CN108020605A - A kind of short-cut method of hair detection Co-Q10 content - Google Patents
A kind of short-cut method of hair detection Co-Q10 content Download PDFInfo
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- CN108020605A CN108020605A CN201610988588.5A CN201610988588A CN108020605A CN 108020605 A CN108020605 A CN 108020605A CN 201610988588 A CN201610988588 A CN 201610988588A CN 108020605 A CN108020605 A CN 108020605A
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- hair
- ethyl alcohol
- absolute ethyl
- ubiquinone
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- 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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Abstract
The invention discloses a kind of short-cut method of hair detection Co-Q10 content.A part of the hair as body, containing cell, can carry out metabolism, and the growth of hair needs nutrition, the quality of hair just in hair cell it is metabolic strong and weak related.And ubiquinone10A kind of and one of element for weighing human health level.The present invention establishes the short-cut method of Co-Q10 content in the Extraction solvent using absolute ethyl alcohol as hair and application high-efficiency liquid chromatography method for detecting detection hair, overcome the extracting method of existing detection method numerous and diverse, organic solvent is using more, method is not sensitive enough, the difficulty such as it is not sufficiently stable, develop and develop a kind of Extraction solvent and be simple and convenient to operate, while the analysis method for the hair Co-Q10 that accuracy is high, analyze speed is fast.
Description
Technical field
The invention belongs to technical field of analysis and detection, is specially a kind of short-cut method of hair detection Co-Q10 content.
Background technology
Co-Q10 is a kind of important liposoluble substance, is present in the advanced animal and plant mitochondria based on the mankind
In film, played in respiratory chain and pass hydrogen carrier effect.It has activation to many enzymes, and can increase the immunity of body.
CoQ10 is also a kind of important antioxidant in body at the same time, and there is natural Green Tea Extract to aoxidize, prevent Cell membrane lipids
Peroxidization, the function of keeping membrane fluidity.But Co-Q10 can be reduced constantly with the increase at disease or age,
Therefore, the content of internal Co-Q10 is detected, is of great significance for the weight and aging state for observing and judging disease.
A part of the hair as body, containing cell, can carry out metabolism, the growth of hair needs nutrition, and the quality of hair is just
To in hair cell it is metabolic strong and weak related.Hair is fairly common as the horizontal application for weighing human elementary, card
It is relevant that the content of element contact with nutrition intake and environment in real hair, and by the use of hair as observe it is micro- in vivo
The biopsy material that metabolism and individual are contacted with environment.And ubiquinone10A kind of and one of element for weighing human health level.Survey
The method of CoQ10 is determined it has been reported that wherein mainly taking spectrophotometry.The sensitivity and specificity of these methods are not
Enough ideals.It is generally more difficult to the measure for being applied to micro C oQ10 in biological sample particularly hair.In recent years high performance liquid chromatography point
Analysis method (HPLC) has started to be applied to CoQ10 assays in blood plasma biological sample, with C oQ10 content phases in plasma sample
Than, CoQ10 contents are lower in hair, there is presently no report, the country is there is not yet correlative study.
The content of the invention
It is an object of the invention to establish a kind of simple and practical Co-Q10 detection method using hair as detection sample,
Overcome the extracting method of existing detection method numerous and diverse, for organic solvent using more, method is not sensitive enough, the difficulty such as is not sufficiently stable,
Develop and develop a kind of Extraction solvent and be simple and convenient to operate, while the hair Co-Q10 that accuracy is high, analyze speed is fast
Analysis method.
The present invention establishes the Extraction solvent using absolute ethyl alcohol as hair and application high-efficiency liquid chromatography method for detecting is examined
The short-cut method of Co-Q10 content in gauge head hair, it is characterised in that：
(1) Co-Q10 in absolute ethyl alcohol extraction hair：Hair is weighed, is placed in round-bottomed flask, adds hair weight
The absolute ethyl alcohol of 8~15 times of amounts, extracts Co-Q10, extracting solution steams recycling by 80 DEG C of rotations again in 80 DEG C of heating water bath 60min
Extraction solvent, the absolute ethyl alcohol dissolving of sediment again, through 0.22 μm of membrane filtration, to obtain the final product；
(2) the Co-Q10 chromatography in hair：The absolute ethyl alcohol extracting solution for taking (1) to obtain, by following chromatography bars
Part is analyzed：Chromatographic column Eclipse Plus C18(4.6*250nm, 5 μm), mobile phase：Methanol: ethanol (v: v)=20: 80, stream
Speed：1ml/min, Detection wavelength：275nm, column temperature：30 DEG C, sampling volume：20μl.
The present invention is by extracting the ubiquinone in hair using absolute ethyl alcohol as extractant10, establish high performance liquid chromatography point
Analysis method is to hair ubiquinone10It is detected, can be right using chromatographic column and methanol-absolute ethyl alcohol (volume ratio 20: 80) mobile phase
Ubiquinone in hair10Carry out quick separating analysis.Method sample preparation processing is simple, and analysis is efficient, as a result accurately, stability
It is good, it is a kind of ubiquinone of simple and practical hair10Detection method.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further.Embodiment is only used for the present invention and is not limited to this
The scope of invention.
1 materials and methods
1.1 reagents and instrument
Absolute ethyl alcohol (analyzes pure, Tianjin great Mao producers)；Absolute ethyl alcohol, methanol (chromatographically pure, Tianjin Ke Miou producers)；It is auxiliary
Enzyme Q10Bulk pharmaceutical chemicals (Guangdong is moistened and bio tech ltd), hair (is collected in the head of Guangdong barber shop of medical university student group
Hair)；High performance liquid chromatograph (Anjelen Sci. ＆ Tech. Inc), Rotary Evaporators (Shanghai Ai Lang Instrument Ltd).
1.2 method
1.2.1HPLC ubiquinone is detected10Chromatographic condition
Chromatographic column Eclipse Plus C18(4.6*250nm, 5 μm), mobile phase：Methanol: ethanol (v: v)=20: 80, stream
Speed：1ml/min, Detection wavelength：275nm, column temperature：30 DEG C, sampling volume：20μl.
1.2.2 the preparation of hair extracting solution and reference substance solution
1. the preparation of test sample
A. the source of hair：It is collected in the hair of 18-23 Sui students of Guangdong barber shop of medical university.
B. test solution：Precision weighs 4.5g hairs, is placed in the round-bottomed flask of 100ml, and the extraction for adding 50ml is molten
Agent, 80 DEG C of heating water bath 60min, after having tested, sample solution are steamed in 80 DEG C of rotations, volatilizes Extraction solvent, then the nothing with 2ml
Water-ethanol dissolves, to be measured through 0.22 μm of membrane filtration.
2. reference substance solution：Precision weighs ubiquinone10Reference substance 25mg, is placed in the volumetric flask of 25ml, adds absolute ethyl alcohol
25ml is settled to, ultrasonic oscillation dissolves 5min, to be measured through 0.22 μm of membrane filtration up to reference substance storing solution after cooling[6]。
1.2.3 ubiquinone10The standard curve of standard items
Precision weighs ubiquinone10Reference substance 25mg, is placed in the volumetric flask of 25ml, adds absolute ethyl alcohol and is settled to 25ml, surpasses
Sound wave shock dissolves 5min, up to reference substance storing solution after cooling, a series of mark product of concentration is diluted to, with ubiquinone10Concentration X
Linear regression is carried out to peak area Y[5-7]。
1.2.4 assay and methodological study
1.2.4.1 stability experiment
1. reference substance stability：Precision weighs ubiquinone10Reference substance 25mg, is placed in the volumetric flask of 25ml, adds absolute ethyl alcohol
25ml is settled to, ultrasonic oscillation dissolving 5min, shakes up a series of pair for up to reference substance storing solution, being diluted to concentration after cooling
According to product solution, take the reference substance solution that concentration is 6.25 μ l/ml, under the conditions of lucifuge (room temperature) respectively at 0,2,4,6,8,10,
12h distinguishes 20 μ l of sample introduction, measures ubiquinone10Main peak area value, calculates ubiquinone10The RSD% of peak area.
2. test sample stability：Precision weighs 4.5g hairs, is placed in the round-bottomed flask of 100ml, adds the absolute ethyl alcohol of 50ml,
80 DEG C of heating water bath 60min, after having tested, sample solution is steamed in 80 DEG C of rotations, volatilizes petroleum ether, then the absolute ethyl alcohol with 2ml
Dissolving, obtains test solution.To same test solution, (room temperature) divides respectively at 0,2,4,6,8,10,12h under the conditions of lucifuge
Other 20 μ l of sample introduction, measure ubiquinone10Main peak area value, calculates ubiquinone10The RSD% of peak area.
1.2.4.2 Precision Experiment
1. reference substance precision：Precision weighs ubiquinone10Reference substance 25mg, is placed in the volumetric flask of 25ml, adds absolute ethyl alcohol
25ml is settled to, ultrasonic oscillation dissolving 5min, shakes up up to reference substance storing solution after cooling, diluted by certain multiple, essence
Close to draw the 20 μ l of reference substance solution that concentration is 6.25 μ l/ml, continuous sample introduction 6 times, is investigated with the main peak area of standard solution
Sample introduction precision.
2. test sample precision：Precision weighs 4.5g hairs, is placed in the round-bottomed flask of 100ml, adds the absolute ethyl alcohol of 50ml,
80 DEG C of heating water bath 60min, after having tested, sample solution are rotated at 80 DEG C, volatilizes absolute ethyl alcohol, then the anhydrous second with 2ml
Alcohol dissolves, and obtains test solution, through 0.22 μm of membrane filtration, accurate 20 μ l of draw solution, continuous sample introduction 6 times, tries to achieve ubiquinone10
Peak area RSD%.
1.2.4.3 repeated experiment
Take with a batch of, 3 parts of sample solution, with legal system available test sample solution, peak face is measured under " 1.2.1 " chromatographic condition
Product.1.2.4.4 sample recovery rate is tested
Precision weighs ubiquinone10Reference substance 25mg, is placed in the volumetric flask of 25ml, adds absolute ethyl alcohol and is settled to 25ml, ultrasound
Ripple concussion dissolving 5min, shakes up after cooling up to reference substance storing solution.Four parts of hair 4.5g are taken to be respectively placed in the circle of four 100ml
In the flask of bottom, the absolute ethyl alcohol of 50ml is separately added into round-bottomed flask, then accurate measure is the average auxiliary of repeated experiment successively
Enzyme Q10The not mark-on of 80%, 100% and 120% above-mentioned reference substance of concentration and one, at 85 DEG C water-bath flow back 60min, cooling
Afterwards, rotated at 80 DEG C, volatilize solution, then dissolved with the absolute ethyl alcohol of 2ml, filtered by 0.22 μm of filter membrane, take subsequent filtrate, treat
Survey.
1.2.4.5 interference is tested
Precision draws absolute ethyl alcohol (analysis is pure) by 20 μ l of chromatographic condition sample introduction injection liquid chromatographs, records chromatogram.
2 results and analysis
2.1 ubiquinone10The standard curve of standard items
Precision weighs ubiquinone10Reference substance 12.5mg, is placed in the volumetric flask of 25ml, and 25ml is settled to absolute ethyl alcohol, first
5 times of dilution, then 2 times of dilution is pressed successively, a series of concentration standard solution (12.5 μ g/ml, 6.25 μ g/ml, 3.125 μ g/ are made
Ml, 1.5625 μ g/ml, 0.78125 μ g/ml), precision draws 20 μ l, injects liquid chromatograph, chromatogram is recorded, by external standard
Method --- calculated by peak area.With ubiquinone10Concentration X carries out linear regression to peak area Y.Standard curve is shown in Fig. 1, and regression equation is：
That is ubiquinone10Concentration X and peak area Y linear relationships in the range of concentration is the μ g/ml of 0.78125 μ g/ml~12.5 are good.It is auxiliary
The standard curve of enzyme Q10 is shown in attached drawing 1.
2.2 system suitabilities are tested
Standard solution, test solution is taken to distinguish sample introduction by chromatogram analysis method.Co-Q10 standard items chromatogram result
Such as Fig. 2, the HPLC chromatogram of hair sample is shown in that Fig. 3, Fig. 4 show that hair is overlapped with the peak area of Co-Q10 standard items, several from this
HPLC chromatogram is opened as it can be seen that ubiquinone10Peak shape is good, theoretical cam curve 8676, ubiquinone10Separating degree with adjacent peak is big
In 1.5, symmetrical factor shows that institute's construction method has good specificity 0.95~1.05.
2.3 interference are tested
Take reference substance, test sample solvent (absolute ethyl alcohol) to inject liquid chromatograph by 20 μ l of chromatographic condition sample introduction, record color
Spectrogram, is shown in Fig. 5.The result shows that solvent is noiseless under this chromatographic condition.
2.4 stability experiment
1. taking reference substance solution (6.25 μ g/ml), (room temperature) divides respectively at 0,2,4,6,8,10,12h under the conditions of lucifuge
Other 20 μ l of sample introduction, measure ubiquinone10Main peak area value, each impurity peaks peak area is substantially unchanged, calculates ubiquinone10Peak area
RSD%.It the results are shown in Table 1.
1 reference substance stability experiment result of table
2. to same test solution, (room temperature) distinguishes sample introduction 20 respectively at 0,2,4,6,8,10,12h under the conditions of lucifuge
μ l, measure ubiquinone10Main peak area value, each impurity peaks peak area is substantially unchanged, calculates ubiquinone10The RSD% of peak area.Knot
Fruit is shown in Table 2.
2 test sample stability experiment result of table
2.5 Precision Experiment
1. reference substance precision is accurate to draw reference substance solution (6.25 μ g/ml) 20 μ l, continuous sample introduction 6 times, with standard items
The main peak area of solution investigates sample introduction precision.It the results are shown in Table 3.
3 reference substance Precision Experiment result of table
2. test sample precision is accurate to draw 20 μ l of test solution, continuous sample introduction 6 times, tries to achieve ubiquinone10Peak area
RSD values are 0.88%, the results are shown in Table 4.Test result indicates that instrument precision is good.
4 test sample Precision Experiment result of table
2.6 repeated experiment
Take with a batch of 3 parts of sample solution, with legal system available test sample solution, peak is measured under " 1.2.1 " chromatographic condition
Area, the results are shown in Table 5.
5 repeated experiment result of table
2.7 sample recovery rates are tested
It (is respectively the average ubiquinone of repeated experiment that precision, which draws basic, normal, high three kinds of concentration,10The 80% of concentration, 100%
With ubiquinone 120%)10Reference substance solution, is separately added into 3 parts of samples of known content, by the optimal preparation of test solution
After prepared by method, analyzed under above-mentioned chromatographic condition, calculate the rate of recovery.It the results are shown in Table 6.Ubiquinone10Average recovery rate be
92.96%.
Ubiquinone in 6 hair of table10Sample-adding recycling measurement result
3 conclusions
This research passes through the ubiquinone in the extracting solution to hair10HPLC analytical method is established, using chromatography
Column and methanol-absolute ethyl alcohol (volume ratio 20: 80) mobile phase can be to the ubiquinones in hair10Carry out quick separating analysis.Method
Sample preparation processing is simple, and analysis is efficient, as a result accurately, the ubiquinone available for hair10Detection.
Brief description of the drawings
Fig. 1 is the HPLC chromatogram of the standard curve of Co-Q10 standard items；
Fig. 2 is the HPLC chromatogram of Co-Q10 standard items；
Fig. 3 is the HPLC chromatogram of hair sample；
Fig. 4 is the HPLC chromatogram that hair is overlapped with the peak area of Co-Q10 standard items；
Fig. 5 is the HPLC chromatogram of absolute ethyl alcohol interference.
Claims (1)
- It is 1. auxiliary in a kind of Extraction solvent and application high-efficiency liquid chromatography method for detecting detection hair using absolute ethyl alcohol as hair The short-cut method of enzyme Q10 contents, it is characterised in that：(1) Co-Q10 in absolute ethyl alcohol extraction hair：Weigh hair, be placed in round-bottomed flask, add hair weight 8~ The absolute ethyl alcohol of 15 times of amounts, extracts Co-Q10, extracting solution steams recycling by 80 DEG C of rotations again and carries in 80 DEG C of heating water bath 60min Take solvent, the absolute ethyl alcohol dissolving of sediment again, through 0.22 μm of membrane filtration, to obtain the final product；(2) the Co-Q10 chromatography in hair：The absolute ethyl alcohol extracting solution for taking (1) to obtain, by following chromatographicconditions point Analysis：Chromatographic column Eclipse Plus C18(4.6*250nm, 5 μm), mobile phase：Methanol: ethanol (v: v)=20: 80, flow velocity： 1ml/min, Detection wavelength：275nm, column temperature：30 DEG C, sampling volume：20μl.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5547580A (en) * | 1992-10-14 | 1996-08-20 | Eisai Chemical Co., Ltd. | Purification method of crude product |
| CN103196860A (en) * | 2013-03-06 | 2013-07-10 | 厦门金达威集团股份有限公司 | Rapid screening method of high-yielding coenzyme Q10 strain |
| CN105467029A (en) * | 2015-11-23 | 2016-04-06 | 威海百合生物技术股份有限公司 | Detection method of coenzyme Q10 in health product |
-
2016
- 2016-11-03 CN CN201610988588.5A patent/CN108020605A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5547580A (en) * | 1992-10-14 | 1996-08-20 | Eisai Chemical Co., Ltd. | Purification method of crude product |
| CN103196860A (en) * | 2013-03-06 | 2013-07-10 | 厦门金达威集团股份有限公司 | Rapid screening method of high-yielding coenzyme Q10 strain |
| CN105467029A (en) * | 2015-11-23 | 2016-04-06 | 威海百合生物技术股份有限公司 | Detection method of coenzyme Q10 in health product |
Non-Patent Citations (4)
| Title |
|---|
| D.D.ANKOLA 等: "Development of potent oral nanoparticulate formulation of coenzyme Q10 for treatment of hypertension: Can the simple nutritional supplements be used as first line therapeutic agents for prophylaxis/therapy?", 《EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS》 * |
| 缪宁梅 等: "高效液相色谱法检查辅酶Q10片中有关物质", 《中国生化药物杂志》 * |
| 郭菊玲 等: "RP-HPLC测定辅酶Q10含量及有关物质的方法学研究", 《海峡药学》 * |
| 闰花丽 等: "高效液相色谱法测定辅酶Q10制剂的含量", 《中国升华药物杂志》 * |
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Application publication date: 20180511 |