CN1924572B - Method for determining hydroxyl citrate acid content - Google Patents

Method for determining hydroxyl citrate acid content Download PDF

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CN1924572B
CN1924572B CN2005100149642A CN200510014964A CN1924572B CN 1924572 B CN1924572 B CN 1924572B CN 2005100149642 A CN2005100149642 A CN 2005100149642A CN 200510014964 A CN200510014964 A CN 200510014964A CN 1924572 B CN1924572 B CN 1924572B
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acid
solution
hydroxycitric acid
hydroxycitric
reference substance
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CN1924572A (en
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李伟
高钧
魏峰
李旭
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Tasly Pharmaceutical Group Co Ltd
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Tianjin Tasly Pharmaceutical Co Ltd
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Abstract

This invention discloses one hydroxyl citrate content testing method, which comprises the following steps: processing the sample by strong alkali solution for thermal process; then using alkali resolution for neutralization till certain volume by dissolvent to process specimen resolution; processing comparing resolution by hydroxyl citrate or hydroxyl citrate; then separately absorbing comparing resolution and test resolution and then injecting effective liquid phase spectrum for measurement.

Description

A kind of content assaying method of hydroxycitric acid
Technical field
The present invention relates to the content assaying method of hydroxycitric acid, relate in particular to the content assaying method of hydroxycitric acid in India's pericarp essence extract and the goods thereof.
Background technology
India's pericarp essence extract (Garcinia Extracts) is the dry pericarp with the Garcinia Cambogia fruit (also being called as Goraka, Tamarind, Malabar or the like in India) that originates in India and Southeast Asia, extract hydroxycitric acid (Hydroxycitric Acid through heating, HCA), concentrate refining forming.According to external many hydroxycitric acids that studies show that the ATP citric acid lyase there is retardation.Hydroxycitric acid is to the affinity of ATP citric acid lyase, and is stronger 100 times than citric acid.Hydroxycitric acid can enter in the liver cell when reaching finite concentration, hinders the activity of ATP citric acid lyase, thereby suppresses the synthetic of fat, reaches the effect of fat-reducing.Mainly contain hydroxycitric acid and other organic acid in India's pericarp essence extract, wherein hydroxycitric acid exists with free acid and two kinds of forms of lactone body, these two kinds of existence forms all have effect of weight reducing, therefore, India's pericarp essence extract often is applied in the diet food, as slim tea, weight-reducing drinks or the like.
There is bibliographical information directly to measure hydroxycitric acid and lactone body thereof in India's pericarp essence extract with high performance liquid chromatography, but when this method is measured in fat-reducing coffee hydroxycitric acid content, Interference Peaks is more, the degree of separation of Interference Peaks and hydroxycitric acid and lactone body thereof is very poor, can not carry out accurate assay; In addition, hydroxycitric acid and lactone body thereof are effective constituent, and hydroxycitric acid lactone body reference substance is difficult for buying, and measures hydroxycitric acid respectively and lactone body difficulty is also bigger, is necessary to explore impurity and disturbs less and more accurate hydroxycitric acid content assaying method.
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.
Fig. 1 is that India's pericarp essence extract of the inventive method is at 210nm place assay chromatogram
Fig. 2 is that the hydroxycitric acid reference substance of the inventive method is at 210nm place assay chromatogram
Fig. 3 is that India's pericarp essence extract of Comparative Examples is at 210nm place assay chromatogram
Fig. 4 is that the hydroxycitric acid reference substance of Comparative Examples is at 210nm place assay chromatogram
Fig. 5 is that the fat-reducing coffee of the inventive method is at 200nm place assay chromatogram
Fig. 6 is that the fat-reducing coffee of the inventive method is at 220nm place assay chromatogram
Fig. 7 is that the fat-reducing coffee of the inventive method is at 210nm place assay chromatogram
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.

Claims (3)

1. the content assaying method of a hydroxycitric acid, comprise India's pericarp essence extract is made need testing solution, hydroxycitric acid or hydroxycitric acid salt are mixed with reference substance solution, draw reference substance solution and need testing solution then respectively, inject high performance liquid chromatograph, measure, it is characterized in that testing sample is first with NaOH or potassium hydroxide solution heat treated, and then it is neutralized with sulfuric acid or perchloric acid solution, water is settled to certain volume, makes need testing solution; Hydroxycitric acid or hydroxycitric acid salt are mixed with reference substance solution with moving phase; The condition of high-performance liquid chromatogram determination is: octadecyl silane is a stationary phase, and the phosphate aqueous solution of 6~7mmol/l or aqueous sulfuric acid or high chloro acid solution are moving phase, and the detection wavelength is 210nm.
2. hydroxycitric acid content assaying method according to claim 1 is characterized in that, described NaOH or potassium hydroxide aqueous solution are 0.1mol/l.
3. hydroxycitric acid content assaying method according to claim 1 is characterized in that, described sulfuric acid or perchloric acid solution are 1mol/l.
CN2005100149642A 2005-09-01 2005-09-01 Method for determining hydroxyl citrate acid content Expired - Fee Related CN1924572B (en)

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CN107356590A (en) * 2017-07-13 2017-11-17 广州安诺食品科学技术有限公司 The method of citric acid in quick detection lotus rhizome
CN111122723A (en) * 2019-01-07 2020-05-08 中国食品发酵工业研究院有限公司 Direct determination of citric acid delta in fruit juice13Method of C value
CN114137117A (en) * 2021-11-29 2022-03-04 上海方予健康医药科技有限公司 Method for determining content of citric acid or citrate in preparation

Citations (2)

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US6160172A (en) * 1997-08-27 2000-12-12 Vittal Mallya Scientific Research Foundation Soluble double metal salt of group IA and IIA of (-) hydroxycitric acid, process of preparing the same and its use in beverages and other food products without effecting their flavor and properties
CN1616962A (en) * 2004-09-07 2005-05-18 常德卷烟厂 Method for detecting tobacco specific nitrosamine by liquid phase chromatography and series mass spectrometry

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赵希荣.羟基柠檬酸的来源、制备方法和性质.中国食品添加剂.2004,(4),59-63. *

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