CN117665154A - Method for detecting voriconazole dextroisomer by adopting reversed phase chromatography - Google Patents
Method for detecting voriconazole dextroisomer by adopting reversed phase chromatography Download PDFInfo
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- CN117665154A CN117665154A CN202311469502.4A CN202311469502A CN117665154A CN 117665154 A CN117665154 A CN 117665154A CN 202311469502 A CN202311469502 A CN 202311469502A CN 117665154 A CN117665154 A CN 117665154A
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- BCEHBSKCWLPMDN-MGPLVRAMSA-N voriconazole Chemical compound C1([C@H](C)[C@](O)(CN2N=CN=C2)C=2C(=CC(F)=CC=2)F)=NC=NC=C1F BCEHBSKCWLPMDN-MGPLVRAMSA-N 0.000 title claims abstract description 94
- 229960004740 voriconazole Drugs 0.000 title claims abstract description 94
- 238000000034 method Methods 0.000 title claims abstract description 46
- 238000004366 reverse phase liquid chromatography Methods 0.000 title claims abstract description 9
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical group O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims abstract description 31
- 238000002360 preparation method Methods 0.000 claims abstract description 22
- 239000003814 drug Substances 0.000 claims abstract description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000000741 silica gel Substances 0.000 claims abstract description 7
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 7
- 239000000945 filler Substances 0.000 claims abstract description 6
- DPUFSSYRLDUXPL-UHFFFAOYSA-N (3-chloro-5-methylphenyl) carbamate Chemical compound CC1=CC(Cl)=CC(OC(N)=O)=C1 DPUFSSYRLDUXPL-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000007983 Tris buffer Substances 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 87
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 55
- 238000002347 injection Methods 0.000 claims description 32
- 239000007924 injection Substances 0.000 claims description 32
- 239000000523 sample Substances 0.000 claims description 28
- 238000001514 detection method Methods 0.000 claims description 21
- 239000012488 sample solution Substances 0.000 claims description 21
- 238000007865 diluting Methods 0.000 claims description 15
- 238000012360 testing method Methods 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000013558 reference substance Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 6
- 239000012085 test solution Substances 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 2
- 238000010829 isocratic elution Methods 0.000 claims description 2
- 229940079593 drug Drugs 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 239000011550 stock solution Substances 0.000 description 20
- 230000006378 damage Effects 0.000 description 9
- 239000007864 aqueous solution Substances 0.000 description 8
- 229920000858 Cyclodextrin Polymers 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 239000013074 reference sample Substances 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000011835 investigation Methods 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000001116 FEMA 4028 Substances 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 235000011175 beta-cyclodextrine Nutrition 0.000 description 4
- 229960004853 betadex Drugs 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
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- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- -1 sulfobutyl Chemical group 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 238000001782 photodegradation Methods 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- RFHAOTPXVQNOHP-UHFFFAOYSA-N fluconazole Chemical compound C1=NC=NN1CC(C=1C(=CC(F)=CC=1)F)(O)CN1C=NC=N1 RFHAOTPXVQNOHP-UHFFFAOYSA-N 0.000 description 1
- 229960004884 fluconazole Drugs 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 150000003852 triazoles Chemical class 0.000 description 1
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- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention provides a method for detecting voriconazole dextroisomer by reversed phase chromatography, which adopts a chromatographic column with amylose-tris (3-chloro-5-methylphenyl carbamate) covalently bonded on the surface of silica gel as a filler, and the mobile phase is acetonitrile-water. The method has the advantages of good durability, strong specificity, high sensitivity, good accuracy and good precision, and is suitable for detecting dextroisomer in voriconazole bulk drugs and preparations.
Description
Technical Field
The invention relates to the field of medicine analysis, in particular to a method for detecting voriconazole dextroisomer by using a reversed phase chromatography.
Background
Voriconazole is a novel high-lipophilicity triazole antifungal drug synthesized on the basis of the structure of fluconazole, and has the characteristics of wide antibacterial spectrum and strong antibacterial effect. Voriconazole is poorly water-soluble, with a solubility in water of only 0.1 mg/ml. The sulfobutyl beta cyclodextrin sodium is a pharmaceutical preparation auxiliary material, belongs to anionic beta cyclodextrin sulfonic acid group derivatives with high water solubility, and has high water solubility. It has special affinity to nitrogen-containing medicine and can form non-covalent compound by inclusion of medicine molecule. The voriconazole freeze-dried preparation for injection is prepared by clathrating the voriconazole with the sodium sulfobutylbetacyclodextrin, has obvious solubilization effect on the voriconazole, and has good water solubility of the finished product of the preparation.
The voriconazole contains two chiral centers, and the dextroisomer shown in the formula 2 is a process byproduct of the voriconazole raw material and degradation impurities of the voriconazole, so that the dextroisomer needs to be controlled in the finished product of the preparation. Referring to the literature and the data, the method for determining the dextroisomer of voriconazole in the 2020 edition of Chinese pharmacopoeia is normal phase chromatography, the solubility property of voriconazole after being included by cyclodextrin is consistent with that of cyclodextrin, and normal phase solvent is difficult to completely dissolve the voriconazole and the dextroisomer in the finished product of the preparation; the method for determining the dextroisomer of the voriconazole in the literature report and other pharmacopoeias of various countries is a reversed phase chromatography and a cyclodextrin chiral chromatographic column, and can be used for determining the voriconazole for injection. According to the separation degree requirement of the pharmacopoeia method, the separation degree of the dextroisomer and the main peak needs to be more than 4.0, but in the using process of the method, the separation degree of the dextroisomer and the main peak is found to be difficult to meet the requirement, and the column efficiency of the chromatographic column is obviously reduced along with the increase of the using times, and the chromatographic column is not durable. For the above reasons, it is of great importance to develop a more robust reverse phase chromatography method for the detection of the dextroisomer in voriconazole.
Disclosure of Invention
Aiming at the situation that the method for measuring the dextroisomer in voriconazole by the existing method is poor in durability, the method for detecting the dextroisomer in voriconazole is good in durability, high in specificity, high in sensitivity, good in accuracy and good in precision, and is suitable for detecting the dextroisomer in voriconazole bulk drugs and preparations.
In order to achieve the above object, the present invention provides the following technical solutions.
The invention provides a method for detecting voriconazole dextroisomer by adopting reverse phase chromatography, which adopts a chromatographic column with amylose-tris (3-chloro-5-methylphenyl carbamate) covalently bonded on the surface of silica gel as a filler, and the mobile phase is acetonitrile-water.
The chromatographic column according to the method is a CHIRALPAK IG column, more preferably a CHIRALPAK IG (Daicel, 4.6 mm. Times.250 mm,5 μm) column.
The volume ratio of acetonitrile to water in the mobile phase of the method is 48: 50-52: 48, preferably acetonitrile-water volume ratio of 50:50.
further, in the method of the present invention, the flow rate is 0.8 to 1.2ml/min, preferably 1.0ml/min.
Further, in the method of the present invention, the detection wavelength is 210 to 300nm, preferably 256nm.
Further, in the method of the invention, the column temperature is 25-35 ℃, preferably 30 ℃;
further, according to the method disclosed by the invention, the sample injection volume is 10-30 mu l, and preferably 20 mu l;
further, in the method of the present invention, the detector is an ultraviolet detector, and the elution mode is isocratic elution.
Further, the method of the present invention described above comprises the steps of:
(1) Preparation of voriconazole dextroisomer control solution: taking a right amount of voriconazole dextroisomer reference substance, adding a right amount of acetonitrile for dissolution, and preparing a reference substance solution by using a 40% acetonitrile water solution;
(2) Preparation of test solution: taking a proper amount of voriconazole for injection, re-dissolving the voriconazole with water, and diluting the voriconazole with 40% acetonitrile water solution to be used as a sample solution;
or, taking a proper amount of voriconazole raw material medicine, adding a proper amount of acetonitrile for ultrasonic dissolution, diluting with 40% acetonitrile aqueous solution, and preparing a test sample solution;
or, taking a proper amount of other voriconazole preparations except voriconazole for injection, and preparing a sample solution;
(3) Taking a voriconazole dextroisomer reference substance solution and a sample solution, injecting into a high performance liquid chromatograph, and performing high performance liquid chromatography analysis according to the chromatographic conditions.
The chromatographic column selected by the invention is CHIRALPAK IG column, the filler is amylose-tris (3-chloro-5-methylphenyl carbamate) covalently bonded on the surface of silica gel, and belongs to polysaccharide bonding chiral column, which is not only suitable for normal phase chromatographic systems, but also can be used under reversed phase chromatographic systems, the chiral compound has distinguishing force, the stability of the chromatographic column is high, the column temperature tolerance range is 0-40 ℃, the pH tolerance range is 2.0-9.0, the upper limit of the column pressure is 30MPa, and the service life is long.
The invention designs a reversed phase liquid chromatography analysis method which takes a chromatographic column with amylose-tris (3-chloro-5-methylphenyl carbamate) covalently bonded on the surface of silica gel as a filler as an analysis column, takes acetonitrile aqueous solution as a mobile phase, realizes the separation of voriconazole and dextroisomer thereof, and is suitable for the analysis of dextroisomer in voriconazole bulk drugs and preparations. The method has the obvious advantages of improving the durability of the method, having high sensitivity, the detection limit reaching 0.25 mug/ml, the quantitative limit reaching 0.15 mug/ml, good specificity, no interference on the measurement of the dextroisomer under each damage condition, simple preparation of a mobile phase system and a sample solution, simple operation, low detection cost, high accuracy of detection results and good repeatability.
Drawings
Fig. 1 shows a mobile phase acetonitrile-water volume ratio of 50 in example 1: adding a standard sample solution chromatogram at the time of 50;
fig. 2 shows a mobile phase acetonitrile-water volume ratio of 52 in example 1: adding a standard sample solution chromatogram at 48 hours;
fig. 3 shows a mobile phase acetonitrile-water volume ratio of 48 in example 1: adding a standard sample solution chromatogram at 52;
FIG. 4 is a chromatogram of a hollow white adjuvant solution of example 2;
FIG. 5 is a chromatogram of the voriconazole acid breaker solution for injection in example 2;
FIG. 6 is a chromatogram of the voriconazole base disruption solution for injection in example 2;
FIG. 7 is a chromatogram of the voriconazole oxidative destruction solution for injection in example 2;
FIG. 8 is a chromatogram of the voriconazole heat-damaging solution for injection in example 2;
FIG. 9 is a chromatogram of the voriconazole photodegradation solution for injection in example 2;
FIG. 10 is a chromatogram of a solution of a test sample for durability inspection in example 5;
FIG. 11 is a chromatogram of a durability test control solution in example 5;
FIG. 12 is a chromatogram of the labeled test solution of comparative example 1;
FIG. 13 is a chromatogram of the control solution of comparative example 1.
Description of the embodiments
The following examples are provided to further illustrate and understand the spirit of the invention, but do not limit the scope of the invention in any way.
Example 1 flow phase investigation
Detector and chromatographic conditions
HPLC instrument: shimadzu, LC-20AD XR A VWD/PDA detector;
chromatographic column: CHIRALPAK IG column (Daicel, 4.6 mm. Times.250 mm,5 μm); column temperature is 30 ℃; the sample feeding disc does not control the temperature; acquisition time: 30min; the flow rate is 1.0ml/min; detection wavelength: 256nm; sample injection amount: 20 μl.
Mobile phase: acetonitrile-water (50:50), (52:48), (48:52)
(1) Sample preparation:
control stock solution: taking a right amount of voriconazole dextroisomer reference substance, precisely weighing, adding acetonitrile for dissolving, and quantitatively diluting with 40% acetonitrile water solution to prepare a solution containing about 10 mug/ml in each 1 ml.
Sample stock solution: the voriconazole sample for injection is taken, dissolved by adding water, and quantitatively diluted by 40% acetonitrile water solution to prepare a solution with the concentration of about 2mg/ml in each 1 ml.
Adding a labeled test sample solution: precisely measuring the stock solution of the test sample and the stock solution of the reference sample, and quantitatively diluting the stock solution with 40% acetonitrile aqueous solution to prepare a mixed solution containing about 0.5mg/ml of voriconazole and 2 mug/ml of voriconazole dextroisomer in each 1 ml.
(2) And (3) detection: taking 20 mu l of the labeled sample solution, injecting into a high performance liquid chromatograph, and recording a chromatogram. Typical chromatograms are shown in FIGS. 1-3 and the results are shown in Table 1.
Table 1 flow comparative investigation results
The results show that: the volume ratio of acetonitrile-water in the mobile phase was 48: 52-52: at 48, voriconazole is well separated from the dextroisomer, and is suitable for detecting the dextroisomer in voriconazole. Of these, acetonitrile-water (50:50) is preferred, and the chromatogram thereof is shown in FIG. 1.
EXAMPLE 2 specificity investigation
Detector and chromatographic conditions:
HPLC instrument: shimadzu, LC-20AT, LC-20AD, VWD detector;
chromatographic column: CHIRALPAK IG column (Daicel, 4.6 mm. Times.250 mm,5 μm); column temperature is 30 ℃; the sample feeding disc does not control the temperature; acquisition time: 30min; the flow rate is 1.0ml/min; detection wavelength: 256nm; sample injection amount: 20 μl; mobile phase: acetonitrile-water (50:50).
(1) Sample preparation:
blank auxiliary material solution: proper amount of sulfobutyl beta cyclodextrin sodium is taken, and 40% acetonitrile water solution is used for preparing blank auxiliary material solution according to the prescription amount.
Voriconazole acid destruction solution for injection: taking a voriconazole 1 bottle for injection, dissolving the voriconazole in 40% acetonitrile water solution, adding 1ml of 0.1M hydrochloric acid solution, standing for 1h at room temperature, adding 1ml of 0.1M sodium hydroxide solution for neutralization, and quantitatively diluting the voriconazole with 40% acetonitrile water solution to prepare a solution with about 0.5mg/ml in each 1 ml.
Voriconazole base destruction solution for injection: taking a voriconazole 1 bottle for injection, dissolving the voriconazole in 40% acetonitrile water solution, adding 1ml of 0.1M sodium hydroxide solution, standing for 10min at room temperature, adding 1ml of 0.1M hydrochloric acid solution for neutralization, and quantitatively diluting the voriconazole with 40% acetonitrile water solution to prepare a solution with about 0.5mg/ml in each 1 ml.
Voriconazole oxidative destruction solution for injection: taking a voriconazole 1 bottle for injection, dissolving the voriconazole in 40% acetonitrile water solution, adding 1ml of 3% hydrogen peroxide solution, standing for 1h at room temperature, and quantitatively diluting the voriconazole with 40% acetonitrile water solution to prepare a solution containing about 0.5mg/ml in each 1 ml.
Voriconazole heat-damaging solution for injection: taking a voriconazole 1 bottle for injection, placing in an oven at 80 ℃ for 6 hours, adding water for dissolution, and quantitatively diluting with 40% acetonitrile water solution to prepare a solution with the concentration of about 0.5mg/ml in each 1 ml.
Voriconazole photodegradation solution for injection: the voriconazole for injection was taken in 1 bottle, placed in an illumination box (25 ℃ C., 4500 Lux) for 24 hours, dissolved in water, and quantitatively diluted with 40% acetonitrile aqueous solution to prepare a solution containing about 0.5mg/ml per 1 ml.
(2) And (3) detection: taking 20 mu l of the blank auxiliary material solution and each destruction solution, injecting the blank auxiliary material solution and each destruction solution into a high performance liquid chromatograph, and recording a chromatogram. Typical chromatograms are shown in fig. 4-9 and the results are shown in table 2.
TABLE 2 results of specificity experiments
The voriconazole for injection is subjected to acid, alkali, oxidation, heat and illumination damage, the interference condition of each degradation impurity on the detection of the dextroisomer is inspected, and the specificity of the method is clarified by combining the interference condition of the hollow white auxiliary material in the preparation. The results show that the blank auxiliary materials do not interfere with the detection of the dextroisomer in the voriconazole for injection; under each destruction condition, voriconazole and each generated degradation impurity do not interfere with the detection of the dextroisomer.
Example 3 limit of detection and limit of quantification investigation
The detector and chromatographic conditions were the same as in example 2.
(1) Sample preparation:
control stock solution: taking a right amount of voriconazole dextroisomer reference substance, precisely weighing, adding acetonitrile for dissolving, and quantitatively diluting with 40% acetonitrile water solution to prepare a solution containing about 10 mug/ml in each 1 ml.
Quantitative limiting solution: the control stock solution was precisely measured and quantitatively diluted with 40% acetonitrile in water to prepare a solution containing about 0.25. Mu.g/ml of voriconazole dextroisomer per 1 ml.
Detection limit solution: precise measurement the quantitative limiting solution was quantitatively diluted with 40% acetonitrile in water to prepare a solution containing about 0.15 μg/ml of voriconazole dextroisomer per 1 ml.
(2) And (3) detection: taking quantitative limiting solution and continuously sampling for 6 times, detecting limiting solution and sampling for 1 time, and recording a chromatogram.
The results showed that in the 6-needle quantitative limiting solution, the dextroisomer peaks S/N were 48, 60, 69, 79, 86, 72, respectively, and the peak area RSD was 1.4%; in the detection limiting solution, the S/N of the dextroisomer peak is 40, and the detection limiting solution and the quantitative limiting solution have higher response. The quantitative limit of the voriconazole dextroisomer in the method is 0.25 mug/ml, and the quantitative limit is 0.15 mug/ml.
Example 4 precision and accuracy investigation
The detector and chromatographic conditions were the same as in example 2.
(1) Sample preparation:
control stock solution: taking a right amount of voriconazole dextroisomer reference substance, precisely weighing, adding acetonitrile for dissolving, and quantitatively diluting with 40% acetonitrile water solution to prepare a solution containing about 10 mug/ml in each 1 ml.
Control solution: the control stock solution was precisely measured and quantitatively diluted with 40% acetonitrile in water to prepare a solution containing about 2. Mu.g/ml of voriconazole dextroisomer per 1 ml.
Sample stock solution: the voriconazole sample for injection is taken, dissolved by adding water, and quantitatively diluted by 40% acetonitrile water solution to prepare a solution with the concentration of about 2mg/ml in each 1 ml.
Blank test solution: the stock solution of the test sample is precisely measured and quantitatively diluted with 40% acetonitrile aqueous solution to prepare a solution containing about 0.5mg/ml voriconazole in 1 ml.
0.2% of labeled test sample solution: precisely measuring the stock solution of the test sample and the stock solution of the reference sample, and quantitatively diluting the stock solution with 40% acetonitrile aqueous solution to prepare a mixed solution containing about 0.5mg/ml of voriconazole and 1 mug/ml of voriconazole dextroisomer in each 1 ml. 3 parts were prepared in parallel.
0.4% of labeled test sample solution: precisely measuring the stock solution of the test sample and the stock solution of the reference sample, and quantitatively diluting the stock solution with 40% acetonitrile aqueous solution to prepare a mixed solution containing about 0.5mg/ml of voriconazole and 2 mug/ml of voriconazole dextroisomer in each 1 ml. 3 parts were prepared in parallel.
0.6% of labeled test sample solution: precisely measuring the stock solution of the test sample and the stock solution of the reference sample, and quantitatively diluting the stock solution with 40% acetonitrile aqueous solution to prepare a mixed solution containing about 0.5mg/ml of voriconazole and 3 mug/ml of voriconazole dextroisomer in each 1 ml. 3 parts were prepared in parallel.
(2) And (3) detection: taking voriconazole reference substance solution, continuously sampling for 6 times, taking each sample solution, injecting into a liquid chromatograph, and recording the chromatogram.
(3) And (3) calculating: and calculating according to a reference external standard method, and counting the recovery rate and the recovery rate RSD of 9 parts of the solution of the standard sample.
TABLE 3 precision and accuracy results
The result shows that the recovery rate of 9 parts of the solution of the standard-added test sample is 10.3.6% -109.9%, the average recovery rate is 106.4%, the recovery rates are all in the range of 80.0% -120.0%, and the RSD (reactive power detector) of the recovery rate is 2.4%, so that the method is accurate and reliable, has good precision and is suitable for detecting the dextroisomer in the voriconazole bulk drug/preparation.
EXAMPLE 5 column durability investigation
The detector and chromatographic conditions were the same as in example 2.
(1) Sample preparation: the solutions used were 0.4% of the standard test solution and the control solution, and were the same as in example 4.
(2) And (3) detection: taking 0.4% of the standard sample solution and the reference sample solution, injecting into a liquid chromatograph, and recording the chromatogram. Typical chromatograms are shown in fig. 10 and 11 and the results are shown in table 4.
Table 4 chromatographic column durability test results
The result shows that the chromatographic system runs continuously for about 50 hours, the chromatograms of the repeated sample injection of the solution of the standard sample and the solution of the reference sample have no obvious difference, the separation degree of the dextroisomer and the voriconazole in the solution of the standard sample is stabilized at 4.0, the peak shape of the solution of the reference sample is symmetrical, and the number of theoretical plates is not less than 7000, thus showing that the chromatographic column is stable in performance and can run continuously for a long time. The method has good durability and is suitable for detecting the dextroisomer in the voriconazole bulk drug and the preparation.
Comparative example 1
Chromatographic conditions of the dextroisomer of voriconazole in the foreign pharmacopoeia (EP, USP, JP) are adopted, and a chromatographic column is adopted: chiraDex (LiChroCART) (250 mm. Times.4 mm,5 μm); mobile phase: 0.77g/L ammonium acetate buffer salt solution (pH 5.0) -acetonitrile (85:15), other chromatographic conditions were the same as in example 2. The solutions used were 0.4% of the standard sample solution and the control solution, and the HPLC patterns are shown in FIG. 10 and FIG. 11, and the sample injection results are shown in Table 5, as in example 4.
TABLE 5 precision and accuracy results
The chromatographic column is beta-cyclodextrin covalent bonding porous silica gel chromatographic column, and is suitable for the chromatographic system. As shown in fig. 10, in the using process of the method, the sample solution to be labeled is injected after the chromatographic system is operated for about 9 hours, the 3-needle chromatographic chart of continuous injection shows great difference, the main peak is deformed and has peak splitting, the dextroisomer and the main peak cannot be completely separated, and the measurement is seriously influenced. FIG. 11 is an overlay of the repeated sample injection chromatograms of the control solution at different time points, the main peak shape is widened along with the increase of the running time, the peak height is reduced, and the theoretical plate number is reduced from 5135 to 3561. The method has limited continuous operation time, and the same situation can still appear when the chromatographic columns with the same specification and the same model are replaced, so that the method has poor durability.
The method selects CHIRALPAK IG column with amylose-tri (3-chloro-5-methyl phenyl carbamate) covalently bonded on the surface of silica gel as a filler, and has better durability.
Claims (10)
1. A method for detecting voriconazole dextroisomer by reverse phase chromatography is characterized in that a chromatographic column with amylose-tris (3-chloro-5-methylphenyl carbamate) covalently bonded on the surface of silica gel as a filler is adopted, the mobile phase is acetonitrile-water, the voriconazole dextroisomer is a compound shown in formula 2,
。
2. the method of claim 1, wherein the chromatographic column is a CHIRALPAK IG column.
3. The process of claim 2, wherein the acetonitrile-water volume ratio in the mobile phase is 48: 50-52: 48, preferably acetonitrile-water volume ratio of 50:50.
4. a method according to claim 3, at a flow rate of 0.8-1.2 ml/min, preferably 1.0ml/min.
5. A method according to claim 3, wherein the detection wavelength is 210-300 nm, preferably 256nm.
6. A process according to claim 3, wherein the column temperature is 25-35 ℃, preferably 30 ℃.
7. A method according to claim 3, wherein the sample volume is 10 to 30 μl, preferably 20 μl.
8. A method according to claim 3, the detector being an ultraviolet detector.
9. A method according to claim 3, the elution mode being isocratic elution.
10. The method according to any one of claims 1-9, characterized in that the method comprises the steps of:
(1) Preparation of voriconazole dextroisomer control solution: taking a right amount of voriconazole dextroisomer reference substance, adding a right amount of acetonitrile for dissolution, and preparing a reference substance solution by using a 40% acetonitrile water solution;
(2) Preparation of test solution: taking a proper amount of voriconazole for injection, re-dissolving the voriconazole with water, and diluting the voriconazole with 40% acetonitrile water solution to be used as a sample solution;
or, taking a proper amount of voriconazole raw material medicine, dissolving and diluting with 40% acetonitrile water solution to prepare a sample solution;
or, taking a proper amount of other voriconazole preparations except voriconazole for injection, and preparing a sample solution;
(3) Taking a voriconazole dextroisomer reference substance solution and a test sample solution, and injecting into a high performance liquid chromatograph for detection.
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