CN110907564A - Method for determining fluorouracil content in blood plasma by HPLC method - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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Abstract
The invention provides a method for determining the content of fluorouracil in blood plasma by an HPLC method, which comprises the following steps: preparing a reference solution, pretreating a sample and measuring the content by using a high performance liquid chromatography; the method provided by the invention has the characteristics of accurate content measurement, high sensitivity and good repeatability.
Description
Technical Field
The invention belongs to the field of content determination, and particularly relates to a method for determining fluorouracil content in blood plasma by an HPLC method.
Background
Fluorouracil is a pyrimidine antimetabolite commonly used in clinic and interferes with DNA synthesis by inhibiting thymidylate synthase, blocking the conversion of deoxynucleotides to thymidine. The fluorouracil is mainly used for treating digestive tract tumors, or treating chorionic epithelial cancers with a large dose, and is also used for treating breast cancer, lung cancer, ovarian cancer, cervical cancer skin cancer and the like.
The fluorouracil has short half-life period in vivo metabolism, high toxicity, irregular absorption and metabolism and easy generation of adverse reactions such as bone marrow transplantation and the like due to overhigh concentration in vivo. And the composition of plasma is extremely complex, including proteins, lipids, inorganic salts, sugars, amino acids, metabolic waste products and large amounts of water; how to detect the drug concentration in plasma is the basis of pharmacokinetic studies; therefore, in order to detect the content of fluorouracil in plasma, a series of methods for determining the content of fluorouracil in plasma are established in the prior art, for example, folka and the like disclose a method for determining the content of fluorouracil in human plasma in journal literature "high performance liquid chromatography for determining the concentration of 5-FU in human plasma", the method comprises the steps of firstly preparing a reference solution, then processing the plasma, using ethyl acetate as an extracting agent in the processing process, then obtaining a sample solution through operations of whirlpool, centrifugation and the like, and finally performing content determination by using an HPLC method.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for determining the content of fluorouracil in blood plasma by an HPLC method.
The specific technical scheme of the invention is as follows:
one embodiment of the present invention provides a method for determining the content of fluorouracil in plasma by an HPLC method, which comprises the following steps:
(1) preparation of control solutions
Dissolving fluorouracil in methanol, and diluting with methanol to a solution containing 0.1mg fluorouracil per mL of methanol as a control solution; aspirating 1mL, diluting with mobile phase to contain 0.25 μ g fluorouracil per mL;
(2) sample pretreatment
Placing blood plasma to be tested in a test tube, adding 0.1mol/L disodium hydrogen phosphate aqueous solution, adjusting pH to 3.2 with phosphoric acid, shaking up, adding an extracting agent for extraction, adjusting pH to 6-7 with 0.5mol/L disodium hydrogen phosphate aqueous solution, shaking in a vortex mode for 2-5min, centrifuging at 3000rpm for 5-15min, taking supernatant, placing in another test tube, adding the extracting agent for secondary extraction, shaking in a vortex mode for 2-5min, and centrifuging at 3000rpm for 5-15 min; collecting supernatant, mixing the two supernatants, blowing at 40-60 deg.C with nitrogen, adding mobile phase to desired volume, mixing, and sampling the supernatant;
(3) determination of content
Measuring content by high performance liquid chromatography.
In a further improved scheme, the mobile phase is 0.1mol/L disodium hydrogen phosphate aqueous solution (phosphoric acid is used for adjusting the pH value to 3.2): acetonitrile 90: 10.
in a further improvement scheme, the volume ratio of the extracting agent is 8: 1: 1 ethyl acetate, acetonitrile and triethylamine.
In a further improved scheme, the chromatographic conditions of the high performance liquid chromatography are as follows:
a chromatographic column: c18A chromatographic column;
mobile phase: 0.1mol/L aqueous disodium hydrogen phosphate solution (phosphoric acid adjusted pH to 3.2): acetonitrile 90: 10;
detection wavelength: 265 nm;
column temperature: 45 ℃;
flow rate: 1.0 mL/min;
isocratic elution.
In a further improved scheme, Kromacil C18Column, 4.6X 250mm, 5 μm.
In a further improved scheme, the sample amount is 10 mu L.
In a further improved scheme, the content measurement further comprises eluting with a mobile phase for 5-10min, and then performing content measurement.
The method for measuring the fluorouracil content in the blood plasma by the HPLC method provided by the invention has the characteristics of accurate content measurement, high sensitivity and good repeatability.
Drawings
FIG. 1 is a chromatogram of blank plasma.
FIG. 2 is a chromatogram of a fluorouracil control solution;
FIG. 3 is a chromatogram for determination of the fluorouracil content in plasma.
Detailed Description
Reagent
Fluorouracil with purity of 99.5%, China institute for food and drug testing;
the rest reagents are commercially available in pure chromatographic form.
Example 1
A method for determining the content of fluorouracil in plasma by an HPLC method, which comprises the following steps:
(1) preparation of control solutions
Dissolving fluorouracil in methanol, and diluting with methanol to obtain a solution containing 0.1mg fluorouracil per mL of methanol as a control solution; aspirate 1mL, dilute to 0.25 μ g fluorouracil per mL with mobile phase of 0.1mol/L aqueous disodium hydrogen phosphate (pH adjusted to 3.2 with phosphoric acid): acetonitrile 90: 10;
(2) sample pretreatment
Taking 100 mu L of blood plasma to be detected, placing the blood plasma in a 5mL test tube, adding 1mL of 0.1mol/L disodium hydrogen phosphate aqueous solution (the pH value is adjusted to 6-7 by phosphoric acid), shaking up, and adding 0.5mL of an extracting agent, wherein the extracting agent is as follows: the volume ratio is 8: 1: 1 mixture of ethyl acetate, acetonitrile and triethylamine; adjusting pH to 6-7 with 0.5mol/L disodium hydrogen phosphate water solution, shaking for 5min, centrifuging at 3000rpm for 10min, collecting supernatant, placing in another test tube, adding 0.5mL extractant, shaking for 2min, and centrifuging at 3000rpm for 10 min; collecting supernatant, mixing the two supernatants, blow-drying at 60 deg.C with nitrogen gas, adding 100 μ L mobile phase, vortex mixing, collecting supernatant 10 μ L, and sampling;
(3) determination of content
Performing content determination by using a high performance liquid chromatography under the following chromatographic conditions:
a chromatographic column: kromacil C18Column, 4.6X 250mm, 5 μm;
mobile phase: 0.1mol/L aqueous disodium hydrogen phosphate solution (phosphoric acid adjusted pH to 3.2): acetonitrile 90: 10;
detection wavelength: 265 nm;
column temperature: 45 ℃;
flow rate: 1.0 mL/min;
sample introduction amount: 10 μ L
Isocratic elution.
Example 2
A method for determining the content of fluorouracil in plasma by an HPLC method, which comprises the following steps:
(1) preparation of control solutions
Dissolving fluorouracil in methanol, and diluting with methanol to obtain a solution containing 0.1mg fluorouracil per mL of methanol as a control solution; aspirate 1mL, dilute to 0.25 μ g fluorouracil per mL with mobile phase of 0.1mol/L aqueous disodium hydrogen phosphate (pH adjusted to 3.2 with phosphoric acid): acetonitrile 90: 10;
(2) sample pretreatment
Taking 100 mu L of blood plasma to be detected, placing the blood plasma in a 5mL test tube, adding 1mL of 0.1mol/L disodium hydrogen phosphate aqueous solution (the pH value is adjusted to 3.2 by phosphoric acid), shaking up, and adding 0.5mL of an extracting agent, wherein the extracting agent is as follows: the volume ratio is 8: 1: 1 mixture of ethyl acetate, acetonitrile and triethylamine; adjusting pH to 6-7, vortex shaking for 3min, centrifuging at 3000rpm for 15min, collecting supernatant, placing in another test tube, adding 0.5mL of extractant, vortex shaking for 5min, and centrifuging at 3000rpm for 5 min; collecting supernatant, mixing the two supernatants, blow-drying at 40 deg.C with nitrogen gas, adding 100 μ L mobile phase, vortex mixing, collecting supernatant 10 μ L, and sampling;
(3) determination of content
Performing content determination by using a high performance liquid chromatography: eluting with mobile phase for 10min, and measuring content with chromatographic conditions:
a chromatographic column: kromacil C18Column, 4.6X 250mm, 5 μm;
mobile phase: 0.1mol/L aqueous disodium hydrogen phosphate solution (phosphoric acid adjusted pH to 3.2): acetonitrile 90: 10;
detection wavelength: 265 nm;
column temperature: 45 ℃;
flow rate: 1.0 mL/min;
sample introduction amount: 10 μ L
Isocratic elution.
Comparative example 1
A method for determining the fluorouracil content of plasma by HPLC, which is substantially the same as in example 1, except that the sample is pretreated as follows:
taking 100 mu L of blood plasma to be detected, placing the blood plasma in a 5mL test tube, adding 1mL of 0.1mol/L disodium hydrogen phosphate aqueous solution (the pH value is adjusted to 3.2 by phosphoric acid), shaking up, and adding 0.5mL of an extracting agent, wherein the extracting agent is as follows: the volume ratio is 8: 1: 1 mixture of ethyl acetate, acetonitrile and triethylamine; adjusting pH to 6-7, vortex shaking for 3min, centrifuging at 3000rpm for 15min, collecting supernatant, placing in another test tube, adding 0.5mL of extractant, vortex shaking for 5min, and centrifuging at 3000rpm for 5 min; collecting supernatant, mixing the two supernatants, blow-drying at 40 deg.C with nitrogen gas, adding 100 μ L mobile phase, vortex mixing, collecting supernatant 10 μ L, and sampling;
comparative example 2
A method of determining the amount of fluorouracil in plasma by HPLC, which method is substantially the same as that of example 1, except that the extractant is an 8: 1 ethyl acetate and acetonitrile.
Comparative example 3
A method for determining the content of fluorouracil in plasma by an HPLC method, which comprises the following steps:
(1) preparation of control solutions
Dissolving fluorouracil in methanol, and diluting with methanol to obtain a solution containing 0.1mg fluorouracil per mL of methanol as a control solution; sucking 1mL, and diluting with a mobile phase to contain 0.25 mu g of fluorouracil per mL, wherein the mobile phase is 10mmol/L dipotassium hydrogen phosphate aqueous solution;
(2) sample pretreatment
Taking 100 mu L of blood plasma to be detected, placing the blood plasma in a 5mL test tube, adding 2mL of ethyl acetate, shaking in a vortex for 2min, centrifuging at 3200rpm for 5min, and taking supernatant to place in another test tube; adding 2mL of ethyl acetate for secondary extraction, shaking for 2min by vortex, and centrifuging for 5min at 3200 rpm; collecting supernatant, mixing the two supernatants, blow-drying at 60 deg.C with nitrogen gas, adding 100 μ L mobile phase, vortex mixing, collecting supernatant 10 μ L, and sampling;
(3) determination of content
Performing content determination by using a high performance liquid chromatography under the following chromatographic conditions:
a chromatographic column: kromacil C18Column, 4.6X 250mm, 5 μm;
mobile phase: 10mmol/L dipotassium hydrogen phosphate aqueous solution;
detection wavelength: 265 nm;
column temperature: 45 ℃;
flow rate: 1.0 mL/min;
sample introduction amount: 10 μ L
Isocratic elution.
Comparative example 4
A method of determining the amount of fluorouracil in plasma by HPLC, which is substantially the same as in example 1, except that the mobile phase used in the determination is: 0.1mol/L aqueous disodium hydrogen phosphate solution (phosphoric acid adjusted pH to 3.2): methanol 90: 10.
specificity experiments
Processing blank plasma according to a sample pretreatment method, and analyzing 10 mu L of solution to obtain a blank plasma chromatogram, which is shown in figure 1; analyzing 10 μ L of the control solution to obtain chromatogram of fluorouracil control, as shown in FIG. 2; 10 mu.L of the test solution was aspirated for analysis, and a plasma fluorouracil chromatogram was obtained, as shown in FIG. 3. The blank plasma chromatogram has no interference on the fluorouracil reference substance solution chromatogram and the plasma fluorouracil chromatogram, and the specificity of the method is proved to be strong.
Linear range experiment
Taking 6 parts of blank plasma, respectively adding reference substance solutions, preparing fluorouracil in each mL of plasma to be 0.25, 1.0, 2.5, 5, 10 and 20 mu g respectively, and precisely sucking 10 mu L of the blank plasma to inject into a chromatograph after sample pretreatment. Taking the peak area A as a vertical coordinate and the concentration c as a horizontal coordinate, and performing linear regression to obtain a fluorouracil regression equation: a is 616.31c-255.48, and a linear correlation coefficient r is 0.9992; wherein, the fluorouracil has good linear relation with the peak area within the range of 0.25-20 mug/mL.
Precision and recovery experiments
Taking 9 parts of blank plasma, respectively adding reference substance solutions, configuring fluorouracil in each mL of plasma to be 1, 5 and 15 mug respectively, respectively carrying out sample pretreatment according to the embodiment 1, the reference example 1 and the reference example 2, respectively and precisely sucking 10 muL of the blank plasma, injecting the blank plasma into a chromatograph, carrying out parallel determination 5 times for each concentration, respectively determining within 1 day and within 3 consecutive days, substituting the measured peak areas into a standard curve to obtain corresponding concentrations, calculating RSD, and obtaining the recovery rate. The results are shown in Table 1.
TABLE 1 precision and recovery test results
As can be seen from the table above, in the content determination provided by the invention, ethyl acetate, acetonitrile and triethylamine are used as extracting agents in the sample pretreatment process, the extraction rate of fluorouracil can be improved, the recovery rate of the method is further improved, and the RSD detected for 3 days continuously is lower than 3%, so that the precision of the method is high.
Sensitivity test
Taking blank plasma, adding a reference substance solution, preparing 0.25 mu g of fluorouracil in each mL of plasma, pretreating according to a sample, and precisely sucking 10 mu L of the plasma for injecting into a chromatograph. According to the chromatographic condition determination under the content determination item, the signal-to-noise ratio of the fluorouracil peak is 14, and the area of the main peak can be accurately integrated, so that the fluorouracil peak can be used as a sensitivity test solution.
Minimum detection limit
Gradually diluting the reference solution, measuring 10 μ L, and injecting into liquid chromatograph with minimum detection limit of fluorouracil of 0.05 μ g/mL.
Stability test
Taking 12 parts of blank plasma, respectively adding reference substance solutions, configuring fluorouracil in each mL of plasma to be 1, 5 and 15 mu g respectively, placing the blank plasma at 30 ℃ for 1 day, performing sample pretreatment according to example 1, example 2, reference example 3 and reference example 4, respectively and precisely sucking 10 mu L of the blank plasma and injecting the blank plasma into a chromatograph, performing parallel measurement on each concentration for 5 times, substituting the measured peak area into a standard curve to obtain the corresponding concentration, and calculating RSD (reference signal decomposition) with the result shown in Table 2.
Taking 12 parts of blank plasma, respectively adding reference substance solutions, preparing fluorouracil in each mL of plasma to be 1, 5 and 15 mu g respectively, freezing and storing for 10 days at the temperature of minus 20 ℃, respectively pretreating the samples according to the examples 1, 2, 3 and 4, respectively, precisely sucking 10 mu L of the samples and injecting the samples into a chromatograph, carrying out parallel measurement for 5 times for each concentration, substituting the measured peak area into a standard curve to obtain the corresponding concentration, and calculating RSD (reference signal decomposition) with the result shown in Table 2.
TABLE 2 stability test results
As can be seen from the above table, the stability of the content determination method provided by the invention is higher than that of the control group, and the stability of the content determination can be further improved after the chromatographic column is washed by the mobile phase.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (7)
1. A method for determining the content of fluorouracil in plasma by an HPLC method, which is characterized by comprising the following steps:
(1) preparation of control solutions
Dissolving fluorouracil in methanol, and diluting with methanol to a solution containing 0.1mg fluorouracil per mL of methanol as a control solution; aspirating 1mL, diluting with mobile phase to contain 0.25 μ g fluorouracil per mL;
(2) sample pretreatment
Placing blood plasma to be tested in a test tube, adding 0.1mol/L disodium hydrogen phosphate aqueous solution, adjusting pH to 3.2 with phosphoric acid, shaking up, adding an extracting agent for extraction, adjusting pH to 6-7 with 0.5mol/L disodium hydrogen phosphate aqueous solution, shaking in a vortex mode for 2-5min, centrifuging at 3000rpm for 5-15min, taking supernatant, placing in another test tube, adding the extracting agent for secondary extraction, shaking in a vortex mode for 2-5min, and centrifuging at 3000rpm for 5-15 min; collecting supernatant, mixing the two supernatants, blowing at 40-60 deg.C with nitrogen, adding mobile phase to desired volume, mixing, and sampling the supernatant;
(3) determination of content
Measuring content by high performance liquid chromatography.
2. The method of claim 1, wherein the mobile phase is a 0.1mol/L aqueous disodium hydrogen phosphate solution (phosphoric acid adjusted to pH 3.2): acetonitrile 90: 10.
3. the method of claim 1, wherein the extractant is a mixture of 8: 1: 1 ethyl acetate, acetonitrile and triethylamine.
4. The method of claim 1, wherein the chromatographic conditions of the high performance liquid chromatography are:
a chromatographic column: c18A chromatographic column;
mobile phase: 0.1mol/L aqueous disodium hydrogen phosphate solution (phosphoric acid adjusted pH to 3.2): acetonitrile 90: 10;
detection wavelength: 265 nm;
column temperature: 45 ℃;
flow rate: 1.0 mL/min;
isocratic elution.
5. The method of claim 3, wherein Kromacil C18Column, 4.6X 250mm, 5 μm.
6. The method of claim 3, wherein the sample size is 10 μ L.
7. The method of claim 3, wherein the assaying further comprises eluting with a mobile phase for 5-10min prior to assaying.
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| CN108956816A (en) * | 2018-07-12 | 2018-12-07 | 西南医科大学 | The HPLC analysis method of curcumin and 5 FU 5 fluorouracil concentration in blood plasma is measured simultaneously |
| US20190049460A1 (en) * | 2016-04-14 | 2019-02-14 | Roche Diagnostics Operations, Inc. | Method for determining a concentration of a target analyte in a sample of bodily fluid |
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