CN109633041A - The method that derivatization HPLC method measures protona in drug - Google Patents
The method that derivatization HPLC method measures protona in drug Download PDFInfo
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
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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
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- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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Abstract
The present invention relates to the methods of protona in a kind of derivatization HPLC method measurement drug, the following steps are included: (1) performs the derivatization reaction to the protona in drug to be measured using benzoyl halogen class compound as derivatization reagent, the sample containing protona derivatization product is obtained；(2) with the sample preparation test solution containing protona derivatization product, the test solution is detected with high performance liquid chromatography, the detector of the high performance liquid chromatography is UV detector.The method of derivatization HPLC-UVD that the present invention establishes measurement protona is simple, versatility is good, can be used for protona preparation of traditional Chinese medicine assay, and the specificity of this method as the result is shown through methodology validation, sensitivity is good, accuracy is high.
Description
Technical field
The present invention relates to drugs analysis detection fields, more particularly to double hydrogen testis in a kind of derivatization HPLC method measurement drug
The method of ketone.
Background technique
Contained in drug imitate ingredient type and content how much be drug quality quality an important indicator, to effective
Ingredient identified and detected be Control of drug quality a kind of effective means.Divide in the analysis of variance in effective ingredient and commonly uses
A kind of method be high performance liquid chromatography (High Performance Liquid Chromatography, HPLC).HPLC
Major defect be the absence of the good detector of high sensitivity versatility, although UV detector (Ultraviolet Detector,
UVD it) is used widely in HPLC with fluorescence detector (Fluorescence Detector, FLD), but still has one
Divide sample that can not examine, because they do not have ultraviolet chromophore or fluorogen.
Microcaulia is not uncommon on Pediatric Clinic, belongs to manlike incomplete most common sign, is generated by androgen insufficient
Cause, it is bad to be often accompanied by other Development of External Genital such as micro-orchidia, cryptorchidism, small scrotum.The development of penis is depended on by testis
Androgen-testosterone of ball interstitial cell secretion, testosterone are converted into protona under 5α-reductase effect, act on male sharp
Plain receptor grows penis.Children microcaulia is treated, at home mostly with testosterone class preparation (testosterone patch, methyltestosterone tablets, ten
One sour testosterone soft capsule, testosterone propionate injection) replacement therapy, and it is external mostly with protona preparation (protona gelling agent,
Protona injection) it treats, since protona cannot be converted to estradiol in vivo, Gynecomastia hair will not be generated
The side effect educated.Therefore, protona preparation has compared with the testosterone of domestic clinical use and its derivative formulations without comparable
Quasi- advantage.
Protona, -5 α of also known as 17 beta-hydroxy-hero [steroid] alkane -3- ketone, 5 α-dihydrotestosterone or androstane-3-one
(stanorone), because in its chemical structure without can produce the chromophore of UV absorption, therefore, protona and its preparation without
Method is domestic also to report without the HPLC measuring method of protona and preparation using conventional HPLC method measurement.Currently, domestic report
Protona measuring method there are two types of: radiation/enzyme-linked immunization, Mass Spectrometry (HPLC-MS method, GC-MS method).
Radiation/enzyme-linked immunization handles sample using specifc immunity kit, in conjunction with radiation immunity arithmometer, microplate reader
Measurement can be used for measuring human body fluid (blood, sperm) or organize the assay of protona in biological samples such as (prostates),
It is mainly used in clinical detection.Wang Xia etc., Yang Liping etc. apply enzyme-linked immunization, measured by radioimmunoassay male's hero to swash respectively
Disposition hair loss patient serum protona is horizontal, and Zhang Xueqing etc. is using measured by radioimmunoassay Oligospermia serum and essence
Protona is horizontal in liquid, and Zhang Yuanfang etc. is using protona content in the different prostata tissues of measured by radioimmunoassay.Exempt from
Epidemic disease method is to generate to precipitate established analytic approach in conjunction with antibody based on antigen, is usually used in enzyme, hormone, protein and other objects
The detection such as matter or element, has the advantages that many, but there is also three broad aspect problems: 1. is the supplementary means diagnosed, easily out
The disadvantages of existing cross reaction, false positive reaction, matrix interference, lack specificity, and sensitivity is lower；2. operating process is cumbersome,
Sample processing time is long, influences vulnerable to degrading enzyme and salt and pH etc.；3. it is biological sample that, which measures sample, cannot detect in preparation
Drug ingedient.
Mass Spectrometry is used using chromatograph (liquid chromatogram HPLC, gas-chromatography GC) and mass spectrograph (MS) combination
Analysis method can combine gas phase (or liquid phase) chromatography with mass spectrographic highly sensitive the advantages of detecting the characteristics of efficiently separating
Come, obtains better analytical effect.Because high sensitivity and high specificity have become complicated substrate sample (such as biological sample) detection
Strong tool, be widely used at present drug surveillance, neonatal screening, toxicology diagnosis etc. various clinicals application and
New drug development field.Chinese patent CN102239266A (passing through the method that mass spectrometry detects protona) discloses measurement
The method of the amount of not derivative protona in humoral sample.The patent passes through Solid Phase Extraction (SPE) from body fluid using ionization
The not derivative protona of Sample Purification on Single, the amount of one or more ions is measured by mass spectrometry, described to purify into one
Step includes being purified with HPLC row, and the humoral sample is blood plasma or serum.Chen Jun etc. establishes one kind suitable for tissue sample
The solid phase extraction method of protona, and developed a kind of GC/MS method based on second order ms technology, before being applied to rat
It arranges in adenoid product, the measurement of protona content.The Mass Spectrometry of above-mentioned report, using HPLC, GC separated plasma, serum and
Protona in prostata tissue biological sample, and be combined mass spectrograph and measure the amount of one or more ions, to calculate biology
The content of protona in sample, has three broad aspects: 1. also needs mass spectrum in addition to needing conventional analysis device instrument HPLC, GC
Instrument (MS) joint-detection therewith, MS instrument, experiment consumptive material expense are high, experiment detection, instrument maintenance it is at high cost；2. is operated
Process is cumbersome, and sample processing time is long, and the sample requirement of GC-MS analysis is organic solution, and the aqueous solution of drug can not measure,
LC-MS analysis mobile phase in should not contain non-volatile salt, 3. measure sample be biological sample, cannot detect preparation of traditional Chinese medicine at
Point.
In conclusion reported protona measuring method, radiation/enzyme-linked immunization are mainly used in clinical detection,
Mass Spectrometry testing cost is high, and there are two main problems: 1. operating process is cumbersome, and sample treatment influence factor is more；
2. measurement sample is biological sample, preparation of traditional Chinese medicine ingredient cannot be detected.This opens the quality analysis of protona preparation, new drug
Hair causes extreme influence, and there is an urgent need to a kind of easy to operate, general protona formulation content measuring methods.
Summary of the invention
Based on this, the present invention provides the methods of protona in a kind of derivatization HPLC method measurement drug.
Specific technical solution is as follows:
A kind of method of protona in measurement drug, comprising the following steps:
(1) protona in drug to be measured is performed the derivatization instead using benzoyl halogen class compound as derivatization reagent
It answers, obtains the sample containing protona derivatization product；
(2) with the sample preparation test solution containing protona derivatization product, by the test solution
It is detected with high performance liquid chromatography, the detector of the high performance liquid chromatography is UV detector.
In wherein some embodiments, the benzoyl halogen class compound be selected from chlorobenzoyl chloride, paranitrobenzoyl chloride and
Any one in DNBC 3,5 dinitrobenzoylchloride.
In wherein some embodiments, the derivative reaction carries out in aprotic polar solvent.
In wherein some embodiments, the aprotic polar solvent be selected from pyridine, methylene chloride, dimethyl sulfoxide, acetone,
At least one of acetonitrile, dimethylformamide and dimethyl acetamide.
In wherein some embodiments, the aprotic polar solvent is the pyridine and methylene chloride that volume ratio is 1:4-6
Mixed solvent.
In wherein some embodiments, the reaction temperature of the derivative reaction is 20-90 DEG C, reaction time 0.5-
48 hours.
In wherein some embodiments, the reaction temperature of the derivative reaction is 20-30 DEG C, reaction time 1.5-
2.5 hour.
In wherein some embodiments, the mass ratio of the drug to be measured and the benzoyl halogen class compound is 1:0.5-
5.0；Concentration of the benzoyl halogen class compound in reaction solution is 10-500mg/mL.
In wherein some embodiments, the mass ratio of the drug to be measured and the benzoyl halogen class compound is 1:1.0-
3.0。
In wherein some embodiments, concentration of the benzoyl halogen class compound in reaction solution is 380-450mg/
mL。
In wherein some embodiments, first drug to be measured is mentioned with organic solvent before performing the derivatization reaction
It takes, then performs the derivatization reaction for extract obtained.
In wherein some embodiments, the number of the extraction is 2-6 times, and each organic solvent used is separately
Selected from ethyl acetate, ethyl alcohol, methanol, n-butanol, hexamethylene.
In wherein some embodiments, include: with the chromatographic condition that high performance liquid chromatography is detected
Chromatographic column: Zorbax Eclipse plus C18 column；
Mobile phase: acetonitrile, tetrahydrofuran and the triethylamine aqueous solution of volume ratio 77: 5: 18, wherein triethylamine aqueous solution
In the triethylamine containing 0.08-0.12wt%；
Or include: with the chromatographic condition that high performance liquid chromatography is detected
Chromatographic column: ZORBAX StableBond-C18 column；
Mobile phase: the methanol and triethylamine aqueous solution of volume ratio 95: 5, wherein triethylamine aqueous solution contains 0.2wt%'s
Triethylamine；
Or include: with the chromatographic condition that high performance liquid chromatography is detected
Chromatographic column: ZORBAX StableBond-C18 column；
Mobile phase: volume ratio is the acetonitrile and ammonia aqueous solution of 80:20.
In wherein some embodiments, include: with the chromatographic condition that high performance liquid chromatography is detected
Chromatographic column: Zorbax Eclipse plus C18 column；
Mobile phase: acetonitrile, tetrahydrofuran and the triethylamine aqueous solution of volume ratio 77: 5: 18, wherein triethylamine aqueous solution
In the triethylamine containing 0.08-0.12wt%；
Column temperature: 24-26 DEG C；
Flow velocity: 0.8mL/min；
Detection wavelength: 235nm；
Or include: with the chromatographic condition that high performance liquid chromatography is detected
Chromatographic column: ZORBAX StableBond-C18 column；
Mobile phase: the methanol and triethylamine aqueous solution of volume ratio 95: 5, wherein triethylamine aqueous solution contains 0.2wt%'s
Triethylamine；
Column temperature: 24-16 DEG C；
Flow velocity: 1.0mL/min；
Detection wavelength: 254nm；
Or include: with the chromatographic condition that high performance liquid chromatography is detected
Chromatographic column: ZORBAX StableBond-C18 column；
Mobile phase: volume ratio is the acetonitrile and ammonia aqueous solution of 80:20；
Column temperature: 34-36 DEG C；
Flow velocity: 1.0mL/min；
Detection wavelength: 230nm.
In wherein some embodiments, the drug is protona gel, protona emulsifiable paste, person's protona ointment
Or protona injection.
The method of protona in HPLC method measurement drug provided by the invention, utilizes most common detector -- ultraviolet inspection
It surveys device (UVD) to be detected, hydroxyl of this method based on protona and the fast reaction of benzoyl halogen, it will be originally without UV absorption
Protona processing is performed the derivatization by benzoyl halogen class compound, due to the presence of nitro electron-withdrawing group on phenyl ring,
The derivatization product of protona is set to have a stronger absorption in ultraviolet visible light region, and the UV absorption of auxiliary material and impurity in drug
It is very weak, to realize qualitative or quantitative detection of the HPLC method to protona.The derivatization HPLC-UVD measurement that the present invention establishes
The method of protona is simple, versatility is good, can be used for protona preparation of traditional Chinese medicine assay, through methodology validation
This method specificity, sensitivity are good as the result is shown, and accuracy is high.
Detailed description of the invention
Fig. 1 is system suitability test result, wherein A is negative controls, B is protona titer, C is double hydrogen
Testosterone standard items derivative, D are the gel derived object of protona.
Specific embodiment
Further details of retouch is done below in conjunction with method of the specific embodiment to protona in measurement drug of the invention
It states.
The proportion of mobile phase refers to volume ratio in following embodiment, and the percentage being not particularly illustrated refers both to quality percentage
Than.
Instrument: Agilent 1200HPLC (Agilent company of the U.S.)；Sartorious BS-224S electronic balance
(Beijing Sai Duolisi balance Co., Ltd)；FA25 homogenizer (German Fu Luke company)；JY92-2D supersonic wave cleaning machine is (peaceful
Bo Xinzhi Biotechnology Co., Ltd)；SH23-2 constant temperature blender with magnetic force (Shanghai Mei Yingpu instrument and meter Manufacturing Co., Ltd).
Agents useful for same: protona raw material (Shandong Boyuan Pharmaceutical Co., Ltd., content > 99.1%)；Injection stage soybean ovum
Phosphatide (Shanghai Taiwei Pharmaceutical Co., Ltd., PC >=92.6%)；Cholesterol (Sinopharm Chemical Reagent Co., Ltd.)；Double hydrogen testis
Ketone gel Andractim (Laboratoires Besins International company, content: 2.5%)；Poloxamer
188, poloxamer188 (upper Hydron is medical auxiliary materials Technology Co., Ltd.)；(U.S. Alfa Aesar is public for paranitrobenzoyl chloride
Department)；Acetonitrile (chromatographically pure, Di Ma company, the U.S.)；Other pharmaceutical adjuncts are pharmaceutical grade, and reagent is that analysis is pure.
Protona gel used in following embodiment is to be prepared by the following method:
It takes Acritamer 940 10g to mix with Tween-80 2g and 800ml distilled water and is swelled into translucent solution, while stirring
It mixes side and triethanolamine 13.5g is added dropwise, obtain gel-type vehicle；It takes the protona 25g sieved by No. nine to be dissolved in ethyl alcohol 50g, has dissolved
Gel-type vehicle is gradually added into after complete to stir evenly, and ethylparaben 1g and distilled water is added to full dose 1000g, stirs evenly, obtains transparent
Protona gel.
Protona ointment used in following embodiment is to be prepared by the following method:
Stearyl alcohol 30g and cera alba 80g is taken to set in container, the stirring of cholesterol 30 is added until molten in the middle fusing in water-bath
Solution, is eventually adding albolene 805g, continues to be heated to 70~80 DEG C, obtain matrix；Take by No. nine sieve protona 25g with
Poly- -200 30g of silicon of diformazan is ground into paste, is slowly added to matrix, and quickly stir, and cooling condensation is under stiring to get double hydrogen
Testosterone ointment.
Protona emulsifiable paste used in following embodiment is to be prepared by the following method:
Stearic acid 120g, single stearic acid glycerine lipoprotein 35g, liquid paraffin 60g, albolene 10g and lanolin 50g is taken to set appearance
In device, fusing is heated in water-bath, continues to be heated to 70~80 DEG C, obtains oily phase；Take the protona 25g and liquid sieved by No. nine
Shape paraffin 60g is ground into paste, is slowly added to oily phase, and quickly stir, and 70~80 DEG C of heat preservations obtain phase containing medicine oil；Separately take three second
Hydramine 4g, glycerol 5g and purified water about 700g, are heated to 70~80 DEG C, slowly pour into water phase in phase containing medicine oil, side edged edge
Same direction stirs rapidly, is stirred continuously a few minutes in water-bath and is stirred continuously at room temperature again, until emulsification condensation is to get double hydrogen
Testosterone O/W type emulsifiable paste.
Protona injection used in following embodiment is to be prepared by the following method:
Protona lipid nano particle prepares Compritol 888 ATO 250g, the lecithin 100g and double hydrogen testis for weighing recipe quantity
Ketone 10g is added in 250mL ethyl alcohol and seals, and makes to melt in 85 DEG C of heating, as oily phase.By pluronic F-68 50g, Tween 80
125g ultrasonic disperse is heated to same temperature as water phase into 150mL water for injection.Water phase is added dropwise under magnetic stirring
In synthermal oil phase, simultaneously stable dispersion l0min is stirred evenly under rate of addition 10mL/min, 2000r/min, forms colostrum.
While hot under room-temperature water bath (20 DEG C) by colostrum ultrasonic cell disruptor redisperse, ultrasonic power 600W, ultrasonic 1sec,
Have a rest l sec, time 10min, cooling with ice-water bath, crosses 0.22 μm of miillpore filter to get protona lipid nano particle liquid.
Whole protona lipid nanometers are made in previous step in the lyophilized technique of protona injection (freeze drying powder injection)
In grain liquid, mannitol 65g is added, adds water for injection to 1000mL, stirs evenly, be sub-packed in ampoule bottle by 2mL/ bottles, use is cold
Dry method is lyophilized and prepares freeze-dried powder.Lyophilized technique process: (1) pre-freeze: the drug dispensed is put on the partition in freeze drying box, in advance
Freeze and starts extremely+25 DEG C~-40 DEG C, 2h；- 40 DEG C, 3h；(2) condenser temperature lyophilization: is down to -45 DEG C hereinafter, starting is true
Sky pump, after vacuum degree reaches certain numerical value, slowly opens butterfly valve, when the vacuum degree in drying box reaches 13.33Pa (0.1mmHg)
Freezing is closed when following, is slowly heated by the heating system under partition, and the temperature of frozen product is made to be gradually increased to -20 DEG C, then
It is gradually increased to 25 DEG C；(3) re-dry: at 25 DEG C, vacuum degree 10Pa or less maintains 5~8h.Then nitrogen charging, tamponade, outlet are used
Aluminium-plastic combined cover rolls mouth to get protona injection (freeze drying powder injection), specification: about 1.2g/ bottles, 20mg/ containing protona
Bottle.
Embodiment 1: chlorobenzoyl chloride derivatization HPLC method measures the content of protona in protona drug
The present embodiment carries out benzoyl using pyridine-chlorobenzoyl chloride as derivatization reagent, to the hydroxyl in protona molecule
After change, measured using RP-HPLC method.Specific step is as follows:
(1) preparation of reference substance solution
Precision weighs protona reference substance 10mg, adds methanol 25mL dissolution as reference substance solution.
(2) preparation of test solution
Precision weighs protona gel 1g (either protona ointment or protona emulsifiable paste 1g or protona
1 bottle of injection), with ethyl acetate 30mL refluxing extraction 10h, after waving most ethyl acetate, then with alcohol reflux extract 12h, recycling
Ethyl alcohol, residue are transferred in 50mL separatory funnel with a small amount of distilled water, are saturated with water extracting n-butyl alcohol 4 times, each 10mL.Merge
N-butanol layer volatilizes, by residue perform the derivatization reaction (specifically: by it is residue obtained be placed in 25mL tool plug round-bottomed flask in, use
Pyridine 5mL dissolution, addition chlorobenzoyl chloride 2.0g is closed at a temperature of ice-water bath, is placed at room temperature for for 24 hours after shaking up, makes fully reacting),
Gained reaction solution is dissolved with acetonitrile, is transferred in 25mL measuring bottle, acetonitrile constant volume, and with 022 μm of filtering with microporous membrane, it is molten to obtain test sample
Liquid.
(3) HPLC-UVD is detected
Taking (2) middle 20 μ L of test solution prepared, sample introduction is analyzed by chromatographic condition.
Chromatographic condition is as follows: chromatographic column: Zorbax Eclipse plus C18 column；Mobile phase: acetonitrile-tetrahydrofuran-
0.1% triethylamine aqueous solution (77: 5: 18)；Column temperature: 25 DEG C: flow velocity: 0.8mL/min；Detection wavelength: 235nm；Sample volume: 20 μ
L。
(4) testing result
Testing result is shown in Table 1.
(n=3) mg/g or mg/ bottles of protona assay result in 1 protona drug of table
Embodiment 2: paranitrobenzoyl chloride derivatization HPLC method measures the content of protona in protona drug
The present embodiment is using methylene chloride-pyridine-paranitrobenzoyl chloride as derivatization reagent, in protona molecule
Hydroxyl carry out benzoylation after, using RP-HPLC method measure.Specific step is as follows:
(1) preparation of reference substance solution
Precision weighs protona reference substance 10mg, adds acetonitrile 25mL dissolution as reference substance solution.
(2) preparation of test solution
Precision weighs protona gel 1g (either protona ointment or protona emulsifiable paste 1g or protona
1 bottle of injection), it takes filter residue to set in conical flask after waving most ethyl acetate with ethyl acetate 30mL refluxing extraction 8h, methanol is added
20mL, ultrasonic extraction 60min, filtering, take filtrate, volatilize, by residue perform the derivatization reaction (specifically: set residue obtained
Have in plug round-bottomed flask in 25mL, dissolved with methylene chloride-pyridine (5:1) 5mL, it is close that paranitrobenzoyl chloride (2.0g) is added
It closes, sets 25℃Ultrasonic 30min stands 2h, water 5mL, ultrasonic 30min is added to be transferred in separatory funnel, stands 1d, takes lower layer clear
Liquid is eluted by large pore resin absorption column (20cm × 2.0cm) with 70% ethanol water 300mL, after with dehydrated alcohol
400mL elution, collects anhydrous ethanol elution liquid, is evaporated spare), it is dissolved, is transferred in 10mL measuring bottle with methanol, be settled to quarter
Degree, with 0.22 μm of filtering with microporous membrane, obtains test solution.
(3) HPLC-UVD is detected
Taking (2) middle 20 μ L of test solution prepared, sample introduction is analyzed by chromatographic condition.
Chromatographic condition is as follows: chromatographic column: ZORBAX StableBond-C18 column (4.4mm × 250mm, 5 μm)；Mobile phase:
- 0.2% triethylamine aqueous solution of methanol (95: 5)；Column temperature: 25 DEG C: flow velocity: 1.0mL/min；Detection wavelength: 254nm；Sample volume:
20μL。
(4) testing result
Testing result is shown in Table 2.
(n=3) mg/g or mg/ bottles of protona assay result in 2 protona drug of table
(5) methodology validation is tested
(5.1) preparation of titer
Precision weighs protona raw material 10.30mg, and methanol 25mL is added to dissolve, and obtains double hydrogen that concentration is 412.00 μ g/mL
Testosterone stock solution.It takes protona stock solution to add methanol dilution, the titer of various concentration is made.
(5.2) preparation of test solution
Test solution is prepared with the method in the present embodiment (2).
(5.3) chromatographic condition
Chromatographic column: ZORBAX StableBond-C18 column (4.4mm × 250mm, 5 μm)；Mobile phase: methanol -0.2% three
Ethylamine solution (95: 5)；Column temperature: 25 DEG C: flow velocity: 1.0mL/min；Detection wavelength: 254nm；Sample volume: 20 μ L.
(5.4) system suitability
By operating under " preparation of (5.1) titer " item, but protona raw material is not added, then presses " (5.2) test solution
Preparation " operate under item, and paranitrobenzoyl chloride is not added, negative controls is made.
By operating under " preparation of (5.1) titer " item, protona titer is obtained.
By being operated under " preparation of (5.1) titer " item, then by being operated under " preparation of (5.2) test solution " item, obtain
Protona standard items derivative.
By operating under " preparation of (5.2) test solution " item, the gel derived object of protona is obtained.
Take negative controls, protona titer, protona standard items derivative and the gel derived object of protona
Feed liquor is mutually pressed to be measured under " (5.3) chromatographic condition " item, and HPLC chromatogram is shown in Fig. 1: under this chromatographic condition, in reference substance and sample
Other components can baseline separation, be all larger than 1.5 with the separating degree of adjacent chromatographic peak, theoretical cam curve, which calculates, is not less than 3000.
(5.5) linear relationship is investigated
Precision pipettes protona stock solution 0.5,1,2,3,1,2ml, is placed in 50,50,50,50,10,10mL measuring bottles,
Add mobile phase to be diluted to scale, shake up, is prepared into concentration and is followed successively by 4.12 μ g/mL, 8.24 μ g/mL, 12.36 μ g/mL, 24.76 μ
G/mL, 41.20 μ g/mL and 82.40 μ g/mL serial standards solution are handled by " preparation of (5.2) test solution " item method
Sample draws 20 μ l and injects liquid chromatograph, by measuring under " (5.3) chromatographic condition " item, records chromatogram and peak area.With peak
Area Y is to mass concentration X (μ gmL-1) linear regression calculating is carried out, obtain the equation of linear regression of protona are as follows: Y=
84517X-29.878 r=0.9997.The result shows that protona is in the concentration range of 4.12~82.40 μ g/mL, it is dense
Degree is in good linear relationship with peak area..
(5.6) precision test
Same standard solution is taken, handles sample, feed liquor chromatography by " preparation of (5.2) test solution " item method
It by being measured under " (5.3) chromatographic condition " item, repeats sample introduction 6 times, the RSD for calculating protona peak area is 0.89%, shows instrument
Device precision is good.
(5.7) repetitive test
It takes with batch of protona gel, handles sample by " preparation of (5.2) test solution " item method, it is parallel to make
Standby 6 parts of samples draw 20 μ l and inject liquid chromatograph, by measuring under " (5.3) chromatographic condition " item, record chromatogram and peak face
Product calculates content.As a result the average content of protona is 24.7276mg/g, and RSD 1.55% shows the survey of the present embodiment
The repeatability of method for testing is good.
(5.8) study on the stability
Precision is drawn to be sampled with a collection of 20 μ L of protona gel test solution respectively at 0,2,4,8,12,16 for 24 hours
Measurement, the RSD for calculating protona peak area is 1.18%, shows that test solution is good in internal stability for 24 hours.
(5.9) sample recovery rate is tested
Precision weighs protona raw material 1.2012g, adds acetonitrile to dissolve, is settled to 50mL, and obtaining concentration is 24.024mg/mL
Protona highly concentrated solution.The same a collection of protona gel of known content is taken, it is accurate respectively that a certain amount of double hydrogen testis are added
Ketone highly concentrated solution handles sample by " preparation of (5.2) test solution " item method, and feed liquor mutually presses " (5.3) chromatographic condition "
Measurement under, calculates sample recovery rate, the results are shown in Table 3.
Table 3 is loaded recovery test result
(5.10) detection limit
Certain density 20 μ L of protona derivative solution is taken, feed liquor is mutually pressed to be measured under " (5.3) chromatographic condition " item.Knot
Fruit is about 4.12ng (S/N=3) to the detection limit of derivative；Certain density protona solution is separately taken, by " (5.2) are for examination
The preparation of product solution " item method handles sample, and 20 μ L feed liquors of absorption are mutually pressed to be measured under " (5.3) chromatographic condition " item, as a result double hydrogen
The detection of testosterone is limited to 41.20ng (S/N=3), after showing with the method for the present embodiment to protona derivatization treatment, inspection
Surveying limit reduces 10 times, can measure the lower protona drug sample of content, and baseline is more steady, measures more quasi-
Really.
Protona contains in embodiment 3:3,5- dinitrobenzoyl chloride derivatization HPLC method measurement protona drug
Amount
The present embodiment with pyridine -3,5- dinitrobenzoyl chloride be derivatization reagent, to the hydroxyl in protona molecule
After carrying out benzoylation, measured using RP-HPLC method.Specific step is as follows:
(1) preparation of reference substance solution
Precision weighs protona reference substance 10mg, adds acetonitrile 25mL dissolution as reference substance solution.
(2) preparation of test solution
Precision weighs protona gel or protona ointment or protona emulsifiable paste 1g or protona note
1 bottle of agent is penetrated, first with ethyl alcohol 30mL refluxing extraction 2h, ethyl alcohol is recycled in filtering, and take filter residue hexamethylene to extract three times, each 15mL,
Merge hexamethylene layer, adds anhydrous Na2SO4About 2g, dehydration filtration, volatilize, by residue perform the derivatization reaction (specifically: by gained
Residue is set in 20mL tool plug test tube, is dissolved with anhydrous pyridine 5mL, and addition 3,5- dinitrobenzoyl chloride (2.0g) is closed, sets 75
Flow back 1h in DEG C water-bath, stands 2h, under being depressurized after taking-up with nitrogen stream dry up to get), dissolved with acetonitrile, be transferred to 10mL amount
In bottle, it is settled to scale, with 0.45 μm of filtering with microporous membrane, obtains test solution.
(3) HPLC-UVD is detected
Taking (2) middle 20 μ L of test solution prepared, sample introduction is analyzed by chromatographic condition.
Chromatographic condition is as follows: chromatographic column: ZORBAX StableBond-C18 column；Mobile phase: acetonitrile-ammonia aqueous solution (80
:20)；Column temperature: 35 DEG C: flow velocity: 1.0mL/min；Detection wavelength: 230nm；Sample volume: 20 μ L.
(4) testing result
Testing result is shown in Table 4.
(n=3) mg/g or mg/ bottles of protona assay result in 4 protona drug of table
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of method of protona in measurement drug, which comprises the following steps:
(1) reaction is performed the derivatization to the protona in drug to be measured using benzoyl halogen class compound as derivatization reagent, obtained
To the sample containing protona derivatization product；
(2) with the sample preparation test solution containing protona derivatization product, the test solution is high
Effect liquid phase chromatogram is detected, and the detector of the high performance liquid chromatography is UV detector.
2. the method for protona in measurement drug according to claim 1, which is characterized in that the benzoyl halogen class
Close any one of object in chlorobenzoyl chloride, paranitrobenzoyl chloride and DNBC 3,5 dinitrobenzoylchloride.
3. the method for protona in measurement drug according to claim 1, which is characterized in that the derivative reaction exists
Carried out in aprotic polar solvent, the aprotic polar solvent be selected from pyridine, methylene chloride, dimethyl sulfoxide, acetone, acetonitrile,
At least one of dimethylformamide and dimethyl acetamide.
4. the method for protona in measurement drug according to claim 1-3, which is characterized in that described spreads out
The reaction temperature of biochemical reaction is 20-90 DEG C, and the reaction time is 0.5-48 hours.
5. the method for protona in measurement drug according to claim 1-3, which is characterized in that described to be measured
The mass ratio of drug and the benzoyl halogen class compound is 1:0.5-5.0；The benzoyl halogen class compound is in reaction solution
Concentration be 10-500mg/mL.
6. the method for protona in measurement drug according to claim 1-3, which is characterized in that carrying out institute
First drug to be measured is extracted with organic solvent before stating derivative reaction, then performs the derivatization reaction for extract obtained.
7. the method for protona in measurement drug according to claim 6, which is characterized in that the number of the extraction is
2-6 times, organic solvent used is separately selected from ethyl acetate, ethyl alcohol, methanol, n-butanol, hexamethylene every time.
8. the method for protona in measurement drug according to claim 1-3, which is characterized in that with efficient liquid
The chromatographic condition that phase chromatography is detected includes:
Chromatographic column: Zorbax Eclipse plus C18 column；
Mobile phase: acetonitrile, tetrahydrofuran and the triethylamine aqueous solution of volume ratio 77: 5: 18 wherein contain in triethylamine aqueous solution
The triethylamine of 0.08-0.12wt%；
Or include: with the chromatographic condition that high performance liquid chromatography is detected
Chromatographic column: ZORBAX StableBond-C18 column；
Mobile phase: the methanol and triethylamine aqueous solution of volume ratio 95: 5, wherein triethylamine aqueous solution contains three second of 0.2wt%
Amine；
Or include: with the chromatographic condition that high performance liquid chromatography is detected
Chromatographic column: ZORBAX StableBond-C18 column；
Mobile phase: volume ratio is the acetonitrile and ammonia aqueous solution of 80:20.
9. it is according to claim 8 measurement drug in protona method, which is characterized in that with high performance liquid chromatography into
Row detection chromatographic condition include:
Chromatographic column: Zorbax Eclipse plus C18 column；
Mobile phase: acetonitrile, tetrahydrofuran and the triethylamine aqueous solution of volume ratio 77: 5: 18 wherein contain in triethylamine aqueous solution
The triethylamine of 0.08-0.12wt%；
Column temperature: 24-26 DEG C；
Flow velocity: 0.8mL/min；
Detection wavelength: 235nm；
Or include: with the chromatographic condition that high performance liquid chromatography is detected
Chromatographic column: ZORBAX StableBond-C18 column；
Mobile phase: the methanol and triethylamine aqueous solution of volume ratio 95: 5, wherein triethylamine aqueous solution contains three second of 0.2wt%
Amine；
Column temperature: 24-16 DEG C；
Flow velocity: 1.0mL/min；
Detection wavelength: 254nm；
Or include: with the chromatographic condition that high performance liquid chromatography is detected
Chromatographic column: ZORBAX StableBond-C18 column；
Mobile phase: volume ratio is the acetonitrile and ammonia aqueous solution of 80:20；
Column temperature: 34-36 DEG C；
Flow velocity: 1.0mL/min；
Detection wavelength: 230nm.
10. the method for protona in measurement drug according to claim 1-3, which is characterized in that the medicine
Object is protona gel, protona emulsifiable paste, protona ointment or protona injection.
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| CN117723679A (en) * | 2024-02-05 | 2024-03-19 | 长沙创新药物工业技术研究院有限公司 | Detection method of R-/S-hydroxypropyl tetrahydropyran triol |
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