CN115128177A - Method for analyzing and determining genotoxic impurities in ganciclovir condensation compound by using HPLC method - Google Patents
Method for analyzing and determining genotoxic impurities in ganciclovir condensation compound by using HPLC method Download PDFInfo
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- CN115128177A CN115128177A CN202110675542.9A CN202110675542A CN115128177A CN 115128177 A CN115128177 A CN 115128177A CN 202110675542 A CN202110675542 A CN 202110675542A CN 115128177 A CN115128177 A CN 115128177A
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- 239000012535 impurity Substances 0.000 title claims abstract description 103
- IRSCQMHQWWYFCW-UHFFFAOYSA-N ganciclovir Chemical compound O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 title claims abstract description 47
- 229960002963 ganciclovir Drugs 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 41
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 39
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 39
- 150000001875 compounds Chemical class 0.000 title claims abstract description 33
- 238000009833 condensation Methods 0.000 title claims abstract description 33
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- VRZVPALEJCLXPR-UHFFFAOYSA-N ethyl 4-methylbenzenesulfonate Chemical compound CCOS(=O)(=O)C1=CC=C(C)C=C1 VRZVPALEJCLXPR-UHFFFAOYSA-N 0.000 claims abstract description 58
- VUQUOGPMUUJORT-UHFFFAOYSA-N methyl 4-methylbenzenesulfonate Chemical compound COS(=O)(=O)C1=CC=C(C)C=C1 VUQUOGPMUUJORT-UHFFFAOYSA-N 0.000 claims abstract description 58
- 238000001514 detection method Methods 0.000 claims abstract description 33
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 11
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- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims abstract description 4
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- 239000002994 raw material Substances 0.000 claims description 7
- FGDQGIKMWOAFIK-UHFFFAOYSA-N acetonitrile;phosphoric acid Chemical group CC#N.OP(O)(O)=O FGDQGIKMWOAFIK-UHFFFAOYSA-N 0.000 claims description 6
- 238000010812 external standard method Methods 0.000 claims description 6
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- 238000002137 ultrasound extraction Methods 0.000 claims description 2
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 abstract description 24
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 238000011160 research Methods 0.000 abstract description 3
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- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 5
- 238000002604 ultrasonography Methods 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 238000013112 stability test Methods 0.000 description 4
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 4
- 230000005526 G1 to G0 transition Effects 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
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- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
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- 238000004811 liquid chromatography Methods 0.000 description 3
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000002211 ultraviolet spectrum Methods 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
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- 239000012895 dilution Substances 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
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- 230000007118 DNA alkylation Effects 0.000 description 1
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- 231100000277 DNA damage Toxicity 0.000 description 1
- 241001233170 Pearcea Species 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
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- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000005456 alcohol based solvent Substances 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 125000005228 aryl sulfonate group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-M benzenesulfonate Chemical compound [O-]S(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-M 0.000 description 1
- 229940077388 benzenesulfonate Drugs 0.000 description 1
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- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 239000012046 mixed solvent 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
- 231100000299 mutagenicity Toxicity 0.000 description 1
- 230000007886 mutagenicity Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- QNXFUWFRTWSSOK-UHFFFAOYSA-N n-acetyl-n-(6-oxo-3,7-dihydropurin-2-yl)acetamide Chemical compound O=C1NC(N(C(C)=O)C(=O)C)=NC2=C1NC=N2 QNXFUWFRTWSSOK-UHFFFAOYSA-N 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- -1 p-toluenesulfonic acid ester Chemical class 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- 150000003460 sulfonic acids Chemical class 0.000 description 1
- YBBRCQOCSYXUOC-UHFFFAOYSA-N sulfuryl dichloride Chemical class ClS(Cl)(=O)=O YBBRCQOCSYXUOC-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012546 transfer Methods 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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- General Health & Medical Sciences (AREA)
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- Pathology (AREA)
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Abstract
The invention discloses a method for analyzing and determining genotoxic impurities in a ganciclovir condensation compound by using an HPLC method. The invention adopts octadecyl bonded silica gel as a fixed phase, and takes 0.1 percent phosphoric acid water solution: the acetonitrile 50:50 is a mobile phase, and an isocratic elution mode is adopted, so that the established high performance liquid analysis method can accurately determine 2 potential genotoxic impurities of methyl p-toluenesulfonate and ethyl p-toluenesulfonate in a ganciclovir condensation compound. The HPLC-UV method is simple to operate, short in analysis time, and capable of reaching the sensitivity of 0.8ppm, and provides reliable basis for improvement of the quality standard of a ganciclovir condensation compound and research on other pyrrosia genotoxic impurities. The invention uses common equipment and instruments, has extremely simple and convenient operation, and greatly saves the detection cost and the detection time.
Description
Technical Field
The invention relates to the technical field of chemical determination, in particular to a method for analyzing and determining genotoxic impurities in a ganciclovir condensation compound by using an HPLC method.
Background
Genotoxic impurities are (GTI) substances that are present in trace levels in drugs and are capable of reacting with DNA, either directly or indirectly, inducing DNA damage, causing genetic mutations or having a carcinogenic propensity. Genotoxic impurities have the characteristic of causing human genetic material damage at an extremely low exposure level, thereby causing serious threat to the safety of medication. The sulfonate is a potential genotoxic impurity, and researches show that the sulfonate has mutagenicity and can directly or indirectly transfer alkyl residues on self structures to electron-rich DNA bases after metabolic activation to cause DNA alkylation, thereby causing damage to genetic materials. Sulfonate impurities are typically derived from side reactions that occur during drug synthesis between sulfonic acids or derivatives, such as sulfonyl chlorides, sulfonic anhydrides, and lower alcohol solvents (e.g., methanol, ethanol, isopropanol, etc.).
The ganciclovir condensation compound is synthesized by ganciclovir side chain and diacetyl guanine, p-toluenesulfonic acid is added as a catalyst, methanol and ethanol are used as solvents in the step of recrystallization, the risk of generating genotoxic impurities of methyl p-toluenesulfonate and ethyl p-toluenesulfonate exists, and the reaction process is as follows:
methyl p-toluenesulfonate and ethyl p-toluenesulfonate belong to aryl sulfonates, most of which have the characteristics of high boiling point and difficult volatility or non-volatility, and can be measured by high performance Liquid Chromatography (LC) and liquid chromatography-tandem mass spectrometry (LC-MS). Tayor et al (see Taylor G E, Gosling M, PearceA. low level determination of p-tolenesulfonate and benzanese sulfonate esters in drug substance by high performance chromatography [ J ] JChromatogra 20061119,1119(1-2): 231. sup. 237) used LC-MS method to detect p-toluenesulfonate and benzenesulfonate in drugs, and ZobaxRC 8(250 mm. times.4.6 mm. times.5 μ M) as chromatographic column, the detection sensitivity was significantly improved; cappiello et al (see Cappiello A, Faminglini G, Palma P, et al. A new liquid chromatography for genetic screening and quantification of potential genetic digestion composition [ J ] JPharm biome dAnal,1255(17):286-290) measured the P-toluenesulfonate in the drug by EI-LC-MS method with a LOD of 0.13 to 1.5 ppm.
Because genotoxic impurities are extremely low in drug content, the determination is difficult, the liquid chromatography-mass spectrometry sensitivity is high, but the price is high, and the popularization and the use of the gene toxicity are limited.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the method for analyzing and determining genotoxic impurities in the ganciclovir condensation compound overcomes the defects of the prior art, establishes a high performance liquid chromatography (HPLC-UV) method, uses a chromatographic column using octadecylsilane chemically bonded silica as a stationary phase, and uses a mixed solvent of an organic phase and a phosphate buffer solution as a mobile phase for isocratic elution, can detect sulfonate impurities, has high sensitivity (the sensitivity can reach 0.8ppm), can simply, conveniently and quickly determine the contents of methyl p-toluenesulfonate and ethyl p-toluenesulfonate in the ganciclovir condensation compound, and effectively controls the quality of raw material medicines.
The genotoxic impurities in ganciclovir include methyl p-toluenesulfonate and ethyl p-toluenesulfonate, wherein the structural formula of the methyl p-toluenesulfonate is shown as formula I, and the molecular formula is C 8 H 10 O 3 S, molecular weight 186.04; the structural formula of the ethyl p-toluenesulfonate is shown as a formula II, and the molecular formula is C 9 H 12 O 3 S, molecular weight 200.05.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for analyzing and determining genotoxic impurities in a ganciclovir condensation compound by using an HPLC method is characterized in that the content of impurities of methyl p-toluenesulfonate and ethyl p-toluenesulfonate in the ganciclovir condensation compound is analyzed and detected by using a high performance liquid chromatography method, and the method comprises the following steps:
(1) and preparing a reference substance solution: taking methyl p-toluenesulfonate and ethyl p-toluenesulfonate reference substances, dissolving and diluting with a diluent to prepare a mixed solution serving as a genotoxic impurity reference substance solution;
(2) and preparing a test solution: putting the raw material drug of the ganciclovir condensation compound into a measuring flask, adding a diluent to a constant volume to scale, ultrasonically extracting, filtering, and sampling the filtrate to be used as a test solution;
(3) and chromatographic conditions: the mobile phase is acetonitrile-phosphoric acid aqueous solution; adopting an isocratic elution mode; flow rate: 1.1-1.3 mL/min; column temperature: 22-32 ℃; detection wavelength: 225 nm; sample introduction amount: 20 mu L of the solution; octadecyl bonded silica gel chromatographic column; the elution time is 30 min;
(4) and detecting a chromatogram map: measuring 20 μ L of each of the sample solution and the reference solution, injecting into a liquid chromatograph, and recording chromatogram;
(5) and analyzing: if the chromatogram of the sample solution does not have a chromatographic peak consistent with the retention time of methyl p-toluenesulfonate and ethyl p-toluenesulfonate, judging that the ganciclovir condensate sample does not contain methyl p-toluenesulfonate and ethyl p-toluenesulfonate;
or if a chromatogram of the sample solution has chromatographic peaks which are consistent with retention time of methyl p-toluenesulfonate and ethyl p-toluenesulfonate, calculating the content of impurities according to a peak area external standard method;
content of impurities (ppm) ═ Cs × A T ×10 6 /(As×C T )；
In the formula A T Is the peak area of each impurity in the test sample; as is the peak area of the impurity reference substance; cs is the concentration of the impurity reference substance; c T Is the concentration of the test sample.
Under the chromatographic detection condition in the step (3), the retention time of the methyl p-toluenesulfonate is 6.4 min; the retention time of ethyl p-toluenesulfonate was 8.5min, and it was found that methyl p-toluenesulfonate and ethyl p-toluenesulfonate could be completely separated under the chromatographic detection conditions.
Further, the diluent in step (1) and step (2) is methanol. The ganciclovir condensation compound, the methyl p-toluenesulfonate and the ethyl p-toluenesulfonate have good solubility in methanol, and stability tests show that the test solution and the reference solution are stable within 24 h. However, the acid hydrolysis reaction of p-toluenesulfonic acid ester impurities in methanol-water solution leads to unstable impurities, so methanol is selected as a diluent in the steps (1) and (2).
Further, the ultrasonic extraction time in the step (2) is 5 minutes; filtering with 0.45 μm microporous membrane. The accuracy test result shows that: the ultrasound 5min and the ultrasound 20min both meet the recovery rate requirement of an external standard method (the determination result of the impurity content with the content of 0.1-1.0 ppm should be between 75-120%, RSD is less than or equal to 8.0%), no obvious difference exists, and the ultrasound 5min is selected in consideration of time cost. The method selects the filtration with the aperture of 0.45 μm and the filtration with the aperture of 0.22 μm, which both meet the recovery rate requirement of an external standard method, and have no obvious difference, the invention uses a high performance liquid chromatograph, and the sample filtered by a filter membrane with the aperture of 0.45 μm can meet the sampling requirement, and the selection is 0.45 μm.
Further, the volume ratio of the acetonitrile-phosphoric acid aqueous solution in the mobile phase in the step (3) is 50: 50; the mass percentage of the phosphoric acid aqueous solution is 0.08-0.12%.
Further, the mass concentration of the phosphoric acid aqueous solution in the step (3) is 0.10%.
Further, the chromatographic column in the step (3) is Agilent Eclipse plus C18, 250X 4.6mm, 5 μm; the flow rate is 1.0 mL/min; column temperature: at a temperature of 27 ℃.
The invention has the beneficial effects that: the invention has reasonable design and the following advantages:
(1) the detection method has good system applicability, and the specificity test shows that the blank solvent has no influence on the detection of impurities and has strong specificity;
(2) the detection method has high sensitivity and good accuracy for detecting the two impurities;
(3) the reference substance solution and the test substance solution adopted by the detection method are placed at room temperature, and the solution stability test shows that the solution stability is good;
(4) the method has the advantages of common instruments, simple operation, great saving of detection cost and detection time, good practical application value, important effect on formulation and improvement of quality standard of raw material medicine of ganciclovir condensation compound, and reference basis for research on genotoxic impurities of other pyrrosia medicines adopting similar starting raw materials and synthetic routes.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a scanning ultraviolet spectrum of methyl p-toluenesulfonate between 190nm and 400 nm;
FIG. 2 is a scanning ultraviolet spectrum of ethyl p-toluenesulfonate at 190nm to 400 nm;
FIG. 3 is a chromatogram of a blank solvent (diluent);
FIG. 4 is a chromatogram of a control mixed solution of genotoxic impurities;
FIG. 5 is a chromatogram of a system suitability solution;
FIG. 6 is a linear result of methyl p-toluenesulfonate;
FIG. 7 is a linear result of ethyl p-toluenesulfonate;
FIG. 8 is a chromatogram of a detection limit test control solution;
FIG. 9 is a chromatogram of a control solution for a limit of quantitation assay.
Detailed Description
The invention will now be further described with reference to the accompanying drawings.
1. Instruments and reagents
The experimental methods used in the detection method of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the detection method of the present invention can be obtained commercially without specific description.
The instruments and reagents used in the detection method of the present invention: agilent 1260 liquid chromatograph (equipped with DAD ultraviolet detector) and OpenLab 2.2 liquid chromatography workstation configured for analytical instrumentation; methanol (chromatographically pure), acetonitrile (chromatographically pure), phosphoric acid (analytically pure), purified water.
2. Determination of the HPLC detection method of the invention
2.1 determination of acquisition wavelength
Carrying out ultraviolet spectrum scanning on methyl p-toluenesulfonate and ethyl p-toluenesulfonate: accurately weighing a proper amount of methyl p-toluenesulfonate and ethyl p-toluenesulfonate reference substances, respectively adding methanol to dissolve and dilute the methyl p-toluenesulfonate and the ethyl p-toluenesulfonate to prepare a solution containing about 80ng/mL of methyl p-toluenesulfonate and ethyl p-toluenesulfonate; the solutions were scanned over a range of 190nm to 400nm, and the results are shown in Table 1, and the UV spectrograms are shown in FIGS. 1 to 2.
TABLE 1 absorption maxima for methyl p-toluenesulfonate and ethyl p-toluenesulfonate
| Name (R) | Maximum absorption wavelength |
| P-toluenesulfonic acid methyl ester | 226nm |
| P-toluenesulfonic acid ethyl ester | 228nm |
As can be seen from Table 1, FIG. 1 and FIG. 2, methyl p-toluenesulfonate and ethyl p-toluenesulfonate both have large absorption at a wavelength near 225nm, indicating that methyl p-toluenesulfonate and ethyl p-toluenesulfonate can be detected by an ultraviolet detector by using an HPLC method, and 225nm can be selected as a detection wavelength.
2.2 determination of flow phase ratio
Solution preparation: taking methyl p-toluenesulfonate and ethyl p-toluenesulfonate reference substances 0.08g respectively, precisely weighing, dissolving with methanol, and gradually diluting to a solution containing 400ng of each impurity per 1mL as impurity reference substance stock solution; accurately weighing 0.5g of ganciclovir condensation compound, placing the ganciclovir condensation compound in a 5mL measuring flask, accurately weighing 1.0mL of impurity reference substance stock solution, adding methanol to a constant volume to scale, carrying out ultrasonic treatment for 5min, filtering through a 0.45 mu m filter membrane, and taking filtrate as system applicability solution.
Preparing a mobile phase: respectively preparing mobile phases with different proportions of 0.1% H 3 PO 4 Aqueous solution: acetonitrile 40:60, 0.1% H 3 PO 4 Aqueous solution: acetonitrile 50:50, 0.1% H 3 PO 4 Aqueous solution: acetonitrile 60: 40.
After being balanced by different mobile phases, 20 μ L of the system applicability solution was precisely taken and injected into a liquid chromatograph, and chromatograms were recorded, with the results shown in table 2.
TABLE 2 System suitability results for different mobile phase ratios
As can be seen from Table 2, increasing the acetonitrile ratio reduced the degree of separation and shortened the retention time, and the peak shape was sharp, whereas increasing the ratio of the phosphoric acid aqueous solution increased the degree of separation and extended the retention time, and the peak shape was broadened. The volume ratio of the acetonitrile-phosphoric acid aqueous solution is 50:50, which can ensure that the test solution does not interfere with the determination of gene impurities, has good separation degree and can obtain proper retention time.
2.3 determination of buffer salt concentration
Solution preparation: taking 0.08g of each of methyl p-toluenesulfonate and ethyl p-toluenesulfonate reference substances, precisely weighing, dissolving with methanol, and gradually diluting to a solution containing 400ng of each impurity per 1mL as an impurity reference substance stock solution; accurately weighing 0.5g of ganciclovir condensation compound, placing the ganciclovir condensation compound in a 5mL measuring flask, accurately weighing 1.0mL of impurity reference substance stock solution, adding methanol to a constant volume to scale, carrying out ultrasonic treatment for 5min, filtering through a 0.45 mu m filter membrane, and taking filtrate as a system applicability solution.
Preparing a mobile phase: buffer salts with different concentrations, 0-0.12% H, are prepared respectively 3 PO 4 Aqueous solution: acetonitrile 50: 50. After being balanced by different mobile phases, 20 μ L of the system applicability solution was precisely taken and injected into a liquid chromatograph, and chromatograms were recorded, with the results shown in table 3.
TABLE 3 System suitability results for different concentrations of buffer salts
As can be seen from Table 3, the addition of phosphoric acid in the mobile phase is beneficial to improving the peak shape and has the function of slightly enhancing the elution capability of the mobile phase, and the good peak shape can be obtained by selecting 0.1% of phosphoric acid aqueous solution by mass percent. Continuing to increase the mass percent of aqueous phosphoric acid did not significantly improve the peak shape and retention time.
2.4 determination of flow Rate
Solution preparation: taking methyl p-toluenesulfonate and ethyl p-toluenesulfonate reference substances 0.08g respectively, precisely weighing, dissolving with methanol, and gradually diluting to a solution containing 400ng of each impurity per 1mL as impurity reference substance stock solution; accurately weighing 0.5g of ganciclovir condensation compound, placing the ganciclovir condensation compound in a 5mL measuring flask, accurately weighing 1.0mL of impurity reference substance stock solution, adding methanol to a constant volume to scale, carrying out ultrasonic treatment for 5min, filtering through a 0.45 mu m filter membrane, and taking filtrate as system applicability solution.
Chromatographic conditions are as follows: the mobile phase is acetonitrile-phosphoric acid aqueous solution; adopting an isocratic elution mode; column temperature: 27 ℃; detection wavelength: 225 nm; sample introduction amount: 20 mu L of the solution; octadecyl bonded silica gel chromatographic column; the elution time is 30 min; setting different flow rates: 1.0-1.4 mL/min;
20. mu.L of the system compatible solution was precisely taken out and injected into a liquid chromatograph, and the chromatogram was recorded, and the results are shown in Table 4.
TABLE 4 System suitability results for different flow rates
As shown in table 4, increasing the flow rate resulted in a decrease in the degree of separation and a decrease in the retention time, and the peak shape was sharp, whereas decreasing the flow rate resulted in an increase in the degree of separation and an increase in the retention time, and the peak shape was broadened. In order to ensure that genotoxic impurities smoothly peak and are effectively separated after the ganciclovir condensate main component peaks, the main component and impurity peaks in the ganciclovir condensate do not interfere, and meanwhile, proper retention time and good peak shape can be obtained, the flow rate is selected to be 1.2 mL/min.
2.5 selection of chromatography columns
Solution preparation: taking 0.08g of each of methyl p-toluenesulfonate and ethyl p-toluenesulfonate reference substances, precisely weighing, dissolving with methanol, and gradually diluting to a solution containing 400ng of each impurity per 1mL as an impurity reference substance stock solution; accurately weighing 0.5g of ganciclovir condensation compound, placing the ganciclovir condensation compound in a 5mL measuring flask, accurately weighing 1.0mL of impurity reference substance stock solution, adding methanol to a constant volume to scale, carrying out ultrasonic treatment for 5min, filtering through a 0.45 mu m filter membrane, and taking filtrate as system applicability solution.
The invention selects chromatographic columns with different stationary phase types, precisely takes 20 mu L of system applicability solution, injects the solution into a liquid chromatograph, records the chromatogram, and the result is shown in Table 5.
TABLE 5 results of System suitability for different chromatography columns
As shown in Table 5, the column Agilent Eclipse plus C18, 250X 4.6mm, 5 μm was selected in combination of column efficiency, peak resolution, peak shape, retention time.
3. Separation and determination of genotoxic impurity in ganciclovir condensate as test sample
3.1 preparation of the relevant solutions
Genotoxic impurity control stock solutions: taking a proper amount of methyl p-toluenesulfonate and ethyl p-toluenesulfonate reference substances, precisely weighing, dissolving by using a diluent, and quantitatively diluting to prepare a solution containing 400ng per 1mL of the solution to serve as a genotoxic impurity reference substance stock solution.
Genotoxic impurity control solution: taking a proper amount of the genotoxic impurity reference substance stock solution, precisely measuring, and diluting with methanol to prepare mixed solutions containing about 80ng of genotoxic impurity reference substance solution in each 1 mL.
System applicability solution: taking 0.5g of raw material drug of the ganciclovir condensation compound, placing the raw material drug into a 5mL measuring flask, precisely weighing 1.0mL of genotoxic impurity reference substance stock solution, adding methanol to a constant volume to scale, carrying out ultrasonic treatment for 5min, filtering the solution through a 0.45 mu m filter membrane, and taking the filtrate for sample injection.
3.2 chromatographic conditions
A chromatographic column: octadecylsilane chemically bonded silica is used as a stationary phase, and an Agilent Eclipse plus C18 chromatographic column with the thickness of 250 multiplied by 4.6mm and the thickness of 5 mu m is used as a chromatographic column;
detection wavelength: 225 nm;
flow rate: 1.2 mL/min;
column temperature: 27 ℃;
sample introduction amount: 20 mu L of the solution;
mobile phase: 0.1% (mass percent);
phosphoric acid water: acetonitrile 50:50 (volume ratio).
3.3, detecting the chromatogram, separating and measuring
Precisely measuring blank solvent (diluent), the genotoxic impurity reference solution, and the system suitability solution by 20 μ L each, injecting into liquid chromatograph, performing isocratic elution, recording chromatogram, and measuring results are shown in Table 6 and chromatograms in FIGS. 3-5.
TABLE 6 System suitability results
| Name(s) | Retention time (min) | Number of column plate (N) | Degree of separation (R) |
| P-toluenesulfonic acid methyl ester | 6.407 | 5288 | / |
| P-toluenesulfonic acid ethyl ester | 8.463 | 5696 | 5.1 |
| Adjacent peaks | 12.041 | 14490 | 8.4 |
As can be seen from the data in Table 6, the system applicability solution sequentially comprises a ganciclovir condensation compound, methyl p-toluenesulfonate and ethyl p-toluenesulfonate in the order of appearance of peaks, and adjacent impurity peaks, wherein the separation degree of each impurity from the main peak is more than 1.5, the separation degree is good, the number of tower plates is high, and the detection requirements are met.
Fig. 3 to 5 are HPLC chromatograms of a blank solvent, a genotoxic impurity control solution and a system suitability solution in this order, and it can be seen that the blank solvent (diluent) does not interfere with the detection of impurities.
4. Precision experiment
Taking a proper amount of methyl p-toluenesulfonate and ethyl p-toluenesulfonate reference substances, precisely weighing, and diluting with methanol to prepare mixed solutions containing about 80ng of each of 1mL of the reference substances as genotoxic impurity reference substance solutions. The control mixed solution (methyl p-toluenesulfonate, ethyl p-toluenesulfonate) was sampled 6 times in sequence, and the precision was calculated, and the results are shown in Table 7.
TABLE 7 precision results
As shown in Table 7, the sample introduction of the control mixed solution was carried out 6 times, and the relative deviations of the peak areas of methyl p-toluenesulfonate and ethyl p-toluenesulfonate were 1.5% and 1.1%, respectively, which met the calculation requirements of the external standard method, so that the chromatographic conditions were accurate.
5. Experiment of linear relationship
Taking a proper amount of methyl p-toluenesulfonate and ethyl p-toluenesulfonate reference substances, and adding methanol to dilute the reference substances to prepare a solution containing about 400ng of each reference substance per 1mL, wherein the solution is used as a linear stock solution; precisely measuring appropriate amount of linear stock solution, and diluting with methanol to obtain solutions with concentration of limit of quantitation, 20ng/mL, 40ng/mL, 80ng/mL, 120ng/mL, and 160ng/mL as linear solutions with various concentrations.
20. mu.L of each solution was measured precisely and injected into a liquid chromatograph, a chromatogram was recorded, and a linear equation was made for the peak area (A) by the concentration C (ng/mL), and the results are shown in tables 8 and 9.
TABLE 8 Linear results for methyl p-toluenesulfonate
TABLE 9 Linear results for ethyl p-toluenesulfonate
As is clear from Table 8, methyl p-toluenesulfonate had a linear relationship in a concentration range of 15.852ng/mL to 158.52ng/mLThe system is good, and the linear equation is: y 0.0591x +0.0275, r 2 0.9992, as shown in fig. 6; as can be seen from Table 9, the linear relationship of ethyl p-toluenesulfonate was good in the concentration range of 15.848 ng/mL-158.48. mu.g/mL, and the linear equation is: 0.0547x +0.0718, r 2 0.9995, as shown in fig. 7.
6. Methodology validation-limit of quantitation and limit of detection
6.1, limit of quantitation
Taking a proper amount of methyl p-toluenesulfonate and ethyl p-toluenesulfonate reference substances, precisely weighing, adding a diluent for dissolving, quantitatively diluting to prepare a solution with a certain concentration, gradually diluting the solution to the proper concentration by using the diluent, and under the condition of genotoxic impurity detection chromatography, performing quantitative analysis on the solution with a signal-to-noise ratio of 10: the detection limit concentration was determined by the amount injected into the chromatograph at around 1, and the results are shown in table 10, and the chromatogram is shown in fig. 8.
TABLE 10 quantitative Limit results
As can be seen from the data in Table 10, the quantitative limit concentrations of the two impurities are respectively 15.852ng/mL and 15.848ng/mL, which are respectively equivalent to 0.16ppm and 0.16ppm of the main component, and the experimental result shows that the product can be quantitatively detected under a relatively high concentration.
The results of the quantitative limits of methyl p-toluenesulfonate and ethyl p-toluenesulfonate show that the method meets the detection requirement of low-concentration quantification and completely meets the detection of the coverage limit of basic toxic impurities of the ganciclovir condensation compound.
6.2, detection Limit
Taking a proper amount of methyl p-toluenesulfonate and ethyl p-toluenesulfonate reference substances, precisely weighing, adding a diluent for dissolving, quantitatively diluting to prepare a solution with a certain concentration, gradually diluting the solution to the proper concentration by using the diluent, and under the condition of genotoxic impurity detection chromatography, obtaining a product with a signal-to-noise ratio of about 3: the detection limit concentration was determined by the amount injected into the chromatograph at around 1, and the results are shown in table 11 and the chromatogram is shown in fig. 9.
TABLE 11 detection Limit results
As can be seen from the data in Table 11, the detection limit concentrations of the two impurities are 4.954ng/mL and 4.952ng/mL respectively, which are respectively equivalent to 0.05ppm and 0.05ppm of the main component, and the experimental result shows that the chromatographic condition of the invention has high sensitivity and is suitable for detecting the two impurities.
7. Accuracy test
To examine the accuracy of the genotoxic impurity measurement method, a recovery rate measurement was performed. Weighing 0.5g of a ganciclovir condensation compound sample, precisely weighing, placing in a 5mL measuring flask, and taking the sample as a blank sample for accuracy verification; 0.08g of methyl p-toluenesulfonate and 0.08g of ethyl p-toluenesulfonate were each taken and diluted with methanol stepwise to prepare a mixed solution containing about 400ng per 1mL as an impurity mixed stock solution. Then, 2.0mL of the impurity-containing mixed stock solution was precisely measured into a 10mL measuring flask, and diluted with methanol to prepare a mixed solution containing about 80ng per 1mL as an impurity-containing mixed solution.
Taking the content of gene impurities (0.8ppm matrix sample concentration) as a reference 100%, precisely measuring 1.0mL of impurity mixed solution, respectively placing 1.0mL and 1.5mL of impurity mixed stock solution in a 5mL measuring flask containing a matrix sample, uniformly mixing, metering volume with methanol, preparing into a solution containing 20%, 100% and 150% of impurities, and paralleling 3 times by the same method to serve as a test solution. The specific formulation is shown in Table 12.
Table 12 accuracy methods of formulating various solutions
Precisely measuring 20 mu L of each solution, respectively injecting into a liquid chromatograph, recording a chromatogram, measuring peak areas (wherein the peak areas of methyl p-toluenesulfonate and ethyl p-toluenesulfonate in a sample matrix need to be deducted from the peak areas of methyl p-toluenesulfonate and ethyl p-toluenesulfonate in the sample matrix), calculating the recovery rate, and obtaining the experimental results shown in tables 13 and 14 below.
The recovery rate calculation formula is as follows:
percent recovery of impurities is measured/added multiplied by 100 percent
The amount added (ng) is the amount of impurity weighed multiplied by PxV Additive for food ×10 9 Dilution factor
Measured quantity (ng) ═ W S ×(A Quasi-miscellaneous -A Supply of impurities ×W Quasi-phase /W Quasi-empty )×10 9 /(A S ×100×100×100)
In the formula: v Additive for food Verifying the volume (mL) of the impurity mixed stock solution or the impurity mixed solution added into the solution for accuracy; p is the content (%) of the impurity reference substance; w S Weighing the impurity reference substance (g); a. the Quasi-miscellaneous Verifying the peak area of the impurity in the solution chromatogram for accuracy; a. the Supply of impurities Verifying the peak area of the impurity in the blank solution chromatogram for accuracy; w is a group of Quasi-drug Sample weighing (g) of ganciclovir condensate solution for accuracy verification of formulation; w Quasi-empty Verifying the weighed amount (g) of the ganciclovir condensate of the blank solution for preparation accuracy; the dilution ratio is 10 for directly adding the mixed stock solution of impurities 6 The impurity mixed liquor is 5 multiplied by 10 6 ；A S The average of the areas of the peaks of the impurities in the control solution was 6 times.
TABLE 13 methyl p-toluenesulfonate accuracy results
TABLE 14 accuracy results for ethyl p-toluenesulfonate
As can be seen from tables 13 to 14, the sample pretreatment adopts ultrasound for 5min, and the average recovery rate of methyl p-toluenesulfonate under each concentration is within the range of 101.3 to 106.0 percent and the RSD is within the range of 1.3 to 2.9 percent under the condition of filtering through a filter membrane of 0.45 mu m; the average recovery rate of the ethyl p-toluenesulfonate at each concentration is within the range of 103.1-106.7%, and the RSD is within the range of 0.8-2.3%;
the sample pretreatment adopts ultrasound for 5min, and the average recovery rate of the methyl p-toluenesulfonate under each concentration is 104.0-106.9% and the RSD is 0.4-1.9% under the condition of filtering by a 0.22 mu m filter membrane; the average recovery rate of the ethyl p-toluenesulfonate at each concentration is within the range of 102.6-106.7%, and the RSD is within the range of 0.5-4.3%;
the sample pretreatment adopts ultrasonic treatment for 20min, and the average recovery rate of the methyl p-toluenesulfonate under each concentration is within the range of 96.6-105.2% and the RSD is within the range of 1.1-1.7% under the condition of filtering by a 0.45-micron filter membrane; the average recovery rate of the ethyl p-toluenesulfonate at each concentration is within the range of 104.4-108.0%, and the RSD is within the range of 1.6-4.4%;
meets the recovery rate requirement of an external standard method (the determination result of the content of impurities with the content of 0.1-1.0 ppm is between 75 and 120 percent, and the RSD is less than or equal to 8.0 percent). The method is proved to have good accuracy in detecting the methyl p-toluenesulfonate and the ethyl p-toluenesulfonate. From the time and cost considerations, the sample pretreatment conditions were selected using sonication for 5min and filtration through a 0.45 μm filter.
8. Stability test
Preparing a reference substance stability test solution: taking 0.08g of methyl p-toluenesulfonate and ethyl p-toluenesulfonate, precisely weighing, placing in a 100mL measuring flask, dissolving with methanol, diluting to scale, shaking, and gradually diluting with methanol to obtain a control solution containing 80ng of the control per 1 mL. Taking the reference solution, standing at normal temperature for 24 hours, injecting 20 μ L of sample at 0h, 2h, 4h, 6.5h, 9h, 12h and 24h respectively, and recording chromatogram; the stability of the control solutions was measured by the peak area change of methyl p-toluenesulfonate and ethyl p-toluenesulfonate, and the results are shown in Table 15.
TABLE 15 control solution stability
As can be seen from Table 15, the peak areas RSD of the control samples of methyl p-toluenesulfonate and ethyl p-toluenesulfonate were 0.8% and 1.0% respectively, both of which were less than 5.0% and did not change much within 24 hours at room temperature; after the ganciclovir condensation compound test solution is placed at the normal temperature for 0h and 24h, methyl p-toluenesulfonate and ethyl p-toluenesulfonate are not detected, which shows that the test solution is stable at room temperature for 24 h.
9. Durability test
In order to examine the tolerance degree of the method for detecting the sample when the chromatographic condition is slightly changed, and examine the durability of the chromatographic condition, the specific parameter changes are as follows:
TABLE 16 chromatographic Condition variation parameters
Preparing a system applicability solution: taking 0.08g of each of methyl p-toluenesulfonate and ethyl p-toluenesulfonate reference substances, precisely weighing, dissolving with methanol, and gradually diluting each 1mL of solution containing 400ng of each impurity as impurity reference substance stock solution; accurately weighing 0.5g of ganciclovir condensation compound, placing the ganciclovir condensation compound in a 5mL measuring flask, accurately weighing 1.0mL of impurity reference substance stock solution, adding methanol to a constant volume to scale, carrying out ultrasonic treatment for 5min, filtering through a 0.45 mu m filter membrane, and taking filtrate as system applicability solution.
The solution was precisely taken at 20. mu.L, injected into a liquid chromatograph, and the durability of each condition was examined, and the results are shown in tables 17 to 20:
TABLE 17 durability different flow rates-System suitability results
TABLE 18 durability different column temperatures-System suitability results
TABLE 19 different mobile phase compositions of durability-System suitability results
Table 20 results of different column-system applicability for durability
From the results, the chromatographic conditions are finely adjusted, and the determination results of all impurities are basically consistent under the conditions of different flow rates, different column temperatures and different chromatographic columns, which shows that the micro-change of the chromatographic conditions in the chromatographic conditions of the method has no influence on the determination of genotoxic impurities in the ganciclovir condensation compound, and the chromatographic conditions of the method have good durability.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (6)
1. A method for analyzing and determining genotoxic impurities in a ganciclovir condensation compound by using an HPLC method is characterized in that: the method for analyzing and detecting the impurity contents of methyl p-toluenesulfonate and ethyl p-toluenesulfonate in a ganciclovir condensation compound by utilizing a high performance liquid chromatography comprises the following steps of:
(1) and preparing a reference substance solution: taking methyl p-toluenesulfonate and ethyl p-toluenesulfonate reference substances, dissolving and diluting with a diluent to prepare a mixed solution serving as a genotoxic impurity reference substance solution;
(2) and preparing a test solution: putting the raw material drug of the ganciclovir condensation compound into a measuring flask, adding a diluent to a constant volume to scale, ultrasonically extracting, filtering, and sampling the filtrate to be used as a test solution;
(3) and chromatographic conditions: the mobile phase is acetonitrile-phosphoric acid aqueous solution; adopting an isocratic elution mode; flow rate: 1.1-1.3 mL/min; column temperature: 22-32 ℃; detection wavelength: 225 nm; sample introduction amount: 20 mu L of the solution; octadecyl bonded silica gel chromatographic column; the elution time is 30 min;
(4) and detecting a chromatogram map: measuring 20 μ L of each of the sample solution and the reference solution, injecting into a liquid chromatograph, and recording chromatogram;
(5) and separation and measurement: if the chromatogram of the sample solution does not have a chromatographic peak consistent with the retention time of methyl p-toluenesulfonate and ethyl p-toluenesulfonate, judging that the ganciclovir condensate sample does not contain methyl p-toluenesulfonate and ethyl p-toluenesulfonate;
or, if the chromatogram of the sample solution has chromatographic peaks consistent with the retention time of methyl p-toluenesulfonate and ethyl p-toluenesulfonate, calculating the impurity content (ppm) ═ Cs × A by a peak area external standard method T ×10 6 /(As×C T )；
In the formula A T Is the peak area of each impurity in the test sample; as is the peak area of the impurity reference substance; cs is the concentration of the impurity reference substance; c T Is the concentration of the test sample.
2. The method of claim 1 for the analytical determination of genotoxic impurities in ganciclovir condensates by HPLC method, characterized in that: and (3) the diluent in the step (1) and the diluent in the step (2) are both methanol.
3. The method of claim 1 for the analytical determination of genotoxic impurities in ganciclovir condensates by HPLC method, characterized in that: the ultrasonic extraction time in the step (2) is 5 minutes; filtering with 0.45 μm microporous membrane.
4. The method of claim 1 for the analytical determination of genotoxic impurities in ganciclovir condensates by HPLC method, characterized in that: the volume ratio of the acetonitrile-phosphoric acid aqueous solution in the mobile phase in the step (3) is 50: 50; the mass percentage of the phosphoric acid aqueous solution is 0.08-0.12%.
5. The method for the analytical determination of genotoxic impurities in ganciclovir condensates by HPLC method according to claim 4, wherein: the mass concentration of the phosphoric acid aqueous solution in the step (3) is 0.10%.
6. The method of claim 1 for the analytical determination of genotoxic impurities in ganciclovir condensates by HPLC method, characterized in that: the chromatographic column in the step (3) is Agilent Eclipse plus C18, 250X 4.6mm, 5 μm; the flow rate is 1.0 mL/min; column temperature: at a temperature of 27 ℃.
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