Method for separating and detecting bis(2-hydroxyethyl) disulfide in calcium folinate

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 folinate by high performance liquid chromatography. The method adopts a chromatographic column with octadecyl bonded silica gel as a filler, adopts a mixed solution of an organic solvent and water as a mobile phase for elution, and separates the bis(2-hydroxyethyl) disulfide from calcium folinate and related substances; the related substances are impurities of calcium folinate; the mobile phase flow rate is 0.9-1.1 ml / min; the column temperature of the chromatographic column is 25-35 DEG C; the wavelength of the detector is 247±10 nm; the detection method detects the bis(2-hydroxyethyl) disulfide in calcium folinate, and other impurities do not interfere with the detection of the target object, the method has good specificity and durability, and accurate impurity content is obtained, and the method is simple, rapid and effective.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and in particular relates to a method for separating and detecting bis(2-hydroxyethyl) disulfide in calcium folinate by using an HPLC method. Background Art

[0002] Calcium folinate, molecular formula is C 20 H 21 CaN7O7, molecular weight: 511.5, Chinese chemical name: N-[4-[(6S)-(2-amino-5-formyl-1,4,5,6,7,8-hexahydro-4-oxo-6-pterydinyl)methyl]amino]benzoyl-L-glutamic acid calcium salt hydrate. Currently, calcium folinate for injection is mainly used to treat gastric cancer and colorectal cancer. Its structural formula is as follows:

[0003]

[0004] Impurities that may exist in calcium folinate: 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; among them, bis(2-hydroxyethyl) disulfide, Chinese chemical name: 2,2'-dithiodiethanol, molecular formula C4H 10 O2S2, molecular weight 154.25, its structural formula is as follows:

[0005]

[0006] However, in current related studies, no detection and analysis of bis(2-hydroxyethyl) disulfide in calcium folinate has been found, which is not conducive to the quality control of calcium folinate drugs. Summary of the invention

[0007] In view of this, one of the objects of the present invention is to provide a method for separating bis(2-hydroxyethyl) disulfide from calcium folinate, which can effectively separate bis(2-hydroxyethyl) disulfide from calcium folinate and other related substances.

[0008] In order to achieve the above object, the technical solution of the present invention is:

[0009] A method for separating bis(2-hydroxyethyl) disulfide from calcium folinate, wherein the method adopts a chromatographic column with octadecyl bonded silica gel as a filler and a mixed solution of an organic solvent and water as a mobile phase for elution to separate the bis(2-hydroxyethyl) disulfide from calcium folinate and related substances; the related substances are one or more of impurities A to I; the structural formulas of calcium folinate, bis(2-hydroxyethyl) disulfide and related substances are as follows:

[0010]

[0011]

[0012] Further, the volume ratio of the organic solution to water is 9-11:91-89;

[0013] Further, the organic solution is one or more of acetonitrile, methanol, and ethanol;

[0014] Specifically, the organic solvent is acetonitrile;

[0015] Further, the flow rate of the mobile phase is 0.9-1.1 ml / min;

[0016] Furthermore, the column temperature of the chromatographic column is 25-35°C.

[0017] The second object of the present invention is to provide a method for identifying whether bis(2-hydroxyethyl) disulfide exists in a calcium folinate test substance by HPLC, which can effectively confirm whether bis(2-hydroxyethyl) disulfide exists in a calcium folinate test substance.

[0018] In order to achieve the above object, the technical solution of the present invention is:

[0019] A method for identifying bis(2-hydroxyethyl) disulfide in calcium folinate, comprising separating the bis(2-hydroxyethyl) disulfide in a calcium folinate test substance from calcium folinate and related substances by the method described in the first objective, passing the bis(2-hydroxyethyl) disulfide into a detector for detection, and comparing the chromatogram obtained by the detection with the chromatogram of a known reference substance of bis(2-hydroxyethyl) disulfide to determine whether the calcium folinate test substance contains the bis(2-hydroxyethyl) disulfide;

[0020] Furthermore, the detector wavelength is set to 247±10 nm.

[0021] The third object of the present invention is to provide a method for determining the content of bis(2-hydroxyethyl) disulfide in calcium folinate by HPLC, which can accurately determine the content of bis(2-hydroxyethyl) disulfide in calcium folinate, and is beneficial to drug quality control.

[0022] In order to achieve the above object, the technical solution of the present invention is:

[0023] A method for determining the content of bis(2-hydroxyethyl) disulfide in calcium folinate, the method specifically comprising the following steps:

[0024] 1) Prepare test solution and reference solution;

[0025] Take the test product and dissolve it in the diluent to obtain a test product solution; take the bis(2-hydroxyethyl) disulfide standard product and dissolve it in the diluent to prepare a reference product solution;

[0026] 2) Separation;

[0027] Using the method described in the first objective, the bis(2-hydroxyethyl) disulfide in the calcium folinate is separated from the calcium folinate and related substances;

[0028] 3) Identification;

[0029] Using the method described in the second objective, identifying the bis(2-hydroxyethyl) disulfide in the calcium folinate;

[0030] 4) Content calculation;

[0031] According to the chromatogram obtained during the identification process, the content of the bis(2-hydroxyethyl) disulfide in the test solution is calculated according to the peak area by the external standard method;

[0032] Furthermore, in step 1), the diluent is water.

[0033] A fourth object of the present invention is to provide a method for analyzing bis(2-hydroxyethyl) disulfide.

[0034] In order to achieve the above object, the technical solution of the present invention is:

[0035] A method for detecting bis(2-hydroxyethyl) disulfide, wherein the chromatographic column used in the method uses octadecyl bonded silica gel as a filler, uses a mixed solution of acetonitrile and water as a mobile phase for elution, separates the bis(2-hydroxyethyl) disulfide, passes it into a detector with a detection wavelength of 247 nm for detection, and measures the content of the bis(2-hydroxyethyl) disulfide by an external standard method; the volume ratio of acetonitrile to water is 1:9; the flow rate of the mobile phase is 0.9-1.1 ml / min; and the column temperature of the chromatographic column is 25-35° C.

[0036] The beneficial effects of the present invention are: using isocratic elution high performance liquid chromatography to detect the content of bis(2-hydroxyethyl) disulfide in calcium folinate, other impurities do not interfere with the detection of the target object, the specificity and durability are good, the accurate impurity content is obtained, and the method is simple, rapid and effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the chromatogram of the reference substance of this method;

[0038] Figure 2 This is the chromatogram of the test product for this method;

[0039] Figure 3 This is the chromatogram of the blank control group of this method;

[0040] Figure 4This is the chromatogram for positioning bis(2-hydroxyethyl) disulfide in this method;

[0041] Figure 5 Position the chromatogram for impurity A in this method;

[0042] Figure 6 The chromatogram for locating impurity B in this method;

[0043] Figure 7 The chromatogram for the location of impurity C in this method;

[0044] Figure 8 The chromatogram for locating impurity D in this method;

[0045] Fig. 9 The chromatogram for locating impurity E in this method;

[0046] Fig.10 The chromatogram for the location of impurity F in this method;

[0047] Fig.11 The chromatogram for the location of impurity G in this method;

[0048] Fig.12 The chromatogram for locating impurity I in this method;

[0049] Fig.13 This is the precision chromatogram of this method;

[0050] Fig.14 Chromatograms of all mixed substances in this method;

[0051] Fig.15 This is the chromatogram of the system suitability of the reference solution of this method;

[0052] Fig.16 This is the chromatogram of the quantitative limit of this method;

[0053] Fig.17 This is the detection limit chromatogram of this method;

[0054] Fig.18 is the stability chromatogram of the control solution 1;

[0055] Fig.19 It is the chromatogram of mixed solution stability 1;

[0056] Fig. 20 is the stability 2 chromatogram of the control solution;

[0057] Fig.21 is the stability 2 chromatogram of the control solution;

[0058] Fig. 22 For durability - mixed solution - normal chromatogram;

[0059] Fig.23Chromatogram for durability-mixed solution-flow rate 0.9;

[0060] Fig.24 Chromatogram for durability - mixed solution - flow rate 1.1;

[0061] Fig.25 For durability - mixed solution - column temperature 25 chromatogram;

[0062] Fig.26 For durability - mixed solution - column temperature 35 chromatogram;

[0063] Fig. 27 For durability - mixed solution - acetonitrile 9% chromatogram;

[0064] Fig.28 This is a chromatogram of durability-mixed solution-acetonitrile 11%. DETAILED DESCRIPTION

[0065] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0066] Example 1

[0067] Main instrument: Shimadzu LC-20A high performance liquid chromatograph.

[0068] Mobile phase: acetonitrile-water (10:90), isocratic elution;

[0069] Acquisition time: record until 1.2 times the retention time of the bis(2-hydroxyethyl) disulfide peak (about 10 min);

[0070] Chromatographic column: octadecyl bonded silica as filler, Agilent ZORBAX SB-C18 4.6mm×250mm, 5μm;

[0071] Flow rate: 1.0ml / min;

[0072] Detection wavelength: 247nm;

[0073] Column temperature: 30°C;

[0074] Diluent: water;

[0075] Injection volume: 20 μl;

[0076] Test solution: 2mg / ml;

[0077] Reference solution: 3 μg / ml;

[0078] Quantitative method: external standard method.

[0079] Example 2

[0080] Solution preparation:

[0081] Test solution: Take an appropriate amount of calcium folinate sample, weigh accurately, add water and ultrasonically dissolve it to make a solution containing about 2 mg per 1 ml, shake well, and the solution is ready.

[0082] Reference solution: Take an appropriate amount of bis(2-hydroxyethyl) disulfide reference substance, dissolve it in water and quantitatively dilute it to make a solution containing about 3 μg per 1 ml, shake well, and the solution is ready.

[0083] Example 3

[0084] Test method:

[0085] Accurately measure 20 μl of the reference solution and the test solution, respectively, and inject them into the liquid chromatograph, and record the chromatogram to 1.2 times the retention time of bis(2-hydroxyethyl) disulfide. Calculate the content of bis(2-hydroxyethyl) disulfide in calcium folinate by the external standard method based on the peak area.

[0086] Take an appropriate amount of this product, accurately weigh it, add water to dissolve it ultrasonically and make a solution containing about 2 mg per 1 ml, shake it well, and use it as the test solution; take an appropriate amount of bis(2-hydroxyethyl) disulfide, add water to dissolve it and quantitatively dilute it to make a solution containing about 3 μg per 1 ml, shake it well, and use it as the reference solution. According to the high performance liquid chromatography method (Chinese Pharmacopoeia 2015 Edition Four General Rules 0512), use octadecylsilane bonded silica gel as the filler (Agilent ZORBAX SB-C18 4.6mm×250mm, 5μm, or a chromatographic column with equivalent performance); acetonitrile-water (10:90) as the mobile phase; detection wavelength 247nm; flow rate 1.0ml per minute; column temperature 30℃. Accurately measure 20μl of the reference solution and the test solution, respectively, and inject them into the liquid chromatograph, and record the chromatogram to 1.2 times the retention time of bis(2-hydroxyethyl) disulfide. Calculate by peak area using the external standard method. (See attached for the obtained chromatogram.) Figure 1 , 2 ). The retention time and separation degree of each impurity are shown in Table 1:

[0087] Table 1 Retention time and separation degree of each impurity

[0088]

[0089] According to this method, the blank control group (see Appendix Figure 3 ), Bis(2-hydroxyethyl) disulfide localization map (see attached Figure 4 , Table 2), Impurity A-Location Map (see Attachment Figure 5-12 , Table 3-10), precision chromatogram (see Appendix Fig.13 , Table 11), chromatogram of all substances mixed (see Appendix Fig.14 , Table 12), System Suitability Chromatogram (see Appendix Fig.15 , Table 13), quantitation limit chromatogram (see attached Fig.16 , Table 14), Detection Limit Chromatogram (see Appendix Fig.17 , Table 15), stability chromatogram of control solution Figure 1 (See attached Fig.18 , Table 16), Mixed solution stability chromatogram Figure 1 (See attached Fig.19 , Table 17), stability chromatogram of control solution Figure 2 (See attached Fig. 20 , Table 18), Mixed solution stability chromatogram Figure 2 (See attached Fig.21 , Table 19), Durability Chromatogram (see Appendix Fig. 22 , Table 20), Durability Flow Rate 0.9 Chromatogram (see Attachment Fig.23 , Table 21), Durability Flow Rate 1.1 Chromatogram (see Appendix Fig.24 , Table 22), Durability Column Temperature 25 Chromatogram (see Appendix Fig.25 , Table 23), Durability Column Temperature 35 Chromatogram (see Appendix Fig.26 , Table 24), Durability 9% acetonitrile chromatogram (see attached Fig. 27 , Table 25), Durability 11% acetonitrile chromatogram (see attached Fig.28 , Table 26).

[0090] Table 2 Bis(2-hydroxyethyl) disulfide localization map integration results

[0091]

[0092] Table 3 Impurity A location map integration results

[0093]

[0094] Table 4. Results of integration of impurity B localization map

[0095]

[0096] Table 5 Impurity C localization map integration results

[0097]

[0098] Table 6 Impurity D localization map integration results

[0099]

[0100] Table 7 Impurity E localization map integration results

[0101]

[0102] Table 8 Impurity F location map integration results

[0103]

[0104] Table 9 Impurity G localization map integration results

[0105]

[0106] Table 10 Impurity I localization map integration results

[0107]

[0108] Table 11 Precision integration results

[0109]

[0110] Table 12 Integration results of mixed chromatograms of all substances

[0111]

[0112]

[0113] Table 13 System adaptability integration results

[0114]

[0115] Table 14 Quantitative limit chromatogram integration results

[0116]

[0117] Table 15 Detection limit chromatogram integration results

[0118]

[0119] Table 16 Control solution stability 1 integral results

[0120]

[0121]

[0122] Table 17 Mixed solution stability 1 integral results

[0123]

[0124] Table 18 Control solution stability 2 integral results

[0125]

[0126] Table 19 Control solution stability 2 integration results

[0127]

[0128] Table 20 Durability mixed solution normal flow rate integral results

[0129]

[0130]

[0131] Table 21 Durability mixed solution 0.9 flow rate integration results

[0132]

[0133] Table 22 Durability mixed solution 1.1 Flow rate integration results

[0134]

[0135] Table 23 Durability mixed solution column temperature 25 integration results

[0136]

[0137]

[0138] Table 24 Durability mixed solution column temperature 35 integration results

[0139]

[0140] Table 25 Durability mixed solution 9% acetonitrile integration results

[0141]

[0142]

[0143] Table 26 Durability mixed solution 11% acetonitrile integration results

[0144]

[0145] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for separating related substances and bis(2-hydroxyethyl) disulfide in calcium folinate, characterized in that: The chromatographic column used in the method uses octadecyl bonded silica gel as a filler, and uses a mixed solution of acetonitrile and water as a mobile phase for elution, and the volume ratio of the acetonitrile to water is 9-11:91-89 to separate the bis(2-hydroxyethyl) disulfide from calcium folinate and related substances; the related substances are impurities A-I; the structural formulas of calcium folinate, bis(2-hydroxyethyl) disulfide and related substances are as follows:

2. The method according to claim 1, characterized in that The flow rate of the mobile phase is 0.9-1.1 ml / min.

3. The method according to claim 1, characterized in that The column temperature of the chromatographic column is 25-35°C.

4. A method for identifying bis(2-hydroxyethyl) disulfide in calcium folinate, characterized in that: The bis(2-hydroxyethyl) disulfide in the test object is separated from calcium folinate and related substances by the method described in claim 1, and the test object is passed into a detector for detection. The chromatogram obtained by the detection is compared with the chromatogram of a known bis(2-hydroxyethyl) disulfide reference substance to determine whether the test object contains the bis(2-hydroxyethyl) disulfide.

5. The method according to claim 4, characterized in that The detector wavelength was set to 247 ± 10 nm.

6. A method for determining the content of bis(2-hydroxyethyl) disulfide in calcium folinate, characterized in that: The method specifically comprises the following steps: 1) Prepare test solution and reference solution; Take the test product and dissolve it in the diluent to obtain a test product solution; take the bis(2-hydroxyethyl) disulfide standard product and dissolve it in the diluent to prepare a reference product solution; 2) Separation; Using the method described in claim 1, the bis(2-hydroxyethyl) disulfide in the calcium folinate is separated from the calcium folinate and related substances; 3) Identification; Using the method described in claim 4, identifying the bis(2-hydroxyethyl) disulfide in the calcium folinate; 4) Calculation of content; According to the chromatogram obtained during the identification process, the content of the bis(2-hydroxyethyl) disulfide in the test solution is calculated according to the peak area using the external standard method.

7. The method according to claim 6, characterized in that In the step 1), the diluent is water.

8. The method according to claim 7, characterized in that The method adopts a chromatographic column with octadecyl bonded silica gel as a filler, and adopts a mixed solution of acetonitrile and water as a mobile phase for elution, separates the bis(2-hydroxyethyl) disulfide, passes it into a detector with a detection wavelength of 247nm for detection, and measures the content of the bis(2-hydroxyethyl) disulfide by an external standard method; The volume ratio of acetonitrile to water is 1:9; the flow rate of the mobile phase is 0.9-1.1 ml / min; The column temperature of the chromatographic column is 25-35°C.

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