Method for quantifying ethanol in water-containing preparations

Abstract

This invention provides a method for quantifying ethanol in water-containing formulations that enables highly reproducible gas chromatography quantitative analysis by removing water in a simple manner without requiring special testing equipment. [Solution] A method for quantifying ethanol in a water-containing formulation, characterized by comprising: 1) a step of uniformly dissolving a water-containing formulation and an organic solvent; 2) a step of adding an inorganic salt or an organic salt to the liquid prepared in step 1 and separating it into an aqueous phase and an organic phase; and 3) a step of separating the organic phase in step 2 and quantitatively analyzing the ethanol contained in the organic phase by gas chromatography analysis.

Description

Technical Field

[0001] The present invention relates to a method for quantifying ethanol in a preparation containing water.

Background Art

[0002] There are products containing ethanol in pharmaceuticals, quasi-drugs, cosmetics, foods, etc., which are manufactured and sold based on each law. Especially in the field of cosmetics, it is used for various purposes such as cleaning, sterilization, astringency, refreshing, solubilization, etc. In the market, there have been cases where product recalls have occurred because the ethanol concentrations displayed on these products are different. From the perspective of consumer protection, accurate quantitative analysis of ethanol for quality control is required.

[0003] However, when a sample solution containing water is analyzed by gas chromatography (GC), events such as the occurrence of ghost peaks, inability to obtain linearity of the calibration curve, and inability to obtain reproducibility occur. The causes include the large vaporization volume of water, the large heat of vaporization causing the inlet temperature to drop during injection of the sample solution, and the inability of the target component to be retained in many siloxane-based liquid phases.

[0004] Conventionally, the headspace injection method has been used for quantitative analysis of ethanol in a preparation containing water (Non-Patent Document 1). The headspace injection method is a method in which a sample solution placed in a sealed container is heated to reach a gas-liquid equilibrium state, and the gas phase part is injected into a GC device. While the preparation of the sample solution is simple, it is difficult to obtain reproducibility because the environment at the time of enclosing the sample solution affects the measured value. Also, multiple injections from the same sample container cannot be performed. Furthermore, since heating of the sample container is required, there is a problem that it takes time for measurement when there are multiple sample solutions.

[0005] Another method that has been used is solid-phase microextraction (SPME) (Patent Document 1). This method involves extracting volatile components into an SPME fiber and injecting it into a GC instrument. While the SPME method offers the advantage of simple sample solution preparation, it has the drawback of requiring the setting of multiple conditions, such as the extraction temperature and time of the target component, the sample volume, and the insertion position of the fiber. Furthermore, both headspace injection and SPME methods require special test equipment, making them less appealing as alternatives.

[0006] In addition, in GC quantitative analysis, there is a method for quantitatively analyzing ethanol in water-containing formulations by increasing the amount of silica wool in the glass insert and raising the wool filling position above the standard position. However, increasing the amount of silica wool presents a challenge in that components in the sample are adsorbed onto the wool, reducing reproducibility. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] PG Whitehead and SI Sandler, Fluid Phase Equilibria,1999,157(1),111-120 [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2016-3923 [Overview of the project] [Problems that the invention aims to solve]

[0009] Therefore, the problem that this invention aims to solve, in view of the problems of the prior art described above, is to provide a method for quantifying ethanol in water-containing formulations that does not require special testing equipment and allows for analysis with high accuracy and reproducibility, without the influence of analytical procedures or the analytical environment on the measured values. Furthermore, it aims to provide a method that allows for the simple preparation of sample solutions and analysis in a short measurement time. [Means for solving the problem]

[0010] The inventors diligently conducted research to solve this problem and succeeded in separating ethanol from water by adding an organic solvent and an inorganic or organic salt to a water-containing formulation and shaking, thereby extracting ethanol into the organic phase. They discovered that ethanol was quantitatively extracted into the separated organic phase and that ethanol could be quantitatively analyzed by GC analysis, thus completing the present invention.

[0011] In other words, the present invention is a method for quantifying ethanol in a water-containing formulation, comprising: (1) a step of uniformly dissolving a water-containing formulation and an organic solvent; (2) a step of adding an inorganic salt or an organic salt to the solution prepared in step 1 and separating it into an aqueous phase and an organic phase; and (3) a step of separating the organic phase in step 2 and quantitatively analyzing the ethanol contained in the organic phase by gas chromatography analysis.

[0012] (2) The present invention provides a method for determining ethanol, characterized in that the inorganic salt or organic salt has a solubility in water at 20°C that is higher than 20% (w / v), as described in (1).

[0013] (3) The method for determining ethanol according to the present invention is the method according to (1), characterized in that the amount of inorganic salt or organic salt added is 50% to 200% (w / v) of the saturation amount relative to water at 20°C. [Effects of the Invention]

[0014] This invention addresses the problems that can occur in GC quantitative analysis when the sample solution contains water, such as the generation of ghost peaks and the inability to obtain linearity of the calibration curve. By removing water during the preparation of the sample solution, highly reproducible and accurate quantitative analysis becomes possible. Furthermore, it has the advantage of not requiring special testing equipment such as SPME fibers. In addition, since ethanol and water can be separated simply by adding an organic solvent and an inorganic or organic salt, the preparation of the sample solution is also very simple. [Brief explanation of the drawing]

[0015] [Figure 1] Ethanol was extracted from a 5% ethanol aqueous solution by adding acetone and potassium carbonate, and the separated organic phase was diluted to obtain the sample solution. The chromatogram obtained by quantitative analysis of ethanol in this sample solution using GC analysis is shown (Example 1). [Figure 2] This figure shows the chromatogram obtained by GC analysis of ethanol in an acetone solution prepared to match the ethanol concentration of the sample solution in Figure 1 (Example 1). [Figure 3] Ethanol was extracted by adding acetone and potassium carbonate to aqueous solutions prepared with ethanol concentrations of 1.0%, 2.5%, 5.0%, 10.0%, 15.0%, and 20.0%, respectively. The separated organic phase was then diluted to the required volume to obtain the sample solution. The calibration curve obtained by quantitative analysis of ethanol in this sample solution using GC analysis is shown (Example 2). [Modes for carrying out the invention]

[0016] The present invention provides a method for quantifying ethanol, which includes the steps of: 1) dissolving a water-containing formulation in an organic solvent; 2) adding an inorganic salt or organic salt to separate it into an aqueous phase and an organic phase; and 3) separating the organic phase containing ethanol and quantitatively analyzing it by GC analysis.

[0017] The organic solvent added in the present invention is not particularly limited as long as it is miscible with water and dissolves uniformly. For example, acetonitrile, methanol, acetone, ethyl methyl ketone, 1,2-dimethoxyethane, acetic acid, dimethylformaldehyde, hexamethylphosphoramide, 1,4-dioxane, pyridine, 1-propanol, 2-propanol, 1-butanol, 2-butanol, ethylene glycol, triethylamine, phenol, tetrahydrofuran, dimethylacetamide, triethanolamine, and dimethyl sulfoxide are preferred, and acetone and acetonitrile are particularly preferred.

[0018] The volume ratio of the preparation containing water to the organic solvent in the present invention is not particularly limited. If necessary, water can be added to the preparation containing water for dilution before use. If the volume of water is too small, it will be difficult to dissolve the sample. If the volume of water is too large, it may reduce the extraction efficiency and lead to a decrease in reproducibility. Therefore, the volume ratio of water to the organic solvent is preferably 2:1 to 1:10, and more preferably 1:1 to 1:4.

[0019] The inorganic salt in the present invention is not particularly limited as long as it can be uniformly dissolved in water, but examples include monovalent inorganic salts such as sodium thiosulfate, sodium tetrahydroborate, sodium fluoride, potassium fluoride, sodium chloride, potassium chloride, ammonium chloride, potassium bromide, sodium iodide, potassium iodide, sodium perchlorate, potassium chlorate, potassium iodate, potassium periodate, potassium bromate, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, sodium nitrite, sodium nitrate, potassium nitrate, ammonium nitrate, sodium sulfite, sodium sulfate, potassium sulfate, potassium hydrogen sulfate, sodium sulfide, ammonium sulfide, sodium carbonate, sodium bicarbonate, potassium carbonate, ammonium carbonate, ammonium bicarbonate, etc., and divalent inorganic salts such as magnesium chloride, magnesium hydrogen phosphate, magnesium nitrate, magnesium sulfate, calcium chloride, etc.

[0020] The organic salt in the present invention is not particularly limited as long as it can be uniformly dissolved in water, but examples include monovalent organic salts such as potassium oxalate, sodium acetate, potassium acetate, ammonium acetate, ammonium formate, etc., and divalent organic salts such as calcium oxalate, magnesium acetate, etc.

[0021] The inorganic salt or organic salt in the present invention is preferably sodium carbonate, potassium carbonate, sodium chloride or potassium chloride, and more preferably potassium carbonate or sodium chloride.

[0022] The solubility of the inorganic or organic salt in water in the present invention is not particularly limited, but it is preferable that the solubility in water at 20°C is higher than 20% (w / v). The amount of inorganic or organic salt added is not particularly limited as long as it is an amount that separates the aqueous phase and the organic phase, but the more inorganic or organic salt is dissolved, the more likely salting out and two-phase separation will occur. Therefore, the amount of inorganic or organic salt added is preferably 50% to 200% (w / v) of the saturation amount in water in the sample solution at 20°C, and more preferably 100% to 200% (w / v) to improve reproducibility.

[0023] When shaking is performed in the step of adding an inorganic salt or organic salt to separate the aqueous phase and the organic phase, the number of shaking cycles is not particularly limited, but if it is too slow, the mixture will not mix well and the extraction rate will decrease, so 100 times / minute or more is preferred, and 200 times / minute or more is particularly preferred to improve reproducibility.

[0024] When shaking is performed in the step of adding an inorganic salt or organic salt to separate the aqueous phase and the organic phase, the shaking time is not particularly limited, but if it is too short, the mixture will not mix well and the extraction rate will decrease, so 5 minutes or more is preferred, and 10 minutes or more is particularly preferred to improve reproducibility.

[0025] When centrifuging is performed in the step of adding an inorganic salt or organic salt to separate the aqueous phase and the organic phase, the centrifugal separation speed is not particularly limited, but if it is too slow, the separation of the two phases will be poor and the extraction rate will decrease, so a speed of 1000 rpm or higher is preferred, and a speed of 2000 rpm or higher is particularly preferred to improve reproducibility.

[0026] When centrifuging is performed in the step of adding an inorganic salt or organic salt to separate the aqueous phase and the organic phase, the centrifugal separation time is not particularly limited, but if it is too short, the separation of the two phases will be poor and the extraction rate will decrease, so 5 minutes or more is preferred, and 10 minutes or more is particularly preferred to improve reproducibility.

[0027] In the step of adding an inorganic salt or organic salt to separate the aqueous phase and the organic phase, the number of extractions is not particularly limited, but if the number of extractions is too many, the operation becomes complicated and reproducibility decreases, so the number of extractions is preferably 1 to 5 times, and more preferably 1 to 3 times.

[0028] In the GC analysis method of the present invention, any column capable of separating ethanol from other components can be used, such as a packed column or a capillary column, but it is preferable to use a capillary column. It is preferable to use a highly polar capillary column.

[0029] The formulations in this invention include pharmaceuticals, quasi-drugs, cosmetics, and foods, and the dosage form is not particularly limited to solid, pressed, oil, liquid, homogeneous gel, paste (balm), mud, lotion, cream, toner, pack, foam (bubble), film, powder, water, pencil, spray (mist), stick, sheet, tablet, etc. [Examples]

[0030] The present invention will be described in more detail below with reference to examples, but these examples are merely illustrative examples for convenience, and the present invention is not limited in any way to these examples.

[0031] Example 1: Quantitative analysis of ethanol in a water-containing formulation (5% ethanol aqueous solution) by adding acetone and potassium carbonate. <Preparation of sample solution> Approximately 2 g of 5% ethanol aqueous solution was accurately weighed into a 50 mL centrifuge tube as a water-containing preparation. 8 mL of water was added, followed by 20 mL of acetone, and the mixture was shaken well. 15 g of potassium carbonate was added and the mixture was shaken well (200 times / min, 10 minutes). The organic phase was then separated by centrifugation (3000 rpm, 10 minutes). 4 g of anhydrous sodium sulfate was added to the separated organic phase and the mixture was shaken. The mixture was then allowed to stand for 15 minutes. After filtration, acetone was added to make exactly 25 mL of solution, which was the sample solution. <Preparation of standard solutions> Precisely weigh approximately 0.1 g of ethanol, add acetone to make the volume exactly 25 mL, and use it as the standard solution.

[0032] <GC Quantitative Analysis> Quantitative analysis of ethanol in the sample solution and the standard solution was performed by GC. The measurement conditions are shown below. Column: TC-WAX capillary column (0.25 mm × 30 m, 0.25 μm) Carrier gas: Helium Detector: Flame ionization detector Linear velocity: 28.6 cm / sec Injection volume: 1 μL Split ratio: 50 Inlet temperature: 250 °C Detector temperature: 250 °C Column temperature: 40 °C (5 minutes) → 5 °C / min (6 minutes) → 30 °C / min (5 minutes)

[0033] The chromatogram of the sample solution is shown in Figure 1, and the chromatogram of the standard solution is shown in Figure 2. From the results of Figure 1 and Figure 2, it can be seen that ethanol can be extracted from a 5% aqueous ethanol solution and can be measured without being affected by water.

[0034] The quantitative analysis results of ethanol are shown in Table 1. Since the measured values are close to the theoretical values and the variation is small, it can be seen that the present invention has high accuracy and reproducibility.

[0035]

Table 1

[0036] Example 2 Quantitative Analysis of Ethanol in 1.0 - 20.0% Aqueous Ethanol Solution by Adding Acetone and Potassium Carbonate <Preparation of Sample Solution> Approximately 2 g of aqueous solutions were precisely weighed into 50 mL centrifuge tubes as preparations containing water, and ethanol concentrations were adjusted to 1.0%, 2.5%, 5.0%, 10.0%, 15.0% and 20.0% respectively. After adding 8 mL of water, 20 mL of acetone was added and shaken well. 15 g of potassium carbonate was added and shaken vigorously (200 times / min, 10 minutes), followed by centrifugation (3000 rpm, 10 minutes) to separate the organic phase. 4 g of anhydrous sodium sulfate was added to the separated organic phase, shaken well, and left standing for 15 minutes. After filtration, acetone was added to make the volume exactly 25 mL, which was used as the sample solution.

[0037] <GC Quantitative Analysis> Quantitative analysis of ethanol in the sample solution by GC was performed to obtain the peak areas at each ethanol concentration. The test conditions were applied by reference to the method of Example 1.

[0038] The results are shown in Figure 3. Since it is a straight line passing as close as possible to the origin and the correlation coefficient is also as close as possible to 1, it can be seen that ethanol can be quantitatively analyzed with high accuracy and reproducibility at least in aqueous solutions with ethanol concentrations of 1.0% - 20.0%.

[0039] Example 3 Ethanol Quantitative Analysis When Changing the Type of Added Salt <Preparation of Sample Solution> Approximately 2 g of 5% ethanol aqueous solution as a preparation containing water was precisely weighed into a 50 mL centrifuge tube. After adding 8 mL of water, 20 mL of acetone was added and shaken well. 15 g of potassium carbonate, 4 g of sodium carbonate, 5 g of potassium chloride, 5 g of sodium chloride, 13 g of calcium chloride, 7 g of sodium acetate, 38 g of potassium acetate, 25 g of dipotassium hydrogen phosphate or 10 g of magnesium chloride was added and shaken vigorously (200 times / min, 10 minutes), followed by centrifugation (3000 rpm, 10 minutes) to separate the organic phase. 4 g of anhydrous sodium sulfate was added to the separated organic phase, shaken well, and left standing for 15 minutes. After filtration, acetone was added to make the volume exactly 25 mL, which was used as the sample solution. <Preparation of Standard Solution> Approximately 0.1 g of ethanol was precisely weighed, and acetone was added to make the volume exactly 25 mL, which was used as the standard solution.

[0040] <GC Quantitative Analysis> Quantitative analysis of ethanol by GC was performed on the sample solution and the standard solution. The test conditions were applied by reference to the method of Example 1.

[0041] The results are shown in Table 2. It can be seen that accurate quantitative analysis can be performed with various salts. Also, although there are differences in the variation of values for each type of salt, it is not large enough to affect the quantitative value, indicating high reproducibility. In particular, high analysis accuracy was achieved when potassium carbonate, sodium carbonate, potassium chloride, or sodium chloride was used.

[0042]

Table 2

[0043] Example 4 Ethanol Quantitative Analysis When Changing the Type of Organic Solvent Added <Preparation of Sample Solution> Approximately 2 g of a 5% ethanol aqueous solution as a preparation containing water was precisely weighed into a 50 mL centrifuge tube. After adding 8 mL of water, 20 mL of acetonitrile, acetone, ethyl methyl ketone, tetrahydrofuran, 1-propanol, or 2-propanol was added and shaken well. 15 g of potassium carbonate or 5 g of sodium chloride was added and shaken (200 times / min, 10 min), followed by centrifugation (3000 rpm, 10 min) to separate the organic phase. 4 g of anhydrous sodium sulfate was added to the separated organic phase, shaken well, and left standing for 15 minutes. After filtration, the same solvent as the organic solvent used was added to make exactly 25 mL, which was used as the sample solution. <Preparation of Standard Solution> Approximately 0.1 g of ethanol was precisely weighed, and acetone was added to make exactly 25 mL, which was used as the standard solution.

[0044] <GC Quantitative Analysis> Quantitative analysis of ethanol by GC was performed on the sample solution and the standard solution. The test conditions were applied by reference to the method of Example 1.

[0045] The results are shown in Table 3. It can be seen that accurate quantitative analysis is possible using various organic solvents. Furthermore, although there are differences in the variability of the values ​​depending on the type of organic solvent, it is not large enough to affect the quantitative values, indicating high reproducibility. In particular, the accuracy of the analysis was high when using acetone and acetonitrile.

[0046] [Table 3]

[0047] Example 5: Quantitative analysis of ethanol in various formulations containing water <Examples of formulations containing water> Quantitative analysis of ethanol was performed on the formulations described in the following prescription examples 1-5.

[0048] (Example prescription 1) Lotion Formulation Content (per portion) 1. Ethanol 5.0 2. Glycerin 2.0 3. Xanthan gum 0.02 4. Citric acid 0.01 5. Sodium citrate 0.1 6.1,3-Butylene glycol 8.0 7. Methyl parahydroxybenzoate 0.1 8. Polyoxyethylene hydrogenated castor oil (40 E.O.) 0.1 9.Fragrance (appropriate amount) 10. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Components 2-6 and 10 and components 1 and 7-9 are uniformly dissolved, the two are mixed, and the mixture is filtered to obtain the formulation.

[0049] (Prescription example 2) Cream Formulation Content (per portion) 1. Ethanol 1.5 2. Olive oil 3.0 3. Stearic acid 2.0 4. Beeswax 2.0 5. Octyldodecyl myristate 3.5 6. Polyoxyethylene cetyl ether (20 E.O.) 3.0 7. Squalane 5.5 8. Glyceryl monostearate 2.5 9.Fragrance 0.1 10. Methyl parahydroxybenzoate 0.2 11.1,3-Butylene glycol 8.5 12. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Heat and dissolve components 1-8 and mix, maintaining the temperature at 70°C to form the oil phase. Heat and dissolve components 10-12 and mix, maintaining the temperature at 75°C to form the aqueous phase. Add the aqueous phase to the oil phase and emulsify, then cool while stirring, add component 9 at 45°C, and cool further to 30°C to form the formulation.

[0050] (Prescription example 3) Emulsion Formulation Content (per portion) 1. Ethanol 1.5 2. Olive oil 5.0 3. Jojoba oil 5.0 4. Squalane 5.0 5. Glyceryl monostearate 2.0 6. Polyoxyethylene cetyl ether (20 E.O.) 3.0 7. Polyoxyethylene sorbitan monooleate (20E.O.) 2.0 8.Fragrance 0.1 9. Propylene glycol 1.0 10. Glycerin 2.0 11. Methyl parahydroxybenzoate 0.2 12. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Heat and dissolve components 1-7, mix, and maintain at 70°C to form the oil phase. Heat and dissolve components 9-12, mix, and maintain at 75°C to form the aqueous phase. Add the aqueous phase to the oil phase and emulsify, then cool while stirring, add component 8 at 45°C, and further cool to 30°C to form the formulation.

[0051] (Prescription example 4) Pack Formulation Content (per portion) 1. Ethanol 5.0 2. Polyvinyl alcohol 12.0 3. 1,3-Butylene glycol 8.0 4. Methyl paraoxybenzoate 0.2 5. Polyoxyethylene hydrogenated castor oil (20 E.O.) 0.5 6. Citric acid 0.1 7. Sodium citrate 0.3 8. Perfume Appropriate amount 9. Make the total amount 100 with purified water [Manufacturing method] Dissolve components (1) to (9) uniformly to obtain a preparation

[0052] (Formulation Example 5) Alcoholic beverage Formulation Content (parts)​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<Comparative Example 1: Preparation of Sample Solution (SPME Method)> Approximately 2g of each formulation from Prescription Examples 1-5 was accurately weighed, dissolved in water, and the total volume was precisely 100mL. 1mL of this solution was accurately placed in a sealed container, 9mL of 10% sodium chloride aqueous solution was added, and the container was covered with aluminum foil to prepare the sample solution. <Comparative Example 1: Preparation of Standard Solution (SPME Method)> Approximately 0.1 g of ethanol was precisely weighed, and water was added to make exactly 100 mL. 1 mL of this solution was accurately placed in a sealed container, 9 mL of 10% sodium chloride aqueous solution was added, and the container was covered with aluminum foil to prepare the standard solution.

[0056] <Comparative Example 1: Ethanol Extraction into SPME Fibers> SPME fibers (Carboxen / PDMS, 80 μm (black)) were inserted into the aluminum foil lids of both the sample solution and the standard solution, and left at 35°C for 10 minutes to extract volatile components from the headspace.

[0057] <Comparative example 1: GC quantitative analysis> Quantitative analysis of ethanol in the headspace extracted from SPME fibers of the sample solution and standard solution was performed by GC. The measurement conditions are as follows. Column: TC-WAX capillary column (0.25mm x 30m, 0.25μm) Carrier gas: Helium Detector: Flame ionization detector Linear speed: 28.6cm / sec Injection amount: Full injection Inlet temperature: 250℃ Detector temperature: 250℃ Column temperature: 40°C (5 min) → 5°C / min (6 min) → 30°C / min (5 min)

[0058] The results are shown in Table 4. It can be seen that ethanol can be accurately quantitatively analyzed in various dosage forms. Furthermore, compared to Comparative Example 1, the present invention yields measured values ​​that are closer to the prescribed amount and show less variability. From these results, it can be seen that the present invention has higher accuracy and reproducibility compared to conventional methods.

[0059] [Table 4] [Industrial applicability]

[0060] The present invention provides a method for quantitatively analyzing ethanol in a water-containing formulation with high accuracy using a simple method that does not require special test equipment.

Claims

1. A method for determining ethanol in a water-containing preparation, comprising: 1) a step of uniformly dissolving the water-containing preparation and an organic solvent; 2) a step of adding an inorganic salt or an organic salt to the solution prepared in step 1 and separating it into an aqueous phase and an organic phase; and 3) a step of separating the organic phase in step 2 and quantitatively analyzing the ethanol contained in the organic phase by gas chromatography analysis.

2. A method for determining ethanol in a water-containing preparation according to claim 1, characterized in that the inorganic salt or organic salt has a solubility in water at 20°C that is higher than 20% (w / v).

3. A method for determining ethanol in a water-containing preparation according to claim 1, characterized in that the amount of inorganic salt or organic salt added is 50% to 200% (w / v) of the saturation amount relative to water at 20°C.

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