with concentrations of 20 g / L and 14 g / L. Draw an appropriate amount of the sample stock solution and dilute with algae culture medium to obtain the test solution.

[0137] (2) Preparation of standard working solution: Accurately weigh 0.01003 g of BOC butyric acid standard, place it in a 100 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well, and prepare a BOC butyric acid standard stock solution with a concentration of 100.2 mg / L. Pipette an appropriate amount of BOC butyric acid standard stock solution and dilute it with algae culture medium to prepare standard working solutions with BOC butyric acid concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, and 1.00 mg / L, for HPLC-MS / MS determination;

[0138] (3) Determination and calculation: Set the operating conditions of the HPLC-MS / MS instrument. After the instrument is stable, perform HPLC-MS / MS determination on the test solution and the standard solution, and calculate the content of BOC butyric acid by the external standard method.

[0139] The operating conditions of HPLC were as follows: Hilic Plus RRHD column, 50 mm × 2.1 mm, 1.8 μm; injection volume was 1.2 μL; mobile phase A was UP water, and mobile phase B was methanol, with a volume ratio of A to B of 10:90; column temperature was 42°C; and flow rate was 0.4 mL / min.

[0140] The operating conditions of the MSD are:

[0141] Ion source: Turbo V ion source (ESI mode);

[0142] Ion source parameters: curtain gas (CUR), 45 psi; collision gas (CAD), 12; ionization voltage (IS), -4500 V; temperature (TEM): 500 °C; spray gas (GS1): 50 psi; auxiliary heating gas (GS2): 50 psi;

[0143] Monitoring mode: MRM.

[0144] The MRM ion acquisition parameters are:

[0145]

[0146] * is the quantitative ion.

[0147] Example 10:

[0148] (1) Preparation of test solution: Accurately weigh 0.2002 g and 1.40141 g of BOC butyric acid sample into 10 mL and 100 mL volumetric flasks, respectively. Dissolve with methanol and dilute to the mark. Shake well to prepare BOC butyric acid sample stock solutions with concentrations of 20 g / L and 14 g / L. Draw an appropriate amount of the sample stock solution and dilute with algae culture medium to obtain the test solution.

[0149] (2) Preparation of standard working solution: Accurately weigh 0.01003 g of BOC butyric acid standard, place it in a 100 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well, and prepare a BOC butyric acid standard stock solution with a concentration of 100.2 mg / L. Pipette an appropriate amount of BOC butyric acid standard stock solution and dilute it with algae culture medium to prepare standard working solutions with BOC butyric acid concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, and 1.00 mg / L, for HPLC-MS / MS determination;

[0150] (3) Determination and calculation: Set the operating conditions of the HPLC-MS / MS instrument. After the instrument is stable, perform HPLC-MS / MS determination on the test solution and the standard solution, and calculate the content of BOC butyric acid by the external standard method.

[0151] The operating conditions of HPLC were as follows: Hilic Plus RRHD column, 50 mm × 2.1 mm, 1.8 μm; injection volume was 1.5 μL; mobile phase A was UP water, and mobile phase B was methanol, with a volume ratio of A to B of 10:90; column temperature was 45°C; and flow rate was 0.5 mL / min.

[0152] The operating conditions of the MSD are:

[0153] Ion source: Turbo V ion source (ESI mode);

[0154] Ion source parameters: curtain gas (CUR), 45 psi; collision gas (CAD), 12; ionization voltage (IS), -4500 V; temperature (TEM): 500 °C; spray gas (GS1): 50 psi; auxiliary heating gas (GS2): 50 psi;

[0155] Monitoring mode: MRM.

[0156] The MRM ion acquisition parameters are:

[0157]

[0158] * is the quantitative ion.

[0159] In order to further confirm the feasibility of the present invention, the inventors conducted the following methodological investigation and experimental research.

[0160] 1. Linearity test

[0161] The standard working solutions of BOC butyric acid concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, and 1.00 mg / L in Examples 1 and 6 were accurately aspirated and assayed according to the method of the present invention. The results are shown in Table 1. A calibration curve was drawn with the concentration of BOC butyric acid as the abscissa and the peak area of ​​BOC butyric acid as the ordinate. When the BOC butyric acid concentration was in the range of 0.100 mg / L to 1.00 mg / L, a linear relationship was observed between y and x. Linear regression of the curve yielded the equation for BOC butyric acid in aerated water: y = 4.71 × 10 7 x+4.57×10 5 The linear correlation coefficient of the curve is 0.9996, and the linear fitting curve is shown in Figure 1 The equation of BOC butyrate in algae culture medium is y = 4.87 × 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 Figure 2 .

[0162] Table 1: Linearity test results

[0163]

[0164] 2. Specificity test

[0165] The detection method of the present invention was used to measure a blank sample of aerated water, a test solution of aerated water, a blank sample of algae culture medium, and a test solution of algae culture medium, and the chromatograms of the blank samples and the test solution were compared. The retention time of BOC butyric acid was 0.4 minutes, and the blank sample had no interfering peak at this time point. The qualitative and quantitative ion pairs were used to achieve qualitative and quantitative determination of BOC butyric acid without interference from other impurities. The results demonstrated that the method has good specificity for BOC butyric acid.

[0166] 3. Repeatability test

[0167] The standard working solution with a concentration of 0.400 mg / L in Example 1 and Example 6 was injected 6 times continuously to determine the precision of the analytical method of the present invention. The RSD of aerated water was R.T. 0.60%, RSD Area The RSD of algae culture medium is 4.05%. The precision test data are shown in Table 2. R.T. 0.43%, RSD Area The precision test data is shown in Table 3. The results show that the analytical method of the present invention meets the analytical precision requirements for the same sample.

[0168] Table 2: Precision test results - aerated water

[0169]

[0170]

[0171] Table 3: Precision test results - culture medium

[0172]

[0173] 4. Detection limit and quantification limit

[0174] Using the method of the present invention, standard working solutions of BOC butyric acid were prepared at a concentration of 0.504 mg / L in aerated water and algae culture medium, respectively. Analysis of the resulting spectra revealed that, at this concentration, the average signal-to-noise ratio (S / N) for the target peak in aerated water was 2002.3, and the average signal-to-noise ratio (S / N) for the target peak in algae culture medium was 1699.8. Based on the formulas LOD = 3 × assay concentration / (S / N) and LOQ = 10 × assay concentration / (S / N), the LOD and LOQ for BOC butyric acid in aerated water using this method were 0.761 μg / L and 2.54 μg / L, respectively. For BOC butyric acid in algae culture medium, the LOD and LOQ were 0.902 μg / L and 3.01 μg / L, respectively. Detailed results are shown in Tables 4 and 5.

[0175] Table 4: Limit of Detection and Limit of Quantitation Test Results - Aerated Water

[0176]

[0177] Table 5: Limit of Detection and Limit of Quantitation Test Results - Culture Medium

[0178]

[0179]

[0180] 5. Recovery rate test

[0181] 5.1 Recovery rate test in aerated water

[0182] Draw 1.00mL embodiment 1 concentration is 14g / L BOC butyric acid sample storing solution and is placed in 100mL volumetric flask, add aeration water and be settled to scale, mix, obtain the aeration water high concentration recovery test sample that concentration is 140mg / L. Add 0.36mL embodiment 1 concentration is 14g / L BOC butyric acid sample storing solution, be settled to scale with aeration water, mix, obtain the low concentration recovery test sample that concentration is 25.2mg / L. Each concentration sample is made 5 parts in parallel, and the high concentration recovery sample is diluted 200 times with aeration water, and the low concentration recovery sample is diluted 50 times with aeration water. After crossing 0.45 μm filter membrane, test is carried out by instrument operating condition of the present invention. According to formula (1), the recovery is calculated, and the recovery relative standard deviation (RSD) is calculated by formula (2) and formula (3).

[0183] Take 1.00 mL of the 20 g / L BOC butyric acid sample stock solution in Example 1 in a 100 mL volumetric flask, add aerated water to the mark, and mix well to obtain a 200 mg / L aerated water high-concentration recovery test sample. Each concentration sample is prepared in parallel with 5 copies. The high-concentration recovery sample is diluted 250 times with aerated water and tested according to the instrument operating conditions of the present invention. The recovery is calculated according to formula (1), and the relative standard deviation (RSD) of the recovery is calculated according to formula (2) and formula (3).

[0184] The analysis results are shown in Table 6. The average recovery rates of three different concentrations of BOC butyric acid in aerated water were 94.9%, 100.3% and 99.4%, respectively. The relative standard deviations of the recoveries were 3.64%, 1.70% and 1.01%, respectively.

[0185]

[0186] Where:

[0187] R——recovery rate, %;

[0188] Cd——measured concentration of target substance, mg / L;

[0189] Ca——theoretical added concentration of target substance, mg / L.

[0190]

[0191] Where:

[0192] S——standard deviation;

[0193] Xi——recovery rate obtained from the i-th measurement, %;

[0194] X——average value of recovery rate, %;

[0195] N——The number of recovery rates involved in the calculation.

[0196]

[0197] Where:

[0198] RSD——Relative standard deviation, %.

[0199] Table 6: Recovery test results in aerated water

[0200]

[0201] 5.2 Recovery rate test in algae culture medium

[0202] 0.79 mL of the 14 g / L BOC butyric acid sample stock solution from Example 1 was placed in a 100 mL volumetric flask, and the volume was adjusted to the mark with algae culture medium. Mixing was done to obtain a high-concentration recovery rate test sample with an algae culture medium concentration of 110.6 mg / L. 0.09 mL of the 14 g / L BOC butyric acid sample stock solution from Example 1 was added to a 500 mL volumetric flask, and the volume was adjusted to the mark with algae culture medium. Mixing was done to obtain a low-concentration recovery rate test sample with a concentration of 2.52 mg / L. Five replicates were prepared for each concentration. The high-concentration recovery rate sample was diluted 200-fold with algae culture medium, and the low-concentration recovery rate sample was diluted 5-fold with algae culture medium. After filtering through a 0.45 μm filter membrane, the sample was tested according to the instrument operating conditions of the present invention. The recovery rate was calculated according to formula (1), and the relative standard deviation (RSD) of the recovery rate was calculated according to formulas (2) and (3).

[0203] 1.00 mL of the 20 g / L BOC butyric acid sample stock solution from Example 1 was placed in a 100 mL volumetric flask. Algae culture medium was added to the volume, and the mixture was mixed thoroughly to obtain a 200 mg / L algae culture medium high-concentration recovery test sample. Five replicates were prepared for each concentration. The high-concentration recovery sample was diluted 250-fold with algae culture medium and tested according to the instrument operating conditions of the present invention. The recovery was calculated according to formula (1), and the relative standard deviation (RSD) of the recovery was calculated according to formulas (2) and (3).

[0204] The analysis results are shown in Table 7. The average recovery rates of three different concentrations of BOC butyric acid added to algae culture medium were 97.4%, 97.9% and 93.8%, respectively, and the relative standard deviations of the recovery rates were 3.49%, 4.95% and 4.12%, respectively.

[0205] Table 7: Recovery rate in algae culture medium

[0206]

[0207] 6. Stability test of test substance

[0208] 1.02 g BOC butyric acid was weighed into a 250 mL conical flask, algae culture medium was added to the volume, the volume was adjusted to the mark, the volume was shaken in a constant temperature incubation shaker at 25 ° C for 24 h, and the insoluble matter was removed by filtering through a 0.45 μm filter membrane to obtain a BOC butyric acid saturated algae culture medium stock solution for use. 1.01 g BOC butyric acid was weighed into a 500 mL conical flask, aerated water was added to the volume, the volume was adjusted to the mark, the volume was shaken in a constant temperature incubation shaker at 25 ° C for 24 h, the insoluble matter was removed by filtering through a 0.45 μm filter membrane to obtain a BOC butyric acid saturated aerated water stock solution for use. The saturated aerated water and algae culture medium stock solutions were respectively diluted 2000 times with methanol, filtered through a 0.45 μm filter membrane, and the test substance concentration in the solution was determined according to the instrument operating conditions of the present invention. Each saturated solution was measured in parallel 3 times.

[0209] The average value of the three measurement results was taken as the saturated stock solution concentration. After measurement, the concentration of BOC butyric acid saturated aerated water stock solution was 890 mg / L, and the concentration of BOC butyric acid saturated algae culture medium stock solution was 288 mg / L.

[0210] 6.1 Daphnia Acute Activity Inhibition Test Stability

[0211] 7.00 mL of saturated aerated water stock solution was added to 100.0 mL of aerated water and mixed thoroughly to obtain a stability sample solution for the Daphnia acute activity inhibition test. This solution was then placed under Daphnia test conditions, with samples taken at intervals. The stability test solution was diluted 100-fold and filtered through a 0.45 μm filter membrane, and concentration was measured according to the instrument operating conditions of the present invention. The stability of the target substance in the sample solution in aerated water was calculated according to formula (4), and the results are shown in Table 8. The results show that the test substance maintained at more than 80% of its initial concentration within 48 hours under the static method conditions of the Daphnia acute activity inhibition test.

[0212] Stability, % = (C nh / C 0h )×100%...........(4)

[0213] Where, C0h: is the measured concentration of the target substance at 0 hours;

[0214] Cnh: is the measured concentration of the target substance at n hours.

[0215] Table 8: Daphnia acute activity inhibition test stability results

[0216]

[0217] 6.2 Stability of fish acute toxicity test

[0218] 200 mL of saturated aerated water stock solution was added to 2800 mL of aerated water and mixed thoroughly to obtain a stability sample solution for the fish acute toxicity test. This solution was then placed under fish test conditions, with samples taken at intervals. The stability test solution was diluted 100-fold and filtered through a 0.45 μm filter membrane, and concentration was measured according to the instrument operating conditions of the present invention. The stability of the target substance in the sample solution in aerated water was calculated according to formula (4), and the results are shown in Table 9. The results show that the test substance maintained at over 80% of its initial concentration within 96 hours under the static method environment of the fish acute toxicity test.

[0219] Table 9: Stability results of fish acute toxicity test

[0220]

[0221]

[0222] 6.3 Stability of algae production inhibition test

[0223] 20.00 mL of saturated algae culture medium stock solution was added to 100.0 mL of aerated water and mixed thoroughly to obtain a stability sample solution for the algae growth inhibition test. This solution was then placed under algae test conditions, with samples taken at intervals. The stability test solution was diluted 100-fold and filtered through a 0.45 μm filter membrane, and concentration was measured according to the instrument operating conditions of the present invention. The stability of the target compound in the algae culture medium in this sample solution was calculated according to formula (4). The results are shown in Table 10. The results show that the test compound maintained at over 80% of its initial concentration within 72 hours under the algae growth inhibition test conditions.

[0224] Table 10: Algae production inhibition test stability parallel design

[0225]

[0226] Stability test results: The analytical method of the present invention can meet the concentration determination requirements of the Daphnia acute activity inhibition test, the algae growth inhibition test and the fish acute toxicity test respectively.

[0227] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for detecting BOC butyric acid in water and algae culture medium using HPLC-MS / MS, characterized in that: The method comprises the following steps: (1) Preparation of test solution: Accurately weigh 0.2002 g and 1.40141 g of BOC butyric acid sample into 10 mL and 100 mL volumetric flasks, respectively. Dissolve with methanol and dilute to the mark. Shake well to prepare BOC butyric acid sample stock solutions with concentrations of 20 g / L and 14 g / L. Draw an appropriate amount of the sample stock solution and dilute with aerated water or algae culture medium to obtain the test solution. (2) Preparation of standard working solution: Accurately weigh 0.01003 g of BOC butyric acid standard, place it in a 100 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well, and prepare a BOC butyric acid standard stock solution with a concentration of 100.2 mg / L. Pipette an appropriate amount of BOC butyric acid standard stock solution and dilute it with aerated water or algae culture medium to prepare standard working solutions with BOC butyric acid concentrations of 0.100 mg / L, 0.200 mg / L, 0.400 mg / L, 0.600 mg / L, 0.800 mg / L, and 1.00 mg / L, for HPLC-MS / MS determination; (3) Determination and calculation: Set the HPLC-MS / MS instrument operating conditions. After the instrument stabilizes, perform HPLC-MS / MS on the test solution and the standard solution, and calculate the content of BOC butyric acid by the external standard method. The operating conditions of the HPLC are: Column: HilicPlusRRHD, 50 mm × 2.1 mm, 1.8 μm; Injection volume: 0.5-1.5 μL; Mobile phase: A is UP water, B is methanol, and the volume ratio of mobile phase A to B is 10:90; Column temperature: 35-45°C; Flow rate: 0.1-0.5 mL / min; The operating conditions of the MS / MS are: Ion source: TurboV ion source, ESI mode; Ion source parameters: curtain gas 45 psi; collision gas 12; ionization voltage -4500 V; temperature 500 °C; spray gas 50 psi; auxiliary heating gas 50 psi; Monitoring mode: MRM; The MRM ion acquisition parameters are: compound name BOC butyric acid, parent ion 332.1, daughter ion quantitative ion 170.9, DP is -48V, EC is -17V, and polarity is negative; parent ion 332.1, daughter ion 257.8, DP is -48V, EC is -17V, and polarity is negative.

2. The method for detecting BOC butyric acid in water and algae culture medium using HPLC-MS / MS according to claim 1, characterized in that: The injection volume is 0.8-1.2 μL, and the flow rate is 0.2-0.4 mL / min.

3. The method for detecting BOC butyric acid in water and algae culture medium using HPLC-MS / MS according to claim 2, characterized in that: The injection volume was 1.0 μL.

4. The method for detecting BOC butyric acid in water and algae culture medium using HPLC-MS / MS according to claim 2, characterized in that: The flow rate was 0.3 mL / min.

5. The method for detecting BOC butyric acid in water and algae culture medium using HPLC-MS / MS according to claim 1, characterized in that: The column temperature is 38-42°C.

6. The method for detecting BOC butyric acid in water and algae culture medium using HPLC-MS / MS according to claim 2, characterized in that: The column temperature was 40°C.

7. The method for detecting BOC butyric acid in water and algae culture medium using HPLC-MS / MS according to any one of claims 1 to 6, characterized in that: The LOD and LOQ of BOC butyrate in aerated water were 0.761 μg / L and 2.54 μg / L, respectively. The LOD and LOQ of BOC butyrate in algae culture medium were 0.902 μg / L and 3.01 μg / L, respectively.

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