CN103293150B - Detection method of nafil medicines and detection kit - Google Patents
Detection method of nafil medicines and detection kit Download PDFInfo
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Abstract
The invention discloses a rapid detection method of nafil medicines, which successively comprises the following steps of: (1) taking a sample, and adding an acid solution to dissolve the sample; (2) adding an extraction agent to extract, and adding a buffer solution into extract; and (3) dropping a colour developing agent, and observing whether the solution is yellow or not, wherein if the solution is yellow, the sample contains nafil medicines, or else the sample does not contain nafil medicines. The invention further provides a special detection kit based on the method. The rapid detection method disclosed by the invention has the advantages of being low in analysis cost, free from special equipment, strong in specificity and high in sensitivity and is applied to testing whether nafil medicines are doped in medicines and health-care products in field; and the disadvantages in the prior art are overcome.
Description
Technical field
The present invention relates to a kind of detection method and detection kit of that non-class medicine.
Background technology
Phosphodiesterase is the negative growth factor of NO-cGMP path, it turns down nitric oxide production effect by the decomposition of catalysis cGMP, it is generally acknowledged that the nitrogen monoxide in body is the factor regulating vascular smooth muscle expansion, the result of thus phosphodiesterase effect is the contraction promoting vascular smooth muscle.That non-class medicine can in the suppression human body of high selectivity 5 type phosphodiesterases (PDE5) active, PDE5 is high in penis sponge Level of Expression of Retinoic Acid, then expresses lower in its hetero-organization of human body and organ.After taking that non-class medicine, the diastole under the effect of medicine of corpora cavernosa penis vascular smooth muscle, blood flow increases, and cavernous body is congested, telotism, thus produces the therapeutic action to Erectile Dysfunction.
The bad reaction that non-class drug main is wanted comes from its inhibiting effect to other position phosphodiesterases of human body.Common bad reaction has: dizzy, nasal obstruction, indigestion and transient paropsia, and this exception may show as indigo plant/green color discrimination exception, light sensation strengthens or blurred vision.These bad reactions are because relevant smooth muscle relaxation causes mostly above.The bad reaction that non-class medicine is more serious in addition also comprises: clinostatism blood pressure drops and cardiac output decline, in addition clinical studies show carries out sexual behaviour after taking that non-class medicine, the probability that heart abnormality occurs increases, the symptoms such as this comprises angina pectoris, dizziness, feel sick, and likely cause sudden cardiac death.
In recent years, in tonifying kidney and strengthening yang class Chinese medicine preparation and health products, the situation of illegal that non-class medicine of interpolation is very serious.Investigating and prosecuting in tonifying kidney and strengthening yang similar drug and health products, add the illegal activities of that non-class medicine without authorization, is a long-term task.Its coverage is wide, and need to carry out a large amount of inspections, carrying out this inspection activity in vast rural area and small and medium-sized cities underdeveloped has larger difficulty.
The method of that non-class medicine of adulterating in existing inspection medicine and health products has:
1, thin-layered chromatography
Composition mixed solvent to be measured [ethanol-normal hexane-ammoniacal liquor (70:30:1)] in sample extracts, and the need testing solution of gained and reference substance solution point, in same chromatographic sheet, launch with developping agent, after drying, examine and show under uviol lamp.Ruo Nafei class drug control product spot relevant position has no chromatogram spot (lower than detection sensitivity), can thinking in product for being mixed with that non-class medicine, if there is obvious spot, should suspects and may be mixed with that non-class medicine.
The advantage of thin-layered chromatography does not need to use expensive analytical instrument.The shortcoming of the method is: (1) chromatographic resolution rate is lower, health products complicated component, disturbing factor is many, and the composition that easily some coexisted is mistaken for that non-class medicine, if some coexists, composition amount is comparatively large and chromatographic mobile value is close with that non-class medicine, can the detecting of jamming target composition.(2) development of chromatogram is long with the time of drying, and can not meet the requirement of Fast Measurement.(3) require that experimenter has good experience.(4) fixing experiment place is needed, the Site Detection that uncomfortable cooperation mobility is large.
2, high performance liquid chromatography
High performance liquid chromatography is conventional Modern Methods.Under identical chromatographic condition, different materials has different chromatographic retentions.Identical be chromatographic condition under the need testing solution sample introduction respectively of that non-class solution control sample solution and sample extraction gained, the content of that non-class medicine can be detected according to chromatographic retention.
The advantage of high performance liquid chromatography is that chromatographic resolution efficiency is high, highly sensitive.Its shortcoming is: (1) instrument price is expensive, and time for sample pretreatment is long, and great majority are for single product, and the mobile phase of use mostly is a certain proportion of methyl alcohol and salt solusion, needs to rinse chromatographic column, chromatographic column vulnerable to pollution with large water gaging after test, and analysis cost is high; (2) when the chromatographic retention of the composition that coexists and the chromatographic retention of silaenafil close to time easily do the judgement that makes mistake; (3) instrument requires high to environment for use, needs fixedly to put, the Site Detection that uncomfortable cooperation mobility is large.
3, tablets by HPLC-MS
Adopt LC-MS technology, mass spectral analyses can be made further to high performance liquid chromatography that non-class medicine peak isolated.Be applicable to complicated component, sample analysis that background interference is serious, this analytical approach can improve the reliability of assay.
Shortcoming: analysis cost is high, complicated operation; Instrument requires higher to environment for use, needs fixedly to put, and the Site Detection that uncomfortable cooperation mobility is large, not easily promotes the use of.
Summary of the invention
The object of the present invention is to provide a kind of shortcoming overcoming prior art and exist, analysis cost is low, do not need to use valuable analytical instrument, specificity strong, highly sensitive, is applicable to field test medicine, method for quick that whether health products mix that non-class medicine.
The method for quick of that non-class medicine of the present invention, in turn includes the following steps:
(1) sample thief adds acid solution extraction;
(2) add extractant extraction, in extract, add damping fluid;
(3) add bromcresol green solution, observe solution and whether show yellow.
That non-class medicine has three nitrogen-atoms that can accept proton, in an acidic solution, generates band three positive charges, kation in free state (with R after accepting three protons
3+represent).The existence of acid solution can strengthen that non-class medicine and generate free state kation (R
3+) ability, make free state kation (R in solution
3+) there is higher concentration.
Acid solution is the one in watery hydrochloric acid, dilute sulfuric acid or phosphoric acid,diluted, and concentration is 0.01 ~ 0.1mol/L, or the mixed solution of aforementioned several acid solution, is preferably 0.05mol/L watery hydrochloric acid.
Containing multiple auxiliary materials or other compositions in medicine, health products etc., the composition in medicine can be minimized after extraction, reach the object eliminating interference.One or more in the preferred phenixin of extractant, chloroform, methylene chloride, wherein chloroform is considered to the extractant of most selectivity and accuracy through experiment.
Described damping fluid is selected from sodium citrate-hydrochloride buffer, glycine-HCI damping fluid or Acetate-acetate buffer solution, and the pH of damping fluid is 1.5 ~ 2.5, preferred Acetate-acetate buffer solution.
In step (3), if display is yellow, then contain that non-class medicine in sample, if do not show yellow, then do not contain that non-class medicine in sample.The speed of colour developing is relevant with the concentration of that non-class medicine in solution.The concentration of that non-class medicine is larger, and the speed of colour developing is faster.
In said method, optimum experimental condition is based upon on basis that each parameter changes separately.
Further, if known to that non-class medicine any, after the solution drying after chromogenic reaction in step (3), use determined by ultraviolet spectrophotometry concentration, namely survey its absorbance at the uv-absorption maximum wavelength place of this medicine, then the concentration of medicine accurately in solution can be obtained according to its concentration-absorbance standard curve.
Another object of the present invention is to the dedicated test kit that said method is provided, described kit comprises: the one in (1) watery hydrochloric acid, dilute sulfuric acid or phosphoric acid,diluted, concentration is 0.01 ~ 0.1mol/L, or the mixed solution of aforementioned several acid solution, is preferably 0.05mol/L watery hydrochloric acid; (2) one or more in phenixin, chloroform, methylene chloride, preferred chloroform; (3) sodium citrate-hydrochloride buffer, glycine-HCI damping fluid or Acetate-acetate buffer solution, pH is 1.5 ~ 2.5, preferred Acetate-acetate buffer solution; (4) bromcresol green solution.
Compared with the existing technology, method of the present invention has following beneficial effect:
Analysis cost is low, do not need Special Equipment, specificity strong, highly sensitive, be applicable to the method for quickly detecting whether mixing that non-class medicine in field test medicine, health products, overcome the shortcoming that prior art exists, meet the needs of medicine, health products supervision and inspection, simultaneously for the specialized laboratories checking medicine, whether health products mix that non-class medicine provides screening test method fast, to reach reduction analysis cost, improve the object of checkability.Meanwhile, it is strong that the present invention also has anti-coexisting substances interference performance, and experimental phenomena is obvious, and check conclusion accuracy is high, fast easy and simple to handle, do not need to use the advantages such as expensive instrument.
The detection kit of that non-class medicine that method according to the present invention is made, can meet the requirement that relevant food supervision and inspection is carried out in vast rural area.
Accompanying drawing explanation
Fig. 1 is silaenafil concentration-ultraviolet absorptivity curve map.
Embodiment
Below in conjunction with embodiment, the present invention is described further, but is not construed as limiting the invention.
Embodiment 1 Libujinqiu capsules detects (capsule, indicating dose is each 2, and high effective liquid chromatography for measuring result this product every is containing that non-class medicine 58mg)
Get 0.5 amount content, grinding evenly, is placed in 50mL beaker, adds 25mL 0.05mol/L watery hydrochloric acid, mix, jolting two minutes, leaves standstill four minutes, filters, and collects filtrate.Filtrate is transferred in 125mL separating funnel, add 5mL chloroform, jolting two minutes, leave standstill one minute.Extract is transferred in the beaker of 50mL, and aqueous phase continues to use 5mL chloroform extraction, combining extraction liquid.Add the Acetate-acetate buffer solution of 3mL pH=2.0 in extract, the agent of 2.5mL visualization Bromocresol green, solution shows yellow immediately, can contain that non-class medicine thus in judgement sample.
Embodiment 2 is hidden whip despot ball and is detected (large honeyed bolus, specification is every ball 10g, and indicating dose is ball every day 1, and the every ball of high effective liquid chromatography for measuring result this product is containing that non-class medicine 176mg)
Sample shreds into grain of rice size, and sampling 0.6g, is placed in 50mL beaker, adds 25mL 0.01mol/L dilute sulfuric acid, mix, jolting two minutes (can smash with glass rod loose if desired), leaves standstill four minutes, collecting by filtration filtrate.Filtrate is transferred in 125mL separating funnel, add 5mL phenixin, jolting two minutes, leave standstill one minute.Extract is transferred in 50mL beaker, and aqueous phase continues to use 5mL carbon tetrachloride extraction, combining extraction liquid.Add the sodium citrate-hydrochloride buffer of 3mLpH=2.5 in extract, the agent of 2.5mL visualization Bromocresol green, solution shows yellow immediately, can contain that non-class medicine thus in judgement sample.
More than operate, replace filtration treatment by centrifugal treating, effect is identical, all can contain that non-class medicine in judgement sample.
Embodiment 3 nine whip ball detects (blue coating tablet, sheet core white look; Indicating dose is each 1; The every sheet of high effective liquid chromatography for measuring result this product is containing that non-class medicine 72mg)
Sample thief 0.5, is placed in 50mL beaker, adds 25mL 0.1mol/L phosphoric acid,diluted, smashes loose, mix with glass rod, jolting two minutes, leaves standstill four minutes, collecting by filtration filtrate.Filtrate is transferred in 125mL separating funnel, add 5mL methylene chloride, jolting two minutes, leave standstill one minute.Extract is transferred in 50mL beaker, and aqueous phase continues to use 5mL dichloromethane extraction, combining extraction liquid.Add the glycine-HCI damping fluid of 3mLpH=1.5 in extract, the agent of 2.5mL visualization Bromocresol green, solution shows yellow immediately, can contain that non-class medicine thus in judgement sample.
The very simple tonic tablet for kidney-reinforcing of embodiment 4 detects (indicating dose is each 5 for tablet, bronzing dressing, and efficient liquid-phase chromatography method detects and confirms not containing that non-class medicine)
Sample thief 1, crushes and is placed in 50mL beaker, add 25mL mixed acid solution (0.05mol/L watery hydrochloric acid: 0.01mol/L dilute sulfuric acid: 0.1mol/L phosphoric acid,diluted volume ratio is 2:2:1), mix, jolting two minutes, leaves standstill four minutes, collecting by filtration filtrate.Filtrate is transferred in 125mL separating funnel, add 5mL chloroform, jolting two minutes, leave standstill one minute.Extract is transferred in 50mL beaker, and aqueous phase continues to use 5mL chloroform extraction, combining extraction liquid.Add the Acetate-acetate buffer solution of 3mLpH=2.0 in extract, the agent of 2.5mL visualization Bromocresol green, solution does not show yellow, leaves standstill and does not still have yellow to show after three minutes, can not contain that non-class medicine thus in judgement sample.
The experiment of that non-class medicine whether is there is in embodiment 5 medicine, health products
The method of embodiment 1 is adopted to detect 10 batches of commercial samples (being denoted as medicine, health products).And adopt LCMS method and testing result comparison, result is as follows:
| Goods # | Explanation | Sampling amount | Colour developing result | Identification result | Mass spectral results |
| Sample 1 | Tablet | 1, grind | Develop the color yellow immediately | Positive | Containing silaenafil |
| Sample 2 | Tablet | 1, grind | Non-displaing yellow in 30 seconds | Negative | Not containing that non-class medicine |
| Sample 3 | Capsule | 1 intragranular is tolerant | Develop the color yellow immediately | Positive | Containing Vardenafil |
| Sample 4 | Capsule | 1 intragranular is tolerant | 30 seconds not interior displaing yellows | Negative | Not containing that non-class medicine |
| Sample 5 | Capsule | 1 intragranular is tolerant | 30 seconds not interior displaing yellows | Negative | Not containing that non-class medicine |
| Sample-6 | Oral liquid | 1/2 | Non-displaing yellow in 30 seconds | Negative | Not containing that non-class medicine |
| Sample 7 | Medicinal tea | 0.5g | Displaing yellow in 20 seconds | Positive | Containing silaenafil |
| Sample 8 | Medicinal tea | 0.5g | Non-displaing yellow in 30 seconds | Negative | Not containing that non-class medicine |
| Sample 9 | Capsule | 1 intragranular is tolerant | Non-displaing yellow in 30 seconds | Negative | Not containing that non-class medicine |
| Sample 10 | Cassia seed | 0.5g | Non-displaing yellow in 30 seconds | Negative | Not containing that non-class medicine |
Above-mentioned experimental result and second order ms the result show, none official holiday of detection method positive report, simultaneously also not undetected any sample containing that non-class medicine, and result accurately and reliably.
By the sampling described in the inventive method measure that non-class drug test above-mentioned be negative reaction 7 samples, and mix silaenafil reference substance 20mg respectively, test by the inventive method, result is all positive, show that this method has good antijamming capability, specificity is strong, has good accuracy.
The detection limit that embodiment 6 synthesizes sample detects
Sample: the silaenafil of massfraction more than 99.5%
In 10mL sample bottle I, II, III, IV, add 2mg, 5mg, 10mg, 20mg silaenafil respectively, add 25mL0.05mol/L watery hydrochloric acid respectively, mix, jolting two minutes, leave standstill four minutes, collecting by filtration filtrate.Filtrate is transferred in 125mL separating funnel, add 5mL chloroform, jolting two minutes, leave standstill one minute.Extract is transferred in 50mL beaker, and aqueous phase continues to use 5mL chloroform extraction, combining extraction liquid.Add the damping fluid of 3mLpH=2.0 in extract, the agent of 2.5mL visualization Bromocresol green, survey its absorbance at 415nm place, according to typical curve, record its exact level and be respectively 0.05mg, 0.489mg, 9.96mg, 18.85mg.
Experimental result:
In color comparison tube I, solution colour does not change, and in color comparison tube II, III, IV, solution display is yellow, and detecting of the method is limited to 5mg as can be seen here.
Embodiment 7 synthesizes the sensitivity technique of sample
Sample: the silaenafil of massfraction more than 99.5%
Adopt the detection method of embodiment 1, prepare the silaenafil solution of a series of variable concentrations, curve as shown in Figure 1 can be obtained according to absorbance.When content is less than 0.5gL
-1time, the absorbance of solution is almost constant, and color does not change substantially, and naked eyes cannot judge; When content is greater than 0.5gL
-1time, concentration becomes good linear relationship with absorbance, and color change obviously, therefore tentatively can determine that the sensitivity of the method is 0.5gL
-1.
Above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although be explained in detail the present invention with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify to technical scheme of the present invention or equivalent replacement, and not depart from essence and the scope of technical solution of the present invention.
Claims (7)
1. a detection method for that non-class medicine, in turn includes the following steps:
(1) sample thief adds acid leach solution;
(2) add extractant extraction, in extract, add damping fluid;
(3) drip bromcresol green solution, observe solution and whether show yellow;
Wherein: described acid solution is the one in watery hydrochloric acid, dilute sulfuric acid or phosphoric acid,diluted, concentration is 0.01 ~ 0.1mol/L, or the mixed solution of aforementioned several acid solution;
Described extractant is one or more in phenixin, chloroform, methylene chloride;
Described damping fluid is sodium citrate-hydrochloride buffer, glycine-HCI damping fluid or Acetate-acetate buffer solution, and the pH of described damping fluid is 1.5 ~ 2.5.
2. the method for claim 1, is characterized in that: described acid solution is 0.05mol/L watery hydrochloric acid.
3. the method for claim 1, is characterized in that: described extractant is chloroform.
4. the method for claim 1, is characterized in that: described damping fluid is Acetate-acetate buffer solution.
5. the method as described in any one of claim 1-4, is characterized in that: described method also comprises: after the solution drying after the chromogenic reaction in described step (3), adopts the concentration of determined by ultraviolet spectrophotometry medicine.
6. the dedicated test kit of the method as described in any one of claim 1-5, it is characterized in that: described kit comprises: (1) concentration is the watery hydrochloric acid of 0.01 ~ 0.1mol/L, dilute sulfuric acid or phosphoric acid,diluted, or the mixed acid solution of aforementioned several acid solution; (2) one or more in phenixin, chloroform, methylene chloride; (3) sodium citrate-hydrochloride buffer, glycine-HCI damping fluid or Acetate-acetate buffer solution, pH1.5 ~ 2.5; (4) bromcresol green solution.
7. kit as claimed in claim 6, is characterized in that: described kit comprises: (1) 0.05mol/L watery hydrochloric acid; (2) chloroform (3) pH1.5 ~ 2.5 Acetate-acetate buffer solution; (4) bromcresol green solution.
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| CN1405314A (en) * | 2001-08-17 | 2003-03-26 | 杭州泰士生物科技有限公司 | Cryptoporus volvatus fermented product, and its preparation method and application |
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| US20050171449A1 (en) * | 2000-03-21 | 2005-08-04 | Suslick Kenneth S. | Method and apparatus for detecting ammonia from exhaled breath |
| CN1275038C (en) * | 2004-03-24 | 2006-09-13 | 广东省药品检验所 | Fast analyzing method for sildenafil citrate doped medicine, health products and foods |
| CN100588954C (en) * | 2007-03-20 | 2010-02-10 | 北京市药品检验所 | Method for assaying cedinafei and derivative thereof |
| FR2965615A1 (en) * | 2010-09-30 | 2012-04-06 | Ceca Sa | SOLVENTS FOR THE DETERMINATION OF AMINES IN WATER |
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| Title |
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| 亚甲基蓝光度法测定西地那非的含量;江虹,等;《分析科学学报 》;20040630;第20卷(第3期);287-289 * |
| 溴甲酚绿分光光度法测定麻黄碱含量的研究;吴英,谢红伟;《分析科学学报》;20070430;第23卷(第2期);246-248 * |
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