CN108152418B - HPLC detection method for ketorolac tromethamine or/and impurities in preparation of ketorolac tromethamine - Google Patents
HPLC detection method for ketorolac tromethamine or/and impurities in preparation of ketorolac tromethamine Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 94
- 238000001514 detection method Methods 0.000 title claims abstract description 65
- BWHLPLXXIDYSNW-UHFFFAOYSA-N ketorolac tromethamine Chemical compound OCC(N)(CO)CO.OC(=O)C1CCN2C1=CC=C2C(=O)C1=CC=CC=C1 BWHLPLXXIDYSNW-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 229960004384 ketorolac tromethamine Drugs 0.000 title claims abstract description 40
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 30
- 239000000243 solution Substances 0.000 claims abstract description 21
- 239000012085 test solution Substances 0.000 claims abstract description 7
- 239000012071 phase Substances 0.000 claims description 40
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- 239000012074 organic phase Substances 0.000 claims description 13
- 238000010828 elution Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000008363 phosphate buffer Substances 0.000 claims description 7
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- 239000002904 solvent Substances 0.000 claims description 3
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- 239000000203 mixture Substances 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
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- UDPWNJVGOHGQFG-UHFFFAOYSA-N azane;phosphoric acid Chemical group [NH4+].OP(O)(O)=O.OP(O)([O-])=O UDPWNJVGOHGQFG-UHFFFAOYSA-N 0.000 claims 1
- 239000007924 injection Substances 0.000 abstract description 18
- 238000002347 injection Methods 0.000 abstract description 18
- 238000012216 screening Methods 0.000 abstract description 10
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- 238000006731 degradation reaction Methods 0.000 abstract description 6
- 238000011002 quantification Methods 0.000 abstract description 2
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- 239000011259 mixed solution Substances 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000000926 separation method Methods 0.000 description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
- 229940079593 drug Drugs 0.000 description 8
- 239000011550 stock solution Substances 0.000 description 8
- 238000004811 liquid chromatography Methods 0.000 description 7
- 239000012488 sample solution Substances 0.000 description 7
- 238000007865 diluting Methods 0.000 description 6
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
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- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 4
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- 238000010521 absorption reaction Methods 0.000 description 3
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- 238000007796 conventional method Methods 0.000 description 3
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- 229960004752 ketorolac Drugs 0.000 description 3
- 239000012452 mother liquor Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- -1 1-ketorolac) Chemical compound 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- OZWKMVRBQXNZKK-UHFFFAOYSA-N ketorolac Chemical compound OC(=O)C1CCN2C1=CC=C2C(=O)C1=CC=CC=C1 OZWKMVRBQXNZKK-UHFFFAOYSA-N 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
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- 239000012088 reference solution Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 229960000281 trometamol Drugs 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 101000671814 Homo sapiens Ubiquitin carboxyl-terminal hydrolase 38 Proteins 0.000 description 1
- 101000777206 Homo sapiens Ubiquitin carboxyl-terminal hydrolase 40 Proteins 0.000 description 1
- 102100040108 Ubiquitin carboxyl-terminal hydrolase 38 Human genes 0.000 description 1
- 102100031284 Ubiquitin carboxyl-terminal hydrolase 40 Human genes 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000000949 anxiolytic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
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- JEGUKCSWCFPDGT-UHFFFAOYSA-N h2o hydrate Chemical compound O.O JEGUKCSWCFPDGT-UHFFFAOYSA-N 0.000 description 1
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- 230000014759 maintenance of location Effects 0.000 description 1
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- 239000000463 material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 description 1
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 239000000932 sedative agent Substances 0.000 description 1
- 230000001624 sedative effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- JAVMESYEMAASLS-UHFFFAOYSA-N tetraazanium dihydrogen phosphate phosphate Chemical group [NH4+].[NH4+].[NH4+].[NH4+].OP(O)([O-])=O.[O-]P([O-])([O-])=O JAVMESYEMAASLS-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an HPLC detection method for ketorolac tromethamine or/and impurities in a preparation thereof. The HPLC impurity detection method for ketorolac tromethamine raw material medicine and related impurities in injection is optimized and improved from the aspects of detection wavelength, determination of a mobile phase system, screening of the concentration of a test solution and the like, and is verified from the aspects of system applicability, degradation specificity, detection limit and quantification limit, linearity and range, precision, accuracy, solution stability, method durability and the like, and the result shows that the method has good impurity detection capability.
Description
Technical Field
The invention belongs to the technical field of substance detection, and particularly relates to an HPLC (high performance liquid chromatography) detection method for ketorolac tromethamine or/and impurities in a preparation thereof.
Background
Ketorolac Tromethamine, the English name Ketorolic Tromethamine, abbreviated as KTT, CAS number 74103-07-4. Ketorolac tromethamine is a non-steroidal anti-inflammatory drug that inhibits prostaglandin biosynthesis, with biological activity related to its S-form. Animal studies have shown that ketorolac tromethamine has analgesic, non-sedative or anxiolytic effects.
According to the KTT synthetic route, the impurities which may appear are mainly degradation impurities, for example, ketorolac reacts with tromethamine to generate impurity A (CAS number 167105-80-8), ketorolac is oxidized in the synthetic process to generate impurity B (CAS number 154476-25-2, 1-hydroxyketorolac), impurity C (CAS number 113502-52-6, 1-ketorolac), impurity D and impurity E (CAS number 80965-09-9), and 5 degradation impurities in the bulk drug need to be controlled. In addition, because ethanol is used as an auxiliary material in the prescription of the KTT injection, acid and alcohol can react to generate ester, 5 degradation impurities in the raw material medicines are controlled in the preparation, and impurity F is also needed (CAS number 108061-03-6).
In the liquid chromatography detection aiming at KTT bulk drug impurities in USP and EP pharmacopoeia, the following defects exist: the salt phase of the mobile phase is 50mmol/L ammonium dihydrogen phosphate (pH is adjusted to 3.0 by phosphoric acid), the organic phase is tetrahydrofuran, and the tetrahydrofuran can cause certain damage to a detection instrument.
Disclosure of Invention
In view of the above, the invention aims to optimize and improve the HPLC detection method for the impurities in the KTT bulk drug and the preparation thereof, and meanwhile, the method can also perform qualitative or quantitative detection on the KTT bulk drug and the KTT in the preparation thereof.
The invention realizes the aim through the following technical scheme:
the invention provides a method for detecting impurities in ketorolac tromethamine or a preparation thereof, which adopts HPLC to detect a solution to be detected, and qualitatively or quantitatively detects the impurities according to a chromatographic result, wherein the chromatographic condition comprises the following steps:
a chromatographic column: c8 silica gel column;
detection wavelength: the detection wavelength is 200-400 nm;
mobile phase and elution procedure: the mobile phase A is a water phase containing a phosphate buffer solution, and the pH value is 4.1-4.3; the mobile phase B is a mixed organic phase of acetonitrile and tetrahydrofuran, and the volume fraction of the tetrahydrofuran in the organic phase is 10-15%; performing gradient elution according to any one of the following gradient 1-3 procedures:
gradient 1:
gradient 2:
gradient 3:
further, the impurities comprise one or a mixture of more than two of impurities A, B, C, D, E and F:
the detection method can effectively separate the A-F impurities from the main component at the same time, and can effectively detect one or more components.
Further, the phosphate buffer solution contains dihydrogen phosphate ions and phosphate ions, and the pH value is 4.1-4.3; further, the phosphate buffer is ammonium dihydrogen phosphate-phosphate buffer; further, the pH was 4.2; further, the concentration of ammonium dihydrogen phosphate was 5.75 g/L.
Further, the specification of the chromatographic column is 4.6X 250mm, 5 μm.
Further, the flow rate of the mobile phase is 0.6 to 1.2ml/min, and is further selected from 0.9 to 1.1 ml/min.
Further, the column temperature is selected from 20-42 ℃, and is further selected from 38-42 ℃.
In the invention, the parameters such as column temperature, flow rate, sample injection amount and the like can be selected in a common range.
Furthermore, the solvent of the solution to be detected is selected from water or acetonitrile water solution with acetonitrile volume fraction of less than 25%; further, the solution to be detected at least comprises a test sample solution; furthermore, the test solution also includes a reference solution.
In the detection method of the present invention, if a reference substance is used, the reference substance corresponds to the target substance. The reference substance is corresponding to the target substance, namely the reference substance corresponding to the compound is selected according to the target substance, and if the target substance is the impurity A, the reference substance is the impurity A; if the target substances are impurities A and D, corresponding impurities A and D are corresponding reference substances.
The invention also provides a method for detecting the ketorolac tromethamine or the ketorolac tromethamine in the preparation thereof, HPLC is adopted to detect the solution to be detected, and the qualitative or quantitative detection is carried out according to the chromatographic result, and the chromatographic conditions comprise:
a chromatographic column: c8 silica gel column;
detection wavelength: the detection wavelength is 200-400 nm;
mobile phase and elution procedure: the mobile phase A is a water phase containing a phosphate buffer solution, and the pH value is 4.1-4.3; the mobile phase B is a mixed organic phase of acetonitrile and tetrahydrofuran, and the volume fraction of the tetrahydrofuran in the organic phase is 10-15%; performing gradient elution according to any one of the following gradient 1-3 procedures:
gradient 1:
gradient 2:
gradient 3:
the wavelengths to be detected in the present invention can be adjusted and selected by conventional means within the range disclosed above. When the optimal detection wavelength is searched, the method can be carried out by using a full-wave-band scanning method and the like which are matched with an ultraviolet spectrophotometry method and HPLC, and then the appropriate detection wavelength is found by adopting a conventional technology by matching with the detection effect of an HPLC detector (such as avoiding solvent interference). In one embodiment of the present invention, the detection wavelength is selected from 311 to 315nm, for example 313 nm.
The research of the invention shows that the detection effect of the invention can be achieved when the volume fraction of the tetrahydrofuran is 10 percent, and the mobile phase with low tetrahydrofuran content is preferentially used in the actual operation.
The qualitative detection in the present invention can be performed by using conventional methods, such as corresponding analysis by external standard method with reference substance, or qualitative analysis by conventional identification means, such as mass spectrum, thin layer, ultraviolet, etc. after separating each component by HPLC.
In the invention, the content can be calculated by using the conventional methods such as an external standard method, an area normalization method and the like.
In addition, the control of the separation degree and the impurity limit in the present invention is adjusted by the conventional technical means in the art, and the impurity limit can be referred to USP or EP pharmacopoeia.
During quantitative analysis, if an external standard method is used, a standard curve is manufactured by a conventional method for calculation; however, in the qualitative analysis, a standard curve is not required to be prepared, and the determination can be made by the retention time.
The invention has the beneficial effects that: the invention optimizes and improves the HPLC detection method for ketorolac tromethamine bulk drug and preparation thereof, particularly relevant impurities in injection, optimizes and improves the detection method from the aspects of detection wavelength, determination of a mobile phase system, screening of the concentration of a test solution and the like, and verifies the detection method from the aspects of system applicability, degradation specificity, detection limit and quantification limit, linearity and range, precision, accuracy, solution stability, method durability and the like.
Drawings
FIG. 1 shows UV absorption spectra of KTT and impurities, wherein a-g shows UV absorption spectra of KTT, impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F; DAD 1,22.252(60.6mAU, -) reference value 21.885&22.719 is mixed solution 1. D;
FIG. 2 is a typical liquid chromatography spectrum of gradient 1;
FIG. 3 is a typical liquid chromatography profile of gradient 2;
FIG. 4 is a typical liquid chromatography profile of gradient 3;
FIG. 5 is a typical liquid chromatography profile of gradient 4;
FIG. 6 is a typical liquid chromatography profile of gradient 5;
figure 7 typical liquid chromatography profile of gradient 6.
Detailed Description
The chromatographic instrument used in the invention is an Agilent 1260 high performance liquid chromatograph.
Ketorolac tromethamine (KTT) and its injection, and the control limits of prior art detection of impurities and individual impurities are shown in table 1.
TABLE 1
Among them, impurity A, B, C, D was obtained by purchase, and impurity E (refer to U.S.,5621115) and impurity F (refer to "Kaneria, Ankur et al, Fe-EROS Encyclopedia of Reagents for Organic Synthesis, Triethyl methyl carboxylate, 2001") were synthesized separately by the laboratory.
The HPLC detection method for ketorolac tromethamine and impurities in the preparation thereof, especially injection or/and ketorolac tromethamine, comprises the following specific steps:
(I) chromatographic conditions
A chromatographic column: ZORBAX Eclipse plus C8 column, 4.6X 250mm, 5 μm, 40 deg.C;
a detector: an ultraviolet detector for detecting the wavelength of 313 nm;
mobile phase and elution procedure: 5.75g/L ammonium dihydrogen phosphate buffer salt solution is used as a mobile phase A, and the pH value is adjusted to 4.2 by phosphoric acid; acetonitrile: tetrahydrofuran 90:10(v/v) as mobile phase B, gradient elution was performed according to the procedure in table 2,
TABLE 2
Flow rate: 1.0 ml/min;
sample introduction amount: 10 μ l.
(II) preparation of reagent
Test solution: precisely measuring 1ml (specification 30mg) or 2ml (specification 15mg) of a test sample, putting the test sample into a 50ml measuring flask, diluting the test sample to a scale with water, and shaking up to obtain a test sample solution; about 0.6mg of ketorolac tromethamine per 1 ml;
control solution: precisely weighing 1ml of reference solution, placing in a 100ml measuring flask, diluting with water to scale, and shaking.
System applicability solution: accurately weighing 10mg of impurity A, impurity B, impurity C, impurity D, impurity E and impurity F, placing in a 10ml measuring flask, dissolving with methanol, diluting to scale, shaking up, and using as a mixed impurity reference mother liquor; precisely measuring 0.6ml of mixed impurity reference mother liquor, placing the mixed impurity reference mother liquor into a 10ml measuring flask, diluting methanol to a scale, and shaking up to be used as impurity reference stock solution; accurately weighing ketorolac tromethamine reference substance 60mg, placing into a 10ml measuring flask, dissolving with methanol, diluting to scale, shaking uniformly, and using as reference substance stock solution; precisely measuring 1ml of each of the impurity reference substance stock solution and the reference substance stock solution, placing the reference substance stock solution and the reference substance stock solution into a 10ml measuring flask, diluting the reference substance stock solution to scales by using water, shaking the reference substance stock solution uniformly to prepare a mixed solution containing about 1.2 mu g of each of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity F and about 0.6mg of ketorolac tromethamine in each 1ml of the mixed solution as a system applicability solution.
(III) sample introduction step
Step one, water is taken and sample introduction is carried out for 1 time;
secondly, sampling a system applicability solution for 1 time;
thirdly, sampling a reference substance solution for 1 time;
and step four, taking a sample solution, and carrying out sample introduction for 1 time.
(IV) calculation of impurity content
Calculated by the following formula:
wherein:
rfi: the correction factor is the peak area of each impurity of ketorolac tromethamine;
ri: peak area for a single impurity;
rs: peak area of the control solution.
Total impurity content (%) — the sum of all impurity contents.
The specific optimization procedure for each condition is as follows.
Determination of detection wavelength
Corresponding ultraviolet absorption spectra are extracted from positioning spectra of KTT, impurities A, B, C, D, E and F, as shown in figure 1, the result shows that the maximum absorption of KTT is achieved at 313nm, and the absorption of impurities A, B, C, D, E and F is greater at 313nm, so 313nm is selected as KTT bulk drugs and injection, including the detection wavelength of impurity determination.
Determination of mobile phase gradient
TABLE 3
The following experiments were carried out using the relevant conditions described in table 3.
Referring to the USP and EP pharmacopoeias, the mobile phase salt phase is 50mmol/L ammonium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid) and the organic phase is tetrahydrofuran. Considering that tetrahydrofuran is harmful to the apparatus, the present invention attempts to reduce the ratio of replacing tetrahydrofuran by adding acetonitrile to the organic phase. Selecting a KTT mixed solution (comprising KTT, impurities A, B, C, D, E and F) to screen a mobile phase. Table 4 shows the chromatographic conditions for screening 1.
Table 4 chromatographic conditions screening 1
The results are shown in fig. 2, and the results show that the impurity detection number of the solution with system applicability is insufficient, impurity peaks may overlap with each other, and impurities may be wrapped in the peak shape of the main peak, and it is preliminarily presumed that the pH value of the mobile phase has a large influence on the peak emergence time of the impurities. The mobile phase was prepared under the same conditions (pH 5.5 as measured in 0.05mol/L ammonium dihydrogen phosphate buffer (pH adjusted to 3.0 with phosphoric acid): tetrahydrofuran (70:30)), so the pH of mobile phase A was adjusted to about 5.5 at the initial ratio of mixing with the organic phase (A: B80: 20) by considering the pH of mobile phase A in the next step and planning to use gradient elution, at which time the pH of mobile phase A was about 4.2. Table 5 shows the chromatographic conditions for the pH of the screened mobile phase and the gradient elution procedure, and the mixed solutions were injected separately.
TABLE 5 chromatographic Condition screening 2
The result of fig. 3 shows that the mixed solution is completely detected in the gradient 2 chromatographic condition, but the impurity C is wrapped by the unknown impurity, and the separation effect of the impurity E and the adjacent unknown impurity is poor; the results in fig. 4 show that the mixed solution was completely purified of impurities under gradient 3 chromatographic conditions, but the main peak was packed with unknown impurities. Therefore, the ratio of the main peak to the mobile phase near the impurity C is changed, the ratio of tetrahydrofuran in the organic phase is slightly increased, and whether the separation effect of the main peak and the impurity B, the impurity C and unknown impurities can be improved or not is considered. Table 6 shows the chromatographic conditions for screening the mobile phase gradients. The sample solutions were analyzed by sample injection under the following 2 chromatographic conditions, respectively.
Table 6 chromatographic conditions screening 3
The result is shown in figure 5, the tailing factor of the main peak of the sample solution under the chromatographic condition of the gradient 4 is 0.9, the chromatographic condition of the gradient 4 is selected for carrying out sample injection analysis on the mixed solution and each impurity positioning solution, the tailing factor of the main peak is 0.9 in the result analysis of the mixed solution, each known impurity is completely separated, the separation degree of each known impurity is larger than 3.0, the separation degree of the impurity C and the adjacent unknown impurity is larger than 1.5, and all impurities are completely separated in 23min, so the second half section of the gradient is considered to be optimized, and the gradient time is shortened. Table 7 shows the chromatographic conditions optimized for gradient 4, and the mixed solution was analyzed by injection.
Table 7 chromatographic condition screening 4
The result is shown in fig. 6, the tailing factor of the main peak of the mixed solution under the chromatographic condition of the gradient 5 is 0.9, the main peak and each known impurity can reach the baseline separation requirement within the range of 0.8-1.5, and the separation degree is more than 3.0; each known impurity is separated from its neighboring unknown impurities by more than 2.0, so this condition is the optimum condition.
Since the mobile phase a is gradually decreased from 15min to 20min and from 20min to 25min in the gradient program, the gradient program of the gradient 5 is optimized. Table 8 analysis of mixed solution injection for gradient 5 optimized chromatographic conditions.
Table 8 chromatographic condition screening 5
The result is shown in fig. 7, the mixed solution is analyzed by sample injection under the optimized chromatographic condition, the tailing factor of the main peak is in the range of 0.8-1.5, the main peak and each known impurity can reach the baseline separation requirement, and the separation degree is more than 3.0, so the condition is determined as the final analysis condition.
Fourthly, screening the concentration of the test solution
By referring to the quality standard of ketorolac tromethamine USP40 bulk drug and considering the detection sensitivity to impurities in related substance detection, the concentration of a test sample solution is preliminarily made to be 0.6mg/ml, and the sample injection volume is 10 mu l. The test result shows that: and analyzing the sample under an optimized condition, wherein the main peak is not overloaded, the tailing factor is in the range of 0.8-1.5, and the theoretical version number is more than 5500, so that the concentration of the sample solution is preliminarily prepared to be 0.6mg/ml, and the sample introduction volume is 10 mu l.
Fifthly, determining sample injection amount
The absolute sample amount (6 mu g) of the test sample is lower than that (20 mu g) of related substances of USP injection, but higher than that (4 mu g) of USP38/40, BP2013 and EP 8.0. Meanwhile, under the selected pH value of the mobile phase, the ultraviolet maximum absorption of the product and various known impurities is near 313nm, and the response values of the main peak and the known impurities at the 313nm detection wavelength are far higher than the response values at the 254nm detection wavelength specified by pharmacopoeia. Therefore, the ability to detect impurities is not affected.
The detection method and the detection method of USP pharmacopoeia are adopted to respectively detect the related substances of 6 batches of pilot plant test finished products, the results of the two methods are compared, the results are shown in tables 9 and 10, and the results show that the detection method of the invention has no inferior impurity detection capability to the detection method of the related substances of USP injection.
TABLE 9 comparative test results of two methods of injection (15mg standard)
Remarking: the test sample was a sample with a long term stability of 9 months.
TABLE 10 comparative test results of two methods of injection (30mg standard)
Remarking: the test sample was a sample with a long term stability of 9 months.
Further, the present invention verifies the detection method in terms of system applicability, degradation specificity, detection and quantitation limits, linearity and range, precision, accuracy, solution stability, method durability, etc., and the verification results are shown in table 11.
TABLE 11
The verification results show that the detection method provided by the invention has good impurity detection capability.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (16)
1. The method for detecting the impurities in the ketorolac tromethamine or the preparation thereof is characterized in that HPLC is adopted to detect the solution to be detected, and the method is qualitative or quantitative according to the chromatographic result, and the chromatographic conditions comprise:
a chromatographic column: c8 silica gel column;
detection wavelength: the detection wavelength is 200-400 nm;
mobile phase and elution procedure: the mobile phase A is a water phase containing a phosphate buffer solution, and the pH value is 4.1-4.3; the mobile phase B is a mixed organic phase of acetonitrile and tetrahydrofuran, and the volume fraction of the tetrahydrofuran in the organic phase is 10-15%; performing gradient elution according to any one of the following gradient 1-3 procedures:
gradient 1: 0min, 75vol.% a-25vol.% B; 5min, 75vol.% a-25vol.% B; 15min, 50vol.% a-50vol.% B; 25min, 40 vol.% a-60vol.% B; 30min, 40 vol.% a-60vol.% B; 32min, 75vol.% a-25vol.% B; 40min, 75vol.% a-25vol.% B;
gradient 2: 0min, 75vol.% a-25vol.% B; 5min, 75vol.% a-25vol.% B; 15min, 50vol.% a-50vol.% B; 20min, 45 vol.% a-55vol.% B; 40min, 30 vol.% a-70vol.% B; 42min, 75vol.% a-25vol.% B;
gradient 3: 0min, 75vol.% a-25vol.% B; 5min, 75vol.% a-25vol.% B; 15min, 50vol.% a-50vol.% B; 20min, 45 vol.% a-55vol.% B; 25min, 40 vol.% a-60vol.% B; 30min, 40 vol.% a-60vol.% B; 32min, 75vol.% a-25vol.% B; 40min, 75vol.% a-25vol.% B;
the impurities comprise one or the mixture of more than two of impurities A, B, C, D, E and F:
2. the detection method according to claim 1, wherein the phosphate buffer contains dihydrogen phosphate ions and phosphate ions, and has a pH of 4.1 to 4.3.
3. The method of claim 2, wherein the phosphate buffer is a monoammonium phosphate-phosphate buffer.
4. The assay of claim 2 or 3, wherein the pH is 4.2.
5. The detection method according to claim 3, wherein the concentration of ammonium dihydrogen phosphate is 5.75 g/L.
6. The detection method according to claim 1, wherein the chromatographic column has a size of 4.6 x 250mm, 5 μm.
7. The detection method according to claim 1, wherein the flow rate of the mobile phase is 0.6 to 1.2 ml/min.
8. The detection method according to claim 7, wherein the flow rate of the mobile phase is 0.9 to 1.1 ml/min.
9. The detection method according to claim 1, wherein the column temperature is selected from the range of 20 to 42 ℃.
10. The detection method according to claim 9, wherein the column temperature is selected from 38 to 42 ℃.
11. The detection method according to claim 1, wherein the solvent of the solution to be detected is selected from water and an acetonitrile aqueous solution having an acetonitrile volume fraction of 25% or less.
12. The detection method according to claim 1, wherein the solution to be detected comprises at least a test solution.
13. The assay of claim 12 wherein the test solution further comprises a control solution.
14. The method for detecting the ketorolac tromethamine or the ketorolac tromethamine in the preparation thereof is characterized in that HPLC is adopted to detect the solution to be detected, and the detection is qualitative or quantitative according to the chromatographic result, and the chromatographic conditions comprise:
mobile phase and elution procedure: the mobile phase A is a water phase containing a phosphate buffer solution, and the pH value is 4.1-4.3; the mobile phase B is a mixed organic phase of acetonitrile and tetrahydrofuran, and the volume fraction of the tetrahydrofuran in the organic phase is 10-15%; performing gradient elution according to any one of the following gradient 1-3 procedures:
gradient 1: 0min, 75vol.% a-25vol.% B; 5min, 75vol.% a-25vol.% B; 15min, 50vol.% a-50vol.% B; 25min, 40 vol.% a-60vol.% B; 30min, 40 vol.% a-60vol.% B; 32min, 75vol.% a-25vol.% B; 40min, 75vol.% a-25vol.% B;
gradient 2: 0min, 75vol.% a-25vol.% B; 5min, 75vol.% a-25vol.% B; 15min, 50vol.% a-50vol.% B; 20min, 45 vol.% a-55vol.% B; 40min, 30 vol.% a-70vol.% B; 42min, 75vol.% a-25vol.% B;
gradient 3: 0min, 75vol.% a-25vol.% B; 5min, 75vol.% a-25vol.% B; 15min, 50vol.% a-50vol.% B; 20min, 45 vol.% a-55vol.% B; 25min, 40 vol.% a-60vol.% B; 30min, 40 vol.% a-60vol.% B; 32min, 75vol.% a-25vol.% B; 40min, 75vol.% A-25vol.% B.
15. The detection method according to claim 1 or 14, characterized in that: the detection wavelength is 311-315 nm.
16. The detection method according to claim 15, characterized in that: the detection wavelength is 313 nm.
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