CN104215711A - Fingerprint determination method of active ingredients in cocoa powder - Google Patents
Fingerprint determination method of active ingredients in cocoa powder Download PDFInfo
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- CN104215711A CN104215711A CN201410470234.2A CN201410470234A CN104215711A CN 104215711 A CN104215711 A CN 104215711A CN 201410470234 A CN201410470234 A CN 201410470234A CN 104215711 A CN104215711 A CN 104215711A
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- epicatechin
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- 235000009470 Theobroma cacao Nutrition 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 20
- 239000000843 powder Substances 0.000 title abstract description 7
- 239000004480 active ingredient Substances 0.000 title abstract 3
- 244000240602 cacao Species 0.000 title 1
- 244000299461 Theobroma cacao Species 0.000 claims abstract description 80
- 238000012360 testing method Methods 0.000 claims abstract description 20
- 238000011156 evaluation Methods 0.000 claims abstract description 8
- 238000010828 elution Methods 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 33
- PFTAWBLQPZVEMU-ZFWWWQNUSA-N (+)-epicatechin Natural products C1([C@@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-ZFWWWQNUSA-N 0.000 claims description 25
- PFTAWBLQPZVEMU-UKRRQHHQSA-N (-)-epicatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-UKRRQHHQSA-N 0.000 claims description 25
- LPTRNLNOHUVQMS-UHFFFAOYSA-N epicatechin Natural products Cc1cc(O)cc2OC(C(O)Cc12)c1ccc(O)c(O)c1 LPTRNLNOHUVQMS-UHFFFAOYSA-N 0.000 claims description 25
- 235000012734 epicatechin Nutrition 0.000 claims description 25
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 230000014759 maintenance of location Effects 0.000 claims description 14
- 239000013558 reference substance Substances 0.000 claims description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- 239000002953 phosphate buffered saline Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 3
- 239000000945 filler Substances 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
- 239000000377 silicon dioxide Substances 0.000 claims description 3
- 238000002137 ultrasound extraction Methods 0.000 claims description 3
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 2
- 238000004587 chromatography analysis Methods 0.000 abstract 1
- 239000003814 drug Substances 0.000 abstract 1
- 238000012544 monitoring process Methods 0.000 abstract 1
- 239000008363 phosphate buffer Substances 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 239000012266 salt solution Substances 0.000 abstract 1
- 239000012085 test solution Substances 0.000 abstract 1
- 241000219161 Theobroma Species 0.000 description 10
- 235000005764 Theobroma cacao ssp. cacao Nutrition 0.000 description 10
- 235000005767 Theobroma cacao ssp. sphaerocarpum Nutrition 0.000 description 10
- 235000001046 cacaotero Nutrition 0.000 description 10
- 239000000047 product Substances 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000473944 Hylaea <moth> Species 0.000 description 1
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- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 235000012970 cakes Nutrition 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 235000019219 chocolate Nutrition 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
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- 230000004438 eyesight Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- 235000015243 ice cream Nutrition 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a fingerprint determination method of active ingredients in cocoa powder. The determination method comprises the following steps: with the cocoa powder as a raw material, analyzing by high performance liquid chromatography, performing gradient elution with an acetonitrile-0.05% phosphate buffer salt solution as an elution system, and testing the fingerprint of a test solution obtained through a high performance liquid chromatograph and a standard fingerprint of the cocoa powder through a traditional Chinese medicine chromatography fingerprint similarity evaluation system, thereby totally controlling the quality of the cocoa powder by virtue of the fingerprint characteristic and guaranteeing the quality and health function of a cocoa product. The method disclosed by the invention has the features of high precision, good stability and good repeatability, and is capable of effectively separating the active ingredients of the cocoa powder and totally monitoring the quality of the cocoa product.
Description
Technical field
The present invention relates to the method for quality control of cocoa power, refer to particularly the finger print measuring method of active component in a kind of cocoa power.
Background technology
Cocoa power is the powder that the seed cocoa (Theobroma cacao Linn.) of Sterculiaceae Theobroma plant cocoa chocolate tree processes from strand.Cocoa chocolate tree originates in the hylaea area of South America Amazon River upstream, and existing 10 ° of the southern equatorial north latitude such as Indonesia, Ghana, Southeast Asia, West Africa of being mainly distributed in are with interior tang, and there is the plantation of introduction in China Hainan.That cocoa power has is anti-oxidant, regulate immunity, regulating blood glucose levels, reduction plasma cholesterol, anti-carious tooth, the multiple physiological function such as aid digestion, has now been widely used in the food service industrys such as chocolate, cake, ice cream, beverage.Modern study shows; in cocoa power, contained epicatechin is one of main effective constituent of its Polyphenols physiological activator; can effectively suppress to comprise the various diseases of diabetes, heart disease, apoplexy, cancer etc., have and the multiple physiological function such as regulate blood platelet, protect cardiovascular system, improve eyesight, be hypotensive, anti-oxidant.
But the performed industry standard of China's cocoa products has only been carried out relevant regulations to cocoa power organoleptic indicator, physical and chemical index and microbiological indicator at present, fails to reflect the inherent quality of cocoa power, can not control effectively to its quality.
Finger-print is as a kind of comprehensive identification of means, the chemical composition systematic study in sample of take is basis, kind and the content that can comprehensively reflect contained chemical composition in sample, authenticity, characteristic and the Optimality etc. that can be used for sample are evaluated, be one of main method of controlling of sample quality, now become one of effective technology of internationally recognized quality control.At present, finger-print is only studied its polysaccharide and lipid component as the quality control method of cocoa power, there is no the correlative study based on main active finger-print in cocoa power.
Summary of the invention
The present invention, for variation and the difference of thoroughly evaluating cocoa power active component, provides the finger print measuring method of active component in a kind of cocoa power.
For achieving the above object, the finger print measuring method of active component in cocoa power provided by the present invention, it comprises the following steps:
(a) preparation of reference substance solution: precision takes epicatechin reference substance, adding methyl alcohol and water volume ratio is the solution dissolving of 1:1, makes every 1mL containing the solution of epicatechin 66 μ g, obtains;
(b) preparation of need testing solution: precision takes cocoa power 0.25～2.5g, adding methyl alcohol and water volume ratio is solution 50～500mL of 1:1, ultrasonic extraction 30～60min filters, and gets subsequent filtrate, obtains;
(c) set chromatographic condition: with octadecylsilane chemically bonded silica, be filler, mobile phase A is acetonitrile, and Mobile phase B is that mass percent is 0.05% phosphate buffered saline(PBS); Adopt gradient elution mode, its elution program is:
0min → 20min, mobile phase A is 10% → 15%, Mobile phase B is 90% → 85%,
20min → 30min, mobile phase A is 15% → 30%, Mobile phase B is 85% → 70%;
Detect wavelength 210nm, flow velocity is 0.8mL/min, 30 ℃ of column temperatures; Theoretical cam curve, by epicatechin peak, is not less than 6000;
(d) acquisition of standard finger-print: accurate reference substance solution and the need testing solution 10 μ L of drawing, according to the chromatographic condition of setting, inject high performance liquid chromatograph, by the chromatographic peak of reference substance epicatechin, determine the epicatechin peak in need testing solution chromatogram, according to the method for chromatogram methodological study, obtain the standard finger-print of cocoa power;
(e) stratographic analysis: the finger-print of the need testing solution of every batch of gained and cocoa power standard finger-print, through similarity evaluation comparison, are determined to the quality of cocoa power.
The standard finger-print of the resulting cocoa power of the present invention detects 5 characteristic fingerprint peaks, and No. 5 peaks are epicatechin,
No. 1 peak: retention time: 4.82 ± 0.2min,
No. 2 peaks: retention time: 6.87 ± 0.2min,
No. 3 peaks: retention time: 14.64 ± 0.2min,
No. 4 peaks; Retention time: 17.52 ± 0.2min,
No. 5 peaks: retention time: 21.13 ± 0.2min.
Beneficial effect of the present invention: the present invention adopts high performance liquid chromatography to be studied the finger-print of cocoa power active component, tests the finger-print of the need testing solution of gained and cocoa power standard finger-print through similarity evaluation.The present invention has the following advantages:
1. the chromatographic process that the present invention sets up has advantages of that precision is high, good stability, reproducible, meets the requirement of finger-print, can be used as cocoa power quality determining method, the quality of overall monitor cocoa power;
2. the present invention can carry out effective separation by cocoa power active component, has built the finger-print of cocoa power, has filled up the blank that cocoa power is studied in active component separation and finger-print;
3. the quality assessment of cocoa power is no longer confined to the research to a certain composition, but from the overall evaluation, employing fingerprint figure spectrum signature is controlled the quality of cocoa power comprehensively, guarantee the health-care efficacy of cocoa power and the quality and safety of cocoa products, the evaluation platform of a kind of effective product quality detection method and technology innovation is provided.
Accompanying drawing explanation
Fig. 1 is the chromatogram of reference substance epicatechin.
Fig. 2 is the standard finger-print of cocoa power.
Fig. 3 is ten crowdes of cocoa power finger-print stacking diagrams.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
In following embodiment, epicatechin is bought in Nat'l Pharmaceutical & Biological Products Control Institute, lot number 110878; Methyl alcohol is chromatographically pure, buys the world company in the U.S..
Embodiment 1
(a) preparation of reference substance solution: precision takes epicatechin, the solution that adds methyl alcohol and water volume ratio and be 1:1 dissolves, mixes, constant volume, makes every 1mL containing the solution of epicatechin 66 μ g, makes reference substance solution.
(b) preparation of need testing solution: get 10 each 0.25g of separate sources cocoa power as shown in table 1, accurately weighed, be placed in tool plug conical flask, adding methyl alcohol and water volume ratio is the solution 50mL of 1:1, and ultrasonic extraction 30min filters, get subsequent filtrate, make need testing solution.
Table 1 cocoa power sample source
| Sample sequence number | Supplier | Qualification result |
| 1 | Shanghai company limited of nest | Theobroma?cacao?Linn. |
| 2 | Malaysia Yi Chang drink industrial group | Theobroma?cacao?Linn. |
| 3 | U.S. Hao Shi cocoa power company | Theobroma?cacao?Linn. |
| 4 | Delif cocoa group | Theobroma?cacao?Linn. |
| 5 | Malaysia cocoa office | Theobroma?cacao?Linn. |
| 6 | Shaoxing Qi Lixingguang cocoa products company | Theobroma?cacao?Linn. |
| 7 | Jiangsu wuxi Ding Yuan cocoa products factory | Theobroma?cacao?Linn. |
| 8 | Jiangsu wuxi Hua Feng cocoa power factory | Theobroma?cacao?Linn. |
| 9 | Wuxi Shang Ke Food Co., Ltd | Theobroma?cacao?Linn. |
| 10 | Wuxi quality supervision and test institute | Theobroma?cacao?Linn. |
(c) chromatographic condition: use the Agilent ZORBAX Extend-C that octadecylsilane chemically bonded silica is filler
18(250mm * 4.6mm, 5 μ m) chromatographic column; Adopt gradient elution, mobile phase A is acetonitrile, and Mobile phase B is that mass percent is 0.05% phosphate buffered saline(PBS), and the program of gradient elution is as shown in table 2:
Table 2 gradient elution program
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0→20 | 10→15 | 90→85 |
| 20→30 | 15→30 | 85→70 |
Detection wavelength is 210nm, and flow velocity is 0.8mL/min, and column temperature is 30 ℃; Theoretical cam curve is calculated and is not less than 6000 by epicatechin peak.
(d) acquisition of standard finger-print: accurate reference substance solution and the need testing solution 10 μ L of drawing, according to the chromatographic condition of setting, inject high performance liquid chromatograph, by the chromatographic peak of reference substance epicatechin, determine the epicatechin peak in need testing solution chromatogram, according to the method for chromatogram methodological study, obtain the standard finger-print of cocoa power.
Fig. 1 is the chromatogram of reference substance epicatechin, the standard finger-print that Fig. 2 is cocoa power.On the standard finger-print of cocoa power, detect 5 characteristic fingerprint peaks, No. 5 peak is epicatechin, as shown in table 3:
The characteristic fingerprint peak of table 3 cocoa power standard finger-print
| Peak sequence number | Retention time (min) | Peak area (mAU*min) |
| 1 | 4.82 | 3.969 |
| 2 | 6.87 | 74.496 |
| 3 | 14.64 | 11.172 |
| 4 | 17.52 | 5.368 |
| 5 | 21.31 | 27.417 |
(e) stratographic analysis: the quality of the evaluation cocoa power that finger-print can be comprehensive, therefore by the cocoa power finger-print of every batch of gained and cocoa power standard finger-print through the comparison of similarity evaluation 2004A version.The similarity of calculating 10 batches of cocoa power chromatographic peaks, similarity is not less than 0.9, illustrates that cocoa power is up-to-standard.10 crowdes of cocoa power finger-print stacking diagrams as shown in Figure 3.
Methodological study of the present invention:
1) precision research: the accurate need testing solution 10 μ L that draw, according to the chromatographic condition of setting, continuous sample introduction 5 times, records its chromatogram.Using epicatechin peak as with reference to peak, and specifying its α value is 1.0000, investigates instrument precision.The RSD value of each characteristic fingerprint peak relative retention time and relative peak area is all less than 3%, meets the requirement of finger-print.
2) stability study: the accurate need testing solution 10 μ L that draw, the chromatographic condition according to setting, detects at 0h, 4h, 8h, 12h, 24h respectively, take epicatechin peak as with reference to peak, and specifying its α value is 1.0000, investigation need testing solution stability.The RSD value of the relative retention time at each characteristic fingerprint peak and relative peak area is all less than 3%, meets the requirement of finger-print.
3) repetitive research: get 5 parts, same batch of cocoa power sample, accurately weighed, by method step (b) Suo Shu, prepare need testing solution, according to the chromatographic condition of setting, measure, take epicatechin peak as with reference to peak, and specifying its α value is 1.0000, the repeatability of investigation method.The RSD value of the relative retention time at each characteristic fingerprint peak and relative peak area is all less than 3%, meets the requirement of finger-print.
Claims (2)
1. a finger print measuring method for active component in cocoa power, is characterized in that, it comprises the following steps:
(a) preparation of reference substance solution: precision takes epicatechin reference substance, adding methyl alcohol and water volume ratio is the solution dissolving of 1:1, makes every 1mL containing the solution of epicatechin 66 μ g, obtains;
(b) preparation of need testing solution: precision takes cocoa power 0.25～2.5g, adding methyl alcohol and water volume ratio is solution 50～500mL of 1:1, ultrasonic extraction 30～60min filters, and gets subsequent filtrate, obtains;
(c) set chromatographic condition: with octadecylsilane chemically bonded silica, be filler, mobile phase A is acetonitrile, and Mobile phase B is that mass percent is 0.05% phosphate buffered saline(PBS); Adopt gradient elution mode, its elution program is:
0min → 20min, mobile phase A is 10% → 15%, Mobile phase B is 90% → 85%,
20min → 30min, mobile phase A is 15% → 30%, Mobile phase B is 85% → 70%;
Detect wavelength 210nm, flow velocity is 0.8mL/min, 30 ℃ of column temperatures; Theoretical cam curve, by epicatechin peak, is not less than 6000;
(d) acquisition of standard finger-print: accurate reference substance solution and the need testing solution 10 μ L of drawing, according to the chromatographic condition of setting, inject high performance liquid chromatograph, by the chromatographic peak of reference substance epicatechin, determine the epicatechin peak in need testing solution chromatogram, according to the method for chromatogram methodological study, obtain the standard finger-print of cocoa power;
(e) stratographic analysis: the finger-print of the need testing solution of every batch of gained and cocoa power standard finger-print, through similarity evaluation comparison, are determined to the quality of cocoa power.
2. the finger print measuring method of active component in cocoa power according to claim 1, is characterized in that: the standard finger-print of resulting cocoa power detects 5 characteristic fingerprint peaks, and No. 5 peaks are epicatechin,
No. 1 peak: retention time: 4.82 ± 0.2min,
No. 2 peaks: retention time: 6.87 ± 0.2min,
No. 3 peaks: retention time: 14.64 ± 0.2min,
No. 4 peaks; Retention time: 17.52 ± 0.2min,
No. 5 peaks: retention time: 21.13 ± 0.2min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107501227A (en) * | 2017-08-15 | 2017-12-22 | 武汉黄鹤楼香精香料有限公司 | A kind of method for preparing epicatechin from cocoa power using high performance liquid chromatography |
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| WO2001097777A1 (en) * | 2000-06-23 | 2001-12-27 | Whan In Pharm. Co. Ltd. | Topical preparation of alprostadil for the treatment of erectile dysfunction |
| CN102680607A (en) * | 2012-06-07 | 2012-09-19 | 江南大学 | Cocoa powder adulteration detection method based on fingerprints |
| CN102778517A (en) * | 2012-07-16 | 2012-11-14 | 无锡市产品质量监督检验中心 | Method for detecting cocoa powder adulteration based on lipid clustering analysis |
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2014
- 2014-09-15 CN CN201410470234.2A patent/CN104215711A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2001097777A1 (en) * | 2000-06-23 | 2001-12-27 | Whan In Pharm. Co. Ltd. | Topical preparation of alprostadil for the treatment of erectile dysfunction |
| CN102680607A (en) * | 2012-06-07 | 2012-09-19 | 江南大学 | Cocoa powder adulteration detection method based on fingerprints |
| CN102778517A (en) * | 2012-07-16 | 2012-11-14 | 无锡市产品质量监督检验中心 | Method for detecting cocoa powder adulteration based on lipid clustering analysis |
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| ELIZABETH M. HUMSTON ETAL.: "Quantitative assessment of moisture damage for cacao bean quality using two-dimensional gas chromatography combined with time-of-flight mass spectrometry and chemometrics", 《JOURNAL OF CHROMATOGRAPHY A》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107501227A (en) * | 2017-08-15 | 2017-12-22 | 武汉黄鹤楼香精香料有限公司 | A kind of method for preparing epicatechin from cocoa power using high performance liquid chromatography |
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Application publication date: 20141217 |