CN102435565A - Quantitative measurement method for theanine - Google Patents
Quantitative measurement method for theanine Download PDFInfo
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- CN102435565A CN102435565A CN2011102863666A CN201110286366A CN102435565A CN 102435565 A CN102435565 A CN 102435565A CN 2011102863666 A CN2011102863666 A CN 2011102863666A CN 201110286366 A CN201110286366 A CN 201110286366A CN 102435565 A CN102435565 A CN 102435565A
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Abstract
The invention discloses a quantitative measurement method for theanine. The method comprises the following steps of: leaching a tea sample by using water, spotting a theanine standard solution, a blank solution and a tea leaching liquor on rectangular filter paper respectively, taking an ethanol-water-ninhydrin mixed solution as a developing agent, performing vertical chromatography, drying, eluting by using an ethanol solution, and measuring the content of the theanine by comparing color at the wavelength of 570nm by using a spectrophotometer. The tea sample is leached by water and the ethanol solution is used for development, so that the stimulation of toxic substances to operators and pollution of the toxic substances on environment are avoided; ninhydrin is mixed into the developing agent, so that operating steps are reduced, and the quantification of development reaction and uniform color spots are ensured; the blank solution and the theanine working solution are spotted and subjected to chromatography, and a standard curve is made, so that a control, a standard substance and a sample are subjected to the same treatment, errors are reduced, and the measurement accuracy is improved; and all measurement steps are simple, the types of related medicines are a few, and the method is economic and environment-friendly.
Description
Technical field
The invention belongs to biochemical field, particularly the plant sample ply of paper is analysed the method for back with the spectrophotometric determination theanine content.
Background technology
The L-theanine has another name called glutamy ethamine, English name L-Theanine, Cas number: 3081-61-6, molecular formula: C
7H
14N
2O
3, the genus amides compound is white or off-white color powdery, fusing point 214-215 degree is very easily water-soluble, has the refreshing flavor of aquatic foods of camerlsed and similar monosodium glutamate.
The L-theanine exists only in Camellia part plant and a kind of gill fungus at nature, account for dry weight of tea leaves 1% ~ 2% between, account for more than 50% of whole free amino acid, be one of main matter that constitutes the tea local flavor, also be widely used in food service industry.Modern medicine confirms that theanine has many-sided health-care effect to human body, especially can regulate neurotransmitter, and gentle people's mood is used to treat anxiety, insomnia, ADD etc.
The method of measuring theanine at present mainly contains high performance liquid chromatography (HPLC) method, capillary electrophoresis, automatic amino acid analyzer method, anion-exchange chromatography-integrated pulse Amperometric Detection Coupled method.These methods have highly sensitive and the strong characteristics of accuracy, but equipment purchasing and maintenance cost are very high, can't satisfy the demand that common lab is measured theanine.
Cheap, the easy operation of spectrophotometer is breadboard standing instrument.Ninhydrin reaction is the most frequently used method of experimental determination amino acid, and its principle is that the product that amino acid and ninhydrin reaction generate has the highest absorbance at the 570nm place, meets Beer law within the specific limits.But what measure in this way is various amino acid whose mixing resultants in the tea.Higher primary school is red to wait people 2005 at the 3rd phase of " spectrographic laboratory " magazine report " theanine in the spectrophotometry tealeaves "; With concentrated hydrochloric acid theanine is resolved into ethamine; Utilize the chlorinated derivative ability oxidation potassium iodide of amine and separate out iodine; After adding people's starch, it is the darkest that the color of solution can reach, and the stable character of 10min internal absorbance is carried out the theanine quantitative measurement.Yet this method relates to NaOH, NaHCO
3, NaClO
3, NaNO
2, many medicines such as starch, KI, operation steps is numerous and diverse, adds NaClO in the middle of especially
3After need to place 1min, add NaNO
25min is placed in the back, and when mensuration had several samples, these times were difficult to control, developing time are more arranged only 10 minutes, are not suitable for a plurality of samples and measure simultaneously.
Ruan Yucheng and Wang Yue root 1981 were published an article " the simplification sizing technique of theanine " in " tealeaves communication " the 3rd phase, use successively ether--ethyl acetate mixed liquor and ethanolic solution lixiviate tea appearance, concentrate leaching liquor; Be dissolved in ethanolic solution; Get supernatant 20-25 μ L point sample on circular filter paper after centrifugal, strong aqua is smoked, blows away ammonia; Use normal butyl alcohol: glacial acetic acid: water=make developing agent at 4: 1: 1, the level exhibition is composed.Drying is opened up spectrum once more with new developing agent.Spray with ninhydrin solution in the dry back of paper spectrum, dry, colour developing, ethanolic solution wash-out, spectrophotometer 570nm place's colorimetric estimation.Measure used standard solution by the pure article preparation of theanine, cut the other place of paper spectrum theanine spot and do blank.The ether highly volatile that this method is used is anesthetic, belongs to dangerous material, and ethyl acetate, normal butyl alcohol, glacial acetic acid, ammoniacal liquor are all human body lower toxicity medicine excitatory; Circular filter paper is shorter because of the limited exhibition spectrum distance of diameter leaves, and needs continuous quadratic exhibition spectrum could amino acid be separated, and causes waste medicine and not free advantage; Chromogenic reaction occurs in ply of paper and analyses triketohydrindene hydrate spraying afterwards; Because the control of the developer amount of not having causes color spot inhomogeneous: typical curve is through formulated, and standard specimen does not carry out ply of paper and analyses step; Handle different with working sample; Produce error at measurment: blank does not have special point sample, arbitrarily cuts, and these cause mensuration accurate inadequately.
Summary of the invention
The object of the invention aims to provide a kind of simple, safe and reliable theanine spectrophotometer detection method, carries out the needs of theanine Biochemistry Experiment and research to satisfy common lab.
For realizing said purpose, the present invention adopts technical scheme following:
Theanine method for quantitatively determining of the present invention comprises the steps:
(1) lixiviate: with the dried tea appearance of 60-100ml boiling water lixiviate 0.2-4.0g 30-60min, be chilled to room temperature, filter, be settled to 100ml, get the tea leaching liquor;
(2) point sample: with theanine standard solution, blank distilled water and tea leaching liquor point sample on filter paper respectively; Said concentration of standard solution is pointed out three points at 0.06-0.2mg/ml and the standard solution of choosing same concentration according to different point sample amounts at least;
(3) exhibition layer: with alcohol-water-triketohydrindene hydrate mixed solution as developing agent, vertical chromatography; In said alcohol-water-triketohydrindene hydrate mixed solution, ethanol volume fraction 70-80%, triketohydrindene hydrate volume fraction 0.1-0.3%;
(4) wash-out: with the filter paper behind the chromatography in 40-80 ℃ of oven dry; Filter paper that the theanine colour developing is with in standard solution theanine color spot filter paper, the tea leaching liquor under cutting out and blank zone filter paper; The ethanolic solution wash-out gets standard specimen eluent, tea appearance eluent and blank eluent;
(5) colorimetric, calculating: with the blank sample is reference, uses spectrophotometer 570nm place to measure the light absorption value of standard specimen solution and tea appearance solution respectively; The production standard curve is obtained regression equation, calculates the theanine content in the tea sample.Computing formula:
Theanine %=
Wherein, the theanine quality (mg) of M for finding, L from typical curve
1Be sample constant volume (mL), L
2Be sample point sample volume (μ L) that W is sample dry weight (g)
Preferably, step (2) empty contrast distilled water is all 20-40 μ l mutually with the point sample volume of tea leaching liquor, and the standard solution of choosing same concentration to point out six points according to different point sample amounts be good.
In the said alcohol-water of step (3)-triketohydrindene hydrate mixed solution, preferred alcohol volume fraction 75%, triketohydrindene hydrate volume fraction 0.2%.
Filter paper is good in 60 ℃ of dry 10-15min in the step (4).
Preferably, the ethanol volumetric concentration of ethanolic solution is 60-70% in the step (4).
It is good placing 40-60 ℃ of water-bath 10-20min during wash-out in the step (4).
The beneficial effect of the invention is following:
1, with flooding tea appearance, ethanolic solution exhibition layer avoids noxious material to operating personnel's stimulation with to the pollution of environment.
2, triketohydrindene hydrate is sneaked into developing agent, has both reduced operation steps, guarantees that also chromogenic reaction quantification, color spot are even.
3, blank and theanine working fluid have guaranteed that through point sample, chromatography production standard curve contrast, standard substance have identical processing with sample, reduce error, improve the accuracy of measuring.
4, triketohydrindene hydrate colour developing keeps stable in a long time, analyzes when can be used for a plurality of sample.
5, whole determination step is simple, and it is few to relate to drug variety, economy, environmental protection.
Description of drawings
Fig. 1 is that tea root, bud-leaf are measured the theanine typical curve;
Fig. 2 measures the theanine typical curve for tealeaves.
Embodiment
Embodiment 1
Clip living then tea tree poplar forest kind water planting in July seedling root system and bud-leaf clean respectively, and oven dry grinds.Weigh up 0.219g root and 0.391g bud-leaf respectively, adding distil water 60ml is as for lixiviate 45min in the boiling water bath, during whenever rock stirring once at a distance from 10min.Lixiviate finishes the back placement and is cooled to room temperature, is settled to 100ml after the filtration.
Chromatography filter paper is cut into suitable big small rectangle, apart from the lower edge 1.5cm place whenever from left to right on horizontal line, draws a point of crossing with pencil drawing one horizontal line at a distance from 2.5cm, from following laterally 10cm picture one mark upwards, be to open up a layer stop flag.
With micropipet draw sample in the filter paper point of crossing point sample 40 μ l, once promptly use drier at every, the sampling point diameter is no more than 0.5cm, repetitive operation is till having put required point sample amount.
At point of crossing point of filter paper distilled water identical with sample volume as blank.
Take by weighing 100 mg purity and be not less than 99% theanine (being accurate to 0 .0000g) and be dissolved in the 100 mL water, accurately draw 5 mL mother liquors, add water and be settled to 50 mL as working fluid as mother liquor.
Draw the above-mentioned theanine working fluid of 20,30,40,50,60,70 μ l respectively with micropipet, according to the sample spot quadrat method respectively at 6 point of crossing point samples of filter paper.
In the time of point sample, on smooth experiment table, spread preservative film, the developing agent (75% ethanolic solution that contains 0.2% triketohydrindene hydrate) that 50ml prepares is poured in the double dish of diameter 15cm; Buckle an enough big beaker; Use the preservative film environmental sealing, fumigate, fumigation time is no less than 40min.After point sample finishes to treat the sampling point bone dry; Chromatography filter paper is crooked along horizontal edge; Two vertical edges are connected (two the limit does not contact) with cotton thread or transparent adhesive tape be rolled into tubular; Remove the large beaker of having fumigated rapidly, the chromatography filter paper of tubular is vertically put into the double dish that developing agent is housed, buckle the large beaker of having fumigated immediately.Exhibition layer about 3h of time arrives the upper edge mark of having finished until the developing agent upper edge and gets final product.
The chromatography filter paper that chromatography is accomplished hangs over dry 10min in 60 ℃ the baking oven rapidly.
Cut out standard solution (being the theanine working fluid of above-mentioned six different point sample amounts) theanine color spot filter paper (1.5cm * 1.5cm) and on the sample chromatogram include filter paper part and the blank zone that the theanine colour developing is with down with onesize, the same position of standard solution; Shred and put into test tube; Add 5ml eluant, eluent (60% ethanol); Jog is placed on 20min in 40 ℃ of water-baths, is placed to the room temperature measuring light absorption value after the taking-up.
Preheating 721 spectrophotometers are reference with the blank sample, measure the light absorption value of standard specimen and each sample at the 570nm place.
Standard specimen theanine content and light absorption value are as shown in table 1:
Table 1 tea root, bud-leaf theanine are measured standard specimen theanine content and light absorption value
| Content (mg) | 0.002 | 0.003 | 0.004 | 0.005 | 0.006 | 0.007 |
| Light absorption value | 0.011 | 0.016 | 0.022 | 0.026 | 0.032 | 0.035 |
The production standard curve is like Fig. 1.Obtaining regression equation is y=0.2022x-0.0003 (r
2=0.9936).The tea root light absorption value is 0.035, and the bud-leaf light absorption value is 0.020, goes out root and the bud-leaf theanine is respectively 0.0068mg and 0.0037mg by regression equation calculation.
Bring correlation values into formula, calculate theanine content:
Root theanine% =
=
= 7.74
Shoots theanine% =
=
= 2.39
Embodiment 2
Take by weighing Xinyang Maojian Tea, Keemun black tea, each 2.4g of Anxi Tieguanyin Tea respectively, oven dry back quality is respectively 2.183g, 2.237 and 2.208g.Grind, adding distil water 100ml is lixiviate 45min in boiling water bath, during every rock stirring once at a distance from 10min.Lixiviate finishes the back placement and is cooled to room temperature, is settled to 100ml after the filtration.
Chromatography filter paper is cut into the rectangle of suitable size, apart from the lower edge 1.5cm place whenever from left to right on horizontal line, draw a point of crossing with pencil drawing one horizontal line at a distance from 2.5cm; From following 10cm picture one mark that laterally makes progress, be an exhibition layer stop flag.
With micropipet draw sample in the filter paper point of crossing point sample 20 μ l, once promptly use drier at every, the sampling point diameter is no more than 0.5cm, repetitive operation is till having put required point sample amount.
At point of crossing point of filter paper and sample with the distilled water of volume as blank.
Take by weighing 100 mg purity and be not less than 99% theanine (being accurate to 0 .0000g) and be dissolved in the 100 mL water, accurately draw 5 mL mother liquors, add water and be settled to 50 mL as working fluid as mother liquor.
Draw the above-mentioned theanine working fluid of 20,30,40,50,60,70 μ l respectively with micropipet, according to the sample spot quadrat method respectively at 6 point of crossing point samples of filter paper.
In the time of point sample, on smooth experiment table, spread preservative film, the developing agent (75% ethanolic solution that contains 0.2% triketohydrindene hydrate) that 50ml prepares is poured in the double dish of diameter 15cm; Buckle an enough big beaker; Use the preservative film environmental sealing, fumigate, fumigation time is no less than 40min.After point sample finishes to treat the sampling point bone dry; Chromatography filter paper is crooked along horizontal edge; Two vertical edges are connected (two the limit does not contact) with cotton thread or transparent adhesive tape be rolled into tubular; Remove the large beaker of having fumigated rapidly, the chromatography filter paper of tubular is vertically put into the double dish that developing agent is housed, buckle the large beaker of having fumigated immediately.Exhibition layer about 3h of time arrives the upper edge mark of having finished until the developing agent upper edge and gets final product.
The chromatography filter paper that chromatography is accomplished hangs over dry 10min in 60 ℃ the baking oven rapidly.
Cut out standard solution theanine color spot filter paper (1.5cm * 1.5cm) and on the sample chromatogram include filter paper part and the blank zone that the theanine colour developing is with down with onesize, the same position of standard solution; Shred and put into test tube; Add 5ml eluant, eluent (60% ethanol); Jog is placed on 20min in 40 ℃ of water-baths, is placed to the room temperature measuring light absorption value after the taking-up.
Preheating 721 spectrophotometers are reference with the blank sample, measure the light absorption value of standard specimen and each sample at the 570nm place.
Standard specimen theanine content and light absorption value are as shown in table 2:
Table 2 tealeaves theanine is measured standard specimen theanine content and light absorption value
| Content (mg) | 0.002 | 0.003 | 0.004 | 0.005 | 0.006 | 0.007 |
| Light absorption value | 0.015 | 0.021 | 0.026 | 0.033 | 0.039 | 0.047 |
The production standard curve is seen Fig. 2.Obtaining regression equation is y=0.1577x-0.0003 (r
2=0.9956). the Xinyang Maojian Tea light absorption value is 0.034, and going out theanine by regression equation calculation is 0.0051mg, and the Keemun black tea light absorption value is 0.035, and calculating theanine by regression equation is 0.0052mg; The light absorption value of Anxi Tieguanyin Tea is 0.017, and trying to achieve theanine content from regression equation is 0.0024mg.
Xinyangmaojian theanine% =
=
= 1.16
Keemun theanine% =
=
= 1.17
Tie Guan Yin theanine% =
=
= 0.54
Claims (6)
1. a theanine method for quantitatively determining is characterized in that: comprise the steps:
(1) lixiviate: with the dried tea appearance of 60-100ml boiling water lixiviate 0.2-4.0g 30-60min, be chilled to room temperature, filter, be settled to 100ml, get the tea leaching liquor;
(2) point sample: with theanine standard solution, blank distilled water and tea leaching liquor point sample on filter paper respectively; Said concentration of standard solution is pointed out three points at 0.08-0.12mg/ml and the standard solution of choosing same concentration according to different point sample amounts at least;
(3) exhibition layer: with alcohol-water-triketohydrindene hydrate mixed solution as developing agent, vertical chromatography; In said alcohol-water-triketohydrindene hydrate mixed solution, ethanol volume fraction 70-80%, triketohydrindene hydrate volume fraction 0.1-0.3%;
(4) wash-out: with the filter paper behind the chromatography in 40-80 ℃ of oven dry; Filter paper that the theanine colour developing is with in standard solution theanine color spot filter paper, the tea leaching liquor under cutting out and blank zone filter paper; With the ethanolic solution wash-out, get standard specimen eluent, tea appearance eluent and blank eluent respectively;
(5) colorimetric, calculating: with the blank sample is reference, uses spectrophotometer 570nm place to measure the light absorption value of standard specimen solution and tea appearance solution respectively; The production standard curve is obtained regression equation, calculates the theanine content in the tea sample.
2. theanine method for quantitatively determining as claimed in claim 1 is characterized in that: step (2) empty contrast distilled water is all 20-40 μ l mutually with the point sample amount of tea leaching liquor, chooses the standard solution of same concentration and points out six points according to different point sample amounts.
3. theanine method for quantitatively determining as claimed in claim 1 is characterized in that: in the said alcohol-water of step (3)-triketohydrindene hydrate mixed solution, and ethanol volume fraction 75%, triketohydrindene hydrate volume fraction 0.2%.
4. theanine method for quantitatively determining as claimed in claim 1 is characterized in that: filter paper is in 60 ℃ of dry 10-15min in the step (4).
5. theanine method for quantitatively determining as claimed in claim 1 is characterized in that: the ethanol volumetric concentration of ethanolic solution is 60-70% in the step (4).
6. theanine method for quantitatively determining as claimed in claim 1 is characterized in that: place 40-60 ℃ of water-bath 10-20min in the step (4) during wash-out.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102928362A (en) * | 2012-07-18 | 2013-02-13 | 新疆天康畜牧生物技术股份有限公司 | Ninhydrin detection method as improvement to free amino quantitative method in solid-phase polypeptide synthesis |
| CN103033578A (en) * | 2012-12-25 | 2013-04-10 | 湖南农业大学 | Method for quickly and preliminarily screening excellent tea tree resource based on contained contents |
| CN107796772A (en) * | 2017-11-10 | 2018-03-13 | 桂林电子科技大学 | A kind of portable instrument and the method for detecting theanine |
| CN108007878A (en) * | 2017-11-10 | 2018-05-08 | 桂林电子科技大学 | A kind of portable theanine detecting instrument and detection method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102095775A (en) * | 2010-12-01 | 2011-06-15 | 江南大学 | Method for efficiently separating and detecting theanine enantiomer by capillary electrophoresis |
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2011
- 2011-09-26 CN CN2011102863666A patent/CN102435565A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102095775A (en) * | 2010-12-01 | 2011-06-15 | 江南大学 | Method for efficiently separating and detecting theanine enantiomer by capillary electrophoresis |
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|---|
| 阮宇成等: "茶氨酸的简化定量法", 《茶叶通讯》, 30 June 1981 (1981-06-30) * |
| 陈舒丽等: "对氨基酸纸层析实验的改进", 《山西医科大学学报》, 26 March 2011 (2011-03-26) * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102928362A (en) * | 2012-07-18 | 2013-02-13 | 新疆天康畜牧生物技术股份有限公司 | Ninhydrin detection method as improvement to free amino quantitative method in solid-phase polypeptide synthesis |
| CN103033578A (en) * | 2012-12-25 | 2013-04-10 | 湖南农业大学 | Method for quickly and preliminarily screening excellent tea tree resource based on contained contents |
| CN103033578B (en) * | 2012-12-25 | 2014-03-05 | 湖南农业大学 | Method for quickly and preliminarily screening excellent tea tree resource based on contained contents |
| CN107796772A (en) * | 2017-11-10 | 2018-03-13 | 桂林电子科技大学 | A kind of portable instrument and the method for detecting theanine |
| CN108007878A (en) * | 2017-11-10 | 2018-05-08 | 桂林电子科技大学 | A kind of portable theanine detecting instrument and detection method |
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Application publication date: 20120502 |