CN112129846B

CN112129846B - High performance liquid chromatography for efficiently separating and detecting p-benzoquinone in hydroquinone and application thereof

Info

Publication number: CN112129846B
Application number: CN202010837731.7A
Authority: CN
Inventors: 苏冠荣; 尹贝立
Original assignee: Guangdong Reekon Pharmaceutical Co ltd
Current assignee: Guangdong Reekon Pharmaceutical Co ltd
Priority date: 2020-08-14
Filing date: 2020-08-19
Publication date: 2021-11-02
Anticipated expiration: 2040-08-19
Also published as: CN112129846A; CN112444575B; CN112444575A

Abstract

The invention relates to a high performance liquid chromatography for efficiently separating and detecting p-benzoquinone in hydroquinone, wherein a chromatographic column in the method is selected from any one of a reverse phase column or a positive phase column which can adopt a reverse phase solvent, and a mobile phase consists of a phase A and a phase B, wherein the phase A is selected from any one of water, a methanol aqueous solution and an acetonitrile aqueous solution, the phase B is selected from any one of methanol and acetonitrile, the detection wavelength is 200-400nm, the sample injection amount is 1-100 mu l, and the column temperature is 10-45 ℃. The detection method provided by the invention realizes effective separation and detection of hydroquinone and related substances, remarkably improves the medication safety of hydroquinone, and has the advantages of simplicity and convenience in operation, high separation degree, good specificity, high sensitivity and the like.

Description

High performance liquid chromatography for efficiently separating and detecting p-benzoquinone in hydroquinone and application thereof

Technical Field

The invention belongs to the technical field of drug analysis, and particularly relates to a high performance liquid chromatography for efficiently separating and detecting the content of p-benzoquinone in hydroquinone and application thereof.

Background

Hydroquinone (also called hydroquinone) is a tyrosinase inhibitor, inhibits tyrosinase activity mainly through complexation, regulates the metabolic process of melanocytes, obviously reduces the number of dopa-reaction-positive melanocytes in epidermis, and generates reversible skin fading. Oettel first proposed in 1936 that hydroquinone had the effect of whitening the skin. Since the 60's of the 20 th century, hydroquinone was used in many countries as a whitening agent for cosmetics and as an external preparation for dermatology for treating pigmentation, spot-removing, etc. However, the amount of hydroquinone to be used is strictly controlled.

Hydroquinone is unstable and is susceptible to oxidative degradation reaction caused by environmental factors such as illumination, temperature and the like. In the preparation process of hydroquinone, process impurities and byproducts are introduced, and oxidative degradation reaction is easy to occur during the preparation and storage of hydroquinone to generate oxidative degradation impurities (such as p-benzoquinone). Studies have shown that p-benzoquinone is a genotoxic substance with stronger photosensitivity than hydroquinone, and light irradiation accelerates the oxidative degradation reaction of hydroquinone. P-benzoquinone is a main oxidative degradation product of hydroquinone, and is easy to form a colored compound with the hydroquinone, so that the purity, quality, stability, property and appearance of the hydroquinone and a product thereof are influenced, and the limit of the hydroquinone and the product thereof needs to be strictly controlled within a safety range. In addition, other researches show that impurities (such as p-benzoquinone, pyrogallol, resorcinol, catechol and the like) generated in the preparation and storage of hydroquinone have strong stimulation effects on skin, mucous membranes and central nervous respiratory systems, and have important clinical value due to the need of quality control.

Therefore, how to scientifically screen a detection method for efficiently separating and detecting hydroquinone and related substances thereof, and further effectively controlling the quality of the medicine so as to guarantee the effectiveness and safety of the medicine becomes a technical problem which needs to be solved urgently.

Disclosure of Invention

The invention provides a high performance liquid chromatography for efficiently separating and detecting p-benzoquinone in hydroquinone, which realizes the efficient separation and detection of hydroquinone and p-benzoquinone by optimizing chromatographic conditions and elution conditions, has the advantages of good specificity, high sensitivity and the like, can be used for the efficient separation and content determination of hydroquinone and p-benzoquinone, realizes the controllable medicine quality and ensures the medication safety.

The invention aims to provide a high performance liquid chromatography for efficiently separating and detecting p-benzoquinone in hydroquinone, wherein a chromatographic column in the method is selected from a reverse phase column or a positive phase column which can adopt a reverse phase solvent, and a mobile phase consists of a phase A and a phase B, wherein the phase A is selected from any one of water, a methanol aqueous solution and an acetonitrile aqueous solution, the phase B is selected from any one of methanol and acetonitrile, the detection wavelength is 200-400nm, the sample introduction amount is 1-100 mu l, and the column temperature is 10-45 ℃.

In a preferred embodiment of the present invention, the phase a contains 0 to 0.5% acid, preferably the acid content in the phase a is 0.02 to 0.3%, and further preferably the acid content in the phase a is 0.05 to 0.1%.

In a preferred embodiment of the present invention, the A phase has a pH of 1.0 to 6.0, preferably a pH of 2.0 to 5.0, more preferably a pH of 2.5 to 4.0, and further preferably a pH of 2.7 to 3.85.

In a preferred embodiment of the present invention, the acid is selected from any one of formic acid, glacial acetic acid, trifluoroacetic acid, phosphoric acid, potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, or a combination thereof.

In a preferred embodiment of the present invention, the volume ratio of the phase A to the phase B in the mobile phase is 99:1 to 70:30, preferably 98:2 to 80:20, and more preferably 97:3 to 90: 10.

In a preferred embodiment of the present invention, the mobile phase elution is selected from the group consisting of isocratic elution and gradient elution.

In the preferred technical scheme of the invention, the flow rate of the mobile phase is 0.6-1.5ml/min, preferably 0.8-1.2ml/min, and more preferably 0.9-1.0 ml/min.

In the preferred technical scheme of the invention, the detection wavelength is 210nm-290nm, preferably 210-230 nm.

In a preferred embodiment of the present invention, the diluting solvent is used for preparing, dissolving, or diluting any one of the sample solution, the control solution, and is preferably selected from any one of water, methanol, acetonitrile, and a mobile phase.

In the preferred technical scheme of the invention, the column temperature is 20-40 ℃, preferably any one of 20 ℃, 25 ℃, 30 ℃ and 35 ℃.

In a preferred embodiment of the present invention, the amount of the sample is 5 to 50. mu.l, preferably 10. mu.l, 15. mu.l, 20. mu.l, 30. mu.l, or 40. mu.l.

In a preferred technical scheme of the invention, the chromatographic column is any one of an octadecyl bonded silica gel reverse phase column, a dodecyl bonded silica gel reverse phase column, an octaalkyl bonded silica gel reverse phase column, a hexaalkyl bonded silica gel reverse phase column, a tetraalkyl bonded silica gel reverse phase column and a propyl amide bonded silica gel column.

In a preferred technical scheme of the invention, the chromatographic column is any one or combination of GL-science, Wondasil, TSKgel, Agela Venusil, Inertsil ODS-P and Xbridge HPLC chromatographic columns.

In the preferable technical scheme of the invention, the diameter of the chromatographic column is 3-5mm, the length of the chromatographic column is 100-300mm, and the particle size of the packing in the chromatographic column is 3-5 μm.

In a preferred technical scheme of the invention, the chromatographic column is selected from any one or a combination of GL-science C18 Wondasil C18 Superb 4.6X 250mm 5 μm, Inertsil ODS-P4.6X 250mm 5 μm, TOSOH TSKgel C184.6X 250mm 5 μm and Agela Venusil C184.6X 250mm 5 μm.

In a preferred embodiment of the present invention, the concentration of the sample is 0.5. mu.g/ml to 3mg/ml, preferably 1. mu.g/ml to 2mg/ml, and more preferably 2. mu.g/ml to 1 mg/ml.

In a preferred embodiment of the present invention, the related substance in hydroquinone is selected from any one or a combination of potassium p-benzoquinone, aniline, phloroglucinol, pyrogallol, resorcinol, catechol, phenol, hydroquinone sulfate.

In a preferred technical scheme of the invention, the chromatographic column is selected from any one of an octadecyl bonded silica gel reverse phase column, a dodecyl bonded silica gel reverse phase column, an octaalkyl bonded silica gel reverse phase column, a hexaalkyl bonded silica gel reverse phase column, a tetraalkyl bonded silica gel reverse phase column and a propyl amide bonded silica gel column, and the phase A in the mobile phase is as follows: the volume ratio of the phase B is 80: 20-95: 5, wherein the phase A is 0.03-0.50% glacial acetic acid solution, the phase B is methanol, isocratic elution is carried out, the detection wavelength is 210-290nm, the sample injection amount is 5-50 mu l, the column temperature is 25-35 ℃, and the flow rate is 0.6-1.5 ml/min.

In a preferred technical scheme of the invention, the phase A in the mobile phase is as follows: the volume ratio of the phase B is 85: 15-92: 8, wherein the phase A is 0.05-0.25% glacial acetic acid solution, the phase B is methanol, isocratic elution is carried out, the detection wavelength is 210-230nm, the sample injection amount is 5-20 mu l, the column temperature is 28-32 ℃, and the flow rate is 0.8-1.2 ml/min.

In a preferred technical scheme of the invention, the phase A in the mobile phase is as follows: the volume ratio of phase B is 90:10, wherein phase A is 0.1% glacial acetic acid solution, phase B is methanol, isocratic elution is carried out, the detection wavelength is any one or combination of 210nm, 220nm and 230nm, the sample injection amount is 10-15 μ l, the column temperature is 30 ℃, and the flow rate is 0.9-1 ml/min.

In a preferred technical scheme of the invention, the chromatographic column is selected from any one of an octadecyl bonded silica gel reverse phase column, a dodecyl bonded silica gel reverse phase column, an octaalkyl bonded silica gel reverse phase column, a hexaalkyl bonded silica gel reverse phase column, a tetraalkyl bonded silica gel reverse phase column and a propyl amide bonded silica gel column, and the phase A in the mobile phase is as follows: the volume ratio of the phase B is 90: 10-99: 1, wherein the phase A is 0.03-0.50% glacial acetic acid solution, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210-290nm, the sample injection amount is 5-50 mu l, the column temperature is 25-35 ℃, and the flow rate is 0.6-1.5 ml/min.

In a preferred technical scheme of the invention, the phase A in the mobile phase is as follows: the volume ratio of the phase B is 95: 5-98: 2, wherein the phase A is 0.05-0.25% glacial acetic acid solution, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210-230nm, the sample injection amount is 5-20 mu l, the column temperature is 28-32 ℃, and the flow rate is 0.8-1.2 ml/min.

In a preferred technical scheme of the invention, the phase A in the mobile phase is as follows: the volume ratio of phase B is 97:3-94:6, wherein phase A is 0.1% glacial acetic acid solution, phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is any one or combination of 210nm, 220nm and 230nm, the sample injection amount is 5-10 μ l, the column temperature is 30 ℃, and the flow rate is 0.9-1.0 ml/min.

In a preferred technical scheme of the invention, the chromatographic column is selected from any one of an octadecyl bonded silica gel reverse phase column, a dodecyl bonded silica gel reverse phase column, an octaalkyl bonded silica gel reverse phase column, a hexaalkyl bonded silica gel reverse phase column, a tetraalkyl bonded silica gel reverse phase column and a propyl amide bonded silica gel column, and the phase A in the mobile phase is as follows: the volume ratio of the phase B is 85: 15-98: 2, wherein the phase A is 0.015-0.035% phosphoric acid solution, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210-290nm, the sample injection amount is 5-50 mu l, the column temperature is 25-35 ℃, and the flow rate is 0.6-1.5 ml/min.

In a preferred technical scheme of the invention, the phase A in the mobile phase is as follows: the volume ratio of the phase B is 90: 10-97: 3, wherein the phase A is 0.020-0.030% phosphoric acid solution, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210-230nm, the sample injection amount is 5-30 mu l, the column temperature is 28-32 ℃, and the flow rate is 0.8-1.2 ml/min.

In a preferred technical scheme of the invention, the phase A in the mobile phase is as follows: and the volume ratio of the phase B is 95:5, wherein the phase A is 0.025 percent phosphoric acid solution, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is any one or the combination of 210nm, 220nm and 230nm, the sample injection amount is 10 mu l-20 mu l, the column temperature is 30 ℃, and the flow rate is 0.9-1.1 ml/min.

In a preferred technical scheme of the invention, the phase A in the mobile phase is as follows: the volume ratio of the phase B is 94:6, wherein the phase A is 0.02% phosphoric acid solution, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210nm, 220nm and 230nm, the sample injection amount is 10-20 mul, the column temperature is 30 ℃, the flow rate is as follows: 0.8-1.2 ml/min.

In the preferred technical scheme of the invention, the chromatographic column is selected from any one of an octadecyl bonded silica gel reverse phase column, a dodecyl bonded silica gel reverse phase column, an octaalkyl bonded silica gel reverse phase column, a hexaalkyl bonded silica gel reverse phase column, a tetraalkyl bonded silica gel reverse phase column and a propyl amide bonded silica gel column, the mobile phase is 0.005-0.05mol/L phosphoric acid aqueous solution, isocratic elution is carried out, the detection wavelength is 210-290nm, the sample introduction amount is 5-50 μ L, the column temperature is 25-35 ℃, and the flow rate is as follows: 0.6-1.2 ml/min.

In the preferred technical scheme of the invention, the mobile phase is 0.01-0.03mol/L phosphoric acid aqueous solution, isocratic elution is carried out, the detection wavelength is 210-230nm, the sample injection amount is 5-30 μ L, the column temperature is 28-32 ℃, the flow rate is as follows: 0.6-1.0 ml/min.

In the preferred technical scheme of the invention, the mobile phase is 0.02mol/L phosphoric acid aqueous solution, isocratic elution is carried out, the detection wavelength is selected from any one or the combination of 210nm, 220nm and 230nm, the sample injection amount is 10-20 mul, the column temperature is 30 ℃, the flow rate is as follows: 0.6-0.8 ml/min.

In a preferred technical scheme of the invention, the chromatographic column is selected from any one of an octadecyl bonded silica gel reverse phase column, a dodecyl bonded silica gel reverse phase column, an octaalkyl bonded silica gel reverse phase column, a hexaalkyl bonded silica gel reverse phase column, a tetraalkyl bonded silica gel reverse phase column and a propyl amide bonded silica gel column, and the phase A in the mobile phase is as follows: the volume ratio of the phase B is 85: 15-98: 2, wherein the phase A is phosphate buffer solution with the pH value of 1-5, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210-290nm, the sample injection amount is 5-50 mu l, the column temperature is 25-35 ℃, and the flow rate is 0.6-1.5 ml/min.

In a preferred technical scheme of the invention, the phase A in the mobile phase is as follows: the volume ratio of the phase B is 90: 10-97: 3, wherein the phase A is phosphate buffer solution with the pH value of 2-4, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210-230nm, the sample injection amount is 5-30 mu l, the column temperature is 28-32 ℃, and the flow rate is 0.8-1.2 ml/min.

In a preferred technical scheme of the invention, the phase A in the mobile phase is as follows: and the volume ratio of the phase B is 95:5, wherein the phase A is potassium dihydrogen phosphate buffer solution with the pH value of 3-3.5, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is any one or the combination of 210nm, 220nm and 230nm, the sample amount is 10-20 mul, the column temperature is 30 ℃, the flow rate: 1 ml/min.

In a preferred technical scheme of the invention, the chromatographic column is selected from any one of an octadecyl bonded silica gel reverse phase column, a dodecyl bonded silica gel reverse phase column, an octaalkyl bonded silica gel reverse phase column, a hexaalkyl bonded silica gel reverse phase column, a tetraalkyl bonded silica gel reverse phase column and a propyl amide bonded silica gel column, and the phase A in the mobile phase is as follows: the volume ratio of the phase B is 85: 15-99: 1, wherein the phase A is glacial acetic acid aqueous solution with the pH value of 1-6, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210-290nm, the sample injection amount is 5-50 mu l, the column temperature is 25-35 ℃, and the flow rate is 0.6-1.5 ml/min.

In a preferred technical scheme of the invention, the phase A in the mobile phase is as follows: the volume ratio of the phase B is 90: 10-98: 2, wherein the phase A is glacial acetic acid aqueous solution with the pH value of 3-4, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210-230nm, the sample injection amount is 5-30 mu l, the column temperature is 28-32 ℃, and the flow rate is 0.8-1.2 ml/min.

In a preferred technical scheme of the invention, the phase A in the mobile phase is as follows: and the volume ratio of the phase B is 97:3, wherein the phase A is glacial acetic acid aqueous solution with the pH value of 3.5-3.85, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is selected from any one or the combination of 210nm, 220nm and 230nm, the sample amount is 10-20 mul, the column temperature is 30 ℃, the flow rate: 1 ml/min.

It is another object of the present invention to provide the high performance liquid chromatography of the present invention for detecting any one of hydroquinone, hydroquinone composition, and hydroquinone preparation, preferably hydroquinone cream.

The invention aims to provide a high-safety hydroquinone composition, wherein the purity of hydroquinone in the hydroquinone composition is not less than 99.0%, and the content of p-benzoquinone in the hydroquinone composition is not more than 0.1%.

In a preferred technical scheme of the invention, the purity of the hydroquinone in the hydroquinone composition is not less than 99.5%, and preferably not less than 99.9%.

In a preferred embodiment of the present invention, the content of p-benzoquinone in the hydroquinone composition is not higher than 0.08%, preferably not higher than 0.05%, and further preferably not higher than 0.03%.

In a preferred embodiment of the present invention, the content of the p-benzoquinone or aniline in the hydroquinone composition is not higher than 0.08%, preferably not higher than 0.05%, and further preferably not higher than 0.03%.

In a preferred embodiment of the present invention, the total content of the relevant substances other than p-benzoquinone and aniline in the hydroquinone composition is not higher than 0.3%, preferably not higher than 0.1%, and further preferably not higher than 0.05%.

In a preferred embodiment of the present invention, the related substance is selected from any one or a combination of p-benzoquinone, aniline, pyrogallol, resorcinol, and catechol.

In a preferred technical scheme of the invention, the related substances are selected from any one or combination of potassium p-benzoquinone, aniline, pyrogallol, resorcinol, catechol, trimellitol, phloroglucinol, phenol and hydroquinone sulfate.

The invention aims to provide a preparation method of a hydroquinone composition with high safety, which comprises the following steps: 1) adding aniline and MnO₂Sulfuric acid according to aniline: MnO₂: adding sulfuric acid into a reaction device at a molar ratio of 1:1-5:2-6, and stirring until the reaction is complete to obtain p-benzoquinone, wherein the feeding temperature is 0-15 ℃, and the reaction temperature is 0-30 ℃; 2) and (3) carrying out steam distillation on the prepared p-benzoquinone reaction liquid, and adding the p-benzoquinone reaction liquid into the collected distillation condensate according to the following steps: adding iron powder at the molar ratio of 1:0.5-1.0, and stirring at 70-100 deg.C until the reaction is complete to obtain hydroquinone.

In a preferred technical scheme of the invention, in the step 1), aniline: MnO₂: the molar ratio of sulfuric acid is 1:1-5:2-6, preferably 1:2-4:3-5, more preferably 1:3: 4.

In the preferred technical scheme of the invention, the feeding temperature of the step 1) is 5-10 ℃.

In the preferred technical scheme of the invention, the reaction temperature of the step 1) is 10-25 ℃.

In the preferred technical scheme of the invention, the step 2) is carried out reduction reaction under the conditions of light protection and no oxygen.

In a preferred technical scheme of the invention, in the step 2), p-benzoquinone: the molar ratio of iron powder is 1:0.6-0.9, preferably 1: 0.7-0.8.

In a preferred embodiment of the invention, the reaction temperature in step 2) is 80 to 100 ℃, preferably 90 to 100 ℃.

In the preferable technical scheme of the invention, after iron oxide slag in the reaction product prepared in the step 2) is separated and removed, the collected separation liquid is subjected to reduced pressure concentration or vacuum concentration to prepare the reaction concentrated liquid.

In the preferable technical scheme of the invention, any one or the combination of a reducing agent, an antioxidant and active carbon is added into the reaction concentrated solution prepared in the step 2), and then the mixture is stirred, separated, filtered, cooled, crystallized, separated, crystallized and dried to prepare the hydroquinone.

In a preferable technical scheme of the invention, in the concentrated solution reaction system in the step 2), the reaction conditions of hydroquinone: the molar ratio of the reducing agent is 1:0.001-0.2, preferably 1:0.002-0.15, more preferably 1: 0.005-0.1.

In a preferred technical scheme of the invention, the reducing agent is selected from any one of iron powder and zinc powder or a combination thereof.

In the preferred technical scheme of the invention, in the concentrated solution reaction system, the antioxidant: the hydroquinone is present in a percentage by weight of between 0.1% and 1.0%, preferably between 0.2% and 0.9%, more preferably between 0.4% and 0.8%.

In the preferred technical scheme of the invention, the antioxidant is selected from sodium metabisulfite, sodium sulfite, sodium bisulfite and V_CAny one or combination thereof.

In the preferred technical scheme of the invention, the crystallization is carried out under the anaerobic condition.

In a preferred embodiment of the present invention, the oxygen-free condition is selected from filling of an inert gas, preferably the inert gas is selected from any one of nitrogen, argon, helium or a combination thereof.

In a preferred embodiment of the present invention, the cooling crystallization is selected from any one of stirring cooling crystallization and standing cooling crystallization or a combination thereof.

In the preferred technical scheme of the invention, the cooling crystallization temperature is-5-25 ℃, preferably 0-20 ℃, and more preferably 5-15 ℃.

In a preferred embodiment of the present invention, the separation is selected from any one of filtration, centrifugation, and membrane separation, or a combination thereof.

In the preferred technical scheme of the invention, the collected crystals are washed and then dried, and the preferred washing solvent is water.

In a preferred embodiment of the present invention, the drying is selected from any one or a combination of hot air drying, vacuum drying, and reduced pressure drying.

In the preferred technical scheme of the invention, the drying temperature is 45-75 ℃, preferably 50-70 ℃, and more preferably 55-65 ℃.

In a preferred technical scheme of the invention, the reaction solvent in the step 1) or the step 2) is water.

In a preferred embodiment of the present invention, the purity of the hydroquinone obtained is not less than 99.0%, preferably not less than 99.5%, more preferably not less than 99.9%.

In a preferred embodiment of the present invention, the content of p-benzoquinone in the obtained hydroquinone is not higher than 0.1%, preferably not higher than 0.08%, further preferably not higher than 0.05%, and more preferably not higher than 0.03%.

In a preferred technical scheme of the invention, the content of aniline in hydroquinone is not higher than 0.08%, preferably not higher than 0.05%, and also preferably not higher than 0.03%.

In a preferred embodiment of the present invention, the total content of the relevant substances other than p-benzoquinone and aniline in hydroquinone is not higher than 0.3%, preferably not higher than 0.1%, and further preferably not higher than 0.05%.

In a preferred technical scheme of the invention, the related substances are selected from any one or combination of pyrogallol, resorcinol and catechol.

In a preferred technical scheme of the invention, the related substances are selected from any one or combination of potassium sulfate of pyrogallol, resorcinol, catechol, trimellitic acid, phloroglucinol, phenol and hydroquinone.

The invention aims to provide a high-safety hydroquinone composition purification method, which comprises the following steps: 1) dissolving hydroquinone to be refined in a crystallization solvent at 80-100 ℃ to prepare a crystallization solution; 2) adding a reducing agent into the prepared crystallization solution, stirring, separating, collecting separated liquid, cooling, crystallizing, separating, collecting crystals, and drying to obtain the crystal.

In a preferred embodiment of the present invention, the crystallization solvent is selected from any one of water, ethanol, isopropanol, n-propanol, and n-butanol, or a combination thereof.

In the preferred technical scheme of the invention, the step 1) and the step 2) are carried out under the conditions of light resistance and no oxygen.

In a preferred embodiment of the present invention, the hydroquinone concentration in the crystallization solution is 5 to 40%, preferably 10 to 35%, more preferably 15 to 30%, still more preferably 20 to 25%.

In the preferable technical scheme of the invention, the temperature of the crystallization solution is 85-95 ℃.

In a preferred technical scheme of the invention, in the crystallization solution in the step 2), the ratio of hydroquinone: the molar ratio of the reducing agent is 1:0.001-0.2, preferably 1:0.002-0.15, more preferably 1: 0.005-0.1.

In a preferred technical scheme of the invention, an antioxidant is optionally added into the crystallization solution in the step 2).

In a preferred technical scheme of the invention, in the crystallization solution in the step 2), the antioxidant: the hydroquinone is present in a percentage by weight of between 0.1% and 1.0%, preferably between 0.2% and 0.9%, more preferably between 0.4% and 0.8%.

In a preferred technical scheme of the invention, activated carbon is optionally added into the crystallization solution in the step 2).

In the preferred technical scheme of the invention, the prepared hydroquinone is stored in a dark place, and the prepared hydroquinone is preferably stored in a dark and anaerobic condition.

In the preferable technical scheme of the invention, the content of any one of p-benzoquinone and aniline in the prepared hydroquinone is not higher than 0.08%, preferably not higher than 0.05%, and further preferably not higher than 0.03%.

In a preferred technical scheme of the invention, the total content of related substances except p-benzoquinone and aniline in the prepared hydroquinone is not higher than 0.3%, preferably not higher than 0.1%, and further preferably not higher than 0.05%.

The invention aims to provide the application of a high-safety hydroquinone composition in preparing a hydroquinone preparation.

In a preferred embodiment of the present invention, the hydroquinone purity of the hydroquinone composition is not less than 99.0%, preferably not less than 99.5%, more preferably not less than 99.9%.

In a preferred embodiment of the present invention, the hydroquinone composition contains p-benzoquinone not higher than 0.1%, preferably not higher than 0.08%, further preferably not higher than 0.05%, and more preferably not higher than 0.03%.

In a preferred embodiment of the present invention, the hydroquinone composition contains neither p-benzoquinone nor aniline in an amount higher than 0.08%, preferably not higher than 0.05%, and further preferably not higher than 0.03%.

In a preferred embodiment of the present invention, the total content of the related substances other than p-benzoquinone and aniline in the hydroquinone composition is not higher than 0.3%, preferably not higher than 0.1%, and further preferably not higher than 0.05%.

In a preferred technical scheme of the invention, the hydroquinone preparation is selected from any one of cream, gel, emulsion, cream, solution, foam, aerosol, spray, liniment and paste.

In a preferred technical scheme of the invention, the hydroquinone preparation is selected from any one of hydroquinone pharmaceutical preparations and hydroquinone cosmetics.

In the preferable technical scheme of the invention, the hydroquinone preparation is used for preparing the medicines for whitening, removing freckles, preventing and treating any disease of skin diseases, color spots and pigmentation diseases or complications thereof.

In a preferred embodiment of the present invention, the stain is selected from any one of freckles, chloasma, age spots, post-inflammatory hyperpigmentation spots, local hyperpigmentation spots, or a complication thereof.

The 'oxygen-free condition' is the reaction condition of isolating oxygen or other oxidants, and is selected from any one or combination of inert gas protection or reducing agent addition.

The invention adopts waters2695 high performance liquid chromatograph and Thermo Fisher U3000 high performance liquid chromatograph.

Unless otherwise indicated, when the present invention relates to percentages between liquids, said percentages are volume/volume percentages; the invention relates to the percentage between liquid and solid, said percentage being volume/weight percentage; the invention relates to the percentages between solid and liquid, said percentages being weight/volume percentages; the balance being weight/weight percent.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention provides the hydroquinone composition with high safety, strictly controls the safety limit of p-benzoquinone and aniline in the hydroquinone composition, promotes the quality upgrade of hydroquinone and a preparation thereof, and obviously improves the stability and the medication safety of the hydroquinone and the preparation thereof.

2. The high performance liquid chromatography of the invention obviously improves the separation degree of hydroquinone and related substances (such as p-benzoquinone, resorcinol, pyrogallol and the like), and realizes the high-efficiency separation and detection of hydroquinone and related substances. The detection method has the advantages of simple and convenient operation, high separation degree, good specificity, high sensitivity and the like, is favorable for better controlling the quality of the hydroquinone and the preparation thereof, ensures the effectiveness and the safety of the medicine, and realizes the controllable quality of the medicine.

3. The preparation method disclosed by the invention is carried out under the conditions of light and oxygen shielding, avoids the occurrence of oxidative degradation reaction, improves the reaction rate, effectively reduces the occurrence of side reaction and the generation of byproducts, improves the reaction yield and the purity and quality of the prepared product, is beneficial to ensuring the medication safety, and has the advantages of simplicity and convenience in operation, environmental friendliness, higher cost, suitability for industrial production and the like.

Drawings

FIG. 1 shows the separation and detection results of hydroquinone, p-benzoquinone, resorcinol, and pyrogallol under water-methanol (70: 30);

FIG. 2 shows the separation and detection results of p-benzoquinone, aniline, hydroquinone, pyrogallol, and resorcinol under water-methanol (70: 30);

FIG. 3 shows the separation and detection of the substances involved in hydroquinone under water-methanol (70:30) conditions;

FIG. 40.1% aqueous glacial acetic acid-methanol (90:10) separation and detection of the substances of interest in hydroquinone;

FIG. 50.1% aqueous glacial acetic acid-acetonitrile (94:6) separation and detection of hydroquinone, p-benzoquinone, pyrogallol, resorcinol;

FIG. 60.025% phosphoric acid aqueous solution-acetonitrile (94:6) separation and detection results of hydroquinone, p-benzoquinone, pyrogallol, and resorcinol;

FIG. 7 shows the separation and detection of related substances in hydroquinone under conditions of pH3.0 potassium dihydrogen phosphate buffer-acetonitrile (95: 5);

FIG. 8 shows the separation and detection of related substances in hydroquinone under the conditions of aqueous glacial acetic acid solution-acetonitrile (97:3) at pH 3.85;

FIG. 90.02 mol/L phosphoric acid aqueous solution separation and detection results of substances involved in hydroquinone;

FIG. 100.1% aqueous glacial acetic acid-methanol (90:10) separation and detection of the substances of interest in hydroquinone;

FIG. 11 separation and detection results of related substances in hydroquinone obtained in example 1 under the condition of 0.1% glacial acetic acid aqueous solution-acetonitrile (97: 3);

FIG. 12 separation and detection results of related substances in hydroquinone obtained in example 2 under the condition of 0.1% glacial acetic acid aqueous solution-acetonitrile (97: 3);

FIG. 13 separation and detection results of related substances in hydroquinone obtained in example 3 under the condition of 0.1% glacial acetic acid aqueous solution-acetonitrile (97: 3).

FIG. 14 shows the separation and detection results of the substances in hydroquinone in the purified product of hydroquinone obtained in example 4 under the conditions of pH3.0 potassium dihydrogen phosphate buffer-acetonitrile (95: 5);

FIG. 15 separation and detection results of hydroquinone related substances in refined hydroquinone product obtained in example 5 under conditions of pH3.0 potassium dihydrogen phosphate buffer-acetonitrile (95: 5);

FIG. 16 separation and detection results of substances related to hydroquinone in purified hydroquinone product obtained in example 6 under conditions of pH3.0 potassium dihydrogen phosphate buffer-acetonitrile (95: 5).

Detailed Description

The preferred embodiments of the present invention will be described below with reference to examples. It should be understood that the examples are for illustrative purposes only and are not intended to limit the invention, and that various modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims.

Example 1Preparation of Hydroquinone according to the invention

The preparation method of hydroquinone comprises the following steps:

1) MnO of₂280g, 430g of 98 percent sulfuric acid and 2.0L of water are added into a reaction bottle, 100g of aniline is slowly dropped into the reaction bottle at the temperature of 5-10 ℃, the mixture is stirred, and the temperature is gradually increased to about 25 ℃ until the reaction is completed. Carrying out steam distillation on the reaction liquid at 60-90 ℃, and introducing the collected p-benzoquinone condensate into another reaction bottle;

2) under the conditions of shading and nitrogen protection, adding 42g of iron powder into the collected p-benzoquinone condensate, stirring and reacting for 3-4 hours at the temperature of 90-100 ℃ until the reaction is complete, filtering, and concentrating the collected filtrate under reduced pressure until the content of hydroquinone in the concentrated solution is 35%;

3) adding 550mg of sodium pyrosulfite, 2.2g of active carbon and 330mg of zinc powder into the concentrated solution, heating to 95 ℃, carrying out hot filtration, collecting filtrate, cooling to 5 ℃, stirring for crystallization, centrifuging, collecting wet product, and carrying out vacuum drying at 60 ℃ to obtain 100.86g of hydroquinone.

Example 2Preparation of Hydroquinone according to the invention

The preparation method of hydroquinone comprises the following steps:

1) MnO of₂350g, 500g of 98 percent sulfuric acid and 2.0L of water are added into a reaction bottle, 100g of aniline is slowly dripped under the condition of 5-8 ℃, stirred and gradually heated to about 25 ℃ until the reaction is completed. Steam distilling the reaction liquid at 60-90 deg.c and introducing the collected p-benzoquinone condensate into the other reaction bottle;

2) under the conditions of shading and nitrogen protection, 60g of iron powder is added into the collected p-benzoquinone condensate, the mixture is stirred and reacted for 2 to 3 hours at the temperature of between 90 and 100 ℃ until the reaction is completed, the mixture is filtered, and the filtrate is collected and concentrated under reduced pressure until the content of hydroquinone in the concentrated solution is 25 percent;

3) adding 750mg of sodium pyrosulfite, 3.1g of active carbon and 500mg of iron powder into the concentrated solution, heating to 90 ℃, filtering, cooling the filtrate to 8 ℃, stirring for crystallization, centrifuging, collecting wet products, and drying in vacuum at 65 ℃ to obtain 96.20g of hydroquinone.

Example 3Preparation of Hydroquinone according to the invention

The preparation method of hydroquinone comprises the following steps:

1) MnO of₂250g, 370g of 98 percent sulfuric acid and 2.5L of water are added into a reaction bottle, 100g of aniline is slowly dropped into the reaction bottle at the temperature of between 6 and 10 ℃, the mixture is stirred, and the temperature is gradually increased to about 25 ℃ until the reaction is completed. Distilling the reaction solution at 70-95 deg.C with steam, and introducing the collected p-benzoquinone condensate into another reaction bottle;

2) under the conditions of shading and nitrogen protection, 31g of iron powder is added into the collected p-benzoquinone condensate, the mixture is stirred and reacted for 2 to 3 hours at the temperature of between 90 and 100 ℃ until the reaction is completed, the mixture is filtered, and the filtrate is collected and concentrated under reduced pressure until the content of hydroquinone in the concentrated solution is 30 percent;

3) adding 550mg of sodium pyrosulfite, 2.20g of active carbon and 350mg of zinc powder into the concentrated solution, heating to 90 ℃, filtering, cooling the filtrate to 10 ℃, stirring for crystallization, centrifuging, collecting wet products, and drying in vacuum at 65 ℃ to obtain 98.43g of hydroquinone.

Example 4Purification of Hydroquinone according to the invention

A process for the purification of hydroquinone comprising the steps of:

dissolving 25g of hydroquinone to be refined in 100ml of 95 ℃ water, adding 140mg of sodium metabisulfite, 600mg of activated carbon and 100mg of zinc powder, stirring for 40min, filtering while hot, collecting filtrate, cooling to 8 ℃, standing for crystallization for 11h, filtering, washing with water, collecting crystals, placing the crystals at 70 ℃ for vacuum drying, internally packaging 22.8g of hydroquinone in a brown glass bottle, externally packaging the hydroquinone in a black bag, and filling nitrogen in the bottle.

Example 5Purification of Hydroquinone according to the invention

A process for the purification of hydroquinone comprising the steps of:

dissolving 40g of hydroquinone to be refined in 120ml of 92 ℃ water, adding 100mg of sodium metabisulfite, 400mg of activated carbon and 60mg of iron powder, stirring for 35min, filtering while hot, collecting filtrate, cooling to 5 ℃, standing for crystallization for 12h, filtering, washing with water, collecting crystals, placing the crystals at 65 ℃ for vacuum drying, wrapping the obtained 36.8g of hydroquinone in a brown glass bottle, wrapping the hydroquinone in a black bag, and filling nitrogen in the bottle.

Example 6Purification of Hydroquinone according to the invention

A process for the purification of hydroquinone comprising the steps of:

dissolving 50g of hydroquinone to be refined in 140ml of 100 ℃ water, adding 175mg of zinc powder, stirring for 30min, adding 1.10g of activated carbon and 275mg of sodium metabisulfite, stirring for 10min, filtering while hot, collecting filtrate, cooling to 10 ℃ under the protection of nitrogen, standing for crystallization for 12h, filtering while hot, washing with water, collecting crystals, placing the crystals at 65 ℃ for vacuum drying, internally packaging 45g of hydroquinone in a brown glass bottle, externally packaging the hydroquinone in a black bag, and filling nitrogen in the bottle.

Comparative examples 1 to 3Detection of impurities in Hydroquinone

1. Chromatographic conditions

Name (R)	Chromatographic parameters
		Chromatographic column	Agela Venusil MP C18 4.6*250mm 5μm
Mobile phase	Water-methanol (70:30)
		Elution gradient	Isocratic elution
Flow rate of flow	1.0ml/min
		Sample volume	10μl
Detection wavelength	220nm
		Column temperature	30℃
Diluting solvent	Water (W)

2. Preparation of the solution

Test solution: a hydroquinone sample was weighed, dissolved in a diluting solvent and diluted to a solution containing about 1mg of hydroquinone per 1 ml.

Control solution: precisely measuring 1ml of the test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to a scale with a diluting solvent, and shaking up to obtain a control solution.

Impurity localization solution: precisely weighing appropriate amount of potassium hydroquinone sulfate, phloroglucinol, pyrogallol, aniline, resorcinol, catechol, p-benzoquinone and phenol, dissolving with a diluting solvent, and quantitatively diluting to obtain a stock solution containing about 1mg of each impurity per 1 ml. An appropriate amount of each impurity stock solution was precisely measured, and diluted with a diluent solvent to prepare a solution containing about 10. mu.g of each impurity per 1ml as a positioning solution.

Mixing impurity solution: a test sample solution and an appropriate amount of an impurity stock solution are precisely measured, placed in the same measuring flask, and quantitatively diluted with a diluting solvent to prepare a mixed solution containing about 10 mug of main components and impurities per 1 ml.

Precisely measuring hydroquinone contrast solution and p-benzoquinone, resorcinol and pyrogallol positioning solution, respectively injecting into a liquid chromatograph, and recording chromatogram. The results are shown in FIG. 1 and Table 1.

TABLE 1

Compound (I)	Retention time	Peak area	Tailing factor	Degree of separation	Number of theoretical plate	Half peak width
							Pyrogallol	4.357	4.3769	1.15	0.20	19200	0.074
Hydroquinone	4.386	4.4919	0.98	9.11	19916	0.073
							Resorcinol	5.953	5.778	0.96	0.92	20179	0.099
P-benzoquinone	6.132	3.6643	1.00	N/A	22688	0.096

Precisely measuring hydroquinone contrast solution and p-benzoquinone, aniline, pyrogallol and resorcinol positioning solution, respectively injecting into a liquid chromatograph, and recording chromatogram. The results are shown in FIG. 2 and Table 2.

TABLE 2

Precisely measuring the p-benzoquinone positioning solution and the impurity mixed solution, respectively injecting into a liquid chromatograph, and recording the chromatogram. The results are shown in FIG. 3 and Table 3.

TABLE 3

Compound (I)	Retention time	Peak area	Tailing factor	Degree of separation	Number of theoretical plate	Half peak width
							Potassium p-dihydroxybenzene sulfate	2.432	1.1498	1.10	19.04	53742	0.025
Trimellit phenol	3.498	0.2532	N/A	0.84	71214	0.031
							Phloroglucinol	3.569	3.2618	1.16	5.93	71214	0.054
Pyrogallol/hydroquinone	4.369	9.3113	1.05	8.82	16292	0.081
							Resorcinol	5.948	5.8085	0.99	0.95	20548	0.098
P-benzoquinone	6.132	3.6643	1.00	7.18	22688	0.096
							Catechol	7.711	4.3501	1.04	7.42	21548	0.124
Aniline	9.936	22.9254	1.07	11.74	17718	0.176
							Phenol and its preparation	14.657	4.1523	0.95	N/A	23203	0.226

Example 7Separation and detection of impurities in hydroquinone

1. Chromatographic conditions are as follows:

name (R)	Chromatographic parameters
		Chromatographic column	TOSOH TSKgel C18 4.6*250mm 5μm
Mobile phase	0.1% glacial acetic acid solution-methanol (90:10)
		Elution gradient	Isocratic elution
Flow rate of flow	1.0ml/min
		Detection wavelength	220nm
Column temperature	30℃
		Sample volume	10μl
Diluting solvent	0.1% glacial acetic acid solution

2. Preparation of the solution

Impurity localization solution: precisely weighing a proper amount of potassium hydroquinone sulfate, phloroglucinol, pyrogallol, aniline, resorcinol, catechol, p-benzoquinone and phenol, dissolving and quantitatively diluting the mixture by using a diluting solvent to prepare a stock solution containing about 1mg of each impurity per 1ml, precisely weighing a proper amount of the stock solution, and respectively diluting the stock solution by using the diluting solvent to prepare a solution containing about 10 mu g of the impurity per 1ml to serve as a positioning solution.

Mixing impurity solution: precisely measuring a test sample solution and an appropriate amount of impurity stock solution, placing into the same measuring flask, and quantitatively diluting with a diluent to obtain a mixed solution containing about 10 μ g of main component and impurity per 1 ml.

Precisely measuring p-benzoquinone positioning solution and mixed impurity solution, respectively injecting into a liquid chromatograph, measuring according to the method, and recording chromatogram. The results are shown in FIG. 4 and Table 4.

TABLE 4

Compound (I)	Retention time	Peak area	Tailing factor	Degree of separation	Number of theoretical plate	Half peak width
							Potassium p-dihydroxybenzene sulfate	4.913	3.3060	3.01	3.52	8362	0.126
Trimellit phenol	5.747	0.7290	1.15	1.28	14722	0.111
							Aniline	6.037	0.6018	1.57	3.82	14905	0.116
Phloroglucinol	6.973	4.2096	1.09	2.24	16163	0.129
							Pyrogallol	7.563	3.9989	1.10	2.69	17777	0.134
Hydroquinone	8.297	6.1492	1.08	14.72	19614	0.139
							Resorcinol	13.420	3.7035	1.07	1.22	22761	0.209
P-benzoquinone	14.003	2.0874	1.31	4.47	15205	0.267
							Catechol	16.310	3.0671	1.06	23.48	23860	0.249
Phenol and its preparation	33.970	2.7211	1.07	N/A	25305	0.503

Example 8Efficient separation and detection of related substances in hydroquinone

1. Chromatographic conditions

2. Preparation of the solution

Precisely weighing the required amount of hydroquinone, p-benzoquinone, pyrogallol and resorcinol, respectively diluting the hydroquinone, p-benzoquinone, pyrogallol and resorcinol with a solvent, dissolving and quantifying to 50ml, preparing into a solution containing 0.3mg of hydroquinone per 1ml, and shaking up to obtain the final product.

Precisely measuring each solution, injecting into liquid chromatograph, measuring according to method, and recording chromatogram. The results are shown in FIG. 5.

ExamplesHigh-efficiency separation and detection of related substances in 9-hydroquinone

1. Chromatographic conditions

Instrument for measuring the position of a moving object	Chromatographic parameters
		Chromatographic column	GL-science，WondaSil C18 Superb 4.6×250mm 5μm
Mobile phase	0.025% aqueous phosphoric acid solution-acetonitrile (94:6)
		Elution gradient	Isocratic elution
Flow rate of flow	1.0ml/min
		Detection wavelength	210nm
Column temperature	30℃
		Sample introduction volume	10μl
Diluting solvent	0.025% aqueous phosphoric acid solution-acetonitrile (94:6)

2. Preparation of the solution

A positioning solution of p-benzoquinone, pyrogallol and resorcinol and a hydroquinone test solution were prepared in accordance with the procedure of example 7. Precisely measuring each solution, injecting into liquid chromatograph, measuring according to method, and recording chromatogram. The results are shown in FIG. 6.

Example 10Efficient separation and detection of related substances in hydroquinone

1. Chromatographic conditions

2. Preparation of the solution

Solvent preparation as in example 7, p-benzoquinone positioning solution and impurity mixed solution were precisely measured, and injected into a liquid chromatograph, measured according to the method, and chromatogram was recorded. The results are shown in FIG. 7 and Table 5.

TABLE 5

Compound (I)	Retention time	Peak area	Tailing factor	Degree of separation	Number of theoretical plate	Half peak width
							Potassium hydrochinone sulfate/aniline	5.527	2.0858	1.72	2.55	15149	0.106
Trimellit phenol	6.070	2.5643	1.07	4.46	17909	0.107
							Phloroglucinol	7.083	3.9310	1.07	4.26	19170	0.12
Pyrogallol	8.157	4.0576	1.08	2.05	21187	0.132
							Hydroquinone	8.703	2.7872	1.03	14.87	23453	0.134
P-benzoquinone	13.847	2.3412	1.00	1.61	23734	0.212
							Resorcinol	14.533	3.6119	1.05	6.97	25544	0.214
Catechol	17.830	3.0695	1.04	23.90	26324	0.259
							Phenol and its preparation	36.570	3.2304	1.03	N/A	26837	0.525

Example 11Efficient separation and detection of related substances in hydroquinone

1. Chromatographic conditions

Name (R)	Chromatographic parameters
		Chromatographic column	Inertsil ODS-P 4.6×250mm 5μm
Mobile phase	Water (glacial acetic acid to pH 3.85) -acetonitrile (97:3)
		Elution gradient	Isocratic elution
Flow rate of flow	1.0ml/min
		Sample volume	10μl
Detection wavelength	220nm
		Column temperature	30℃
Diluting solvent	Water (glacial acetic acid to pH 3.85) -acetonitrile (97:3)

2. Preparation of the solution

Solvent preparation as in example 7, p-benzoquinone positioning solution and impurity mixed solution were precisely measured, and injected into a liquid chromatograph, measured according to the method, and chromatogram was recorded. The results are shown in FIG. 8 and Table 6.

TABLE 6

Compound (I)	Retention time	Peak area	Tailing factor	Degree of separation	Number of theoretical plate	Half peak width
							Potassium p-dihydroxybenzene sulfate	5.471	2.5864	1.52	4.85	5419	0.175
Trimellit phenol	6.829	3.4308	1.12	8.00	23412	0.105
							Phloroglucinol	8.700	5.8666	1.04	1.59	25387	0.129
Pyrogallol	9.133	5.6819	1.04	3.42	22724	0.143
							Hydroquinone	10.117	4.4139	1.07	1.59	27025	0.145
Aniline	10.612	3.0498	1.11	14.86	22351	0.167
							P-benzoquinone	16.883	3.3365	1	1.10	24315	0.255
Resorcinol	17.437	5.8329	1.05	5.81	27238	0.249
							Catechol	20.671	4.6992	1.01	23.90	24873	0.308
Phenol and its preparation	42.492	4.0239	1.04	N/A	27316	0.605

Example 12Efficient separation and detection of related substances in hydroquinone

1. Chromatographic conditions

Name (R)	Chromatographic parameters
		Chromatographic column	Inertsil ODS-P 4.6×250mm 5μm
Mobile phase	0.02mol/L phosphoric acid aqueous solution (pH2.7)
		Elution gradient	Isocratic elution
Flow rate of flow	0.6ml/min
		Detection wavelength	210nm
Sample volume	10μl
		Detection wavelength	210nm
Column temperature	30℃
		Diluting solvent	0.02mol/L phosphoric acid aqueous solution (pH2.7)

2. Preparation of the solution

Solvent preparation as in example 7, precisely measuring each impurity localization solution, control solution, and mixed impurity solution, respectively injecting into liquid chromatograph, measuring by method, and recording chromatogram. The results are shown in FIG. 9 and Table 7.

TABLE 7

The chromatographic conditions realize effective separation and detection of effective separation of chromatographic peaks between hydroquinone and various impurities, and realize medicine quality control.

Example 13Efficient separation and detection of related substances in hydroquinone

1. Chromatographic conditions

Instrument for measuring the position of a moving object	Chromatographic parameters
		Chromatographic column	GL-science，WondaSil C18 Superb 4.6×250mm 5μm
Mobile phase	0.1% aqueous glacial acetic acid-methanol (90:10)
		Elution gradient	Isocratic elution
Flow rate of flow	1.0ml/min
		Detection wavelength	220nm
Column temperature	30℃
		Sample introduction volume	10μl
Diluting solvent	0.1% aqueous glacial acetic acid

2. Preparation of the solution

Solvent preparation as in example 7, precisely measuring each impurity localization solution, control solution, and mixed impurity solution, respectively injecting into liquid chromatograph, measuring according to the method, and recording chromatogram, the results are shown in FIG. 10, Table 8, and Table 9.

TABLE 8 results of impurity localization

TABLE 9 examination of mixed impurity solutions

Serial number	Mixed impurities	Retention time	Relative retention	Peak area	Degree of separation	Number of theoretical plates
							1	Potassium p-dihydroxybenzene sulfate	4.913	0.59	3.306	4.13	8362
2	Trimellit phenol	5.747	0.69	0.729	1.5	14722
							3	Aniline	6.037	0.73	0.6018	4.5	14905
4	Phloroglucinol	6.973	0.84	4.2096	2.65	16163
							5	Pyrogallol	7.563	0.91	3.9989	3.17	17777
6	Hydroquinone	8.297	——	6.1492	17.33	19614
							7	Resorcinol	13.42	1.62	3.7035	7.45	22761
8	Catechol	16.31	1.97	3.0671	27.74	23860
							9	Phenol and its preparation	33.97	4.09	2.7211	N/A	25305

Examples 7-13 methodological validation results:

quantitative limit and detection limit

The detection limit of 10 substances is 0.32-1.65ng, and the quantification limit is 0.64-3.30 ng.

Linearity and range

In the range from the limit concentration of 10 substances to the quantitative limit concentration, the linear relation between each impurity and a main peak is good, the correlation coefficients are all larger than 0.998, and the intercept is all smaller than the response value of the limit concentration by 25%.

Precision degree

Repeatability: the same batch of samples are repeatedly measured for 6 times, and the results of 6 times of measurement have no obvious difference, which indicates that the method has good repeatability.

Recovery rate

The method measures that the recovery rate results of 9 samples with the concentrations of 3 impurities (50%, 100% and 150% of the limit concentration) are all in the range of 90-110%, and the RSD of the recovery rate results of 9 samples is less than 5.0%, so that the results meet the verification requirements, and the method has good accuracy.

Stability of solution

The sample injection detection of the related substance test solution is carried out for 0h, 2h, 4h, 6h, 8h, 15h and 24h at room temperature, the detection amount of each impurity has no obvious change, no new impurity is detected, and the product is placed for 24h at room temperature and has good solution stability.

Durability

The product has no obvious change in the detected amount of impurities and the number of impurities after changing the column temperature, the flow rate, the buffer salt concentration and the organic phase ratio and changing chromatographic columns of different manufacturers; the system has good applicability and the separation between the impurities and the main components in the solution is good. It is understood from this that the measurement of the relevant substance is not affected by the fine adjustment of the conditions, and the durability of the method is good.

Example 14Compatibility of p-benzoquinone

The phloroglucinol, pyrogallol and phloroglucinol were combined with p-benzoquinone and hydroquinone, respectively, and the peak area changes in the solution were examined, with the results shown in Table 10.

TABLE 10 compatibility stability test results (Peak area) for p-benzoquinone

Example 15Stability survey

1. Chromatographic conditions

Instrument for measuring the position of a moving object	Chromatographic parameters
		Chromatographic column	GL-science，WondaSil C18 Superb 4.6×250mm 5μm
Mobile phase	0.025% aqueous phosphoric acid solution-acetonitrile (95:5)
		Elution gradient	Isocratic elution
Flow rate of flow	1.0ml/min
		Detection wavelength	210nm
Column temperature	30℃
		Sample introduction volume	10μl
Diluting solvent	0.025% aqueous phosphoric acid solution-acetonitrile (95:5)

2. Preparation of the solution

Dissolving hydroquinone bulk drug and p-benzoquinone reference substance with diluent solvent respectively, and diluting to obtain test solution and reference solution.

Analyzing and detecting the component changes of the sample solution and the reference solution which are configured and placed for 0h, 2h, 4h, 6h and 8 h. The results are shown in tables 12 and 13.

TABLE 12 hydroquinone stability test results

	0h	2h	4h	6h	8h
						Main peak	577.3538	578.3176	577.5982	577.0191	577.2214
P-benzoquinone	Not detected out	0.1776	0.2002	0.2212	0.2112
						Peak area%	Not detected out	0.03	0.03	0.04	0.04

TABLE 13 stability test results for p-benzoquinone

	0h	2h	4h	6h	8h
						P-benzoquinone	2.4340	2.4121	2.3862	2.3828	2.4728

Examples 16 to 18Hydroquinone purity detection of the present invention

1. Chromatographic conditions

2. Preparation of the solution

Detecting a sample: examples 1-3 Hydroquinone was produced.

Blank solvent: 0.1% aqueous glacial acetic acid-acetonitrile (97: 3).

Impurity localization solution: precisely weighing a proper amount of the p-benzoquinone, adding water to dissolve and dilute the p-benzoquinone into a solution containing 10 mu g of the benzoquinone per 1ml, and shaking up to obtain the benzoquinone.

Test solution: precisely weighing an appropriate amount of hydroquinone, adding a diluting solvent to dissolve and dilute the hydroquinone into a solution containing about 1mg per 1ml, and shaking up to obtain the product.

Control solution: precisely measuring 1ml of the test solution, placing the test solution in a 100ml measuring flask, diluting with water to scale, and shaking up to obtain the final product.

Taking 10 μ l of each of the blank solvent, the impurity locating solution, the sample solution and the reference solution, injecting into a liquid chromatograph, recording chromatogram, and testing product content, wherein the results are shown in figures 11-13.

The hydroquinone obtained in examples 1-3 had a purity of 99.95%, 99.93% and 99.96%, respectively.

Examples 19 to 21Hydroquinone purity detection of the present invention

1. Chromatographic conditions

Instrument for measuring the position of a moving object	Chromatographic parameters
		Chromatographic column	GL-science，WondaSil C18 Superb 4.6*250mm 5μm
Mobile phase	pH3.0 Potassium dihydrogen phosphate buffer-acetonitrile (95:5)
		Elution gradient	Isocratic elution
Flow rate of flow	1.0ml/min
		Detection wavelength	220nm
Column temperature	30℃
		Sample introduction volume	10μl
Diluting solvent	pH3.0 Potassium dihydrogen phosphate buffer-acetonitrile (95:5)

2. Preparation of the solution

Detecting a sample: the purified hydroquinone obtained in example 4 to 6.

Blank solvent: pH3.0 Potassium dihydrogen phosphate buffer acetonitrile (95: 5).

Test solution: precisely weighing appropriate amount of hydroquinone refined product, adding diluting solvent to dissolve and dilute into solution containing 1mg per 1ml, and shaking.

And (3) taking 10 mu l of each of the blank solvent, the test solution and the reference solution, injecting the blank solvent, the test solution and the reference solution into a liquid chromatograph, recording a chromatogram, and testing the content of the product, wherein the results are shown in figures 14-16.

The purity of hydroquinone obtained in examples 4 to 6 was 100%, 100% and 100%, respectively.

The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined in the appended claims.

Claims

1. The high performance liquid chromatography for separating and detecting related substances in hydroquinone with high efficiency is characterized in that a chromatographic column in the method is selected from an octadecyl bonded silica gel reverse phase column, and a mobile phase consists of a phase A and a phase B, wherein the phase A is selected from water, the phase A contains acid with the concentration of 0-0.5% or phosphate with the concentration of 0-0.5%, the phase B is selected from any one of methanol and acetonitrile, and the volume ratio of the phase A to the phase B in the mobile phase is 99: 1-90: 10; the detection wavelength is 200-400nm, the sample injection amount is 1-100 μ l, the column temperature is 10-45 ℃, the flow rate is 0.6-1.5ml/min, and related substances in hydroquinone comprise p-benzoquinone, catechol, phenol and resorcinol; the related substances also comprise any one or the combination of aniline, phloroglucinol, pyrogallol and potassium hydroquinone sulfate.

2. The high performance liquid chromatography of claim 1, wherein the phase a has a ph of 2.0 to 5.0.

3. The high performance liquid chromatography of claim 2, wherein the phase a has a ph of 2.5 to 4.0.

4. The high performance liquid chromatography of claim 3, wherein the phase A has a pH of 2.7 to 3.85.

5. The high performance liquid chromatography of claim 1, wherein the a phase comprises an acid at a concentration of 0.02-0.3%.

6. The high performance liquid chromatography of claim 5, wherein the A phase contains an acid at a concentration of 0.05-0.1%.

7. The high performance liquid chromatography according to claim 1, wherein the phosphate is selected from any one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, or a combination thereof.

8. The high performance liquid chromatography of claim 1, the mobile phase elution being isocratic.

9. The high performance liquid chromatography of claim 1, wherein the mobile phase has a flow rate of 0.8-1.2 ml/min.

10. The high performance liquid chromatography of claim 9, wherein the mobile phase has a flow rate of 0.9-1.0 ml/min.

11. The high performance liquid chromatography as claimed in claim 1, wherein the detection wavelength is 210-290 nm.

12. The high performance liquid chromatography as claimed in claim 11, wherein the detection wavelength is 210-230 nm.

13. The high performance liquid chromatography according to claim 1, wherein the diluting solvent is used for preparing, dissolving, and diluting any one of a test solution and a control solution.

14. The high performance liquid chromatography according to claim 13, wherein the diluent solvent is selected from any one of water, methanol, acetonitrile, and a mobile phase.

15. The high performance liquid chromatography of claim 1, wherein the column temperature is 20-40 ℃.

16. The high performance liquid chromatography according to claim 15, wherein the column temperature is any one of 25 ℃, 30 ℃ and 35 ℃.

17. The high performance liquid chromatography of claim 1, wherein the sample is added in an amount of 5-50 μ l.

18. The high performance liquid chromatography according to claim 17, wherein the sample amount is any one of 10. mu.l, 15. mu.l, 20. mu.l, 30. mu.l, and 40. mu.l.

19. The high performance liquid chromatography method of claim 1, wherein the column is selected from any one of GL-science, WondaSil, TSKgel, Agela Venusil, insetsil ODS-P, XBridge HPLC column, or a combination thereof.

20. The high performance liquid chromatography as claimed in claim 1, wherein the diameter of the column is 3-5mm, the length of the column is 100-300mm, and the particle size of the packing in the column is 3-5 μm.

21. The high performance liquid chromatography method of claim 20, wherein the column is selected from Shimadzu GL-science C184.6 x 250mm 5 μm; wondasil C18 Superb 4.6X 250mm 5 μm; TOSOH TSKgel C184.6X 250mm 5 μm; inertsil ODS-P4.6X 250mm 5 μm, Agela Venusil C184.6X 250mm 5 μm, or a combination thereof.

22. The high performance liquid chromatography of claim 13, wherein the sample concentration is 0.5 μ g/ml to 3 mg/ml.

23. The high performance liquid chromatography of claim 22, wherein the sample concentration is 1 μ g/ml to 2 mg/ml.

24. The high performance liquid chromatography of claim 23, wherein the sample concentration is 2 μ g/ml to 1 mg/ml.

25. The high performance liquid chromatography of claim 1, wherein the ratio of phase a: the volume ratio of the phase B is 90: 10-97: 3, wherein the phase A is 0.020-0.030% phosphoric acid solution, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210-230nm, the sample injection amount is 5-30 mu l, the column temperature is 28-32 ℃, and the flow rate is 0.8-1.2 ml/min.

26. The high performance liquid chromatography of claim 25, wherein phase a: and the volume ratio of the phase B is 95:5, wherein the phase A is 0.025 percent phosphoric acid solution, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is any one or the combination of 210nm, 220nm and 230nm, the sample injection amount is 10 mu l-20 mu l, the column temperature is 30 ℃, and the flow rate is 0.9-1.1 ml/min.

27. The high performance liquid chromatography of claim 25, wherein phase a: the volume ratio of the phase B is 94:6, wherein the phase A is 0.02% phosphoric acid solution, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210nm, 220nm and 230nm, the sample injection amount is 10-20 mul, the column temperature is 30 ℃, the flow rate is as follows: 0.8-1.2 ml/min.

28. The high performance liquid chromatography of claim 1, wherein the ratio of phase a: the volume ratio of the phase B is 90: 10-97: 3, wherein the phase A is phosphate buffer solution with the pH value of 2-4, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210-230nm, the sample injection amount is 5-30 mu l, the column temperature is 28-32 ℃, and the flow rate is 0.8-1.2 ml/min.

29. The high performance liquid chromatography of claim 28, phase a in the mobile phase: and the volume ratio of the phase B is 95:5, wherein the phase A is potassium dihydrogen phosphate buffer solution with the pH value of 3-3.5, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is any one or the combination of 210nm, 220nm and 230nm, the sample amount is 10-20 mul, the column temperature is 30 ℃, the flow rate: 1 ml/min.

30. The high performance liquid chromatography for high performance separation and detection of relevant matter in hydroquinone is characterized in that a chromatographic column in the method is selected from an octadecyl bonded silica gel reverse phase column, a mobile phase is 0.005-0.05mol/L phosphoric acid water solution, isocratic elution is carried out, the detection wavelength is 210-290nm, the sample injection amount is 5-50 mu L, the column temperature is 25-35 ℃, the flow rate is as follows: 0.6-1.2ml/min, wherein the related substances in the hydroquinone comprise p-benzoquinone, catechol, phenol and resorcinol; the related substances also comprise any one or the combination of aniline, phloroglucinol, pyrogallol and potassium hydroquinone sulfate.

31. The high performance liquid chromatography as claimed in claim 30, wherein the mobile phase is 0.01-0.03mol/L phosphoric acid aqueous solution, isocratic elution is performed, the detection wavelength is 210-230nm, the sample amount is 5-30 μ L, the column temperature is 28-32 ℃, the flow rate: 0.6-1.0 ml/min.

32. The high performance liquid chromatography of claim 31, wherein the mobile phase is 0.02mol/L phosphoric acid aqueous solution, the elution is carried out at equal intervals, the detection wavelength is selected from any one or combination of 210nm, 220nm and 230nm, the sample amount is 10-20 μ L, the column temperature is 30 ℃, and the flow rate: 0.6-0.8 ml/min.

33. The high performance liquid chromatography of any one of claims 1-32, wherein the hydroquinone is not less than 99.0% pure and the hydroquinone is not more than 0.1% para-benzoquinone.

34. The high performance liquid chromatography of claim 33, wherein the hydroquinone in the hydroquinone is not less than 99.5% pure.

35. The high performance liquid chromatography of claim 34, wherein the hydroquinone is not less than 99.9% pure.

36. The high performance liquid chromatography of any one of claims 1-32, wherein the p-benzoquinone is present in an amount of no more than 0.08%.

37. The high performance liquid chromatography of claim 36, wherein the p-benzoquinone is present in an amount of no more than 0.05%.

38. The high performance liquid chromatography of claim 37, wherein the p-benzoquinone is present in an amount of no more than 0.03%.

39. The high performance liquid chromatography method of any one of claims 1-32, wherein neither p-benzoquinone nor aniline is present in the hydroquinone in an amount greater than 0.08%.

40. The high performance liquid chromatography of claim 39, wherein neither p-benzoquinone nor aniline is present in the hydroquinone of greater than 0.05%.

41. The high performance liquid chromatography of claim 40, wherein neither p-benzoquinone nor aniline is present in the hydroquinone of greater than 0.03%.

42. The high performance liquid chromatography of any one of claims 1-32, wherein the hydroquinone is not more than 0.3% of total content of related substances other than p-benzoquinone and aniline.

43. The high performance liquid chromatography of claim 42, wherein the hydroquinone is not more than 0.1% by weight of the total content of the relevant substances except p-benzoquinone and aniline.

44. The high performance liquid chromatography of claim 43, wherein the hydroquinone contains not more than 0.05% of other related substances except p-benzoquinone and aniline.

45. The high performance liquid chromatography for high performance separation and detection of relevant matter in hydroquinone is characterized in that a chromatographic column is an octadecyl bonded silica gel reversed phase column, a mobile phase consists of a phase A and a phase B, and the phase A in the mobile phase is as follows: the volume ratio of the phase B is 99: 1-90: 10, wherein, phase A is selected from any one of 0-0.5% phosphoric acid solution and 0-0.5% phosphate solution or the combination thereof, phase A has pH of 1.0-pH6.0, phase B is selected from any one of methanol and acetonitrile, the detection wavelength is 200-400nm, the sample injection amount is 1-100 μ l, the column temperature is 10-45 ℃, the flow rate is 0.6-1.5ml/min, the relevant substances in the hydroquinone comprise p-benzoquinone and resorcinol, and the relevant substances also comprise any one of aniline, trimellitic acid, phloroglucinol, potassium hydroquinone sulfate or the combination thereof.

46. The high performance liquid chromatography of claim 45, wherein the phase A has a pH of 2.0 to 5.0.

47. The high performance liquid chromatography of claim 46, wherein the phase A has a pH of 2.5 to 4.0.

48. The high performance liquid chromatography of claim 47, wherein the phase A has a pH of 2.7 to 3.85.

49. The high performance liquid chromatography of claim 45, wherein the A phase contains an acid at a concentration of 0.02-0.3%.

50. The high performance liquid chromatography of claim 49, wherein the A phase comprises an acid at a concentration of 0.05-0.1%.

51. The high performance liquid chromatography of claim 45, wherein the phosphate is selected from any one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, or a combination thereof.

52. The high performance liquid chromatography of claim 45, the mobile phase elution being an isocratic elution.

53. The high performance liquid chromatography of claim 45, wherein the mobile phase has a flow rate of 0.8-1.2 ml/min.

54. The high performance liquid chromatography of claim 53, wherein the mobile phase has a flow rate of 0.9-1.0 ml/min.

55. The high performance liquid chromatography as claimed in claim 45, wherein the detection wavelength is 210-290 nm.

56. The high performance liquid chromatography as claimed in claim 55, wherein the detection wavelength is 210-230 nm.

57. The high performance liquid chromatography of claim 45, wherein the diluent solvent is used for preparing, dissolving, or diluting a sample solution or a control solution.

58. The high performance liquid chromatography of claim 57, wherein the diluent solvent is selected from any one of water, methanol, acetonitrile, and mobile phase.

59. The high performance liquid chromatography of claim 45, wherein the column temperature is 20-40 ℃.

60. The high performance liquid chromatography of claim 59, wherein the column temperature is any one of 25 ℃, 30 ℃ and 35 ℃.

61. The high performance liquid chromatography of claim 45, wherein the sample is added in an amount of 5-50 μ l.

62. The high performance liquid chromatography of claim 61, wherein the sample is any one of 10 μ l, 15 μ l, 20 μ l, 30 μ l, and 40 μ l.

63. The high performance liquid chromatography method of claim 45, wherein the column is selected from any one of GL-science, Wondasil, TSKgel, Agela Venusil, Inertsil ODS-P, Xbridge HPLC column, or a combination thereof.

64. The high performance liquid chromatography as claimed in claim 45, wherein the diameter of the chromatographic column is 3-5mm, the length of the chromatographic column is 100-300mm, and the particle size of the packing in the chromatographic column is 3-5 μm.

65. The high performance liquid chromatography method of claim 64, wherein the column is selected from Shimadzu GL-science C184.6 x 250mm 5 μm; wondasil C18 Superb 4.6X 250mm 5 μm; TOSOH TSKgel C184.6X 250mm 5 μm; inertsil ODS-P4.6X 250mm 5 μm, Agela Venusil C184.6X 250mm 5 μm, or a combination thereof.

66. The high performance liquid chromatography of claim 57 wherein the sample concentration is between 0.5 μ g/ml and 3 mg/ml.

67. The high performance liquid chromatography of claim 66, wherein the sample concentration is 1 μ g/ml to 2 mg/ml.

68. The high performance liquid chromatography of claim 67, wherein the sample concentration is between 2 μ g/ml and 1 mg/ml.

69. The high performance liquid chromatography of claim 45, wherein phase A: the volume ratio of the phase B is 90: 10-97: 3, wherein the phase A is 0.020-0.030% phosphoric acid solution, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210-230nm, the sample injection amount is 5-30 mu l, the column temperature is 28-32 ℃, and the flow rate is 0.8-1.2 ml/min.

70. The high performance liquid chromatography of claim 69, phase A in the mobile phase: and the volume ratio of the phase B is 95:5, wherein the phase A is 0.025 percent phosphoric acid solution, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is any one or the combination of 210nm, 220nm and 230nm, the sample injection amount is 10 mu l-20 mu l, the column temperature is 30 ℃, and the flow rate is 0.9-1.1 ml/min.

71. The high performance liquid chromatography of claim 69, phase A in the mobile phase: the volume ratio of the phase B is 94:6, wherein the phase A is 0.02% phosphoric acid solution, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210nm, 220nm and 230nm, the sample injection amount is 10-20 mul, the column temperature is 30 ℃, the flow rate is as follows: 0.8-1.2 ml/min.

72. The high performance liquid chromatography of claim 45, wherein phase A: the volume ratio of the phase B is 90: 10-97: 3, wherein the phase A is phosphate buffer solution with the pH value of 2-4, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is 210-230nm, the sample injection amount is 5-30 mu l, the column temperature is 28-32 ℃, and the flow rate is 0.8-1.2 ml/min.

73. The high performance liquid chromatography of claim 72, phase A: and the volume ratio of the phase B is 95:5, wherein the phase A is potassium dihydrogen phosphate buffer solution with the pH value of 3-3.5, the phase B is acetonitrile, isocratic elution is carried out, the detection wavelength is any one or the combination of 210nm, 220nm and 230nm, the sample amount is 10-20 mul, the column temperature is 30 ℃, the flow rate: 1 ml/min.

74. The high performance liquid chromatography for high performance separation and detection of relevant substances in hydroquinone is characterized in that a chromatographic column is selected from an octadecyl bonded silica gel reverse phase column, a mobile phase is 0.005-0.05mol/L phosphoric acid aqueous solution, isocratic elution is carried out, the detection wavelength is 210-290nm, the sample injection amount is 5-50 mu L, the column temperature is 25-35 ℃, the flow rate is as follows: 0.6-1.2ml/min, wherein the related substances in the hydroquinone comprise p-benzoquinone and resorcinol, and the related substances also comprise any one or the combination of aniline, trimellitol, phloroglucinol, pyrogallol and potassium hydroquinone sulfate.

75. The high performance liquid chromatography as claimed in claim 74, wherein the mobile phase is 0.01-0.03mol/L phosphoric acid aqueous solution, isocratic elution is performed, the detection wavelength is 210-230nm, the sample amount is 5-30 μ L, the column temperature is 28-32 ℃, the flow rate: 0.6-1.0 ml/min.

76. The high performance liquid chromatography of claim 75, wherein the mobile phase is 0.02mol/L phosphoric acid water solution, the elution is carried out at equal intervals, the detection wavelength is any one or the combination of 210nm, 220nm and 230nm, the sample amount is 10-20 μ L, the column temperature is 30 ℃, and the flow rate: 0.6-0.8 ml/min.

77. The high performance liquid chromatography of any one of claims 45-76, wherein the hydroquinone is not less than 99.0% pure and the hydroquinone is not more than 0.1% para-benzoquinone.

78. The high performance liquid chromatography of claim 77, wherein the hydroquinone in the hydroquinone is not less than 99.5% pure.

79. The high performance liquid chromatography of claim 78, wherein the hydroquinone in the hydroquinone is not less than 99.9% pure.

80. The high performance liquid chromatography of any one of claims 45-76, wherein the p-benzoquinone is present in an amount of no greater than 0.08%.

81. The high performance liquid chromatography of claim 80, wherein the p-benzoquinone is present in an amount of no more than 0.05%.

82. The high performance liquid chromatography of claim 81, wherein the p-benzoquinone is present in an amount of no more than 0.03%.

83. The high performance liquid chromatography of any one of claims 45-76, wherein neither p-benzoquinone nor aniline is present in the hydroquinone in an amount greater than 0.08%.

84. The high performance liquid chromatography of claim 83, wherein neither p-benzoquinone nor aniline is present in the hydroquinone above 0.05%.

85. The high performance liquid chromatography of claim 84, wherein neither p-benzoquinone nor aniline is present in the hydroquinone of greater than 0.03%.

86. The high performance liquid chromatography of any one of claims 45-76, wherein the hydroquinone is not more than 0.3% of total content of related substances other than p-benzoquinone and aniline.

87. The high performance liquid chromatography of claim 86, wherein the hydroquinone is not more than 0.1% by weight of the total content of the relevant substances except p-benzoquinone and aniline.

88. The high performance liquid chromatography of claim 87 wherein the hydroquinone is not more than 0.05% total content of related substances other than p-benzoquinone and aniline.

89. Use of high performance liquid chromatography according to any one of claims 1-88 for the detection of any one of hydroquinone, hydroquinone compositions, hydroquinone preparations.

90. The use according to claim 89, wherein said hydroquinone preparation is a hydroquinone cream.

91. The use according to claim 89, wherein said hydroquinone preparation is selected from any one of hydroquinone pharmaceutical preparations and hydroquinone cosmetics.

92. The use according to claim 89 of a hydroquinone preparation for the preparation of any one of a medicament for depigmenting, whitening or otherwise treating skin disorders, pigmentary spots or pigmentation disorders.

93. The use according to claim 92, wherein the stain is selected from any one of freckles, chloasma, age spots, post-inflammatory hyperpigmentation spots, local pigmentation-induced stains.