CN1619304A

CN1619304A - Method of determining mycophenolic acid in human blood plasma and its metabolite

Info

Publication number: CN1619304A
Application number: CN 200410066519
Authority: CN
Inventors: 郁韵秋; 焦正
Original assignee: Fudan University; Huashan Hospital of Fudan University
Current assignee: Fudan University; Huashan Hospital of Fudan University
Priority date: 2004-09-20
Filing date: 2004-09-20
Publication date: 2005-05-25
Anticipated expiration: 2024-09-20
Also published as: CN100383520C

Abstract

The present invention relates to a method for determining mycophenolic acid in human body plasma and its metabolite. Said method includes the following steps: pretreating sample to be determined, utilizing the characteristics of MPA which has strong fluorescence absorption property under the alkaline condition to make analytical chromatographic column separation, then making derivatization and using fluorescence detector to make detection. Said method can simultaneously determine total and free MPA and MPAG.

Description

A kind of method of measuring mycophenolic acid in the human plasma and metabolin thereof

Technical field

The invention belongs to field of medical examination, relate to the analysis determining method of drug disposition, be specifically related to a kind of method of measuring mycophenolic acid in the human plasma and metabolin thereof.

Background technology

Mycophenolate (Mycophenolate Mofetil, MMF, RS61443; Trade name: Cellcept, MMF) is a kind of novel anti-metabolism immunodepressant, is widely used in the anti-repelling treatment after the organ transplant clinically.This medicine is that (Mycophenolic Acid, 2-ethyl ester pro-drug MPA) can significantly improve bioavilability in the body of MPA to mycophenolic acid.The oral back of MMF absorbs rapidly and is complete, be hydrolyzed rapidly in vivo and take off ester and be converted into active metabolite MPA, the latter combines with glucuronic acid then, be converted into and do not have active metabolic product glucoside acidifying thing (Mycophenolic acid glucuronide, MPAG), its main metabolic pathway as shown in Figure 1.MPAG concentration ratio MPA in vivo is much higher, and can be hydrolyzed to MPA once more by liver sausage circulation, reenters in the body and plays a role, and can be electric the combination with the MPA protein combination competitively, and therefore, MPAG can influence pharmacokinetics in the body of MPA.Simultaneously, the average blood plasma protein binding rate of MPA reaches 97.5%, so measure total and free MPA simultaneously and the drug concentration of MPAG has the important clinical meaning.

At present, the external total concentrations that adopt in immunization or the high-efficient liquid phase technique mensuration blood plasma more, high performance liquid chromatogram mass spectrometric hyphenated technique (HPLC/MS) or improved immunization are measured free drug concentration.Simultaneously MPA and MPAG are measured, the experiment condition of bibliographical information often adopts two cover chromatographic systems or adopts gradient elution, ion-pairing agent etc.There are all drawbacks in above-mentioned method: low as efficient, the mensuration cycle is long, and required instrument and equipment or reagent cost an arm and a leg, the maintenance cost height, and the analysis cost height is not suitable for extensively carrying out.

Summary of the invention

The method that the purpose of this invention is to provide mycophenolic acid in a kind of quick, sensitive mensuration human plasma and metabolin thereof.This method need not expensive equipment and reagent, can measure total and free MPA and MPAG simultaneously.Sample sampling of the present invention is few, and pre-treatment is simple, quick, and analytical cycle is short, and cost is low, is suitable for routine clinical detection.

The inventive method through after the pre-service, utilizes MPA that the feature of strong fluorescent absorption is arranged under alkali condition to testing sample, analyzing chromatographic column separation back derivatization, detects with fluorescence detector then.This method can make the sensitivity that detects of MPA improve two orders of magnitude, can measure free concentration.

This method adopts derivatization reagent behind an additional high-pressure pump and the threeway joint pin, and connects UV-detector and fluorescence detector with series system, carries out the sample post column derivatization, can make the sample after the separation that fluorescence signal is arranged.

This method can be used in blood plasma, the serum quantitative measurement of total MPA and MPAG and free MPA and MPAG concentration.Wherein,

The concentration determination of total MPA and MPAG: quantitatively divide and get blood plasma or blood serum sample, add quantitative protein precipitant mixing, centrifugal, get the supernatant sample introduction, at first detect MPAG, carry out detecting MPA with fluorescence detector behind the derivatization with NaOH solution again through UV-detector.In the said determination method, the protein precipitant of adding is methyl alcohol, acetonitrile or the two potpourri.

The concentration determination of free MPA and MPAG: quantitatively divide and get blood plasma or blood serum sample, isolate ultrafiltrate, separate, at first detect MPAG, carry out detecting MPA with fluorescence detector behind the derivatization with NaOH solution again through UV-detector through chromatographic column with ultrafiltration.

In the said determination method, adopt the peak area of UV-detector and fluorescence detector detection MPAG and MPA respectively, be converted into the concentration of MPAG and MPA with the typical curve equation.

In the method for said determination MPA and MPAG concentration, adopt universal liquid-phase chromatographic column, its filler is that octadecyl or octyl bonding phase silica gel are the liquid-phase chromatographic column of filler.The high performance liquid chromatogram system adopts universal ultraviolet and fluorescence detector and high-pressure pump, and moving phase adopts isocratic elution.

The inventive method quick and precisely, and is sensitive reliable, and analysis cost is low, is suitable for routine clinical detection and Study on pharmacokinetics.Have following advantage: under the inventive method condition determination, human body endogenous material and drug combination commonly used be interference measurement not all.The total MPA and the range of linearity of MPAG are respectively 0.1～40 μ g/ml, 10～250 μ g/ml; The free MPA and the range of linearity of MPAG are respectively 0.01～1 μ g/ml, and 2.5～100 μ g/ml, linearly dependent coefficient are greater than 0.997, and the minimum detectable activity of MPA and MPAG is respectively 10ng and 50pg, and the method recovery is greater than 92%, and RSD is less than 12%.

Description of drawings

Fig. 1 is the main metabolic pathway of mycophenolate,

Wherein, mycophenolate mofetil: mycophenolate; Mycophenolic acid: mycophenolic acid;

MPAG: glucuronic acid mycophenolic acid.

Fig. 2 is a chromatographic system device synoptic diagram.

Fig. 3 is the typical color spectrogram

A wherein, blank human plasma ultrafiltrate chromatogram: (a) ultraviolet (b) fluorescence;

Actual renal transplant recipients is taken the chromatogram of MMF 750mg bid, prednisone and ciclosporin A:

(c) ultraviolet (d) fluorescence; 1:MPAG; 2: interior mark; 3:MPA.

B, blank human plasma ultrafiltrate chromatogram (a) ultraviolet (b) fluorescence; Actual renal transplant recipients is taken chromatogram (c) ultraviolet (d) fluorescence of MMF750mg bid, prednisone and ciclosporin A; 1:MPAG; 2:MPA.

Embodiment

Embodiment 1: total MPA and MPAG measure

Chromatographic condition

(system comprises the LC-10AD pump to day island proper Tianjin LC-10A of company high performance liquid chromatogram instrument, LC-10AT pump, SPD-10A UV-detector, the RF-10AXL fluorescence detector, SIL-10A automatic sampler, CBM-10A system communication device, column oven, Class-LC10 Version 1.63 chromatographic work stations); Chromatographic column adopting KromasilC8 (150mm * 4.6mm i.d., 5 μ m); Column temperature: 30 ℃; Moving phase: methyl alcohol-0.1% trifluoroacetic acid aqueous solution (55: 45); Flow velocity: 1.2ml/min; Add 0.2M NaOH 0.15ml/min behind the post.Ultraviolet detection wavelength: 295nm; Fluorescence: Ex=325nm, Em=435nm is during sensitivity (sensitivity) is made as (medium).

Specimen preparation

Get 100 μ l samples, add the acetonitrile solution 300 μ l of mark Propafenone 150 μ g/ml in containing, the vortex mixing in 12000xg, below 10 ℃, centrifugal 10 minutes, was got supernatant 20 μ l sample introductions after 15 seconds.

Linear test

Get blank plasma, add an amount of MPA and MPAG standard reserving solution, be mixed with and contain MPA (0.1,0.5,10.0 respectively, 20.0,30.0,40.0 μ g/ml), MPAG (10,50,100,150,200,250 μ g/ml) blood plasma standard items are by last joint " specimen preparation " processing down.Comparison sample concentration with sample peak area and interior mark peak area carries out linear regression, and the linear equation of MPA and MPAG is respectively C=0.000705 Ratio+0.001946 (r=0.9991), and C=3.1434Ratio+0.2113 (r=0.9994) (n=5).

Precision and recovery test

Get blank plasma, add an amount of MPA and MPAG standard reserving solution, be mixed with and contain MPA (0.25,15.0,35.0 μ g/ml) respectively, the blood plasma quality-control product of MPAG (25,125,225 μ g/ml) is handled down by last joint " specimen preparation ".Calculate its measured concentration according to equation of linear regression, calculate the actual measurement mean value and the relative standard deviation of every kind of concentration, precision is with relative standard deviation (RSD) expression, and (C actual measurement/C theory) x100% is the recovery.

Embodiment 2: free MPA and MPAG measure

Chromatographic condition

Measure part with MPA total among the embodiment 1 and MPAG, fluorescence detector sensitivity (sensitivity) is made as height (high).

Specimen preparation

Get 500 μ l blood plasma, place the ultrafiltration pipe (Amicon, Danvers, MA, USA) in, the molecular weight that dams of ultrafiltration pipe filter membrane is 10 000 Da.In 25 ℃, 12000xg centrifugal 40 minutes, can obtain nearly 200 μ l ultrafiltration blood plasma, sample introduction 20 μ l.

Linear test

Get the blank plasma after the blank ultrafiltration, add an amount of MPA and MPAG standard reserving solution, be mixed with and contain MPA respectively, the standard items of MPAG are handled down by last joint " specimen preparation ".With the sample peak area sample concentration is carried out linear regression, the linear equation of MPA and MPAG is respectively C=0.00006187Area-4.31441 (r=0.9997), and C=0.0002478Area-0.331591 (r=0.9997) (n=5).

Precision and recovery test

Get blank plasma, add an amount of MPA and MPAG standard reserving solution, be mixed with and contain MPA respectively (20,400,800ng/ml), the blood plasma quality-control product of MPAG (5,40,80 μ g/ml) is handled down by last joint " specimen preparation ".Calculate its measured concentration according to equation of linear regression, calculate the actual measurement mean value and the relative standard deviation of every kind of concentration, precision is with relative standard deviation (RSD) expression, and (C actual measurement/C theory) x100% is the recovery.

Table 1 be in the blood plasma total MPA in a few days, day to day precision and the method recovery.

Table 2 be in the blood plasma total MPAG in a few days, day to day precision and the method recovery.

Table 3 be in the blood plasma free MPA in a few days, day to day precision and the method recovery.

In table 4 blood plasma free MPAG in a few days, day to day precision and the method recovery.

Table 1

Withinday precision (n=6) day to day precision (n=6)

Theoretical concentration

Measured value RSD measured value RSD method recovery %

μg·mL ^-1

μg·mL ^-1 ％ μg·mL ^-1 ％

0.25 0.2497 5.89 0.2435 6.34 97.4％

15 15.82 2.60 14.85 3.18 99.0％

30 29.91 1.71 28.78 1.32 95.9％

Table 2

Withinday precision (n=6) day to day precision (n=6)

Theoretical concentration

Measured value RSD measured value RSD method recovery %

μg·mL ^-1

μg·mL ^-1 ％ μg·mL ^-1 ％

25 23.39 5.75 24.07 6.34 96.3％

125 123.98 3.19 136.67 3.18 109.3％

225 212.67 3.23 236.11 1.32 104.9％

Table 3

Withinday precision (n=6) day to day precision (n=6)

Theoretical concentration

Measured value RSD measured value RSD method recovery %

ng·mL ^-1

μg·mL ^-1 ％ μg·mL ^-1 ％

20 19.31 5.95 18.79 6.22 94.0％

400 399.02 5.64 429.83 3.80 107.5％

800 738.61 3.20 773.14 2.77 96.6％

Table 4

Withinday precision (n=6) day to day precision (n=6)

Theoretical concentration

Measured value RSD measured value RSD method recovery %

μg·mL ^-1

μg·mL ^-1 ％ μg·mL ^-1 ％

5 4.96 1.50 4.87 2.29 97.4％

40 38.00 1.25 37.15 0.64 92.9％

80 78.13 1.09 75.81 0.68 94.8％

Claims

1, a kind of method of measuring mycophenolic acid in the human plasma and metabolin thereof is characterized in that testing sample after pre-service, separates the back derivatization analyzing chromatographic column, detects with fluorescence detector, may further comprise the steps:

(1) sample pretreatment

Measure total concentration: get testing sample, add methyl alcohol, acetonitrile or both mixed liquors, carry out albumen precipitation, measure Cf: get testing sample, 25 ℃, 12000xg carries out with ultrafiltration;

(2) sample separation and analysis

Adopt universal liquid-phase chromatographic column, its filler is that octadecyl or octyl bonding phase silica gel are the liquid-phase chromatographic column of filler, the high performance liquid chromatogram system adopts universal ultraviolet and fluorescence detector and high-pressure pump, moving phase adopts isocratic elution, adopt the peak area of UV-detector and fluorescence detector detection MPAG and MPA respectively, be converted into the concentration of MPAG and MPA with the typical curve equation;

(3) sample post column derivatization

Adopt derivatization reagent behind an additional high-pressure pump and the threeway joint pin, and connect UV-detector and fluorescence detector, make the sample after the separation that fluorescence signal be arranged with series system.

2, the method for mycophenolic acid and metabolin thereof in the mensuration human plasma according to claim 1, the testing sample that it is characterized in that described step (1) is human plasma or serum.

3, the method for mycophenolic acid and metabolin thereof in the mensuration human plasma according to claim 1, the chromatographic condition that it is characterized in that described step (2) is chromatographic column adopting Kromasil C8 (150mm * 4.6mm i.d., 5 μ m) wherein; Column temperature is 30 ℃; Moving phase is methyl alcohol-0.1% trifluoroacetic acid aqueous solution (55: 45); Flow velocity is 1.2ml/min; Add 0.2M NaOH 0.15ml/min behind the post.