CN108593827B - Method for detecting sorbic acid content in levocarnitine oral solution - Google Patents
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Abstract
The invention belongs to the field of drug analysis, and relates to a method for detecting the sorbic acid content in a levocarnitine oral solution, which comprises the following steps (1) of chromatographic conditions: octadecylsilane chemically bonded silica chromatographic column; wavelength of ultraviolet detector: 257 nm; mobile phase: a mixed solution of phosphate buffer solution and methanol; (2) preparing a test solution: (3) preparation of control solutions: (4) the determination method comprises the following steps: precisely measuring the reference solution, injecting into a liquid chromatograph, repeating for 3-6 times, and recording chromatogram, wherein the relative standard deviation of sorbic acid peak area should be less than or equal to 2.0%. Precisely measuring a test solution, injecting the test solution into a liquid chromatograph, recording a chromatogram, and calculating according to the peak area by an external standard method to obtain the test solution; the method has the advantages of short detection period, satisfactory reproducibility, accuracy, stability and durability, low cost and the like.
Description
Technical Field
The invention relates to a method for detecting the sorbic acid content in a levocarnitine oral solution in the field of pharmaceutical analysis.
Background
Sorbic acid (English name: Sorbicic), 2, 4-hexadienoic acid and 2-propenyl acrylic acid. Sorbic acid is an efficient and safe preservative, is widely applied to industries such as food, beverage, tobacco, pesticide, cosmetics and the like, and can also be applied to industries such as resin, spice and rubber. The application amount in developed countries in the west is large, but the application range in China is not wide. Sorbic acid is an unsaturated fatty acid, which, like other natural fatty acids, participates in metabolic processes in the human body and is digested and absorbed by the human body to produce carbon dioxide and water. In terms of safety, sorbic acid is an internationally recognized as safe (GRAS) preservative and has high safety. The food and agriculture organization, the world health organization and the FDA in the United states all give a positive on the safety of the food and agriculture organization and the world health organization. Sorbic acid can effectively inhibit the activity of mould, microzyme and aerobic bacteria and prevent the growth and reproduction of harmful microorganisms such as botulium, staphylococcus and salmonella, but has no effect on beneficial microorganisms such as anaerobic bacillus and lactobacillus acidophilus, and the growth inhibition effect of the sorbic acid is stronger than the sterilization effect, thereby effectively prolonging the storage time of food and keeping the flavor of the original food. The preservative effect is 5-10 times of that of sodium benzoate of the same kind of products, the toxic and side effects are lower than those of benzoic acid, vitamin C and salt, and the toxicity is only one fourth of that of benzoic acid and one half of that of salt. Sorbic acid has no carcinogenic and teratogenic effects on human bodies. The replacement of sodium benzoate by sorbic acid is a trend in the development of the food industry. However, if the sorbic acid added in the food is too high seriously, the bone growth can be inhibited to a certain extent and the health of the kidney and the liver is damaged if the sorbic acid is taken by consumers for a long time. At present, no relevant literature and patent about the detection of the sorbic acid content in the levocarnitine oral solution are found, so that the development of a method for detecting the sorbic acid content in the levocarnitine oral solution is a new problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a method for detecting the sorbic acid content in a levocarnitine oral solution, which adopts a high performance liquid chromatography for detection, establishes a method for detecting the sorbic acid content in the levocarnitine oral solution by optimizing chromatographic conditions, selecting component proportion in a mobile phase and selecting the mobile phase, and has the advantages of short detection period, satisfactory reproducibility, accuracy, stability and durability, low cost and the like.
The purpose of the invention is realized as follows: a method for detecting the sorbic acid content in a levocarnitine oral solution comprises the following steps:
(1) chromatographic conditions are as follows:
octadecylsilane chemically bonded silica chromatographic column;
wavelength of ultraviolet detector: 257 nm;
mobile phase: a mixed solution of phosphate buffer solution and methanol;
(2) preparing a test solution:
(3) preparation of control solutions:
(4) the determination method comprises the following steps:
precisely measuring the reference solution, injecting into a liquid chromatograph, repeating for 3-6 times, and recording chromatogram, wherein the relative standard deviation of sorbic acid peak area should be less than or equal to 2.0%. Precisely measuring a test solution, injecting the test solution into a liquid chromatograph, recording a chromatogram, and calculating according to the peak area by an external standard method to obtain the test solution;
(5) formula for calculation
In the formula:
Ato pair: area of main peak of control solution;
Asample (A): test solutionThe area of the main peak;
Wto pair: weight of control, mg;
p: purity of control,%;
in the mobile phase, the volume ratio of the phosphate buffer solution to the methanol is 65-75: 25-35; in the mobile phase, the volume ratio of the phosphate buffer solution to the methanol is 70: 30, of a nitrogen-containing gas; the preparation method of the phosphate buffer solution comprises the following steps: taking 11.5ml of phosphoric acid, adding water to dilute to 1900ml, adjusting the pH to 2.0-3.0 by using 1mol/L sodium hydroxide solution, adding 1.1g of sodium heptanesulfonate, and shaking to dissolve; the octadecylsilane bonded silica gel chromatographic column is selected from one of Agilent TC-C18 chromatographic column, Agilent zorbax SB-C18 chromatographic column, Waters symmetry shield RP18 chromatographic column, Diamonsil C18 chromatographic column, Agilent eclipse XDB-C18 chromatographic column, Agilent HC-C18 chromatographic column, Agilent Extend C18 chromatographic column, Ecosil C18 chromatographic column, SHISEIDO MG II C18 chromatographic column, SHISEIDO MG C18 chromatographic column, SHISEDO C18 chromatographic column, Waters XTERRAMS C18 chromatographic column, Waters XTERRARP 18 chromatographic column, Waters Spherissorb 2 chromatographic column; the temperature of the chromatographic column is 20-40 ℃, and the flow rate of the mobile phase is 0.5-2.0 ml/min; the specification of the chromatographic column is one of 250 multiplied by 4.6mm, 5 μm, 150 multiplied by 4.6mm, 3.5 μm, 250 multiplied by 4.6mm and 3.5 μm; the method for preparing the test solution comprises the following steps: precisely measuring 2ml of levocarnitine oral solution, placing the levocarnitine oral solution into a 100ml volumetric flask, diluting with water to scale, and shaking up to obtain a test solution; the method for preparing the reference substance solution comprises the following steps: accurately weighing sorbic acid reference substance 15mg, placing into a 100ml measuring flask, adding appropriate amount of ethanol for dissolving, diluting with water to scale, and shaking; precisely measuring 2ml, placing in a 10ml volumetric flask, diluting with water to scale, and shaking up to obtain a reference solution; the sample amount of the test solution and the reference solution is 5-100 mul, and the preferable sample amount is 5-20 mul; the reference solution is precisely measured and injected into the liquid chromatograph, and the process is repeated for 5 times.
The key point of the invention is that the method adopts HPLC for detection, a chromatographic column with octadecylsilane chemically bonded silica as a filler is adopted, the wavelength is scanned by an ultraviolet-visible spectrophotometer to select the maximum absorption wavelength, and the method for detecting the sorbic acid content in the levocarnitine oral solution is established by optimizing chromatographic conditions, selecting a mobile phase and selecting the component proportion in the mobile phase; the result of methodology verification shows that blank auxiliary materials (other components except sorbic acid in the technical formula of the levocarnitine oral solution) have no interference on sorbic acid detection, and the blank auxiliary materials have strong specificity, the reproducibility result RSD is 0.7%, the accuracy detection result recovery rate is 100.5%, the RSD is 0.3%, the solution stability result RSD is 0.1%, and the durability result RSD is 0.2%, and all the blank auxiliary materials can meet the requirements.
Compared with the prior art, the method for detecting the sorbic acid content in the levocarnitine oral solution has the advantages of simple, convenient and accurate operation, low detection cost and the like. Will be widely applied in the field of pharmaceutical analysis.
Drawings
The present invention will be described in detail below with reference to the accompanying drawings and examples.
FIG. 1 is an HPLC chromatogram of a blank solution of the present invention.
FIG. 2 is an HPLC chromatogram of a test solution of the present invention.
FIG. 3 is an HPLC chromatogram of a control solution of the present invention.
In the figure, 1 is sorbic acid, and 2 is levocarnitine.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to facilitate a better understanding of the present invention, but the present invention is not limited to the following examples.
Example one
1. Chromatographic conditions
A chromatographic column: diamonsil C18(2) 250X 4.6mm 5 μm, column temperature: 35 ℃, flow rate: 1.0ml/min, detection wavelength: 257nm, sample injection amount: 20 μ l.
2. Preparation of solutions
2.1 mobile phase: a mixture of 70 volumes of phosphate buffer and 30 volumes of methanol.
2.2 phosphate buffer: 11.5ml of phosphoric acid was taken, diluted to 1900ml with water, adjusted to pH 2.0-3.0 with about 100ml of sodium hydroxide solution (1mol/L), and dissolved with 1.1g of sodium heptanesulfonate with shaking.
2.3 test solution: 2 samples of the sample are taken, 2ml of each sample is precisely measured, and the samples are respectively placed in 100ml volumetric flasks and are diluted by water to scale and shaken up to be used as sample solutions 1 and 2.
2.4 control solutions: accurately weighing sorbic acid reference substances 15.58mg and 15.25mg, placing in a 100ml measuring flask, adding an appropriate amount of ethanol for dissolving, diluting with water to scale, and shaking up; precisely measure 2ml of the solution and place the solution in a 10ml volumetric flask, dilute the solution to the scale with water, shake the solution evenly, and use the solution as a reference substance solution 1 and 2.
3. Measurement method
Injecting the reference substance solution 1 into a liquid chromatograph, carrying out parallel sample injection for 5 needles, carrying out sample injection amount of 20 mu l, recording a spectrogram, and calculating the relative standard deviation RSD of the sorbic acid peak area to be less than or equal to 2.0%; injecting the reference solution 2 into a liquid chromatograph, and injecting 2 needles in parallel with the sample injection amount of 20 mul. The sample solutions 1 and 2 are injected into a liquid chromatograph, the sample amount is 20 mu l, and the chromatogram is recorded.
The peak areas of 5 consecutive needles of control solution 1 are:
A1＝7995.1 A2＝7993.5 A3＝7991.5 A4＝7993.6
A5＝7992.9 Ato 1Average 7993.3 RSD 0.1%
The peak area of control solution 2 was:
A1＝7825.5 A2＝7840.0 Ato 2Average 7832.8
4. Sample assay
Injecting the sample solution into a liquid chromatograph, and recording the chromatogram once again.
Main peak area of test solution 1: a 1-7593.3 a 2-7584.2 aSample 1Average 7588.8
Main peak area of test solution 2: a 1-7594.7 a 2-7595.6 aSample 2Average 7595.2
5. Calculation results
In the formula:
Ato pair: area of main peak of control solution;
Asample (A): the main peak area of the test solution;
Wto pair: weight of control, mg;
p: purity of control,%.
Test solution 1:
sample solution 2:
example selection of the component ratios in the two flowing phases
1. A chromatographic column: diamonsil C18(2) 250X 4.6mm 5 μm, column temperature: 35 ℃, flow rate: 1.0ml/min, detection wavelength: 257nm, sample injection amount: 20 μ l.
2. Preparation of solutions
2.1 mobile phase: a mixture of 72 volumes of phosphate buffer and 28 volumes of methanol.
2.2 phosphate buffer: 11.5ml of phosphoric acid was taken, diluted to 1900ml with water, adjusted to pH 2.0-3.0 with about 100ml of sodium hydroxide solution (1mol/L), and dissolved with 1.1g of sodium heptanesulfonate with shaking.
2.3 test solution: 2 samples of the sample are taken, 2ml of each sample is precisely measured, and the samples are respectively placed in 100ml volumetric flasks and are diluted by water to scale and shaken up to be used as sample solutions 3 and 4.
2.4 control solutions: accurately weighing sorbic acid reference substances 15.28mg and 15.62mg, placing in a 100ml measuring flask, adding an appropriate amount of ethanol for dissolving, diluting with water to scale, and shaking up; 2ml of the solution was precisely measured and placed in a 10ml volumetric flask, diluted to the mark with water and shaken up to give control solutions 3 and 4.
3. Measurement method
Injecting the reference solution 3 into a liquid chromatograph, and injecting sample 5 needles in parallel with the sample injection amount of 20 mul. Recording a spectrogram, and calculating the relative standard deviation RSD of the sorbic acid peak area to be less than or equal to 2.0 percent; injecting the reference substance solution 4 into a liquid chromatograph, and parallelly injecting 2 needles with the injection amount of 20 mul. The sample solutions 3 and 4 are injected into a liquid chromatograph, the sample amount is 20 mu l, and the atlas is recorded.
The peak areas of 5 consecutive needles of control solution 3 are:
A1＝8004.1 A2＝8007.8 A3＝8005.2 A4＝8006.0
A5＝7999.6 Ato 1Average 8004.5 RSD 0.1%
The peak area of control solution 4 was:
A1＝8161.1 A2＝8163.8 Ato 2Average 8162.4
4. Sample assay
Injecting the sample liquid into a liquid chromatograph, and recording the chromatogram once again.
Main peak area of test solution 3: a 1-7755.0 a 2-7759.3 aSample 1Average 7757.2
Main peak area of test solution 4: a 1-7751.9 a 2-7756.3 aSample 2Average 7754.1
5. Calculation results
In the formula:
Ato pair: area of main peak of control solution;
Asample (A): the main peak area of the test solution;
Wto pair: weight of control, mg;
p: purity of control,%.
Test solution 3:
test solution 4:
example selection of tristimulus columns
1. A chromatographic column: agilent zorbax SB-C18250X 4.6mm 5 μm, column temperature: 35 ℃, flow rate: 1.0ml/min, detection wavelength: 257nm, sample injection amount: 20 μ l.
2. Preparation of solutions
2.1 mobile phase: a mixture of 70 volumes of phosphate buffer and 30 volumes of methanol.
2.2 phosphate buffer: 11.5ml of phosphoric acid was taken, diluted to 1900ml with water, adjusted to pH 2.0-3.0 with about 100ml of sodium hydroxide solution (1mol/L), and dissolved with 1.1g of sodium heptanesulfonate with shaking.
2.3 test solution: 2 samples of the sample are taken, 2ml of each sample is precisely measured, and the samples are respectively placed in 100ml volumetric flasks and are diluted by water to scale and shaken up to be used as sample solutions 5 and 6.
2.4 control solutions: accurately weighing sorbic acid reference substances 15.05mg and 14.48mg, placing in a 100ml measuring flask, adding appropriate amount of ethanol for dissolving, diluting with water to scale, and shaking; 2ml of the solution was precisely measured and placed in a 10ml volumetric flask, diluted to the mark with water and shaken up to give control solutions 5 and 6.
3. Measurement method
Injecting the control solution 5 into a liquid chromatograph, and injecting sample 5 needles in parallel with the sample injection amount of 20 μ l. Recording a spectrogram, and calculating the relative standard deviation RSD of the sorbic acid peak area to be less than or equal to 2.0 percent; injecting the reference solution 6 into a liquid chromatograph, and injecting 2 needles in parallel with the sample injection amount of 20 mul. The sample solutions 5 and 6 are injected into a liquid chromatograph, the sample amount is 20 mu l, and the chromatogram is recorded.
The peak areas of 5 consecutive needles of control solution 5 are:
A1＝7892.6 A2＝7894.3 A3＝7894.3 A4＝7890.5
A5＝7897.0 Ato 1Average 7893.7 RSD 0.1%
The peak area of control solution 6 was:
A1＝7536.4 A2＝7534.1 Ato 2Average 7535.2
4. Sample assay
Injecting the sample liquid into a liquid chromatograph, and recording the chromatogram once again.
Main peak area of test solution 5: a 1-7751.9 a 2-7758.8 aSample 1Average 7755.4
Main peak area of test solution 6: a 1-7755.3 a 2-7763.0 aSample 2Average 7759.2
5. Calculation results
In the formula:
Ato pair: area of main peak of control solution;
Asample (A): the main peak area of the test solution;
Wto pair: weight of control, mg;
p: purity of control,%.
Sample solution 5:
test solution 6:
note: all the samples to be tested in the specification are levocarnitine oral solution produced by Shenyang first pharmaceutical company Limited of northeast pharmaceutical group, and the specifications are as follows: 10 ml: 1g, approved article No.: the national drug standard H19990372; the batch number used: 170508.
the chromatographic column using octadecylsilane chemically bonded silica as a filler has the following sources:
Claims (4)
1. a method for detecting the sorbic acid content in a levocarnitine oral solution is characterized by comprising the following steps: the detection method comprises the following steps:
(1) chromatographic conditions are as follows:
octadecylsilane chemically bonded silica chromatographic column;
wavelength of ultraviolet detector: 257 nm;
mobile phase: a mixed solution of phosphate buffer solution and methanol;
(2) preparing a test solution:
(3) preparation of control solutions:
(4) the determination method comprises the following steps:
precisely measuring the reference solution, injecting into a liquid chromatograph, repeating for 3-6 times, and recording chromatogram, wherein the relative standard deviation of sorbic acid peak area should be less than or equal to 2.0%; precisely measuring a test solution, injecting the test solution into a liquid chromatograph, recording a chromatogram, and calculating according to the peak area by an external standard method to obtain the test solution;
(5) formula for calculation
In the formula:
Ato pair: area of main peak of control solution;
Asample (A): the main peak area of the test solution;
Wto pair: weight of control, mg;
p: purity of control,%;
in the mobile phase, the volume ratio of phosphate buffer solution to methanol is 70-75: 25-30;
the preparation method of the phosphate buffer solution comprises the following steps: taking 11.5ml of phosphoric acid, adding water to dilute to 1900ml, adjusting the pH to 2.0-3.0 by using 1mol/L sodium hydroxide solution, adding 1.1g of sodium heptanesulfonate, and shaking to dissolve;
the octadecylsilane bonded silica gel chromatographic column is selected from one of Agilent TC-C18 chromatographic column, Agilent zorbax SB-C18 chromatographic column, Waters symmetry shield RP18 chromatographic column, Diamonsil C18 chromatographic column, Agilent eclipse XDB-C18 chromatographic column, Agilent HC-C18 chromatographic column, Agilent Extend C18 chromatographic column, Ecosil C18 chromatographic column, SHISEIDO MG II C18 chromatographic column, SHISEDO MG C18 chromatographic column, SHISEDO C18 chromatographic column, Waters XTERRA MS C18 chromatographic column, Waters XTERRA RP18 chromatographic column, Waters SpherissordODS b 2 chromatographic column; the specification of the chromatographic column is 250 multiplied by 4.6mm and 5 mu m;
the temperature of the chromatographic column is 20-40 ℃;
the method for preparing the test solution comprises the following steps: precisely measuring 2ml of levocarnitine oral solution, placing the levocarnitine oral solution into a 100ml volumetric flask, diluting with water to scale, and shaking up to obtain a test solution; the method for preparing the reference substance solution comprises the following steps: accurately weighing sorbic acid reference substance 15mg, placing into a 100ml measuring flask, adding appropriate amount of ethanol for dissolving, diluting with water to scale, and shaking; precisely measuring 2ml, placing in a 10ml volumetric flask, diluting with water to scale, shaking up, and using as a reference solution.
2. The method for detecting the sorbic acid content of the levocarnitine oral solution as claimed in claim 1, wherein the flow rate of the mobile phase is 0.5-2.0 ml/min.
3. The method for detecting the sorbic acid content in the levocarnitine oral solution as claimed in claim 1, wherein the sample volume of the test solution and the control solution is 5-100 μ l; the reference solution is precisely measured and injected into the liquid chromatograph, and the process is repeated for 5 times.
4. The method for detecting the sorbic acid content of the levocarnitine oral solution as claimed in claim 3, wherein the sample amount of the test solution and the control solution is 5-20 μ l.
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| US4055072A (en) * | 1975-09-19 | 1977-10-25 | Nasa | Apparatus for measuring a sorbate dispersed in a fluid stream |
| JPH03186760A (en) * | 1989-12-15 | 1991-08-14 | Shimadzu Corp | Simultaneous analysis of benzoic acid, sorbic acid and dehydroacetic acid |
| CA2792390C (en) * | 2008-09-11 | 2016-10-25 | The Iams Company | Animal feed kibble with protein-based core and related methods |
| CN101526509B (en) * | 2009-05-04 | 2012-11-21 | 佛山市海天调味食品股份有限公司 | Method for rapidly determining content of preservatives in condiment |
| CN101564378B (en) * | 2009-05-19 | 2011-11-30 | 邵爱霞 | levocarnitine oral solution and preparation method thereof |
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