CN109633041B - Method for measuring dihydrotestosterone in medicine by derivatization HPLC method - Google Patents
Method for measuring dihydrotestosterone in medicine by derivatization HPLC method Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Abstract
The invention relates to a method for measuring dihydrotestosterone in a medicament by a derivatization HPLC method, which comprises the following steps of (1) performing derivatization reaction on the dihydrotestosterone in the medicament to be measured by taking a benzoyl halide compound as a derivatization reagent to obtain a sample containing a derivative product of the dihydrotestosterone; (2) preparing a test solution by using the sample containing the derivative product of the dihydrotestosterone, and detecting the test solution by using high performance liquid chromatography, wherein a detector of the high performance liquid chromatography is an ultraviolet detector. The method for measuring dihydrotestosterone by derivatization HPLC-UVD established by the invention is simple and good in universality, can be used for measuring the content of the drug in the dihydrotestosterone preparation, and the result verified by methodology shows that the method has specificity, good sensitivity and high accuracy.
Description
Technical Field
The invention relates to the field of drug analysis and detection, in particular to a method for determining dihydrotestosterone in a drug by a derivatization HPLC method.
Background
The type and content of the effective components in the medicine are an important index of the quality of the medicine, and the identification and detection of the effective components are an effective means for controlling the quality of the medicine. One method commonly used in the separation and analysis of pharmaceutically active ingredients is High Performance Liquid Chromatography (HPLC). The major drawback of HPLC is the lack of highly sensitive and versatile detectors, although Ultraviolet (UVD) and Fluorescence (FLD) detectors are widely used in HPLC, a fraction of the samples are not detectable because they do not have uv chromophores or fluorophores.
The penis is not uncommon in clinical pediatrics, belongs to the most common physical sign of male insufficiency, is caused by insufficient production of androgen, and is often accompanied by hypoplasia of other external genitalia such as small testis, cryptorchidism, small scrotum and the like. The development of the penis is mainly dependent on testosterone, an androgen secreted by the leydig cells, which is converted to dihydrotestosterone by the action of 5 α -reductase and then acts on the androgen receptor to grow the penis. For the treatment of children's penis, testosterone preparations (testosterone patch, testosterone tablet, testosterone undecanoate soft capsule and testosterone propionate injection) are used for substitution treatment at home, and dihydrotestosterone preparations (dihydrotestosterone gel and dihydrotestosterone injection) are used at abroad, and as dihydrotestosterone cannot be converted into estradiol in vivo, the side effect of male and female breast development cannot be generated. Therefore, the dihydrotestosterone preparation has incomparable advantages compared with the testosterone and the derivative preparation thereof used clinically in China.
Dihydrotestosterone, also known as 17 beta-hydroxy-5 alpha-androstane-3-one, 5 alpha-dihydrotestosterone or androstanone (stanorone) has no chromogen group capable of generating ultraviolet absorption in the chemical structure, so that dihydrotestosterone and preparations thereof cannot be measured by a conventional HPLC method, and no HPLC measuring method for dihydrotestosterone and preparations is reported in China. At present, there are two methods for measuring dihydrotestosterone reported in China: a radiation/enzyme linked immunization method and a mass spectrum combined method (HPLC-MS method and GC-MS method).
The radio/enzyme linked immunosorbent assay adopts a special immunoassay kit to process samples, combines a radio-immune counter and an enzyme-linked immunosorbent assay to determine, can be used for determining the content of dihydrotestosterone in biological samples such as human body fluid (blood, semen) or tissues (prostate), and is mainly applied to clinical detection. The method comprises the steps of respectively measuring the serum dihydrotestosterone level of male androgenetic alopecia patients by using an enzyme-linked immunosorbent assay and a radioimmunoassay, measuring the dihydrotestosterone level in the serum and the semen of oligospermia patients by using a radioimmunoassay, such as the eucrypti, and the like, and measuring the dihydrotestosterone content in different prostate tissues by using a radioimmunoassay, such as the Zhanguan, and the like. The immunoassay is an analytical method established based on the formation of a precipitate by combining an antigen and an antibody, is commonly used for detecting enzymes, hormones, proteins and other substances or elements, and has a plurality of advantages, but has three general problems: the method is only an auxiliary means for diagnosis, and has the defects of easy occurrence of cross reaction, false positive reaction, matrix interference, lack of specificity, low sensitivity and the like; the operation process is more complicated, the sample treatment time is long, and the sample is easily influenced by degrading enzyme, salt, pH and the like; and thirdly, determining that the sample is a biological sample and cannot detect the medicinal components in the preparation.
The mass spectrum combined method adopts an analysis method of combining a chromatograph (liquid chromatogram HPLC, gas chromatogram GC) and a Mass Spectrometer (MS), can combine the characteristics of high-efficiency separation of gas (or liquid) chromatogram with the advantages of high-sensitivity detection of mass spectrum, and obtains better analysis effect. Because of high sensitivity and strong specificity, the method becomes a powerful tool for detecting complex matrix samples (such as biological samples), and is widely applied to the fields of various clinical applications such as drug monitoring, newborn screening, toxicology diagnosis and the like and new drug research and development at present. Chinese patent CN102239266A (method for detecting dihydrotestosterone by mass spectrometry) discloses a method for determining the amount of underivatized dihydrotestosterone in a body fluid sample. This patent uses ionization of underivatized dihydrotestosterone purified by Solid Phase Extraction (SPE) from a body fluid sample, which is plasma or serum, and determination of the amount of one or more ions by mass spectrometry, said purification further comprising purification by HPLC. Chenjun et al established a solid phase extraction method suitable for dihydrotestosterone in tissue samples, and developed a GC/MS method based on a secondary mass spectrometry technology, which is applied to the determination of dihydrotestosterone content in rat prostate samples. The mass spectrometry method reported above, which uses HPLC and GC to separate dihydrotestosterone from plasma, serum and prostate tissue biological samples, and uses a mass spectrometer to measure the amount of one or more ions, so as to calculate the dihydrotestosterone content in the biological samples, has three broad aspects: the method needs a Mass Spectrometer (MS) and a conventional analyzer for joint detection besides HPLC and GC, and has high expense of MS instruments and experimental consumables and high cost of experimental detection and instrument maintenance; the operation process is complicated, the sample processing time is long, the sample of GC-MS analysis requires organic solution, the aqueous solution of the medicine cannot be measured, the mobile phase of LC-MS analysis does not contain volatile salt, and the measured samples are all biological samples and cannot detect the medicine components in the preparation.
In summary, the reported methods for measuring dihydrotestosterone, namely the radio/enzyme-linked immunosorbent assay, are mainly applied to clinical detection, and the detection cost of the mass spectrometry is high, so that two main problems exist: firstly, the operation process is complicated, and the sample treatment influence factors are more; ② the measured sample is a biological sample, and the medicinal components in the preparation can not be detected. The method has great influence on the quality analysis of the dihydrotestosterone preparation and the development of new drugs, and the method for measuring the content of the dihydrotestosterone preparation, which is simple to operate and is universal, is urgently needed.
Disclosure of Invention
Based on the method, the invention provides a method for determining dihydrotestosterone in a medicament by a derivatization HPLC method.
The specific technical scheme is as follows:
a method for determining dihydrotestosterone in a medicament, comprising the steps of:
(1) performing derivatization reaction on dihydrotestosterone in a to-be-detected medicament by taking a benzoyl halide compound as a derivatization reagent to obtain a sample containing a dihydrotestosterone derivatization product;
(2) preparing a test solution by using the sample containing the derivative product of the dihydrotestosterone, and detecting the test solution by using high performance liquid chromatography, wherein a detector of the high performance liquid chromatography is an ultraviolet detector.
In some of these embodiments, the benzoyl halide compound is selected from any one of benzoyl chloride, p-nitrobenzoyl chloride, and 3, 5-dinitrobenzoyl chloride.
In some of these embodiments, the derivatization reaction is performed in an aprotic polar solvent.
In some of these embodiments, the aprotic polar solvent is selected from at least one of pyridine, dichloromethane, dimethylsulfoxide, acetone, acetonitrile, dimethylformamide, and dimethylacetamide.
In some of these embodiments, the aprotic polar solvent is a mixed solvent of pyridine and dichloromethane in a volume ratio of 1: 4-6.
In some of these embodiments, the derivatization reaction is carried out at a temperature of 20 to 90 ℃ for a time of 0.5 to 48 hours.
In some of these embodiments, the derivatization reaction is carried out at a temperature of 20 to 30 ℃ for a time of 1.5 to 2.5 hours.
In some embodiments, the mass ratio of the drug to be tested to the benzoyl halide compound is 1: 0.5-5.0; the concentration of the benzoyl halide compound in the reaction liquid is 10-500 mg/mL.
In some embodiments, the mass ratio of the drug to be tested to the benzoyl halide compound is 1: 1.0-3.0.
In some embodiments, the concentration of the benzoyl halide compound in the reaction solution is 380-450 mg/mL.
In some embodiments, the test drug is extracted with an organic solvent before the derivatization reaction, and the resulting extract is subjected to the derivatization reaction.
In some of these embodiments, the number of extractions is from 2 to 6, and the organic solvent used for each extraction is independently selected from ethyl acetate, ethanol, methanol, n-butanol, cyclohexane.
In some of these embodiments, the chromatographic conditions for detection by high performance liquid chromatography include:
a chromatographic column: a Zorbax Eclipse plus C18 column;
mobile phase: acetonitrile, tetrahydrofuran and triethylamine water solution with the volume ratio of 77: 5: 18, wherein the triethylamine water solution contains 0.08-0.12 wt% of triethylamine;
or the chromatographic conditions for detection by high performance liquid chromatography include:
a chromatographic column: ZORBAX STAbleBond-C18 column;
mobile phase: methanol and triethylamine aqueous solution with the volume ratio of 95: 5, wherein the triethylamine aqueous solution contains 0.2 wt% of triethylamine;
or the chromatographic conditions for detection by high performance liquid chromatography include:
a chromatographic column: ZORBAX STAbleBond-C18 column;
mobile phase: acetonitrile and aqueous ammonia in a volume ratio of 80: 20.
In some of these embodiments, the chromatographic conditions for detection by high performance liquid chromatography include:
a chromatographic column: a Zorbax Eclipse plus C18 column;
mobile phase: acetonitrile, tetrahydrofuran and triethylamine water solution with the volume ratio of 77: 5: 18, wherein the triethylamine water solution contains 0.08-0.12 wt% of triethylamine;
column temperature: 24-26 ℃;
flow rate: 0.8 mL/min;
detection wavelength: 235 nm;
or the chromatographic conditions for detection by high performance liquid chromatography include:
a chromatographic column: ZORBAX STAbleBond-C18 column;
mobile phase: methanol and triethylamine aqueous solution with the volume ratio of 95: 5, wherein the triethylamine aqueous solution contains 0.2 wt% of triethylamine;
column temperature: 24-16 ℃;
flow rate: 1.0 mL/min;
detection wavelength: 254 nm;
or the chromatographic conditions for detection by high performance liquid chromatography include:
a chromatographic column: ZORBAX STAbleBond-C18 column;
mobile phase: acetonitrile and ammonia water solution with the volume ratio of 80: 20;
column temperature: 34-36 ℃;
flow rate: 1.0 mL/min;
detection wavelength: 230 nm.
In some of these embodiments, the drug is dihydrotestosterone gel, dihydrotestosterone cream, dihydrotestosterone ointment, or dihydrotestosterone injection.
The method for determining dihydrotestosterone in the medicament by the HPLC method provided by the invention utilizes the most commonly used detector, namely an ultraviolet detector (UVD), for detection, and is based on the rapid reaction of hydroxyl of the dihydrotestosterone and benzoyl halide, the originally ultraviolet-absorption-free dihydrotestosterone is subjected to derivatization treatment by benzoyl halide compounds, and due to the existence of nitro electron-withdrawing groups on a benzene ring, the derivatization product of the dihydrotestosterone has strong absorption in an ultraviolet visible light region, and the ultraviolet absorption of auxiliary materials and impurities in the medicament is weak, so that the qualitative or quantitative detection of the dihydrotestosterone by the HPLC method is realized. The method for measuring dihydrotestosterone by derivatization HPLC-UVD established by the invention is simple and good in universality, can be used for measuring the content of the drug in the dihydrotestosterone preparation, and the result verified by methodology shows that the method is special, good in sensitivity and high in accuracy.
Drawings
Fig. 1 shows the results of the system adaptability test, wherein a is a negative control solution, B is a dihydrotestosterone standard solution, C is a dihydrotestosterone standard derivative, and D is a dihydrotestosterone gel derivative.
Detailed Description
The method for measuring dihydrotestosterone in a drug according to the present invention is further described in detail with reference to the following specific examples.
The proportions of the mobile phases in the following examples are volume ratios, and the percentages not specifically mentioned are mass percentages.
The apparatus used was: agilent 1200HPLC (Agilent, usa); sartorious BS-224S electronic balance (beijing sidoris balance, ltd); FA25 homogenizer (fleuke, germany); JY92-2D ultrasonic cleaning machine (Ningbo Xinzhi Biotechnology Co., Ltd.); SH23-2 constant temperature magnetic stirrer (Shanghai Meipu Instrument manufacturing Co., Ltd.).
The reagents used were: dihydrotestosterone (Shandong platinum source pharmaceutical Co., Ltd., content > 99.1%); injection-grade soybean lecithin (Shanghai Taiwei pharmaceutical Co., Ltd., PC not less than 92.6%); cholesterol (national drug group chemical agents limited); andractim dihydrotestosterone gel (laboratory Besins International, 2.5%); poloxamer 188 and poloxamer 407 (Shanghai Chang is a pharmaceutical adjuvant technology company, Ltd.); p-nitrobenzoyl chloride (Alfa Aesar, usa); acetonitrile (chromatographically pure, dima, usa); the auxiliary materials of other preparations are all medicinal grade, and the reagents are all analytically pure.
The dihydrotestosterone gel used in the following examples was prepared by the following method:
mixing 94010 g of carbomer, 802 g of polysorbate and 800ml of distilled water, swelling to form a semitransparent solution, and dropwise adding 13.5g of triethanolamine while stirring to obtain a gel matrix; dissolving 25g of dihydrotestosterone passing through a No. nine sieve in 50g of ethanol, gradually adding the gel matrix after complete dissolution, stirring uniformly, adding 1g of ethylparaben and distilled water to the total weight of 1000g, and stirring uniformly to obtain transparent dihydrotestosterone gel.
The dihydrotestosterone ointment used in the following examples was prepared by the following method:
placing 30g of stearyl alcohol and 80g of white beeswax in a container, melting in a water bath, adding 30g of cholesterol, stirring until the cholesterol is dissolved, finally adding 805g of white vaseline, and continuously heating to 70-80 ℃ to obtain a matrix; grinding 25g of dihydrotestosterone passing through a No. nine sieve and 20030 g of dimethyl silicone polymer into paste, slowly adding the matrix, rapidly stirring, cooling and coagulating under stirring to obtain dihydrotestosterone ointment.
The dihydrotestosterone cream used in the following examples was prepared by the following method:
putting 120g of stearic acid, 35g of glycerin monostearate, 60g of liquid paraffin, 10g of white vaseline and 50g of lanolin in a container, heating and melting in a water bath, and continuously heating to 70-80 ℃ to obtain an oil phase; grinding 25g of dihydrotestosterone passing through a No. nine sieve and 60g of liquid paraffin into paste, slowly adding an oil phase, rapidly stirring, and keeping the temperature at 70-80 ℃ to obtain a medicine-containing oil phase; and heating 4g of triethanolamine, 5g of glycerol and about 700g of purified water to 70-80 ℃, slowly pouring the water phase into the medicine-containing oil phase, rapidly stirring along the same direction while adding, continuously stirring for several minutes on a water bath, and continuously stirring at room temperature until emulsification and condensation are carried out, thus obtaining the dihydrotestosterone O/W type cream.
The dihydrotestosterone injection used in the following examples was prepared by the following method:
preparation of the dihydrotestosterone lipid nanoparticle, 250g of glyceryl behenate, 100g of lecithin and 10g of dihydrotestosterone in the formula amount are weighed and added into 250mL of ethanol for sealing, and the mixture is heated at 85 ℃ to be melted to be used as an oil phase. Pluronic F-6850 g and Tween 80125 g were ultrasonically dispersed in 150mL of water for injection, and heated to the same temperature to be used as a water phase. Dropwise adding the water phase into the oil phase at the same temperature under magnetic stirring at a dropping speed of 10mL/min, uniformly stirring at 2000r/min, and stably dispersing for l0min to form primary emulsion. And (3) redispersing the colostrum by using an ultrasonic cell crusher at room temperature water bath (20 ℃), carrying out ultrasonic treatment with the ultrasonic power of 600W for 1sec, carrying out intermittent l sec for 10min, cooling by using an ice water bath, and filtering by using a 0.22 mu m microporous membrane to obtain the dihydrotestosterone lipid nanoparticle solution.
Freeze-drying process of dihydrotestosterone injection (freeze-dried powder injection), in the last step, all dihydrotestosterone lipid nanoparticle solution is prepared, 65g of mannitol is added, injection water is added till 1000mL, the mixture is stirred uniformly, 2 mL/bottle is subpackaged into ampoule bottles, and freeze-dried powder is prepared by adopting a freeze-drying method. The freeze-drying process flow comprises the following steps: (1) pre-freezing, namely putting the subpackaged medicines on a partition plate in a freeze-drying box, and pre-freezing to +25 to-40 ℃ for 2 hours; -40 ℃,3 h; (2) sublimation drying, namely reducing the temperature of a condenser to below-45 ℃, starting a vacuum pump, slowly opening a butterfly valve after the vacuum degree reaches a certain value, closing the refrigerator when the vacuum degree in a drying box reaches below 13.33Pa (0.1mmHg), slowly heating the refrigerator by a heating system below a partition plate to gradually increase the temperature of a frozen product to-20 ℃, and then gradually increasing the temperature to 25 ℃; (3) and drying, namely maintaining the vacuum degree below 10Pa for 5-8 h at 25 ℃. Then filling nitrogen, pressing, taking out of the box, and rolling the opening by using an aluminum-plastic combined cover to obtain the dihydrotestosterone injection (freeze-dried powder injection), wherein the specification is as follows: about 1.2 g/bottle, containing 20 mg/bottle of dihydrotestosterone.
Example 1: benzoyl chloride derivatization HPLC method for determining content of dihydrotestosterone in dihydrotestosterone medicine
In the embodiment, pyridine-benzoyl chloride is used as a derivatization reagent, and after benzoylation is carried out on hydroxyl in dihydrotestosterone molecules, RP-HPLC is adopted for determination. The method comprises the following specific steps:
(1) preparation of control solutions
10mg of dihydrotestosterone reference substance is precisely weighed and dissolved by adding 25mL of methanol to serve as a reference substance solution.
(2) Preparation of test solution
Precisely weighing 1g of dihydrotestosterone gel (or dihydrotestosterone ointment or dihydrotestosterone cream or dihydrotestosterone injection 1 bottle), reflux-extracting with ethyl acetate 30mL for 10h, volatilizing ethyl acetate, reflux-extracting with ethanol for 12h, recovering ethanol, transferring the residue with a small amount of distilled water to a 50mL separating funnel, and extracting with water saturated n-butanol for 4 times, 10mL each time. And (3) combining the n-butanol layers, volatilizing, performing derivatization reaction on the residues (specifically, putting the obtained residues into a25 mL round-bottom flask with a stopper, dissolving the residues with 5mL pyridine, adding 2.0g benzoyl chloride at the temperature of ice-water bath, sealing, shaking uniformly, standing at room temperature for 24h to complete the reaction), dissolving the obtained reaction liquid with acetonitrile, transferring the reaction liquid into a25 mL measuring flask, fixing the volume with the acetonitrile, and filtering with a 022 mu m microporous filter membrane to obtain a sample solution.
(3) HPLC-UVD detection
And (3) taking 20 mu L of the sample solution prepared in the step (2) and carrying out sample injection analysis according to chromatographic conditions.
The chromatographic conditions were as follows: a chromatographic column: a Zorbax Eclipse plus C18 column; mobile phase: acetonitrile-tetrahydrofuran-0.1% aqueous triethylamine (77: 5: 18); column temperature: 25 ℃ of: flow rate: 0.8 mL/min; detection wavelength: 235 nm; the sample injection amount is 20 mu L.
(4) The result of the detection
The results are shown in Table 1.
TABLE 1 determination of dihydrotestosterone content in dihydrotestosterone drugs (n-3) mg/g or mg/bottle
Example 2: method for determining content of dihydrotestosterone in dihydrotestosterone medicine by using p-nitrobenzoyl chloride derivatization HPLC (high performance liquid chromatography)
In the embodiment, dichloromethane-pyridine-p-nitrobenzoyl chloride is used as a derivatization reagent, and after benzoylation is carried out on hydroxyl in a dihydrotestosterone molecule, RP-HPLC method is adopted for determination. The method comprises the following specific steps:
(1) preparation of control solutions
10mg of dihydrotestosterone reference substance is precisely weighed, and 25mL of acetonitrile is added to be dissolved to be used as a reference substance solution.
(2) Preparation of test solution
Precisely weighing 1g dihydrotestosterone gel (or dihydrotestosterone ointment or dihydrotestosterone cream 1g or dihydrotestosterone injection 1 bottle), reflux-extracting with ethyl acetate 30mL for 8h, volatilizing ethyl acetate, placing the residue in a conical flask, adding methanol 20mL, ultrasonically extracting for 60min, filtering, collecting the filtrate, volatilizing, performing derivatization reaction on the residue (specifically, placing the residue in a25 mL round-bottomed flask with a plug, dissolving with dichloromethane-pyridine (5:1)5mL, adding p-nitrobenzoyl chloride (2.0g), sealing, and placing 25mL round-bottomed flask with a plug, adding p-nitrobenzoyl chloride (2.0g), sealing, and packaging℃Performing ultrasonic treatment for 30min, standing for 2h, adding 5mL of water, performing ultrasonic treatment for 30min, transferring to a separating funnel, standing for 1d, taking the lower clear liquid, eluting with 300mL of 70% ethanol aqueous solution through a macroporous adsorption resin column (20cm multiplied by 2.0cm), then eluting with 400mL of absolute ethanol, collecting the absolute ethanol eluate, evaporating to dryness for later use, dissolving with methanol, transferring to a 10mL measuring flask, fixing the volume to the scale, and filtering with a 0.22 mu m microporous membrane to obtain a sample solution.
(3) HPLC-UVD detection
And (3) taking 20 mu L of the sample solution prepared in the step (2) and carrying out sample injection analysis according to chromatographic conditions.
The chromatographic conditions were as follows: a chromatographic column: ZORBAX STAbleBond-C18 column (4.4 mm. times.250 mm,5 μm); mobile phase: methanol-0.2% aqueous triethylamine (95: 5); column temperature: 25 ℃ of: flow rate: 1.0 mL/min; detection wavelength: 254 nm; the sample injection amount is 20 mu L.
(4) The result of the detection
The results are shown in Table 2.
TABLE 2 determination of dihydrotestosterone content in dihydrotestosterone drugs (n-3) mg/g or mg/bottle
(5) Methodology validation experiment
(5.1) preparation of Standard solution
10.30mg of dihydrotestosterone raw material is precisely weighed, and 25mL of methanol is added for dissolution, so as to obtain a dihydrotestosterone stock solution with the concentration of 412.00 mu g/mL. And (4) adding methanol into the dihydrotestosterone stock solution for dilution to prepare standard solutions with different concentrations.
(5.2) preparation of test solution
A test solution was prepared by the method in this example (2).
(5.3) chromatographic conditions
A chromatographic column: ZORBAX STAbleBond-C18 column (4.4 mm. times.250 mm,5 μm); mobile phase: methanol-0.2% aqueous triethylamine (95: 5); column temperature: 25 ℃ of: flow rate: 1.0 mL/min; detection wavelength: 254 nm; the sample injection amount is 20 mu L.
(5.4) System Adaptation
The negative control solution was prepared by the procedure of "(5.1) preparation of standard solution without adding dihydrotestosterone, and then by the procedure of" (5.2) preparation of test solution without adding p-nitrobenzoyl chloride.
The standard solution of dihydrotestosterone is obtained by the operation under the item of (5.1) preparation of the standard solution.
And (5) operating under the item of (5.1) preparation of standard solution, and then operating under the item of (5.2) preparation of test solution to obtain the dihydrotestosterone standard product derivative.
The dihydrotestosterone gel derivative is obtained by the operation under the item of (5.2) preparation of a test solution.
Taking the negative control solution, the dihydrotestosterone standard product derivative and the dihydrotestosterone gel derivative, performing liquid phase determination under the (5.3) chromatographic condition, and determining an HPLC chromatogram as shown in figure 1: under the chromatographic condition, other components in the reference substance and the sample can be separated from a base line, the separation degree of the other components from adjacent chromatographic peaks is more than 1.5, and the number of theoretical plates is not less than 3000.
(5.5) examination of Linear relationship
Precisely transferring 0.5,1,2,3,1,2mL of dihydrotestosterone stock solution, placing the dihydrotestosterone stock solution into a 50,50,50,50,10,10mL measuring flask, adding a mobile phase to dilute the dihydrotestosterone stock solution to a scale, shaking the dihydrotestosterone stock solution to prepare series of standard solution with the concentration of 4.12 mu g/mL, 8.24 mu g/mL, 12.36 mu g/mL, 24.76 mu g/mL, 41.20 mu g/mL and 82.40 mu g/mL in sequence, treating the sample according to the item of preparation of a test solution of (5.2), sucking 20 mu l of the standard solution, injecting the sample into a liquid chromatograph, measuring under the chromatographic condition of (5.3), and recording a chromatogram and a peak area. By peak area Y versus mass concentration X (. mu.g.mL)-1) Linear regression calculation is carried out, and the linear regression equation of the dihydrotestosterone is shown in the specification of Y-84517X-29.878 and r-0.9997. The result shows that the concentration of the dihydrotestosterone is in a good linear relation with the peak area within the concentration range of 4.12-82.40 mu g/mL. .
(5.6) precision test
And (3) taking the same standard substance solution, processing the sample according to the item of preparation of the test sample solution (5.2), determining by a liquid chromatograph according to the item of chromatographic condition (5.3), repeatedly injecting samples for 6 times, and calculating the RSD of the peak area of the dihydrotestosterone to be 0.89%, thereby indicating that the precision of the instrument is good.
(5.7) repeatability test
Taking the same batch of dihydrotestosterone gel, processing samples according to the item of preparation of the test solution (5.2), preparing 6 samples in parallel, sucking 20 mul of dihydrotestosterone gel, injecting into a liquid chromatograph, measuring according to the item of chromatographic condition (5.3), recording a chromatogram and peak area, and calculating the content. As a result, the average dihydrotestosterone content was 24.7276mg/g, and the RSD was 1.55%, indicating that the test method of this example has good reproducibility.
(5.8) stability examination
Precisely sucking 20 mu L of dihydrotestosterone gel sample solution of the same batch, sampling and measuring in 0,2,4,8,12,16 and 24 hours respectively, and calculating the RSD of the dihydrotestosterone peak area to be 1.18 percent, which indicates that the sample solution has good stability in 24 hours.
(5.9) sample application recovery test
1.2012g of dihydrotestosterone raw material is precisely weighed, acetonitrile is added for dissolution, the volume is determined to be 50mL, and a dihydrotestosterone high-concentration solution with the concentration of 24.024mg/mL is obtained. Taking the same batch of dihydrotestosterone gel with known content, respectively and precisely adding a certain amount of dihydrotestosterone high-concentration solution, processing the sample according to the item of the preparation of the (5.2) test sample solution, measuring the liquid phase according to the (5.3) chromatographic condition, and calculating the sample adding recovery rate, wherein the result is shown in the table 3.
TABLE 3 sample recovery test results
(5.10) detection Limit
Taking 20 mu L of dihydrotestosterone derivative solution with certain concentration, and determining the solution by liquid phase according to the chromatographic condition of (5.3). As a result, the detection limit of the derivative was about 4.12ng (S/N — 3); in addition, a dihydrotestosterone solution with a certain concentration is taken, a sample is processed according to the item of preparation of the test sample solution (5.2), 20 mu L of feed liquid is absorbed, and the sample is determined under the item of chromatographic condition (5.3), and the detection limit of the dihydrotestosterone is 41.20ng (S/N is 3), which shows that the detection limit is reduced by 10 times after the dihydrotestosterone is subjected to derivatization treatment by using the method of the embodiment, the dihydrotestosterone drug sample with lower content can be determined, the base line is more stable, and the determination is more accurate.
Example 3: method for determining content of dihydrotestosterone in dihydrotestosterone medicine by 3, 5-dinitrobenzoyl chloride derivatization HPLC (high performance liquid chromatography)
In the embodiment, pyridine-3, 5-dinitrobenzoyl chloride is used as a derivatization reagent, and after benzoylation is carried out on hydroxyl in dihydrotestosterone molecules, RP-HPLC method is adopted for determination. The method comprises the following specific steps:
(1) preparation of control solutions
10mg of dihydrotestosterone reference substance is precisely weighed, and 25mL of acetonitrile is added to be dissolved to be used as a reference substance solution.
(2) Preparation of test solution
Precisely weighing 1g of dihydrotestosterone gel or dihydrotestosterone ointment or dihydrotestosterone cream or 1 bottle of dihydrotestosterone injection, reflux-extracting with 30mL of ethanol for 2h, filtering, recovering ethanol, extracting the residue with cyclohexane for three times (15 mL each time), mixing cyclohexane layers, adding anhydrous Na2SO4About 2g, dehydrating, filtering, volatilizing, performing derivatization reaction on the residue (specifically, the obtained residue is placed in a 20mL test tube with a plug, dissolved by 5mL of anhydrous pyridine, added with 3, 5-dinitrobenzoyl chloride (2.0g) for sealing, placed on a 75 ℃ water bath for refluxing for 1h, placed for 2h, taken out and dried by nitrogen flow under reduced pressure to obtain the product), dissolved by acetonitrile, transferred into a 10mL measuring flask, fixed to the volume to the scale, and filtered by a 0.45 mu m microporous membrane to obtain a sample solution.
(3) HPLC-UVD detection
And (3) taking 20 mu L of the sample solution prepared in the step (2) and carrying out sample injection analysis according to chromatographic conditions.
The chromatographic conditions were as follows: a chromatographic column: ZORBAX STAbleBond-C18 column; mobile phase: acetonitrile-aqueous ammonia (80: 20); column temperature: 35 ℃ of: flow rate: 1.0 mL/min; detection wavelength: 230 nm; the sample injection amount is 20 mu L.
(4) The result of the detection
The results are shown in Table 4.
TABLE 4 determination of dihydrotestosterone content in dihydrotestosterone drugs (n-3) mg/g or mg/bottle
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (7)
1. A method for determining dihydrotestosterone in a medicament, which is characterized by comprising the following steps:
(1) performing derivatization reaction on dihydrotestosterone in a to-be-detected medicament by taking a benzoyl halide compound as a derivatization reagent to obtain a sample containing a dihydrotestosterone derivatization product;
(2) preparing a test solution from the sample containing the derivative product of dihydrotestosterone, and detecting the test solution by using high performance liquid chromatography, wherein a detector of the high performance liquid chromatography is an ultraviolet detector;
the benzoyl halide compound is selected from any one of benzoyl chloride, paranitrobenzoyl chloride and 3, 5-dinitrobenzoyl chloride;
the chromatographic conditions for detection by the high performance liquid chromatography include:
a chromatographic column: a Zorbax Eclipse plus C18 column;
mobile phase: acetonitrile, tetrahydrofuran and triethylamine water solution with the volume ratio of 77: 5: 18, wherein the triethylamine water solution contains 0.08-0.12 wt% of triethylamine;
column temperature: 24-26 ℃;
flow rate: 0.8 mL/min;
detection wavelength: 235 nm.
2. The method for measuring dihydrotestosterone in a drug according to claim 1, wherein said derivatization reaction is performed in an aprotic polar solvent selected from at least one of pyridine, dichloromethane, dimethylsulfoxide, acetone, acetonitrile, dimethylformamide, and dimethylacetamide.
3. The method for determining dihydrotestosterone in a drug according to claim 1 or 2, wherein the reaction temperature of the derivatization reaction is 20-90 ℃ and the reaction time is 0.5-48 hours.
4. The method for determining dihydrotestosterone in drugs according to claim 1 or 2, wherein the mass ratio of the drug to be tested to the benzoyl halide compound is 1: 0.5-5.0; the concentration of the benzoyl halide compound in the reaction liquid is 10-500 mg/mL.
5. The method for determining dihydrotestosterone in a drug according to claim 1 or 2, wherein the drug to be tested is extracted with an organic solvent before the derivatization reaction, and the obtained extract is subjected to the derivatization reaction.
6. The method for detecting dihydrotestosterone in a medicament according to claim 5, wherein the number of times of extraction is 2-6, and the organic solvent used in each time is independently selected from ethyl acetate, ethanol, methanol, n-butanol and cyclohexane.
7. The method for measuring dihydrotestosterone in a drug according to claim 1 or 2, wherein the drug is dihydrotestosterone gel, dihydrotestosterone cream, dihydrotestosterone ointment or dihydrotestosterone injection.
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| CN114414721B (en) * | 2021-12-03 | 2023-06-09 | 浙江新安化工集团股份有限公司 | Method for measuring content of 2-nitro-4-methylsulfonyl benzoyl chloride and preparation method of derivative thereof |
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