CN104569206B - A kind of measure blood plasma middle reaches ionization compound concentration, logD and the method for compound protein combination rate - Google Patents

Abstract

The invention discloses and a kind of measure blood plasma middle reaches ionization compound concentration, log D and the method for compound protein combination rate, measure blood plasma middle reaches ionization compound concentration and the method for log D, step: (1), hatches containing compound and the donor phase of blood plasma with phosphate buffer solution preparation；(2) n-octyl alcohol is injected in hollow-fibre membrane；Adding phosphate buffer solution in bag filter, hollow-fibre membrane and bag filter are put in the solution that step (1) obtains, extraction makes balance；(3) take out liquid in bag filter, analyze the concentration [D] of compound in buffer solution with HPLC_d, this concentration is equivalent to ionization compound concentration in blood plasma middle reaches during balance；Take out liquid in hollow-fibre membrane, compound concentration [D] in n-octyl alcohol when analyzing balance with HPLC_a, calculate the log D of compound in blood plasma；It is few that the present invention analyzes speed biological sample amount fast, that need, sensitive, easy and simple to handle, low cost, need not the instrument of costliness, environmental protection.

Description

A kind of measure blood plasma middle reaches ionization compound concentration, log D and the method for compound protein combination rate

Technical field

The present invention relates to a kind of blood plasma middle reaches ionization compound concentration of mensuration simultaneously, log D and the method for compound protein combination rate.

Technical background

Along with developing rapidly, for the screening of compound of the subjects such as human activities environment, protein science, combinatorial chemistry, computer Provide thousands of new model, novel targets, new technique, promote the high development of screening compound research.Screening compound Be from synthesis or natural product compound to possibly as candidate compound carry out preliminary Pharmacological Activity Screening process.Chemical combination In thing screening, need substantial amounts of candidate compound to be carried out analysis of physical and chemical property, such as dissolubility, infiltration coefficient, compound protein Combination rate, log D and the mensuration of log P.Wherein the lipotropy of compound is an important physicochemical property of compound, it with Compound absorption in vivo, distribution, metabolism, excretion, toxicological profile, even with blood brain barrier permeability and compound Removing have important relation.The Determination of oil-water partition coefficient of compound is often used as the lipophilic parameter of characterization compound, and it is fixed Justice is the logarithm of compound partition coefficient between n-octyl alcohol and water, i.e. log D.

At present, the method measuring compound Determination of oil-water partition coefficient is a lot, including fask oscillating method, chromatography, pH value measure, fixes Changing synthetic membrane and dialysis tube method, wherein, classical way is fask oscillating method, and common method is chromatography.Fask oscillating method is the simplest and normal Method, but need to consume substantial amounts of organic reagent in mensuration, be unfavorable for environmental protection, and this method is bothersome, laborious, Sample is asked to have higher purity and good stability.It addition, in experiment shakes procedure, organic facies and aqueous phase are easily generated Emulsion, causes the biphase result thoroughly not occurring mistake when separating.Measure the chromatography of Determination of oil-water partition coefficient, including Thin layer chromatography, reversed phase high performance liquid phase method, hydrophilic compound-formation chromatography, counter current chromatography and microemulsion electrokinetic chromatography.Chromatography It it is the relational model of chromatographic system based on the compound setting up a series of known Determination of oil-water partition coefficient experiment value.Being total to of these methods It is that these assay methods can have similar functional groups or belong to the profit distribution of isonomic compound by Accurate Determining with feature Coefficient.But, when not having the identical compound of structure, the result that the method obtains is the most inaccurate.

The seminar of Bao Jianmin professor in 2006 proposes hollow fiber membrane liquid-phase micro extraction method and measures the profit distribution of compound Coefficient (Guo Y G, Zhang J, Liu D N, Fu H F.Determination of n-octanol-water partition coefficients by hollow-fiber membrane solvent microextraction coupled with HPLC,Anal.Bioanalytical Chem. 386 (7-8), 2193-2198 (2006) .), the method can directly measure the Determination of oil-water partition coefficient of compound.

Meanwhile, the mensuration of compound protein combination rate is one of very important content in screening compound and clinical evaluation of new drug, Its substantial connection is to the pharmacological action intensity of compound.If the protein binding rate of compound is higher, long half time in blood And few side effects；If the protein binding rate of compound is low, half-life short internal elimination in blood is fast, and effect is held time Short.The assay method of common compounds protein binding rate has equilibrium dialysis, ultrafiltration, microdialysis, affinity chromatography, efficiently Molecular exclusion chromatography, high performance capillary electrophoresis, spectrographic method, nuclear magnetic resonance method, mass spectrography, Solid-phase Microextraction and doughnut Film liquid-phase micro-extraction method etc..These compound protein combine assay method and are respectively arranged with pluses and minuses, wherein hollow fiber membrane liquid-phase micro extraction Method is a kind of novel, and collection is sampled, extracted and concentrate in Sample Pretreatment Technique integrally, and Preliminary Applications is in compound egg The white mensuration combined.

The seminar of Tatjana Trtic '-Petrovic in 2005 professor (T,J Determination of drug–protein binding using supported liquid membrane extraction under Equilibrium conditions.J.Chromatogr.B 814 (2), 375-384 (2005) .) use hollow fiber membrane liquid-phase micro extraction method Success measures the compound protein binding characteristics such as lignocaine, ropivacaine, prilocaine, bupivacaine.The method is simple, Quickly, inexpensively, it is the new technique measuring compound protein binding characteristic, but this technology can not directly measure in blood plasma free Compound concentration and protein binding rate.Seminar (Fu H, Guan J, the James J.Bao.A of Bao Jianmin professor in 2006 hollow fiber solvent microextraction approach to measure drug–protein binding.Anal.Sci.22(12), 1565-1569 (2006) .) use hollow-fibre membrane liquid-liquid microextraction method to measure the protein binding rate of hydrocortisone, the method is suitable for Accept phase volume in the sufficiently bulky of donor phase less, i.e. the extraction of organic facies does not affect the reversible balance that compound protein combines. And the method is relatively accurate to the measurement result of high protein combination rate compound, but the free chemical combination in blood plasma cannot be measured simultaneously Substrate concentration and log D.Therefore need one badly and can measure blood plasma middle reaches ionization compound concentration, log D and protein binding rate simultaneously, and And the volume of donor phase does not affect the technology of measurement result with the size accepting phase volume.

Summary of the invention

It is an object of the invention to overcome the deficiencies in the prior art, it is provided that free in a kind of easily operated, quick, Accurate Determining blood plasma Compound concentration and the method for log D.

Second object of the present invention is to provide a kind of method measuring compound protein combination rate.

Technical scheme is summarized as follows:

A kind of method measuring blood plasma middle reaches ionization compound concentration and log D, comprises the steps:

(1) with concentration be 0.02-0.3M, pH be the phosphate buffer solution preparation solute of 7.4 be the solution 5mL of compound and blood plasma For donor phase, the concentration of described donor middle compound mutually is 0.2-100 μ g/mL, Plasma volumes concentration is 10-50%, by donor Put into mutually in container, in 37 DEG C of water-baths, hatch 0.5-2 hour；

(2) 50-100 μ L n-octyl alcohol is injected in the hollow-fibre membrane inner chamber that one end has sealed, heats the opening of hollow-fibre membrane End seals；The phosphate buffer solution that concentration is 0.02-0.3M of 0.5mL pH=7.4 is added, by closed at both ends in bag filter Hollow-fibre membrane and bag filter put in the solution that step (1) obtains, extract and make balance in 75-300 minute；

(3) take out liquid in bag filter, analyze the concentration [D] of compound in buffer solution with HPLC_d, when this concentration is equivalent to balance Blood plasma middle reaches ionization compound concentration；Take out liquid in hollow-fibre membrane, compound concentration in n-octyl alcohol when analyzing balance with HPLC [D]_a, calculate the log D of compound in blood plasma；

The computational methods of log D are Formulas I:

$\log D=\log \frac{\left[D\right]a}{{\left[D\right]}_d}$

Formulas I.

The molecular cut off of described bag filter is 7000-10000.

The material of hollow-fibre membrane is Kynoar, politef, polypropylene or poly-carbonic acid cellulose.

Hollow-fibre membrane uses methanol ultrasonic cleaning before use, removes the impurity on hollow-fibre membrane surface, naturally dries.

A kind of method measuring compound protein combination rate, comprises the steps:

(1) with concentration be 0.02-0.3M, pH be the phosphate buffer solution preparation solute of 7.4 be the solution 5mL of compound and blood plasma For donor phase, the concentration of described donor middle compound mutually is 0.2-100 μ g/mL, Plasma volumes concentration is 10-50%, by donor Put into mutually in container, in 37 DEG C of water-baths, hatch 0.5-2 hour；

(2) 50-100 μ L n-octyl alcohol is injected in the hollow-fibre membrane inner chamber that one end has sealed, heats the opening of hollow-fibre membrane End seals；The phosphate buffer solution that concentration is 0.02-0.3M of 0.5mL pH=7.4 is added, by closed at both ends in bag filter Hollow-fibre membrane and bag filter put in the solution that step (1) obtains, extract and make balance in 75-300 minute；

(3) take out liquid in bag filter, analyze the concentration [D] of compound in buffer solution with HPLC_d, when this concentration is equivalent to balance Blood plasma middle reaches ionization compound concentration；Take out liquid in hollow-fibre membrane, compound concentration in n-octyl alcohol when analyzing balance with HPLC [D]_a, calculate, obtain compound protein combination rate；

The computational methods of compound protein combination rate are Formula II:

$\mathrm{CPB}\%=\frac{\left[\mathrm{DP}\right]}{{\left[D\right]}_0}\×100\%=\frac{{\left[D\right]}_0{\left[V\right]}_0-{\left[D\right]}_d{\left[V\right]}_d-{\left[D\right]}_a{\left[V\right]}_a}{{\left[D\right]}_0{\left[V\right]}_0}\×100\%$

Formula II；

Wherein CPB% is compound protein combination rate, [V]_dFor donor phase during balance and overall solution volume in bag filter；[V]_aFor N-octyl alcohol volume in hollow-fibre membrane during balance；[DP] is that compound protein combines concentration；[V]₀For donor phase initial volume；[D]₀ For donor middle compound initial concentration mutually.

The molecular cut off of described bag filter is 7000-10000.

The material of described hollow-fibre membrane is Kynoar, politef, polypropylene or poly-carbonic acid cellulose.

Hollow-fibre membrane uses methanol ultrasonic cleaning before use, removes the impurity on hollow-fibre membrane surface, naturally dries.

Advantages of the present invention:

(1) method that hollow fiber membrane liquid-phase micro extraction method and bag filter technology combine is applied simultaneously to plasma sample by the present invention first The mensuration field of middle reaches ionization compound concentration, log D and protein binding rate thereof.

(2) analyze speed biological sample amount fast, that need few, and the quick of free cpds and binding compounds in blood plasma can be realized Separate.

(3) present invention is quick, sensitive, easy and simple to handle, low cost, need not the instrument of costliness, environmental protection.

(4) be widely used, to be widely portable in the complex samples such as biological sample, environmental sample and food samples free little molecule dense Degree measures.

Accompanying drawing explanation

Fig. 1 is the installation drawing of the method use of the present invention.In figure, 1 is container；3 is hollow-fibre membrane；4 is bag filter；5 For donor phase.

Fig. 2 be donor mutually in three kinds of balance schematic diagrams.

Detailed description of the invention

The derivation of compound protein combination rate computational methods is as follows:

It is thermodynamical equilibrium that protein is combined with compound:

In above formula, [P], [D] and [DP] represents floating preteins concentration, free cpds concentration and compound protein respectively and combines concentration. Wherein equilibrium constant K_PIt is:

$K_P=\frac{\left[\mathrm{DP}\right]}{\left[D\right]\left[P\right]}$

Assume [D₀] it is precursor compound concentration, according to mass conservation law:

[DP]+[D]=[D]₀

The computing formula of the free cpds quality in this experiment is as follows:

[D]₀[V]₀=[D]_d[V]_d+[D]_a[V]_a

[D]₀For donor middle compound initial concentration mutually；[V]₀For donor phase initial volume；[D]_dBlood plasma middle reaches ionization when being balance Compound concentration, is equivalent to during balance in bag filter compound concentration in buffer；[V]_dMolten with in bag filter for donor phase during balance Liquid cumulative volume；[D]_aCompound concentration in n-octyl alcohol in hollow-fibre membrane during balance；[V]_aFor the most pungent in hollow-fibre membrane during balance Alcohol volume；

The protein bound method of computerized compound is obtained according to above-mentioned formula, as follows:

$\mathrm{CPB}\%=\frac{\left[\mathrm{DP}\right]}{{\left[D\right]}_0}\×100\%=\frac{{\left[D\right]}_0{\left[V\right]}_0-{\left[D\right]}_d{\left[V\right]}_d-{\left[D\right]}_a{\left[V\right]}_a}{{\left[D\right]}_0{\left[V\right]}_0}\×100\%$

In formula, CPB% is compound protein combination rate.

Compound, plasma solutions exist three kinds of balances and sees Fig. 2.

First it is balance A between conjunction type compound and free cpds, next to that free cpds is fine in donor phase and hollow Partition equilibrium B between n-octyl alcohol in dimension film, be finally donor mutually in free cpds and bag filter in flat between buffer solution Weighing apparatus C.Conjunction type compound is quickly combined/dissociates reversible dynamic equilibrium between free cpds.The micro-extraction of hollow-fibre membrane liquid phase Taking in partition equilibrium, compound is extracted in the organic facies that chemical potential is low under the driving force of chemical potential, and final compound is being given Body phase and accepting reaches chemical potential balance between phase.Bag filter buffer solution and donor mutually in compound at the driving force of concentration Under, be distributed in the bag filter that compound concentration is little buffer solution mutually from the donor that compound concentration is big, until donor mutually in In free cpds concentration and bag filter, in buffer solution, compound concentration is equal.

The preferred Kynoar of material of hollow-fibre membrane, polypropylene, experiment proves that politef or poly-carbonic acid cellulose also may be used For the present invention, hollow-fibre membrane uses methanol ultrasonic cleaning before use, to remove the impurity in hollow-fibre membrane surface and fenestra, Naturally dry.

The present invention is further illustrated with specific embodiment below in conjunction with the accompanying drawings.

Embodiment 1

Free cpds (acetaminophen) concentration, log D and compound (acetaminophen) egg in a kind of mensuration blood plasma The method of white combination rate, comprises the steps:

(1) to 4.5mL concentration be 0.1M, pH be 7.4 phosphate buffer solution in add 2.5 μ g acetaminophen and 0.5mL Ox blood plasma (Pu Zhen bio tech ltd, Shanghai), with the solute of gained 5mL as acetaminophen with the phosphoric acid of Ox blood plasma Buffer solution is donor phase 5, and the concentration of described donor middle acetaminophen mutually is 0.5 μ g/mL, Plasma volumes concentration is 10%, Donor is put into mutually in container 1, in 37 DEG C of water-baths, hatches 0.5 hour；

(2) 50 μ L n-octyl alcohols are injected in hollow-fibre membrane 3 inner chamber that one end has sealed, heat the opening of hollow-fibre membrane Seal；The phosphate buffer solution that concentration is 0.1M of 0.5mL, pH=7.4 is added, by closed at both ends in bag filter 4 Hollow fiber film and bag filter are put in the solution that step (1) obtains, and extract and make balance in 90 minutes；The molecular cut off of bag filter It is 10000；The material of hollow-fibre membrane is polypropylene；

(3) take out liquid in bag filter, extract liquid out with HPLC syringe, analyze acetaminophen in buffer solution with HPLC Concentration [D]_dBeing 0.321 μ g/mL, be equivalent to dissociate in blood plasma during balance Determination of Acetaminophen；Hollow-fibre membrane is taken out, Cut one end, with liquid in HPLC syringe taking-up hollow-fibre membrane, acetparaminosalol in n-octyl alcohol when analyzing balance with HPLC Phenol concentration is 0.656 μ g/mL, with [D]_aRepresent, calculate the log D of acetaminophen in blood plasma；

The computational methods of log D are:

$\log D=\log \frac{\left[D\right]a}{{\left[D\right]}_d}$

Formulas I,

Being computed acetaminophen log D value is 0.31, list of references value 0.34.

Calculating the protein binding rate of acetaminophen, its computational methods are:

$\mathrm{CPB}\%=\frac{\left[\mathrm{DP}\right]}{{\left[D\right]}_0}\×100\%=\frac{{\left[D\right]}_0{\left[V\right]}_0-{\left[D\right]}_d{\left[V\right]}_d-{\left[D\right]}_a{\left[V\right]}_a}{{\left[D\right]}_0{\left[V\right]}_0}\×100\%$

Formula II,

Wherein CPB% is compound (acetaminophen) protein binding rate；[V]_dMolten with in bag filter for donor phase during balance Liquid cumulative volume 5.5mL；[V]_aFor n-octyl alcohol volume 50 μ L in hollow-fibre membrane during balance；[V]₀For donor phase initial volume 5mL； [D]₀For donor middle acetaminophen initial concentration 0.5 μ g/mL mutually.

Substituting into Formula II, obtaining acetaminophen protein binding rate is 18.58%, and list of references value is 20%.

Embodiment 2

Free cpds (dexamethasone acetate) concentration, log D and compound (dexamethasone acetate) egg in a kind of mensuration blood plasma The method of white combination rate, comprises the steps:

(1) to 2.5mL concentration be 0.3M, pH be 7.4 phosphate buffer solution in add 500 μ g dexamethasone acetate and 2.5mL Ox blood plasma (Pu Zhen bio tech ltd, Shanghai), with gained 5mL solute as dexamethasone acetate and Ox blood plasma phosphoric acid delay Dissolved liquid is donor phase, and described donor middle dexamethasone acetate concentration mutually is 100 μ g/mL, Plasma volumes concentration is 50%, will Donor is put in container mutually, hatches 2 hours in 37 DEG C of water-baths；

(2) 100 μ L n-octyl alcohols are injected in the hollow-fibre membrane inner chamber that one end has sealed, heat the opening of hollow-fibre membrane Seal；The phosphate buffer solution that concentration is 0.3M of 0.5mL pH=7.4 is added, by closed at both ends in bag filter 4 Hollow fiber film and bag filter are put in the solution that step (1) obtains, and extract and make balance in 300 minutes；The molecular cut off of bag filter It is 7000；The material of hollow-fibre membrane is politef；

(3) take out liquid in bag filter, extract liquid out with HPLC syringe, analyze dexamethasone acetate in buffer solution with HPLC Concentration [D]_dBeing 7.9 μ g/mL, be equivalent to dissociate in blood plasma during balance dexamethasone acetate concentration；Hollow-fibre membrane is taken out, cuts One end, with liquid in HPLC syringe taking-up hollow-fibre membrane, dexamethasone acetate in n-octyl alcohol when analyzing balance with HPLC Concentration is 558.3 μ g/mL, with [D]_aRepresent, calculate the log D of dexamethasone acetate in blood plasma by Formulas I；Dexamethasone acetate Log D value is 1.80, list of references value 1.83.

Dexamethasone acetate protein binding rate, [V] is calculated by Formula II_dFor donor phase during balance and overall solution volume 5.5 in bag filter mL；[V]_aFor n-octyl alcohol volume 100 μ L in hollow-fibre membrane during balance；[V]₀For donor phase initial volume 5mL；[D]₀For giving Body middle dexamethasone acetate initial concentration 100 μ g/mL mutually.

Substituting into Formula II, obtaining dexamethasone acetate protein binding rate is 79.04%, and list of references value is 75%.

Embodiment 3

Free cpds (hydrocortisone) concentration, log D and compound (hydrocortisone) albumen knot in a kind of mensuration blood plasma The method of conjunction rate, comprises the steps:

(1) to 4.5mL concentration be 0.02M, pH value be 7.4 phosphate buffer solution in add 2.5 μ g hydrocortisone and 0.5mL Ox blood plasma (Pu Zhen bio tech ltd, Shanghai), with the solute of gained 5mL as hydrocortisone with the solution of Ox blood plasma is Donor phase, described donor middle hydrocortisone concentration mutually is 0.5 μ g/mL, Plasma volumes concentration is 10%, and donor is put into appearance mutually In device, in 37 DEG C of water-baths, hatch 1 hour；

(2) being injected in the hollow-fibre membrane inner chamber that one end has sealed by 50 μ L n-octyl alcohols, heating is by close for the opening of hollow-fibre membrane Envelope；The phosphate buffer solution that concentration is 0.02M of 0.5mL, pH=7.4 is added, by closed at both ends in bag filter 4 Hollow fiber film and bag filter are put in the solution that step (1) obtains, and extract and make balance in 75 minutes；The molecular cut off of bag filter It is 7000；The material of hollow-fibre membrane is Kynoar；

(3) take out liquid in bag filter, extract liquid out with HPLC syringe, analyze hydrocortisone in buffer solution with HPLC dense Degree [D]_dBeing 0.023 μ g/mL, be equivalent to dissociate in blood plasma during balance hydrocortisone concentration；Hollow-fibre membrane is taken out, cuts one End, by liquid in HPLC syringe taking-up hollow-fibre membrane, hydrocortisone concentration in n-octyl alcohol when analyzing balance with HPLC It is 29.560 μ g/mL, with [D]_aRepresent, calculate the log D of donor middle hydrocortisone mutually by Formulas I；Calculate hydrocortisone Log D value is 3.11, list of references value 3.11.

Hydrocortisone protein binding rate, [V] is calculated by Formula II_dFor donor phase during balance and overall solution volume 5.5mL in bag filter； [V]_aFor n-octyl alcohol volume 50 μ L in hollow-fibre membrane during balance；[V]₀For donor phase initial volume 5mL；[D]₀For donor phase Middle hydrocortisone initial concentration 0.5 μ g/mL.

Substituting into Formula II, obtaining hydrocortisone protein binding rate is 93.01%, and list of references value is 95%.

It is demonstrated experimentally that substitute the Kynoar of the present embodiment with poly-carbonic acid cellulose, other same the present embodiment, its result and basis Embodiment is similar.

Embodiment 4

The precision of the inventive method is investigated: with hydrocortisone for test compound, this method is carried out precision investigation.

The method of embodiment 3, measures under 40 μ g/mL, 6 μ g/mL, tri-concentration of 0.2 μ g/mL in bag filter and hollow every day The concentration of hydrocortisone in n-octyl alcohol in fibrous membrane, the 5 groups of parallel laboratory tests of each concentration, calculate the log D value of hydrocortisone And protein binding rate, METHOD FOR CONTINUOUS DETERMINATION three days, the day to day precision (RSD%) of log D value is respectively 4.42%, 4.56% and 3.18%, The day to day precision (RSD%) of protein binding rate is respectively 9.29%, 8.93% and 9.67%, and the difference between parallel group is less.

By above method the result it can be seen that the mensuration compound log D that sets up of the present invention and protein binding rate method Result is relatively stable.

Claims (3)

1. measure blood plasma middle reaches ionization compound concentration, log D and a method for compound protein combination rate simultaneously, it is characterized in that including Following steps:

(1) with concentration be 0.02-0.3M, pH be the phosphate buffer solution preparation solute of 7.4 be the solution 5mL of compound and blood plasma For donor phase, the concentration of described donor middle compound mutually is 0.2-100 μ g/mL, Plasma volumes concentration is 10-50%, by donor Put into mutually in container, in 37 DEG C of water-baths, hatch 0.5-2 hour；

(2) 50-100 μ L n-octyl alcohol is injected in the hollow-fibre membrane inner chamber that one end has sealed, heats the opening of hollow-fibre membrane End seals；The phosphate buffer solution that concentration is 0.02-0.3M of 0.5mL pH=7.4 is added, by closed at both ends in bag filter Hollow-fibre membrane and bag filter put in the solution that step (1) obtains, extract and make balance in 75-300 minute；

(3) take out liquid in bag filter, analyze the concentration [D] of compound in buffer solution with HPLC_d, when this concentration is equivalent to balance Blood plasma middle reaches ionization compound concentration；Take out liquid in hollow-fibre membrane, compound concentration in n-octyl alcohol when analyzing balance with HPLC [D]_a, calculate log D and the compound protein combination rate of compound in blood plasma；

The computational methods of log D are Formulas I:

$\log D=log\frac{\[D\]a}{{\[D\]}_d}$

Formulas I；

The computational methods of compound protein combination rate are Formula II:

$CPB\%=\frac{\[DP\]}{{\[D\]}_0}\×100\%=\frac{{\[D\]}_0{\[V\]}_0-{\[D\]}_d{\[V\]}_d-{\[D\]}_a{\[V\]}_a}{{\[D\]}_0{\[V\]}_0}\×100\%$

Formula II；

Wherein CPB% is compound protein combination rate, [V]_dFor donor phase during balance and overall solution volume in bag filter；[V]_aFor N-octyl alcohol volume in hollow-fibre membrane during balance；[DP] is that compound protein combines concentration；[V]₀For donor phase initial volume；[D]₀ For donor middle compound initial concentration mutually.

Method the most according to claim 1, is characterized in that the molecular cut off of described bag filter is 7000-10000.

Method the most according to claim 1, it is characterized in that the material of described hollow-fibre membrane be Kynoar, politef, Polypropylene or poly-carbonic acid cellulose.