CN109100435B - HPLC analysis method for determining curcumin and paclitaxel content in biological sample - Google Patents

Abstract

The invention relates to the field of pharmacokinetic analysis, in particular to an HPLC (high performance liquid chromatography) method for determining the content of curcumin and paclitaxel in a biological sample. The method sequentially comprises two steps of standard curve establishment and sample detection, and is characterized in that the conditions of the HPLC analysis method are as follows: a Luna 5 mu m C18(4.6mm multiplied by 250mm) chromatographic analysis column is adopted, and a gradient elution wavelength-changing method is adopted. The method realizes the synchronous determination of the contents of the curcumin and the paclitaxel in the biological sample by adopting HPLC, quickly improves the pharmacokinetics research efficiency of the combined medication of the curcumin and the paclitaxel, can quickly promote the development process of the combined medication of the curcumin and the paclitaxel, and greatly reduces the time, the labor and the economic cost for research and development enterprises and scientific research institutions.

Description

HPLC analysis method for determining curcumin and paclitaxel content in biological sample

Technical Field

The invention relates to the field of pharmacokinetic analysis, in particular to an HPLC (high performance liquid chromatography) method for determining the content of curcumin and paclitaxel in a biological sample.

Background

Curcumin (CU) is a monomer separated and purified from traditional Chinese medicines, is a pigment with diketone which is rare in the plant world,

is diketone compound, orange yellow crystal powder, and is insoluble in water. Has pharmacological actions of resisting inflammation, resisting tumor, resisting aging and the like, has high efficiency and low toxicity, and has good prospect as a new generation of antitumor drugs. The anti-tumor effect of the polypeptide is realized mainly from the aspects of blocking a signal path to conduct NF-kappa B, STAT3 and MAPK, regulating and controlling related tumor genes and protein expression BcL-2, p53 and the like, regulating mediation of adhesion molecules, inhibiting tumor angiogenesis and the like.

Paclitaxel (PTX) is a natural diterpenoid with anticancer activity. The traditional Chinese medicine composition is a first-line chemotherapeutic medicine for clinically treating breast cancer for years, and has obvious curative effect on various refractory malignant tumors, such as testicular embryonic carcinoma, gall bladder cancer, non-small cell lung cancer and ovarian cancer. But has strong toxic and side effects after long-term application, and can cause severe hypersensitivity, multidrug resistance and gastrointestinal tract stimulation, even bone marrow suppression. In clinical application, a chemotherapy scheme mainly based on cisplatin and PTX is widely applied to the treatment of malignant tumors, but the toxic and side effects are not relieved. In recent years researchers have tended to find an attenuated and synergistic agent for the treatment of malignancies in combination with PTX.

In recent years, researches show that CU can slow down the removal speed of cells on PTX, increase the effective concentration of PTX and reverse the drug resistance of the cells on the PTX, and the combination of the two medicines is proved to enhance the killing effect on endometrial cancer cells, have obvious effect on metastatic lung cancer of mice and remarkably increase the curative effect on human laryngeal cancer and liver cancer. The two medicines are combined to be researched on the aspects of liver cancer, lung cancer, ovarian cancer, oral cancer and the like, and certain achievements are achieved, so that the research on the pharmacokinetic analysis method of the two medicines is of great significance.

Currently, for pharmacokinetic studies on CU and PTX, HPLC methods for measuring the concentrations of biological samples thereof are all single methods for measuring the drug concentrations thereof, and no pharmacokinetic evaluation analysis methods for combined use thereof are available (zhuangyan, gompheyan, sojoram, etc. curcumin in turmeric capsule is pharmacokinetic and tissue distribution in rat [ J ] chinese pharmacist, 2018(1): 65-68; xu juan, yellow-brilliant, threong, etc.. paclitaxel liposome pharmacokinetic studies for injection [ C ] national treatment drug monitoring academic annual meeting, 2014). In addition, there are studies to establish UPLC-MS/MS for separately determining CU and PTX and simultaneously determining the drug concentration of both biological samples, but the processing operations for biological samples are cumbersome and the requirements for the instruments used are high (li zhining, wei yue, zhuye, etc.. ultra performance liquid chromatography-mass spectrometry for determination of curcumin [ J ] in rat plasma, hennan science, 2017,35(8):1252 + 1257; Shi Yonghui, Yuxian, Luli, etc.

HPLC-MS/MS method for determining paclitaxel content [ J ] in rat plasma, 2017(11): 735-738; plum-honeysuckle, atrial crystalloid, plum literature, and the like, the pharmacokinetics and tissue distribution of paclitaxel in animal bodies are analyzed at high throughput [ J ]. Chinese medicine, 2014,9(12): 1833-1837). Pharmacokinetic studies of CU and PTX the prior art still suffers from the following disadvantages: the existing method can only separately measure the content of CU and PTX biological samples, and is difficult to realize the combination of CU and PTX; when the UPLC-MS/MS method is adopted to simultaneously determine the content of CU and PTX, sample pretreatment and determination operations are complicated, time consumption is long, and requirements on instruments and equipment are high.

Disclosure of Invention

The invention provides an HPLC analysis method for measuring the content of curcumin and paclitaxel in a biological sample, which can synchronously measure the content of curcumin and paclitaxel without being combined with a mass spectrometer, is simple and efficient to operate, has low requirements on pretreatment of the sample, and has no fussy pretreatment process.

An HPLC analysis method for determining the content of curcumin and paclitaxel in a biological sample sequentially comprises two steps of establishing a standard curve and detecting the sample, and is characterized in that the conditions of the HPLC analysis method are as follows: by using

5 μm C18(4.6 mm. times.250 mm) chromatography column using gradient elution wavelength method: when the time is 0-9.5min, the detection wavelength is 425nm, and the mobile phase is acetonitrile: phosphate buffer 55:45 (v/v); and when 9.51-11.5min, the detection wavelength is 227nm, and the mobile phase is acetonitrile: phosphate buffer 40:60 (v/v); the column temperature was 30 ℃ and the mobile phase flow rate was 1 mL/min.

Preferably, the conditions of the HPLC analytical method further comprise: at 11.51-19.0min, the detection wavelength is 425nm, and the mobile phase is acetonitrile: phosphate buffer 55:45 (v/v).

Preferably, the phosphate buffer is a mixture of phosphate and triethylamine at a pH of 3.5.

Preferably, the establishment of the standard curve comprises the following steps:

(1) preparing a reference substance solution: curcumin, paclitaxel and curcumin and paclitaxel are respectively dissolved in methanol to prepare curcumin stock solution, paclitaxel stock solution and mixed stock solution of curcumin and paclitaxel with the same concentration, and then methanol is used for diluting to obtain a series of standard solutions with gradient concentration.

(2) Establishment of a standard curve: curcumin, paclitaxel and a standard solution mixed with curcumin and paclitaxel with series concentrations are respectively taken and mixed with a blank biological sample corresponding to a sample to be detected to obtain a mixed solution, the mixed solution is subjected to sample pretreatment to obtain a test solution, then the content is measured by HPLC under the condition of the HPLC analysis method, linear regression is carried out by taking the concentration of the sample as a horizontal coordinate and taking a peak area as a vertical coordinate, and a standard curve is established.

Preferably, the sample detection step is to take a sample to be detected to perform sample pretreatment to obtain a test solution, then determine the content by using HPLC under the condition of the HPLC analysis method, and calculate the concentrations of curcumin and paclitaxel in the sample according to the standard curve and the peak area.

Preferably, the citric acid buffer solution is added into the mixed solution in the sample pretreatment process, the mixed solution is uniformly mixed, the extraction solvent with the volume 8-12 times that of the mixed solution is added, the mixed solution is uniformly mixed and centrifuged, the organic layer is taken, nitrogen is volatilized, the composite solvent is added, the mixed solution is uniformly mixed, and the supernatant is taken in the centrifugation process to obtain the test solution.

Preferably, the composite solvent is prepared from acetonitrile and a phosphate buffer solution, and the volume ratio of the acetonitrile to the phosphate buffer solution is 75: 25.

Preferably, the extraction solvent is prepared from ethyl acetate and methanol, and the volume ratio of the ethyl acetate to the methanol is 90: 10.

Preferably, when the sample to be detected is a rabbit sample, the volume ratio of the standard solution to the blank biological sample is 1:10, and when the sample to be detected is a rat sample, the volume ratio of the standard solution to the blank biological sample is 1: 2.

More preferably, the specific method of sample pretreatment is as follows:

a quantitative mixture was placed in a 5mL centrifuge tube, 25. mu.L of CA buffer (pH 3.6) was added, vortex-mixed for 30s, a quantitative extractant (ethyl acetate: methanol: 90:10) was added, vortex-mixed for 3min, and centrifuged for 3min (8000rpm/min), and the supernatant was collected. Adding the extractant again to extract for one time repeatedly, and combining the supernatants for 2 times. Blow-drying with nitrogen, adding 200 μ L of a double solvent [ acetonitrile: phosphoric acid (pH 3.5) ], vortex mixing for 4min, ultrasonic treating for 4min, centrifuging for 10min (10000rpm/min), and taking 20 μ L of supernatant for HPLC detection.

The invention has the beneficial effects that:

1. under the condition of not using a mass spectrometer together, the method realizes the synchronous determination of the contents of the curcumin and the paclitaxel in the biological sample by only adopting HPLC, quickly improves the efficiency of the pharmacokinetic research of the curcumin and the paclitaxel combined drug, can quickly promote the development process of the curcumin and the paclitaxel combined drug, and greatly reduces the time, the labor and the economic cost for research and development enterprises and scientific research institutions.

2. The method does not need to use ultra-high performance liquid chromatography, and effectively simplifies the pretreatment step of the sample; meanwhile, the requirement of the eluent of HPLC on purity is far lower than that of the ultra-high performance liquid chromatography, so that the running cost of the instrument is far lower than that of the ultra-high performance liquid chromatography.

3. The method has high sensitivity, accuracy and stability, and can completely satisfy pharmacokinetic research of raw material medicines and preparations of curcumin, taxol and combined medicine of curcumin and taxol.

Drawings

FIG. 1 is a standard curve for measuring blood concentrations of CU and PTX in rat plasma CU single product, PTX single product and CU combined PTX compound product, which is established in example 1.

FIG. 2 is an HPLC chromatogram of rat plasma in example 1, wherein (1) is a CU single HPLC chromatogram, (2) is a PTX single HPLC chromatogram, and (3) is a CU combined PTX composite HPLC chromatogram.

FIG. 3 is an HPLC chromatogram of the rat plasma spiked sample established in example 2, wherein (1) is a CU single spiked sample HPLC chromatogram, (2) is a PTX single spiked sample HPLC chromatogram, and (3) is a CU combined PTX composite spiked sample HPLC chromatogram.

FIG. 4 is a standard curve established in example 3 for measuring blood concentrations of CU and PTX in rabbit plasma CU single products, PTX single products and CU combined PTX compound products.

FIG. 5 is an HPLC chromatogram of rabbit plasma obtained in example 3, wherein (1) is a CU single HPLC chromatogram, (2) is a PTX single HPLC chromatogram, and (3) is a CU combined PTX composite HPLC chromatogram.

FIG. 6 is the HPLC chromatogram for detecting rabbit plasma spiked samples established in example 4, wherein (1) is the CU single spiked sample HPLC chromatogram, (2) is the PTX single spiked sample HPLC chromatogram, and (3) is the CU combined PTX compound spiked sample HPLC chromatogram. .

FIG. 7 is a standard curve for testing tissue drug concentrations of CU and PTX in rat heart single products, PTX single products and CU combined PTX compound products, which is established in example 5.

FIG. 8 is a standard curve for testing tissue drug concentrations of CU and PTX in rat stomach single products, PTX single products and CU combined PTX compound products, which is established in example 5.

FIG. 9 is a standard curve for testing tissue drug concentrations of CU and PTX in rat liver CU single products, PTX single products and CU combined PTX composite products, which are established in example 5.

FIG. 10 is a standard curve for testing tissue drug concentrations of CU and PTX in rat spleen CU single product, PTX single product and CU combined PTX composite product, which is established in example 5.

FIG. 11 is a standard curve for testing tissue drug concentrations of CU and PTX in rat lung CU single products, PTX single products and CU combined PTX composite products, which is established in example 5.

FIG. 12 is a standard curve for testing tissue drug concentrations of CU and PTX in rat kidney CU single product, PTX single product and CU combined PTX composite product, which is established in example 5.

FIG. 13 is a standard curve for testing tissue drug concentrations of CU and PTX in rat brain CU single product, PTX single product and CU combined PTX composite product, which is established in example 5.

FIG. 14 is the HPLC chromatogram for detecting rat hearts established in example 6, wherein (1) is a CU single HPLC chromatogram, (2) is a PTX single HPLC chromatogram, and (3) is a CU joint PTX composite HPLC chromatogram. .

FIG. 15 is a graph showing the results of testing single tail vein injections of CU single product, PTX single product, CU and PTX compound product and their formulations for the plasma drug after CU and PTX in rats established in example 7.

FIG. 16 is a graph showing the results of testing plasma drug after CU and PTX in rabbit ear vein injection CU single product, PTX single product and CU combined PTX compound product (mass ratio of 1:1 to 1:2) established in example 8.

Detailed Description

The invention is further described below with reference to the drawings and specific examples in the description, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The HPLC is a short name of a high performance liquid chromatography technology; CU is curcumin for short; PTX is paclitaxel for short.

Example 1: establishing a standard curve for measuring the concentrations of CU and PTX in the single CU product, the single PTX product and the combined PTX compound CU product in rat plasma

1. Preparing a standard solution:

(1) preparing CU and PTX standard solutions: precisely weighing 4.00mg of CU and PTX reference substances, respectively placing the CU and the PTX reference substances in a 50mL volumetric flask, adding methanol to dissolve, shaking up, and fixing the volume to 50mL to obtain stock solutions of CU and PTX with the concentration of 80 mu g/mL, and diluting the stock solutions with methanol to respectively prepare standard solutions of CU and PTX with the concentrations of 0.02, 0.04, 0.08, 0.16, 0.32, 0.80, 1.60, 3.20, 4.80, 6.60 and 8.00 mu g/mL.

(2) Preparing a CU and PTX compound control solution: newly prepared CU and PTX reference substance stock solutions are diluted by methanol to prepare CU and PTX compound reference substance solutions with the concentrations of 0.02, 0.04, 0.08, 0.16, 0.32, 0.80, 1.60, 3.20, 4.80, 6.60 and 8.00 mu g/mL respectively.

2. Establishment of a Standard Curve

50 μ L of each of the serial standard solutions was pipetted precisely, and added to a 5mL centrifuge tube, 100 μ L of rat blank plasma was added, vortexed for 30s, 25 μ L of CA buffer (pH 3.6) was added, vortexed for 30s, 0.5mL of an extractant (ethyl acetate: methanol ═ 90:10) was added, vortexed for 3min, centrifuged for 3min (8000rpm/min), and the supernatant was collected. The extraction was repeated once more by adding 0.5mL of extractant and 2 supernatants were combined. Blow-drying with nitrogen, adding 200 μ L of a double solvent [ acetonitrile: phosphoric acid (pH 3.5) ], vortex mixing for 4min, ultrasonic mixing for 4min, high speed centrifuging at 10000rpm/min for 10min, taking 160 mu L of supernatant, and performing HPLC detection.

And performing linear regression by taking CU and PTX blood concentration C with different concentrations as abscissa and taking peak areas Y of CU and PTX with different concentrations as ordinate to obtain a regression equation and R2. The standard curve results are shown in figure 1.

The conditions of the HPLC analysis method are as follows:

use of

5 μm C18(4.6 mm. times.250 mm) chromatography column, detection wavelength 425nm at 0-9.5min, mobile phase acetonitrile: phosphate buffered saline 55:45 (v/v); when the time is 9.51-11.5min, the detection wavelength is 227nm, and the mobile phase is acetonitrile; phosphate buffered saline 40:60 (v/v); at 11.51-19.0min, the detection wavelength is 425nm, and the mobile phase is acetonitrile: phosphate buffered solution 55:45 (v/v). The column temperature was 30 ℃ and the flow rate was 1 mL/min. The amount of sample was 20. mu.L.

Example 2: sample detection

1. Spiked rat plasma sample detection

(1) Pretreatment of blank rat plasma and preparation of test solution

About 500. mu.L of blood was collected from the heart of a blank rat. The blood sample was placed in a heparinized EP tube and whole blood was centrifuged at 54000rpm for 3min to aspirate the upper plasma, i.e. the blank rat plasma, for use.

Adding CU, PTX and a CU and PTX compound control solution into blank rat plasma to obtain CU concentrations of 0.1124, 0.9050 and 3.062 mu g/mL; PTX concentrations were 0.1005, 0.4726, 1.4204. mu.g/mL; and the concentrations of CU in the compound of CU and PTX are 0.1872, 0.7027 and 3.1453 mu g/mL, and the concentration of PTX is 0.1097, 0.3086 and 1.2386 mu g/mL.

After 100. mu.L of the standard plasma sample was treated according to the plasma sample pretreatment method of example 1, 160. mu.L of the supernatant was transferred to a sample bottle.

(2) Spiked plasma sample detection

A sample of 20. mu.L was introduced and subjected to HPLC analysis under the same conditions as in example 1. Calculating the concentrations of CU and PTX plasma samples in the CU single sample, the PTX single sample and the CU and PTX composite sample according to the standard curve respectively as follows: 0.0952 +/-0.0124, 0.8549 +/-0.08678 and 2.7337 +/-0.1800; 0.0752 plus or minus 0.0055, 0.3875 plus or minus 0.0095 and 1.2640 plus or minus 0.1377; 0.1439 + -0.0059, 0.6851 + -0.0129, 2.9135 + -0.1994, 0.0800 + -0.0178, 0.2866 + -0.0096 and 1.1258 + -0.0541, and the sample chromatogram is shown in FIG. 3.

The determination method is proved to be capable of accurately determining and analyzing the concentrations of the PTX and the compound of the CU and the PTX in the rat plasma, and the CU and the PTX in the chromatogram are smooth and symmetrical in peak shape and free of interference, and the CU and the PTX are mutually separated and free of interference, the chromatogram is stable and reliable, the method is efficient and stable, and the requirement of pharmacokinetic evaluation of the rat plasma CU, the PTX and the combination of the CU and the PTX can be well met.

2. In-vivo sample detection:

(1) the test animals Sprague-DawLey rats were selected.

Fasting was performed for 12h before administration, and water was freely available.

Preparation of injection: the powder of CU or PTX was weighed precisely, and extracted with absolute ethanol: ultrasonic dissolving polyoxyethylene castor oil (EL-35) (1:1, v/v) to obtain clear liquid, filtering with sterile filter with pore size of 0.22 μm in ultra-clean bench, and making into injection.

Experiment design: after grouping, blood is collected from the heart at 5min, 10min, 15min, 30min, 45min, 60min, 120min, 240min, 480min and 1440min after administration by tail vein (administration scheme is shown in fig. 15 and 16). The whole blood (about 500. mu.L) was removed and placed in a centrifuge tube containing heparin sodium, centrifuged for 3min (5000rpm/min), and the supernatant plasma was taken for processing.

(2) The pretreatment method of the rat plasma sample comprises the following steps: rat plasma (100. mu.L) was taken, placed in a 2mL centrifuge tube, 25. mu.L (pH 3.6) of citric acid buffer was added, vortexed for 30s, then 0.5mL of an extractant (ethyl acetate: methanol: 90:10) was added, vortexed for 3min, and centrifuged for 3min (8000rpm/min), and the supernatant was taken. The extraction was repeated once more by adding 0.5mL of extractant and 2 supernatants were combined. After blowing at 35 ℃ with nitrogen, 200. mu.L of a double solvent [ phosphoric acid: and (3) acetonitrile 75:25(pH 3.5) ], vortex mixing for 4min, carrying out ultrasonic treatment for 4min, centrifuging for 10min (10000rpm/min), taking 160 mu L of supernatant into a sample injection bottle, and carrying out HPLC sample injection detection.

The results are shown in FIG. 2.

The detection method is not only suitable for in vitro sample labeling detection, but also can ensure that a spectrogram is clean and free of impurity peaks when detecting the in vivo sample, and is not interfered by other metabolites in the in vivo sample.

Example 3: establishing a standard curve for measuring the concentrations of CU and PTX in the single CU product, the single PTX product and the combined PTX compound CU product in rabbit plasma

1. Preparing a standard solution:

(1) preparing CU and PTX standard solutions: precisely weighing 4.00mg of CU and PTX reference substances, respectively placing the CU and the PTX reference substances in a 50mL volumetric flask, adding methanol to dissolve, shaking and shaking up, and fixing the volume to 50mL to obtain 80 mu g/mL CU and PTX stock solutions, and then diluting with methanol to respectively prepare CU and PTX standard substance solutions with the concentrations of 0.008, 0.016, 0.032, 0.08, 0.16, 0.8, 5, 10, 15, 20, 25 and 30 mu g/mL.

(2) Preparing a CU and PTX composite standard solution: newly prepared CU and PTX reference substance stock solutions are diluted by methanol to prepare CU and PTX composite standard solutions with the concentrations of 0.008, 0.016, 0.032, 0.08, 0.16, 0.64, 0.8, 5, 10, 15, 20, 25 and 30 mug/mL respectively.

2. Establishing a standard curve

Separately, 50. mu.L of the standard solution was pipetted precisely, and the pipetted solution was added to a 5mL centrifuge tube, 500. mu.L of rabbit blank plasma was added, vortexed for 30s, 25. mu.L of CA buffer (pH 3.6) was added, vortexed for 30s, 3mL of an extractant (ethyl acetate: methanol: 90:10) was added, vortexed for 3min, and centrifuged for 3min (8000rpm/min), and the supernatant was collected. The extraction was repeated once more by adding 3mL of extractant and 2 supernatants were combined. Blow-drying with nitrogen, adding 200 μ L of a double solvent [ acetonitrile: phosphoric acid (pH 3.5) ], vortex mixing for 4min, ultrasonic treating for 4min, centrifuging for 10min (10000rpm/min), collecting supernatant 160 μ L, and performing

And (5) HPLC detection.

Taking CU and PTX blood concentration C with different concentrations as abscissa and peak area Y of CU and PTX with different concentrations as ordinate to perform linear regression to obtain regression equation and R². The standard curve results are shown in figure 4.

The process conditions for the above HPLC analysis were as follows:

using a reversed phase chromatographic analysis column, wherein the detection wavelength is 425nm at 0-9.5min, and the mobile phase is acetonitrile: phosphate buffered saline 55:45 (v/v); when the time is 9.51-11.5min, the detection wavelength is 227nm, and the mobile phase is acetonitrile; phosphate buffered saline 40:60 (v/v); at 11.51-19.0min, the detection wavelength is 425nm, and the mobile phase is acetonitrile: phosphate buffered solution 55:45 (v/v). The column temperature was 30 ℃ and the flow rate was 1 mL/min. The amount of sample was 20. mu.L.

Example 4: sample detection

1. Rabbit plasma labeled sample detection

(1) Pretreatment of blank rabbit plasma and preparation of test solution

Blood was collected from the heart of a blank rabbit at approximately 1.5 mL. The blood sample was placed in a heparinized EP tube and whole blood was centrifuged at 5000rpm for 3min to aspirate the upper plasma, the blank rat plasma, for use.

Adding CU, PTX and a standard solution of the compound of CU and PTX into blank rabbit plasma to obtain CU concentration of 0.1124, 0.9050 and 3.062 mu g/mL; PTX concentrations were 0.0586, 0.4726, 1.4204. mu.g/mL; and the concentrations of CU in the CU and PTX compound solution are 0.1872, 0.7027 and 3.1453 mu g/mL, and the concentration of PTX is 0.0697, 0.3086 and 1.2386 mu g/mL.

After 100. mu.L of the standard plasma sample was treated according to the plasma sample pretreatment method of example 3, 160. mu.L of the supernatant was transferred to a sample bottle.

(2) Spiked plasma sample detection

A sample of 20. mu.L was introduced and subjected to HPLC analysis under the same conditions as in example 1. Calculating the concentrations of CU and PTX plasma samples in the CU single sample, the PTX single sample and the CU and PTX composite sample according to the standard curve respectively as follows: 0.0875 +/-0.0016, 0.8421 +/-0.0478 and 2.6819 +/-0.0174; 0.0444 plus or minus 0.0086, 0.3778 plus or minus 0.0163 and 1.2824 plus or minus 0.0858; 0.1394 + -0.0102, 0.7906 + -0.0213, 2.7781 + -0.2852, 0.0456 + -0.0045, 0.2759 + -0.0266 and 0.9773 + -0.0151, and the sample chromatogram is shown in FIG. 6.

The determination method is proved to be capable of accurately determining and analyzing the concentrations of the PTX and the compound of the CU and the PTX in the rabbit plasma, and the CU and the PTX in the chromatogram are smooth and symmetrical in peak shape and free of interference peaks, and the CU and the PTX are mutually separated and free of interference, the chromatogram is stable and reliable, the method is efficient and stable, and the requirement of pharmacokinetic evaluation of the rabbit plasma CU, the PTX and the CU and the PTX in combination can be well met.

2. Establishing a rabbit plasma sample chromatogram of an HPLC analysis method:

(1) selecting tested animals of rabbits.

Fasting was performed for 12h before administration, and water was freely available.

Preparation of injection:

polyoxyethylene castor oil EL-35: anhydrous ethanol: the sodium chloride injection (1:1:8, v/v/v) is dissolved with CU and/or PTX to prepare a clear liquid with corresponding administration dosage, and the clear liquid is prepared before use and is used after being filtered by a sterile filter with the pore size of 0.2 mu m.

Experiment design:

after grouping, blood was taken at 0.083h, 0.167h, 0.25h, 0.5h, L h, 2h, 4h, 6h, 8h, 12h and 24h after administration via auricular intravenous injection (the administration mode is shown in figures 5 and 6). Whole blood (about 1.5mL) was removed and placed in a heparin sodium-containing blood collection tube and centrifuged at 5000rpm/min for 3min to collect plasma for processing.

(2) The pretreatment method of the rabbit plasma sample comprises the following steps: mu.L of rabbit plasma was taken, placed in a 5mL centrifuge tube, 25. mu.L (pH 3.6) hj of citric acid buffer was added, vortexed for 30s, then 3mL of an extractant (ethyl acetate: methanol: 90:10) was added, vortexed for 3min, and centrifuged for 3min (8000rpm/min) to obtain a supernatant. The extraction was repeated once more by adding 0.5mL of extractant and 2 supernatants were combined. After blowing at 35 ℃ with nitrogen, 200. mu.L of a double solvent [ phosphoric acid: and (3) acetonitrile 75:25(pH 3.5) ], vortex mixing for 4min, performing ultrasonic treatment for 4min, transferring to a 2mL centrifuge tube, centrifuging for 10min (10000rpm/min), taking 160 mu L of supernatant into a sample injection bottle, and performing HPLC sample injection detection.

The results are shown in FIG. 5.

The detection method is not only suitable for in vitro sample labeling detection, but also can ensure that a spectrogram is clean and free of impurity peaks when detecting the in vivo sample, and is not interfered by other metabolites in the in vivo sample.

Example 5: establishing standard curves for measuring CU (heart, liver, spleen, lung, kidney and brain) single products, PTX (total protein X) single products and CU (total protein X) combined PTX compound products in rat tissues (heart, liver, spleen, lung, kidney and brain)

1. Preparing a reference substance solution:

preparing CU and PTX standard solutions: precisely weighing 4.00mg of CU and PTX reference substances, respectively placing the CU and the PTX reference substances in a 50mL volumetric flask, adding methanol to dissolve, shaking up, and fixing the volume to 50mL to obtain 80 mu g/mL CU and PTX standard stock solutions, and diluting with methanol to respectively prepare 0.01, 0.08, 0.16, 0.4, 0.8, 1.6, 2.4, 3.2 and 4 mu g/mL CU, PTX and CU and PTX composite standard solutions with the concentrations respectively.

2. Establishing a standard curve

Separately, 50. mu.L of the standard solution was pipetted precisely, and the pipetted solution was added to a 5mL centrifuge tube, 100. mu.L of rat blank homogenate (tissue: physiological saline: 1, w/v) was added, vortexed for 30s, 25. mu.L of CA buffer (pH 3.6) was added, vortexed for 30s, 0.5mL of an extractant (ethyl acetate: methanol: 90:10) was added, vortexed for 3min, centrifuged for 3min (8000rpm/min), and the supernatant was collected. The extraction was repeated once more by adding 0.5mL of extractant and 2 supernatants were combined. Blow-drying with nitrogen, adding 200 μ L of a double solvent [ acetonitrile: phosphoric acid (pH 3.5) ], vortex mixing for 4min, ultrasonic mixing for 4min, high speed centrifuging at 10000rpm/min for 10min, taking 160 mu L of supernatant, and performing HPLC detection.

And performing linear regression by taking CU and PTX blood concentration C with different concentrations as abscissa and taking peak areas Y of CU and PTX with different concentrations as ordinate to obtain a regression equation and R2. The standard curve results are shown in figure 7.

The conditions of the HPLC analysis method described above were as in example 1 for rat plasma samples.

Example 6: spiked sample detection

1. Pretreatment of labeled rat tissue sample

(1) Taking blank rats, killing the rats after neck breaking, taking out heart, liver, spleen, lung, kidney and brain, adding physiological saline (tissue: physiological saline is 1: 1; w/v), and grinding into homogenate to obtain blank biological samples of each tissue.

CU, PTX and CU and PTX composite standard solution were added to 100. mu.L of blank rat tissue sample (taking heart as an example herein) to obtain spiked plasma samples with CU, PTX and CU-PTX composite concentrations of 0.4000, 1.0000, 3.0000. mu.g/mL, respectively.

(2) After 100. mu.L of the sample of the labeled tissue was treated according to the method for pretreatment of the rat plasma sample described in example 2, 160. mu.L of the supernatant was transferred to a sample bottle.

2. Spiked plasma sample detection

A sample of 20. mu.L was introduced and subjected to HPLC analysis under the same conditions as in example 1. Calculating the concentrations of CU and PTX plasma samples in the CU single sample, the PTX single sample and the CU and PTX composite sample according to the standard curve respectively as follows: 0.3411 + -0.0021, 1.0057 + -0.0089, 3.1916 + -0.0740; 0.3260 + -0.0054, 0.8148 + -0.0250, 2.7434 + -0.1303; 0.2696 + -0.0053, 0.6488 + -0.0274, 1.9858 + -0.2104; 0.4472 + -0.0067, 1.0745 + -0.0651 and 2.7712 + -0.1062, and the sample chromatogram is shown in FIG. 8.

The determination method is proved to be capable of accurately determining and analyzing the concentrations of the PTX and the compound of the CU and the PTX in the rat plasma, and the CU and the PTX in the chromatogram are smooth and symmetrical in peak shape and free of interference, and the CU and the PTX are mutually separated and free of interference, the chromatogram is stable and reliable, the method is efficient and stable, and the requirement of combined pharmacokinetic assessment of the CU, the PTX and the CU and the PTX in the rat tissue can be well met.

Example 7: sample detection

1. Plasma sample pretreatment for pharmacokinetic studies after single tail vein injection of CU, PTX, CU and PTX complexes and formulations thereof in Sprague-DawLey rats.

(1) After grouping, blood was taken at 0.083h, 0.167h, 0.25h, 0.5h, L h, 2h, 4h, 6h, 8h, 12h, 24h, respectively, after administration according to the experimentally designed dosing regimen (see figures 5, 6). Whole blood (about 1.5mL) was removed and placed in a heparin sodium-containing blood collection tube and centrifuged at 5000rpm/min for 3min to collect plasma for processing.

(2) After 100. mu.L of the above plasma sample to be treated was treated according to the plasma sample pretreatment method of example 1, 160. mu.L of the supernatant was transferred to a sample bottle.

2. Plasma sample detection for pharmacokinetic studies of single tail vein injections of CU, PTX, CU and PTX compound and preparation thereof in Sprague-DawLey rats. A sample of 20. mu.L was introduced and subjected to HPLC analysis under the same conditions as in example 1. And calculating the blood concentration according to a standard curve.

The results show that the concentrations of CU and PTX drugs at each time point in the plasma of tail vein injection Sprague-DawLey rats are shown in FIG. 9. As can be seen from the figure, the method of the invention is rapid, accurate, high in sensitivity and simple and convenient to operate, and can provide a basis for determining the blood concentration of rats after CU, PTX and preparations thereof are singly or jointly administered.

Example 8: sample detection

1. The pretreatment of plasma samples is researched in pharmacokinetics after single ear edge intravenous injection of CU, PTX and a CU-PTX compound product (the mass ratio is 1:1 and 1:2) of rabbits.

(1) After grouping, following administration according to the experimentally designed dosing schedule, the rat heart was bled at approximately 1.5mL according to the experimentally designed sampling time schedule. The blood sample is placed in a heparinized EP tube, whole blood is centrifuged for 3min at 5000rpm to absorb upper plasma, and the plasma sample is pretreated.

(2) After 500. mu.L of the standard plasma sample was treated according to the plasma sample pretreatment method of example 3, 160. mu.L of the supernatant was transferred to a sample bottle.

2. Performing pharmacokinetic study on plasma sample detection by injecting CU, PTX and a compound product of CU and PTX (in a mass ratio of 1:1 and 1:2) into ear margin of a rabbit. A sample of 20. mu.L was introduced and subjected to HPLC analysis under the same conditions as in example 1. And calculating the blood concentration according to a standard curve.

The results show that the concentrations of CU and PTX drugs at each time point in plasma of ear-margin intravenous rats are shown in fig. 10. As can be seen from the figure, the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and can provide a basis for determining the blood concentration of the rabbit after CU and PTX are independently administered or are jointly administered.

Claims (7)

1. An HPLC analysis method for determining the content of curcumin and paclitaxel in a biological sample sequentially comprises two steps of establishing a standard curve and detecting the sample, and is characterized in that the conditions of the HPLC analysis method are as follows: a4.6 mm × 250mm Luna 5 μm C18 chromatographic analysis column is adopted, and a gradient elution wavelength-changing method is adopted: when the time is 0-9.5min, the detection wavelength is 425nm, the mobile phase is acetonitrile and phosphate buffer solution, and the volume ratio of the acetonitrile to the phosphate buffer solution is 55: 45; when the detection time is 9.51-11.5min, the detection wavelength is 227nm, the mobile phase is acetonitrile and phosphate buffer solution, and the volume ratio of the acetonitrile to the phosphate buffer solution is 40: 60; the column temperature is 30 ℃, and the flow rate of the mobile phase is 1 mL/min; the phosphate buffer is a mixed solution with pH =3.5 prepared from phosphoric acid and triethylamine;

the method also comprises a sample preparation step to be detected, and the specific operation is as follows: adding a citric acid buffer solution into a plasma sample or an animal tissue sample after homogenization, uniformly mixing, adding an extraction solvent with the volume being 8-12 times that of the plasma sample, uniformly mixing and centrifuging, taking an organic layer, volatilizing nitrogen, adding a composite solvent, uniformly mixing, centrifuging, and taking a supernatant as a test solution; the extraction solvent is prepared from ethyl acetate and methanol, and the volume ratio of the ethyl acetate to the methanol is 90: 10.

2. The HPLC analytical method of claim 1, wherein the conditions of the HPLC analytical method further comprise: and when the time is 11.51-19.0min, the detection wavelength is 425nm, the mobile phase is acetonitrile and phosphate buffer, and the volume ratio of the acetonitrile to the phosphate buffer is 55: 45.

3. The HPLC analysis method according to claim 1 or 2, wherein the establishment of said standard curve comprises the following steps:

(1) preparing a reference substance solution: respectively dissolving curcumin, paclitaxel and curcumin and paclitaxel in methanol to obtain curcumin stock solution, paclitaxel stock solution and mixed stock solution of curcumin and paclitaxel with the same concentration, and diluting with methanol to obtain standard solution with series gradient concentration;

(2) establishment of a standard curve: curcumin, paclitaxel and a standard solution mixed with curcumin and paclitaxel with series concentrations are respectively taken and mixed with a blank biological sample corresponding to a sample to be detected to obtain a mixed solution, the mixed solution is subjected to sample pretreatment to obtain a test solution, then the content is measured by HPLC under the condition of the HPLC analysis method, linear regression is carried out by taking the concentration of the sample as a horizontal coordinate and taking a peak area as a vertical coordinate, and a standard curve is established.

4. The HPLC analysis method as claimed in claim 3, wherein the sample detection step is to take a sample to be tested, perform sample pretreatment to obtain a test solution, measure the content by HPLC under the conditions of the HPLC analysis method, and calculate the concentrations of curcumin and paclitaxel in the sample according to the standard curve and peak area.

5. The HPLC analysis method of claim 4, wherein the complex solvent is prepared from acetonitrile and phosphate buffer at a volume ratio of 75: 25.

6. The HPLC analysis method according to claim 5, wherein the specific method of sample pretreatment is as follows:

putting the quantitative mixed solution into a 5mL centrifuge tube, adding 25 mu L of CA buffer solution with pH of 3.6, carrying out vortex mixing for 30s, and adding a quantitative extracting agent, wherein the volume ratio of the extracting agent is 90:10, performing vortex for 3min, centrifuging at 8000rpm/min for 3min, and taking supernatant; adding the extractant again for repeated extraction once, and merging the supernatants for 2 times; blowing the mixture to dry by nitrogen, adding 200 mu L of composite solvent which is acetonitrile and phosphoric acid with pH of 3.5, mixing for 4min by vortex, carrying out ultrasonic treatment for 4min, centrifuging at 10000rpm/min for 10min, and taking 20 mu L of supernatant for HPLC detection.

7. The HPLC analysis method of claim 1, wherein the volume ratio of the standard solution to the blank biological sample is 1:10 when the sample to be tested is a rabbit sample, and the volume ratio of the standard solution to the blank biological sample is 1:2 when the sample to be tested is a rat sample.

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