CN108956816B - HPLC analysis method for simultaneously determining curcumin and 5-fluorouracil concentration in plasma - Google Patents

Abstract

The invention belongs to the technical field of pharmacokinetic analysis, and relates to an HPLC (high performance liquid chromatography) analysis method for simultaneously determining the concentrations of curcumin and 5-fluorouracil in animal plasma. The method comprises the following steps of preparation of a standard substance solution, pretreatment of a sample, separation of the sample, detection of the peak area of the sample and preparation of a standard curve. The method overcomes the difficulties that the polarity difference of curcumin and 5-fluorouracil is large and the elution and separation are not easy in the same chromatographic system, and can simultaneously determine the concentrations of curcumin and 5-fluorouracil in blood plasma.

Description

HPLC analysis method for simultaneously determining curcumin and 5-fluorouracil concentration in plasma

Technical Field

The invention belongs to the technical field of pharmacokinetic analysis, relates to an in vivo drug analysis and determination method, and more particularly relates to an HPLC (high performance liquid chromatography) analysis method for simultaneously determining the concentrations of curcumin and 5-fluorouracil in animal plasma.

Background

Curcumin (CU) is a phenolic pigment extracted from rhizome of Curcuma rhizome of Zingiberaceae, and is orange yellow crystalline powder, slightly bitter in taste, and insoluble in water. Modern researches find that curcumin has the effects of reducing blood fat, resisting tumors, resisting inflammation, benefiting gallbladder, resisting oxidation and the like, and has good practicability and application prospect in the field of anticancer. 5-fluorouracil (5-FU) is a pyrimidine anticancer drug, is also a first-line antitumor drug used clinically, has better curative effect on various cancers (such as liver, stomach, pancreatic cancer and breast cancer), but has serious adverse reactions such as gastrointestinal toxicity, bone marrow suppression and the like, so that the clinical use of the 5-fluorouracil is limited. According to the combination principle of chemotherapeutic drugs, the curcumin with spectral antitumor activity and 5-fluorouracil are combined for application, and the synergistic effect is expected to be achieved.

Pharmacokinetic evaluation is to quantitatively research the absorption, distribution, metabolism and excretion rules of drugs in organisms and to illustrate the change rule of blood drug concentration with time by using mathematical principles and methods. With the development of pharmaceutical chemistry and the continuous improvement of human health level, the requirements on the pharmacokinetic properties of the drugs are higher and higher, and the application prospect, particularly the market prospect, of one drug is judged, which is not only strong in curative effect and small in toxic and side effects, but also needs to have good pharmacokinetic properties. The detection technology of the concentration of the drug in the blood plasma is an important prerequisite technology for the pharmacokinetic analysis and evaluation.

Because the polarities of curcumin and 5-fluorouracil are greatly different, elution and separation are not easy to occur in the same chromatographic system, and no literature report exists at present for an analysis method for simultaneously determining the contents of curcumin and 5-fluorouracil in a biological sample in the same chromatographic system. Among the analytical methods used are more common: and respectively detecting the content of curcumin and 5-fluorouracil in the biological sample by using HPLC or liquid chromatography tandem mass spectrometry (LC-MS/MS). Such as:

1. placing the plasma of the experimental mouse in a centrifugal tube containing citric acid glucose, carrying out vortex centrifugation, sucking the supernatant, placing the supernatant in another centrifugal tube, and storing the supernatant in a refrigerator at the temperature of-20 ℃ for later use. Plasma samples were aspirated with precision, precipitated with methanol, treated overnight at-20 ℃, centrifuged (4000rpm, 15min), and appropriate amounts of supernatant were taken for HPLC analysis. HPLC chromatographic conditions for detecting curcumin are as follows: a chromatographic column: luna5 μm C18; mobile phase: acetonitrile: acetic acid (75: 25); detection wavelength: 429 nm. HPLC chromatographic conditions for detecting 5-fluorouracil are as follows: a chromatographic column: qualisil BDS C18 column. (5 μm, 4.6X 250 mm); mobile phase: methanol: water (1: 9); detection wavelength: 262 nm. The flow rate is 1.0 mL/min; column temperature: 30 ℃; sample introduction volume: 20 mu L of the solution; the measurement was carried out under the chromatographic conditions described above.

2. And establishing a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to determine the concentration of 5-fluorouracil in human plasma. Taking 50 mu L of plasma sample of a subject, adding 50 mu L of internal standard solution (5-bromouracil solution of 500 ng/mL), adding methanol to 1000 mu L, vortex mixing for 30s, centrifuging at the low temperature of 15000r/min at 4 ℃ for 10min, taking supernatant, drying with nitrogen, redissolving with 100 mu L of mobile phase, and carrying out sample injection analysis. Chromatographic conditions are as follows: an A gilent-C a8 reverse phase chromatographic column (2.1 mm. times.150 mm, 3.5 μm) was used, methanol-deionized water (0.1% formic acid) (80: 20) was used as the mobile phase, the column temperature was 20 ℃, the flow rate was 0.2 mL/min, and the sample volume was 1 μ L.

Mass spectrum conditions, namely quantitative analysis is carried out by adopting electrospray negative ionization and Multiple Reaction Monitoring (MRM); the ion injection voltage is 5.5kV, and the ion source temperature is 550 ℃; the pressure of gas 1(GS1) in the source is 310kPa, the pressure of gas 2(GS2) in the source is 55lkPa, the pressure of gas curtain is 103kPa, and the DP voltages (cluster-splitting voltage) of 5-fluorouracil and internal standard 5-bromouracil are 65V respectively; the ion reaction pairs used for the quantitative analysis were mass to charge ratio m/z 128.70-m/z 42.0 (5-fluorouracil) and m/z 188.60-m/z 42.0 (5-bromouracil, internal standard), respectively.

Although the HPLC-MS/MS method has advantages of complementary advantages of the chromatography and the mass spectrum, and combines the advantages of high separation capability of the chromatography on complex samples, high selectivity and high sensitivity of the MS and capability of providing relative molecular mass and structural information compared with the HPLC method, the biggest defect is that the above analysis methods can only be used for detecting the content of curcumin and 5-fluorouracil in different chromatographic systems respectively, but not for analyzing together in the same chromatographic system.

Disclosure of Invention

The invention aims to provide an HPLC analysis method for simultaneously determining the concentrations of curcumin and 5-fluorouracil in plasma, which overcomes the difficulties that the curcumin and 5-fluorouracil have large difference in polarity and are not easy to elute and separate in the same chromatographic system, and can simultaneously determine the concentrations of the curcumin and 5-fluorouracil in plasma.

The HPLC analysis method for simultaneously determining the concentration of curcumin and 5-fluorouracil in plasma comprises the following steps:

step one, preparation of standard solution

Mixing curcumin standard substance and 5-fluorouracil standard substance according to a molar ratio of 1:1, dissolving with methanol to obtain mixed standard substance mother liquor of curcumin and 5-fluorouracil, and diluting with methanol to obtain mixed standard substance solutions of curcumin and 5-fluorouracil with a series of concentrations.

Step two, sample pretreatment

Taking a series of equivalent animal blank blood plasma, respectively adding equivalent curcumin and 5-fluorouracil mixed standard substance solution of the series of concentrations in the step I to obtain a series of animal blood plasma samples to be detected, adding citric acid solution or tartaric acid solution into each animal blood plasma sample to be detected, carrying out vortex, adding an extracting agent, carrying out vortex and centrifugation, taking an upper layer organic solution, carrying out nitrogen blow drying to obtain a dried substance, adding a re-solvent into the dried substance, carrying out vortex and centrifugation, and taking a supernatant to obtain a sample solution.

Step three, sample separation

The chromatographic column is Inertsil ODS-SP C18, 5 μm,4.6 × 250mm, guard column Phenomenex C18, 4.0 × 3.0 mm; the column temperature is 25 ℃, the sample injection amount is 20 mu L, and the gradient elution is carried out on each sample solution by respectively taking a mixed solution of methanol and water, a mixed solution of acetonitrile and a buffer solution as mobile phases.

Step four, detecting the peak area of the sample

Detecting peak areas of a sample solution subjected to HPLC analysis by using a universal ultraviolet detector, detecting the peak area of curcumin at a wavelength of 425nm, detecting the peak area of 5-fluorouracil at a wavelength of 265nm, and performing HPLC analysis for 25-30 minutes to obtain the peak areas of curcumin and 5-fluorouracil.

Step five, making a standard curve

Respectively taking the concentrations of curcumin and 5-fluorouracil in an animal plasma sample as horizontal coordinates and peak areas of corresponding curcumin and 5-fluorouracil as vertical coordinates, and drawing a standard curve to obtain a standard curve equation; substituting the peak areas of curcumin and 5-fluorouracil into a standard curve equation to convert into the concentrations of curcumin and 5-fluorouracil.

The concentration of the citric acid solution or the tartaric acid solution is 0.03-0.18 g/ml.

The extractant is a mixed solution of ethyl acetate and methanol, and the volume ratio of the ethyl acetate to the methanol is 9: 1.

The double solvent is a mixed solution of methanol and 2% Tween 80, and the volume ratio is 3: 7.

Centrifuging at 8000r/min and 3min before adding the double solvent in the step II; centrifuging at 10000r/min for 10min after adding the double solvent.

The procedure of gradient elution in step three included: according to the volume percentage, 0-12.4min, methanol: water (1:99, v/v) is used as a mobile phase, the flow rate is 0.8ml/min, and the detection wavelength is 265 nm; 12.5-22.5min, acetonitrile: 0.1% phosphate buffer (65:35, v/v) as mobile phase, flow rate of 1.0ml/min, detection wavelength of 425 nm; 22.6-30min, methanol: water (1:99, v/v) was used as the mobile phase, the flow rate was 0.8ml/min, and the detection wavelength was 265 nm.

The 0.1% phosphate buffer was a 0.1% phosphate solution adjusted to pH 3.5 with triethylamine.

The invention has the following beneficial effects:

1. and (4) simultaneously measuring. Only one method is needed, the curcumin content and the 5-fluorouracil content in a biological sample can be measured in the same chromatographic system, and the problems that the curcumin and the 5-fluorouracil have large polarity difference and are not easy to elute and separate in the same chromatographic system are overcome.

2. The pretreatment method is simple and convenient. Adopts a one-step protein precipitation method, and is simple and easy to implement. Mixing the following raw materials in percentage by weight of ethyl acetate: the effect of methanol (9:1, v/v) as an extractant for precipitating protein is better than that of ethyl acetate, isopropanol, ethyl acetate and the like. The method comprises the following steps of mixing methanol: the 2% Tween 80 aqueous solution (3:7, v/v) as a double solvent can overcome the defects of 5-FU peak splitting and low CU recovery rate caused by too large organic phase proportion.

3. The sampling amount is small. Only 100. mu.l of the biological sample is required for the determination of one sample.

4. Strong selectivity and accurate content measurement. Improving the stability of the drug during the analysis process is the key to ensure the accuracy of the plasma concentration determination method. In the pretreatment process of the plasma biological sample, the citric acid or tartaric acid solution is added, so that the influence of plasma endogenous substances on the stability of curcumin and 5-fluorouracil is eliminated, the CU extraction recovery rate is improved, and meanwhile, the impurity peak and the 5-FU peak can be well separated, so that the determination is more accurate.

5. Saving analysis time and cost. The method does not need to use a large-scale precise-tip instrument or prepare an internal standard solution, has simple process, convenient and simple operation, lower cost and less environmental pollution, simultaneously measures the contents of the two medicines, simplifies the analysis steps, shortens the time for respectively measuring the contents, and saves the analysis time.

6. The method is rapid, accurate, high in sensitivity and simple and convenient to operate. The linear range of the CU, 5-FU plasma standard curve of the method is 0.01-20 mu g/ml and 0.056-7.062 mu g/ml. The relative recovery rate of CU and 5-FU is 86.29-106.05%, and the absolute recovery rate is 70.90-93.20%. The precision RSD of CU, 5-FU, CU/5-FU in-day and in-day is less than 9.29%.

Drawings

FIGS. 1 to 4 are specific chromatogram for investigation by the HPLC analysis method established in example 1. Wherein FIG. 1 is a blank plasma chromatogram of a rat; FIG. 2 is a chromatogram of rat blank plasma after addition of CU standard solution; FIG. 3 is a chromatogram of rat blank plasma after addition of 5-FU standard solution; FIG. 4 is a chromatogram of rat blank plasma after addition of a mixed standard solution of CU and 5-FU.

FIG. 5 is a standard curve diagram of CU after adding CU and 5-FU mixed standard solution to the blank plasma established in example 1.

FIG. 6 is a standard curve diagram of 5-FU after addition of a mixed standard solution of CU and 5-FU to a blank plasma established in example 1.

FIG. 7 is a graph showing the time course of 5-fluorouracil assay in rats after a single caudal vein injection of curcumin and 5-fluorouracil mixed standard solution, which is established in example 1.

FIG. 8 is a graph of curcumin drug timing after single tail vein injection of curcumin and 5-fluorouracil mixed standard solution in rats established in example 1.

FIG. 9 is the drug timing graph of curcumin at various ratios of drug in SD rat plasma established in example 2.

FIG. 10 is the time-series graph of 5-fluorouracil assay established in example 2 for various ratios of drugs in SD rat plasma.

FIGS. 11 to 14 are the chromatogram for investigation on the specificity of the HPLC analysis method established in example 3. Wherein FIG. 11 is a rabbit blank plasma chromatogram; FIG. 12 is a chromatogram of rabbit plasma after addition of CU standard solution; FIG. 13 is a chromatogram of rabbit blank plasma after addition of 5-FU standard solution; FIG. 14 is a chromatogram of rabbit blank plasma after addition of a mixed standard solution of CU and 5-FU;

FIG. 15 is a standard curve graph of CU after the addition of the mixed standard solutions of CU and 5-FU to rabbit blank plasma established in example 3.

FIG. 16 is a standard curve graph of 5-FU after addition of a mixed standard solution of CU and 5-FU to rabbit blank plasma established in example 3.

FIG. 17 is a graph of the drug timing for the determination of 5-fluorouracil in rabbit plasma, as established in example 3.

FIG. 18 is the graph of curcumin in rabbit plasma determined according to example 3.

FIGS. 19 to 22 are specific investigation chromatograms of the HPLC analysis method established in example 4. Wherein FIG. 19 is an empty white spectrum of rat liver tissue fluid; FIG. 20 is a chromatogram of rat liver tissue fluid after addition of CU standard solution; FIG. 21 is a chromatogram of rat liver tissue fluid after addition of 5-FU standard solution; FIG. 22 is a chromatogram of rat liver tissue fluid after addition of a mixed standard solution of CU and 5-FU.

Fig. 23 is a standard graph established in example 4 for determining curcumin distribution in rat liver.

FIG. 24 is a standard graph established in example 4 to determine the distribution of 5-fluorouracil in rat liver.

Detailed description of the preferred embodiments

In order to make the technical process of the present invention clearer, the present invention will now be further described with reference to examples. Unless otherwise indicated, all reagents and equipment used in the present invention are conventional in the art.

The SD rat experimental animal licenses used: SCXK 2013-17. CU, 99.59% by Kymenster Biotech, Inc.; 5-FU in an amount of 99.40% provided by Chongqing Laimei pharmaceutical Co.

Example 1 determination of curcumin, 5-fluorouracil concentration in SD rat plasma.

1. Preparation of Standard solutions

Preparing a curcumin standard substance solution: the curcumin standard substance is precisely weighed, dissolved in methanol solution and shaken up to obtain curcumin standard solution of 80 mug/ml.

Preparation of 5-fluorouracil standard solution: precisely weighing 5-fluorouracil standard substance, dissolving in methanol solution, shaking and shaking up to obtain 28.2500 mug/ml 5-fluorouracil standard solution.

Preparation of a mixed standard solution of curcumin and 5-fluorouracil: preparing a CU/5-FU standard mixed product solution according to the molar ratio of CU to 5-FU of 1 to 1. Namely: mu.g of CU and 706.25 mu.g of 5-FU are precisely weighed and placed in a 50ml volumetric flask, and 50ml of methanol is added to dissolve the CU and 5-FU to obtain a CU/5-FU mixture mother liquor, wherein the CU and 5-FU concentrations are 40 mu.g/ml and 14.1250 mu.g/ml respectively. Then diluted by methanol to prepare 6 mixed solution with different concentrations, wherein the concentration of CU in each mixed solution is 0.3200, 1.6000, 3.200, 4.8000, 6.4000, 40.0000 mug/ml and the concentration of 5-FU is 0.1130, 0.5710, 1.1300, 1.6950, 2.2600 and 14.1250 mug/ml respectively.

2. Liquid chromatography conditions:

a chromatographic column: inertsil ODS-SP C18, 5 μm, 4.6X 250 mm; protection of the column: phenomenex C18, 4.0X 3.0 mm; column temperature: 25 ℃; sample introduction amount: 20 μ L. And respectively taking a mixed solution of methanol and water, a mixed solution of acetonitrile and a buffer solution as mobile phases, and carrying out gradient elution on each sample solution. The gradient elution procedure was: 0-12.4min, methanol: water (1:99, v/v) is used as a mobile phase, the flow rate is 0.8ml/min, and the detection wavelength is 265 nm; 12.5-22.5min, acetonitrile: 0.1% phosphate buffer (pH 3.5, 65:35, v/v) as mobile phase, flow rate of 1.0ml/min, detection wavelength of 425 nm; 22.6-30min, methanol: water (1:99, v/v) was used as the mobile phase, the flow rate was 0.8ml/min, and the detection wavelength was 265 nm. Total analysis time 30 min.

3. Plasma sample pretreatment

Taking 100 mu L of SD rat plasma sample in a 5ml EP tube, precisely adding 50 mu L of CA solution (0.05g/ml), vortex mixing for 1min, adding 1.5ml of extracting agent (ethyl acetate: methanol, 9:1, v/v), vortex mixing for 3min, centrifuging at 8000rpm/min for 3min, taking supernatant, drying with nitrogen, adding 200 mu L of redissolving solvent (methanol: 2% Tween 80 aqueous solution, 3:7, v/v) into the dried residue, vortex mixing for 4min, centrifuging at 10000rpm/min for 10min, taking supernatant, injecting sample and analyzing.

4. Specificity

And (3) respectively taking 100 mu l of rat blank plasma pretreated by a plasma sample, rat blank plasma + CU standard solution, rat blank plasma +5-FU standard solution and rat blank plasma + CU/5-FU mixture standard solution, and obtaining chromatograms shown in figures 1-4 by HPLC analysis according to the chromatographic conditions. The results show that under the HPLC conditions used in this experiment, CU and 5-FU were completely separated, the peak shape was good, the baseline was smooth, the two drugs did not interfere with each other, and were not interfered by endogenous substances in plasma. The 5-FU retention time is about 9min and the CU retention time is about 20 min. There are slight differences in the retention times of the drug due to factors such as the column pressure of the chromatographic system.

5. Standard curve

(1) A series of concentrations of 50. mu.l each of CU/5-FU standard solutions were added to rat plasma blanks, and the procedures were performed according to the "pretreatment of plasma samples".

(2) Drawing a standard curve: and (3) drawing a standard curve by taking the concentration C of the drug in the plasma sample as a horizontal coordinate (X) and the corresponding peak area A of the drug as a vertical coordinate (Y), and calculating to obtain a linear regression equation and a linear correlation coefficient. And calculating by using the signal-to-noise ratio S/N-3 to obtain the lowest detection concentration of the method.

The plasma biological sample standard curve is shown in table 1 and fig. 5-6.

TABLE 1 plasma biological sample Standard Curve and its Linear Range

Medicine	Standard curve	Linear Range (μ g/ml)	Correlation coefficient R2
				CU in the mixture	y＝1.0357x-0.0077	0.0100-20.0000	0.9998
5-FU in a blend	y＝0.5208x+0.013	0.0560-7.0620	0.9999

The results in Table 1 show the CU Standard Curve in the CU/5-FU blend: y 1.0357x-0.0077, linear range: 0.01-20 μ g/ml (R2 ═ 0.9998); 5-FU Standard Curve in CU/5-FU blend: y 0.5208x +0.013, linear range: 0.056-7.062 mug/ml (R2 ═ 0.9999), and the linear correlation coefficients are all larger than 0.999. Under the condition of the invention, the lower limit of the quantitative determination of the blood concentration of 5-FU is 0.0560 mu g/ml, and the lower limit of the quantitative determination of CU is 0.0100 mu g/ml.

6. Recovery utilization rate

(1) Relative recovery experiments: preparing low, medium and high concentration CU/5-FU solutions respectively, wherein each concentration is 3 parts of parallel samples, and detecting according to the method under the item of 'pretreatment of plasma samples'. And calculating respective concentrations according to the standard curves, and calculating the ratio of the obtained drug concentration to the concentration of the corresponding drug standard solution to obtain the relative recovery rate, wherein the relative recovery rate is the accuracy. The relative recovery results are shown in table 2.

(2) Absolute recovery experiments: a low, medium and high concentration of CU/5-FU solution was prepared, 3 samples of each concentration were prepared, and the samples were examined according to the method of "pretreatment of plasma sample" in example 1. Taking 18 centrifuge tubes, and grouping two centrifuge tubes; adding a drug standard solution which is the same as the prepared plasma standard solution into each group, drying by using nitrogen, re-dissolving by using 200 mu l of re-solvent, and taking 20 mu l of sample injection for detection; and recording the peak areas of the drugs with different concentrations, namely the peak areas of the pure standard substances with the concentrations. And calculating the ratio of the peak area of the plasma sample with the same concentration to the peak area of the pure standard substance, namely the absolute recovery rate. The absolute recovery results are shown in table 2.

Table 2 recovery of drug (x ± s, n ═ 3)

The results in Table 2 show that the relative recovery rate of CU and 5-FU in the mixture is 86.29-106.05%, and the absolute recovery rate is 70.90-93.20%.

7. Precision degree

Preparing low, medium and high three-concentration CU/5-FU solutions respectively, measuring 3 parts of parallel samples with each concentration in the same day after processing under the item of 'pretreatment of plasma samples' in example 1, calculating the detection concentration according to a corresponding standard curve, and calculating the precision in the day; the same procedure was carried out for three consecutive days, and the precision between days was calculated. The results are shown in Table 3.

Table 3 precision of drugs in rat plasma (x ± s, n ═ 3)

The results in Table 3 show that the precision RSD of CU, 5-FU, CU/5-FU in-day and in-day is less than 9.29%,

8. stability of

(1) And (3) investigating freezing stability: respectively preparing low, medium and high concentration CU/5-FU solutions, wherein each concentration is 3 parts of parallel samples; after the drug solution was added to the plasma, it was frozen for one day and subjected to 3 freeze-thaw cycles, and then tested for stability by treatment under the "pretreatment of plasma sample" in example 1. The results are shown in Table 4.

TABLE 4 Freeze storage stability of drugs (x. + -.s, n ═ 3)

As shown in Table 4, the RE and CV of the low, medium and high 3 concentrations are less than 14.82%, which indicates that the freeze-thaw conditions have no obvious influence on the detection result of the plasma sample within one day, and the analysis method has good stability. Through investigation, the concentration of the plasma biological sample is obviously reduced when the plasma biological sample is frozen for more than two days, and RE is more than 15%, which indicates that under the condition, the CU, 5-FU rat plasma biological sample can not be stored for more than one day under the freezing condition.

(2) Stability study at room temperature for 24 h: CU/5-FU solutions with low, medium and high concentrations are prepared respectively, and the stability of the solutions at room temperature for 24 hours is examined according to the treatment detection under the item of 'pretreatment of plasma samples' in example 1. The results are shown in Table 5.

24h stability of the samples in Table 5 (x. + -.s, n. RTM. 3)

As shown in Table 5, the CV of the mixture of CU, 5-FU at low, middle and high concentrations is less than 12.42%, which indicates that the detection results of the plasma samples have no obvious difference within 24 h. That is, the stability of the assay was good within one day of freezing and 24 hours at room temperature.

9. Determination of curcumin and 5-fluorouracil metabolic condition in plasma after single tail vein injection of curcumin and 5-fluorouracil mixed solution in SD rat

SD rats were fasted for 12h (without water deprivation) before dosing, and after grouping, dosing was performed according to the experimentally designed dosing schedule. Blood was drawn after dosing according to the design of the experiment sampling time. The concentrations of curcumin and 5-fluorouracil at each time point are plotted as drug-forming curves. The results are shown in FIGS. 7 to 8.

Example 2 the metabolism of curcumin and 5-fluorouracil mixtures in SD rat plasma was determined at different ratios.

After the mixed solution of CU/5-FU with the molar ratio of 1:1, 1:2 and 1:4 is injected into the tail vein of the SD rat, the content of CU and 5-FU in blood plasma is detected by the analysis method.

(1) SD rats were fasted for 12h (without water deprivation) before dosing, and after grouping, dosing was performed according to the experimentally designed dosing schedule. Blood was drawn after dosing according to the design of the experiment sampling time. Placing the blood sample in a heparin tube, centrifuging whole blood at 5000rpm/min for 3min, collecting supernatant and plasma, packaging into 100 μ l, and storing in a refrigerator at-80 deg.C until determination.

(2) After 100. mu.L of rat plasma was treated according to the "pretreatment of plasma sample" method described in example 1, 100. mu.L of the supernatant was transferred to a sample bottle for HPLC analysis, and the plasma sample concentration was calculated according to the standard curve in the same manner as in example 1.

The drug metabolism time curve of curcumin at each ratio in the plasma of SD rat is shown in FIG. 9. The metabolic profile of 5-fluorouracil is shown in FIG. 10.

Example 3 the concentration of curcumin, 5-fluorouracil in rabbit plasma was determined.

1. Preparation of Standard solutions

Preparation of curcumin standard solution: the curcumin standard substance is precisely weighed, dissolved in methanol solution and shaken up to obtain curcumin standard solution of 80 mug/ml.

Preparation of 5-fluorouracil standard solution: precisely weighing 5-fluorouracil standard substance, dissolving in methanol solution, shaking and shaking up to obtain 28.2500 mug/ml 5-fluorouracil standard solution.

Preparation of curcumin/5-fluorouracil mixed standard substance solution: preparing a CU/5-FU standard mixed product solution according to the molar ratio of CU to 5-FU of 1 to 1. Namely: mu.g of CU and 706.25 mu.g of 5-FU are precisely weighed and placed in a 50ml volumetric flask, and 50ml of methanol is added to dissolve the CU and 5-FU to obtain a CU/5-FU mixture mother liquor, wherein the CU and 5-FU concentrations are 40 mu.g/ml and 14.1250 mu.g/ml respectively. Then diluted by methanol to prepare 6 mixed solution with different concentrations, wherein the concentration of CU in each mixed solution is 0.3200, 1.6000, 3.200, 4.8000, 6.4000, 40.0000 mug/ml and the concentration of 5-FU is 0.1130, 0.5710, 1.1300, 1.6950, 2.2600 and 14.1250 mug/ml respectively.

2. Chromatographic conditions are as follows:

a chromatographic column: inertsil ODS-SP C18, 5 μm, 4.6X 250 mm; protection of the column: phenomenex C18, 4.0X 3.0 mm; column temperature: 25 ℃; sample introduction amount: 20 μ L. And respectively taking a mixed solution of methanol and water, a mixed solution of acetonitrile and a buffer solution as mobile phases, and carrying out gradient elution on each sample solution. The gradient elution procedure was: 0-12.4min, methanol: water (1:99, v/v) is used as a mobile phase, the flow rate is 0.8ml/min, and the detection wavelength is 265 nm; 12.5-22.5min, acetonitrile: 0.1% phosphate buffer (pH 3.5, 65:35, v/v) as mobile phase, flow rate of 1.0ml/min, detection wavelength of 425 nm; 22.6-30min, methanol: water (1:99, v/v) was used as the mobile phase, the flow rate was 0.8ml/min, and the detection wavelength was 265 nm. Total analysis time 30 min.

3. Pretreatment of rabbit plasma sample

Adding 100 μ L of clean-grade rabbit medicated plasma into 5ml EP tube, adding 50 μ L CA solution (0.05g/ml) precisely, mixing for 1min by vortex, adding 1.5ml extractant (ethyl acetate: methanol, 9:1, v/v), mixing for 3min by vortex, centrifuging at 8000rpm/min for 3min, taking supernatant, drying with nitrogen, adding 200 μ L redissolution solvent (methanol: 2% Tween 80 aqueous solution, 3:7, v/v) into dry residue, mixing for 4min by vortex, centrifuging at 10000rpm/min for 10min, sampling supernatant, and analyzing.

4. Specificity

And respectively taking 100 mu l of clean-grade rabbit blank plasma subjected to plasma sample pretreatment, clean-grade rabbit blank plasma + CU standard solution, clean-grade rabbit blank plasma +5-FU standard solution, and clean-grade rabbit blank plasma + CU/5-FU mixed product standard solution, and performing specificity evaluation according to the chromatographic conditions. The chromatogram is shown in FIGS. 11-14. No interference of the biological matrix on the determination of curcumin and 5-fluorouracil is found. The chromatographic retention time of curcumin and 5-fluorouracil is 9min and 20min respectively.

5. Linear test

Taking blank rabbit plasma, respectively adding 50 μ l of CU/5-FU standard solution with the above series of concentrations, and operating according to the method of rabbit plasma sample pretreatment. And (3) drawing a standard curve by taking the drug concentration C in the tissue sample as a horizontal coordinate (X) and the corresponding drug peak area A as a vertical coordinate (Y), and calculating to obtain a linear regression equation and a linear correlation coefficient. The regression equations of the standard curves in fig. 15 to 16 are respectively: CU y 0.9884x-0.074, R2 0.9999; 5-FU y is 0.451x +0.0011, and R2 is 0.9997.

6. Accuracy and precision

Taking blank plasma, preparing low, medium and high concentration CU/5-FU plasma quality control samples, wherein each concentration is 3 parts of parallel samples, and inspecting day precision and accuracy by the operation of 'pretreatment of rabbit plasma samples'. The respective concentrations were calculated from the standard curves, the relative recovery was calculated, and the absolute recovery was the accuracy. The recovery and precision results are shown in tables 6 and 7.

Table 6 recovery of drug from rabbit plasma (x ± s, n ═ 3)

The results in Table 6 show that the relative recovery rate of CU and 5-FU in the single product and the mixed product is 86.69-104.50%, and the absolute recovery rate is 78.41-93.03%.

TABLE 7 precision of drugs in rabbit plasma (x. + -. s, n ═ 3)

The results in Table 7 show that the precision RSD of CU, 5-FU, CU/5-FU in-day and in-day is less than 10.15%,

the concentrations of curcumin and 5-fluorouracil at each time point in the plasma of the clean rabbit injected by the marginal ear vein are drawn into a drug time curve. The results are shown in FIGS. 17 to 18.

Example 4 curcumin and 5-fluorouracil were assayed in rat liver.

1. Preparation of Standard solutions

Preparation of curcumin standard solution: the curcumin standard substance is precisely weighed, dissolved in methanol solution and shaken up to obtain curcumin standard solution of 80 mug/ml.

Preparation of 5-fluorouracil standard solution: precisely weighing 5-fluorouracil standard substance, dissolving in methanol solution, shaking and shaking up to obtain 28.2500 mug/ml 5-fluorouracil standard solution.

Preparation of curcumin/5-fluorouracil mixed standard solution: preparing a CU/5-FU standard mixed product solution according to the molar ratio of CU to 5-FU of 1 to 1. Namely: mu.g of CU and 706.25 mu.g of 5-FU are precisely weighed and placed in a 50ml volumetric flask, and 50ml of methanol is added to dissolve the CU and 5-FU to obtain a CU/5-FU mixture mother liquor, wherein the CU and 5-FU concentrations are 40 mu.g/ml and 14.1250 mu.g/ml respectively. Then diluted by methanol to prepare 6 mixed solution with different concentrations, wherein the concentration of CU in each mixed solution is 0.3200, 1.6000, 3.200, 4.8000, 6.4000, 40.0000 mug/ml and the concentration of 5-FU is 0.1130, 0.5710, 1.1300, 1.6950, 2.2600 and 14.1250 mug/ml respectively.

2. Chromatographic conditions

A chromatographic column: inertsil ODS-SP C18, 5 μm, 4.6X 250 mm; protection of the column: phenomenex C18, 4.0X 3.0 mm; column temperature: 25 ℃; sample introduction amount: 20 μ L. And respectively taking a mixed solution of methanol and water, a mixed solution of acetonitrile and a buffer solution as mobile phases, and carrying out gradient elution on each sample solution. The gradient elution procedure was: 0-12.4min, methanol: water (1:99, v/v) is used as a mobile phase, the flow rate is 0.8ml/min, and the detection wavelength is 265 nm; 12.5-22.5min, acetonitrile: 0.1% phosphate buffer (pH 3.5, 65:35, v/v) as mobile phase, flow rate of 1.0ml/min, detection wavelength of 425 nm; 22.6-30min, methanol: water (1:99, v/v) was used as the mobile phase, the flow rate was 0.8ml/min, and the detection wavelength was 265 nm. Total analysis time 30 min.

3. Tissue sample pretreatment

Taking 100 mu L of blank homogenate of the liver group of SD rat in a 5ml EP tube, precisely adding 50 mu L of CA solution (0.05g/ml), vortex and mixing uniformly for 1min, adding 1.5ml of extracting agent (ethyl acetate: methanol, 9:1, v/v), vortex and mixing uniformly for 3min, centrifuging at 8000rpm/min for 3min, taking supernatant and drying by nitrogen, adding 200 mu L of redissolving solvent (methanol: 2% Tween 80 aqueous solution, 3:7, v/v) into dried residue, vortex and mixing uniformly for 4min, centrifuging at 10000rpm/min for 10min, taking supernatant and sampling for analysis.

4. Specificity

Taking 100 mul of rat blank liver tissue homogenate, rat blank liver tissue homogenate + CU standard solution, rat blank liver tissue homogenate +5-FU standard solution, and rat blank liver tissue homogenate + CU/5-FU mixture standard solution after tissue sample pretreatment, and performing specificity evaluation according to the chromatographic conditions. The chromatogram is shown in 19-22. No interference of the biological matrix on the determination of curcumin and 5-fluorouracil is found. The chromatographic retention time of curcumin and 5-fluorouracil is 9min and 20min respectively.

5. Linear test

Taking blank liver homogenate, respectively adding 50 μ l of CU/5-FU standard solution with the above series of concentrations, and operating according to the method of 'tissue sample pretreatment'. And (3) drawing a standard curve by taking the drug concentration C in the tissue sample as a horizontal coordinate (X) and the corresponding drug peak area A as a vertical coordinate (Y), and calculating to obtain a linear regression equation and a linear correlation coefficient. The regression equations of the standard curves in fig. 23 to 24 are respectively: CU y 1.142x-0.384, R2 0.9993; 5-FU y-0.4529 x-0.0377, and R2-0.9993.

6. Accuracy and precision

Taking blank liver tissue homogenate, preparing CU/5-FU tissue quality control samples with low, medium and high concentrations, preparing 3 parts of parallel samples of each concentration, and inspecting day precision and accuracy according to the 'pretreatment of tissue samples'. The respective concentrations were calculated from the standard curves, the relative recovery was calculated, and the absolute recovery was the accuracy. The recovery and precision results are shown in tables 8 and 9.

Table 8 recovery of drug in rat liver tissue (x ± s, n ═ 3)

The results in Table 8 show that the relative recovery rate of CU and 5-FU in the single product and the mixed product is 87.12-103.03%, and the absolute recovery rate is 78.22-91.63%.

TABLE 9 precision of drugs in rat liver tissue (x. + -. s, n ═ 3)

The results in Table 9 show that the daily precision RSD of CU, 5-FU, CU/5-FU is less than 11.42%.

Claims (2)

1. An HPLC analysis method for simultaneously determining the concentrations of curcumin and 5-fluorouracil in plasma, characterized by comprising the following steps:

step one, preparation of standard solution

Mixing a curcumin standard substance and a 5-fluorouracil standard substance according to a molar ratio of 1:1, dissolving with methanol to obtain a mixed standard substance mother liquor of curcumin and 5-fluorouracil, and diluting with methanol to obtain a series of mixed standard substance solutions of curcumin and 5-fluorouracil with a concentration;

step two, sample pretreatment

Taking a series of equivalent animal blank blood plasma, respectively adding equivalent curcumin and 5-fluorouracil mixed standard substance solution with the series of concentrations in the step I to obtain a series of animal blood plasma samples to be detected, adding citric acid solution or tartaric acid solution into each animal blood plasma sample to be detected, carrying out vortex, adding an extracting agent, carrying out vortex and centrifugation, taking an upper layer organic solution, carrying out nitrogen blow drying to obtain a dried substance, adding a re-solvent into the dried substance, carrying out vortex and centrifugation, and taking a supernatant to obtain a sample solution;

step three, sample separation

The chromatographic column is Inertsil ODS-SP C18, 5 μm,4.6 × 250mm, guard column Phenomenex C18, 4.0 × 3.0 mm; carrying out gradient elution on each sample solution by taking a mixed solution of methanol and water, a mixed solution of acetonitrile and a buffer solution as mobile phases respectively at the column temperature of 25 ℃ and the sample injection amount of 20 mu L;

step four, detecting the peak area of the sample

Detecting peak areas of a sample solution subjected to HPLC analysis by using a universal ultraviolet detector, detecting the peak area of curcumin at a wavelength of 425nm, detecting the peak area of 5-fluorouracil at a wavelength of 265nm, and performing HPLC analysis for 25-30 minutes to obtain the peak areas of curcumin and 5-fluorouracil;

step five, making a standard curve

Respectively taking the concentrations of curcumin and 5-fluorouracil in an animal plasma sample as horizontal coordinates and peak areas of corresponding curcumin and 5-fluorouracil as vertical coordinates, and drawing a standard curve to obtain a standard curve equation; substituting the peak areas of curcumin and 5-fluorouracil into a standard curve equation to convert to obtain the concentrations of curcumin and 5-fluorouracil;

the concentration of the citric acid solution or the tartaric acid solution is 0.03-0.18 g/ml;

the double solvent is a mixed solution of methanol and 2% Tween 80, and the volume ratio is 3: 7;

the procedure of gradient elution in step three included: according to the volume percentage, 0-12.4min, methanol: water is 1:99, v/v is a mobile phase, the flow rate is 0.8ml/min, and the detection wavelength is 265 nm; 12.5-22.5min, acetonitrile: 0.1% phosphate buffer 65:35, v/v as mobile phase, flow rate of 1.0ml/min, detection wavelength 425 nm; 22.6-30min, methanol: water is 1:99, v/v is a mobile phase, the flow rate is 0.8ml/min, and the detection wavelength is 265 nm;

the 0.1% phosphoric acid buffer solution is a 0.1% phosphoric acid solution with pH adjusted to 3.5 by triethylamine;

the extractant is a mixed solution of ethyl acetate and methanol, and the volume ratio of the ethyl acetate to the methanol is 9: 1.

2. The method of claim 1, wherein: centrifuging at 8000r/min and 3min before adding the double solvent in the step II; centrifuging at 10000r/min for 10min after adding the double solvent.

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