Method for quantitatively analyzing total paclitaxel and free paclitaxel in human plasma

Abstract

The invention belongs to the field of medical examination, and relates to analysis and determination of biological samples, in particular to a method for quantitatively analyzing total paclitaxel and free paclitaxel in human plasma. According to the method, a sample is pretreated through a protein precipitation method and an ultrafiltration method, and then the total paclitaxel concentration and the free paclitaxel concentration in the biological sample are analyzed and detected through a high performance liquid chromatography-mass spectrometry tandem method. Compared with the prior art, the method has the advantages that the sample pretreatment method is simple, high in accuracy, wide in measurement range, good in reproducibility and suitable for clinical detection of large-batch samples.

Description

Technical Field

[0001] The invention relates to the technical field of plasma analysis, and in particular to a method for quantitatively analyzing total paclitaxel and free paclitaxel in human plasma. Background Art

[0002] The molecular formula of paclitaxel is C 47 H 51 NO 14 , the relative molecular mass is 853.91. It is a tetracyclic diterpene compound, its N is in an amide state, there is an ester bond, it is unstable under alkaline conditions, and it is obviously neutral. The taxane ring structure is large and complex, and contains a large number of hydrophobic functional groups, so it has a strong affinity for fat.

[0003] Studies have shown that paclitaxel can promote the polymerization of tubulin and inhibit the degradation of microtubules. When the concentration is n-molar, paclitaxel can inhibit the cleavage of intracellular microtubules and significantly increase the number and quality of intracellular microtubules. Under the action of paclitaxel, the microtubule structure is stable, which can affect the microtubule dynamics in cell division and key intermediate stages, and then affect the formation of spindles, thereby preventing cell division and causing cell death.

[0004] Paclitaxel is a commonly used anti-tumor drug. It can be used alone or in combination with other anti-tumor drugs such as cisplatin, carboplatin and cyclophosphamide to treat breast cancer and head and neck cancer. There are currently three dosage forms of paclitaxel in clinical use: paclitaxel injection, paclitaxel liposome injection and albumin-bound paclitaxel injection. Albumin paclitaxel is a nanoparticle with an average particle size of about 130nm, which is formed by coupling paclitaxel and albumin. It is a new type of tumor-targeted drug. Compared with traditional injection preparations such as paclitaxel and paclitaxel liposomes, it has two significant advantages: first, it does not contain polyethylene castor oil; since this preparation does not contain polyethylene castor oil, it can greatly reduce the patient's allergic reaction, no preoperative medication is required, the medication time can be shortened to 30 minutes, and no special intravenous injection is required. Second, it uses the unique transport mode of albumin (gp60-hydrogenin-SPARC) to increase the distribution of paclitaxel in the tumor, increase its content in the tumor, and ensure the safety and effectiveness of the drug.

[0005] Paclitaxel exists in three forms in human plasma: free paclitaxel, paclitaxel bound to exogenous albumin, and paclitaxel bound to endogenous albumin. Because free paclitaxel has pharmacological activity, in addition to detecting total paclitaxel, it is also important to detect the content of free paclitaxel in clinical practice.

[0006] At present, there are studies on the determination of the blood concentration of paclitaxel in patient plasma by high performance liquid chromatography (HPLC), reverse phase high performance liquid chromatography (BR-HPLC), gas chromatography, microbiological determination and polarimetry. However, the above methods have certain differences in sensitivity and specificity, large sample volume required, cumbersome sample processing process, low extraction recovery rate, long detection time, interference by endogenous substances, and the disadvantages of not being suitable for detecting large batches of samples, and being susceptible to interference by endogenous substances. Therefore, it is necessary to establish a detection method with high sensitivity, high accuracy, small sample volume required, and not easily interfered by endogenous substances, and capable of simultaneously detecting the total paclitaxel concentration and free paclitaxel concentration of albumin paclitaxel in patient plasma. Summary of the invention

[0007] In order to overcome the shortcomings of the prior art, the present invention provides a method for quantitatively analyzing total paclitaxel and free paclitaxel in human plasma, which is simple, highly sensitive and accurate. The present invention does not require special instruments and reagents, requires a small amount of sample, has simple and rapid pretreatment, simple analysis operation and good reproducibility, and is suitable for clinical monitoring of the total paclitaxel concentration and free paclitaxel concentration after injection of albumin paclitaxel.

[0008] The present invention is implemented by the following technical solutions:

[0009] A method for quantitatively analyzing total paclitaxel and free paclitaxel in human plasma comprises the following steps:

[0010] S1. Sample pretreatment for total paclitaxel concentration detection: plasma and organic reagent are mixed in proportion, and after protein precipitation by centrifugation, a plasma sample as supernatant is obtained;

[0011] S2. Sample pretreatment for free paclitaxel concentration detection: adding the plasma sample to an ultrafiltration tube, ultrafiltration to obtain an ultrafiltrate, mixing the ultrafiltrate with an organic reagent in proportion, and centrifuging to obtain a supernatant sample to be tested;

[0012] S3. High performance liquid chromatography-mass spectrometry tandem analysis: The samples to be tested are analyzed using a high performance liquid chromatography-mass spectrometer to detect the chromatographic peaks of paclitaxel and internal standard docetaxel in the samples, and the total paclitaxel and free paclitaxel concentrations in the plasma are calculated based on these peaks.

[0013] In the present invention, a method for determining the total paclitaxel concentration in a plasma sample is provided, and the pretreatment of the plasma sample is the key to the detection process. Generally, solid phase extraction, liquid-liquid extraction, and protein precipitation methods can be used to process the plasma sample. However, the solid phase extraction method has a high cost of consumables and requires corresponding extraction columns and instruments; the liquid-liquid extraction method is complicated to operate and introduces high uncertainty to the detection; the protein precipitation method is the simplest and cheapest, but often due to unclean sample processing, impurities are introduced into the detection, reducing the detection sensitivity. However, the present invention realizes the simplest protein precipitation method to pre-treat the plasma sample, so that the detection obtains a higher sensitivity.

[0014] The present invention also provides a method for determining the concentration of free paclitaxel in a plasma sample. It is critical to determine the free concentration of the drug but not to destroy the normal plasma protein binding of the drug, and to select a method for separating the free drug. Existing technologies include equilibrium dialysis and ultrafiltration. The equilibrium dialysis method requires a dialysis membrane for separation, and has the disadvantages of long equilibrium time (4-48h), high environmental requirements, expensive consumables, and low flux. Ultrafiltration often destroys the balance between the drug and the protein due to improper operation. In the present invention, appropriate ultrafiltration conditions are adopted, and plasma quality control samples at low, medium, and high concentration levels are used for quality control, so that the drug free rate obtained by ultrafiltration is consistent with that reported by the equilibrium dialysis method, and the free rate is kept consistent throughout the test batch, ensuring that the test operation does not destroy the binding balance between the drug and the protein.

[0015] Further, in step S1, plasma and an organic reagent are mixed in a volume ratio of 1:6, and the mixture is centrifuged at 14,500 rpm for 10 minutes to precipitate proteins.

[0016] Furthermore, in step S1, the organic reagent is a 100 ng / ml docetaxel methanol solution.

[0017] In the present invention, the pretreatment of total paclitaxel in step S1 is preferably carried out by protein precipitation, preferably by methanol, and most preferably by a methanol solution containing 100 ng / ml docetaxel as an internal standard. Plasma and 100 ng / ml docetaxel methanol solution are vortex-mixed at a volume ratio of 1:6 for 1 minute, and then centrifuged at 14500 rpm for 10 minutes for protein precipitation, and the supernatant is aspirated for sampling and analysis.

[0018] Further, in step S2, the specific steps are as follows: take 500uL of plasma sample in a Centrifree ultrafiltration device, ultrafilter for 15min at 25°C and 1000g centrifugal force, take the ultrafiltrate and mix it with 100ng / mL docetaxel methanol solution in a volume ratio of 1:1, centrifuge at 14500 rpm for 10 minutes, and obtain the supernatant sample to be tested.

[0019] In the present invention, the separation of free paclitaxel in step S2 is preferably carried out by ultrafiltration, and the ultrafiltration is carried out by Centrifree ultrafiltration device. The ultrafiltration step can be carried out with 150-1000 μL of biological sample, preferably 500 μL of biological sample, and the amount of ultrafiltrate obtained can meet the requirements of subsequent analysis. The ultrafiltration step can be centrifuged at 1000-2000g, preferably at 1000g. The ultrafiltration step is preferably centrifuged for 15min. In a preferred embodiment of the present invention, the ultrafiltrate is mixed with 100ng / ml of docetaxel methanol solution in a volume ratio of 1:1, and the supernatant can be drawn for analysis at 14500r / min centrifugation for 10min.

[0020] In the present invention, the prior art can ultrafilter 150-1000 μL of biological samples at a speed of 1000-2000g, but does not specify the ultrafiltration time and ultrafiltration temperature. The present invention finds that the free concentration of the drug is not affected when the centrifugation time is 15-30min, the centrifugal force is 1000-2000g, and the centrifugal temperature is 25-37°C. Therefore, the present invention determines the shortest centrifugation time, the most easily achievable test temperature, and the lowest centrifugal force conditions for testing, and examines that the accuracy, precision, stability, matrix effect, etc. under the conditions determined by the present invention can meet the CFDA requirements for biological sample analysis. And significantly shorten the ultrafiltration equilibrium time, ultrafiltration time, simplify the sample processing after ultrafiltration.

[0021] Therefore, the present invention provides an analytical method for determining the concentrations of total paclitaxel and free paclitaxel in plasma after the use of albumin paclitaxel injection, comprising a plasma sample processing step of total paclitaxel, an ultrafiltration step of free paclitaxel and an analysis step of a high performance liquid chromatography-mass spectrometer.

[0022] Further, in step S3, the liquid chromatography determination conditions are:

[0023] Chromatographic column: Agilent Poroshell 120EC-C18 (4.6×50 mm, 2.7 μm);

[0024] Chromatographic column temperature: 25°C;

[0025] Mobile phase: 0.1% formic acid water: methanol = 20:80 for isocratic elution;

[0026] Flow rate: 0.4 mL / min;

[0027] Injection volume: 5 μL.

[0028] In the present invention, the liquid mobile phase is preferably 0.1% formic acid water (aqueous phase)-methanol (organic phase). This mobile phase formula is simple, but can ensure that the analyte has a good chromatographic peak shape. The chromatographic column uses an Agilent Poroshell 120EC-C18 chromatographic column (4.6×50mm, 2.7μm). This chromatographic column has high separation efficiency, and the retention time of paclitaxel in the plasma sample is only 2.4min, which can meet the analysis of large-scale clinical samples. The present invention adopts an Agilent 6420 mass spectrometry detection system, and the quantitative lower limit of free paclitaxel can reach 0.5ng / mL, which can meet clinical detection needs.

[0029] Further, in step S3, the mass spectrometry conditions are: electrospray ion source, ion source temperature of 300°C, positive ion mode, selective ion monitoring scanning mode, paclitaxel collision energy: 30ev; docetaxel collision energy: 22ev; paclitaxel fragmentation voltage: 200V; docetaxel fragmentation voltage: 220V; electron multiplier voltage: 300V; paclitaxel: m / z876.4→m / z308.1; docetaxel: m / z830.4→m / z304.2; capillary voltage: 4000V.

[0030] Further, in step S3, the internal standard method is used, with paclitaxel as the analyte and docetaxel as the internal standard. The horizontal axis of the standard curve is the paclitaxel concentration, and the vertical axis is the ratio of the peak area of ​​paclitaxel to that of docetaxel. The weighted curve (W = 1 / x 2 ) The least squares method was used for regression calculation.

[0031] In a preferred embodiment of the present invention, the concentration range of total paclitaxel in the detected plasma is 20-10000 ng / mL.

[0032] In a preferred embodiment of the present invention, the concentration range of free paclitaxel in the detected plasma is 0.5-1000 ng / mL.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The biological sample pretreatment method for the total paclitaxel detection method is simple, using the protein precipitation method, and only requires mixing and centrifugation to perform the analysis, which meets the sensitivity required for the detection.

[0035] (2) The separation of free paclitaxel uses a Centrifree ultrafiltration device, which only requires a small amount of sample. After one centrifugation, it can be mixed with the internal standard solution and then injected for analysis. There is no need for cumbersome extraction or nitrogen drying and re-dissolution steps.

[0036] (3) Docetaxel was used as the internal standard. Docetaxel has a similar structure to paclitaxel and can well simulate the behavior of paclitaxel in chromatography and mass spectrometry. In addition, docetaxel is easily available and inexpensive, making it suitable for the promotion and use of the method.

[0037] (4) The method of the present invention is rapid, simple, highly accurate and sensitive, and provides a method for the determination of total paclitaxel and free paclitaxel in clinical samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is the parent ion scan mass spectrum of paclitaxel;

[0039] Figure 2 is the product ion scan mass spectrum of paclitaxel;

[0040] Figure 3 is the precursor ion scan mass spectrum of docetaxel;

[0041] Figure 4 is the product ion scan mass spectrum of docetaxel;

[0042] Figure 5 is the chromatogram of total paclitaxel after adding albumin paclitaxel to blank plasma;

[0043] Figure 6 is the chromatogram of free paclitaxel after adding albumin paclitaxel to blank plasma;

[0044] Figure 7 is the standard curve of total paclitaxel;

[0045] Figure 8 is the standard curve of free paclitaxel. DETAILED DESCRIPTION

[0046] In order to more clearly illustrate the present invention, the following specific examples will be described in detail. However, it will be understood that the following examples are only used for illustrative purposes of the present invention, rather than any limitation of the present invention, and the materials, reagents, instruments and operations used therein are only representative and are not limited to the embodiments. Those skilled in the art can make changes and improvements to the present invention without departing from the scope of protection defined by the claims of the invention through the following description, and these changes and improvements also fall within the scope of the protection claimed by the present invention.

[0047] 1. Test instruments:

[0048] Table 1 Instrument name

[0049] name model factory LC-MS / MS Agilent 6420 Agilent Technologies Electronic analytical balance MettlesToledoXS205DU Mettler Vortex mixer vortex-7 Haimen Qilin Belle Instrument Manufacturing Co., Ltd. Pipette (10-100ul) researchplus EBEND China Co., Ltd. Pipette (100-1000ul) researchplus EBEND China Co., Ltd. Pipette (10-200ul) researchplus EBEND China Co., Ltd. High-speed refrigerated centrifuge centrifuge5424R Eppendorf China Ltd. 4℃ refrigerator HY-310S Haier Refrigerator -20℃ Refrigerator DWYL450 Zhongke Meiling Ultrapure water machine simplicityuv Merck KGaA Ultrasonic Cleaner South China HNY-350L Guangzhou Huanan Ultrasonic Equipment Co., Ltd. Low speed centrifuge South China BY-600A Beijing Baiyang Medical Equipment Co., Ltd.

[0050] 2. Test materials

[0051] Paclitaxel reference substance (Batch No.: 100382-201904, China Food and Drug Administration, Figure 1 and Figure 2 ), docetaxel reference substance (Batch No.: 100666-202005, China Food and Drug Administration, Figure 3 and Figure 4 ), methanol (Fisher Scientific, USA), formic acid (Fisher Scientific, USA), Centrifree ultrafiltration device (model: 4104, MERCK)

[0052] 3. Chromatographic conditions

[0053] Chromatographic column: Agilent Poroshell 120EC-C18 (4.6×50 mm, 2.7 μm); mobile phase: 0.1% formic acid water: methanol = 20:80; flow rate: 0.4 ml / min; column temperature 25°C; injection volume 5 μl.

[0054] 4. Mass spectrometry conditions

[0055] Ion Source: Electrospray ionization source (ESI + ); ion source temperature: 300°C; gas flow rate: 11.0 l / min; ion polarity: positive ion mode; scanning mode: selective ion monitoring; paclitaxel collision energy: 30 ev; docetaxel collision energy: 22 ev; paclitaxel fragmentation voltage: 200 V; docetaxel fragmentation voltage: 220 V; electron multiplier voltage: 300 V; paclitaxel: m / z 876.4→m / z 308.1; docetaxel: m / z 830.4→m / z 304.2; capillary voltage: 4000 V.

[0056] 5. Total Paclitaxel Sample Pretreatment

[0057] 50 μl of plasma was drawn, 300 μl of 100 ng / mL docetaxel methanol internal standard solution was added, the mixture was inserted into a vortex mixer and vortexed for 1 min, then placed in a centrifuge and centrifuged at 14,500 rpm for 10 min, 200 μl of the supernatant was drawn for injection, the mass chromatogram was recorded, and LC-MS / MS analysis was performed using the internal standard method.

[0058] 6. Pretreatment of free paclitaxel samples

[0059] Take 500 μl of plasma and add it to the Centrifree ultrafiltration device. Centrifuge it at 1000g for 15 minutes at 25°C using a fixed-angle rotor centrifuge. Accurately aspirate 100 μl of the ultrafiltrate, add 100 μl of 100 ng / mL docetaxel methanol internal standard solution, vortex for 1 minute, centrifuge at 14500 r / min for 10 minutes, aspirate 160 μl of the supernatant for injection, record the mass chromatogram, and perform LC-MS / MS analysis using the internal standard method.

[0060] 7. Results

[0061] 7.1 In order to ensure that the most convenient and most achievable operating conditions are obtained without affecting the plasma protein binding equilibrium of the drug, the present invention uses the method of controlling variables to verify and screen the key conditions of ultrafiltration: centrifugation time, centrifugation temperature, and centrifugal force. The test results are shown in Table 2-4. Finally, the centrifugation time is within the range of 15-30min, which has no effect on the free concentration. Therefore, in order to save test time, the most preferred condition is 15min. There is no difference in the free concentration results when centrifuged at 25℃ and 37℃. Considering the instrument environment control, 25℃ is easier to reach and maintain than 37℃, so the most preferred condition is 25℃. There is no statistically significant difference in the free paclitaxel concentration when centrifuged at 1000g and 2000g. Therefore, considering the comprehensive test instrument conditions, a centrifuge that can reach a centrifugal force of 1000g is easier to obtain, so 1000g is preferred as the centrifugation condition for ultrafiltration.

[0062] Table 2. Comparison of different centrifugation times at 1000 g and 37 °C

[0063]

[0064] Table 3. Comparison of different centrifugation temperatures at 1000g, 15min

[0065]

[0066] Table 4. Comparison of centrifugal force under the conditions of 25℃, 15min centrifugation

[0067]

[0068] 7.2 Methodological Validation

[0069] 7.2.1 Specificity Verification

[0070] Six blank plasmas from different sources were added with albumin paclitaxel and processed according to the items “5 Pretreatment of total paclitaxel samples” and “6 Pretreatment of free paclitaxel samples”, and analyzed according to the items “3 Chromatographic conditions” and “4 Mass spectrometry conditions”. The obtained chromatograms are shown in Figure 5 and Figure 6 No interference peaks appeared within the retention time range of 2.4 min of paclitaxel, indicating that this method is specific for the analysis of total paclitaxel and free paclitaxel in plasma.

[0071] 7.2.2 Standard curve

[0072] The horizontal axis of the standard curve is the concentration of paclitaxel, and the vertical axis is the ratio of the peak area of ​​paclitaxel and docetaxel. The weighted (W=1 / x2) least square method is used for regression calculation. The standard curve of total paclitaxel is obtained by adding paclitaxel standard solution to blank plasma and processing and analyzing according to "5 Total Paclitaxel Sample Pretreatment". Table 5 shows 5 total paclitaxel standard curves obtained after 5 analysis batches, among which, Figure 7 The standard curve of total paclitaxel is y=2.8562x-0.004210, and the linear range is 20-10000ng / mL. The standard curve of free paclitaxel is obtained by adding the standard solution of paclitaxel to the blank ultrafiltrate and analyzing it according to "6 free paclitaxel sample pretreatment". Table 6 shows 5 standard curves of free paclitaxel obtained after 5 analysis batches, among which, Figure 8 The standard curve of free paclitaxel is y=21.3891x+0.001838, and the linear range is 0.5-1000 ng / mL.

[0073] Table 5 Standard curve of total paclitaxel

[0074] Total Paclitaxel Equation Linear coefficient y=2.8562x-0.004210 <![CDATA[r 2 =0.9985]]> y=3.2061x-2.5008e-5 <![CDATA[r 2 =0.9949]]> y=3.1483x+0.003764 <![CDATA[r 2 =0.9982 <!-- 7 -->]]> y=2.4773x-0.002468 <![CDATA[r 2 =0.9981]]> y=2.7275x+0.004476 <![CDATA[r 2 =0.9955]]>

[0075] Table 6 Standard curve of free paclitaxel

[0076] Ultrafiltration Paclitaxel Equation Linear coefficient y=15.6839x-5.4389e-4 <![CDATA[r 2 =0.9962]]> y=21.3891x+0.001838 <![CDATA[r 2 =0.9978]]> y=28.5064x+0.039741 <![CDATA[r 2 =0.9946]]> y=25.7167x+0.005512 <![CDATA[r 2 =0.9955]]> y=21.2540x+0.005735 <![CDATA[r 2 =0.9949]]>

[0077] 7.2.3 Precision and accuracy

[0078] Take human blank plasma to dilute the paclitaxel stock solution, prepare three different concentrations (80, 800, 8000 ng / mL) of mass samples, low concentration, medium concentration, and high concentration, prepare 6 samples for analysis at each concentration, and measure continuously for 3 days. Based on the corresponding standard curve, each concentration is calculated, and then the accuracy and precision of the method are evaluated based on the obtained concentration. The specific results are shown in Table 7. As shown in the table, the intra-batch and inter-batch precision (RSD) is <8.80%. It can be seen from the results that the method established in the experiment has high precision and high accuracy, which is consistent with the requirements of the current "Guidelines for Confirmation of Quantitative Analysis Methods of Biological Samples" and can be used for the quantitative detection of paclitaxel in blood.

[0079] 10 μl of paclitaxel working solution was added to 90 μl of blank ultrafiltrate to prepare three different concentrations (8, 80, 400 ng / mL) of free paclitaxel quality control samples, low concentration, medium concentration, and high concentration. Six samples were prepared for each concentration for three days of analysis. According to the corresponding standard curve, each concentration was calculated, and then the accuracy and precision of the method were evaluated based on the obtained concentration. The specific results are shown in Table 8. The intra-day and inter-day precision (RSD) were both <9.97%, indicating that the method used in this experiment has good precision and accuracy in determining free paclitaxel in human plasma, which meets the relevant regulations and can be applied to the quantitative analysis of free paclitaxel in plasma.

[0080] Table 7 Total paclitaxel method validation accuracy and precision

[0081]

[0082]

[0083] Table 8 Accuracy and precision of free paclitaxel

[0084]

[0085]

[0086] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for quantitatively analyzing total paclitaxel and free paclitaxel in human plasma, characterized in that: The following steps are involved: S1. Sample pretreatment for total paclitaxel concentration detection: plasma and organic reagent are mixed in proportion, and after protein precipitation by centrifugation, a plasma sample as supernatant is obtained; S2. Sample pretreatment for free paclitaxel concentration detection: adding the plasma sample to an ultrafiltration tube, ultrafiltration to obtain an ultrafiltrate, mixing the ultrafiltrate with an organic reagent in proportion, and centrifuging to obtain a supernatant sample to be tested; S3. High performance liquid chromatography-mass spectrometry tandem analysis: The samples to be tested are analyzed using a high performance liquid chromatography-mass spectrometer to detect the chromatographic peaks of paclitaxel and internal standard docetaxel in the samples, and the total paclitaxel and free paclitaxel concentrations in the plasma are calculated based on these peaks.

2. The method for quantitatively analyzing total paclitaxel and free paclitaxel in human plasma according to claim 1, characterized in that: In step S1, plasma and an organic reagent are mixed at a volume ratio of 1:6 and centrifuged at 14,500 rpm for 10 minutes to precipitate proteins.

3. The method for quantitative analysis of total paclitaxel and free paclitaxel in human plasma according to claim 2, characterized in that: In step S1, the organic reagent is a 100 ng / ml docetaxel methanol solution.

4. The method for quantitatively analyzing total paclitaxel and free paclitaxel in human plasma according to claim 1, characterized in that: In step S2, the specific steps are as follows: 500 μL of plasma sample is taken into a Centrifree ultrafiltration device, ultrafiltration is performed for 15 minutes at 25° C. and 1000 g centrifugal force, the ultrafiltrate is mixed with 100 ng / mL docetaxel methanol solution in a volume ratio of 1:1, and centrifuged at 14500 rpm for 10 minutes to obtain the supernatant sample to be tested.

5. The method for quantitative analysis of total paclitaxel and free paclitaxel in human plasma according to claim 1, characterized in that: In step S3, the liquid chromatography determination conditions are: Chromatographic column: Agilent Poroshell 120EC-C18 (4.6×50 mm, 2.7 μm); Chromatographic column temperature: 25°C; Mobile phase: 0.1% formic acid water: methanol = 20:80 for isocratic elution; Flow rate: 0.4 mL / min; Injection volume: 5 μL.

6. The method for quantitative analysis of total paclitaxel and free paclitaxel in human plasma according to claim 1, characterized in that: In step S3, the mass spectrometry conditions are: electrospray ion source, ion source temperature of 300°C, positive ion mode, selective ion monitoring scanning mode, paclitaxel collision energy: 30ev; docetaxel collision energy: 22ev; paclitaxel fragmentation voltage: 200V; docetaxel fragmentation voltage: 220V; electron multiplier voltage: 300V; paclitaxel: m / z876.4→m / z308.1; Docetaxel: m / z 830.4→m / z 304.2; capillary voltage: 4000V.

7. The method for quantitatively analyzing total paclitaxel and free paclitaxel in human plasma according to claim 1, characterized in that: In step S3, the internal standard method is used, with paclitaxel as the analyte and docetaxel as the internal standard. The horizontal axis of the standard curve is the paclitaxel concentration, and the vertical axis is the ratio of the peak areas of paclitaxel and docetaxel. The weighted (W = 1 / x 2 ) The least squares method was used for regression calculation.

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