CN107167546B - A kind of liquid phase analysis method for the Fluconazole optical isomer that ends - Google Patents
A kind of liquid phase analysis method for the Fluconazole optical isomer that ends Download PDFInfo
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- CN107167546B CN107167546B CN201710526473.9A CN201710526473A CN107167546B CN 107167546 B CN107167546 B CN 107167546B CN 201710526473 A CN201710526473 A CN 201710526473A CN 107167546 B CN107167546 B CN 107167546B
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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Abstract
The invention discloses a kind of liquid phase analysis methods of Fluconazole optical isomer that ends.The stationary phase of liquid chromatography is octadecylsilane chemically bonded silica, mobile phase is acetonitrile solution, acetonitrile concentration expressed in percentage by volume is 25-45%, L-threonine and D-Thr containing effective concentration in the acetonitrile solution, and the molar ratio of L-threonine and D-Thr is 2-4:1.Liquid phase analysis method separating effect provided by the invention is excellent, and without chiral chromatographic column, application cost is low, easy to spread.
Description
Technical field
The invention belongs to Pharmaceutical Analysis fields, and in particular to a kind of liquid phase analysis method for the Fluconazole optical isomer that ends.
Background technique
End Fluconazole, entitled (2R, 3R) -2- (2,4- difluorophenyl) -3- (4- methylenepiperidines -1- the base) -1- of chemistry
(1H-1,2,4- triazol-1-yls) -2- butanol, is first external application antifungal drug in triazole class, public by Canadian Valeant pharmacy
Department's research and development obtain FDA approval listing, trade name Jublia on June 6th, 2014.The solution that the medicine is 10%, part, which applies, to be used, mainly
For treating the hand as caused by Trichophyton rubrum, trichophyton gypseum, tinea pedis, mechanism of action is to inhibit fungi 14- demethyl
Change enzyme, to interfere the synthesis of ergosterol.There are 2 chiral centres in the Fluconazole structure that ends, and share 4 kinds of optical isomers,
In addition to the 2R/3R configuration (1) with pharmacological activity, there are also 2S/3S configuration (2), 2R/3S configuration (3) and 2S/3R configurations (4).
The chemical structural formula of four kinds of various configurations is as follows:
According to the associated specifications of research and development stereoisomer new drug, it is necessary to establish a set of perfect analysis method to Chinese mugwort
The optical isomer impurity of Fluconazole carries out quality control and Product Quality Evaluation.Li Peijie is provided 4 in a kind of Chinese mugwort Fluconazole
The HPLC measuring method (Chinese Journal of Pharmaceuticals, 2016,47) of kind optical isomer, but this method is using chiral color
It composes column (Daicel Chiralpak IC chiral column).It is well known that chiral chromatographic column is costly, and service life is short, uses
The liquid phase analysis method application cost with higher of chiral chromatographic column exploitation.
Summary of the invention
The purpose of the present invention is to provide a kind of liquid phases point for being more economically separated analysis Chinese mugwort Fluconazole and its isomers
Analysis method.
The present invention is achieved by following technical solution:
A kind of liquid phase analysis method for the Fluconazole optical isomer that ends, the stationary phase of liquid chromatography is octadecylsilane
Bonded silica gel, mobile phase are acetonitrile solution, and acetonitrile concentration expressed in percentage by volume is 25-45%, are contained in the acetonitrile solution effective
The molar ratio of the L-threonine and D-Thr of concentration, L-threonine and D-Thr is 2-4:1.
Preferably, L-threonine, D-Thr molar ratio are 3:1.
Preferably, the sum of molar concentration of the L-threonine, D-Thr is 0.3-0.5 μm of ol/L.
Preferably, the liquid phase analysis method includes following chromatographic parameter:
Chromatographic column: octadecylsilane chemically bonded silica (C18) chromatographic column；
Mobile phase A phase: 25% acetonitrile solution, L-threonine, D-Thr are respectively 0.3 μm of ol/L, 0.1 μm of ol/L；
Mobile phase B phase: 45% acetonitrile solution, L-threonine, D-Thr are respectively 0.3 μm of ol/L, 0.1 μm of ol/L；
Elution program: 0-5min, 0%B phase；5-8min, 0% → 50%B phase；8-10min, 50% → 100%B phase；10-
25min, 100%B phase；Flow velocity: 0.7-0.9mL/min；
Column temperature: 33-37 DEG C；
Detection wavelength: 208-212nm.
Preferably, flow velocity 0.8mL/min.
Preferably, column temperature is 35 DEG C.
Preferably, Detection wavelength 210nm.
Preferably, the specification of octadecylsilane chemically bonded silica chromatographic column is long 250mm, internal diameter 4.6mm, 5 μ of packing material size
m。
Preferably, the preferred Thermo Scientific of the octadecylsilane chemically bonded silica chromatographic columnTM AcclaimTM
120 C18。
Advantages of the present invention:
For liquid phase analysis method provided by the invention without chiral chromatographic column, application cost is low, easy to spread.
Detailed description of the invention
Fig. 1 is the chemical structural formula of Chinese mugwort Fluconazole and its isomers；
Fig. 2 is the separating effect figure that mixed reference substance solution uses 1 liquid phase analysis method of embodiment；
Fig. 3 is the separating effect figure that test solution uses 1 liquid phase analysis method of embodiment；
Fig. 4 is the separating effect figure that mixed reference substance solution uses 2 liquid phase analysis method of embodiment；
Fig. 5 is the separating effect figure that mixed reference substance solution uses 3 liquid phase analysis method of embodiment.
Specific embodiment
It is further described technical solution of the present invention combined with specific embodiments below.
The liquid phase analysis method of the Chinese mugwort Fluconazole optical isomer of embodiment 1
One, experimental material
Shimadzu LC-20A high performance liquid chromatograph；
CPA225D electronic balance (Beijing Sai Duolisi instrument system Co., Ltd)；
Chromatographic column: Thermo ScientificTM AcclaimTM120 C18；
End the self-control of Fluconazole bulk pharmaceutical chemicals, end Fluconazole (1) and its 2S/3S (2), 2R/3S (3), 2S/3R (4) isomers
Reference substance self-control (purity is greater than 98%), the chemical structural formula of Chinese mugwort Fluconazole and its isomers is as shown in Figure 1；
D-Thr (CAS 632-20-2, lark prestige Science and Technology Ltd., purity 98%)；
L-threonine (CAS 6028-28-0, Chengdu bass spy's reagent Co., Ltd, purity 99%)；
Acetonitrile is chromatographically pure, and water is deionized water, remaining reagent is that analysis is pure.
Two, experimental method and result
1, solution is prepared
Diluents: 25% acetonitrile solution, the D-Thr of L-threonine containing 0.3 μm of ol/L, 0.1 μm of ol/L.
Reference substance solution: precision weighs 1-4 reference substance 10mg respectively, respectively sets in 100ml measuring bottle, is dissolved with diluents
And constant volume, shake up the 1-4 reference substance solution for being 100 μ g/ml to get concentration.
Mixed reference substance solution: 1-4 reference substance solution is mixed in equal volume, obtains the mixing reference substance of each 25 μ g/ml of concentration
Solution.
Test solution: precision weighs Chinese mugwort Fluconazole bulk pharmaceutical chemicals 100mg, sets in 100ml measuring bottle, is dissolved with diluents
And constant volume, shake up the test solution for being 1mg/ml to get concentration.
2, chromatographic condition
Chromatographic column: Thermo ScientificTM AcclaimTM120 C18；
Mobile phase A phase: 25% acetonitrile solution, L-threonine, D-Thr are respectively 0.3 μm of ol/L, 0.1 μm of ol/L；
Mobile phase B phase: 45% acetonitrile solution, L-threonine, D-Thr are respectively 0.3 μm of ol/L, 0.1 μm of ol/L；
Elution program: 0-5min, 0%B phase；5-8min, 0% → 50%B phase；8-10min, 50% → 100%B phase；10-
25min, 100%B phase；Flow velocity: 0.8mL/min；
Column temperature: 35 DEG C；
Detection wavelength: 210nm；
Sample volume: 20 μ L.
3, system suitability
Accurate respectively to measure 1-4 reference substance solution and mixed reference substance solution, sample introduction measurement records chromatogram, and appearance is suitable
Sequence is followed successively by 1,2,3,4, and number of theoretical plate is all larger than 10000, and separating degree is all larger than 1.5, and tailing factor is respectively less than 1.15, and blank is molten
Interference measurement, mixed reference substance solution chromatogram are not shown in Fig. 2 for agent.
4, linear relationship is investigated
It is appropriate to weigh 1-4 reference substance for precision respectively, is dissolved and is diluted respectively with mobile phase, be made 0.5,5,10,20,50,
80, the series of concentrations solution of 100 and 120 μ g/ml, sample introduction measurement.Using peak area as ordinate, concentration is abscissa, carries out line
Property return, obtain regression equation.Isomers 1-4 linear relationship in above-mentioned concentration range is good, R2> 0.9999.
5, sensitivity is investigated
Precision weighs that 1-4 isomer control product are each appropriate, and adding dilution to dissolve and being diluted to 4 kinds of isomers peak heights is about base
3 times of line noise.As a result the minimum detectability of Isomers is each about 1.1 × 10-3μ g, high sensitivity.
6, sample recovery rate is tested
Determination of recovery rates solution is prepared according to a conventional method, calculates the rate of recovery of Isomers, as a result proves 4 kinds of Fluconazole of Chinese mugwort
The rate of recovery of isomers (RSD < 1.5%, n=3) between 98-102%, meets the requirements.
7, precision and stability test
Above-mentioned mixed reference substance solution and test solution are taken, continuous sample introduction 6 times respectively, records chromatogram.As a result RSD is equal
Less than 2.0% (n=6), show that this method precision is good.
Take above-mentioned mixed reference substance solution and test solution respectively, in being placed at room temperature for, respectively at 0,4,8,12, for 24 hours into
Sample measurement, records chromatogram.As a result the peak area of Chinese mugwort 4 kinds of isomers of Fluconazole is respectively less than 2.0% (n=5) in RSD interior for 24 hours,
It is stable for 24 hours to show that mixed reference substance solution is placed at room temperature for.
8, bulk pharmaceutical chemicals measure
It takes sample appropriate, prepares mixed reference substance solution and test solution according to the above method, respectively sample introduction measurement, record
Chromatogram.If any identical miscellaneous with Isomers retention time in mixed reference substance solution chromatogram in test solution chromatogram
Mass peak, by external standard method with the content of calculated by peak area Isomers.Test solution chromatogram is as shown in Figure 3.In bulk pharmaceutical chemicals only
2S/3R (4) isomers is only detected, the content in three batches of samples is respectively 0.04%, 0.05%, 0.04%.
2 comparative example of embodiment, mobile phase only add L-threonine
One, experimental material
Shimadzu LC-20A high performance liquid chromatograph；
CPA225D electronic balance (Beijing Sai Duolisi instrument system Co., Ltd)；
Chromatographic column: Thermo ScientificTM AcclaimTM120 C18；
End the self-control of Fluconazole bulk pharmaceutical chemicals, end Fluconazole (1) and its 2S/3S (2), 2R/3S (3), 2S/3R (4) isomers
Reference substance self-control (purity is greater than 98%), the chemical structural formula of Chinese mugwort Fluconazole and its isomers is as shown in Figure 1；
L-threonine (CAS 6028-28-0, Chengdu bass spy's reagent Co., Ltd, purity 99%)
Acetonitrile is chromatographically pure, and water is deionized water, remaining reagent is that analysis is pure.
Two, experimental method and result
1, solution is prepared
Diluents: 25% acetonitrile solution, the L-threonine containing 0.3 μm of ol/L.
Reference substance solution: precision weighs 1-4 reference substance 10mg respectively, respectively sets in 100ml measuring bottle, is dissolved with diluents
And constant volume, shake up the 1-4 reference substance solution for being 100 μ g/ml to get concentration.
Mixed reference substance solution: 1-4 reference substance solution is mixed in equal volume, obtains the mixing reference substance of each 25 μ g/ml of concentration
Solution.
2, chromatographic condition
Chromatographic column: Thermo ScientificTM AcclaimTM120 C18；
Mobile phase A phase: 25% acetonitrile solution, L-threonine are 0.3 μm of ol/L；
Mobile phase B phase: 45% acetonitrile solution, L-threonine are 0.3 μm of ol/L；
Elution program: 0-5min, 0%B phase；5-8min, 0% → 50%B phase；8-10min, 50% → 100%B phase；10-
25min, 100%B phase；Flow velocity: 0.8mL/min；
Column temperature: 35 DEG C；
Detection wavelength: 210nm；
Sample volume: 20 μ L.
3, system suitability
Precision measure mixed reference substance solution, sample introduction measurement, record chromatogram, as a result end Fluconazole (1), 2S/3S (2),
2R/3S (3), 2S/3R (4) isomers can not achieve and separate two-by-two, and mixed reference substance solution chromatogram is shown in Fig. 4.
3 comparative example of embodiment, mobile phase only add D-Thr
One, experimental material
Shimadzu LC-20A high performance liquid chromatograph；
CPA225D electronic balance (Beijing Sai Duolisi instrument system Co., Ltd)；
Chromatographic column: Thermo ScientificTM AcclaimTM120 C18；
End the self-control of Fluconazole bulk pharmaceutical chemicals, end Fluconazole (1) and its 2S/3S (2), 2R/3S (3), 2S/3R (4) isomers
Reference substance self-control (purity is greater than 98%), the chemical structural formula of Chinese mugwort Fluconazole and its isomers is as shown in Figure 1；
D-Thr (CAS 632-20-2, lark prestige Science and Technology Ltd., purity 98%)；
Acetonitrile is chromatographically pure, and water is deionized water, remaining reagent is that analysis is pure.
Two, experimental method and result
1, solution is prepared
Diluents: 25% acetonitrile solution, the D-Thr containing 0.1 μm of ol/L.
Reference substance solution: precision weighs 1-4 reference substance 10mg respectively, respectively sets in 100ml measuring bottle, is dissolved with diluents
And constant volume, shake up the 1-4 reference substance solution for being 100 μ g/ml to get concentration.
Mixed reference substance solution: 1-4 reference substance solution is mixed in equal volume, obtains the mixing reference substance of each 25 μ g/ml of concentration
Solution.
2, chromatographic condition
Chromatographic column: Thermo ScientificTM AcclaimTM120 C18；
Mobile phase A phase: 25% acetonitrile solution, D-Thr are 0.1 μm of ol/L；
Mobile phase B phase: 45% acetonitrile solution, D-Thr are 0.1 μm of ol/L；
Elution program: 0-5min, 0%B phase；5-8min, 0% → 50%B phase；8-10min, 50% → 100%B phase；10-
25min, 100%B phase；Flow velocity: 0.8mL/min；
Column temperature: 35 DEG C；
Detection wavelength: 210nm；
Sample volume: 20 μ L.
3, system suitability
Precision measure mixed reference substance solution, sample introduction measurement, record chromatogram, as a result end Fluconazole (1), 2S/3S (2),
2R/3S (3), 2S/3R (4) isomers can not achieve and separate two-by-two, and mixed reference substance solution chromatogram is shown in Fig. 5.
For liquid phase analysis method provided by the invention without chiral chromatographic column, application cost is low, easy to spread.
Claims (2)
1. a kind of liquid phase analysis method for the Fluconazole optical isomer that ends, which is characterized in that including following chromatographic parameter:
Chromatographic column: octadecylsilane chemically bonded silica chromatographic column, specification are long 250mm, 4.6 mm of internal diameter, 5 μm of packing material size；
Mobile phase A phase: the acetonitrile solution that concentration expressed in percentage by volume is 25%, wherein being respectively containing L-threonine, D-Thr
0.3 μmol/L,0.1 μmol/L；
Mobile phase B phase: the acetonitrile solution that concentration expressed in percentage by volume is 45%, wherein being respectively containing L-threonine, D-Thr
0.3 μmol/L,0.1 μmol/L；
Elution program: 0-5min, 0% B phase；5-8min, 0% → 50% B phase；8-10min, 50% → 100% B phase；10-25min,
100% B phase；Flow velocity: 0.8 mL/min；
Column temperature: 35 DEG C；
Detection wavelength: 210nm.
2. liquid phase analysis method according to claim 1, it is characterised in that: the octadecylsilane chemically bonded silica chromatography
Column is 120 C18 chromatographic column of Thermo Scientific Acclaim.
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