CN113281425A - Method for detecting free fatty acid in polyoxyethylene (35) castor oil - Google Patents
Method for detecting free fatty acid in polyoxyethylene (35) castor oil Download PDFInfo
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Abstract
The invention belongs to the technical field of analysis and detection, and particularly relates to a method for detecting free fatty acid in polyoxyethylene (35) castor oil. The detection method adopts a liquid chromatography-mass spectrometry combined method to detect the polyoxyethylene (35) castor oil sample, wherein the chromatographic conditions are as follows: detecting a polyoxyethylene (35) castor oil sample by adopting a liquid chromatography-mass spectrometry combined method, wherein the chromatographic conditions are as follows: the mobile phase is composed of organic solution containing 0.9-1.1g/L ammonium acetate and water according to the volume ratio of (85-95) to (15-5), and the chromatographic column is a C18 column. The detection method provided by the invention has good accuracy and repeatability and low detection limit, and is suitable for detecting the content of free fatty acid in a polyoxyethylene (35) castor oil sample. Quality control for free fatty acids in polyoxyethylene (35) castor oil is achieved.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a method for detecting free fatty acid in polyoxyethylene (35) castor oil.
Background
Paclitaxel is considered to act as a mitotic spindle toxin and a potent inhibitor of cell replication, a pharmaceutically active substance with anti-tumor activity. One dosage form of paclitaxel is an injection, which is commercialized by the company Mimex Meishibaobao under the trade name TAXOL (TAXOL). Advantages of TAXOL include strong stability, however, the composition of TAXOL and its preparation method are not disclosed and do not belong to the prior art.
Paclitaxel is a compound which is difficult to dissolve in water, and in order to develop a new paclitaxel injection, two solvents are used together to dissolve paclitaxel, wherein ethanol is used as a polar solvent, and polyoxyethylene (35) castor oil is used as a solubilizer.
The polyoxyethylene (35) castor oil is obtained by reacting glycerol ricinoleate with ethylene oxide, contains a small amount of polyethylene glycol ricinoleate and free glycol, is a mixture and has complex components. Among these, polyoxyethylene (35) castor oil of standard quality is commercially available under the name Cremophor EL from BASF, and the chemical component is polyoxyethylene triglyceride.
The content of various components (such as acidic compounds) in the existing polyoxyethylene (35) castor oil product can affect the performance of the formulated paclitaxel, so in order to establish quality standards and perform quality control, a suitable method needs to be developed to detect the content of the acidic compounds in the polyoxyethylene (35) castor oil. Such a detection method has not been established in the prior art.
The acidic compounds in polyoxyethylene (35) castor oil are mainly free fatty acids. These fatty acids themselves have high boiling points and no ultraviolet absorption, and the prior art methods for detecting fatty acids are essentially derived as esters and measured by gas chromatography, however, this method is not very practical for use with free fatty acids in polyoxyethylated (35) castor oil. This is due to the low free fatty acid content of polyoxyethylene (35) castor oil, which makes it difficult to detect low levels of free fatty acids in many esters, including polyoxyethylene (35) castor oil, even after the ester has been derived, and the interference is large. Meanwhile, a common derivatization reagent may destroy polyoxyethylene (35) castor oil to generate ricinoleic acid, and the detected result is not reliable because free ricinoleic acid or destroyed ricinoleic acid cannot be judged.
Patent "AU 2003256786B 2" discloses a method for purifying polyoxyethylene (35) castor oil by separating impurities by liquid chromatography using alcohol and water as solvents. However, the polyoxyethylene (35) castor oil has very complicated components, many types of free fatty acids and very low content, and it is very difficult to separate the free fatty acids one by one to realize quantitative detection. Thus, the isolation methods and conditions given in this patent are not applicable to the detection of free fatty acids in polyoxyethylene (35) castor oil samples.
Disclosure of Invention
Aiming at the difficulties in the prior art, the invention provides a method for detecting free fatty acid in polyoxyethylene (35) castor oil, aiming at: the detection of the content of free fatty acid in polyoxyethylene (35) castor oil applied to the preparation of paclitaxel injection is realized.
A polyoxyethylene (35) castor oil is detected by a liquid chromatography-mass spectrometry combined method by taking heptadecanoic acid as an internal standard substance, and free fatty acid and relative retention time thereof are detected as follows: ricinoleic acid 0.327 + -5%; 0.372 plus or minus 5 percent of lauric acid; 0.526 percent of tetradecanoic acid plus or minus 5 percent; linolenic acid 0.538 +/-5%; linoleic acid 0.667 + -5%; 0.801 + -5% palmitic acid; oleic acid 0.878 +/-5%; 1.295 +/-5% of stearic acid; cis-11-carbon decaenoic acid 1.385 plus or minus 5%; 2.135 +/-5% of arachidic acid;
wherein, the chromatographic conditions are as follows: the mobile phase is composed of organic solution containing 0.9-1.1g/L ammonium acetate and water according to the volume ratio of (85-95) to (15-5), and the chromatographic column is a C18 column.
A method for detecting free fatty acid in polyoxyethylene (35) castor oil is used for detecting a polyoxyethylene (35) castor oil sample by adopting a liquid chromatography-mass spectrometry combined method, wherein the chromatographic conditions are as follows: the mobile phase is composed of organic solution containing 0.9-1.1g/L ammonium acetate and water according to the volume ratio of (85-95) to (15-5), and the chromatographic column is a C18 column.
Preferably, the solvent of the organic solution is selected from at least one of methanol, ethanol, ethyl acetate or acetonitrile, preferably methanol; and/or the concentration of the ammonium acetate is 1 g/L.
Preferably, the organic solution and water form a mobile phase according to the volume ratio of 90: 10.
Preferably, the polyoxyethylene (35) castor oil sample is diluted with an organic solution containing 0.05 to 0.2 wt.% formic acid, preferably with a methanol solution containing 0.1 wt.% formic acid, before injection, and/or the polyoxyethylene (35) castor oil sample and the methanol solution are used in a ratio of 4mg to (0.5 to 2) ml, preferably 4mg to 1 ml.
Preferably, the chromatographic conditions further comprise: the chromatographic column is YMC ODS-A, and/or the temperature of the chromatographic column is 30-40 ℃, preferably 35 ℃, and/or the elution mode is isocratic elution, and/or the flow rate is 0.4-0.6ml/min, preferably 0.5ml/min, and/or the elution time of the mobile phase is 25-50min, preferably 35min, and/or the sample amount is 1-10 mul, preferably 5 mul.
Preferably, the mass spectrometry conditions comprise: the detection mode of the mass spectrum is SIM.
Preferably, the mass spectrometry conditions further comprise: the ion source is an ESI source, and/or the drying gas temperature is 280-320 ℃, preferably 300 ℃, and/or the flow rate is 8-12L/min, preferably 10L/min, and/or the atomizer pressure is 55-65psi, preferably 60psi, and/or the sheath gas temperature is 330-370 ℃, preferably 350 ℃, and/or the flow rate is 9-13L/min, preferably 11L/min, and/or the capillary voltage is 3000-4000V, preferably 3500V, and/or the nozzle voltage is 400-600V, preferably 500V, and/or the residence time is 80-120, preferably 100, and/or the Fragmentor is 110-160V, preferably 135V, and/or the acceleration voltage is 4-6V, preferably 5V.
Preferably, the free fatty acid is selected from at least one of ricinoleic acid, lauric acid, myristic acid, linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid, cis-11-carbodecaenoic acid, and arachidic acid.
Preferably, the relative retention time of the free fatty acids is as follows, using heptadecanoic acid as an internal standard: ricinoleic acid 0.327 + -5%; 0.372 plus or minus 5 percent of lauric acid; 0.526 percent of tetradecanoic acid plus or minus 5 percent; linolenic acid 0.538 +/-5%; linoleic acid 0.667 + -5%; 0.801 + -5% palmitic acid; oleic acid 0.878 +/-5%; 1.295 +/-5% of stearic acid; cis-11-carbon decaenoic acid 1.385 plus or minus 5%; 2.135 +/-5% of arachidic acid.
Preferably, the mass-to-charge ratio of the free fatty acid is: ricinoleic acid 297.1, lauric acid 199.1, myristic acid 227.1, linolenic acid 277.1, linoleic acid 279.1, palmitic acid 255.1, oleic acid 281.1, stearic acid 283.1, cis-11-carbo-decaenoic acid 309.1, arachidic acid 311.1.
By adopting the technical scheme, the fatty acids such as ricinoleic acid, oleic acid and palmitic acid can be effectively separated without being interfered by the main component of polyoxyethylene (35) castor oil, and the content of free fatty acid in the polyoxyethylene (35) castor oil can be accurately detected, so that the quality of the polyoxyethylene (35) castor oil can be effectively controlled.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a TIC graph of a control positioning solution of example 1; the peak-out sequence is ricinoleic acid, lauric acid, myristic acid, linolenic acid, linoleic acid, palmitic acid, oleic acid, heptadecanoic acid, stearic acid, cis-11-carbon decaenoic acid and arachidic acid in sequence;
FIG. 2 is a ricinoleic acid EIC profile of a control positioning solution of example 1;
FIG. 3 is a graph of lauric acid EIC in a control localization solution of example 1;
FIG. 4 is a graph of myristic acid EIC of a control positioning solution in example 1;
FIG. 5 is a graph of linolenic acid EIC in the localization solution of the control in example 1;
FIG. 6 is a graph of linoleic acid EIC in a control localization solution of example 1;
FIG. 7 is a graph of EIC palmitate in a control positioning solution of example 1;
FIG. 8 is a graph of oleic acid EIC of a control positioning solution of example 1;
FIG. 9 is an EIC map of heptadecanoic acid of the control localization solution of example 1;
FIG. 10 is a stearic acid EIC pattern of a control positioning solution of example 1;
FIG. 11 is a drawing of the EIC of cis-11-carbon decadecenoic acid in the control localization solution of example 1;
FIG. 12 is an EIC profile of eicosanoids from a control positioning solution of example 1;
FIG. 13 is a TIC graph of the labeled test article solution in example 1;
FIG. 14 is a ricinoleic acid EIC profile of the spiked test sample solution of example 1;
FIG. 15 is a graph showing EIC of lauric acid in the test sample solution added with a standard in example 1;
FIG. 16 is a diagram of the EIC of tetradecanoic acid of the labeled test article solution in example 1;
FIG. 17 is a chart of linolenic acid EIC in the solution of the spiked test sample in example 1;
FIG. 18 is a graph showing EIC of linoleic acid in the test sample solution added with a standard in example 1;
FIG. 19 is a graph of EIC palmitate in the solution of the test sample added with the standard in example 1;
FIG. 20 is a graph of oleic acid EIC of a solution of the spiked test article of example 1;
FIG. 21 is an EIC diagram of heptadecanoic acid in the test sample solution added with a label in example 1;
FIG. 22 is an EIC chart of stearic acid in the test sample solution of example 1;
FIG. 23 is an EIC chart of cis-11-carbon decadecenoic acid in the test sample solution to be labeled in example 1;
FIG. 24 is an EIC plot of eicosanoids in a test sample solution of example 1;
FIG. 25 is a chromatogram of an air-white solution of comparative example 1;
FIG. 26 is a chromatogram of a control solution of comparative example 1; the peak is ricinoleic acid, myristic acid, palmitic acid, heptadecanoic acid, oleic acid and stearic acid in sequence;
FIG. 27 is a perspective view of the sample solution of comparative example 1;
FIG. 28 is a partial view of a sample solution in comparative example 1;
FIG. 29 is a chromatogram of the palmitic acid localization solution of comparative example 2;
FIG. 30 is a chromatogram of the sample solution in comparative example 2;
FIG. 31 is a chromatogram of a solution of the labeled test sample in comparative example 2;
FIG. 32 is a chromatogram of a 0.1. mu.g/ml control solution of comparative example 3;
FIG. 33 is a chromatogram of a 0.01. mu.g/ml control solution of comparative example 3;
FIG. 34 is a chromatogram of a 0.1. mu.g/ml control solution and a chromatogram of a 0.01. mu.g/ml control solution in comparative example 3;
FIG. 35 is a chromatogram of a 0.1. mu.g/ml control solution of comparative example 4;
FIG. 36 is a chromatogram of a 0.1. mu.g/ml control solution of comparative example 5;
FIG. 37 is a chromatogram of a 0.1. mu.g/ml control solution of comparative example 6;
FIG. 38 is a chromatogram of a 0.1. mu.g/ml control solution of comparative example 7.
Detailed Description
The test materials adopted by the invention are all common commercial products and can be purchased in the market. In the present invention, the polyoxyethylene (35) castor oil is Cremophor EL-P from Pasteur.
The invention is further illustrated by the following examples:
example 1
1. Preparing a solution:
control positioning solution: ricinoleic acid, lauric acid, myristic acid, linolenic acid, linoleic acid, palmitic acid, heptadecanoic acid, oleic acid, stearic acid, cis-11-carbodiimide and arachidic acid are precisely weighed, a diluent (methanol solution containing 0.1 wt.% formic acid) is used for preparing a solution containing 0.4 mu g of fatty acid for each 1ml, and the solution is shaken up.
Adding a standard test solution: cremophor EL-P, ricinoleic acid, lauric acid, myristic acid, linolenic acid, linoleic acid, palmitic acid, heptadecanoic acid (internal standard), oleic acid, stearic acid, cis-11-carbon decaenoic acid and arachidic acid were precisely weighed, prepared into a solution containing about 4mg of polyoxyethylene (35) castor oil per 1ml, and about 0.4. mu.g of each fatty acid, with a diluent (0.1 wt.% formic acid in methanol), and shaken well.
2. The above solutions were tested using the following chromatographic and mass spectrometric conditions:
chromatographic conditions are as follows:
the chromatographic column is YMC ODS-A, 4.6X 150mm, 3 μm; the column temperature was 35 ℃;
taking 1g/L of ammonium acetate in methanol and water at a ratio of 90:10 as a mobile phase; the flow rate was 0.5ml per minute; the amount of sample was 5. mu.l.
Detecting by using a triple quadrupole mass spectrometer, wherein the mass spectrometry conditions are as follows:
the ion source is an ESI source, the anion mode is the SIM acquisition mode, the temperature of the drying gas is 300 ℃, the flow rate is 10L/min, the pressure of the atomizer is 60psi, the temperature of the sheath gas is 350 ℃, the flow rate is 11L/min, the voltage of the capillary tube is 3500V, the voltage of the nozzle is 500V, the residence time is 100, the fragment is 135V, and the acceleration voltage is 5V. The collection time is 4 min-35 min.
And a segmented acquisition means is adopted to cut off all polyoxyethylene (35) castor oil peaks before a ricinoleic acid peak (retention time is 5.1min), so that the polyoxyethylene (35) castor oil with large concentration is prevented from entering a mass spectrum, and pollution is avoided.
3. The result of the detection
The mass spectrograms of the reference substance localization solution are shown in FIGS. 1-12, and the mass spectrograms of the labeled test substance solution are shown in FIGS. 13-24. As can be seen from the figure, ricinoleic acid, lauric acid, myristic acid, linolenic acid, linoleic acid, palmitic acid, heptadecanoic acid, oleic acid, stearic acid, cis-11-carbon decaenoic acid and arachidic acid peak in sequence without interference, and have strong specificity and high sensitivity.
The mass-to-charge ratio of each fatty acid is ricinoleic acid (297.1), lauric acid (199.1), myristic acid (227.1), linolenic acid (277.1), linoleic acid (279.1), palmitic acid (255.1), oleic acid (281.1), stearic acid (283.1), cis-11-carbodecaenoic acid (309.1), and arachidic acid (311.1).
The retention time of each fatty acid is 5.1 min; lauric acid 5.8 min; myristic acid 8.2 min; linolenic acid for 8.4 min; linoleic acid 10.4 min; palmitic acid 12.5 min; oleic acid 13.7 min; internal standard (heptadecanoic acid) 15.6 min; stearic acid for 20.2 min; cis-11-carbon decaenoic acid for 21.6 min; eicosanoid for 33.3 min.
In the method of this example, the type of each fatty acid was qualitatively determined from the retention time and the mass-to-charge ratio. The content of each fatty acid can also be quantitatively determined by the prior art methods such as an external standard method, an internal standard method, a standard addition method and the like. In this example, ricinoleic acid, lauric acid, myristic acid, linolenic acid, linoleic acid, oleic acid, cis-11-carbon decaenoic acid, and arachidic acid were preferably quantified by the internal standard method, and the internal standard was heptadecanoic acid.
Experimental example 1 verification of chromatographic accuracy
According to the chromatographic-mass spectrometric conditions and concentration requirements of example 1, recovery rate experiments are performed, wherein the recovery rate experiments are performed respectively at concentrations of 0.08 μ g/ml, 0.2 μ g/ml, 0.4 μ g/ml and 0.6 μ g/ml for each fatty acid with a polyoxyethylene (35) castor oil concentration of 4mg/ml and a fatty acid control solution concentration of 0.4 μ g/ml, the recovery rates are all in the range of 80-120%, and the RSD is less than 8%, which indicates that the accuracy of the detection method meets the application requirements, and the results are as follows:
experimental example 2 chromatographic reproducibility verification
According to the chromatographic-mass spectrometric conditions and concentration requirements of example 1, repeated experimental investigation is carried out, and the content RSD of each fatty acid is measured to be in the range of 0.4-8.5% by using the polyoxyethylene (35) castor oil with the concentration of 4mg/ml, the fatty acid control solution with the concentration of 0.4 mug/ml and the standard fatty acid concentration in 6 repeated solutions with the concentration of 0.4 mug/ml, which indicates that the repeatability of the detection method meets the application requirements, and the results are as follows:
experimental example 3 quantitative Limit determination
In the experimental example, the quantitative limit of palmitic acid and stearic acid is calculated according to a standard addition method, namely 8 needles of sample are continuously fed when the quantitative limit is measured, and then the quantitative limit is obtained by derivation according to a linear slope. The limits of quantitation for other free fatty acids were determined as follows: each fatty acid standard solution was gradually diluted according to the chromatography-mass spectrometry conditions and concentration requirements of example 1, and concentrations with a signal-to-noise ratio greater than 10 were considered to be above the detection limit, with the results as follows:
from the data in the above table it can be seen that the limit of quantitation for the majority of free fatty acids is below 0.02. mu.g/ml.
Experimental example 4 durability examination
The conditions of the chromatography-mass spectrometry of example 1 were adjusted within a certain range, and the durability was examined, and it was found that the method of the present application had good durability. The specific results are shown in the following table:
experimental example 5 detection of sample to be measured
The polyoxyethylene (35) castor oil product Cremophor EL-P was tested as established in example 1 and the results are given in the following table:
from the above results, it can be seen that the method provided by the present invention can detect polyoxyethylene (35) castor oil and accurately quantify the free fatty acids contained therein.
Comparative example 1
Determination of free fatty acids in polyoxyethylated (35) castor oil using a CAD detector
1. Preparing sample and control solutions:
control solution: respectively taking appropriate amount of ricinoleic acid, myristic acid, palmitic acid, heptadecanoic acid, oleic acid and stearic acid, and respectively preparing into single standard solution with the amount of about 0.01mg per 1 ml. The solvent was a methanol solution containing 0.1 wt.% formic acid.
Sample solution: an appropriate amount of Cremophor EL-P was weighed and prepared into about 10mg per 1ml solution. The solvent was a methanol solution containing 0.1 wt.% formic acid.
Blank solution: methanol solution containing 0.1 wt.% formic acid.
2. Detecting each fatty acid single-standard solution and each sample solution by adopting a CAD detector method, wherein the chromatographic parameters are as follows:
a chromatographic column: ACE Super C18，4.6×250mm，5μm
Mobile phase: 0.1% aqueous formic acid/acetonitrile 15: 85, isocratic elution.
Atomizer temperature is low (40 deg.C)
Single-label solutions and sample solutions of each fatty acid were injected and chromatograms (fig. 25-28) show that each fatty acid can be separated efficiently, but the peak of polyoxyethylene (35) castor oil severely interferes with the detection of free fatty acids.
Comparative example 2
Determination of free fatty acids in polyoxyl (35) castor oil using liquid chromatography-mass spectrometry
1. Preparing a sample and adding a standard sample solution and a palmitic acid positioning solution:
palmitic acid localization solution: an appropriate amount of palmitic acid was taken and prepared into a solution of about 0.01mg per 1 ml. The solvent was a methanol solution containing 0.1 wt.% formic acid.
Sample solution: an appropriate amount of Cremophor EL-P was weighed and prepared into about 2mg per lml of solution. The solvent was a methanol solution containing 0.1 wt.% formic acid.
Adding a standard test solution: a proper amount of Cremophor EL-P and palmitic acid are taken to prepare a solution containing about 2mg of Cremophor EL-P and about 0.01mg of palmitic acid per 1 ml. The solvent was a methanol solution containing 0.1 wt.% formic acid.
2. The following chromatographic conditions were used for the detection of the samples
The chromatographic column is YMC ODS-A, 4.6X 150mm, 3 μm; the column temperature was 35 ℃;
using 40mmol/L ammonium formate aqueous solution and acetonitrile 15: 85 as mobile phase; the flow rate was 0.5ml per minute; the amount of sample was 5. mu.l.
And detecting by using a triple quadrupole mass spectrometer, and only collecting the mass-to-charge ratio of the palmitic acid.
The sample is injected with the palmitic acid positioning solution, the sample solution and the standard sample solution, and the chromatogram (figure 29-31) shows that the palmitic acid can effectively generate a peak, but the response value is low, and the palmitic acid is not easy to detect
Comparative example 3
Determination of free fatty acids in polyoxyl (35) castor oil using liquid chromatography-mass spectrometry
1. Prepare 0.1. mu.g/ml control solution and 0.01. mu.g/ml control solution:
0.1 μ g/ml control solution: taking proper amount of palmitic acid and heptadecanoic acid, and preparing into solution containing 0.1 μ g of palmitic acid and 0.1 μ g of heptadecanoic acid per 1 ml. The solvent was a methanol solution containing 0.1 wt.% formic acid.
0.01 μ g/ml control solution: taking appropriate amount of palmitic acid and heptadecanoic acid, and preparing into solution containing 0.01 μ g palmitic acid and 0.01 μ g heptadecanoic acid per 1 ml. The solvent was a methanol solution containing 0.1 wt.% formic acid.
2. The following chromatographic conditions were used for the detection of the samples
The chromatographic column is YMC ODS-A, 4.6X 150mm, 3 μm; the column temperature was 35 ℃;
methanol and ultrapure water are taken as mobile phases at a ratio of 90: 10; the flow rate was 0.5ml per minute; the amount of sample was 5. mu.l.
Detection with triple quadrupole mass spectrometer
When 0.1. mu.g/ml of the control solution and 0.01. mu.g/ml of the control solution were injected, chromatograms (FIGS. 32-34) were obtained in which palmitic acid and heptadecanoic acid were effectively peaked, but the peak areas were not linear and could be related to the ionization efficiency, suggesting that acid was added to the mobile phase.
Comparative example 4
Determination of free fatty acids in polyoxyl (35) castor oil using liquid chromatography-mass spectrometry
1. Prepare 0.1 μ g/ml control solution:
0.1 μ g/ml control solution: taking proper amount of palmitic acid and heptadecanoic acid, and preparing into solution containing 0.1 μ g of palmitic acid and 0.1 μ g of heptadecanoic acid per 1 ml. The solvent was a methanol solution containing 0.1 wt.% formic acid.
2. The following chromatographic conditions were used for the detection of the samples
The chromatographic column is ZORBAX Ecilpse Plus C18, 2.1X 50mm, 1.8 μm; the column temperature was 35 ℃;
methanol and ultrapure water are taken as mobile phases, wherein the ratio of the methanol to the ultrapure water is 85: 15; the flow rate was 0.5ml per minute; the amount of sample was 5. mu.l.
Detection with triple quadrupole mass spectrometer
When a control solution of 0.1. mu.g/ml was injected, the chromatogram (FIG. 35) showed that palmitic acid and heptadecanoic acid were effective but had poor peak shapes.
Comparative example 5
Determination of free fatty acids in polyoxyl (35) castor oil using liquid chromatography-mass spectrometry
1. Preparing a reference solution:
control solution: respectively taking appropriate amount of ricinoleic acid, myristic acid, palmitic acid, heptadecanoic acid, oleic acid and stearic acid, and respectively preparing into single standard solution with the amount of about 0.01mg per 1 ml. The solvent was a methanol solution containing 0.1 wt.% formic acid.
2. The following chromatographic conditions were used for the detection of the samples
The chromatographic column is YMC ODS-A, 4.6X 150mm, 3 μm; the column temperature was 35 ℃;
taking 0.1% methanoic acid methanol and 0.1% formic acid water solution as mobile phase 90: 10; the flow rate was 0.5ml per minute; the amount of sample was 5. mu.l.
Detection with triple quadrupole mass spectrometer
The reference solution was injected, and the chromatogram (fig. 36) showed low response values for the remaining free fatty acids, except for the higher response value for ricinoleic acid.
Comparative example 6
Determination of free fatty acids in polyoxyl (35) castor oil using liquid chromatography-mass spectrometry
1. Preparing a reference solution:
control solution: respectively taking appropriate amount of ricinoleic acid, myristic acid, palmitic acid, heptadecanoic acid, oleic acid and stearic acid, and respectively preparing into single standard solution with the amount of about 0.01mg per 1 ml. The solvent was a methanol solution containing 0.1 wt.% formic acid.
2. The following chromatographic conditions were used for the detection of the samples
The chromatographic column is YMC ODS-A, 4.6X 150mm, 3 μm; the column temperature was 35 ℃;
taking 1g/L of ammonium acetate methanol solution and 0.1% formic acid aqueous solution as a mobile phase, wherein the ratio of the mobile phase to the formic acid aqueous solution is 90: 10; the flow rate was 0.5ml per minute; the amount of sample was 5. mu.l.
Detection with triple quadrupole mass spectrometer
When a reference solution is injected, the chromatogram (figure 37) shows that the response of palmitic acid is high and the response values of the rest free fatty acids are low.
Comparative example 7
Determination of free fatty acids in polyoxyl (35) castor oil using liquid chromatography-mass spectrometry
1. Preparing a reference solution:
control solution: respectively taking appropriate amount of ricinoleic acid, myristic acid, palmitic acid, heptadecanoic acid, oleic acid and stearic acid, and respectively preparing into single standard solution with the amount of about 0.01mg per 1 ml. The solvent was a methanol solution containing 0.1 wt.% formic acid.
2. The following chromatographic conditions were used for the detection of the samples
The chromatographic column is YMC ODS-A, 4.6X 150mm, 3 μm; the column temperature was 35 ℃;
taking 1g/L of ammonium acetate methanol solution, 1g/L of ammonium acetate and 1ml/L of glacial acetic acid aqueous solution as mobile phases, wherein the ratio of the mobile phases to the glacial acetic acid aqueous solution is 90: 10; the flow rate was 0.5ml per minute; the amount of sample was 5. mu.l.
Detection with triple quadrupole mass spectrometer
When a reference solution is injected, the chromatogram (figure 38) shows that the response values of stearic acid and palmitic acid are higher, and the response values of the rest free fatty acids are lower.
It can be seen from the above comparative examples that only under the preferred mobile phase and mixing ratio thereof in the present application, each free fatty acid can give consideration to better peak shape, resolution and high response value, thereby facilitating the realization of accurate content analysis.
In conclusion, the detection method provided by the invention has good accuracy and repeatability and low detection limit, and is suitable for detecting the content of free fatty acid in a polyoxyethylene (35) castor oil sample. The effective quality control detection method for the free fatty acid in the polyoxyethylene (35) castor oil is realized.
Claims (10)
1. A polyoxyethylene (35) castor oil, characterized by: detecting by using a liquid chromatography-mass spectrometry combined method by using heptadecanoic acid as an internal standard substance, and detecting free fatty acid and the relative retention time as follows: ricinoleic acid 0.327 + -5%; 0.372 plus or minus 5 percent of lauric acid; 0.526 percent of tetradecanoic acid plus or minus 5 percent; linolenic acid 0.538 +/-5%; linoleic acid 0.667 + -5%; 0.801 + -5% palmitic acid; oleic acid 0.878 +/-5%; 1.295 +/-5% of stearic acid; cis-11-carbon decaenoic acid 1.385 plus or minus 5%; 2.135 +/-5% of arachidic acid;
wherein, the chromatographic conditions are as follows: the mobile phase is composed of organic solution containing 0.9-1.1g/L ammonium acetate and water according to the volume ratio of (85-95) to (15-5), and the chromatographic column is a C18 column.
2. The method for detecting the free fatty acid in the polyoxyethylene (35) castor oil is characterized in that a liquid chromatography-mass spectrometry combined method is adopted to detect a polyoxyethylene (35) castor oil sample, wherein the chromatographic conditions are as follows: the mobile phase is composed of organic solution containing 0.9-1.1g/L ammonium acetate and water according to the volume ratio of (85-95) to (15-5), and the chromatographic column is a C18 column.
3. The detection method according to claim 2, characterized in that: the solvent of the organic solution is selected from at least one of methanol, ethanol, ethyl acetate or acetonitrile; and/or the concentration of the ammonium acetate is 1 g/L; and/or the organic solution and water form a mobile phase according to the volume ratio of 90: 10.
4. The detection method according to claim 2, characterized in that: the polyoxyethylene (35) castor oil sample is diluted with an organic solution containing 0.05-0.2 wt.% formic acid, preferably with a methanol solution containing 0.1 wt.% formic acid, before injection, and/or the ratio of the amount of the polyoxyethylene (35) castor oil sample to the methanol solution is 4mg (0.5-2) ml, preferably 4mg:1 ml.
5. The detection method according to claim 2, characterized in that: the chromatographic conditions further comprise: the chromatographic column is YMC ODS-A, and/or the temperature of the chromatographic column is 30-40 ℃, preferably 35 ℃, and/or the elution mode is isocratic elution, and/or the flow rate is 0.4-0.6ml/min, preferably 0.5ml/min, and/or the elution time of the mobile phase is 25-50min, preferably 35min, and/or the sample amount is 1-10 mul, preferably 5 mul.
6. The detection method according to claim 2, characterized in that: the mass spectrometry conditions include: the detection mode of the mass spectrum is SIM.
7. The detection method according to claim 6, characterized in that: the mass spectrometry conditions further comprise: the ion source is an ESI source, and/or the drying gas temperature is 280-320 ℃, preferably 300 ℃, and/or the flow rate is 8-12L/min, preferably 10L/min, and/or the atomizer pressure is 55-65psi, preferably 60psi, and/or the sheath gas temperature is 330-370 ℃, preferably 350 ℃, and/or the flow rate is 9-13L/min, preferably 11L/min, and/or the capillary voltage is 3000-4000V, preferably 3500V, and/or the nozzle voltage is 400-600V, preferably 500V, and/or the residence time is 80-120, preferably 100, and/or the Fragmentor is 110-160V, preferably 135V, and/or the acceleration voltage is 4-6V, preferably 5V.
8. The detection method according to claims 2 to 7, characterized in that: the free fatty acid is at least one selected from ricinoleic acid, lauric acid, myristic acid, linolenic acid, linoleic acid, palmitic acid, oleic acid, stearic acid, cis-11-carbodecaenoic acid, and arachidic acid.
9. The detection method according to claim 8, characterized in that: relative retention times of the free fatty acids with heptadecanoic acid as an internal standard were as follows: ricinoleic acid 0.327 + -5%; 0.372 plus or minus 5 percent of lauric acid; 0.526 percent of tetradecanoic acid plus or minus 5 percent; linolenic acid 0.538 +/-5%; linoleic acid 0.667 + -5%; 0.801 + -5% palmitic acid; oleic acid 0.878 +/-5%; 1.295 +/-5% of stearic acid; cis-11-carbon decaenoic acid 1.385 plus or minus 5%; 2.135 +/-5% of arachidic acid;
and/or the mass-to-charge ratio of the free fatty acid is: ricinoleic acid 297.1, lauric acid 199.1, myristic acid 227.1, linolenic acid 277.1, linoleic acid 279.1, palmitic acid 255.1, oleic acid 281.1, stearic acid 283.1, cis-11-carbo-decaenoic acid 309.1, arachidic acid 311.1.
10. The detection method according to claim 2, characterized in that: and detecting the content of the free fatty acid by an external standard method, an internal standard method or a standard addition method.
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