Summary of the invention
The purpose of this invention is to provide the hydroxycitric acid content assaying method that a kind of impurity disturbs less, degree of accuracy is high.This method can accurately be measured the content of hydroxycitric acid in India's pericarp essence extract and the goods thereof.
The inventive method can realize by following measures:
Testing sample is used the strong base solution heat treated earlier, and then with strong acid solution it is neutralized, and is settled to certain volume with solvent, makes need testing solution; Hydroxycitric acid or hydroxycitric acid salt are mixed with reference substance solution with solvent; Draw reference substance solution and need testing solution respectively, inject high performance liquid chromatograph, measure, promptly.
Above-mentioned testing sample is India's pericarp essence extract, perhaps for being that the various goods made of active component are as fat-reducing coffee, slim tea, weight-reducing drinks etc. with India's pericarp essence extract.
Above-mentioned is inorganic base with the highly basic in the strong base solution heat treated, and the best is NaOH or potassium hydroxide solution; Above-mentioned strong acid in strong acid solution it being neutralized is mineral acid, and the best is sulfuric acid or perchloric acid solution.Above-mentioned solvent is water or moving phase solution.
The condition of above-mentioned high-performance liquid chromatogram determination: octadecyl silane is a stationary phase, and the acid solution of water cut more than 90% is moving phase.The acid solution of described water cut more than 90% is water or water-methanol or water-acetonitrile, as formic acid, glacial acetic acid, phosphoric acid, hydrochloric acid, sulfuric acid, perchloric acid etc. it is adjusted to pH1~6 with acid.Described water cut acid solution the best more than 90% is a water, perhaps water-methanol=99: 1, and perhaps water-acetonitrile=99: 1 is adjusted to pH1~6 as formic acid, glacial acetic acid, phosphoric acid, hydrochloric acid, sulfuric acid, perchloric acid etc. with it with acid., above-mentioned detection wavelength is 200~220nm, the best is 210nm.
The present invention adopts highly basic that India's pericarp essence extract and goods thereof are carried out basic hydrolysis earlier, the form that makes free acid in the hydroxycitric acid and two kinds of forms of lactone body all change into free acid, and other Interference Peaks is destroyed with the strong base solution heat treated time, thereby eliminated the interference of Interference Peaks, use in the strong acid then and superfluous alkali lye, measure with the high-efficient liquid phase color instrument.The inventive method not only accuracy height, specificity is strong, can well hydroxycitric acid and other impurity peaks be separated, thereby can measure the content of hydroxycitric acid exactly, and applicability is wide, can be widely used in the assay of India's pericarp essence extract and goods thereof.
Enumerate embodiment below and further describe the present invention, this embodiment only is used to the present invention is described and the present invention is not limited.
Embodiment one
Chromatographic condition and system flexibility chromatographic column Agilent Zorbax SB-C
18Post (4.6 * 250mm, 5 μ m); The moving phase aqueous sulfuric acid of 6mmol/l; Flow velocity is 0.8ml/min; The ultraviolet detection wavelength is 210nm; Column temperature is 30 ℃; Theoretical cam curve is pressed the hydroxycitric acid peak and is calculated, and should be not less than 2000.
The preparation precision of reference substance solution takes by weighing hydroxycitric acid reference substance 12mg, puts in the 25ml measuring bottle, adds an amount of mutual-assistance dissolving of flowing, and adds moving phase again to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing India pericarp essence extract 100mg (Beijing Rivalcine Bioengineering Technology Co., Ltd provides), put in the 100ml round-bottomed flask, the potassium hydroxide 50ml that adds 0.1mol/l, boiling water bath heating 30 minutes, be cooled to room temperature, the perchloric acid solution 5ml that adds 1mol/l, shake up, this liquid is transferred in the 100ml measuring bottle, round-bottomed flask washes with water 3 times, each 10ml, washing lotion is incorporated in the measuring bottle, and water is settled to scale, shakes up, cross 0.45 μ m miillpore filter, promptly.
The accurate respectively reference substance solution 10 μ l that draw of determination method, need testing solution 10 μ l inject high performance liquid chromatograph, measure.
The hydroxycitric acid content of this India's pericarp essence extract is 55.74% as a result.The test sample chromatogram is seen Fig. 1, and the reference substance chromatogram is seen Fig. 2.
Comparative Examples
Chromatographic condition and system flexibility chromatographic column Agilent Zorbax SB-Aq post (4.6 * 250mm, 5 μ m); The moving phase aqueous sulfuric acid of 6mmol/l; Flow velocity is 0.5ml/min; The ultraviolet detection wavelength is 210nm; Column temperature is 30 ℃; Theoretical cam curve is pressed the hydroxycitric acid peak and is calculated, and should be not less than 2000.
The preparation precision of reference substance solution takes by weighing hydroxycitric acid reference substance 15mg, puts in the 25ml measuring bottle, adds an amount of mutual-assistance dissolving of flowing, and adds moving phase again to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing India pericarp essence extract 100mg (Beijing Rivalcine Bioengineering Technology Co., Ltd provides), puts in the 100ml round-bottomed flask, and it is an amount of to add moving phase, ultrasonicly make molten loose, be settled to scale with moving phase again, shake up, cross 0.45 μ m miillpore filter, promptly.
The accurate respectively reference substance solution 10 μ l that draw of determination method, need testing solution 10 μ l inject high performance liquid chromatograph, measure.
The result by Fig. 3 and Fig. 4 as seen, with this understanding, test sample chromatogram impurity peaks is more, and seriously disturbs the mensuration of hydroxycitric acid, can not satisfy the requirement of quantitative test.
Embodiment two
Chromatographic condition and system flexibility chromatographic column Agilent Zorbax SB-Aq post (4.6 * 250mm, 5 μ m); The moving phase phosphate aqueous solution of 6mmol/l; Flow velocity is 0.8ml/min; The ultraviolet detection wavelength is 220nm; Column temperature is 30 ℃; Theoretical cam curve is pressed the hydroxycitric acid peak and is calculated, and should be not less than 2000.
The preparation precision of reference substance solution takes by weighing hydroxycitric acid calcium reference substance 15mg, puts in the 25ml measuring bottle, adds an amount of mutual-assistance dissolving of flowing, and adds moving phase again to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing India pericarp essence extract 100mg (Beijing Rivalcine Bioengineering Technology Co., Ltd provides), put in the 100ml round-bottomed flask, the NaOH 50ml that adds 0.1mol/l, boiling water bath heating 30 minutes, be cooled to room temperature, the sulfuric acid solution 5ml that adds 1mol/l, shake up, this liquid is transferred in the 100ml measuring bottle, round-bottomed flask washes with water 3 times, each 10ml, washing lotion is incorporated in the measuring bottle, and water is settled to scale, shakes up, cross 0.45 μ m miillpore filter, promptly.
The accurate respectively reference substance solution 10ul that draws of determination method, need testing solution 10 μ l inject high performance liquid chromatograph, measure (being equivalent to the 0.7847mg hydroxycitric acid with 1mg hydroxycitric acid calcium calculates).
The hydroxycitric acid content of this India's pericarp essence extract is 55.24% as a result.
Embodiment three
Chromatographic condition and system flexibility chromatographic column Agilent Zorbax SB-Aq post (4.6 * 250mm, 5 μ m); The moving phase phosphate aqueous solution of 7mmol/l; Flow velocity is 0.8ml/min; The ultraviolet detection wavelength is 200nm; Column temperature is 30 ℃; Theoretical cam curve is pressed the hydroxycitric acid peak and is calculated, and should be not less than 2000.
The preparation precision of reference substance solution takes by weighing hydroxycitric acid calcium reference substance 15mg, puts in the 25ml measuring bottle, adds an amount of mutual-assistance dissolving of flowing, and adds moving phase again to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing fat-reducing coffee 300mg, and (Tianjin Jin Shili health products company limited provides, form by instant coffee, India's pericarp essence extract etc.), put in the 100ml round-bottomed flask, the NaOH 50ml that adds 0.1mol/l, boiling water bath heating 30 minutes, be cooled to room temperature, add the sulfuric acid solution 5ml of 1mol/l, shake up, this liquid is transferred in the 100ml measuring bottle, round-bottomed flask washes with water 3 times, each 10ml, and washing lotion is incorporated in the measuring bottle, water is settled to scale, shake up, cross 0.45 μ m miillpore filter, promptly.
The accurate respectively reference substance solution 10 μ l that draw of determination method, need testing solution 10 μ l inject high performance liquid chromatograph, measure (being equivalent to the 0.7847mg hydroxycitric acid with 1mg hydroxycitric acid calcium calculates).
Should the lose weight hydroxycitric acid content of coffee of result is 16.51%.The test sample chromatogram is seen Fig. 5.
Embodiment four
Chromatographic condition and system flexibility chromatographic column Agilent Zorbax SB-C18 post (4.6 * 250mm, 5 μ m); The moving phase aqueous sulfuric acid of 6mmol/l; Flow velocity is 0.8ml/min; The ultraviolet detection wavelength is 220nm; Column temperature is 30 ℃; Theoretical cam curve is pressed the hydroxycitric acid peak and is calculated, and should be not less than 2000.
The preparation precision of reference substance solution takes by weighing hydroxycitric acid reference substance 12mg, puts in the 25ml measuring bottle, adds an amount of mutual-assistance dissolving of flowing, and adds moving phase again to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing fat-reducing coffee 300mg, and (Tianjin Jin Shili health products company limited provides, form by instant coffee, India's pericarp essence extract etc.), put in the 100ml round-bottomed flask, the potassium hydroxide 50ml that adds 0.1mol/l, boiling water bath heating 30 minutes, be cooled to room temperature, add the perchloric acid solution 5ml of 1mol/l, shake up, this liquid is transferred in the 100ml measuring bottle, round-bottomed flask washes with water 3 times, each 10ml, and washing lotion is incorporated in the measuring bottle, water is settled to scale, shake up, cross 0.45 μ m miillpore filter, promptly.
The accurate respectively reference substance solution 10 μ l that draw of determination method, need testing solution 10 μ l inject high performance liquid chromatograph, measure.
Should the lose weight hydroxycitric acid content of coffee of result is 15.75%.The test sample chromatogram is seen Fig. 6.
Embodiment five
Chromatographic condition and system flexibility chromatographic column Waters μ-BondapackTM C18 post (4.6 * 250mm, 5 μ m); Moving phase is used the high chloro acid solution of 6mmol/l; Flow velocity is 0.8ml/min; The ultraviolet detection wavelength is 210nm; Column temperature is 30 ℃; Theoretical cam curve is pressed the hydroxycitric acid peak and is calculated, and should be not less than 2000.
The preparation precision of reference substance solution takes by weighing hydroxycitric acid calcium reference substance 15mg, puts in the 25ml measuring bottle, adds an amount of mutual-assistance dissolving of flowing, and adds moving phase again to scale, shakes up, promptly.
The preparation precision of need testing solution takes by weighing fat-reducing coffee 150mg, and (Tianjin Jin Shili health products company limited provides, form by instant coffee, India's pericarp essence extract etc.), put in the 100ml round-bottomed flask, the NaOH 50ml that adds 0.1mol/l, boiling water bath heating 30 minutes, be cooled to room temperature, add the perchloric acid solution 5ml of 1mol/l, shake up, this liquid is transferred in the 100ml measuring bottle, round-bottomed flask washes with water 3 times, each 10ml, and washing lotion is incorporated in the measuring bottle, water is settled to scale, shake up, cross 0.45 μ m miillpore filter, promptly.
The accurate respectively reference substance solution 10 μ l that draw of determination method, need testing solution 10 μ l inject high performance liquid chromatograph, measure (being equivalent to the 0.7847mg hydroxycitric acid with 1mg hydroxycitric acid calcium calculates).
Should the lose weight hydroxycitric acid content of coffee of result is 14.73%.The test sample chromatogram is seen Fig. 7.