A method for detecting halogenated plant alcohol content by high performance liquid chromatography

Abstract

The present invention relates to a method for detecting the content of halogenated plant alcohols using high-performance liquid chromatography, comprising the following steps: 1) derivatizing the halogenated plant alcohols with a derivatization reagent at a molar ratio of 1:1.2-1.5 to obtain a derivatized product having a conjugated group; and 2) preparing the derivatized product obtained in step 1) into a test solution, which is then detected using a high-performance liquid chromatograph. The method has the advantage that the halogenated plant alcohols are derivatized with a derivatization reagent, resulting in conjugated groups that absorb in the ultraviolet region, thereby enabling detection using a conventional high-performance liquid chromatograph.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, in particular to a method for detecting the content of halogenated plant alcohols by high performance liquid chromatography. Background Art

[0002] The halogenated plant alcohols involved in the present invention are key intermediates in the synthesis process of vitamin K1. According to relevant literature, the detection method of the compound has not been reported. Gas chromatography, liquid chromatography-mass spectrometry and other methods are used for detecting similar compounds. However, the compound has a high boiling point and cannot be detected by gas chromatography. Conventional liquid chromatography is generally an ultraviolet detector, and there is no large conjugated system in the structure, no ultraviolet absorption, and conventional liquid chromatography cannot be used for detection. The differential refractive index detector has high requirements for system stability and is greatly affected by the external temperature and environment. It is not suitable for operation and control. The liquid chromatography-mass spectrometer is relatively expensive and is not suitable for detection methods during large-scale production. The development of halogenated plant alcohol detection has always been a difficult problem for analytical developers. In order to meet the CFDA's requirements for drug supervision and control of raw materials from the source, it is urgent to develop a detection method that can accurately quantify halogenated plant alcohols to achieve quality control in the process of vitamin K1 drug development and production. Summary of the Invention

[0003] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for detecting the content of halogenated plant alcohols by high performance liquid chromatography. The halogenated plant alcohols are derivatized with a derivatization reagent so that they have conjugated groups that absorb in the ultraviolet region, and can then be detected using a conventional high performance liquid chromatograph.

[0004] The present invention is achieved through the following technical solutions: On the one hand, a method for detecting the content of halogenated plant alcohols by high performance liquid chromatography is provided, comprising the following steps:

[0005] Step 1) derivatizing the halogenated plant alcohol with a derivatization reagent at a molar ratio of 1:1.2-1.5 to obtain a derivatized product having a conjugated group;

[0006] Step 2) The derivatized product obtained in step 1) is prepared into a test solution, and detected by high performance liquid chromatography.

[0007] Through the above technical solution, it is well known to those skilled in the art that halogenated plant alcohols have a high boiling point, no large conjugated structure, no obvious characteristic absorption in the ultraviolet region (200-400nm), only terminal absorption, and cannot be directly detected using liquid phase HPLC-UV. The present invention derivatizes halogenated plant alcohols with a derivatization reagent in a molar ratio of 1:1.2 to 1.5, so that the derivatives of halogenated plant alcohols have large conjugated groups. Based on the absorption of the conjugated groups in the ultraviolet region, they can be detected using a conventional high performance liquid chromatograph. This method breaks through the technical difficulty that conventional methods cannot detect this compound. The derivatization step is easy to operate, has strong specificity, high accuracy, is easy to master, has high precision, and the content determination results are accurate and reproducible. It can be used for the content determination of halogenated plant alcohols, a key intermediate of vitamin K1.

[0008] Furthermore, in step 1), the derivatization reagent includes any one of pyridine, 4-methylpyridine, and quinoline.

[0009] Through the above technical solution, pyridine has a structure close to a regular hexagon, similar to benzene, and has the same electronic structure. Due to the electron-withdrawing effect of the nitrogen atom in the ring, the electron cloud density at positions 2, 4, and 6 is lower than that at positions 3 and 5. In an acidic medium, electrophilic substitution reactions occur at positions 3 and 5, and nucleophilic reactions such as amination, alkylation, arylation, and acylation occur at positions 2, 4, and 6. The present invention uses pyridine, 4-methylpyridine, and quinoline as derivatization reagents because the derivatization products thereof with halogenated plant alcohols have chromogenic groups, that is, the -halogen auxiliary color group contained in the halogenated plant alcohol is connected to the chromophore contained in the derivatization reagent, which can shift the absorption peak of the chromophore to the long-wave direction and increase the absorption intensity, thereby overcoming the technical difficulty of having no absorption zone in the ultraviolet region.

[0010] The chromophore of the derivatization reagent is the reagent itself: for example, pyridine has a structure close to a regular hexagon, similar to benzene, and has the same electronic structure; preferably, pyridine and 4-methylpyridine, the derivatized product is relatively stable, the chromophore and the auxiliary color group can be better matched and combined, thereby better overcoming the technical difficulties of the present invention, and at the same time, the absorption is stronger when performing liquid phase detection.

[0011] Furthermore, in step 1), the derivatization temperature is 30-60° C., and the derivatization reaction time is 1.0 h-2.0 h.

[0012] Through the above technical solution, when the halogenated plant alcohol and the derivatization reagent undergo derivatization reaction within the above range, the derivatization can be effectively guaranteed to be complete and the sensitivity of detection can be increased.

[0013] Furthermore, in step 2), the chromatographic conditions of the high performance liquid chromatograph are as follows:

[0014] The stationary phase of the chromatographic column: bonded octadecylsilane bonded silica gel is selected as the filler;

[0015] Detection wavelength: 230-265nm;

[0016] Flow rate: 0.8-1.2 mL / min;

[0017] Column temperature: 30-40°C;

[0018] Mobile phase: By volume, an aqueous solution of triethylamine in cetyltrimethylammonium bromide, adjusted to pH 8.0-9.5 with acid, is used as mobile phase A. Acetonitrile:methanol = (50-70): (30-50) (V / V) is used as mobile phase B. Elution is performed using the following gradient program:

[0019] Time (min) A％ B％ 0 25 75 25 10 90 30 25 75 .

[0020] Through the above technical solution, the derivatized product is used for the above high performance liquid chromatography detection, which effectively improves the thermal stability and molecular weight of the halogenated plant alcohol and improves the detection sensitivity.

[0021] Furthermore, in the mobile phase, the concentration of triethylamine is 2.0%-3.0%, the concentration of hexadecyltrimethylammonium bromide is 8-12 mmol / l, and the acid used is one of acetic acid and phosphoric acid.

[0022] Furthermore, the detector used in the high performance liquid chromatograph is a UV detector or a diode array detector.

[0023] Furthermore, the halogenated plant alcohol has the following structure,

[0024]

[0025] Wherein, X is one of Br or Cl.

[0026] The beneficial effects of the present invention are as follows: the present invention derivatizes the compound by using a suitable derivatization reagent so that the compound has a conjugated group that absorbs in the ultraviolet region, and can then be detected using a conventional high-performance liquid chromatography. This method overcomes the technical difficulty that the conventional method cannot detect the compound. The derivatization step is easy to operate, has strong specificity, high accuracy, is easy to master, has high precision, and the content determination results are accurate and reproducible. The method can be used to determine the content of halogenated plant alcohols, a key intermediate of vitamin K1. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a chromatogram showing the suitability of the brominated plant alcohol system in Example 1;

[0028] Figure 2is the liquid phase spectrum of the pyridine-underivatized brominated phytol in Example 1;

[0029] Figure 3 is the liquid phase spectrum of the pyridine-derived brominated plant alcohol in Example 1;

[0030] Figure 4 This is the liquid chromatography detection chart of the underivatized product in Comparative Example 1;

[0031] Figure 5 is the liquid phase spectrum of the chlorophytol not underivatized with 4-picoline in Example 2;

[0032] Figure 6 is the liquid phase spectrum of the chlorophytol after derivatization with 4-picoline in Example 2;

[0033] Figure 7 is the liquid phase spectrum of the quinoline-underivatized brominated plant alcohol in Example 3;

[0034] Figure 8 This is the liquid phase spectrum of the brominated plant alcohol after quinoline derivatization in Example 3. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions or parts are by weight.

[0036] Example 1

[0037] Derivatization: Take 1.64 g of brominated plant alcohol (content 80%), place it in a 50 mL conical flask, add 12 mL of acetonitrile and 0.35 g of pyridine, then derivatize in a digital water bath constant temperature oscillator at 40°C for 1.5 hours, cool to room temperature, filter, and take the filtrate.

[0038] Chromatographic conditions: The stationary phase of the chromatographic column was octadecylsilane bonded silica gel; detection wavelength: 250 nm; flow rate: 1.2 ml / min; column temperature: 30°C; mobile phase: 2.0% triethylamine in a 10 mmol / L hexadecyltrimethylammonium bromide aqueous solution, pH adjusted to 9.5 with acetic acid as mobile phase A, acetonitrile:methanol = 50:50 (v / v) as mobile phase B, eluted using the following gradient program:

[0039] Time (min) A％ B％ 0 25 75 25 10 90 30 25 75

[0040] like Figure 2 and Figure 3 As shown in the derived graph, it can be seen that Figure 4 (There is no UV absorption and quantitative analysis cannot be performed using liquid phase) Compared with the derivatized brominated plant alcohol, the pyridine group has obvious absorption characteristics in the ultraviolet region, giving a more accurate detection result.

[0041] Example 2

[0042] Derivatization: Take 1.44 g of chlorophytol (80% content), place it in a 50 mL conical flask, add 12 mL of acetonitrile and 0.44 g of 4-methylpyridine, then derivatize in a digital water bath constant temperature oscillator at 60°C for 1 hour, cool to room temperature, filter, and take the filtrate.

[0043] Chromatographic conditions: The stationary phase of the chromatographic column was octadecylsilane bonded silica gel; detection wavelength: 265 nm; flow rate: 1.0 ml / min; column temperature: 35°C; mobile phase: 3.0% triethylamine in 8 mmol / L hexadecyltrimethylammonium bromide aqueous solution, pH adjusted to 8.0 with phosphoric acid as mobile phase A, acetonitrile:methanol = 70:30 (v / v) as mobile phase B, eluted using the following gradient program:

[0044] Time (min) A％ B％ 0 25 75 25 10 90 30 25 75

[0045] like Figure 5 、 Figure 6 It can be seen from the derived graph that Figure 4 (There is no UV absorption and quantitative analysis cannot be performed using liquid phase) Compared with the derivatized chlorophytol, the pyridine group has obvious absorption characteristics in the ultraviolet region, giving a more accurate detection result.

[0046] Example 3

[0047] Derivatization: Take 1.64 g of chlorophytol (80% content), place it in a 50 mL conical flask, add 12 mL of acetonitrile and 0.81 g of quinoline, then derivatize in a digital water bath constant temperature oscillator at 40°C for 1.5 hours, cool to room temperature, filter, and take the filtrate.

[0048] Chromatographic conditions: The stationary phase of the chromatographic column was octadecylsilane bonded silica gel; detection wavelength: 259 nm; flow rate: 2.0 ml / min; column temperature: 40°C; mobile phase: 2.5% triethylamine in a 12 mmol / L hexadecyltrimethylammonium bromide aqueous solution, pH adjusted to 9.0 with phosphoric acid (mobile phase A); acetonitrile:methanol = 65:35 (v / v) (mobile phase B), eluted using the following gradient program:

[0049] Time (min) A％ B％ 0 25 75 25 10 90 30 25 75

[0050] like Figure 7 and Figure 8 It can be seen from the derived spectrum that Figure 4 (There is no UV absorption and quantitative analysis cannot be performed using liquid phase) Compared with the derivatized brominated plant alcohol, the quinoline group has obvious absorption characteristics in the ultraviolet region, giving a more accurate detection result.

[0051] Comparative Example 1

[0052] Solution preparation: Take an appropriate amount of brominated plant alcohol, accurately weigh it, dissolve it in acetonitrile and quantitatively dilute it to make a solution containing about 1 mg per 1 ml, which is used as the test solution.

[0053] Chromatographic conditions: The stationary phase of the chromatographic column was octadecylsilane bonded silica gel; detection wavelength: 250 nm; flow rate: 1.2 ml / min; column temperature: 30°C; mobile phase: 2.0% triethylamine in a 10 mmol / L hexadecyltrimethylammonium bromide aqueous solution, pH adjusted to 9.5 with acetic acid as mobile phase A, acetonitrile:methanol = 50:50 (v / v) as mobile phase B, eluted using the following gradient program:

[0054] Time (min) A％ B％ 0 25 75 25 10 90 35 25 75

[0055] Underivatized brominated plant alcohols have no UV absorption and cannot be quantitatively analyzed by liquid phase. Example 1: System suitability experiment

[0056] Test solution: Take 0.1 mL of the filtrate and place it in a 20 mL volumetric flask. Add acetonitrile to dilute to the mark and mix well.

[0057] Reference substance solution: Take an appropriate amount of reference substance, weigh accurately, dissolve it in acetonitrile and quantitatively dilute it to make a solution containing about 1 mg per 1 ml as the reference substance solution.

[0058] Blank solution: Take 12 mL of acetonitrile and the corresponding acylating agent, place them in a 50 mL conical flask and mix well. Accurately measure 0.1 mL and place it in a 20 mL volumetric flask. Add acetonitrile to dilute to the mark and mix well to obtain a blank solution.

[0059] Take 25 μl of each of the blank solution, reference solution, and test solution and inject them into the HPLC instrument. Record the chromatogram. Inject at least one injection of the blank solution and two parallel reference solution injections: one for five injections and the other for two injections. Record the chromatograms to assess system suitability.

[0060] Take the blank solution, blank interference solution, sensitivity solution, and test solution and record the chromatogram. Inject each solution once. Observe whether the blank solution and blank interference solution have interference.

[0061] Table 1 Specificity and sensitivity test results

[0062]

[0063] Effect Example 2: Linearity and Range Test

[0064] Linearity stock solution: This is the reference stock solution under "Specificity and System Suitability". (20 mg / mL)

[0065] Preparation of linear solutions: Accurately measure an appropriate amount of linear stock solution and place it in a corresponding volumetric flask. Add acetonitrile and dilute to the mark to obtain solutions at 40%, 60%, 80%, 100%, and 120%. Shake well to obtain the linear solutions. Measure the linear solutions at each concentration level and inject them into the HPLC. Repeat the injection twice for each level and record the chromatogram. The results are shown in Table 2.

[0066] Table 2 Linear regression

[0067]

[0068] Effect Example 3: Accuracy Test

[0069] The filtrate was taken, and an appropriate amount of the corresponding recovery stock solution was added, diluted to the mark with acetonitrile, and mixed to obtain solutions at various recovery levels. The results are shown in Table 3.

[0070]

[0071] In summary, the recoveries of the nine sample solutions were all above 98%, and the RSD% was 0.7, indicating that the accuracy of this method was good.

[0072] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting the content of halogenated plant alcohols by high performance liquid chromatography, characterized in that: The following steps are involved: Step 1) derivatizing the halogenated plant alcohol with a derivatization reagent at a molar ratio of 1:1.2-1.5 to obtain a derivatized product having a conjugated group; Step 2) preparing a test solution with the derivatized product obtained in step 1) and detecting it using a high performance liquid chromatography; In step 1), the halogenated plant alcohol has the following structure, ， Wherein, X is one of Br or Cl; In step 1), the derivatization reagent includes any one of pyridine, 4-methylpyridine, and quinoline; In step 2), the chromatographic conditions of the high performance liquid chromatograph are as follows: The stationary phase of the chromatographic column: bonded octadecylsilane bonded silica gel is selected as the filler; Detection wavelength: 230-265nm; Mobile phase: By volume, an aqueous solution of triethylamine and cetyltrimethylammonium bromide, adjusted to pH 8.0-9.5 with acid, is used as mobile phase A. Acetonitrile and methanol are used as mobile phase B in a volume ratio of 50-70:30-50. Elution is performed using the following gradient program: 。 2. The method for detecting the content of halogenated plant alcohols by high performance liquid chromatography according to claim 1, wherein In step 1), the derivatization temperature is 30-60° C., and the derivatization reaction time is 1.0 h-2.0 h.

3. The method for detecting the content of halogenated plant alcohols by high performance liquid chromatography according to claim 1, wherein The chromatographic conditions of the high performance liquid chromatograph also include a flow rate of 0.8 to 1.2 mL / min and a column temperature of 30 to 40°C.

4. The method for detecting the content of halogenated plant alcohols by high performance liquid chromatography according to claim 3, wherein In the mobile phase, the concentration of triethylamine is 2.0%-3.0%, the concentration of hexadecyltrimethylammonium bromide is 8-12 mmol / l, and the acid used is acetic acid or phosphoric acid.

5. The method for detecting the content of halogenated plant alcohols by high performance liquid chromatography according to claim 3, wherein: The detector used in the high performance liquid chromatograph is an ultraviolet detector or a diode array detector.

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