CN109100435A - The HPLC analysis method of curcumin and content of taxol in a kind of measurement biological sample - Google Patents

Abstract

The present invention relates to pharmacokinetic analysis fields, and in particular to a kind of HPLC method for measuring curcumin and content of taxol in biological sample.This method successively includes two steps of foundation and sample detection of standard curve, which is characterized in that the condition of HPLC analysis method are as follows: uses 5 μm of C18(4.6mm × 250mm of Luna) chromatographic column, wavelength method is become using gradient elution.This method is the Simultaneous Determination for realizing curcumin and content of taxol in biological sample only with HPLC, quickly improve the efficiency of the pharmacokinetic of curcumin and taxol drug combination, the development process of curcumin and taxol drug combination can have been rapidly promoted, has significantly reduced time, manpower and economic cost for the enterprise of research and development and scientific research institution.

Description

The HPLC analysis method of curcumin and content of taxol in a kind of measurement biological sample

Technical field

The present invention relates to pharmacokinetic analysis fields, and in particular to curcumin and taxol in a kind of measurement biological sample The HPLC method of content.

Background technique

Curcumin (CU) is a kind of monomer of separating-purifying from Chinese medicine, is the very rare color with diketone of plant kingdom Element,

It is orange-yellow crystalline powder for cyclohexadione compounds, it is not soluble in water.With the pharmacology such as anti-inflammatory, antitumor, anti-aging Effect, high-efficiency low-toxicity have good prospects as anti-tumor drug of new generation.Mainly from disabling signal access conduction NF- κ B, STAT3 and MAPK regulates and controls related neoplasms gene and protein expression BcL-2, p53 etc., adjusts the mediation of adhesion molecule, inhibits tumour Angiogenesis etc. realizes its antineoplastic action.

Taxol (PTX) is the natural diterpene compound with anticancer activity.It is for many years clinical treatment mammary gland Front-line chemotherapeutic agents of cancer, and effective in cure significant to a variety of refractory malignant tumours, such as embryonal carcinoma of testis, gallbladder cancer, non- Small Cell Lung Cancer, oophoroma.But its prolonged application toxic side effect is stronger, can lead to serious hypersensitivity, multidrug resistance stomach function regulating Enteron aisle stimulation or even bone marrow suppression.Apply more treatment malignant tumour in clinic is the change based on cisplatin combined PTX Treatment scheme, but toxic side effect is not eased.Recent study person tend to find it is a kind of can attenuation synergistic with PTX combination Substance treats malignant tumour.

Recent study shows that CU can slow down cell to the removing speed of PTX, increases the effective concentration of PTX, reverses thin Born of the same parents generate the drug resistance of PTX, and have proven to two medicines combination enhancing to the lethal effect of endometrial carcinoma cell, shift to mouse Property lung cancer also has obvious effect, dramatically increases to human laryngeal cancer and therapeutic efficacy for hepatic carcinoma.Two medicines are combined in liver cancer, lung cancer, oophoroma, oral cavity Cancer etc. research is more, and the analysis method for having obtained certain achievement, therefore having studied pharmacokinetics associated with two medicines has important Meaning.

At present for the pharmacokinetic studies of CU and PTX, the HPLC method of its biological sample concentration is measured, is individually to measure it Drug concentration has no that pharmacokinetic evaluation analysis method associated with it (opens one to sing, Gong Yuan, Fang Huiqiong wait turmeric in turmeric capsule Element is in rat Internal pharmacokinetics and Tissue distribution [J] Chinese pharmacists, 2018 (1): 65-68；Xu Dujuan, Huang Can, Su Yong wait to infuse It penetrates with Paclitaxel liposome pharmacokinetic [C] whole nation Therapeutic Drug Monitoring Annual Conference .2014).In addition, also grinding Study carefully the UPLC-MS/MS for establishing and both measuring CU and PTX respectively and measure simultaneously biological sample drug concentration, but for biology The processing operation of sample is cumbersome and (Li Zhi is peaceful, Wei Yue, Zhu Jie, waits ultra performance liquid chromatography-to used instrument requirements height The Henan curcumin [J] science in mass spectrometric determination rat plasma, 2017,35 (8): 1252-1257；Shi Yonghui, Yu Xiaoxia, Lv Li waits

HPLC-MS/MS method measures content [J] pharmacy today of taxol in rat plasma, 2017 (11): 735-738； Li Jinyin, Fang Jing, Li Wenxue wait traditional Chinese medical science in the pharmacokinetics and Tissue distribution [J] of high throughput analysis taxol in animal body Medicine, 2014,9 (12): 1833-1837).There are still following disadvantages for the pharmacokinetic studies prior art of CU and PTX: existing method is only CU and PTX biological sample content can individually be measured, it is difficult to realize CU and PTX combination；And it is surveyed simultaneously using UPLC-MS/MS method When determining CU and PTX content, sample pretreatment and measurement are cumbersome, and time-consuming, require instrument and equipment high.

Summary of the invention

The present invention provides the HPLC of curcumin and content of taxol in a kind of measurement biological sample and divides in order to solve the above problem Analysis method, above-mentioned HPLC method be not necessarily to mass spectrograph combination can Simultaneous Determination curcumin and taxol content, this method behaviour It is simple and efficient, and low to the friendship of the pre-processing requirements of sample, without cumbersome preprocessing process.

The HPLC analysis method of curcumin and content of taxol in a kind of measurement biological sample, successively including standard curve It establishes and two steps of sample detection, which is characterized in that the condition of HPLC analysis method are as follows: use5μm C18 (4.6mm × 250mm) chromatographic column becomes wavelength method using gradient elution: when 0~9.5min, Detection wavelength 425nm, Mobile phase is acetonitrile: phosphate buffer=55:45 (v/v)；When 9.51~11.5min, Detection wavelength 227nm, mobile phase is Acetonitrile: phosphate buffer=40:60 (v/v)；30 DEG C of column temperature, flow rate of mobile phase 1mL/min.

Preferably, the condition of the HPLC analysis method further include: in 11.51~19.0min, Detection wavelength is 425nm, mobile phase are acetonitrile: phosphate buffer=55:45 (v/v).

Preferably, the phosphate buffer is the mixed liquor of the pH=3.5 prepared by phosphoric acid and triethylamine.

Preferably, the standard curve foundation the following steps are included:

(1) preparation of reference substance solution: being dissolved in methanol for curcumin, taxol and curcumin and taxol respectively, preparation Stock solution, again with methanol dilution are mixed at the curcumin stock solution of same concentrations, taxol stock solution and curcumin and taxol For the standard solution of graded series concentration.

(2) foundation of standard curve: curcumin, taxol and the curcumin of series of concentrations and taxol is taken to mix respectively Standard solution blank biological sample corresponding with sample to be tested mixes to obtain mixed liquor, and carrying out sample pre-treatments to mixed liquor must be for Then test solution measures content using HPLC under conditions of the HPLC analysis method, using sample concentration as abscissa, with peak face Product is that ordinate carries out linear regression, establishes standard curve.

Preferably, the sample detection step is to take sample to be tested to carry out sample pre-treatments to obtain test liquid, then in institute Content is measured using HPLC under conditions of the HPLC analysis method stated, according to ginger in above-mentioned standard curve and calculated by peak area sample The concentration of flavine and taxol.

Preferably, it is directed toward in mixed liquor when the sample pre-treatments and citrate buffer solution is added, be uniformly mixed, addition 8~ The extractant of 12 times of volume is mixed and is centrifuged, and takes organic layer, and nitrogen volatilizes, and double solvents is added, and is mixed, in centrifuging and taking Clear liquid is test liquid.

Preferably, the double solvents is that acetonitrile and phosphate buffer are formulated, and the two volume ratio is 75:25.

Preferably, the extractant is that ethyl acetate and methanol configure, and the volume ratio of the two is 90:10.

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

It is furthermore preferred that the specific method is as follows for sample pre-treatments:

It takes quantitative mixed liquor to be placed in 5mL centrifuge tube, 25 μ L CA buffers (pH 3.6) is added, vortex mixed 30s adds Enter quantified extract agent (ethyl acetate: methanol=90:10), vortex 3min, is centrifuged 3min (8000rpm/min), takes supernatant.Again Secondary addition extractant repeats to extract once, merges 2 supernatants.It is dried with nitrogen, 200 μ L double solvents [acetonitriles: phosphoric acid (pH is added 3.5)], vortex mixed 4min, ultrasonic 4min are centrifuged 10min (10000rpm/min), and 20 μ L of supernatant is taken to carry out HPLC detection.

The beneficial effects of the present invention are:

1. without under the conditions of being combined mass spectrometric, this method realized in biological sample only with HPLC curcumin and The Simultaneous Determination of content of taxol quickly improves the effect of the pharmacokinetic of curcumin and taxol drug combination Rate can rapidly promote the development process of curcumin and taxol drug combination, a large amount of for the enterprise and scientific research institution of research and development Reduce time, manpower and economic cost.

2. this method without using ultra performance liquid chromatography, effectively simplifies the pre-treatment step of sample；Simultaneously as The eluant, eluent of HPLC is far below ultra performance liquid chromatography to purity requirement, so that the operating cost of instrument, which is far below, uses ultra high efficiency Liquid chromatogram.

3. this method has high sensitivity, accuracy and stability, be fully able to meet curcumin, taxol and The pharmacokinetic of the bulk pharmaceutical chemicals and preparation of curcumin and taxol drug combination.

Detailed description of the invention

Fig. 1 is that detection rat plasma CU single-item, PTX single-item and the CU that embodiment 1 is established combine CU, PTX in PTX combination product Blood concentration standard curve.

Fig. 2 is the HPLC chromatogram for the detection rat plasma that embodiment 1 is established, wherein (1) is CU single-item HPLC chromatogram Figure, (2) are PTX single-item HPLC chromatogram, and (3) are that CU combines PTX combination product HPLC chromatogram.

Fig. 3 is the HPLC chromatogram for the rat plasma mark-on sample that embodiment 2 is established, wherein (1) is CU single-item mark-on sample Product HPLC chromatogram, (2) are PTX single-item mark-on sample HPLC chromatogram, and (3) are that CU combines PTX combination product mark-on sample HPLC Chromatogram.

Fig. 4 is that detection plasma in rabbit CU single-item, PTX single-item and the CU that embodiment 3 is established combine CU, PTX in PTX combination product Blood concentration standard curve.

Fig. 5 is the HPLC chromatogram for the detection plasma in rabbit that embodiment 3 is established, wherein (1) is CU single-item HPLC chromatogram Figure, (2) are PTX single-item HPLC chromatogram, and (3) are that CU combines PTX combination product HPLC chromatogram.

Fig. 6 is the HPLC chromatogram for the detection plasma in rabbit mark-on sample that embodiment 4 is established, wherein (1) adds for CU single-item Standard specimen product HPLC chromatogram, (2) are PTX single-item mark-on sample HPLC chromatogram, and (3) are that CU combines PTX combination product mark-on sample HPLC chromatogram.

Fig. 7 is the group that detection rat heart single-item, PTX single-item and the CU that embodiment 5 is established combine CU, PTX in PTX combination product Knit the standard curve of drug concentration.

Fig. 8 is the group that detection rat stomach single-item, PTX single-item and the CU that embodiment 5 is established combine CU, PTX in PTX combination product Knit the standard curve of drug concentration.

Fig. 9 is that detection rats'liver CU single-item, PTX single-item and the CU that embodiment 5 is established combine CU, PTX in PTX combination product The standard curve of Tissue.

Figure 10 is that detection rat spleen CU single-item, PTX single-item and the CU that embodiment 5 is established combine CU, PTX in PTX combination product Tissue standard curve.

Figure 11 is that detection induced lung CU single-item, PTX single-item and the CU that embodiment 5 is established combine CU, PTX in PTX combination product Tissue standard curve.

Figure 12 is that detection Rat renal CU single-item, PTX single-item and the CU that embodiment 5 is established combine CU, PTX in PTX combination product Tissue standard curve.

Figure 13 is that detection rat brain CU single-item, PTX single-item and the CU that embodiment 5 is established combine CU, PTX in PTX combination product Tissue standard curve.

Figure 14 is the HPLC chromatogram for the detection rat heart that embodiment 6 is established, wherein (1) it is CU single-item HPLC chromatogram, It (2) is PTX single-item HPLC chromatogram, (3) are that CU combines PTX combination product HPLC chromatogram.

Figure 15 is detection rat single tail vein injection CU single-item, the PTX single-item, CU and PTX combination product that embodiment 7 is established And its blood plasma Drug-time curve figure after CU, PTX in preparation.

Figure 16 is that detection rabbit auricular vein injection CU single-item, PTX single-item and CU joint PTX that embodiment 8 is established are compound Blood plasma Drug-time curve figure after CU, PTX in product (mass ratio is 1:1 and 1:2).

Specific embodiment

The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not with any shape Formula limits the present invention.Except special instruction, reagent that the present invention uses, method and apparatus is the art conventional reagents, method And equipment.

Except special instruction, reagent used in following embodiment and material can be bought by commercial sources and be obtained.

HPLC of the present invention is the abbreviation of high-efficient liquid phase chromatogram technology；CU is curcumin abbreviation；PTX is taxol letter Claim.

Embodiment 1: CU single-item, PTX single-item and CU in measurement rat plasma are established and combines CU, PTX concentration in PTX combination product Standard curve

1, standard solution is prepared:

(1) CU, PTX standard solution are prepared: precision weighs CU, PTX reference substance 4.00mg, is respectively placed in 50mL volumetric flask In, add methanol dissolution concussion to shake up, is settled to 50mL to get the stock solution of CU, PTX of 80 μ g/mL, again with methanol dilution divides Not preparing concentration is respectively 0.02,0.04,0.08,0.16,0.32,0.80,1.60,3.20,4.80,6.60,8.00 μ g/mL CU, PTX standard solution solution.

(2) CU and PTX combination product contrast solution are prepared: CU, PTX the reference substance stock solution newly prepared are taken, with methanol dilution, Preparation concentration is respectively the CU of 0.02,0.04,0.08,0.16,0.32,0.80,1.60,3.20,4.80,6.60,8.00 μ g/mL With PTX combination product reference substance solution.

2, the foundation of standard curve

It is accurate respectively to draw 50 μ L serial standards solution, it is added in 5mL centrifuge tube, is separately added into rat blank plasma 100 μ L, vortex mixed 30s, are added 25 μ L CA buffers (pH 3.6), and 0.5mL extractant (acetic acid is added in vortex mixed 30s Ethyl ester: methanol=90:10), vortex 3min is centrifuged 3min (8000rpm/min), takes supernatant.0.5mL extractant is added again It repeats to extract once, merges 2 supernatants.It is dried with nitrogen, is added 200 μ L double solvents [acetonitrile: phosphoric acid (pH 3.5)], be vortexed mixed 4min, ultrasonic 4min, 10000rpm/min high speed centrifugation 10min are closed, 160 μ L of supernatant is taken, carries out HPLC detection.

It is vertical sit with the peak area Y of CU, PTX of various concentration using CU, PTX blood concentration C of various concentration as abscissa Mark carries out linear regression, obtains regression equation and R2.Standard curve result is as shown in Figure 1.

Above-mentioned HPLC analysis method condition is as follows:

It uses5 μm of C18 (4.6mm × 250mm) chromatographic columns, when 0-9.5min, Detection wavelength 425nm, Mobile phase is acetonitrile: phosphoric acid delays solution=55:45 (v/v)；When 9.51-11.5min, Detection wavelength 227nm, mobile phase is second Nitrile；Phosphoric acid delays solution=40:60 (v/v)；When 11.51-19.0min, Detection wavelength 425nm, mobile phase is acetonitrile: phosphoric acid delays molten Liquid=55:45 (v/v).Column temperature is 30 DEG C, flow velocity 1mL/min.Sample volume is 20 μ L.

Embodiment 2: sample detection

1, mark-on rat plasma pattern detection

(1) blank rat plasma pretreatment and test liquid preparation

From about 500 μ L of blank rat Culling heart blood.Blood sample is placed in test tube of hepari EP pipe, and whole blood is centrifuged through 54000rpm 3min draws upper plasma, i.e. blank rat plasma, for use.

Blank rat plasma is added in CU, PTX and CU and PTX combination product contrast solution, obtaining CU concentration is 0.1124, 0.9050,3.062 μ g/mL；PTX concentration is 0.1005,0.4726,1.4204 μ g/mL；CU concentration is in CU and PTX combination product The spiked plasma sample of 0.1097,0.3086, the 1.2386 μ g/mL of concentration of 0.1872,0.7027,3.1453 μ g/mL, PTX.

100 μ L of spiked plasma sample is taken to take supernatant after being handled according to the plasma sample pre-treating method of embodiment 1 160 μ L are moved into sample injection bottle.

(2) spiked plasma pattern detection

20 μ L of sample introduction carries out HPLC analysis, and condition is the same as embodiment 1.According to standard curve calculate CU single-item, PTX single-item and CU is respectively as follows: 0.0952 ± 0.0124,0.8549 ± 0.08678,2.7337 with CU, PTX plasma sample concentration in PTX combination product ±0.1800；0.0752 ± 0.0055,0.3875 ± 0.0095,1.2640 ± 0.1377；0.1439 ± 0.0059,0.6851 ± 0.0129,2.9135 ± 0.1994,0.0800 ± 0.0178,0.2866 ± 0.0096,1.1258 ± 0.0541, sample chromatogram As shown in Figure 3.

Show this measuring method and can accurately measure very much in analyzing rat blood plasma, the concentration of PTX and CU and PTX combination product And CU, PTX peak shape are smoothly symmetrical in chromatogram, noiseless peak, and two peaks CU and PTX are separated from each other, noiseless, chromatogram is steady Fixed reliable, method is efficient, stablizes, and can meet rat plasma CU, PTX and CU and PTX combination pharmcokinetic evaluation well It needs.

2, vivo sample detects:

(1) animal subject Sprague-DawLey rat is chosen.

Fasting 12h, free water before being administered.

The preparation of injection: precision weighs the powder of CU or PTX, with dehydrated alcohol: Emulsifier EL-60 (EL-35) (1:1, v/v) ultrasonic dissolution, after being configured to clear liquid, then with aperture be 0.22 μm sterilizing filter in superclean bench It uses after filtering, now matches before injection.

Experimental design: after grouping, after being administered using tail vein mode (dosage regimen is shown in Figure 15,16), respectively at 5min, Culling heart blood when 10min, 15min, 30min, 45min, 60min, 120min, 240min, 480min, 1440min.That takes out is complete Blood (about 500 μ L) is put in centrifuge tube containing heparin sodium, is centrifuged 3min (5000rpm/min), and take supernatant blood plasma to be processed.

(2) rat plasma sample preprocess method: taking 100 μ L of rat plasma, be placed in 2mL centrifuge tube, and 25 μ L (pH are added 3.6) citrate buffer solution after vortex 30s, is added 0.5mL extractant (ethyl acetate: methanol=90:10), vortex 3min, It is centrifuged 3min (8000rpm/min), takes supernatant.It is primary that 0.5mL extractant repetition extracting operation is added again, merges on 2 times Clear liquid.It 35 DEG C, after being dried with nitrogen, is added 200 μ L double solvents [phosphoric acid: acetonitrile=75:25 (pH 3.5)], vortex mixed 4min, Ultrasonic 4min is centrifuged 10min (10000rpm/min), takes 160 μ L of supernatant in sample injection bottle, HPLC sample detection.

As a result as shown in Figure 2.

This detection method is applicable not only to external sample mark-on detection and still ensures that when detecting biological vivo sample Spectrogram is completely without miscellaneous peak, not by the interference of other metabolins in vivo sample.

Embodiment 3: CU single-item, PTX single-item and CU in measurement plasma in rabbit are established and combines CU, PTX concentration in PTX combination product Standard curve

1, standard solution is prepared:

(1) CU, PTX standard solution are prepared: precision weighs CU, PTX reference substance 4.00mg, is respectively placed in 50mL volumetric flask In, add methanol dissolution concussion to shake up, is settled to 50mL to get CU, PTX stock solution of 80 μ g/mL, again with methanol dilution, difference Preparation concentration is respectively CU, PTX mark of 0.008,0.016,0.032,0.08,0.16,0.8,5,10,15,20,25,30 μ g/mL Quasi- product solution.

(2) the compound standard solution of CU and PTX is prepared: CU, PTX the reference substance stock solution newly prepared is taken, with methanol dilution, system Standby concentration be respectively 0.008,0.016,0.032,0.08,0.16,0.64,0.8,5,10,15,20,25,30 μ g/mL CU with The compound standard solution of PTX.

2, standard curve is established

It is accurate respectively to draw 50 μ L standard solution, it is added in 5mL centrifuge tube, is separately added into 500 μ L of rabbit blank plasma, whirlpool Rotation mixing 30s, 25 μ L CA buffers (pH 3.6) of addition, vortex mixed 30s, addition 3mL extractant (ethyl acetate: methanol= 90:10), vortex 3min is centrifuged 3min (8000rpm/min), takes supernatant.3mL extractant is added again to repeat to extract once, Merge 2 supernatants.It is dried with nitrogen, is added 200 μ L double solvents [acetonitrile: phosphoric acid (pH 3.5)], vortex mixed 4min, ultrasound 4min is centrifuged 10min (10000rpm/min), takes 160 μ L of supernatant, carries out

HPLC detection.

It is vertical sit with the peak area Y of CU, PTX of various concentration using CU, PTX blood concentration C of various concentration as abscissa Mark carries out linear regression, obtains regression equation and R².Standard curve result is as shown in Figure 4.

The method condition of above-mentioned HPLC analysis is as follows:

Using reverse-phase chromatography analytical column, when 0-9.5min, Detection wavelength 425nm, mobile phase is acetonitrile: phosphoric acid delays solution =55:45 (v/v)；When 9.51-11.5min, Detection wavelength 227nm, mobile phase is acetonitrile；Phosphoric acid delays solution=40:60 (v/ v)；When 11.51-19.0min, Detection wavelength 425nm, mobile phase is acetonitrile: phosphoric acid delays solution=55:45 (v/v).Column temperature is 30 DEG C, flow velocity 1mL/min.Sample volume is 20 μ L.

Embodiment 4: sample detection

1, plasma in rabbit mark-on sample detection

(1) pretreatment of blank plasma in rabbit and test liquid preparation

From blank Rabbit Heart blood sampling about 1.5mL.Blood sample is placed in test tube of hepari EP pipe, and whole blood is centrifuged through 5000rpm 3min draws upper plasma, i.e. blank rat plasma, for use.

Blank plasma in rabbit is added in CU, PTX and CU and PTX combination product standard solution, obtaining CU concentration is 0.1124, 0.9050,3.062 μ g/mL；PTX concentration is 0.0586,0.4726,1.4204 μ g/mL；CU and CU concentration in PTX composite solution The spiked plasma sample for 0.0697,0.3086, the 1.2386 μ g/mL of concentration for being 0.1872,0.7027,3.1453 μ g/mL, PTX.

100 μ L of spiked plasma sample is taken to take supernatant after being handled according to the plasma sample pre-treating method of embodiment 3 160 μ L are moved into sample injection bottle.

(2) spiked plasma pattern detection

20 μ L of sample introduction carries out HPLC analysis, and condition is the same as embodiment 1.According to standard curve calculate CU single-item, PTX single-item and CU is respectively as follows: 0.0875 ± 0.0016,0.8421 ± 0.0478,2.6819 with CU, PTX plasma sample concentration in PTX combination product ±0.0174；0.0444 ± 0.0086,0.3778 ± 0.0163,1.2824 ± 0.0858；0.1394 ± 0.0102,0.7906 ± 0.0213,2.7781 ± 0.2852,0.0456 ± 0.0045,0.2759 ± 0.0266,0.9773 ± 0.0151, sample chromatogram As shown in Figure 6.

Show this measuring method and can accurately measure very much in analysis plasma in rabbit, the concentration of PTX and CU and PTX combination product And CU, PTX peak shape are smoothly symmetrical in chromatogram, noiseless peak, and two peaks CU and PTX are separated from each other, noiseless, chromatogram is steady Fixed reliable, method is efficient, stablizes, and can meet plasma in rabbit CU, PTX and CU and PTX combination pharmcokinetic evaluation well It needs.

2, the plasma in rabbit sample chromatogram of HPLC analysis method is established:

(1) animal subject rabbit is chosen.

Fasting 12h, free water before being administered.

The preparation of injection:

Emulsifier EL-60 EL-35: dehydrated alcohol: sodium chloride injection (1:1:8, v/v/v) dissolves CU and/or PTX, It is configured to the clear liquid of corresponding dosage, is used after the sterilizing filter filtering that prepared before use is 0.2 μm with aperture.

Experimental design:

After grouping, after auricular vein drug administration by injection (administration mode is referring to Fig. 5,6), respectively in 0.083h, 0.167h, 0.25h, 0.5h, L h, 2h, 4h, 6h, 8h, 12h, for 24 hours when take blood.It takes out whole blood (about 1.5mL) and is put in heparin tube containing heparin sodium In, 5000rpm/min centrifugation 3min takes blood plasma to be processed.

(2) plasma in rabbit sample pretreating method: taking 500 μ L of plasma in rabbit, be placed in 5mL centrifuge tube, and 25 μ L (pH are added 3.6) citrate buffer solution of hj after vortex 30s, is added 3mL extractant (ethyl acetate: methanol=90:10), vortex 3min, It is centrifuged 3min (8000rpm/min), takes supernatant.It is primary that 0.5mL extractant repetition extracting operation is added again, merges on 2 times Clear liquid.It 35 DEG C, after being dried with nitrogen, is added 200 μ L double solvents [phosphoric acid: acetonitrile=75:25 (pH 3.5)], vortex mixed 4min, Ultrasonic 4min is transferred in 2mL centrifuge tube, is centrifuged 10min (10000rpm/min), is taken 160 μ L of supernatant in sample injection bottle, HPLC sample detection.

As a result as shown in Figure 5.

This detection method is applicable not only to external sample mark-on detection and still ensures that when detecting biological vivo sample Spectrogram is completely without miscellaneous peak, not by the interference of other metabolins in vivo sample.

Embodiment 5: CU single-item in measurement rat tissue (heart, liver, spleen, lung, kidney, brain), PTX single-item and CU joint are established The standard curve of CU, PTX concentration in PTX combination product

1, reference substance solution is prepared:

CU, PTX standard solution are prepared: precision weighs CU, PTX reference substance 4.00mg, is respectively placed in 50mL volumetric flask, adds Methanol dissolution concussion shakes up, and is settled to 50mL to get CU, PTX standard reserving solution of 80 μ g/mL, again with methanol dilution is made respectively Standby concentration is respectively CU, PTX and CU and the compound mark of PTX of 0.01,0.08,0.16,0.4,0.8,1.6,2.4,3.2,4 μ g/mL Quasi- solution.

2, standard curve is established

It is accurate respectively to draw 50 μ L standard solution, it is added in 5mL centrifuge tube, is separately added into rat blank tissue homogenate (group Knit: physiological saline=1:1, w/v) 100 μ L, vortex mixed 30s, 25 μ L CA buffers (pH 3.6) of addition, vortex mixed 30s, It is added 0.5mL extractant (ethyl acetate: methanol=90:10), vortex 3min, is centrifuged 3min (8000rpm/min), takes supernatant Liquid.0.5mL extractant is added again to repeat to extract once, merges 2 supernatants.It is dried with nitrogen, 200 μ L double solvents [second is added Nitrile: phosphoric acid (pH 3.5)], vortex mixed 4min, ultrasonic 4min, 10000rpm/min high speed centrifugation 10min takes 160 μ of supernatant L carries out HPLC detection.

It is vertical sit with the peak area Y of CU, PTX of various concentration using CU, PTX blood concentration C of various concentration as abscissa Mark carries out linear regression, obtains regression equation and R2.Standard curve result is as shown in Figure 7.

Above-mentioned HPLC analysis method condition, according to 1 rat plasma sample HPLC analysis method condition of embodiment.

Embodiment 6: mark-on sample detection

1, mark-on rat tissue Sample pretreatment

(1) blank rat is taken, the neck that breaks is put to death, and the heart, liver, spleen, lung, kidney, brain are taken out, and physiological saline (tissue: physiology salt is added Water=1:1；W/v it) is ground into homogenate, obtains each tissue blank biological sample.

By the blank rat tissue sample of 100 μ L of the compound standard solution addition of CU, PTX and CU and PTX, (this sentences the heart Example), respectively obtain the spiked plasma sample that CU, PTX and CU-PTX combination product concentration are 0.4000,1.0000,3.0000 μ g/mL Product.

(2) 100 μ L of mark-on tissue sample is taken, after being handled according to the rat plasma sample pre-treating method of embodiment 2, 160 μ L of supernatant is taken to move into sample injection bottle.

2, spiked plasma pattern detection

20 μ L of sample introduction carries out HPLC analysis, and condition is the same as embodiment 1.According to standard curve calculate CU single-item, PTX single-item and CU is respectively as follows: 0.3411 ± 0.0021,1.0057 ± 0.0089,3.1916 with CU, PTX plasma sample concentration in PTX combination product ±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 ± 0.0651,2.7712 ± 0.1062, sample chromatogram As shown in Figure 8.

Show this measuring method and can accurately measure very much in analyzing rat blood plasma, the concentration of PTX and CU and PTX combination product And CU, PTX peak shape are smoothly symmetrical in chromatogram, noiseless peak, and two peaks CU and PTX are separated from each other, noiseless, chromatogram is steady Fixed reliable, method is efficient, stablizes, and can meet CU, PTX and CU and PTX combination pharmcokinetic evaluation in rat tissue well Needs.

Embodiment 7: pattern detection

1, medicine is dynamic after Sprague-DawLey rat single tail vein injection CU, PTX, CU and PTX combination product and its preparation Learn research plasma sample pre-treatment.

(1) be grouped after, according to experimental design Dosage Regimens Dosage after (dosage regimen is referring to Fig. 5,6), respectively in 0.083h, 0.167h, 0.25h, 0.5h, L h, 2h, 4h, 6h, 8h, 12h, for 24 hours when take blood.Whole blood (about 1.5mL) is taken out to be put in In heparin tube containing heparin sodium, 5000rpm/min centrifugation 3min takes blood plasma to be processed.

(2) above-mentioned 100 μ L of plasma sample to be processed is taken, is handled according to the plasma sample pre-treating method of embodiment 1 Afterwards, 160 μ L of supernatant is taken to move into sample injection bottle.

2, medicine is dynamic after Sprague-DawLey rat single tail vein injection CU, PTX, CU and PTX combination product and its preparation Learn research plasma sample detection.20 μ L of sample introduction carries out HPLC analysis, and condition is the same as embodiment 1.It is dense that blood medicine is calculated according to standard curve Degree.

The results show that each time point CU, PTX drug concentration is as schemed in tail vein injection Sprague-DawLey rat plasma Shown in 9.As seen from the figure, the method for the present invention quickly, it is accurate, high sensitivity, easy to operate, can individually be given for CU, PTX and its preparation The measurement of serum level provides foundation after medicine or administering drug combinations.

Embodiment 8: pattern detection

1, rabbit single auricular vein injects CU, PTX, CU and PTX combination product (mass ratio is 1:1 and 1:2) pharmacokinetics afterwards Study plasma sample pre-treatment.

(1) after being grouped, after the Dosage Regimens Dosage of experimental design, according to experimental design sampling time scheme, rat Culling heart blood about 1.5mL.Blood sample is placed in test tube of hepari EP pipe, and whole blood draws upper plasma through 5000rpm centrifugation 3min, to blood Slurry samples pretreatment.

(2) 500 μ L of spiked plasma sample is taken to take after being handled according to the plasma sample pre-treating method of embodiment 3 Clear 160 μ L is moved into sample injection bottle.

2, rabbit auricular vein injection CU, PTX, CU and PTX combination product (mass ratio is 1:1 and 1:2) pharmacokinetic studies blood Slurry samples detection.20 μ L of sample introduction carries out HPLC analysis, and condition is the same as embodiment 1.Blood concentration is calculated according to standard curve.

The results show that each time point CU, PTX drug concentration is as shown in Figure 10 in auricular vein injection rat plasma.By scheming It is found that the method for the present invention quickly, it is accurate, high sensitivity, easy to operate, can be administered alone or both and to be administered in combination for CU, PTX The measurement of rabbit blood concentration provides foundation afterwards.

Claims (9)

1. the HPLC analysis method of curcumin and content of taxol, successively includes building for standard curve in a kind of measurement biological sample Vertical and two steps of sample detection, which is characterized in that the condition of HPLC analysis method are as follows: use 5 μm of C18(4.6 of Luna The mm of mm × 250) chromatographic column, wavelength method is become using gradient elution: when 0 ~ 9.5 min, Detection wavelength 425 Nm, mobile phase are acetonitrile: phosphate buffer=55:45(v/v)；When 9.51 ~ 11.5min, Detection wavelength is 227 nm, flowing It is mutually acetonitrile: phosphate buffer=40:60(v/v)；30 DEG C of column temperature, flow rate of mobile phase is 1 mL/min.

2. HPLC analysis method as described in claim 1, which is characterized in that the condition of the HPLC analysis method further include: In 11.51 ~ 19.0 min, Detection wavelength is 425 nm, and mobile phase is acetonitrile: phosphate buffer=55:45(v/v).

3. HPLC analysis method as described in claim 1, which is characterized in that the phosphate buffer is by phosphoric acid and triethylamine The mixed liquor of pH=3.5 of preparation.

4. such as the described in any item HPLC analysis methods of claim 1 ~ 3, which is characterized in that the foundation of the standard curve includes Following steps:

(1) preparation of reference substance solution: curcumin, taxol and curcumin and taxol are dissolved in methanol respectively, are prepared into phase Mix stock solution with the curcumin stock solution of concentration, taxol stock solution and curcumin and taxol, again with methanol be diluted to be The standard solution of column gradient concentration；

(2) foundation of standard curve: curcumin, taxol and the curcumin of series of concentrations and the standard of taxol mixing are taken respectively Solution blank biological sample corresponding with sample to be tested mixes to obtain mixed liquor, and carrying out sample pre-treatments to mixed liquor must be for examination Then liquid measures content using HPLC under conditions of the HPLC analysis method, using sample concentration as abscissa, with peak area Linear regression is carried out for ordinate, establishes standard curve.

5. HPLC analysis method as claimed in claim 4, which is characterized in that the sample detection step be take sample to be tested into Row sample pre-treatments obtain test liquid, then measure content using HPLC under conditions of the HPLC analysis method, according to The concentration of curcumin and taxol in above-mentioned standard curve and calculated by peak area sample.

6. HPLC analysis method as claimed in claim 5, which is characterized in that be directed toward in mixed liquor and add when the sample pre-treatments Enter citrate buffer solution, be uniformly mixed, the extractant of 8~12 times of volume is added, mixes and is centrifuged, take organic layer, nitrogen It volatilizes, double solvents is added, mix, centrifuging and taking supernatant is test liquid.

7. HPLC analysis method as claimed in claim 6, which is characterized in that the double solvents is acetonitrile and phosphate buffer It is formulated, the two volume ratio is 75:25；Preferably, the extractant is that ethyl acetate and methanol are formulated, the two Volume ratio is 90:10.

8. HPLC analysis method as claimed in claim 8, which is characterized in that the specific method is as follows for sample pre-treatments:

It takes quantitative mixed liquor to be placed in 5 mL centrifuge tubes, 25 μ L CA buffers (pH 3.6) is added, 30 s of vortex mixed adds Enter quantified extract agent (ethyl acetate: methanol=90:10), be vortexed 3 min, is centrifuged 3 min(8000 rpm/min), take supernatant； Extractant is added again to repeat to extract once, merges 2 supernatants；It is dried with nitrogen, 200 μ L double solvents [acetonitriles: phosphorus is added Sour (pH 3.5)], 4 min of vortex mixed, 4 min of ultrasound are centrifuged 10 min(10000 rpm/min), take 20 μ L of supernatant Carry out HPLC detection.

9. HPLC analysis method as described in claim 1, which is characterized in that described when the sample to be tested is rabbit sample The volume ratio of standard solution and blank biological sample is 1:10, when the sample to be tested is rat sample, the standard solution with The volume ratio of blank biological sample is 1:2.