A method for detecting diethyl (3-pyridyl)-borane by ultra-high performance liquid chromatography

Abstract

The present invention discloses a method for detecting diethyl (3-pyridyl) borane by ultra-high performance liquid chromatography. The method comprises treating the test product diethyl (3-pyridyl) borane with tetrahydrofuran; detecting the sample by ultra-high performance liquid chromatography, using an Agilent 1260 Infinuty II ultra-high performance liquid chromatograph and a VWD detector, with a detection wavelength of 270 nm, a mobile phase of acetonitrile:ethanol = 60:40 (v / v), a flow rate of 0.5 ml / min, an injection volume of 40 μL, and a column temperature of 40°C. The method has the advantages of a simple method, good separation effect, high sensitivity, and good reproducibility.

Description

Technical Field

[0001] The present invention relates to the field of chemical analysis, and in particular to a method for detecting diethyl (3-pyridyl)-borane by ultra-high performance liquid chromatography. Background Art

[0002] Diethyl(3-pyridyl)borane is used as a pharmaceutical intermediate, particularly in the Suzuki reaction and other C-C couplings. Its most widely known application is as a key intermediate in the synthesis of abiraterone, a targeted drug for the treatment of advanced prostate cancer. Research on diethyl(3-pyridyl)borane has been gaining increasing attention. Diethyl(3-pyridyl)borane is irritating to the eyes and skin, and may also cause respiratory irritation. Contact with it can cause eye and skin irritation. Due to its ecotoxicity, persistence, and potential for bioaccumulation, quality analysis of diethyl(3-pyridyl)borane is crucial for its application.

[0003] At present, my country has not yet established a standard detection method for diethyl (3-pyridyl) borane, and there are few related reports in the literature.

[0004] [1] Zhang Jing and Jin Fengmin, Synthesis of diethyl (3-pyridyl) borane, Chemical Industry and Engineering, 2013, 30(03), 24-27. The paper discloses a method for structural characterization of diethyl (3-pyridyl) borane using infrared spectroscopy and mass spectrometry, but does not perform quantitative analysis and cannot effectively control the quality of diethyl (3-pyridyl) borane.

[0005] [2] Gong Aiqin, Rapid determination of related substances in diethylpyridine borane by HPLC, Guangdong Chemical Industry, 2010, 37(7). This paper discloses an analytical method for determining related substances in diethylpyridine borane by high performance liquid chromatography. The chromatographic conditions of this method are as follows: the chromatographic column is phere Clone 3pSilica (150mmx4.60I / IlTI, 3micron), the mobile phase is n-hexane, the detection wavelength is 268nm, the flow rate is 1.0mL / min, the column temperature is room temperature, and the injection volume is 20uL. Preparation of the test solution: weigh about 20mg of diethylpyridine borane with batch number 20090701, accurately weigh it, place it in a 100mL volumetric flask, dissolve it in n-hexane and dilute it to the mark, and shake it evenly (concentration is 0.2mg / mL). Preparation of the control solution: Accurately measure 1.0 mL of the test solution into a 100 mL volumetric flask, dilute to the mark with n-hexane, and shake well (concentration is 2.0 μg / mL). The detection limit for diethylpyridine borane in this method is 1.0 μg / mL, and the limit of quantification is 4.0 μg / mL. Lower concentrations of diethylpyridine borane will not achieve the desired detection objective. Furthermore, in actual application, this method has been found to be not very sensitive and is not fully adequate for trace or ultratrace detection. Summary of the Invention

[0006] The present invention aims to overcome the problems of high detection limit and low sensitivity in the prior art and provide a method for detecting diethyl (3-pyridyl) -borane by ultra-high performance liquid chromatography. The method comprises the following steps: 1) preparing a standard stock solution: weighing 0.01022 g of a diethyl (3-pyridyl) -borane standard sample into a 10.00 mL volumetric flask, adding tetrahydrofuran to the scale, shaking well, and preparing a diethyl (3-pyridyl) -borane standard stock solution with a concentration of 1021 mg / L; 2) preparing a standard working solution: taking an appropriate amount of the standard stock solution obtained in step 1), diluting it with a 50% tetrahydrofuran aqueous solution to prepare a standard working solution with a concentration of 1.02 μg / L to 20.4 μg / L for ultra-high performance liquid chromatography determination; 3) determining the solution: setting instrument parameters, and after the instrument stabilizes, performing ultra-high performance liquid chromatography determination on the series of standard working solutions in step 2), and recording the chromatogram.

[0007] Preferably, the method comprises the following steps: 1) preparation of a standard stock solution: weighing 0.01022 g of a diethyl (3-pyridyl) -borane standard sample into a 10.00 mL volumetric flask, adding tetrahydrofuran to the mark, shaking, and preparing a diethyl (3-pyridyl) -borane standard stock solution with a concentration of 1021 mg / L; 2) preparation of a standard working solution: taking 1.00 mL of the diethyl (3-pyridyl) -borane standard stock solution into a 100.0 mL volumetric flask, adding tetrahydrofuran to the mark, mixing, and preparing a diethyl (3-pyridyl) -borane standard solution with a concentration of 10.2 mg / L; taking 1.00 mL of the diethyl (3-pyridyl) -borane standard stock solution into a 100.0 mL volumetric flask, adding tetrahydrofuran to the mark, mixing, and preparing a diethyl (3-pyridyl) -borane standard solution with a concentration of 10.2 mg / L; A 10.2 mg / L diethyl (3-pyridyl)-borane standard solution was diluted to the mark with tetrahydrofuran in 100.0 mL and shaken to prepare a 102 μg / L diethyl (3-pyridyl)-borane standard solution. An appropriate amount of the 102 μg / L diethyl (3-pyridyl)-borane standard solution was diluted with 50% tetrahydrofuran aqueous solution to prepare a standard working solution with a concentration of 1.02 μg / L to 20.4 μg / L for ultra-high performance liquid chromatography (UPLC) determination. 3) Determination: Set the instrument parameters. After the instrument stabilizes, perform UPLC determination on the series of standard working solutions in step 2) and record the chromatogram.

[0008] In the step 2), the gradient concentration of the standard working solution is 1.02 μg / L, 2.04 μg / L, 5.10 μg / L, 10.2 μg / L, and 20.4 μg / L.

[0009] In the ultra-high performance liquid chromatography determination, the chromatographic conditions are:

[0010] Ultra-high performance liquid chromatograph (with VWD detector): Agilent 1260 Infinuty II, HFC0166;

[0011] Chromatographic column: Poroshell 120EC-C18, 150 mm × 3.0 mm, 2.7 μm, SN: USCFW19686;

[0012] Injection volume: 40.00 μL;

[0013] Flow rate: 0.5 mL / min;

[0014] Mobile phase: A is acetonitrile, B is ethanol;

[0015] Column temperature: 40°C;

[0016] Detection wavelength: 270nm;

[0017] Retention time: about 3.7 minutes;

[0018] Running time: 10.0min.

[0019] In one embodiment, the mobile phase A:B=60:40 (v / v).

[0020] In one embodiment, the detection limit of the detection method is 0.149 μg / L.

[0021] In one embodiment, the limit of quantification of the detection method is 1.02 μg / L.

[0022] In one embodiment, the average spike recovery rate of the detection method is 93.6%.

[0023] In one embodiment, the average spike recovery rate of the detection method is 96.1%.

[0024] Beneficial effects

[0025] 1. The verification test of the detection method of the present invention shows that the injection concentration of diethyl (3-pyridyl) -borane is in the range of 1.02 μg / L to 20.4 μg / L and shows a good linear relationship. The linear equation is y = 0.2832x + 0.1058 and the linear correlation coefficient R = 0.9999. The average recovery rate of diethyl (3-pyridyl) -borane in aerated water at high and low concentrations is 96.1% and 93.6% respectively. The RSD of the target peak at a concentration of 5.10 μg / L is 0. R.T. 0.03%, RSD Area The linear relationship of the present invention is good, the recovery rate meets the requirements, and the repeatability is high.

[0026] 2. By comparing the spectra of the aerated water blank sample and the sample with added recovery rate, the present invention found that diethyl (3-pyridyl) -borane peaked at about 3.7 min, and the blank sample had no interfering peak at this time point. The results showed that this method has good specificity for diethyl (3-pyridyl) -borane.

[0027] 3. The verification test of the present invention shows that the LOD of diethyl (3-pyridyl) -borane in aerated water is 0.149 μg / L; the LOQ is 1.02 μg / L. This method has high sensitivity and can meet the detection requirements even when the concentration of diethyl (3-pyridyl) -borane is low. This solves the problem that the detection method in the prior art is not sensitive and cannot fully meet the requirements for trace or ultra-trace detection.

[0028] 4. The present invention utilizes ultra-high performance liquid chromatography to detect diethyl (3-pyridyl) -borane by systematically and rationally controlling the chromatographic conditions. Methodological validation tests have shown that this method has strong specificity, good repeatability, and a recovery rate that meets the requirements. The operation is simple and can quickly and accurately achieve qualitative and quantitative detection of diethyl (3-pyridyl) -borane. This method saves detection time and cost and improves detection efficiency.

[0029] 5. In view of the current situation that there are few means for detecting diethyl (3-pyridyl) -borane, the present invention provides a new detection and analysis method for diethyl (3-pyridyl) -borane, which has guiding significance for the quality control in the subsequent application process of diethyl (3-pyridyl) -borane. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the linear fitting curve of diethyl (3-pyridyl) -borane;

[0031] Figure 2 This is a typical spectrum of diethyl(3-pyridyl)-borane (1.02 μg / L);

[0032] Figure 3 This is a typical spectrum of diethyl(3-pyridyl)-borane (2.04 μg / L);

[0033] Figure 4 This is a typical spectrum of diethyl(3-pyridyl)-borane (5.10 μg / L);

[0034] Figure 5 This is a typical spectrum of diethyl(3-pyridyl)-borane (10.2 μg / L);

[0035] Figure 6 This is a typical spectrum of diethyl(3-pyridyl)-borane (20.4 μg / L);

[0036] Figure 7This is a typical spectrum of the recovery of the spiked solution of diethyl(3-pyridyl)-borane in aerated water (low concentration);

[0037] Figure 8 Typical graph of the recovery of diethyl(3-pyridyl)-borane spiked solution in aerated water (high concentration)

[0038] Figure 9 This is a typical spectrum of tetrahydrofuran blank solvent. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not limit the scope of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the raw materials and equipment used in this embodiment are conventional commercially available raw materials and equipment in the art.

[0040] The instruments and reagents involved in the following examples include but are not limited to:

[0041] Instruments and reagents used in the examples:

[0042] 1. Reagents and solvents

[0043] Tetrahydrofuran: HPLC grade, Tianjin Kemeiou Chemical Reagent Co., Ltd., 20211010;

[0044] Acetonitrile: HPLC grade, Shanghai Anpu Laboratory Technology Co., Ltd., W5641440;

[0045] Ethanol: HPLC grade, Shanghai Anpu Laboratory Technology Co., Ltd., V0610432;

[0046] UP water: resistivity, 18.2 MΩ×cm;

[0047] Aerated water: homemade in the laboratory;

[0048] Diethyl(3-pyridyl)-borane: Chongqing Boteng Pharmaceutical Technology Co., Ltd.

[0049] 2. Main instruments and equipment

[0050] Ultra-high performance liquid chromatograph (with VWD detector): Agilent 1260 Infinuty II, HFC0166;

[0051] Electronic balance: XSE205DU, LHY213;

[0052] Chromatographic column: Poroshell 120EC-C18, 150 mm × 3.0 mm, 2.7 μm, SN: USCFW19686.

[0053] Example 1

[0054] HPLC operating conditions:

[0055] Injection volume: 40.00 μL;

[0056] Flow rate: 0.5 mL / min;

[0057] Mobile phase: A acetonitrile; B ethanol; A:B = 60:40 (v / v);

[0058] Column temperature: 40°C;

[0059] Detection wavelength: 270nm;

[0060] Retention time: about 3.7 minutes;

[0061] Running time: 10.0min.

[0062] Preparation of standard stock solution: Weigh 0.01022 g of diethyl (3-pyridyl)-borane standard into a 10.00 mL volumetric flask, add tetrahydrofuran to the mark, and shake well to prepare a diethyl (3-pyridyl)-borane standard stock solution (STD) with a concentration of 1021 mg / L.

[0063] Preparation of standard working solution: take 1.00mL of diethyl (3-pyridyl) - borane standard stock solution (STD) in a 100.0mL volumetric flask, dilute to the mark with tetrahydrofuran, mix well, and prepare a diethyl (3-pyridyl) - borane standard solution (STD-0) with a concentration of 10.2mg / L; take 1.00mL of diethyl (3-pyridyl) - borane standard solution (STD-0) in a 100.0mL volumetric flask, dilute to the mark with tetrahydrofuran, mix well, and prepare a diethyl (3-pyridyl) - borane standard solution (STD-0) with a concentration of 10.2mg / L. Dilute the volume of furan to the mark and shake well to prepare a diethyl(3-pyridyl)-borane standard solution (STD-100) with a concentration of 102 μg / L. Take an appropriate amount of the standard solution (STD-100) and dilute it with 50% tetrahydrofuran aqueous solution according to Table 1 to prepare standard working solutions with concentrations of 1.02 μg / L, 2.04 μg / L, 5.10 μg / L, 10.2 μg / L, and 20.4 μg / L, respectively, for ultra-performance liquid chromatography determination.

[0064] Table 1: Preparation record of diethyl (3-pyridyl)-borane standard solution

[0065]

[0066] Determination: Under the above operating conditions, after the instrument is stable, continuously inject several needles of standard working solution until the relative change in the peak area of ​​two adjacent needles of standard sample is less than 1.5%, and then perform the determination. The test results are shown in Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 and Figure 6 .

[0067] Comparative Example

[0068] For the same sample to be tested, the method of the present invention and the high performance liquid chromatography method mentioned in the background art [2] Gong Aiqin, HPLC method for rapid determination of diethylpyridine borane related substances were used for testing, and the results were as follows:

[0069] The method of the present invention is used to detect a diethyl (3-pyridyl)-borane standard (ω=99.9%) produced by Tianjin Komiou Chemical Reagent Co., Ltd. The results show that diethyl (3-pyridyl)-borane peaks at about 3.7 minutes, and the blank sample has no interfering peak at this time point. The method of the present invention has good separation and strong specificity.

[0070] The following method was developed by combining the detection method in the background technology with the actual situation of our laboratory:

[0071] Chromatographic conditions: The chromatographic column is Poroshell 120EC-C18, 150mm×3.0mm, 2.7μm, SN: USCFW19686, the mobile phase is n-hexane, the detection wavelength is 268nm, the flow rate is 1.0mL / min, the column temperature is room temperature, and the injection volume is 20uL. Preparation of the test solution: Weigh 20mg of diethylpyridine borane sample, place it in a 100mL volumetric flask, add n-hexane to dissolve and dilute to the scale, and shake well (concentration is 0.2mg / mL). Preparation of the control solution: Accurately measure 1.0mL of the test solution, place it in a 100mL volumetric flask, add n-hexane to dilute to the scale, and shake well (concentration is 2.0ug / mL). The results show that the response value of this method is low and the detection effect is poor, so the present invention does not adopt this method.

[0072] Experimental example

[0073] In order to prove the scientificity and rationality of the present invention, the following methodological experimental studies were carried out:

[0074] 1. Linearity test

[0075] The standard working solutions of diethyl (3-pyridyl) -borane with concentrations of 1.02 μg / L, 2.04 μg / L, 5.10 μg / L, 10.2 μg / L, and 20.4 μg / L in Example 1 were accurately drawn and subjected to ultra-high performance liquid chromatography according to the detection method of the present invention. A calibration curve was drawn with the concentration of diethyl (3-pyridyl) -borane as the abscissa and the peak area of ​​diethyl (3-pyridyl) -borane as the ordinate. The curve was subjected to linear regression to obtain an equation of diethyl (3-pyridyl) -borane: y = 0.2832x + 0.1058. The linear correlation coefficient of the curve was R = 0.9999. The results are shown in Tables 2 and Figure 1 .

[0076] Table 2: Analysis results of diethyl (3-pyridyl)-borane working solutions at different concentrations

[0077]

[0078] 2. Specificity test

[0079] The detection method of the present invention was used to measure the aeration water blank sample and the aeration water recovery sample, and the chromatograms of the blank sample and the recovery sample were compared. The retention time of diethyl (3-pyridyl)-borane was approximately 3.7 minutes, and the blank sample had no interfering peak at this time point. The results indicate that the method has good specificity for diethyl (3-pyridyl)-borane.

[0080] 3. Repeatability test

[0081] The diethyl (3-pyridyl) - borane standard working solution (STD-5) with a concentration of 5.10 μg / L in Example 1 was selected for the method repeatability test, and the injection was repeated 6 times to calculate the relative standard deviation of retention time and peak area, RSD R.T. 0.03%, RSD Area The RSDs of retention time and peak area were ≤5%, indicating that the analytical precision of this method for the same sample met the requirements. The results are shown in Table 3.

[0082] Table 3: Precision results of analytical methods

[0083]

[0084] 4. Detection limit and quantification limit

[0085] The spectrum obtained from the 1.02 μg / L diethyl(3-pyridyl)-borane standard working solution (STD-1) in Example 1 was analyzed. At this concentration, the average signal-to-noise ratio (S / N) of the target peak was 20.5, and this concentration was the LOQ of the detection method. According to LOD=3×analytical concentration / (S / N), the LOD of this method was 0.149 μg / L. The results are shown in Table 4.

[0086] Table 4: LOD results of analytical methods

[0087]

[0088] 5. Recovery rate

[0089] Take 5.00 mL of the diethyl (3-pyridyl) -borane standard solution (STD-0) with a concentration of 10.2 mg / L in Example 1 in a 100.0 mL volumetric flask, dilute to the mark with tetrahydrofuran to prepare a 510 μg / L diethyl (3-pyridyl) -borane recovery test stock solution (STD-HSL), take 1.00 mL of the recovery test stock solution (STD-HSL) in a 25.00 mL volumetric flask, dilute to the mark with aerated water, mix well, and prepare a high concentration aeration water recovery test sample of 20.4 μg / L diethyl (3-pyridyl) -borane.

[0090] Take 0.40mL of recovery test stock solution (STD-HSL) in a 100.0mL volumetric flask, dilute to the mark with aerated water, and mix evenly to obtain a low-concentration aerated water low-concentration recovery test sample with a concentration of 2.04μg / L; 5 replicates of each concentration sample were prepared in parallel.

[0091] The samples with high recovery concentration were diluted 2 times with tetrahydrofuran, and the samples with low recovery concentration were directly sampled and measured by liquid chromatography. The concentration of the test substance in the solution was analyzed and the recovery rate was calculated.

[0092] The detection method of the present invention was used for detection, and the recovery rate was calculated according to formula (1). The relative standard deviation (RSD) of the recovery rate was calculated according to formula (2) and formula (3). The average addition recovery rates of two different concentrations of diethyl (3-pyridyl) -borane in aerated water were 96.1% and 93.6%, respectively, and the relative standard deviations of the recovery rates were 0.47% and 2.49%, respectively. The analysis results are shown in Table 5. Figure 7 、 Figure 8 .

[0093]

[0094] Where:

[0095] R——recovery rate, %;

[0096] Cd ——Measured concentration of target substance, μg / L;

[0097] C a ——Theoretical added concentration of target substance, μg / L.

[0098]

[0099] Where:

[0100] S——standard deviation;

[0101] Xi——recovery rate obtained from the i-th measurement, %;

[0102] ——Average value of recovery rate, %;

[0103] N——The number of recovery rates involved in the calculation.

[0104]

[0105] Where:

[0106] RSD——Relative standard deviation, %.

[0107] Table 5: Recovery test results

[0108]

[0109] 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 diethyl (3-pyridyl) -borane by ultra performance liquid chromatography, characterized in that, The method comprises the following steps: 1) Preparation of Standard Stock Solution: Weigh 0.01022 g of diethyl(3-pyridyl)-borane standard into a 10.00 mL volumetric flask, add tetrahydrofuran to the mark, and shake well to prepare a 1021 mg / L diethyl(3-pyridyl)-borane standard stock solution. 2) Preparation of standard working solution: Take an appropriate amount of the standard stock solution obtained in step 1) and dilute it with 50% tetrahydrofuran aqueous solution to prepare a standard working solution with a concentration of 1.02 μg / L to 20.4 μg / L for ultra-high performance liquid chromatography determination; 3) Determination: Set the instrument parameters, wait for the instrument to stabilize, and then perform ultra-high performance liquid chromatography on the series of standard working solutions in step 2) and record the chromatogram; In the step 3), the chromatographic conditions of the ultra-high performance liquid chromatography determination are: The ultra-high performance liquid chromatograph has a VWD detector: Agilent 1260 Infinuty II; Chromatographic column: Poroshell 120EC-C18, 150 mm × 3.0 mm, 2.7 μm, SN: USCFW19686; Injection volume: 40.00 μL; Flow rate: 0.5 mL / min; Mobile phase: A is acetonitrile, B is ethanol; Column temperature: 40°C; Detection wavelength: 270nm; Retention time: 3.7 min; Run time: 10.0 min; In the chromatographic conditions, the volume ratio of the mobile phase is A:B=60:

40.

2. The method for detecting diethyl (3-pyridyl) -borane by ultra-high performance liquid chromatography according to claim 1, characterized in that: In step 2), the concentrations of the standard working solution are 1.02 μg / L, 2.04 μg / L, 5.10 μg / L, 10.2 μg / L, and 20.4 μg / L.

3. The method for detecting diethyl (3-pyridyl) -borane by ultra-high performance liquid chromatography according to any one of claims 1 to 2, characterized in that: The detection limit of the method was 0.149 μg / L.

4. The method for detecting diethyl (3-pyridyl) -borane by ultra-high performance liquid chromatography according to any one of claims 1 to 2, characterized in that: The limit of quantification of the method was 1.02 μg / L.

5. The method for detecting diethyl (3-pyridyl) -borane by ultra-high performance liquid chromatography according to any one of claims 1 to 2, characterized in that: The average recovery rate of aeration water added in the method is 93.6% to 96.1%.

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