CN114280167A - Method for separating and detecting bis (2-hydroxyethyl) disulfide in calcium levofolinate - Google Patents
Method for separating and detecting bis (2-hydroxyethyl) disulfide in calcium levofolinate Download PDFInfo
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- CN114280167A CN114280167A CN202011032523.6A CN202011032523A CN114280167A CN 114280167 A CN114280167 A CN 114280167A CN 202011032523 A CN202011032523 A CN 202011032523A CN 114280167 A CN114280167 A CN 114280167A
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Abstract
The invention belongs to the field of analytical chemistry, and particularly relates to a method for separating and detecting bis (2-hydroxyethyl) disulfide in calcium levofolinate by high performance liquid chromatography, wherein a chromatographic column adopted in the method takes octadecyl bonded silica gel as a filler, and a mixed solution of an organic solvent and water as a mobile phase for elution, so that the bis (2-hydroxyethyl) disulfide is separated from the calcium levofolinate and related substances; the related substances are impurities of calcium levofolinate; the flow rate of the mobile phase is 0.9-1.1 ml/min; the temperature of the chromatographic column is 25-35 ℃; the wavelength of the detector is 247 +/-10 nm; the detection method is used for detecting the bis (2-hydroxyethyl) disulfide in the calcium levofolinate, other impurities do not interfere with the detection of the target object, the specificity and the durability are good, the accurate impurity content is obtained, and the method is simple, rapid and effective.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a method for separating and detecting bis (2-hydroxyethyl) disulfide in calcium levofolinate by an HPLC (high performance liquid chromatography) method.
Background
Calcium levofolinate with molecular formula C20H21CaN7O7Molecular weight: 511.5, Chinese cultural name: n- [4- [ (6S) - (2-amino-5-formyl-1, 4,5,6,7, 8-hexahydro-4-oxo-6-pteridinyl) methyl group]Amino group]benzoyl-L-glutamate calcium salt hydrate, calcium levofolinate for injection at present is mainly used for treating gastric cancer and colorectal cancer, and the structural formula is as follows:
impurities that may be present in calcium levofolinate: bis (2-hydroxyethyl) disulfide, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, mercaptoethanol and other impurities; wherein, the bis (2-hydroxyethyl) disulfide, the Chinese cultural name: 2, 2' -dithiodiethanol, formula C4H10O2S2Molecular weight 154.25, having the formula:
however, in the related research, the detection analysis of the bis (2-hydroxyethyl) disulfide in the calcium levofolinate is not found, which is not beneficial to the quality control of the calcium levofolinate drug.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide a method for separating bis (2-hydroxyethyl) disulfide from calcium levofolinate, which can effectively separate bis (2-hydroxyethyl) disulfide from calcium levofolinate and other related substances.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for separating bis (2-hydroxyethyl) disulfide from calcium levofolinate adopts a chromatographic column which takes octadecyl bonded silica gel as a filler and adopts a mixed solution of an organic solvent and water as a mobile phase for elution, and the bis (2-hydroxyethyl) disulfide is separated from the calcium levofolinate and related substances; the related substances are one or more of impurities A to I; the structural formulas of the levofolinic acid calcium, the bis (2-hydroxyethyl) disulfide and the related substances are as follows:
further, the volume ratio of the organic solution to water is 9-11: 91-89;
further, the organic solution is one or more of acetonitrile, methanol and ethanol;
specifically, the organic solution is acetonitrile;
further, the flow velocity of the mobile phase is 0.9-1.1 ml/min;
further, the temperature of the chromatographic column is 25-35 ℃.
The invention also aims to provide a method for identifying whether the double (2-hydroxyethyl) disulfide exists in the calcium levofolinate detection object by an HPLC method, and the method can effectively confirm whether the double (2-hydroxyethyl) disulfide exists in the calcium levofolinate detection object.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for identifying bis (2-hydroxyethyl) disulfide in calcium levofolinate, wherein the method in the first objective is utilized to separate the bis (2-hydroxyethyl) disulfide from calcium levofolinate and related substances in a calcium levofolinate detector, a detector is introduced for detection, a chromatogram obtained by detection is compared with a chromatogram of a known reference substance of bis (2-hydroxyethyl) disulfide, and whether the calcium levofolinate detector contains the bis (2-hydroxyethyl) disulfide is judged;
further, the detector wavelength is set to 247 ± 10 nm.
The invention also aims to provide a method for determining the content of the bis (2-hydroxyethyl) disulfide in the calcium levofolinate by an HPLC method, which can accurately determine the content of the bis (2-hydroxyethyl) disulfide in the calcium levofolinate and is beneficial to the quality control of medicines.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for determining the bis (2-hydroxyethyl) disulfide content in calcium levofolinate, the method comprising the steps of:
1) preparing a detection product solution and a reference product solution;
dissolving the detection product in a diluent to obtain a detection product solution; dissolving a bis (2-hydroxyethyl) disulfide standard substance with a diluent to prepare a reference substance solution;
2) separating;
separating the bis (2-hydroxyethyl) disulfide from the calcium levofolinate and related materials using the method described in objective one;
3) identifying;
identifying bis (2-hydroxyethyl) disulfide in the calcium levofolinate using the method described in objective two;
4) calculating the content;
according to the chromatogram obtained in the identification process and the peak area, calculating the content of the bis (2-hydroxyethyl) disulfide in the detection product solution according to an external standard method;
further, the diluent in the step 1) is water.
It is a fourth object of the present invention to provide a method for analyzing bis (2-hydroxyethyl) disulfide.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the method for detecting the bis (2-hydroxyethyl) disulfide comprises the steps of eluting a chromatographic column by using octadecyl bonded silica gel as a filler and using a mixed solution of acetonitrile and water as a mobile phase, separating the bis (2-hydroxyethyl) disulfide, introducing the separated bis (2-hydroxyethyl) disulfide into a detector with a detection wavelength of 247nm for detection, and measuring the content of the bis (2-hydroxyethyl) disulfide by an external standard method; the volume ratio of the acetonitrile to the water is 1: 9; the flow rate of the mobile phase is 0.9-1.1 ml/min; the temperature of the chromatographic column is 25-35 ℃;
the invention has the beneficial effects that: the content of the bis (2-hydroxyethyl) disulfide in the calcium levofolinate is detected by isocratic elution high performance liquid chromatography, the detection of the target object is not interfered by other impurities, the specificity and the durability are good, the accurate impurity content is obtained, and the method is simple, rapid and effective.
Drawings
FIG. 1 is a chromatogram of a control of the present method;
FIG. 2 is a chromatogram of a sample of the method;
FIG. 3 is a chromatogram of a blank control of the method;
FIG. 4 is a bis (2-hydroxyethyl) disulfide mapping chromatogram of the present method;
FIG. 5 is a chromatogram for locating the impurity A in the method;
FIG. 6 is a chromatogram for locating the impurity B in the method;
FIG. 7 is a chromatogram for locating the impurity C in the method;
FIG. 8 is a chromatogram for locating the impurity D in the method;
FIG. 9 is a chromatogram for locating the impurity E in the method;
FIG. 10 is a chromatogram for locating the impurity F in the method;
FIG. 11 is a chromatogram for locating the impurity G in the method;
FIG. 12 is a chromatogram for locating the impurity I in the present method;
FIG. 13 is a precision chromatogram of the present method;
FIG. 14 is a chromatogram of all mixtures of the present method;
FIG. 15 is a systematic chromatogram of a control solution of the present method;
FIG. 16 is a limit chromatogram of the method;
FIG. 17 is a detection limit chromatogram of the present method;
FIG. 18 is a control solution stability 1 chromatogram;
FIG. 19 is a mixed solution stability 1 chromatogram;
FIG. 20 is a control solution stability 2 chromatogram;
FIG. 21 is a control solution stability 2 chromatogram;
FIG. 22 is a durability-mixed solution-normal chromatogram;
FIG. 23 is a durability-mixed solution-flow rate 0.9 chromatogram;
FIG. 24 is a durability-mixed solution-flow rate 1.1 chromatogram;
FIG. 25 is a durability-mixed solution-column temperature 25 chromatogram;
FIG. 26 is a durability-mixed solution-column temperature 35 chromatogram;
fig. 27 is a durability-mixed solution-acetonitrile 9% chromatogram;
fig. 28 is a durability-mixed solution-acetonitrile 11% chromatogram.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example 1
The main apparatus is as follows: shimadzu LC-20A high performance liquid chromatograph.
Mobile phase: acetonitrile-water (10:90), isocratic elution;
collecting time: record to 1.2 times the retention time of the peak of bis (2-hydroxyethyl) disulfide (about 10 min);
a chromatographic column: octadecyl bonding silica gel is used as filler, Agilent ZORBAX SB-C184.6mm multiplied by 250mm, 5 μm;
flow rate: 1.0 ml/min;
detection wavelength: 247 nm;
column temperature: 30 ℃;
diluent agent: water;
sample introduction volume: 20 mu l of the mixture;
test solution: 2 mg/ml;
control solution: 3 mu g/ml;
the quantitative method comprises the following steps: and (4) an external standard method.
Example 2
Solution preparation:
test solution: taking a proper amount of calcium levofolinate sample, precisely weighing, adding water, ultrasonically dissolving, releasing into a solution containing about 2mg per 1ml, and shaking up to obtain the calcium levofolinate.
Control solution: taking a proper amount of a bis (2-hydroxyethyl) disulfide reference substance, adding water, quantitatively diluting to prepare a solution containing about 3 mu g of the bis (2-hydroxyethyl) disulfide in each 1ml, and shaking up to obtain the product.
Example 3
The test method comprises the following steps:
precisely measuring 20 μ l of each of the reference solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatogram until the retention time of the bis (2-hydroxyethyl) disulfide is 1.2 times. The content of bis (2-hydroxyethyl) disulfide in the levofolinic acid calcium is calculated by peak area according to an external standard method.
Taking a proper amount of the product, precisely weighing, adding water, ultrasonically dissolving, releasing to obtain a solution containing 2mg per 1ml, and shaking up to obtain a test solution; taking a proper amount of bis (2-hydroxyethyl) disulfide, adding water, quantitatively diluting to obtain a solution containing about 3 mu g of bis (2-hydroxyethyl) disulfide per 1ml, and shaking up to obtain a control solution. Performing high performance liquid chromatography (0512 in the four-part general regulation of the Chinese pharmacopoeia 2015 edition) with octadecylsilane chemically bonded silica as filler (Agilent ZORBAX SB-C184.6 mm × 250mm, 5 μm, or column with equivalent performance); acetonitrile-water (10:90) is used as a mobile phase; the detection wavelength is 247 nm; the flow rate was 1.0ml per minute; the column temperature was 30 ℃. Precisely measuring 20 μ l of each of the reference solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatogram until the retention time of the bis (2-hydroxyethyl) disulfide is 1.2 times. Calculated as peak area by external standard method. (the chromatogram obtained is shown in FIGS. 1 and 2). The retention time and the degree of separation of each impurity are shown in table 1:
TABLE 1 retention time and degree of separation of the respective impurities
Sequentially determining blank reference group (shown in figure 3), bis (2-hydroxyethyl) disulfide mapping chart (shown in figure 4 and table 2), impurity A-mapping chart (shown in figures 5-12 and tables 3-10), precision chromatogram (shown in figure 13 and table 11), all-substance mixed chromatogram (shown in figure 14 and table 12), system applicability chromatogram (shown in figure 15 and table 13), quantitative limit chromatogram (shown in figure 16 and table 14), detection limit chromatogram (shown in figure 17 and table 15), reference solution stability chromatogram 1 (shown in figure 18 and table 16), mixed solution stability chromatogram 1 (shown in figure 19 and table 17), reference solution stability chromatogram 2 (shown in figure 20 and table 18), mixed solution stability chromatogram 2 (shown in figure 21 and table 19), durability chromatogram (shown in figure 22 and table 20), Durability flow 0.9 chromatogram (see FIG. 23, Table 21), durability flow 1.1 chromatogram (see FIG. 24, Table 22), durability column temperature 25 chromatogram (see FIG. 25, Table 23), durability column temperature 35 chromatogram (see FIG. 26, Table 24), durability 9% acetonitrile chromatogram (see FIG. 27, Table 25), durability 11% acetonitrile chromatogram (see FIG. 28, Table 26).
TABLE 2 integration of bis (2-hydroxyethyl) disulfide mapping
TABLE 3 integration results of impurity A mapping
TABLE 4 integration of impurity B mapping
TABLE 5 integration of the localization patterns for impurity C
TABLE 6 integration results of impurity D mapping
TABLE 7 integration results of impurity E mapping
TABLE 8 integration results of impurity F mapping
TABLE 9 integration results of localization patterns for impurity G
TABLE 10 integration of the localization patterns for impurity I
TABLE 11 results of integration of precision
TABLE 12 integration results of all substance mixture chromatograms
TABLE 13 System adaptive integration results
TABLE 14 integration results of quantitative limiting chromatograms
TABLE 15 integration results of the detection limit chromatogram
TABLE 16 stability of control solution 1 integration results
TABLE 17 stability of mixed solution 1 integration results
TABLE 18 control solution stability 2 integration results
TABLE 19 control solution stability 2 integration results
TABLE 20 durability Mixed solution Normal flow integration results
TABLE 21 durability Mixed solution 0.9 flow Rate integration results
TABLE 22 integrated results of flow rate 1.1 for durable mixed solutions
TABLE 23 durability hybrid sol column temperature 25 integration results
TABLE 24 durability hybrid sol column temperature 35 integration results
TABLE 25 durability Mixed solution 9% acetonitrile integration results
TABLE 26 durability Mixed solution 11% acetonitrile integration results
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (10)
1. The method for separating the bis (2-hydroxyethyl) disulfide from the calcium levofolinate is characterized in that a chromatographic column adopted by the method takes octadecyl bonded silica gel as a filler, a mixed solution of an organic solvent and water is taken as a mobile phase for elution, and the bis (2-hydroxyethyl) disulfide is separated from the calcium levofolinate and related substances; the related substances are one or more of impurities A to I; the structural formulas of the levofolinic acid calcium, the bis (2-hydroxyethyl) disulfide and the related substances are as follows:
2. the method of claim 1, wherein the volume ratio of the organic solution to the water is 9-11: 91-89.
3. The method of claim 1, wherein the organic solution is one or more of acetonitrile, methanol, and ethanol.
4. The method of claim 1, wherein the mobile phase flow rate is 0.9 to 1.1 ml/min.
5. The method of claim 1, wherein the column temperature of the chromatographic column is 25 to 35 ℃.
6. A method for identifying bis (2-hydroxyethyl) disulfide in calcium levofolinate, which is characterized in that the method in claim 1 is utilized to separate the bis (2-hydroxyethyl) disulfide from calcium levofolinate and related substances in a detection object, the detection object is introduced into a detector for detection, a chromatogram obtained by the detection is compared with a chromatogram of a known bis (2-hydroxyethyl) disulfide reference substance, and whether the bis (2-hydroxyethyl) disulfide is contained in the detection object or not is judged.
7. The method of claim 6, wherein the detector wavelength is set at 247 ± 10 nm.
8. Method for determining the content of bis (2-hydroxyethyl) disulfide in calcium levofolinate, characterized in that it comprises the following steps:
1) preparing a detection product solution and a reference product solution;
dissolving the detection product in a diluent to obtain a detection product solution; dissolving a bis (2-hydroxyethyl) disulfide standard substance with a diluent to prepare a reference substance solution;
2) separating;
separating bis (2-hydroxyethyl) disulfide from said calcium levofolinate and related materials using the method of claim 1;
3) identifying;
identifying said bis (2-hydroxyethyl) disulfide in said calcium levofolinate using the method of claim 6;
4) calculating the content;
and calculating the content of the bis (2-hydroxyethyl) disulfide in the detection product solution according to the peak area and the chromatogram obtained in the identification process and an external standard method.
9. The method as claimed in claim 8, wherein the diluent in the step 1) is water.
10. The method for detecting the bis (2-hydroxyethyl) disulfide is characterized in that a chromatographic column adopted by the method uses octadecyl bonded silica gel as a filler, mixed solution of acetonitrile and water is used as a mobile phase for elution, the bis (2-hydroxyethyl) disulfide is separated, the mixture is introduced into a detector with the detection wavelength of 247nm for detection, and the content of the bis (2-hydroxyethyl) disulfide is measured by an external standard method; the volume ratio of the acetonitrile to the water is 1: 9; the flow rate of the mobile phase is 0.9-1.1 ml/min; the temperature of the chromatographic column is 25-35 ℃.
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