CN101713736B - Method for testing aureomycin medicaments by rare-earth complex fluorescent probe - Google Patents
Method for testing aureomycin medicaments by rare-earth complex fluorescent probe Download PDFInfo
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Abstract
The invention discloses a method for testing aureomycin medicaments by a rare-earth complex fluorescent probe, which comprises the steps of: mixing amion acetic acid-NaOH buffer solution, Tb3+stock solution, aqueous solution of sodium dodecyl benzene sulfonate and a to-be-tested sample containing the aureomycin medicaments, adding water to a fixed volume, uniformly shaking, and standing the mixture for 15 to 60 minutes as test solution; and in a fluorescent analyzer of which the excitation wavelength is between 290 and 300 nm, the scanning speed is between 300 and 1,000 nm/minute and the excitation slit width and the emission slit width are between 5 and 15 nm, testing the fluorescence intensity at a position where the emission wavelength of the test solution is between 540 and 550 nm. By adding the sodium dodecyl benzene sulfonate, the fluorescence intensity of a system is improved by 3 times, the sensitivity of detection is improved, the operation is simple, quick and accurate, and the method has high sensitivity and good selectivity, is wide in application range, and is an ideal method for testing the aureomycin medicaments.
Description
Technical field
The present invention relates to the detection method of aureomycin medicine, be specifically related to the assay method of a kind of rare earth coordination compound fluorescent probe, belong to the Pharmaceutical Analysis technical field the aureomycin medicine.
Background technology
Aureomycin (chlortetracycline, CTC), chemical name: 6-methyl-4-(dimethylamino)-3,6,10,12,12a ,-penta hydroxy group-1,11-dioxo-7-chloro-1,4,4a, 5,5a, 6,11,12a-octahydro-2-aphthacene carboxamide hydrochloride.It is the broad-spectrum antibiotic that extracts from the nutrient solution of golden streptomysin, and gram-positive bacteria, negative bacterium, Richettsia and large-scale virus are had the ability that suppresses widely.Though the aureomycin drug effect is obvious, it can enrichment in liver, and the toxicity reflection mainly shows as the infringement to internal organ; Easily in bone and tooth, deposit, cause tooth to dye lastingly; Symptoms such as DOMS also can occur, even cause effects such as allergic reaction, superinfection.Therefore aureomycin generally is not used in human body, and is widely used as antibiotic feed additive for promoting growth.
Aureomycin is approved by the world as feed addictive, and its effect also is definite.But excessive interpolation can cause residual in the animal tissue, and to the exhaust emission of peripheral soil and water body, most bacterial classification is developed immunity to drugs, thereby be detrimental to health, and therefore many countries are to the residual routine monitoring of CTC.China in nineteen ninety-five version pharmacopeia regulation aureomycin content assaying method be the microbial detection method, but this method complex steps, experimental period is long.The European Community has all adopted high performance liquid chromatography (HPLC) method at present.The HPLC method detects reliable, but still comparatively complicated after all by the process of liquid chromatographic detection.In addition, also just like dual-wavelength spectrophotometry, spot gold immunofiltration assay, the report of methods such as ELISA method.
The fluorophotometric method because of its have highly sensitive, advantage has been widely used in the detection of such medicine fast, when but fluorescence method contains the component of multiple fluorescent material in analysis, when particularly having the overlapping material of wave band, can cause interference to target product, can't obtain gratifying result, therefore, need to explore new aureomycin detection method.
Summary of the invention
The invention provides the assay method of a kind of quick, sensitive rare earth coordination compound fluorescent probe, utilize rare earth terbium ion (Tb the aureomycin medicine
3+) forming multicomponent complex with aureomycin, neopelex (SDBS), the fluorescence that terbium ion is sent strengthens greatly, and the fluorescence that this multicomponent complex system of SDBS selectivity enhanced sensitivity is sent, thereby the selective determination of aureomycin medicine in the realization sample.
A kind of rare earth coordination compound fluorescent probe may further comprise the steps the assay method of aureomycin medicine:
(1) preparation Tb
3+Stock solution: with terbium oxide (Tb
4O
7) be dissolved in the hydrochloric acid, add the water constant volume, preparation Tb
3+Concentration is 1.0 * 10
-3The Tb of mol/L
3+Stock solution;
(2) with the mass percentage concentration of 0.2mL～2.0mL pH=7.5～8.0, aminoacetic acid (being glycocoll) be aminoacetic acid-NaOH buffer solution of 2%～10%, 0.5mL～2.0mL Tb
3+Stock solution, 0.5mL～2.0mL 5.0 * 10
-3The SDBS aqueous solution of mol/L is mixed with the testing sample that contains the aureomycin medicine, adds water and is settled to 10mL, shakes up the back and places 15min～60min in room temperature, obtains Tb
3+Concentration is 5.0 * 10
-5Mol/L～2.0 * 10
-4The test liquid of mol/L;
(3) test liquid is placed fluorescence analyser, the setting excitation wavelength is 290nm～300nm, sweep velocity is 300nm/min～1000nm/min, excites slit width and emission slit width to be 5nm～15nm, measures the fluorescence intensity at emission wavelength 540nm～550nm place.
Preparation Tb
3+During stock solution, the consumption of hydrochloric acid needn't be too much, as long as can make Tb
4O
7Dissolving gets final product, and concrete operations can comprise: terbium oxide is dissolved in the hydrochloric acid, and heating evaporation is done near, is dissolved in water and constant volume.
The influence of pH: pH＜7.5 of aminoacetic acid-NaOH buffer solution o'clock, with the increase of pH, the fluorescence intensity of test system strengthens, when pH=7.5～8.0, the fluorescence intensity of test system reaches maximum and keeps stable, and the increase fluorescence intensity with pH when pH＞8.0 dies down gradually.Therefore, select the aminoacetic acid-NaOH buffer solution of pH=7.5～8.0 for use, regulate the pH value of test system.
Because conventional buffer solution such as phosphate buffer solution, borax buffer solution, Tris buffer solution etc. have the effect of extinguishing fluorescence, the present invention selects for use aminoacetic acid-NaOH buffer solution to regulate the pH value of test liquid, test liquid pH value is tended towards stability, and can not influence test result.
The preparation of described aminoacetic acid-NaOH buffer solution: aminoacetic acid is soluble in water, regulate the pH value with NaOH and get final product.
Tb in the test liquid
3+The influence of concentration: Tb in the test liquid
3+Concentration is lower than 1.0 * 10
-4During mol/L, the fluorescence intensity of test system is along with Tb
3+Concentration increases and significantly strengthens, and works as Tb
3+Concentration reaches 1.0 * 10
-4During mol/L, the system fluorescence intensity reaches maximum and keeps stable, and therefore, the present invention guarantees Tb in the test liquid
3+Concentration is 5.0 * 10
-5Mol/L～2.0 * 10
-4Mol/L, Tb in the preferred test liquid
3+Concentration is 1.0 * 10
-4Mol/L is to reach good detection effect.
The influence of standing time: need during the preparation test liquid shaking up back placement certain hour, the present invention finds standing time 〉=15min, fluorescence intensity will keep stable always, be at least 45min stabilization time, therefore, the present invention places 15min～60min with test liquid, most preferably places to carry out fluorescence intensity behind the 20min and detect, to guarantee reaching good detection effect when saving detection time.
As preferably, described rare earth coordination compound fluorescent probe may further comprise the steps the assay method of aureomycin medicine:
(1) preparation Tb
3+Stock solution: terbium oxide is dissolved in the hydrochloric acid, adds the water constant volume, preparation Tb
3+Concentration is 1.0 * 10
-3The Tb of mol/L
3+Stock solution;
(2) with 1.0mL pH=7.7, aminoacetic acid mass percentage concentration be aminoacetic acid-NaOH buffer solution of 5%, 1.0mL Tb
3+Stock solution, 1.0mL 5.0 * 10
-3The SDBS aqueous solution of mol/L is mixed with the testing sample that contains the aureomycin medicine, adds water and is settled to 10mL, shakes up the back and places 20min in room temperature, obtains Tb
3+Concentration is 1.0 * 10
-4The test liquid of mol/L;
(3) test liquid is placed fluorescence analyser, the setting excitation wavelength is 296nm, and sweep velocity is 300nm/min～1000nm/min, excites slit width and emission slit width to be 10nm, measures the fluorescence intensity at emission wavelength 545nm place.
Determining of linear relationship (being standard working curve): it is a series of by the CTC standard solution of low concentration to high concentration to get the water-soluble preparation of aureomycin standard items, add other reagent identical and under identical fluoroscopic examination condition, measure fluorescence intensity respectively by the method for the invention with test liquid, the concentration that with the fluorescence intensity is ordinate, aureomycin is horizontal ordinate drawing standard working curve, and the concentration of determining aureomycin is 2.0 * 10
-8Mol/L～2.0 * 10
-6During mol/L, fluorescence intensity (in order to reduce error, generally adopt the fluorescence intensity of fluorescence intensity of fluorescence intensity and blank test poor) is linear with CTC concentration.
Routine operation standard according to this area analytical test, generally also need to do blank test, promptly except not adding testing sample or standard solution, with the experiment of handling and testing with the same method of testing sample or standard solution, test result is deducted the result of blank test, testing result to testing sample or standard solution is proofreaied and correct, the response error that reduces systematic error and bring because of the impurity in institute's water, the reagent in analyzing and instrument.
The operation of the inventive method blank test comprises: other get with test liquid in identical aminoacetic acid-NaOH buffer solution, Tb
3+Stock solution and SDBS aqueous solution add water and are settled to 10ml after the mixing, shake up and place the identical time, and as blank solution, the fluorescence intensity of blank testing liquid gets final product under fluoroscopic examination condition of the present invention.
During concrete calculating, the fluorescence intensity of fluorescence intensity gained that the fluorescence intensity of the above-mentioned testing sample that records is deducted blank solution is poor, draws the concentration of aureomycin in the test liquid in conjunction with above-mentioned linear relationship.
Determining of the inventive method detectability: select fluorescence intensity near blank CTC standard solution, promptly CTC concentration is 2.0 * 10
-8The CTC standard solution of mol/L is measured fluorescence intensity under the preferred optimum experimental condition of the present invention, METHOD FOR CONTINUOUS DETERMINATION 11 times is calculated by computing method (the 3s/x) * c that is similar to detectability, obtains its detection and is limited to 4.0 * 10
-9Mol/L (S/N=3); Wherein s represents that the standard deviation that calculated by the fluorescence intensity that records for 11 times, x represent the mean value of the fluorescence intensity that records for 11 times, and c represents the concentration of the CTC standard solution selected, is 2.0 * 10
-8Mol/L.
The metallic ion interference test:
In concentration is 1.0 * 10
-6Add metallic ion in the CTC standard solution of mol/L as testing sample, adopt the inventive method to detect, investigate multiple common metal ion Tb
3+The influence of-CTC-SDBS system fluorescence intensity, the result is as shown in table 1.
Table 1 metallic ion is to the influence of system fluorescence intensity
| Metallic ion | Concentration (mol/L) | ΔIF(％) |
| K + | 5.0×10 -4 | +4.3 |
| Na + | 5.0×10 -4 | -5.9 |
| Ba 2+ | 1.5×10 -4 | +5.2 |
| Ca 2+ | 1.0×10 -5 | -7.5 |
| Cu 2+ | 2.5×10 -5 | -6.8 |
| Zn 2+ | 2.5×10 -6 | -7.8 |
| Mg 2+ | 5.0×10 -5 | +0.2 |
| Al 3+ | 2.5×10 -5 | -1.9 |
| La 2+ | 1.0×10 -5 | -8.3 |
| Gd 2+ | 5.0×10 -5 | -6.5 |
| Ce 3+ | 5.0×10 -5 | -6.1 |
| Dy 3+ | 2.5×10 -6 | +1.0 |
Remarks: Δ If represents the fluorescence deviation in the table, and "+" expression strengthens, and "-" expression weakens.
By table 1 as seen, most of metallic ion need reach higher concentration just can be to the Tb in the inventive method
3+-CTC-SDBS system produces to be disturbed, and shows that the inventive method has selectivity preferably.
The medicine interference test:
In concentration is 1.0 * 10
-6Add the normal medicine that uses with the CTC compatibility in the CTC standard solution of mol/L as testing sample, adopt the inventive method to detect, investigates several often and the medicine of CTC compatibility use to Tb
3+The influence of-CTC-SDBS system fluorescence intensity, result such as table 2 descend.
Table 2 other medicines are to Tb
3+The influence of-CTC-SDBS system fluorescence intensity
| Medicine | Concentration (mol/L) | ΔIF(％) |
| Chloromycetin | 5.0×10 -5 | -0.6 |
| Roxithromycin | 1.0×10 -5 | -0.1 |
| Cefalexin | 2.5×10 -5 | +5.5 |
| Cefradine | 1.0×10 -5 | -4.8 |
| The Amoxicillin | 5.0×10 -6 | +8.8 |
| Paracetamol | 1.0×10 -6 | -3.8 |
| Dopamine | 1.0×10 -6 | -7.0 |
| Adrenaline | 1.0×10 -6 | -3.8 |
| Norepinephrine | 1.0×10 -6 | +3.5 |
| Tetracycline | 5.0×10 -5 | -6.7 |
| Terramycin | 1.0×10 -6 | -13.1 |
| Fortimicin | 2.5×10 -6 | -8.4 |
Remarks: Δ If represents the fluorescence deviation in the table, and "+" expression strengthens, and "-" expression weakens.
By table 2 as seen, Chang Yong compatibe drug disturbs less to the mensuration of CTC; Under same concentrations, the interference of different tetracycline medications differs greatly, and wherein tetracycline and fortimicin are little to the influence of system fluorescence intensity; Terramycin then has certain interference.
The used reagent of the present invention all selects for use analysis pure, and used water is all selected pure water or distilled water for use.
The present invention has following beneficial effect:
1. by adding SDBS, make the fluorescence intensity of system improve 3 times, improved the sensitivity that detects, detectability reaches 4.0 * 10
-9Mol/L.
2. the inventive method is simple, quick, accurate, has high sensitivity and good selectivity, have wide range of applications, and be the Perfected process of CTC drug monitoring.
3. the present invention adopts rare earth coordination compound fluorescent probe can realize selective determination to the aureomycin medicine, and the interference of the normal medicine with the use of CTC compatibility of metallic ion and other is less.
Description of drawings
The fluorescence spectrum synoptic diagram that Fig. 1 detects for the present invention; Wherein, 1 expression Glycine-Tb
3+2 expression Glycine-CTC; 3 expression Glycine-Tb
3+-SDBS; 4 expression Glycine-CTC-Tb
3+5 expression Glycine-CTC-Tb
3+-SDBS, i.e. the fluorescence spectrum synoptic diagram of embodiment 1 detection; Experiment condition is: C
Tb 3+=1.0 * 10
-4Mol/L, C
CTC=1.0 * 10
-6Mol/L, C
SDBS=5.0 * 10
-4Mol/L, C
Glycine=0.5% (mass percentage concentration); C indicated concentration, Glycine are glycocoll;
Fig. 2 is the standard working curve in the embodiment of the invention 1.
Embodiment
Embodiment 1
By best test condition: with 1.0mL pH=7.7, aminoacetic acid mass percentage concentration is aminoacetic acid-NaOH buffer solution of 5%, 1.0mL 1.0x10
-3The Tb of mol/L
3+Stock solution, 1.0mL5.0x10
-3The SDBS aqueous solution of mol/L and testing sample mix, and add water and are settled to 10mL, shake up the back and place 20min in room temperature, obtain test liquid.
Test liquid is placed the quartzy colorimetric pool of 1cm of fluorescence analyser, and settings excitation wavelength is 296nm, and sweep velocity is 300nm/min, excites slit width and emission slit width to be 10nm, the fluorescence intensity at mensuration emission wavelength 545nm place.
Other get with test liquid in identical aminoacetic acid-NaOH buffer solution, Tb
3+Stock solution and SDBS aqueous solution add water and are settled to 10ml after the mixing, shake up and place the identical time, as blank solution, and the fluorescence intensity of blank testing liquid under above-mentioned fluoroscopic examination condition.
According to the method for drafting of standard working curve, it is a series of by the CTC standard solution of low concentration to high concentration: 2.0x10 to get the water-soluble preparation of CTC standard items
-8Mol/L, 5.0x10
-8Mol/L, 1.0x10
-7Mol/L, 2.0x10
-7Mol/L, 5.0x10
-7Mol/L, 1.0x10
-6Mol/L, 1.5 * 10
-6Mol/L, 2.0x10
-6The CTC standard solution of mol/L, respectively above-mentioned CTC standard solution is replaced testing sample by the fluorescence intensity of measuring each CTC standard solution under above-mentioned best test condition and the fluoroscopic examination condition, with the fluorescence intensity of each the CTC standard solution concentration that to deduct the fluorescence poor (Δ F) of the fluorescence intensity of blank solution be ordinate, CTC is horizontal ordinate drawing standard working curve, as Fig. 2.
Get 1.0mL aureomycin hydrochloride eye ointment (Baijingyu Pharmaceutical Co., Ltd., Nanjing) as testing sample, disposal route operation by above-mentioned best test condition, and under above-mentioned fluoroscopic examination condition, measure the fluorescence intensity of system, it is poor that the fluorescence intensity that this fluorescence intensity is deducted blank solution obtains fluorescence, draw the concentration of CTC in the 1.0mL aureomycin hydrochloride eye ointment by the typical curve of Fig. 2, further calculate the content (by percentage to the quality) of CTC; Measurement result sees Table 3.
Table 3
| Measured value (‰) | Mean value, x ± s | Sign value (‰) | RSD(％) |
| 5.10，5.06，5.01，4.99，5.16 | 5.06±0.071 | 5 | 1.41 |
Be 5.0 * 10 at Cefradine and cefalexin drug concentration
-7Adopt standard addition method to carry out the recovery test of CTC in the aqueous solution of mol/L, each concentration is set five parallel sample, and measurement result is as shown in table 4.
Table 4
From the recovery of standard addition of table 4, all between 97.0%～111.4%, the recovery is good for the recovery, shows that this detection method interference resistance is better, and method is reliable, the accuracy height.
Claims (4)
1. a rare earth coordination compound fluorescent probe may further comprise the steps the assay method of aureomycin medicine:
(1) preparation Tb
3+Stock solution: terbium oxide is dissolved in the hydrochloric acid, adds the water constant volume, preparation Tb
3+Concentration is 1.0 * 10
-3The Tb of mol/L
3+Stock solution;
(2) with 0.2mL～2.0mL pH=7.5～8.0, aminoacetic acid mass percentage concentration be aminoacetic acid-NaOH buffer solution of 2%～10%, 0.5mL～2.0mLTb
3+Stock solution, 0.5mL～2.0mL 5.0 * 10
-3The sodium dodecyl benzene sulfonate aqueous solution of mol/L mixes with the testing sample that contains the aureomycin medicine, adds water and is settled to 10mL, shakes up the back and places 15min～60min in room temperature, obtains Tb
3+Concentration is 5 * 10
-5Mol/L～2 * 10
-4The test liquid of mol/L;
(3) test liquid is placed fluorescence analyser, the setting excitation wavelength is 290nm～300nm, sweep velocity is 300nm/min～1000nm/min, excites slit width and emission slit width to be 5nm～15nm, measures the fluorescence intensity at emission wavelength 540nm～550nm place.
2. assay method as claimed in claim 1 is characterized in that, in the step (2), be 20min described standing time.
3. assay method as claimed in claim 1 is characterized in that, in the step (2), and Tb in the test liquid
3+Concentration is 1 * 10
-4Mol/L.
4. assay method as claimed in claim 1 is characterized in that, may further comprise the steps:
(1) preparation Tb
3+Stock solution: terbium oxide is dissolved in the hydrochloric acid, adds the water constant volume, preparation Tb
3+Concentration is 1.0 * 10
-3The Tb of mol/L
3+Stock solution;
(2) with 1.0mL pH=7.7, aminoacetic acid concentration expressed in percentage by weight be aminoacetic acid-NaOH buffer solution of 5%, 1.0mL Tb
3+Stock solution, 1.0mL 5.0 * 10
-3The sodium dodecyl benzene sulfonate aqueous solution of mol/L mixes with the testing sample that contains the aureomycin medicine, adds water and is settled to 10mL, shakes up the back and places 20min in room temperature, obtains Tb
3+Concentration is 1.0 * 10
-4The test liquid of mol/L;
(3) test liquid is placed fluorescence analyser, the setting excitation wavelength is 296nm, and sweep velocity is 300nm/min～1000nm/min, excites slit width and emission slit width to be 10nm, measures the fluorescence intensity at emission wavelength 545nm place.
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| CN103076316A (en) * | 2013-01-07 | 2013-05-01 | 桂林理工大学 | Method for detecting trace amount of oxytetracycline by utilizing CdTe quantum dot fluorescent probe |
| CN105300946B (en) * | 2015-11-12 | 2018-03-23 | 广西师范大学 | A kind of hypaphorine concentration measuring method based on fluorescent quantitation |
| CN108181275A (en) * | 2017-12-21 | 2018-06-19 | 安徽神剑新材料股份有限公司 | Cd in a kind of preparation method of terbium sulphonic acid complex, water2+Detection method |
| CN115406978A (en) * | 2022-02-17 | 2022-11-29 | 山东省畜产品质量安全中心(山东省畜禽屠宰技术中心) | Method for detecting terramycin and aureomycin in mixed feed additive |
| CN116087165B (en) * | 2022-12-22 | 2023-07-28 | 南京林业大学 | Method for simply and rapidly detecting MTC and/or OTC and/or TC |
| CN116087164B (en) * | 2022-12-22 | 2023-07-18 | 南京林业大学 | Method for simply and rapidly detecting aureomycin |
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