CN107884498A - Liquid phase chromatography analytical method that is a kind of while determining procaine, penicillin and content of streptomycin - Google Patents
Liquid phase chromatography analytical method that is a kind of while determining procaine, penicillin and content of streptomycin Download PDFInfo
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Abstract
Procaine is determined the invention discloses a kind of simultaneously, the liquid phase chromatography analytical method of penicillin and content of streptomycin, this method first prepares procaine respectively, penicillin and streptomysin reference substance standard liquid, then prepare and contain procaine, the need testing solution of penicillin and/or streptomysin composition, reuse the liquid chromatogram that liquid chromatogram obtains and draw procaine respectively, the standard curve of penicillin and streptomysin, finally detect need testing solution, and utilize procaine, the standard curve of penicillin and streptomysin calculates procaine, the content of penicillin/or streptomysin.The method provided by the present invention can accurately detect the content of procaine, penicillin, streptomysin, method specificity is strong, precision is high, stability is good, the degree of accuracy is high, the quality control of procaine penicillin-streptomysin folk prescription and compound product suitable for veterinary drug industry, can effectively save testing cost and time.
Description
Technical field
The invention belongs to technical field of analytical chemistry, and in particular to a kind of to determine procaine, penicillin and strepto- simultaneously
The liquid phase chromatography analytical method of cellulose content.
Background technology
At present, American Pharmacopeia has included the quality standard of " procaine penicillin-dihydrostreptomycin sulfate parenteral solution ",
In American Pharmacopeia, European Pharmacopoeia, British Pharmacopoeia to preparation in Penicillin Content measure use iodimetric titration, the assay method is endless
Kind, poor selectivity, disturbing factor is more, the domestic content measuring standard method for not yet formulating penicillin compound preparation.And iodimetric titration
It is the principle based on redox titration, it is desirable to titration process is comparatively fast completed, if the antioxidant or other auxiliary materials that are used in formula
Titration process and result are may interfere with, starch indicator solution, which adds the assurance at time point etc., can increase the error of titration results.
Streptomysin uses the nephelometry in antibiotic microorganism identification method to be measured.Content of streptomycin principle is that detection supplies
The antibacterial activity of reference culture is calculated test product compared with standard items, and its detection time length (>=24h), content detection are missed
It is poor big, it is unfavorable for the quality control of semi-finished product.
The content of the invention
For deficiencies of the prior art, it is an object of the invention to provide it is a kind of determine simultaneously procaine,
The liquid phase chromatography analytical method of penicillin and content of streptomycin, it is intended to when solving to detect inaccuracy, detection in existing detection method
Between the technical problem such as long.
To achieve these goals, the technical solution adopted by the present invention is as follows：
Liquid phase chromatography analytical method that is a kind of while determining procaine, penicillin and content of streptomycin, including following step
Suddenly：
1) respectively by the standard items of procaine, penicillin and streptomysin flowing phased soln, being configured to concentration respectively is
2mg·mL-1、2mg·mL-1And 2mgmL-1Solution, then filter off impurity respectively, procaine, penicillin and streptomysin be made
Reference substance standard liquid is stand-by.
2) by the test sample of procaine, penicillin and/or streptomysin flowing phased soln, it is configured to concentration 1.0mg/ml
Solution, filtering, it is stand-by that need testing solution is made.
3) procaine, penicillin and the streptomysin reference substance standard liquid for taking step 1) to prepare respectively, it is dense to prepare gradient
The standard liquid of degree；Then analyzed respectively using liquid chromatogram under 190~250nm of Detection wavelength, and according to analyzing
To liquid chromatogram draw the standard curve of procaine, penicillin and streptomysin respectively.
4) detecting step 2 under the identical liquid phase chromatogram condition of step 3)) made from need testing solution, recycle step 3)
The standard curve of obtained procaine, penicillin and streptomysin calculates procaine, penicillin/or the content of streptomysin.
Wherein：In step 1), described mobile phase is organic phase and the mixture of the phosphate buffer containing ion pair；Its
In, organic phase and the volume ratio of the phosphate buffer containing ion pair are (5~30)：(70~95), described organic phase are acetonitrile
Or methanol, the phosphate buffer containing ion pair are the mixing water of sodium sulphate, perfluorooctane sulfonate, potassium dihydrogen phosphate and phosphoric acid
Solution.
The compound method of the described phosphate buffer containing ion pair is：By 15.91g anhydrous sodium sulfates, 1.7g octane sulphurs
Sour sodium is dissolved in 700mL water, adds 57mL phosphate buffer solutions, is added water to be settled to 1000mL and is made；Wherein, phosphate
Cushioning liquid is that concentration is that the phosphorus acid for adjusting pH that 27.2g/L potassium dihydrogen phosphates are 22.5g/L with concentration is obtained to 3.0.
Preferably, organic phase and the volume ratio of the phosphate buffer containing ion pair are 12：88.
In step 3) and 4) in liquid chromatogram, flow velocity is 0.3~1.5mL/min, and column temperature is 25~40 DEG C, and sample size is
20μL。
Preferably, described Detection wavelength is 205nm；Described flow velocity is 1.0mL/min.Described column temperature is 40 DEG C.
In step 3) and 4) in liquid chromatogram, chromatographic column is using octadecylsilane chemically bonded silica as filler.
In order to improve accuracy, repeat step 4) once more than, average carry out calculating procaine, penicillin/or
The content of streptomysin.
The present invention is filler with common octadecylsilane chemically bonded silica, and stream is used as using ion pair buffer-organic phase
Three kinds of dynamic relative procaine, penicillin, streptomysin compositions carry out isocratic elution, because procaine is organic base, it is blue or green
Mycin is organic acid, and streptomysin is organic bases ionic compound, and its streptomycin ionizing power is strong, in Reversed-phase Chromatographic System
It is situated between without reserve.Therefore the chromatographic condition of selection streptomysin is one of key technology of the present invention.The present invention is tried from ion pair
Agent effect and streptomysin, enable streptomysin to retain on reverse-phase chromatographic column, while common by flowing phase pH value, organic Phase Proportion
Streptomysin is set to reach optimal separation and reservation；Procaine is organic base, can quickly be eluted in acid condition, is reduced organic
Phase Proportion extends the retention time of procaine；Penicillin is that organic acid also can obtain very well in acid mobile phase；Three kinds of compositions
In, the ultraviolet feature of streptomysin is most weak, and through experiment sieving 205nm as Detection wavelength, procaine, penicillin are in 205nm wavelength
There is good response；First filtered out in the present invention from sodium pentanesulfonate, sodium heptanesulfonate, perfluorooctane sulfonate suitable for streptomysin
The ion-pairing agent of separation, finally but perfluorooctane sulfonate as analysis streptomysin ion-pairing agent；Alkali ion is to reagent
The retention time of procaine can be extended by adding, and added about 15% acetonitrile not influenceing streptomysin retention time, strengthened Proca
The elution of cause and penicillin.
Compared with prior art, the present invention has the advantages that：
1st, the present invention utilizes high performance liquid chromatography, coordinates UV-detector, to procaine, penicillin, content of streptomycin
Analyzed, alleviate prior art and antibiosis is used to penicillin method measure imperfection, iodimetric titration poor selectivity and streptomysin
Nephelometry detection time, the technical problem of poor accuracy in plain microbial identification methods.
2nd, the content of penicillin, procaine, streptomysin can be accurately detected using the present invention, as a result specificity is strong, accurate
Degree is high, stability is good, the degree of accuracy is high, the matter for the procaine penicillin-streptomysin injection products being used in veterinary drug industry
Amount control, can effectively save testing cost and time.
3rd, common octadecylsilane chemically bonded silica is used as filler, and flowing is used as using ion pair buffer-organic phase
Isocratic elution is carried out with respect to three kinds of procaine, penicillin, streptomysin compositions, the intact separation of composition in can be achieved 3 in 60 minutes
And quantitative analysis.
Brief description of the drawings
Fig. 1 is the chromatogram of reference substance solution；
Fig. 2 is penicillin linear standard curve；
Fig. 3 is streptomysin linear standard curve；
Fig. 4 is procaine linear standard curve；
Fig. 5 is the chromatogram of commercially available procaine penicillin+dihydrostreptomycin sulfate suspension injection detection；
Fig. 6 is the chromatogram of commercially available procaine benzylpenicillin injection detection；
Fig. 7 is commercially available procaine penicillin for injection-streptomycin sulphate powder pin.
Embodiment
The present invention is described in further detail with reference to specific embodiment.
Embodiment one
Procaine, penicillin and content of streptomycin are determined by this implementation, and verify its specificity.
1st, testing conditions are as follows：
Chromatographic column：Octadecylsilane chemically bonded silica is filler (C18 250mm × 4.6, μm)；
Mobile phase：With cocktail buffer-acetonitrile (88：12) (detailed compound method is shown in quality standard)；
Flow velocity：1.0mL/min；
Sample size：20μL；
Detection wavelength：205nm；
Column temperature：40℃；
2nd, contrast solution is as follows：
Penicillin reference substance solution (calls a liquid in the following text)：Penicillin reference substance adds flowing phased soln that 2mgmL is made in right amount-1It is right
According to product solution.
Streptomysin reference substance solution (calls b liquid in the following text)：Streptomysin reference substance adds flowing phased soln that 2mgmL is made in right amount-1It is right
According to product solution.
Procaine reference substance solution (calls c liquid in the following text)：Procaine reference substance adds flowing phased soln that 0.5mg is made in right amount
mL-1Reference substance solution.
3rd, assay method is：The μ L of need testing solution (detecting sample) 20 injection liquid chromatographs are measured, by foregoing chromatogram
Condition is measured, and records chromatogram；Another precision measures 20uL reference substance solutions injection liquid chromatograph, is measured in the same method；By outer
Mark method is with each component content of calculated by peak area.
4th, specificity is investigated
Each 20 μ L of negative need testing solution of reference substance solution and 3 kinds of compositions are taken respectively, injecting chromatograph, record chromatogram
Figure.The chromatogram (see Fig. 1) of reference substance solution, the chromatogram of each negative test sample by comparing reference substance solution and 3 kinds of compositions
Figure, the retention time of each composition is consistent, illustrates that each composition negative test sample on retention time position does not significantly interfere with, enters one
Under chromatographic condition of the present invention, three kinds of compositions are effectively maintained step explanation, and separation is intact.Penicillin, streptomysin, Pu Lu
The chromatographic peak Average residence time of cacaine is as shown in table 1.
The penicillin of table 1, streptomysin, the chromatographic peak Average residence time statistical form of procaine
| Composition | Average residence time (min) |
| Penicillin | 11.2 |
| Streptomysin | 41.5 |
| Procaine | 50.1 |
Embodiment two
Using the testing conditions in embodiment one, contrast solution and assay method, single component linear relationship investigation is carried out
Experiment.
1st, penicillin linear relationship
Accurate a liquid of drawing is appropriate respectively, adds mobile phase to be made respectively containing penicillin 0.2,0.4,0.6,1.0,2.0mg
mL-1Reference substance solution, be measured by chromatographic condition under assay item, with peak area (A) to concentration (C) carry out return point
Analysis, as shown in table 2, Fig. 2.
The penicillin linear relationship result of table 2
It can be seen that penicillin concn in the range of 0.26016---2.6016mg/mL, has good linear relationship, phase relation
Number r=0.9999.
2nd, streptomysin linear relationship
Accurate b liquid of drawing is appropriate respectively, adds mobile phase to be made respectively containing streptomysin 0.2,0.4,0.6,1.0,2.0mg
mL-1Reference substance solution, be measured by chromatographic condition under assay item, with peak area (A) to concentration (C) carry out return point
Analysis, as shown in table 3, Fig. 3.
The streptomysin linear relationship result of table 3
It can be seen that streptomysin concentration in the range of 0.2577---2.577mg/mL, there is preferable linear relationship, correlation coefficient r
=0.9999.
3rd, procaine linear relationship
Accurate c liquid of drawing is appropriate respectively, adds mobile phase to be made respectively containing procaine 0.1,0.2,0.3,0.4,0.5mg
mL-1Reference substance solution, be measured by chromatographic condition under assay item, with peak area (A) to concentration (C) carry out return point
Analysis, as shown in table 4, Fig. 4.
The procaine linear relationship result of table 4
It can be seen that streptomysin concentration in the range of 0.1108---0.5771mg/mL, there is preferable linear relationship, coefficient correlation
R=0.9999.
Embodiment three
Using the testing conditions in embodiment one, contrast solution and assay method, investigated on the basis of implementing one, two accurate
Exactness, stability and precision.
1st, the degree of accuracy
Average recovery is tested：Precision measures known each 9 parts of component content need testing solution, and every 3 parts are one group, and every group equal
Contrast solution 1mL, 3mL, 5mL are separately added into according to basic, normal, high three concentration, shakes up, is diluted to graduation mark, precision measures 20 μ
L decibel injecting chromatographs, chromatogram is recorded, calculate procaine, penicillin, the mean sample recovery rate and RSD of streptomysin, knot
Fruit is shown in Table 4, and the mean sample recovery rate of three kinds of compositions is all higher than 97%, RSD less than 2.0% as shown in Table 4, illustrates standard of the invention
Exactness is high.
Result is investigated in the degree of accuracy of table 4
2nd, stability
Reference substance solution is prepared, the μ L injecting chromatographs of reference substance solution 20 are taken respectively at 0,1,2,3,4,6,8,12h precisions,
Chromatogram is recorded, each time point carries out sample 3 times, calculates RSD values, the results are shown in Table 5, as shown in Table 5, three kinds of Component peak areas
RSD meets the requirements, and illustrates that the present invention has good stability.
The study on the stability result of table 5
| Composition | RSD (%) |
| Penicillin | 1.89 |
| Streptomysin | 1.23 |
| Procaine | 1.07 |
3rd, precision is investigated
Precision measures the μ L of reference substance solution 20 injection liquid chromatographs, records chromatogram, is repeated 6 times.Measured with each composition
Calculated by peak area relative standard deviation (RSD) result, be shown in Table 6, as shown in Table 6, three kinds of Component peak area RSD meet precision
It is required that illustrate that precision of the present invention is good.
The precision of table 6 investigates result
| Composition | RSD (%) |
| Penicillin | 1.28 |
| Streptomysin | 1.67 |
| Procaine | 1.97 |
Example IV
Sample is measured using the testing conditions in embodiment one, contrast solution and assay method.
1st, commercially available procaine penicillin+dihydrostreptomycin sulfate suspension injection (Penstrep Britain)
Using commercially available procaine penicillin+dihydrostreptomycin sulfate suspension injection as test sample, its labelled amount contains per mL
Penicillin 200,000IU, 200mg containing streptomysin, containing procaine it must not exceed 20mg.
It is prepared by need testing solution：Precision absorption test sample is appropriate, puts in 200mL volumetric flasks, adds mobile phase ultrasound 5min,
Add mobile phase to be diluted to graduation mark, shake up, filter, gained filtrate is as test sample.
Procaine, penicillin, content of streptomycin in the suspension injection are determined by foregoing content assaying method, is as a result seen
Fig. 2, as shown in Figure 5, it is respectively containing procaine, penicillin, streptomysin, its sign content in the parenteral solution：Per mL containing green grass or young crops
Mycin 199,894IU, 198mg containing streptomysin, 16mg containing procaine.
2nd, commercially available procaine benzylpenicillin injection
Using commercially available Procaine benzylpenicillin suspension injection as test sample, its labelled amount contains penicillin 300 per mL, 000IU,
120mg is must not exceed containing procaine.
It is prepared by need testing solution：Precision absorption test sample is appropriate, puts in 200mL volumetric flasks, adds mobile phase ultrasound 5min,
Add mobile phase to be diluted to graduation mark, shake up, filter, gained filtrate is as test sample.
Procaine, Penicillin Content in the suspension injection are determined by foregoing content assaying method, as a result sees Fig. 6, by
Fig. 6 is understood, is respectively containing procaine, penicillin, its sign content in the parenteral solution：Per mL 299,877IU containing penicillin,
118mg containing procaine.
3rd, commercially available procaine penicillin for injection-streptomycin sulphate powder pin
Using commercially available procaine penicillin for injection-streptomycin sulphate powder pin as test sample, its labelled amount contains penicillin per g
2000IU, 2mg containing streptomysin, containing procaine it must not exceed 0.2mg.
It is prepared by need testing solution：Precision absorption test sample is appropriate, puts in 200mL volumetric flasks, adds mobile phase ultrasound 5min
Dissolving, adds mobile phase to be diluted to graduation mark, shakes up, and filters, gained filtrate is as test sample.
Procaine, penicillin, content of streptomycin in the suspension injection are determined by foregoing content assaying method, is as a result seen
Fig. 4, as shown in Figure 7, it is respectively containing procaine, penicillin, streptomysin, its sign content in the parenteral solution：Contain mould per g
Plain 1988IU, 1.97mg containing streptomysin, 0.18mg containing procaine.
The above embodiment of the present invention is only example to illustrate the invention, and is not the implementation to the present invention
The restriction of mode.For those of ordinary skill in the field, other can also be made not on the basis of the above description
With the change and variation of form.Here all embodiments can not be exhaustive.It is every to belong to technical scheme
Row of the obvious changes or variations amplified out still in protection scope of the present invention.
Claims (10)
1. liquid phase chromatography analytical method that is a kind of while determining procaine, penicillin and content of streptomycin, it is characterised in that bag
Include following steps：
1) respectively by the standard items of procaine, penicillin and streptomysin flowing phased soln, it is 2mg to be configured to concentration respectively
mL-1、2mg·mL-1And 2mgmL-1Solution, then filter off impurity respectively, procaine, penicillin and streptomysin control be made
Product standard liquid is stand-by；
2) by the test sample of procaine, penicillin and/or streptomysin flowing phased soln, it is configured to the molten of concentration 1.0mg/mL
Liquid, filtering, it is stand-by to be made need testing solution；
3) procaine, penicillin and the streptomysin reference substance standard liquid for taking step 1) to prepare respectively, prepare gradient concentration
Standard liquid；Then analyzed respectively using liquid chromatogram under 190~250nm of Detection wavelength, and obtained according to analysis
Liquid chromatogram draws the standard curve of procaine, penicillin and streptomysin respectively；
4) detecting step 2 under the identical liquid phase chromatogram condition of step 3)) made from need testing solution, recycle step 3) obtain
Procaine, the standard curve of penicillin and streptomysin calculate procaine, penicillin/or the content of streptomysin.
2. liquid-phase chromatographic analysis side that is according to claim 1 while determining procaine, penicillin and content of streptomycin
Method, it is characterised in that the mobile phase described in step 1) is organic phase and the mixture of the phosphate buffer containing ion pair；Its
In, organic phase and the volume ratio of the phosphate buffer containing ion pair are (5~30)：(70~95), described organic phase are acetonitrile
Or methanol, the phosphate buffer containing ion pair are the mixing water of sodium sulphate, perfluorooctane sulfonate, potassium dihydrogen phosphate and phosphoric acid
Solution.
3. liquid-phase chromatographic analysis side that is according to claim 2 while determining procaine, penicillin and content of streptomycin
Method, it is characterised in that the compound method of the phosphate buffer containing ion pair is：By 15.91g anhydrous sodium sulfates, 1.7g
Perfluorooctane sulfonate is dissolved in 700mL water, adds 57mL phosphate buffer solutions, is added water to be settled to 1000mL and is made；Wherein,
Phosphate buffer solution is that concentration is that the phosphorus acid for adjusting pH that 27.2g/L potassium dihydrogen phosphates are 22.5g/L with concentration is obtained to 3.0.
4. liquid-phase chromatographic analysis side that is according to claim 2 while determining procaine, penicillin and content of streptomycin
Method, it is characterised in that the volume ratio of organic phase and the phosphate buffer containing ion pair is 12：88.
5. liquid-phase chromatographic analysis side that is according to claim 1 while determining procaine, penicillin and content of streptomycin
Method, it is characterised in that in liquid chromatogram in step 3) and 4), flow velocity is 0.3~1.5mL/min, and column temperature is 25~40 DEG C, is entered
Sample amount is 20 μ L.
6. liquid-phase chromatographic analysis side that is according to claim 5 while determining procaine, penicillin and content of streptomycin
Method, it is characterised in that described Detection wavelength is 205nm.
7. liquid-phase chromatographic analysis side that is according to claim 5 while determining procaine, penicillin and content of streptomycin
Method, it is characterised in that described flow velocity is 1.0mL/min.
8. liquid-phase chromatographic analysis side that is according to claim 5 while determining procaine, penicillin and content of streptomycin
Method, it is characterised in that described column temperature is 40 DEG C.
9. liquid-phase chromatographic analysis side that is according to claim 1 while determining procaine, penicillin and content of streptomycin
Method, it is characterised in that in liquid chromatogram in step 3) and 4), chromatographic column is using octadecylsilane chemically bonded silica as filler.
10. liquid-phase chromatographic analysis side that is according to claim 1 while determining procaine, penicillin and content of streptomycin
Method, it is characterised in that repeat step 4) once more than, average and carry out containing for calculating procaine, penicillin/or streptomysin
Amount.
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| CN109917038B (en) * | 2019-03-26 | 2022-04-01 | 广西壮族自治区食品药品检验所 | HPLC detection method of procaine hydrochloride related substances |
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| CN114814067B (en) * | 2022-06-24 | 2022-09-13 | 珠海溪谷医疗科技有限公司 | Method for detecting content of heptasodium diethylenetriamine penta (methylene phosphonic acid) by HPLC (high performance liquid chromatography) |
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