CN103512961B - Method for determining free analyte in biological sample and determining drug protein binding ratio - Google Patents
Method for determining free analyte in biological sample and determining drug protein binding ratio Download PDFInfo
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Abstract
The invention discloses a method for determining a free analyte in a biological sample. The method comprises the following steps: adopting the solvent bar hollow fiber liquid-phase micro-extraction technology, determining a time constant of the solvent bar hollow fiber liquid-phase micro-extraction through a concentration curve method in the reverse extraction process, and further computing the concentration of the free analyte in the actual sample through the analyte pre-equilibrium concentration obtained through determining the pre-equilibrium solvent bar hollow fiber liquid-phase micro-extraction. In the biological sample containing protein, the drug protein binding ratio can be computed according to the free drug concentration. According to the method, the sample pretreatment device is simple and convenient to operate. The deuterated material is not required, the property of the biological sample is not changed, the content of the free analyte in the biological sample can be determined simply, quickly and accurately.
Description
Technical field
The present invention relates to chemical analysis method, particularly a kind of method measuring free analyte in biological sample and the method measuring percentage of protein binding.
Background technology
Free drug receives increasing concern and attention in recent years, and has become one of study hotspot of pharmacokinetics and Therapeutic Drug Monitoring.Medicine has two kinds of existence forms after entering body blood.One is mating type medicine; Weak acidic drug is combined with albumin, and weakly basic drugs and 1-acidoglycoprotein (AGP) combine.Another kind is Free drug.Free drug in blood plasma produces the part of pharmacodynamics effect, free drug concentration and receptor site drug balance, thus itself and drug effect or adverse drug reaction closely related, also only have Free drug to be transformed by body and to drain.
Hollow fiber liquid-phase micro-extraction is to the extracting and developing of microcomponent in complex sample, enrichment and concentratedly play an important role.Hollow fiber has can make drug molecule pass through, the intransitable special construction of biomacromolecule of biomacromolecule or bound drug.The research utilizing the architectural characteristic of hollow fiber to measure free drug concentration and percentage of protein binding has been reported, and above-mentioned all working is all adopt balance extraction.And the relatively long balance extraction time topmost method defect of hollow fiber liquid-phase micro-extraction just.For Pharmaceutical Analysis, long extraction time can affect the balance of free drug and bound drug, and then can produce some illusions in the analysis of biological sample and affect accuracy and the precision of result.In addition, the loss of extraction solvent may be caused long analysis time; And biological sample such as blood plasma etc. also can cause the change of matrix due to factors such as the metabolism of enzyme.
Vibrate to sample or stir, the sample high to viscosity such as blood plasma dilutes, and extraction phase applies the extraction time that voltage etc. all can shorten hollow fiber liquid-phase micro-extraction.These disposal routes are limited on the impact of extraction rate on the one hand, add difficulty and the operating parameter of instrument on the other hand.In addition, these methods are all to improve mass transfer rate, but for the low analysis thing of partition factor, mass transfer is not the rate-limiting step of hollow fiber liquid-phase micro-extraction.
Kinetic proofreading in micro-extraction is proposed in solid-phase microextraction by Chen etc. the earliest, be a kind of micro-extraction quantitative technique utilizing internal standard compound desorption to carry out calibration analyte absorption, have broad application prospects in environmental analysis, animals and plants In vivo detection and Pharmaceutical Analysis etc.The analysis of kinetic proofreading to following sample based on pre-equilibration has outstanding advantage: 1. the sample grown very much of equilibration time; 2. sample concentration is too high; 3. matrix composition is unstable, as biological sample etc.Ouyang etc. once the differential of kinetic proofreading in liquid-phase micro-extraction.Kinetic proofreading's method of above-mentioned foundation all needs deuterated thing as the internal standard compound of stripping process.Deuterated thing is expensive, and analytical instrument needs the instrument of this kind of high separating efficiency of mass spectrum (can compartment analysis test analyte and deuterated thing thereof).These factors limit the application of pre-equilibration kinetic proofreading method in micro-extraction technique, also limit the application of pre-equilibration kinetic proofreading hollow fiber liquid-phase microextraction method in free drug express-analysis.
Summary of the invention
The technical matters that the present invention solves is to provide a kind of method measuring free analyte in biological sample, to overcome the difficult point of existing free analyte sample pre-treatments, provide a kind of simple, fast, the method for Accurate Determining free analyte in biological sample.
Another technical matters that the present invention solves is to provide a kind of method measuring percentage of protein binding, with provide one simply, assay method fast and accurately.
In order to solve the problems of the technologies described above, the embodiment of the present invention proposes a kind of method measuring free analyte in biological sample, is to utilize pre-equilibration solvent rod hollow fiber liquid-phase micro extraction technique, comprises the following steps:
Step one: make solvent rod hollow-fibre membrane micro-extraction device;
Step 2: the extraction equipment of step one that utilizes analyzing thing standard items with variable concentrations carries out stripping process, obtains the time constant of solvent rod hollow fiber liquid-phase micro-extraction by concentration curve method; Described concentration curve method uses formula (1):
（1）
Wherein, q0 is the quality of the analysis thing standard items be added in advance in extraction solvent, Q is the quality of the analysis thing standard items residual in extraction solvent when the pre-equilibration of extraction time t, Ve is the volume of extraction solvent, Vs is the volume of blank biological sample, Kes analyzes the partition factor of thing between extraction solvent and sample solution, and t is extraction time; And
Step 3: utilize the extraction equipment of step one to extract analysis thing in biological sample to be measured, obtain the amount of the analysis thing of pre-equilibration extraction, according to the time constant obtained in the amount of analysis thing of pre-equilibration extraction and step 2, calculate the content of free analyte in actual sample.
Preferably, the following formula of described step 3 middle reaches compartment analysis substrate concentration computing formula (2):
（2）
Wherein, Cf is the concentration of free analyte in biological sample solution, n is the quality of the analysis thing of the extraction solvent extraction when the pre-equilibration of extraction time t, Ve is the volume of extraction solvent, Vs is the volume of blank biological sample, Kes analyzes the partition factor of thing between extraction solvent and sample solution, and t is extraction time.
Preferably, described step one is that Pvdf Microporous Hollow Fiber Membrane is cut into suitable segment, dries after cleaning, and the Single port of closed hollow tunica fibrosa.
Preferably, in described step 2, will be q0 containing analyzing thing standard items quality and the extractant that volume is Ve injects solvent rod and by solvent rod sealing two ends; Again solvent rod is immersed in the blank biological sample solution that volume is Vs completely; After oscillation extraction a period of time t; Taking-up solvent rod, cuts off one end of solvent rod after leaving standstill, extract extraction solvent and carry out instrumental analysis acquisition Q value; According to formula (1), take Q as ordinate, q0 is horizontal ordinate, carries out linear regression, slope of a curve namely:
，
At Kes, when Ve, Vs and extraction time t are known, calculate time constant a by slope value.
Described step 2 is further comprising the steps:
(A) extraction solvent containing variable concentrations analysis thing standard items of 50-80 μ L is added in the inner chamber of the solvent rod hollow-fibre membrane closed one end, leaves standstill;
(B) other end of solvent rod is closed;
(C) described hollow-fibre membrane is put in sample bottle, the blank biological sample solution of 1.8-3.8 mL is added with in sample bottle, vortex oscillation, extract, in extraction process, analyze thing standard items meeting back extraction in sample solution, extraction time t decides according to the related coefficient of concentration curve and the sensitivity of instrumental analysis;
(D) after having extracted, take out hollow fiber membrane solvent rod, get the extraction solvent 25-40 μ L in fibre membrane lumens, inject instrument and carry out assay, obtain the Q value in formula (1), the surplus of target analytes in solvent rod when being t that Q is back-extraction time, carries out linear regression according to formula (1) and calculates time constant a.
Described step 3 is further comprising the steps:
(A) the blank extraction solvent of 50-80 μ L is added in the inner chamber of the solvent rod hollow-fibre membrane closed one end, leaves standstill;
(B) other end of solvent rod is closed;
(C) above-mentioned hollow-fibre membrane is put in sample bottle, be added with the biological sample containing test analyte of 1.8-3.8 mL in sample bottle, vortex oscillation, extracts, and extraction time decides according to the sensitivity of instrumental analysis;
(D) after having extracted, take out hollow fiber membrane solvent rod, the extraction solvent 25-40 μ L got in fibre membrane lumens carries out assay, obtains the n value in formula (2), according to formula (2), the concentration of actual free analyte in biological sample namely can be obtained.
Preferably, described extraction solvent is selected from least one in n-octyl alcohol, P-xylene, n-hexyl ether.
Preferably, the analysis thing of the extraction of pre-equilibration described in described step 2 or step 3 or the amount of analyzing thing standard items carry out assay by chromatograph; Kes refers to and analyzes the partition factor of thing between extraction solvent and sample solution, and By consulting literatures or fask oscillating method measure and obtain.
In order to solve the problems of the technologies described above, the embodiment of the present invention also proposes a kind of method of mensuration percentage of protein binding, comprises and calculates binding of drug to plasma proteins rate according to following formula (3):
（3）
Wherein, PB (%) is percentage of protein binding; Ct is the medicine total concentration added in plasma sample; Cf is the concentration of free drug in sample solution, is the content of the free analyte calculated by the method for described mensuration free analyte in biological sample.
Preferably, described educt is selected from least one in lidocaine hydrochloride, bupivacaine HCl or tetracaine hydrochloride; Described biological sample solution is the phosphate buffered solution of phosphate buffered solution or bovine serum albumin(BSA).
Compared with existing analytical approach, the method tool of the mensuration free analyte in biological sample that the present invention relates to has the following advantages and marked improvement:
(1) correct its extraction process with the desorption process of target analytes self, avoid the use of deuterated thing.This method be a kind of need not the pre-equilibration bearing calibration of deuterated analysis thing.
(2) time constant utilizing back extraction to obtain, can Cf in rapid and accurate determination biological sample.
(3) without the need to the pretreating device of complexity, solvent rod hollow fiber membrane device is simple, and cost is low, does not have the phenomenons such as non-specific adsorption.
(4) solvent rod pre-equilibration extraction biological sample, extraction area is large, and extraction time is short, need not carry out the change of sample pH, sample can keep physiological condition always, avoids the change (Drug-protein binding, pH value etc.) of change to properties of samples of physiological condition.Improve the accuracy of analytical approach.
Accompanying drawing explanation
In conjunction with embodiment, the invention will be further described with reference to the accompanying drawings.
Fig. 1 is device and the technique of the stripping process of embodiment of the present invention standard analysis thing, wherein: A uses tack micro syringe the extraction solvent containing standard analysis thing to be added the hollow-fibre membrane closed one end; B is the solvent rod hollow fiber micro-extraction device of liquid phase having added extraction solvent and closed another port; C carries out extraction process the solvent rod sample bottle put into containing blank sample solution of figure B; D uses tack micro syringe to extract extraction solvent after pre-equilibration extraction.
Fig. 2 is device and the technique that embodiment of the present invention pre-equilibration solvent rod liquid-phase micro-extraction analyzes actual biological sample free analyte, wherein: A uses tack micro syringe extraction solvent to be added the hollow-fibre membrane closed one end; B is the solvent rod hollow fiber micro-extraction device of liquid phase having added extraction solvent and closed another port; C carries out extraction process the solvent rod sample bottle put into containing actual biological sample solution of figure B; D uses tack micro syringe to extract extraction solvent after pre-equilibration extraction.
Embodiment
The method of a kind of Fast Measurement free analyte in biological sample of the present invention: be adopt solvent rod hollow-fibre membrane micro-extraction device; First analyzed the stripping process of thing by variable concentrations in extraction solvent, obtained the time constant of solvent rod hollow fiber liquid-phase micro-extraction by concentration curve method; Then the analysis thing in pre-equilibration solvent rod hollow fiber liquid-phase micro extraction technique extraction actual sample is utilized, by instrumental analysis such as chromatograms, obtain the amount of the analysis thing of pre-equilibration extraction, according to amount and the time constant of the analysis thing of pre-equilibration extraction, calculate the content of free analyte in actual sample.
Described solvent rod hollow-fibre membrane micro-extraction device, Kynoar (PVDF) hollow-fibre membrane is cut into suitable segment, and methyl alcohol ultrasonic cleaning, dries naturally.With the tweezers of the heating port by pressure and heat closed hollow tunica fibrosa.
The following formula of formula (1) that described concentration curve method uses:
（1）
Wherein, q0 representative is added to the quality (known quantity) of the analysis thing standard items in extraction solvent in advance, the quality (result that stratographic analysis obtains) of the analysis thing standard items that Q remains when representing pre-equilibration in extraction time t extraction solvent, Ve represents the volume of extraction solvent, Vs represents the volume of blank biological sample, the partition factor of thing between extraction solvent and sample solution is analyzed in Kes representative, and (some medicine can obtain this value by By consulting literatures, literature search less than can measure according to fask oscillating method), t represents extraction time.
According to formula (1), take Q as ordinate, q0 is horizontal ordinate, carries out linear regression, slope of a curve namely:
，
At Kes, when Ve, Vs and extraction time t are known, just can obtain time constant a by slope value.
The following formula of free analyte concentration computing formula of the present invention (2):
（2）
Wherein, Cf represents the concentration of free analyte in biological sample solution, in the quality (numerical value that stratographic analysis obtains) of the analysis thing of extraction time t extraction solvent extraction when n represents pre-equilibration, Ve represents the volume of extraction solvent, Vs represents the volume of blank biological sample, Kes representative analyze the partition factor of thing between extraction solvent and sample solution (some medicine can obtain this value by By consulting literatures, literature search less than can measure according to fask oscillating method), t represents extraction time.
According to formula (2), then for the concentration determination of free analyte in biological sample, the amount of the actual sample analysis thing of the time constant that can obtain according to back extraction concentration curve method and pre-equilibration extraction calculates the concentration obtaining actual free analyte in biological sample, shortens analysis time.
Further, the present invention also and then can calculate binding of drug to plasma proteins rate according to formula (3):
（3）
In formula (3), PB (%) represents percentage of protein binding, the total concentration that Ct represents plasma sample drug in solution (adds the drug concentration in plasma sample, known), the concentration of free drug (analysis thing) in Cf representative sample solution, namely according to the free drug concentration that formula (2) calculates.
The method of Fast Measurement free analyte in biological sample of the present invention, specifically comprises following operation steps:
Step one, the making of solvent rod sample pretreatment device:
Described solvent rod Sample Pretreatment Technique is solvent rod hollow fiber liquid-phase micro extraction technique; Further, described solvent rod sample pre-treatments adopts the solvent rod hollow fiber liquid-phase micro extraction technique of pre-equilibration extraction; As embodiment, this method for making is:
A. hollow-fibre membrane segment is made: Pvdf Microporous Hollow Fiber Membrane is cut the segment of growing into 20-50 mm, with methyl alcohol ultrasonic cleaning 2 min, to remove the impurity on hollow-fibre membrane surface, dried by hollow-fibre membrane in fuming cupboard, for subsequent use;
B. (conventional is n-octyl alcohol to inject extraction solvent, P-xylene, n-hexyl ether etc.) and seal: ready hollow-fibre membrane one end tweezers are added heat-seal 2-3 mm, in hollow-fibre membrane, appropriate extraction solvent (as n-octyl alcohol) is slowly injected subsequently with tack micro syringe, leave standstill a few minutes, make the cinclides of the full hollow-fibre membrane of filled with organic solvent, to cut higher than the hollow-fibre membrane outside liquid level 2-3 mm place (avoiding sorption extraction solvent and target analytes in extraction experiments process), then add heat-seal with tweezers openend and be about 2-3 mm, prepared by solvent rod,
As embodiment, 0.2 μm, the aperture of Pvdf Microporous Hollow Fiber Membrane in described step a, internal diameter 1.2 mm, external diameter 1.4 mm, and be cut into the segment of 30 mm, methyl alcohol ultrasonic cleaning 2 minutes, dries naturally;
In described step b, the preparation of solvent rod needs first hollow-fibre membrane one end tweezers to be added heat-seal 2 mm, in hollow-fibre membrane, 50 μ L extraction solvents (as n-octyl alcohol) are slowly injected subsequently with 50 μ L tack micro syringes, leave standstill 10 min, then cut higher than the hollow-fibre membrane outside liquid level 2 mm, then add heat-seal about 2 mm with tweezers openend.
Step 2, by the stripping process of variable concentrations standard analysis thing, the time constant of solvent rod hollow fiber liquid-phase micro-extraction is obtained: be placed in the sample bottle loading 1-5 mL sample solution by the solvent prepared in step one rod by concentration curve method, solvent rod is immersed in sample substrate completely: such as, described sample bottle can be 2 mL sizes, and sample solution is 1.8 mL, ensure that solvent rod is immersed in sample solution completely like this; Sample bottle is placed on after on vortex oscillation instrument, open immediately, 400-800rpm, extract after 5-20 minute, solvent rod is taken out from sample solution, leave standstill a few minutes, cut off one end of solvent rod, extract extraction solvent with tack micro syringe, injection liquid chromatography is analyzed, such as, the rotating speed of turbula shaker is 600 rpm, extracts 20 minutes.
Please refer to Fig. 1, described step 2 operation steps is as follows:
(A) extraction solvent (conventional be n-octyl alcohol, P-xylene, n-hexyl ether etc.) analyzing thing standard items (amount of the analysis thing standard items of interpolation is the q0 in formula (1)) containing variable concentrations of 50-80 μ L (Ve value) is added in the inner chamber of the solvent rod hollow-fibre membrane closed one end (please refer to Figure 1A), leaves standstill 10 minutes;
(B) other end (please refer to Figure 1B) of solvent rod is closed;
(C) above-mentioned hollow-fibre membrane is put in the sample bottle of 2-4 mL, the blank biological sample (please refer to Fig. 1 C) that Vs is 1.8-3.8 mL is added with in sample bottle, vortex oscillation, extract, in extraction process, analyze thing standard items meeting back extraction in sample solution, extraction time t is 5-20 minute (mainly deciding extraction time according to the related coefficient of concentration curve and the sensitivity of instrumental analysis);
(D) after having extracted, take out hollow fiber membrane solvent rod, Fig. 1 D is please refer to) with the extraction solvent 25-40 μ L(that tack micro syringe is got in fibre membrane lumens, injecting chromatograph carries out assay, obtain the Q value in formula (1), the surplus of target analytes in solvent rod when being t that Q is back-extraction time, according to formula (1):
（1）
Take Q as ordinate, q0 is horizontal ordinate, carries out linear regression, slope of a curve namely:
At Kes, when Ve, Vs and extraction time t are known, just can obtain time constant a by slope value.
Step 3: pre-equilibration solvent rod liquid-phase micro-extraction analyzes actual biological sample free analyte: the method for operating of this step is identical with step 2 method of operating, and difference is to use blank extraction solvent in step step (A) and in step (C) is the biological sample of actual test analyte.
Please refer to Fig. 2 (A)-(D), the concrete operations of step 3 are as follows:
(A) the blank extraction solvent (conventional be n-octyl alcohol, P-xylene, n-hexyl ether etc.) being 50-80 μ L Ve is added in the inner chamber of the solvent rod hollow-fibre membrane closed one end, leaves standstill 10 minutes;
(B) other end of solvent rod is closed;
(C) above-mentioned hollow-fibre membrane is put in the sample bottle of 2-4 mL, the biological sample containing test analyte that Vs is 1.8-3.8 mL is added with in sample bottle, vortex oscillation, extracts, and extraction time t is 5-20 minute (sensitivity according to instrumental analysis decides extraction time);
(D), after having extracted, take out hollow fiber membrane solvent rod, get the extraction solvent 25-40 μ L in fibre membrane lumens with tack micro syringe, injecting chromatograph carries out assay, obtains the n value in formula (2), according to formula (2):
（2）
Namely the concentration of actual free analyte in biological sample can be obtained.
Form is described in further detail foregoing of the present invention again by the following examples, but this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment, all technology realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1:
Kynoar (PVDF) hollow-fibre membrane (0.2 μm, aperture, internal diameter 1.2 mm, external diameter 1.4 mm) is cut into the segment of 30 mm, with methyl alcohol ultrasonic cleaning 2 min, to remove the impurity on hollow-fibre membrane surface, in fuming cupboard, hollow-fibre membrane is dried, for subsequent use.
In phosphate buffered solution, the concentration curve method of time constant measures: ready hollow-fibre membrane one end tweezers are added heat-seal 2 mm, in hollow-fibre membrane, slowly injecting Ve with 50 μ L tack micro syringes is subsequently that 50 μ L contain variable concentrations local anesthetic (0.5, 1, 2, 3, 4, the lidocaine hydrochloride of 5 μ g/mL, bupivacaine HCl and tetracaine hydrochloride) Extraction with n-Octanol solution (as shown in Figure 1A), leave standstill 10 min, extraction solution fills the cinclides of full hollow-fibre membrane, to cut higher than the hollow-fibre membrane outside liquid level 2 mm place (avoiding sorption extraction solvent and target analytes in extraction experiments process), then heat-seal about 2 mm is added with tweezers openend.Solvent rod has been prepared (as shown in Figure 1B).The solvent prepared rod is placed on to load Vs be that in 2 mL sample bottles of 1.8 mL phosphate buffered solution, solvent rod is immersed in (as shown in Figure 1 C) in sample substrate completely.Sample bottle is placed on after on vortex oscillation instrument, opens immediately, oscillation rate is 600 rpm.Extraction t is after 12 minutes, solvent rod is taken out from sample solution, leave standstill 10 min, cut off one end of solvent rod, extract 25 μ L n-octyl alcohols (as shown in figure ip) with 25 μ L tack micro syringes, be injected into liquid chromatography and carry out analyzing the Q value (quality of the analysis thing standard items residual in extraction solvent when extraction time is 12 minutes) of trying to achieve each local anesthetic standard items.
According to above-mentioned formula (1), take Q as ordinate, q0 is horizontal ordinate, carries out linear regression, and the linear regression curves equation of each local anesthetic is respectively as follows:
Lidocaine hydrochloride: y=0.7850x-0.0166 (R2=0.9982);
Bupivacaine HCl: y=0.9184x-0.0328 (R2=0.9976);
Tetracaine hydrochloride: y=0.8435x-0.0996 (R2=0.9956).
Slope of a curve value (slope value of lidocaine hydrochloride, bupivacaine HCl and tetracaine hydrochloride is 0.7850,0.9184 and 0.8435 respectively) is namely:
，
Kes, (lidocaine hydrochloride, bupivacaine HCl and the tetracaine hydrochloride partition factor in n-octyl alcohol and phosphate buffered solution is respectively 44.1,166.8 and 76.0), Ve (50 μ L), Vs (1800 μ L) and extraction time t(12 min) when being all known, the time constant a that just can be obtained time constant a(lidocaine hydrochloride, bupivacaine HCl and tetracaine hydrochloride by slope value is 0.0542,0.0507 and 0.0556 respectively).
The actual sample analysis of local anesthetic phosphate buffered solution: as the device of Fig. 2.Ready hollow-fibre membrane one end tweezers are added heat-seal 2 mm, in hollow-fibre membrane, 50 μ L Extraction with n-Octanols solvent (as shown in Figure 2 A) are slowly injected subsequently with 50 μ L tack micro syringes, leave standstill 10 min, n-octyl alcohol fills the cinclides of full hollow-fibre membrane, to cut higher than the hollow-fibre membrane outside liquid level 2 mm place (avoiding sorption extraction solvent and target analytes in extraction experiments process), then add heat-seal about 2 mm with tweezers openend.Solvent rod has been prepared (as shown in Figure 2 B).Be placed on by the solvent prepared rod and load 1.8 mL and contain in 2 mL sample bottles of the phosphate buffered solution of local anesthetic, solvent rod is immersed in (as shown in Figure 2 C) in sample substrate completely.Sample bottle is placed on after on vortex oscillation instrument, opens immediately, oscillation rate is 600 rpm.Extract after 12 minutes, solvent rod is taken out from sample solution, leave standstill 10 min, cut off one end of solvent rod, extract 25 μ L n-octyl alcohols (as shown in Figure 2 D) with 25 μ L tack micro syringes, be injected into liquid chromatography and carry out analyzing the n value quality of the local anesthetic that Extraction with n-Octanol solvent extraction is arrived (when extraction time is 12 minutes) of trying to achieve each local anesthetic standard items.
According to above-mentioned formula (2), utilize the n value of each local anesthetic standard items recorded, namely each narcotic concentration in actual sample can be obtained, the method is as follows for detecting the narcotic recovery of recovery: the recovery of lidocaine hydrochloride, bupivacaine HCl and tetracaine hydrochloride is respectively 98.2%, 98.3% and 98.5%.
Embodiment 2:
The preparation of solvent rod hollow fiber micro-extraction device of liquid phase and operating process are with embodiment 1.First, respectively by the phosphate buffered solution (wherein in phosphate buffered solution the concentration of bovine serum albumin(BSA) be 1 %(w/v) of the solvent of the Extraction with n-Octanol solution containing 10,20,25,30,40,50 μ g/mL local anesthetics (lidocaine hydrochloride, bupivacaine HCl and tetracaine hydrochloride) rod at the blank bovine serum albumin(BSA) of blank 1.8 mL) middle back extraction 12 min, draw Q-q0 reextraction concentration curve, obtain a linear equation, utilize the slope of linear equation, obtain time constant a according to formula (1); Then be that Ct(Ct is known with blank solvent rod containing local anesthetic total concentration) 1.8 mL 1 %(w/v) bovine serum albumin(BSA) phosphate buffered solution in extract 12 min, obtain the extraction quantity n of local anesthetic; Finally time constant a value and extraction quantity n are substituted into formula (2), try to achieve the Cf Cf of local anesthetic in bovine serum albumin solution.
1 %(w/v) in bovine serum albumin solution, the linear regression curves equation of each local anesthetic back extraction is respectively as follows:
Lidocaine hydrochloride: y=0.8380x-0.0467 (R2=0.9976);
Bupivacaine HCl: y=0.9460x-0.0148 (R2=0.9985);
Tetracaine hydrochloride: y=0.8838x-0.0771 (R2=0.9965).
Slope of a curve value (slope value of lidocaine hydrochloride, bupivacaine HCl and tetracaine hydrochloride is 0.8380,0.9460 and 0.8838 respectively) is namely:
，
Kes, (lidocaine hydrochloride, bupivacaine HCl and the tetracaine hydrochloride partition factor in n-octyl alcohol and phosphate buffered solution is respectively 44.1,166.8 and 76.0), Ve (50 μ L), Vs (1800 μ L) and extraction time t(12 min) when being all known, the time constant a that just can be obtained time constant a(lidocaine hydrochloride, bupivacaine HCl and tetracaine hydrochloride by slope value is 0.0372,0.0302 and 0.0374 respectively).
1 %(w/v) in bovine serum albumin solution, according to above-mentioned formula (2), utilize the n value of each local anesthetic standard items recorded, namely can obtain each narcotic Cf in actual sample.And then calculate binding of drug to plasma proteins rate according to formula 3:
（3）
In formula (3), PB (%) represents percentage of protein binding, Ct represents the total concentration (adding the drug concentration in plasma sample) of plasma sample drug in solution, the concentration of free drug (analysis thing) in Cf representative sample solution, namely according to the free drug concentration that formula (3) calculates.
1 %(w/v according to the method records) in bovine serum albumin solution, the percentage of protein binding of each local anesthetic is as follows: the percentage of protein binding of lidocaine hydrochloride, bupivacaine HCl and tetracaine hydrochloride is respectively 70.0 %, 91.2 % and 74.2 %.
Be appreciated that, the size that the large I of the rod of solvent described in above-mentioned all embodiments hollow fiber micro-extraction device of liquid phase is measured per sample carries out respective handling, Hollow-fibre membranes material is selected according to analysis thing, and those skilled in the art can carry out selecting and making according to prior art.
It should be noted that in addition, concentration and the extraction time of analyzing thing in concentration curve method in extraction solvent will design according to the range of linearity of subsequent instrumentation analysis and sensitivity etc., and analytical instrument used is also not limited to chromatograph.
The method of solvent rod Fast Measurement free analyte in biological sample of the present invention, the concentration curve method of minute constant is wherein used to be adopt the back extraction analyzing thing to carry out, do not need deuterated analysis thing, without the need to the pretreating device of complexity, solvent rod hollow fiber membrane device is simple, cost is low, does not have the phenomenons such as non-specific adsorption.Solvent rod pre-equilibration extraction biological sample, extraction area is large, and extraction time is short, and need not carry out the change of sample pH, sample can keep physiological condition always, avoids the change (Drug-protein binding, pH value etc.) of change to properties of samples of physiological condition.Improve the accuracy of analytical approach.
The above is the specific embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
Claims (8)
1. measure a method for free analyte in biological sample, be utilize pre-equilibration solvent rod hollow fiber liquid-phase micro extraction technique, comprise the following steps:
Step one: make solvent rod hollow-fibre membrane micro-extraction device;
Step 2: the extraction equipment of step one that utilizes analyzing thing standard items with variable concentrations carries out stripping process, obtains the time constant of solvent rod hollow fiber liquid-phase micro-extraction by concentration curve method; Described concentration curve method uses formula (1):
Wherein, q
0be the quality of the analysis thing standard items be added in advance in extraction solvent, Q is the quality of the analysis thing standard items residual in extraction solvent when the pre-equilibration of extraction time t, V
ethe volume of extraction solvent, V
sthe volume of blank biological sample, K
esbe analyze the partition factor of thing between extraction solvent and sample solution, t is extraction time; In this step: will be q containing analysis thing standard items quality
0and volume is V
eextractant inject solvent rod and by solvent rod sealing two ends, then solvent rod to be immersed in completely volume be V
sblank biological sample solution, after oscillation extraction a period of time t, take out solvent rod, cut off one end of solvent rod after leaving standstill, extracting extraction solvent and carry out instrumental analysis and obtain Q value, according to formula (1), take Q as ordinate, q
0for horizontal ordinate, carry out linear regression, slope of a curve namely:
At Kes, Ve, V
swhen being all known with extraction time t, calculate time constant a by slope value;
Step 3: utilize the extraction equipment of step one to extract analysis thing in biological sample to be measured, obtain the amount of the analysis thing of pre-equilibration extraction, according to the time constant obtained in the amount of analysis thing of pre-equilibration extraction and step 2, calculate the content of free analyte in actual sample, the following formula of free analyte concentration computing formula (2):
Wherein, C
fbe the concentration of free analyte in biological sample solution, n is the quality of the analysis thing of the extraction solvent extraction when the pre-equilibration of extraction time t, V
ethe volume of extraction solvent, V
sthe volume of blank biological sample, K
esbe analyze the partition factor of thing between extraction solvent and sample solution, t is extraction time.
2. the method for mensuration free analyte in biological sample according to claim 1, is characterized in that: described step one is that Pvdf Microporous Hollow Fiber Membrane is cut into suitable segment, dries after cleaning, and the Single port of closed hollow tunica fibrosa.
3. the method for mensuration free analyte in biological sample according to claim 1, is characterized in that: described step 2 is further comprising the steps:
(A) 50 ?the extraction solvent analyzing thing standard items containing variable concentrations of 80 μ L be added in the inner chamber of the solvent rod hollow-fibre membrane closed one end, leave standstill;
(B) other end of solvent rod is closed;
(C) described hollow-fibre membrane is put in sample bottle, be added with in sample bottle 1.8 ?the blank biological sample solution of 3.8mL, vortex oscillation, extract, in extraction process, analyze thing standard items meeting back extraction in sample solution, extraction time t decides according to the related coefficient of concentration curve and the sensitivity of instrumental analysis;
(D) after having extracted, take out hollow fiber membrane solvent rod, get the extraction solvent 25-40 μ L in fibre membrane lumens, inject instrument and carry out assay, obtain the Q value in formula (1), the surplus of target analytes in solvent rod when being t that Q is back-extraction time, carries out linear regression according to formula (1) and calculates time constant a.
4. the method for mensuration free analyte in biological sample according to claim 1, is characterized in that: described step 3 is further comprising the steps:
(A) 50 ?the blank extraction solvent of 80 μ L be added in the inner chamber of the solvent rod hollow-fibre membrane closed one end, leave standstill; (B) other end of solvent rod is closed;
(C) above-mentioned hollow-fibre membrane is put in sample bottle, be added with in sample bottle 1.8 ?3.8ml containing the biological sample of test analyte, vortex oscillation, extracts, and extraction time decides according to the sensitivity of instrumental analysis;
(D) after having extracted, take out hollow fiber membrane solvent rod, the extraction solvent 25 ?40 μ L got in fibre membrane lumens carries out assay, obtains the n value in formula (2), according to formula (2), the concentration of actual free analyte in biological sample namely can be obtained.
5. the method for mensuration free analyte in biological sample according to claim 1, is characterized in that: described extraction solvent is selected from least one in n-octyl alcohol, P-xylene, n-hexyl ether.
6. the method for mensuration free analyte in biological sample according to claim 1, is characterized in that: the analysis thing of the extraction of pre-equilibration described in described step 2 or step 3 or the amount analyzing thing standard items carry out assay by chromatograph; K
esrefer to and analyze the partition factor of thing between extraction solvent and sample solution, By consulting literatures or fask oscillating method measure and obtain.
7. measure a method for percentage of protein binding, comprise and calculate binding of drug to plasma proteins rate according to following formula (3):
Wherein, PB% is percentage of protein binding; C
tit is the medicine total concentration added in plasma sample; C
fthe concentration of free drug in sample solution, C
fthe content of the free analyte calculated by the method for the mensuration free analyte in biological sample according to any one of claim 1 to 6.
8. the method for mensuration percentage of protein binding according to claim 7, is characterized in that: described educt is selected from least one in lidocaine hydrochloride, bupivacaine HCl or tetracaine hydrochloride; Described biological sample solution is the phosphate buffered solution of phosphate buffered solution or bovine serum albumin(BSA).
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