CN103487521A - Detection method for dextroisomer of pazufloxacin mesilate injection - Google Patents
Detection method for dextroisomer of pazufloxacin mesilate injection Download PDFInfo
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Abstract
The invention belongs to the field of medicine analysis, and particularly relates to a detection method for the dextroisomer of pazufloxacin mesilate injection. The invention aims at providing a method which is simple and convenient to operate, and capable of rapidly and accurately detecting a dextroisomer, wherein HPLC (high performance liquid chromatography) detection conditions are as follows: octadecylsilane chemically bonded silica is used as a filler in a stationary phase; a mobile phase A is an L-phenylalanine-copper sulphate solution; a mobile phase B is methanol, wherein the L-phenylalanine-copper sulphate solution contains 0.3-3 mg/ml L-phenylalanine, and 0.2-2 mg/ml copper sulphate; the pH value is adjusted to 2.0-5.0 by 1 mol/L sodium hydroxide solution; a mobile phase is prepared from the mobile phase A and the mobile phase B in a volume ratio of (40 to 90): (60 to 10); a flow speed is 0.6-1.0 ml/min; a detection wavelength is 330 nm; and the number of theoretical plates is not less than 2500 counted by the peaks of the laevoisomer of pazufloxacin, and a separation degree between the laevoisomer and the dextroisomer of pazufloxacin needs to meet requirements.
Description
Technical field
The invention belongs to the Pharmaceutical Analysis field, be specifically related to the detection method of pazufloxacin mesylate injection dextroisomer.
Technical background
QNS is the antibacterials that develop rapidly in recent years, world's QNS average annual growth rate 8%～10%, and sales volume reaches more than 8,000,000,000 dollars.Domestic 14 Urban Statisticals, anti-infectives accounts for purchases 29.54% of medicine total value, and wherein QNS accounts for 14.89% of anti-infectious agent total value, occupies the 3rd.And the cephalo-type of front two and penicillins downtrending are obvious, and quinolones is powerful ascendant trend, approaches the share of penicillins medicine.Pazufloxacin Mesilate is fourth generation quinolone, now has been widely used in the middle of all kinds of bacterial infections of clinical treatment, has obtained good clinical efficacy.
Pazufloxacin Mesilate has broad spectrum antibiotic activity, gram-negative bacteria, positive bacteria are all had to stronger lethality, antibacterial efficacy is strong, toxic and side effect is lower, be difficult for to produce drug resistance, clinical practice has good clinical efficacy and security for the responsive microbial respiratory system for the treatment of, the genito-urinary system skin soft-tissue infection that unifies.
Impurity that may exist of record in " pharmaceuticals ィ ソ タ PVC ユ mono-Off オ mono-system hospital in Japan pharmacists understand IF record main points ", the structural formula of Pazufloxacin Mesilate and the impurity that may exist thereof is respectively:
Wherein, formula I is Pazufloxacin Mesilate; Formula II is its dextroisomer, originates and brings into racemization in synthetic and degrade for raw material; Formula III is its synthetic intermediate, the generation of also may degrading in the preservation process; Formula IV～IX is its catabolite.
Contain a chiral center in the Pazufloxacin Mesilate molecule, have a pair of isomeride, what generally play clinically therapeutic action at present is its laevoisomer.Dextroisomer is without therapeutic action, and mentions it in " pharmaceuticals ィ ソ タ PVC ユ mono-Off オ mono-system hospital in Japan pharmacists understands IF record main points " and have toxic and side effect.At present, in domestic many raw materials, preparation quality standard, the control to this kind impurity mainly concentrates on the control to this isomeride.
The inventor produces the dextroisomer that in pazufloxacin mesylate injection, one of major impurity is Pazufloxacin Mesilate, chemical name is (+)-(3R)-10-(the amino cyclopropyl of 1-)-9-fluoro-2,3-dihydro-3-methyl-7-oxygen-7H-pyrido [1,2,3-de]-[1,4] benzoxazines-6-carboxylic acid mesylate, molecular formula is C
16h
15fN
2o
4cH
4o
3s.
In view of between the enantiomorph of many medicines, in pharmacology, toxicity and even clinical properties, existing larger difference, it is necessary that some chiral drug is carried out to the enantiomeric purity inspection.
High performance liquid chromatography can split enantiomorph quickly and effectively, and in enantiomeric purity checks, application is very extensive at present.Mainly can be divided into two approach: indirectly with direct.Indirect method (CDR) relates to the enantiomter column front derivation is become to diastereo-isomerism, needs the chiral derivatization reagent of high-optical-purity, operates more loaded down with trivial details.Direct method is to create chiral environment in chromatographic system, is divided into again Chiral mobile phase additives (CMPA) and Chiral Stationary Phases (CSP), and Chiral mobile phase additives adopts common chromatographic column, does in mobile phase and is added; And Chiral Stationary Phases development is swift and violent, existing tens of kinds of CSP commodity posts up to now, these two kinds of methods are in the situation that experiment condition is satisfied, operates very easyly, are widely used in the optical antipode detection.
Commonly used as the optically active amino acids or derivatives thereof of the chiral ligand (aglucon) of Mobile Phase Additives have L-PROLINE, L-Histidine, an ILE etc., and coordination of metal ion commonly used has Cu
2+, Zn
2+, Ni
2+deng.
The chromatographic column type of chiral stationary phase is divided into: brush type (Prikle type), cellulose type, cyclodextrin, macrocyclic antibiotic type, protein type, ligand exchange type, crown ether type.Commercial chiral chromatographic column relatively common are Daicel company the cellulose derivative class as Chiralcel OJ, carbamate cellulose derivant class as Chiralcel OD, polysaccharide derivates class as Chiralpak AD etc., and corresponding reversed-phase column, and the Chiralpak WM of compatibility exchange class and the Chiralpak CR of crown ether-like etc.
The existing detection method to Pazufloxacin Mesilate and preparation optical isomer thereof, the chirality aglucon exchange mobile phase additive methods that belong to Chiral mobile phase additives that adopt more, the mechanism of the method is for being used common octadecyl silane as fixing phase, add chiral ligand in mobile phase, chiral ligand mostly is the optically active amino acids or derivatives thereof, form again the coordination compound of chelating with bivalent metal ion, with suitable CONCENTRATION DISTRIBUTION in mobile phase, can form corresponding diastereomer coordination compound pair while running into the medicine raceme, then complete fractionation on common forward post or reversed-phase column.
For more convenient, detect the dextroisomer in pazufloxacin mesylate injection exactly, the inventor provides a kind of outer marking quantitative detection method based on chirality aglucon exchange Mobile Phase Additives, easy to realize, quick, as accurately to control pazufloxacin mesylate injection product quality purpose.
Summary of the invention
Technical matters solved by the invention is to provide a kind of easy and simple to handle, detection method fast and accurately, for detection of the dextroisomer in pazufloxacin mesylate injection.
Pazufloxacin Mesilate dextroisomer of the present invention, its chemical name is that (+)-(3R)-10-(1-amino cyclopropyl)-9-is fluoro-2,3-dihydro-3-methyl-7-oxygen-7H-pyrido [1,2,3-de]-[Isosorbide-5-Nitrae] benzoxazine-6-carboxylic acid mesylate, molecular formula is C
16h
15fN
2o
4cH
4o
3s.
The detection method of dextroisomer of the present invention adopts liquid chromatographic detection, the detection method based on chirality aglucon exchange mobile phase additive method, and the HPLC testing conditions is as follows:
Fixing phase: take octadecylsilane chemically bonded silica as filling agent;
Mobile phase A: L-Phe-copper-bath; Mobile phase B: methyl alcohol;
Wherein, L-Phe-copper-bath contains L-Phe 0.3mg/ml to 3mg/ml, preferably 1.32mg/ml; Sulfur acid copper 0.2mg/ml to 2mg/ml, preferably 1mg/ml; Regulating pH with the 1mol/L sodium hydroxide solution is 2.0 to 5.0, preferably pH3.5;
With mobile phase A, Mobile phase B is 40～90:60～10 preparation mobile phases by volume, and preferred proportion is 75:25;
Flow velocity: 0.6～1.0ml/min;
Detect wavelength: 310nm to 350nm, preferably 330nm;
Number of theoretical plate calculates and is not less than 2500 by Pazufloxacin laevoisomer peak, and the left and right degree of separation of revolving between the body isomeride of Pazufloxacin should meet the requirements.
The processing of experiment material:
(1) preparation of need testing solution: get pazufloxacin mesylate injection 1ml, add 25～100 times of volumes (preferably 50 times) that the sample solvent is settled to pazufloxacin mesylate injection, shake up, as need testing solution.
(2) preparation of reference substance solution: it is appropriate that precision takes the Pazufloxacin Mesilate reference substance, dissolves and dilute with sample solvent and make every 1ml approximately containing the preferred 3 μ g of Pazufloxacin 1～6 μ g() solution, shake up, in contrast product solution.
(3) preparation of prerun solution: get the Pazufloxacin Mesilate raceme appropriate, add sample dissolution with solvents dilution and make every 1ml approximately containing the preferred 6 μ g of 2～12 μ g() solution of Pazufloxacin, shake up, as prerun solution.
Before detection, according to the HPLC testing conditions, precision measures prerun solution 20 μ l injection liquid chromatographies, and peak sequence is followed successively by laevoisomer and the dextroisomer of Pazufloxacin.Get again reference substance solution 20 μ l injection liquid chromatographies, regulate detection sensitivity, make the peak height at major component peak be about 20% of full scale.
The advantage of this detection method is: easy and simple to handle; Adopt reference substance external standard method quantitative measurement result accurately and reliably; The main peak retention time is between 8～20 minutes, and detection time is shorter.
The accompanying drawing explanation
Fig. 1 measures linear graph
Embodiment
Below by specific description of embodiments of the present invention the explanation but do not limit the present invention.
Prior art is:
1, Pazufloxacin Mesilate quality standard YBH04972007, the assay method condition determination of Pazufloxacin Mesilate dextroisomer provides as follows:
Get Pazufloxacin Mesilate raceme reference substance appropriate, adding mobile phase dissolves and dilutes and make every 1ml approximately containing the solution of 20 μ g, get 10 μ l injection liquid chromatographies, record chromatogram, its retention time is followed successively by laevoisomer and dextroisomer, and number of theoretical plate should be not less than 2000 by laevoisomer; The degree of separation of levo form and d-isomer should meet the requirements.
Get the about 30mg of this product, put in the 100ml measuring bottle, add mobile phase and dissolve and be diluted to scale, shake up, as need testing solution; Precision measures need testing solution 0.5ml, puts in the 100ml measuring bottle, adds mobile phase and is diluted to scale, and solution, get contrast solution 10 μ l injection liquid chromatographies in contrast, regulates detection sensitivity, and the peak height that makes the major component chromatographic peak is full scale 10%～20%; Precision measures contrast solution and each 10 μ l of need testing solution again, and the injection liquid chromatography, record chromatogram respectively.In the chromatogram of need testing solution, as aobvious dextroisomer impurity peaks, its peak area must not be greater than contrast solution main peak area (0.5%).
The chromatographic condition wherein adopted is is filling agent with octadecylsilane chemically bonded silica; (0.12%L-phenylalanine-0.08% copper sulfate solution)-methyl alcohol (4:1) is mobile phase; Flow velocity 1.0ml/min; Detect wavelength 321nm.
The method mobile phase is prepared the weighing solute in number percent, not science; The mobile phase pH value of chirality aglucon exchange mobile phase additive method is very large on the impact separated, and the method not flow phase pH is carried out clearly.Above factor makes the pazufloxacin mesylate injection quality controllability be affected.
It is below pazufloxacin mesylate injection method for detecting impurities testing conditions screening experiment of the present invention.
1, determine sample solvent
Pazufloxacin Mesilate is easily molten under sour environment, under neutral and slight alkali environment, can separate out precipitation.In Pazufloxacin Mesilate quality standard YBH04972007, under the dissolubility item, the regulation Pazufloxacin Mesilate is easily molten in the 0.1mol/L hydrochloric acid solution.Select mobile phase, 0.1mol/L hydrochloric acid solution and the 0.01mol/L hydrochloric acid solution of YBH04972007 regulation as sample solvent, the dextroisomer testing conditions that adopts Pazufloxacin Mesilate quality standard YBH04972007 to provide carries out contrast test to same lot number Pazufloxacin Mesilate raceme with a collection of pazufloxacin mesylate injection sample, the results are shown in Table 1:
Table 1 sample solvent contrast test
Three kinds of sample solvents are dissolution sample preferably all, the percentage no significant difference of testing result dextroisomer in the left-right rotary isomeride.For saving mobile phase (consuming the solute amount in mobile phase larger), avoid injury and the larger solvent peak of generation to chromatographic column simultaneously, determine and adopt the sample solvent of 0.01mol/L hydrochloric acid solution as the inventive method.
2, determine and detect wavelength
It is appropriate that precision takes the Pazufloxacin Mesilate raceme, take the 0.01mol/L hydrochloric acid solution as dissolution with solvents and be diluted to the solution of 9 μ g/ml, in 200～400nm wavelength coverage, scanned, and maximum absorption wavelength is 247.0nm and 331.0nm.For the interference of avoiding low wavelength place's phenylalanine and other impurity to detect isomeride as far as possible, selecting to detect wavelength is 330nm.
3, determine mobile phase
Chromatographic condition
Fixing phase: take octadecylsilane chemically bonded silica as filling agent;
Flow velocity: 0.8ml/min;
Column temperature: room temperature (25 ℃);
Detect wavelength: 330nm;
Number of theoretical plate calculates and should be not less than 2500 by Pazufloxacin laevoisomer peak, and the left and right degree of separation of revolving between the body isomeride of Pazufloxacin should meet the requirements.
The preparation of need testing solution: precision takes Pazufloxacin raceme 7.26mg, puts in the 50ml measuring bottle, with the 0.01mol/L dissolve with hydrochloric acid solution and be diluted to scale, shakes up, and as need testing solution, gets 20 μ l injection liquid chromatographies.
The mobile phase the selection result is in Table 2.
Table 2 mobile phase filtered list
The screening that 1～No. 4 mobile phase condition is flow phase ratio, left-right rotary isomeride peak all can reach fully and separate, and considers analysis time and separating effect, determines the ratio of alternative condition 4; 4～No. 5 mobile phase conditions are the screening (chiral ligand and coordination of metal ion ratio are by usually being set to 2:1) to the chirality ligand concentration, the degree of separation of low concentration 4mmol/L is lower than the degree of separation of high concentration 8mmol/L, two concentration retention times are more or less the same, therefore select the concentration of high concentration 8mmol/L as the chiral ligand L-Phe; 4,6～No. 7 mobile phase conditions are for screening the pH of mobile phase, and degree of separation can be issued to maximum in specific pH value, and pH raises or reduces and all can reduce degree of separation, and pH3.5 is best separation pH.To sum up, select No. 4 mobile phase conditions as the mobile phase condition.For convenience of operation, its description is changed into to (get L-Phe 1.32g, copper sulphate 1g, after adding water 1000ml dissolving, regulate pH value to 3.5 with the sodium hydroxide solution of 1mol/L)-methyl alcohol (75:25).
Below for after the chromatographic condition of detection method of the present invention determines, verify that it measures the experiment of effect.
1, specificity research
Do not destroy test solution: precision measures pazufloxacin mesylate injection 1ml, puts in the 50ml measuring bottle, with the 0.01mol/L hydrochloric acid solution, is diluted to scale, obtains.
Alkali destroys test solution: precision measures pazufloxacin mesylate injection 1ml, puts in the 50ml measuring bottle, adds 1mol/L sodium hydroxide solution 2ml, under 60 ℃, place 5 hours, add again 1mol/L hydrochloric acid solution 2ml neutralization, with the 0.01mol/L hydrochloric acid solution, be diluted to scale, obtain.
Acid destroys test solution: precision measures pazufloxacin mesylate injection 1ml, puts in the 50ml measuring bottle, adds 1mol/L hydrochloric acid solution 2ml, under 60 ℃, place 5 hours, add again 1mol/L sodium hydroxide solution 2ml neutralization, with the 0.01mol/L hydrochloric acid solution, be diluted to scale, obtain.
The Oxidative demage test solution: precision measures pazufloxacin mesylate injection 1ml, puts in the 50ml measuring bottle, adds 30% hydrogen peroxide 2ml, places 5 hours under 60 ℃, with the 0.01mol/L hydrochloric acid solution, is diluted to scale, obtains.
High temperature destroys test solution: precision measures pazufloxacin mesylate injection 4ml, places 5 hours under 100 ℃, moves in the 50ml measuring bottle, with the 0.01mol/L hydrochloric acid solution, is diluted to scale, obtains.
Illumination destroys test solution: get pazufloxacin mesylate injection, under uviol lamp, illumination is 48 hours, gets 1ml and puts in the 50ml measuring bottle, with the 0.01mol/L hydrochloric acid solution, is diluted to scale, obtains.
Above each solution, respectively get 20 μ l injection liquid chromatographies, records chromatogram.Test findings shows, each failure condition does not all produce isomeride is detected to noisy impurity peaks, and this method specificity is good.By dextroisomer peak area and left and right isomeride peak area sum relatively (normalization method), each failure condition with destroy and compare, the not significant change of dextroisomer content.Visible, this product configuration under each failure condition is relatively stable, and under illumination condition, isomeride increases to some extent, and obvious change of configuration does not occur other condition.The results are shown in Table 3.
Dextroisomer table as a result under each failure test condition of table 3
| Failure condition | Do not destroy | Alkali destroys | Acid destroys | Oxidation is broken | High temperature is broken | Illumination is broken |
| Dextroisomer | 0.01 | 0.02 | 0.02 | 0.01 | 0.01 | 0.03 |
2, linear test
Precision takes the Pazufloxacin Mesilate reference substance, and (this reference substance is laevoisomer, containing Pazufloxacin 75.3%, lower same, the left-right rotary isomeride has identical uv absorption) 11.12mg, put in the 25ml measuring bottle, with the 0.01mol/L dissolve with hydrochloric acid solution and be diluted to scale, shake up, as stock solution.Precision measures stock solution 1ml, puts in the 50ml measuring bottle, with the 0.01mol/L hydrochloric acid solution, is diluted to scale, shakes up, as linear need testing solution 1..1. linear need testing solution is diluted respectively to 2,4,8,80 times with the 0.01mol/L hydrochloric acid solution, as linear need testing solution 2., 3., 4., 5..Respectively get 20 μ l injection liquid chromatographies with the Linear need testing solution, record chromatogram.
The Pazufloxacin concentration (μ g/ml) of take is horizontal ordinate, and peak area is ordinate, carries out linear regression, and obtaining regression equation is y=6 * 10
7x+2327.6, correlation coefficient r=0.9998.Test findings shows, Pazufloxacin concentration is in 0.084 μ g/ml～6.699 μ g/ml scopes, and the concentration of its peak area and mensuration is good linear relationship.
The results are shown in Table 4, linear graph is shown in Fig. 1.
Table 4 linear test is table as a result
| Numbering | 1 | 2 | 3 | 4 | 5 |
| Pazufloxacin concentration (μ g/ml) | 6.699 | 3.349 | 1.675 | 0.837 | 0.084 |
| Peak area | 411202 | 203251 | 102369 | 58210 | 5907 |
[0077]3, sample introduction precision test
Line taking test item lower linear need testing solution 2., 5., 20 μ l inject the HPLC instrument respectively, each concentration continuous sample introduction 6 pin, record chromatogram, investigate sample introduction precision, test findings shows, the method in the range of linearity under variable concentrations the sample introduction precision of (contrast concentration and low concentration) good.The results are shown in Table 5.
Table 5 sample introduction Precision test result table (peak area)
| Test sample | 1 | 2 | 3 | 4 | 5 | 6 | RSD(%) |
| Linear need testing solution 2. | 202956 | 201585 | 205048 | 199145 | 202121 | 201937 | 0.95 |
| Linear need testing solution 5. | 5549 | 5465 | 5314 | 5360 | 5332 | 5286 | 1.89 |
4, detectability and quantitative limit
Precision measures 5. 1ml of linear need testing solution, puts in the 5ml measuring bottle, with the 0.01mol/L hydrochloric acid solution, is diluted to scale, shakes up, and gets 20 μ l injection liquid chromatographies, obtains signal to noise ratio (S/N ratio) and is about 10, quantitatively is limited to 0.34ng(in Pazufloxacin).
Get above-mentioned dilute solution 5 μ l injection liquid chromatographies, obtain signal to noise ratio (S/N ratio) and be about 3, detect and be limited to 0.08ng(in Pazufloxacin).
5, replica test
Precision takes Pazufloxacin Mesilate reference substance 10.87mg, puts in the 25ml measuring bottle, with the 0.01mol/L dissolve with hydrochloric acid solution and be diluted to scale, shake up, precision measures 1ml, puts in the 100ml measuring bottle, be diluted to scale with the 0.01mol/L hydrochloric acid solution, shake up, in contrast product solution.Precision measures pazufloxacin mesylate injection 1ml, is placed in the 50ml measuring bottle, with the 0.01mol/L hydrochloric acid solution, is diluted to scale, shake up, and as need testing solution, 6 parts of same batch of parallel preparations.Reference substance solution and need testing solution are respectively got 20 μ l injection liquid chromatographies, record chromatogram, the content by external standard method with dextroisomer in the calculated by peak area need testing solution.Test findings shows, it is good that the method detects the dextroisomer repeatability of pazufloxacin mesylate injection.The results are shown in Table 6.
Table 6 replica test is table as a result
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | RSD（%） |
| The dextroisomer peak area | 7270 | 7427 | 7600 | 7207 | 7327 | 7633 | 2.37 |
| Dextroisomer (%) | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | / |
6, sample solution stability test
Precision takes Pazufloxacin Mesilate reference substance 10.89mg, puts in the 25ml measuring bottle, with the 0.01mol/L dissolve with hydrochloric acid solution and be diluted to scale, shake up, precision measures 1ml, puts in the 100ml measuring bottle, be diluted to scale with the 0.01mol/L hydrochloric acid solution, shake up, as need testing solution 1..Precision measures pazufloxacin mesylate injection 1ml, is placed in the 50ml measuring bottle, with the 0.01mol/L hydrochloric acid solution, is diluted to scale, shakes up, as need testing solution 2..Get need testing solution 1. and 2. each 20 μ l injection liquid chromatographies respectively at 0h, 2h, 4h, 6h, 8h, record chromatogram, investigate sample solution stability in 8 hours.Test findings shows, need testing solution 1. (contrast laevoisomer) and need testing solution 2. (dextroisomer in sample) peak area in 8 hours, have no significant change, the left-right rotary isomeride transforms mutually, more stable.The results are shown in Table 7.
The stability test of table 7 sample solution is table as a result
7, accuracy test
Raceme is pressed in sample to amount containing dextroisomer 0.5% as 100%, is mixed with respectively respective concentration and adds in sample solution, make to be respectively 0.4%(80% containing dextroisomer in sample solution), 0.5%(100%), 0.6%(120%).
Precision takes Pazufloxacin Mesilate reference substance 11.73mg, puts in the 25ml measuring bottle, with the 0.01mol/L dissolve with hydrochloric acid solution and be diluted to scale, shake up, precision measures 1ml, puts in the 100ml measuring bottle, be diluted to scale with the 0.01mol/L hydrochloric acid solution, shake up, in contrast product solution.
Precision takes Pazufloxacin Mesilate raceme 9.28mg, puts in the 25ml measuring bottle, with the 0.01mol/L dissolve with hydrochloric acid solution and be diluted to scale, shakes up, as raceme solution.
Precision measures pazufloxacin mesylate injection 1ml and raceme solution 0.8ml, puts in the 50ml measuring bottle, with the 0.01mol/L hydrochloric acid solution, is diluted to scale, shakes up, as 80% recovery need testing solution.
Precision measures pazufloxacin mesylate injection 1ml and raceme solution 1.0ml, puts in the 50ml measuring bottle, with the 0.01mol/L hydrochloric acid solution, is diluted to scale, shakes up, as 100% recovery need testing solution.
Precision measures pazufloxacin mesylate injection 1ml and raceme solution 1.2ml, puts in the 50ml measuring bottle, with the 0.01mol/L hydrochloric acid solution, is diluted to scale, shakes up, as 120% recovery need testing solution.
Above reference substance solution and need testing solution are respectively got 20 μ l injection liquid chromatographies, record chromatogram, by external standard method with the calculated by peak area application of sample after the content of dextroisomer in need testing solution, the own content of dextroisomer in the deduction sample, try to achieve the dextroisomer amount that adds, and with theoretical addition relatively (raceme by left and right isomeride each 50%).Test findings shows, in sample, adds dextroisomer, surveys its recovery, and repeatability is good.The results are shown in Table 8.
Table 8 recovery test result
8, dextroisomer assay
It is appropriate that precision measures this product, and the hydrochloric acid solution that adds 0.01mol/L quantitatively dilutes and makes the solution that approximately contains Pazufloxacin 0.6mg in every 1ml, as need testing solution; It is appropriate that precision takes the Pazufloxacin Mesilate reference substance, add the 0.01mol/L dissolve with hydrochloric acid solution and quantitatively dilution make every 1ml approximately containing the solution of Pazufloxacin 3 μ g, product solution in contrast.According to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2010), measure.Determine chromatographic condition according to the present invention, get prerun solution and (get the Pazufloxacin Mesilate raceme appropriate, add 0.01mol/L dissolve with hydrochloric acid solution dilution and make every 1ml approximately containing the solution of 6 μ g Pazufloxacins) 20 μ l injection liquid chromatographies, peak sequence is followed successively by laevoisomer and the dextroisomer of Pazufloxacin, number of theoretical plate calculates and should be not less than 2500 by Pazufloxacin laevoisomer peak, and the left and right degree of separation of revolving between the body isomeride of Pazufloxacin should meet the requirements.Get reference substance solution 20 μ l injection liquid chromatographies, regulate detection sensitivity, make the peak height at major component peak be about 20% of full scale, precision measures need testing solution and each 20 μ l of reference substance solution again, difference injection liquid chromatography, record chromatogram, in the need testing solution chromatogram, if any the chromatographic peak consistent with the dextroisomer retention time, calculate respectively the content of dextroisomer by external standard method, normalization method, major component Self-control method.The results are shown in Table 9.
Table 9 dextroisomer measurement result
Result shows: adopt external standard method to calculate dextroisomer and normalization method and Self-control method relatively, result is consistent.Visible, for the optical isomer with identical uv absorption, three kinds of methods are calculated no significant difference to result, but consider that external standard method calculating has more accuracy, therefore adopt the external standard method dextroisomer.
Pazufloxacin mesylate injection dextroisomer test case
Determination method: it is appropriate that precision measures pazufloxacin mesylate injection, and the hydrochloric acid solution that adds 0.01mol/L quantitatively dilutes and makes the solution that approximately contains Pazufloxacin 0.6mg in every 1ml, as need testing solution; It is appropriate that precision takes the Pazufloxacin Mesilate reference substance, add the 0.01mol/L dissolve with hydrochloric acid solution and quantitatively dilution make every 1ml approximately containing the solution of Pazufloxacin 3 μ g, product solution in contrast.According to high performance liquid chromatography (two appendix V D of Chinese Pharmacopoeia version in 2010), measure.With octadecylsilane chemically bonded silica, it is filling agent; (get L-Phe 1.32g with copper sulphate L-Phe solution, copper sulphate 1g, after adding water 1000ml dissolving, regulate pH value to 3.5 with the sodium hydroxide solution of 1mol/L)-methyl alcohol (75:25) is mobile phase, flow velocity 0.8ml/min(can suitably adjust), the detection wavelength is 330nm.Get prerun solution and (get the Pazufloxacin Mesilate raceme appropriate, add 0.01mol/L dissolve with hydrochloric acid solution dilution and make every 1ml approximately containing the solution of 6 μ g Pazufloxacins) 20 μ l injection liquid chromatographies, peak sequence is followed successively by laevoisomer and the dextroisomer of Pazufloxacin, number of theoretical plate calculates and should be not less than 2500 by Pazufloxacin laevoisomer peak, and the left and right degree of separation of revolving between isomeride of Pazufloxacin should meet the requirements.Get reference substance solution 20 μ l injection liquid chromatographies, regulate detection sensitivity, make the peak height at major component peak be about 20% of full scale, precision measures need testing solution and each 20 μ l of reference substance solution again, respectively the injection liquid chromatography, record chromatogram, in the need testing solution chromatogram if any the chromatographic peak consistent with the dextroisomer retention time, with calculated by peak area, containing dextroisomer, should cross 0.3% of Pazufloxacin labelled amount by external standard method.
The pazufloxacin mesylate injections of being produced by " the abundant scientific and technological pharmaceutical Co. Ltd in Chengdu hundred " according to 3 batches of above detections and the commercially available product of another two producers (trade name is respectively: fado beautiful jade, the rising sun are former), measurement result is in Table 10.
Table 10 dextroisomer testing result table
To sum up, detection method provided by the invention, can be easy, quickly and accurately the pazufloxacin mesylate injection dextroisomer is quantitatively detected.
Claims (8)
1. the detection method of pazufloxacin mesylate injection dextroisomer is characterized in that: adopt the liquid chromatographic detection based on chirality aglucon exchange mobile phase additive method, the HPLC testing conditions is as follows:
Fixing phase: take octadecylsilane chemically bonded silica as filling agent;
Mobile phase A: L-Phe-copper-bath; Mobile phase B: methyl alcohol;
Wherein, L-Phe-copper-bath is containing L-Phe 0.3mg/ml to 3mg/ml, sulfur acid copper 0.2mg/ml to 2mg/ml; Regulating the pH value with the 1mol/L sodium hydroxide solution is 2.0 to 5.0;
With mobile phase A, Mobile phase B is 40～90:60～10 preparation mobile phases by volume;
Flow velocity: 0.6～1.0ml/min;
Detect wavelength: 310nm to 350nm;
Number of theoretical plate calculates and is not less than 2500 by Pazufloxacin laevoisomer peak, and the left and right degree of separation of revolving between the body isomeride of Pazufloxacin should meet the requirements.
2. the detection method of pazufloxacin mesylate injection dextroisomer according to claim 1 is characterized in that: L-Phe-copper-bath is containing L-Phe 1.32mg/ml, sulfur acid copper 1mg/ml; Regulate pH value 3.5 with the 1mol/L sodium hydroxide solution.
3. the detection method of pazufloxacin mesylate injection dextroisomer according to claim 1 is characterized in that: with mobile phase A, and Mobile phase B 75:25 preparation by volume mobile phase.
4. the detection method of pazufloxacin mesylate injection dextroisomer according to claim 1, it is characterized in that: the detection wavelength is 330nm.
5. according to the detection method of the described pazufloxacin mesylate injection dextroisomer of claim 1-4 any one, it is characterized in that: adopt and take the external standard method that the Pazufloxacin Mesilate reference substance is contrast and quantitatively detect, implementation step is:
(1) preparation of need testing solution: get pazufloxacin mesylate injection 1ml, add 25～100 times of volumes that the sample solvent is settled to pazufloxacin mesylate injection, shake up, as need testing solution;
(2) preparation of reference substance solution: it is appropriate that precision takes the Pazufloxacin Mesilate reference substance, dissolves and dilute with sample solvent and make 1ml approximately containing the solution of Pazufloxacin 1～6 μ g, shakes up, in contrast product solution;
(3) preparation of prerun solution: get the Pazufloxacin Mesilate raceme appropriate, add sample dissolution with solvents dilution and make every 1ml approximately containing the solution of 2～12 μ g Pazufloxacins, shake up, as prerun solution;
Before detection, according to the HPLC testing conditions, precision measures prerun solution 20 μ l injection liquid chromatographies, carries out location, enantiomter peak; Get again reference substance solution 20 μ l injection liquid chromatographies, regulate detection sensitivity, make the peak height at major component peak be about 20% of full scale; Precision measures need testing solution and each 20 μ l of reference substance solution again, and the injection liquid chromatography, record chromatogram respectively, in the need testing solution chromatogram, if any the chromatographic peak consistent with the dextroisomer retention time, presses external standard method with calculated by peak area.
6. the detection method of pazufloxacin mesylate injection dextroisomer according to claim 5 is characterized in that:
(1) preparation of need testing solution: get pazufloxacin mesylate injection 1ml, add 50 times of volumes that the sample solvent is settled to pazufloxacin mesylate injection, shake up, as need testing solution;
(2) preparation of reference substance solution: it is appropriate that precision takes the Pazufloxacin Mesilate reference substance, dissolves and dilute with sample solvent and make 1ml approximately containing the solution of Pazufloxacin 3 μ g, shakes up, in contrast product solution;
(3) preparation of prerun solution: get the Pazufloxacin Mesilate raceme appropriate, add sample dissolution with solvents dilution and make every 1ml approximately containing the solution of 6 μ g Pazufloxacins, shake up, as prerun solution.
7. the detection method of pazufloxacin mesylate injection dextroisomer according to claim 5, it is characterized in that: sample solvent comprises: concentration is 0.05mol/L-0.1mol/L hydrochloric acid solution or mobile phase.
8. the detection method of pazufloxacin mesylate injection dextroisomer according to claim 7, it is characterized in that: sample solvent comprises the 0.01mol/L hydrochloric acid solution.
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| CN108593795A (en) * | 2018-04-25 | 2018-09-28 | 邯郸学院 | A kind of UPLC methods of three kinds of astaxanthin optical isomers of separation and the application in field of food |
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| CN115290800A (en) * | 2022-10-08 | 2022-11-04 | 江西省药品检验检测研究院 | Method for splitting antofloxacin enantiomer by chiral stationary phase method |
| CN115290800B (en) * | 2022-10-08 | 2023-01-13 | 江西省药品检验检测研究院 | Method for splitting antofloxacin enantiomer by chiral stationary phase method |
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