Summary of the invention:
Technical matters to be solved by this invention is to design a kind of to carrying out the method for barbaloin content separation detection in the compound aloe capsule, thereby controls product quality effectively.
The invention provides barbaloin content detecting method in a kind of compound aloe capsule.
The object of the invention can be realized by following proposal:
One, instrument, reagent and confession test agent
Instrument: UV-200II type high performance liquid chromatograph, UV-200II type ultraviolet variable-wavelenght detector, Echrom98 chromatographic data work of treatment station
Reagent: barbaloin standard items, acetonitrile, methyl alcohol are chromatographically pure level reagent, and other reagent is analyzes pure level reagent.
Supply test agent: compound aloe capsule
Chromatographic condition
Chromatographic column: Hyper 0DS2C18,4.6 * 250mm
Moving phase: acetonitrile-0.1% trifluoracetic acid-tetrahydrofuran (150-250: 750-850: 2-5)
Flow velocity: 1ml/min
Detect wavelength: 355nm
Column temperature: room temperature.
Two, test method and result
(1) preparation of reference substance solution:
Precision takes by weighing barbaloin reference substance 12.5mg, puts in the 25ml volumetric flask, with dissolve with methanol and be diluted to scale, shakes up; Precision is measured 5ml, puts in the 50ml volumetric flask, with dissolve with methanol and be diluted to scale, shakes up, promptly;
(2) preparation of need testing solution
Get the content of 10 of compound aloe capsules, mix; Get 0.2 gram, the accurate title, decide, and puts in the tool plug conical flask, the accurate methyl alcohol 20ml that adds, close plug claims to decide weight, and sonicated 30min is put cold, claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol, filter, discard filtrate just, precision is measured subsequent filtrate 5ml, put in the 50ml volumetric flask, with dissolve with methanol and be diluted to scale, shake up, filter with miillpore filter (0.45um), get subsequent filtrate, promptly;
(3) assay method
Accurate respectively reference substance and each 20u1 sample introduction of need testing solution drawn injects high performance liquid chromatograph, measures, and calculates, promptly.
Three, the technical standard of Jian Ceing
Every (0.5g) compound aloe capsule contains barbaloin 〉=7.5mg
Four, the feasibility of this detection method can be described by following test
1. the preparation of negative control solution
According to the prescription of compound aloe capsule, the analog sample that the technology preparation lacks aloe, make the compound aloe capsule negative control solution according to the need testing solution preparation method.
2. the investigation of linear relationship
Precision takes by weighing the barbaloin reference substance, adds the solution that methyl alcohol is mixed with 0.5mg/m1, and it is an amount of to get this solution, is diluted to 0.01,0.02,0.05,0.1,0.2 with methyl alcohol, the solution of 0.3mg/ml.Get above-mentioned solution 20ul sample introduction, measure according to the chromatographic condition worked out, record peak area and sample size are horizontal ordinate with the reference substance concentration of sample introduction, are ordinate with the peak area, the drawing standard curve.Regression equation is Y=365195X-1322.3 R=0.9993, becomes good linear relationship between the range of linearity 0.2-10ug. linear relationship is investigated and be the results are shown in Table 1, the typical curve See Figure
Table 1 barbaloin linear relationship is investigated the result
Barbaloin reference substance concentration (mg/ml) |
0.01 |
0.02 |
0.05 |
0.1 |
0.2 |
0.3 |
0.5 |
The peak area of measuring |
3459.52 |
7355.75 |
17771.60 |
34841.38 |
69714.04 |
104008.94 |
184522.95 |
Get same reference substance solution and need testing solution, respectively 0,2, sample introduction was measured in 4,6,20,24 hours.RSD to the product peak area is 0.33% (n=6), and the RSD of test sample peak area is 0.29% (n=6), illustrates that barbaloin is stable in 24 hours at least, and stability test the results are shown in Table 2
Table 2 stability test result
Time (hour) |
Barbaloin reference substance peak area |
The test sample peak area |
0 |
17052.32 |
12123.97 |
2 |
17079.19 |
12083.13 |
4 |
17078.44 |
12039.67 |
6 |
17142.32 |
12014.69 |
20 |
17027.42 |
12037.61 |
24 |
17166.04 |
12069.81 |
Mean value |
17090.955 |
12061.48 |
RSD |
0.33% |
0.29% |
3. precision test
Accurate reference substance solution and each 20ul of need testing solution (lot number 050611) of drawing repeats sample introduction 5 times, measures peak area, and the result shows, the RSD=0.43% of reference substance peak area (n=5); Test sample peak area RSD=0.81% (n=5). the result shows that degree of accuracy is good.The degree of accuracy test findings sees Table 3.
Table 3 Precision test result
Mensuration number of times numbering |
Barbaloin reference substance peak area |
The test sample peak area |
1 |
16918.8 |
12193.25 |
2 |
17076.83 |
12019.77 |
3 |
17052.32 |
12218.29 |
4 |
17109.36 |
12068.04 |
5 |
17077.25 |
12011.01 |
Mean value |
17046.912 |
12102.072 |
RSD |
0.43% |
0.81% |
4. replica test
Get compound aloe capsule (lot number 050611) and carry out parallel assay 5 times, calculate content, recording every, to contain barbaloin mean value be 17.602mg, and RSD=0.26% (n=5) illustrates this assay method good reproducibility.Replica test the results are shown in Table 4
Table 4 compound aloe capsule replica test result
Numbering |
Barbaloin content (mg/ grain) |
1 |
17.54 |
2 |
17.57 |
3 |
17.65 |
4 |
17.63 |
5 |
17.62 |
Mean value |
17.602 |
RSD |
0.26% |
5. specificity test
Under the chromatographic condition of drafting, accurate respectively reference substance solution and each 20ul of need testing solution of drawing injects high performance liquid chromatograph respectively, measures.The result shows that the test sample degree of separation is better, and retention time is moderate, and barbaloin is consistent in the retention time of reference substance and peak and the test sample.
The accurate negative control product solution 20ul that draws injects high performance liquid chromatograph, measures.The result shows, in the negative control chromatogram, with the relevant position at barbaloin reference substance peak on, noiseless peak occurs, and illustrates that the medicinal material of other except that aloe does not have influence to measurement result in the compound aloe capsule, promptly negative control is noiseless.
6. average recovery test
Take the test of application of sample absorption method.Get the compound aloe capsule (lot number 050613 of known content, content 21.13mg/ grain) 10 intragranulars are tolerant, mix, get above-mentioned medicinal powder 0.2g, the accurate title, decide, 5 parts of nominals, and each adds a certain amount of barbaloin reference substance respectively, make the application of sample need testing solution according to the need testing solution preparation method, measure.Test findings shows that five times average recovery rate is 100.33%, and RSD=0.44% illustrates that this assay method measurement result is accurate.The average recovery test findings sees Table 5
Table 5 barbaloin application of sample reclaims experimental result
This assay method specificity is strong, and the linearity of method, stability, reappearance, average recovery all reach technical requirement preferably.The inventive method can effectively be controlled the product quality of compound aloe capsule.
Embodiment
One, instrument, reagent and confession test agent
Instrument: UV-200II type high performance liquid chromatograph, UV-200II type ultraviolet variable-wavelenght detector, Echrom98 chromatographic data work of treatment station
Reagent: barbaloin standard items, acetonitrile, methyl alcohol are chromatographically pure level reagent, and other reagent is analyzes pure level reagent.
Supply test agent: the compound aloe capsule sample
Chromatographic condition
Chromatographic column: Hyper 0DS2C18,4.6 * 250mm
Moving phase: acetonitrile-0.1% trifluoracetic acid-tetrahydrofuran (180: 820: 2.5)
Flow velocity: 1ml/min
Detect wavelength: 355nm
Column temperature: room temperature.
Two, test method and result
1. the preparation of reference substance solution:
Precision takes by weighing barbaloin reference substance 12.5mg, puts in the 25ml volumetric flask, with dissolve with methanol and be diluted to scale, shakes up; Precision is measured 5ml, puts in the 50ml volumetric flask, with dissolve with methanol and be diluted to scale, shakes up, promptly.
2. the preparation of need testing solution
Get the content of 10 of compound aloe capsules, mix; Get 0.2 gram, the accurate title, decide, and puts in the tool plug conical flask, the accurate methyl alcohol 20ml that adds, close plug claims to decide weight, and sonicated 30min is put cold, claim again to decide weight, supply the weight that subtracts mistake, shake up with methyl alcohol, filter, discard filtrate just, precision is measured subsequent filtrate 5ml, put in the 50ml volumetric flask, with dissolve with methanol and be diluted to scale, shake up, filter with miillpore filter (0.45um), get subsequent filtrate, promptly.
3. assay method
Respectively accurate reference substance and each 20ul sample introduction of need testing solution drawn injects high performance liquid chromatograph, measures, and calculates, and gets promptly that to contain barbaloin content be 8.5mg.