CN117030871B - Method for detecting palmitic acid in paliperidone palmitate - Google Patents
Method for detecting palmitic acid in paliperidone palmitate Download PDFInfo
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- CN117030871B CN117030871B CN202310792968.1A CN202310792968A CN117030871B CN 117030871 B CN117030871 B CN 117030871B CN 202310792968 A CN202310792968 A CN 202310792968A CN 117030871 B CN117030871 B CN 117030871B
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- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 title claims abstract description 164
- 235000021314 Palmitic acid Nutrition 0.000 title claims abstract description 82
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 title claims abstract description 82
- VOMKSBFLAZZBOW-UHFFFAOYSA-N 3-{2-[4-(6-fluoro-1,2-benzoxazol-3-yl)piperidin-1-yl]ethyl}-2-methyl-4-oxo-6,7,8,9-tetrahydropyrido[1,2-a]pyrimidin-9-yl hexadecanoate Chemical compound FC1=CC=C2C(C3CCN(CC3)CCC3=C(C)N=C4N(C3=O)CCCC4OC(=O)CCCCCCCCCCCCCCC)=NOC2=C1 VOMKSBFLAZZBOW-UHFFFAOYSA-N 0.000 title claims abstract description 45
- 229960000635 paliperidone palmitate Drugs 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 36
- 239000002904 solvent Substances 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 239000007789 gas Substances 0.000 claims abstract description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000010828 elution Methods 0.000 claims abstract description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000000105 evaporative light scattering detection Methods 0.000 claims abstract description 6
- 238000002347 injection Methods 0.000 claims abstract description 6
- 239000007924 injection Substances 0.000 claims abstract description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000945 filler Substances 0.000 claims abstract description 4
- 235000019253 formic acid Nutrition 0.000 claims abstract description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 4
- 239000007864 aqueous solution Substances 0.000 claims abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 63
- 239000000243 solution Substances 0.000 claims description 52
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 44
- 239000013558 reference substance Substances 0.000 claims description 27
- 239000011550 stock solution Substances 0.000 claims description 16
- 238000009210 therapy by ultrasound Methods 0.000 claims description 15
- 238000007865 diluting Methods 0.000 claims description 14
- 239000000523 sample Substances 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- 238000005303 weighing Methods 0.000 claims description 11
- 238000012417 linear regression Methods 0.000 claims description 8
- 239000012488 sample solution Substances 0.000 claims description 5
- 239000012085 test solution Substances 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 3
- 238000011003 system suitability test Methods 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 20
- FPWNQPQTICPCOM-UHFFFAOYSA-N acetonitrile;propan-2-ol Chemical compound CC#N.CC(C)O FPWNQPQTICPCOM-UHFFFAOYSA-N 0.000 abstract description 11
- 239000012071 phase Substances 0.000 description 7
- 238000011084 recovery Methods 0.000 description 7
- 230000004807 localization Effects 0.000 description 3
- 230000001154 acute effect Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 201000000980 schizophrenia Diseases 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- PMXMIIMHBWHSKN-UHFFFAOYSA-N 3-{2-[4-(6-fluoro-1,2-benzoxazol-3-yl)piperidin-1-yl]ethyl}-9-hydroxy-2-methyl-6,7,8,9-tetrahydropyrido[1,2-a]pyrimidin-4-one Chemical compound FC1=CC=C2C(C3CCN(CC3)CCC=3C(=O)N4CCCC(O)C4=NC=3C)=NOC2=C1 PMXMIIMHBWHSKN-UHFFFAOYSA-N 0.000 description 1
- OUCSEDFVYPBLLF-KAYWLYCHSA-N 5-(4-fluorophenyl)-1-[2-[(2r,4r)-4-hydroxy-6-oxooxan-2-yl]ethyl]-n,4-diphenyl-2-propan-2-ylpyrrole-3-carboxamide Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@H]2OC(=O)C[C@H](O)C2)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 OUCSEDFVYPBLLF-KAYWLYCHSA-N 0.000 description 1
- -1 6-fluoro-1, 2-benzisoxazol-3-yl Chemical group 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- IPCSVZSSVZVIGE-UHFFFAOYSA-M hexadecanoate Chemical compound CCCCCCCCCCCCCCCC([O-])=O IPCSVZSSVZVIGE-UHFFFAOYSA-M 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000011866 long-term treatment Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 229960001057 paliperidone Drugs 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-N pyridine Substances C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
-
- 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
- G01N2030/042—Standards
- G01N2030/047—Standards external
Abstract
The invention discloses a method for detecting palmitic acid in paliperidone palmitate, which uses octadecylsilane chemically bonded silica as a filler, and uses a high performance liquid chromatography-evaporative light detector method to detect, and uses 0.05% formic acid aqueous solution as a mobile phase A; methanol is used as a mobile phase B, gradient elution is carried out, and the elution time is 50 minutes; flow rate: 0.5-1.0ml/min, column temperature: 10-30 ℃, sample injection amount: 20 μl, solvent: isopropanol-acetonitrile (1:1), evaporative light scattering detector conditions: the atomizing gas is nitrogen, the flow rate of the atomizing gas is 1.7L per minute, and the temperature of the drift tube is 60 ℃. The method has good specificity: the blank solvent, other impurities and main components do not interfere with palmitic acid detection, so that the palmitic acid response value in the paliperidone palmitate is greatly improved compared with an ultraviolet detector method, and the paliperidone palmitate can be effectively separated from other impurities.
Description
Technical Field
The invention relates to a palmitic acid detection method, in particular to a method for detecting palmitic acid in paliperidone palmitate.
Background
Paliperidone palmitate injection is mainly used for essenceThe treatment of the acute phase and the maintenance phase of the schizophrenia is the only novel anti-schizophrenia drug which can be used for rapidly acting in the acute phase and effectively preventing relapse in long-term treatment in China at present and is injected once a month. Paliperidone palmitate, chemical name: (±) -3-2- [4- (6-fluoro-1, 2-benzisoxazol-3-yl) -1-piperidine]Ethyl group]-6,7,8, 9-tetrahydro-2-methyl-4-oxo-4H-pyridine [1,2-a ]]Pyrimidine-9-yl palmitate, having the formula: c (C) 39 H 57 FN 4 O 4。 Palmitic acid is the starting material for the synthesis of paliperidone palmitate, which also degrades during placement to produce palmitic acid, and therefore reasonable quality control of palmitic acid in paliperidone palmitate is required. At present, the existing palmitic acid detection method mostly adopts a high-performance liquid-ultraviolet detector method, and a great number of defects are found in the experimental groping process: (1) palmitic acid absorbs terminally in the uv detector and responds little even at the lower detection wavelength of 210 nm;
(2) in this system, the palmitic acid peak shape is poor, and palmitic acid is interfered by the solvent peak;
(3) by adopting a high performance liquid-ultraviolet detector method, a sample solution with larger concentration needs to be prepared, the quantity of other impurities in the sample is larger, the sample solution cannot be effectively separated from palmitic acid, and the detection of the palmitic acid can be interfered.
To solve the above problems, the present application proposes an analytical method for detecting palmitic acid in paliperidone palmitate using an evaporative light scattering detector. After the evaporative light scattering detector is used, the palmitic acid response is obviously improved, and a good peak shape is obtained. The conditions of liquid phase gradient elution program, different brands of chromatographic columns replacement and the like are adjusted to enable the palmitic acid peak and other impurity peaks to achieve baseline separation, and the temperature of a drift tube in an evaporation light detector and parameters of a gas flow detector are further optimized, so that the detection of the palmitic acid is well reproduced. In summary, the optimized method of the evaporative light detector is used, so that the influence of other impurities can be effectively eliminated. The method has strong specificity and high accuracy, and can effectively control the quality of paliperidone palmitate.
The structure of palmitic acid and other partial impurities in paliperidone palmitate is as follows:
disclosure of Invention
The inventor groves a method capable of effectively detecting the palmitic acid in the paliperidone palmitate through a large number of experiments, and ensures that the quality of the paliperidone palmitate is controllable.
The invention is realized by the following technical scheme:
a method of detecting palmitic acid in paliperidone palmitate, the method comprising:
s1: chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler, and detection is carried out by a high performance liquid chromatography-evaporative light detector method, wherein the specification of a C18 chromatographic column is 4.6X250 mm and 5 mu m; taking 0.05% formic acid aqueous solution as a mobile phase A; methanol is used as a mobile phase B, gradient elution is carried out, and the elution time is 50 minutes; flow rate: 0.5-1.0ml/min, column temperature: 10-30 ℃, sample injection amount: 20 μl, solvent: isopropanol-acetonitrile (1:1), evaporative light scattering detector conditions: the atomizing gas is nitrogen, the flow rate of the atomizing gas is 1.7L per minute, and the temperature of the drift tube is 60 ℃;
s2: preparing a palmitic acid reference stock solution: taking a proper amount of palmitic acid reference substance, precisely weighing, adding isopropanol with the volume of 50% of a measuring flask, performing ultrasonic treatment to dissolve, diluting with acetonitrile to prepare a solution with the concentration of 150 mug in each 1ml, and shaking uniformly;
s3: control solutions (1) to (5): precisely measuring the palmitic acid reference stock solution, quantitatively diluting with isopropanol-acetonitrile (1:1) to obtain a solution containing 6-45 μg of palmitic acid in each 1 ml;
s4: test solution: taking proper amount of paliperidone palmitate, precisely weighing, adding isopropanol with 50% of the volume of a measuring flask, performing ultrasonic treatment to dissolve, diluting with acetonitrile to prepare a solution containing 10mg per 1ml, shaking uniformly, centrifuging for 10 minutes at 10000 revolutions per minute for 2 times, and taking supernatant;
s5: assay: precisely measuring the reference substance solutions (1) to (5), respectively injecting into a liquid chromatograph, recording the chromatograms, calculating a linear regression equation according to the logarithmic value of the concentration of the reference substance solution and the logarithmic value of the corresponding peak area, wherein the correlation coefficient is not less than 0.99, precisely measuring the sample solution, injecting into the liquid chromatograph, recording the chromatograms, and calculating the palmitic acid content in the sample according to the linear regression equation.
Preferably, the flow rate is 0.5ml/min.
Preferably, the column temperature is 30 ℃.
Compared with the prior art, the invention has the following advantages: the invention discloses a method for using a high performance liquid chromatography-evaporative light detector, which has good specificity: the blank solvent, other impurities and main components do not interfere with palmitic acid detection, so that the palmitic acid response value in the paliperidone palmitate is greatly improved compared with an ultraviolet detector method, and the paliperidone palmitate can be effectively separated from other impurities.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a specific pattern of the present invention, which includes impurity mixing;
FIG. 2 is a sample map;
FIG. 3 is a graph of the linear relationship between pre-validation of palmitic acid method in paliperidone palmitate and palmitic acid
Detailed Description
The invention will be described in further detail with reference to the drawings and the specific examples.
Example 1
The specific analysis method is as follows:
chromatographic column: octadecylsilane chemically bonded silica is used as filler (thermo Hypersil GOLD) TM C18，4.6×250mm，5μm)；
Mobile phase a:0.05% formic acid in water;
mobile phase B: methanol;
a detector: evaporative light scattering detector (detector condition: atomizing gas is nitrogen, atomizing gas flow rate is 1.7L per minute, drift tube temperature is 60 ℃);
flow rate: 0.5ml/min-1.0ml/min, the flow rate in this example is 0.5ml/min;
column temperature: the column temperature in this example is 30 ℃ at 10-30 ℃;
sample injection amount: 20 μl;
solvent: isopropanol-acetonitrile (1:1).
Gradient elution was performed as follows:
test solution: taking proper amount of paliperidone palmitate, precisely weighing, adding isopropanol with 50% of the volume of a measuring flask, performing ultrasonic treatment to dissolve, diluting with acetonitrile to prepare a solution containing 10mg per 1ml, shaking uniformly, centrifuging for 10 minutes at 10000 revolutions per minute for 2 times, and taking supernatant;
palmitic acid control stock solution: taking proper amount of palmitic acid reference substance, precisely weighing, adding 50% of isopropanol with volume of a measuring flask, performing ultrasonic treatment to dissolve, diluting with acetonitrile to obtain solution containing about 150 μg per 1ml, and shaking.
Control solutions (1) to (5): precisely weighing the palmitic acid reference stock solution, quantitatively diluting with isopropanol-acetonitrile (1:1) to obtain solutions containing 6 μg, 9 μg, 15 μg, 21 μg and 30 μg of palmitic acid in each 1ml, wherein the palmitic acid can be set to be 6-45 μg of palmitic acid in each 1 ml.
Assay: precisely measuring the reference substance solutions (1) to (5), respectively injecting into a liquid chromatograph, and recording the chromatograms. And calculating a linear regression equation by using the logarithmic value of the concentration of the reference substance solution and the logarithmic value of the corresponding peak area, wherein the correlation coefficient is not less than 0.99. And precisely measuring the solution of the sample, injecting the solution into a liquid chromatograph, recording the chromatogram, and calculating the palmitic acid content in the sample by a linear regression equation.
Example 2
Specialization of
Blank solvent: isopropanol-acetonitrile (1:1).
Test solution: proper amount of paliperidone palmitate is taken, precisely weighed, added with isopropanol with 50 percent of the volume of a measuring flask for ultrasonic dissolution, diluted with acetonitrile to prepare a solution containing 10mg per 1ml, shaken uniformly, centrifuged for 10 minutes at 10000 revolutions per minute, centrifuged for 2 times, and the supernatant is taken.
Palmitic acid localization solution: taking proper amount of palmitic acid reference substance, precisely weighing, adding 50% of isopropanol with volume of a measuring flask, performing ultrasonic treatment to dissolve, diluting with acetonitrile to obtain a solution containing about 15 μg per 1ml, and shaking uniformly.
Each impurity localization solution: taking the appropriate amounts of the reference substances of impurities SM1-D, impurity A, impurity C, impurity E, impurity F, impurity G and impurity H, respectively adding solvents to dissolve and dilute the reference substances to prepare solutions with the concentration of about 0.3mg in each 1 ml.
Mixing solution: taking proper amounts of paliperidone palmitate and impurities, precisely weighing, adding isopropanol with 50% of the volume of a measuring flask for ultrasonic dissolution, diluting with acetonitrile to prepare a solution containing 10mg of paliperidone palmitate and 15 mug of impurities in each 1ml, shaking uniformly, centrifuging for 10 minutes at 10000 revolutions per minute, centrifuging for 2 times, and taking supernatant.
Precisely measuring 20 μl of each solution, respectively injecting into a liquid chromatograph, and recording the chromatogram. The test results are shown in Table 1, and the test results show that the method has good specificity, and the blank solvent, other impurities and main components do not interfere with palmitic acid detection, and the results are shown in the accompanying figures 1-2.
TABLE 1 palmitic acid method validation-specificity test results in paliperidone palmitate
Example 3
System precision
Blank solvent: isopropanol-acetonitrile (1:1).
System precision solution: taking proper amount of palmitic acid reference substance, precisely weighing, adding 50% of isopropanol with volume of a measuring flask, performing ultrasonic treatment to dissolve, diluting with acetonitrile to obtain a solution containing about 15 μg per 1ml, and shaking uniformly.
And precisely measuring 20 mu l of system precision solution, injecting the solution into a liquid chromatograph, continuously injecting the sample for 6 times, and recording a chromatogram. The test results are shown in Table 2, the system precision of the method is good, the RSD of the retention time of the palmitic acid peak is 0.02%, and the RSD of the peak area is 0.70%.
TABLE 2 palmitic acid method validation-System precision test results in paliperidone palmitate
Example 4
Linearity and range
Blank solvent: isopropanol-acetonitrile (1:1).
Palmitic acid control stock solution: about 15mg of palmitic acid reference substance is taken, precisely weighed, placed in a 100ml measuring flask, added with 50ml of isopropanol and subjected to ultrasonic treatment to dissolve, diluted to a scale with acetonitrile and shaken well.
Control solutions of different concentrations of palmitic acid: 2ml, 3ml, 5ml, 7ml and 10ml of palmitic acid reference substance stock solution are respectively measured precisely, respectively, and are put into 50ml measuring flasks, diluted to the scale with solvent, and shaken uniformly to obtain linear relation solutions 1,2, 3, 4 and 5.
And precisely measuring 20 mu l of each linear relation solution, respectively injecting into a liquid chromatograph, and recording a chromatogram. And (3) plotting the logarithmic value of the concentration of the linear solution by using the logarithmic value of the peak area, and calculating a linear regression equation, a correlation coefficient, a residual square sum and a residual standard deviation by using a least square method. The results are shown in Table 3, FIG. 3. As is clear from the test results, palmitic acid has a good logarithmic linear relationship between the logarithmic concentration and the logarithmic peak area in the concentration range of 6.058. Mu.g/ml to 30.289. Mu.g/ml.
TABLE 3 palmitic acid method validation-Linear test results in paliperidone palmitate
Example 5
Quantitative limit
Quantitative limiting solution: and (3) taking a proper amount of palmitic acid reference substance, adding a proper amount of isopropanol to dissolve, and gradually diluting to a concentration with a signal to noise ratio S/N of more than or equal to 10 by using isopropanol-acetonitrile (1:1).
And precisely measuring 20 μl of the quantitative limiting solution, injecting into a liquid chromatograph, repeating sample injection for 6 times, and recording a chromatogram. The test results are shown in tables 4 and 5.
TABLE 4 palmitic acid method validation-quantitative limiting solution precision test results in paliperidone palmitate
TABLE 5 palmitic acid method validation-quantitative limit test results in paliperidone palmitate
Example 6
Accuracy of
Blank solvent: isopropanol-acetonitrile (1:1).
Palmitic acid control stock solution: about 15mg of palmitic acid reference substance is taken, precisely weighed, placed in a 100ml measuring flask, added with 50ml of isopropanol and subjected to ultrasonic treatment to dissolve, diluted to a scale with acetonitrile and shaken well.
Control solutions of different concentrations of palmitic acid: 2ml, 3ml, 5ml, 7ml and 10ml of palmitic acid reference substance stock solution are respectively measured precisely, respectively, and are put into 50ml measuring flasks, diluted to the scale with solvent, and shaken uniformly to obtain linear relation solutions 1,2, 3, 4 and 5.
Background value solution: about 100mg of paliperidone palmitate is taken, precisely weighed, placed in a 10ml measuring flask, added with 5ml of isopropanol, subjected to ultrasonic treatment for 10 minutes, added with acetonitrile to dilute to a scale, uniformly shaken, centrifuged for 10 minutes at 10000 revolutions per minute, centrifuged for 2 times, and the supernatant is taken to obtain the paliperidone palmitate.
Quantitative limit concentration recovery rate solution: about 500mg of paliperidone palmitate is taken, precisely weighed, placed in a 50ml measuring flask, added with 25ml of isopropanol, subjected to ultrasonic treatment for 10 minutes, precisely added with 2ml of palmitic acid reference substance stock solution, diluted to a scale with acetonitrile, shaken uniformly, centrifuged for 10 minutes at 10000 RPM), centrifuged for 2 times, and the supernatant is taken to obtain the paliperidone palmitate.
50% recovery solution: about 500mg of paliperidone palmitate is taken, precisely weighed, placed in a 50ml measuring flask, added with 25ml of isopropanol, subjected to ultrasonic treatment for 10 minutes, precisely added with 2.5ml of palmitic acid reference substance stock solution, diluted to a scale with acetonitrile, shaken uniformly, centrifuged for 10 minutes at 10000RPM, centrifuged for 2 times, and the supernatant is taken to obtain the paliperidone palmitate.
100% recovery solution: about 500mg of paliperidone palmitate is taken, precisely weighed, placed in a 50ml measuring flask, added with 25ml of isopropanol, subjected to ultrasonic treatment for 10 minutes, precisely added with 5ml of palmitic acid reference substance stock solution, diluted to a scale with acetonitrile, shaken uniformly, centrifuged for 10 minutes at 10000RPM, centrifuged for 2 times, and the supernatant is taken to obtain the paliperidone palmitate.
150% recovery solution: about 500mg of paliperidone palmitate is taken, precisely weighed, placed in a 50ml measuring flask, added with 25ml of isopropanol, subjected to ultrasonic treatment for 10 minutes, precisely added with 7.5ml of palmitic acid reference substance stock solution, diluted to a scale with acetonitrile, shaken uniformly, centrifuged for 10 minutes at 10000RPM, centrifuged for 2 times, and the supernatant is taken to obtain the paliperidone palmitate.
Precisely measuring 20 μl of each solution, respectively injecting into a liquid chromatograph, and recording the chromatogram. And calculating a linear regression equation by using the logarithmic value of the concentration of the reference substance solution and the logarithmic value of the corresponding peak area, and calculating the background quantity and the measured quantity of the palmitic acid in the sample solution and the accuracy solution by using the regression equation. The test results are shown in Table 6. From the test results, (1) the quantitative concentration limit recovery rate was good: the limit recovery rate of palmitic acid is 106.4 percent (which is 80 to 120 percent); (2) the method has good accuracy: the recovery rate of 50%, 100% and 150% of palmitic acid is 105.88% -111.05% (according to 80% -120%), and RSD is 2.56% and less than 5.0%.
TABLE 6 palmitic acid method validation-accuracy test results in paliperidone palmitate
Example 7
Durability of
Blank solvent: isopropanol-acetonitrile (1:1).
Palmitic acid localization solution: taking about 3mg of palmitic acid reference substance, precisely weighing, placing into a 10ml measuring flask, adding 5ml of isopropanol, performing ultrasonic treatment to dissolve, diluting to scale with acetonitrile, and shaking to obtain palmitic acid reference substance stock solution; precisely measuring 1ml of palmitic acid reference substance stock solution, placing into a 20ml measuring flask, adding solvent to dilute to scale, and shaking.
Test solution: proper amount of paliperidone palmitate is taken, precisely weighed, added with isopropanol with 50 percent of the volume of a measuring flask for ultrasonic dissolution, diluted with acetonitrile to prepare a solution containing 10mg per 1ml, shaken uniformly, centrifuged for 10 minutes at 10000 revolutions per minute, centrifuged for 2 times, and the supernatant is taken.
Precisely measuring 20 μl of each solution, respectively injecting into liquid chromatograph under standard conditions and different batch chromatographic column conditions, and recording chromatogram. The test results are shown in Table 7, and the method has good durability, basically consistent retention time of palmitic acid peak under the conditions of standard chromatographic conditions and the conditions of changing different batches of chromatographic columns of the same brand, and no interference of the palmitic acid detection by blank solvents, other impurities and main components.
TABLE 7 palmitic acid method validation in paliperidone palmitate-durability test results
Claims (3)
1. A method of detecting palmitic acid in paliperidone palmitate, the method comprising:
s1: chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler, and detection is carried out by a high performance liquid chromatography-evaporative light detector method, wherein the specification of a C18 chromatographic column is 4.6X250 mm and 5 mu m; taking 0.05% formic acid aqueous solution as a mobile phase A; methanol is used as a mobile phase B, gradient elution is carried out, and the elution time is 50 minutes; flow rate: 0.5-1.0ml/min, column temperature: 10-30 ℃, sample injection amount: 20 μl of isopropanol as solvent: acetonitrile = 1:1, evaporative light scattering detector conditions: the atomizing gas is nitrogen, the flow rate of the atomizing gas is 1.7L per minute, and the temperature of the drift tube is 60 ℃; the gradient elution procedure was as follows:
s2: preparing a palmitic acid reference stock solution: taking a proper amount of palmitic acid reference substance, precisely weighing, adding isopropanol with the volume of 50% of a measuring flask, performing ultrasonic treatment to dissolve, diluting with acetonitrile to prepare a solution with the concentration of 150 mug in each 1ml, and shaking uniformly;
s3: control solutions (1) to (5): precisely measuring a palmitic acid reference substance stock solution, and using isopropanol: acetonitrile = 1:1 quantitatively diluting to prepare a solution containing 6-45 mug of palmitic acid in each 1 ml;
s4: test solution: taking paliperidone palmitate, precisely weighing, adding isopropanol with 50% of the volume of a measuring flask for ultrasonic dissolution, diluting with acetonitrile to prepare a solution containing 10mg per 1ml, shaking uniformly, centrifuging for 10 minutes at 10000 revolutions per minute for 2 times, and taking supernatant;
s5: assay: precisely measuring the reference substance solutions (1) to (5), respectively injecting into a liquid chromatograph, recording the chromatograms, calculating a linear regression equation according to the logarithmic value of the concentration of the reference substance solution and the logarithmic value of the corresponding peak area, wherein the correlation coefficient is not less than 0.99, precisely measuring the sample solution, injecting into the liquid chromatograph, recording the chromatograms, and calculating the palmitic acid content in the sample according to the linear regression equation.
2. The method of detecting palmitic acid in paliperidone palmitate of claim 1, wherein the flow rate is 0.5ml/min.
3. The method of detecting palmitic acid in paliperidone palmitate of claim 1, wherein the column temperature is 30 ℃.
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