CN110308232A - A kind of method of uricase in HPLC method analyzing rat blood plasma - Google Patents
A kind of method of uricase in HPLC method analyzing rat blood plasma Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention belongs to bioassay technique field, the method that specifically discloses uricase in a kind of HPLC method analyzing rat blood plasma, comprising the following steps: S1, sample acquisition；S2, sample pretreatment；S3, HPLC measurement: Mobile phase B: the aqueous solution containing 0.1% acetic acid: methanol (9:1, v:v)；Mobile phase C: methanol；Using gradient elution；S4, quantitative analysis: the standard curve of uric acid concentration and peak area and the standard curve of uric acid enzyme concentration and urea consumption are established, the concentration of uricase is gone out according to regression equation calculation.Uricase concentration analysis of the invention has the wider array of range of linearity, and easy to operate, sensitivity and precision height, specificity and favorable reproducibility, the rate of recovery are high, the quantitative analysis quickly and accurately to uricase in plasma sample may be implemented, it all can accurate quantitative analysis within the scope of 0.25~20.0 μ g/mL.
Description
Technical field
The present invention relates to bioassay technique fields, and in particular to the side of uricase in a kind of HPLC method analyzing rat blood plasma
Method.
Background technique
Lack uricase in human and ape class body, is expelled directly out in the form of uric acid external.In water due to uric acid and its esters
Solubility is very low, and under certain pathologic conditions, due to purine metabolic disturbance, uric acid in blood accumulation is excessive, will lead to high lithemia
Mass formed by blood stasis then causes gout syndrome.Uricase (EC1.7.3.3, urate oxidase) is purine degradation metabolism way in organism
Uric acid can be oxidized to the higher allantoin of solubility and hydrogen peroxide by one of diameter enzyme, no longer absorbed and drained by renal tubule,
There is good effect to hyperuricemia caused by nodositas gout, urinary calculus and nephrosis functional failure.But native uricase is due to immunogene
Property is strong, significantly limits its clinical application.PEGylated recombination pig-human urate oxidase fusion protein is high due to stablizing.Immunogene
Property is low, and what is obtained is widely applied.Currently, the pharmacokinetics of PEGylated recombination pig-human urate oxidase is often adopted in assessment blood plasma
Uric acid enzyme concentration is measured with urine ultraviolet spectrometry degree, but this method sensitivity is low, if measuring samples impurity is excessive, uric acid after reaction
Testing result may be interfered easily by impurity；And the range of linearity is relatively narrow, detection limit for height, therefore urine in accurate, quick measurement organism
Sour enzyme concentration becomes a difficult point of medicine generation/Drug Pharmacokinetics research.
China Patent No. CN107515265A discloses a kind of active measuring method of urate oxidase, is detected by HPLC
Uric acid concentration before enzyme reaction and after reaction, calculates reaction speed V according to uric acid concentration [S], establishes Michaelis-Menten equation, find out most
Big reaction speed Vmax and meter Shi constant Km, according to the content of total protein, the molal weight of uricase and uric acid in uric acid enzyme solution
Enzyme solution volume calculates uricase concentration [E], and process is more complex, is not easy to the concentration of fast quantification uricase.Model, which is opened et al., to be opened
Sent out uricase concentration mensuration RP-HPLC method (model is opened, Ma Xuefeng, Xie Min, et al.RP-HPLC method measure blood plasma in poly- second
Method [J] the Chongqing University of Technology journal of glycol uric acid enzyme concentration: natural science, 2010 (5): 29-32.), pass through uricase
In the presence of the reduction of uric acid concentration calculate enzyme concentration；But this method uses isocratic elution, uric acid appearance time is 4.5min,
Sample detection time is long, is not easy to the concentration of fast quantification uricase, and there are stronger matrix effect, will affect measurement result
Reply accuracy.
Summary of the invention
For disadvantages mentioned above present in the prior art, the present invention provides uric acid in a kind of HPLC method analyzing rat blood plasma
The method of enzyme.
A kind of method of uricase in HPLC method analyzing rat blood plasma, comprising the following steps:
S1, sample acquisition: it is centrifuged after acquisition whole blood, obtains plasma sample；
S2, sample pretreatment: the plasma sample of step S1: 1:3 is mixed uric acid solution by volume, water-bath 10min,
The perchloric acid of 1/2 volume is added to terminate reaction, immediately ice bath 5min, then low-temperature and high-speed is centrifuged, and is taken supernatant, is obtained sample to be tested；
S3, HPLC measurement: chromatographic condition are as follows:
Chromatographic column: SymmetryC18；
Flow velocity: 1.20mL/min；
Detection wavelength: 288nm；
Mobile phase B: the aqueous solution containing 0.1% acetic acid: methanol (9:1, v:v)；
Mobile phase C: methanol；
Using gradient elution, the program of gradient elution are as follows:
0-0.80min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
0.80-1.80min:90% (vt) (vt) mobile phase of Mobile phase B+10% C；
1.80-2.80min:90% (vt) (vt) mobile phase of Mobile phase B+10% C；
2.80-3.50min:85% (vt) (vt) mobile phase of Mobile phase B+15% C；
3.50-3.60min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
3.60-5.50min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
S4, quantitative analysis: the standard curve and uric acid enzyme concentration and urea consumption of uric acid concentration and peak area are established
Standard curve, the concentration of uricase is gone out according to regression equation calculation.
Make uric acid that there is extremely short detection duration using above-mentioned gradient elution, uric acid retention time is 2.6min, uric acid
Good separating effect, analyte retention is suitable for and peak shape is preferable, is conducive to improve and analyzes quantitative accuracy and accuracy.
Further, in step S2, supernatant is taken, is added the aqueous solution of 0.1% acetic acid of 2 times of volumes: methanol (9:1, v:
V), sample to be tested is obtained.
Further, in step S2, the uric acid solution concentration is 1000 μM.
Further, in step S2, the low-temperature and high-speed centrifugal condition is 10000r/min, 4 DEG C.
Further, in step S4, the foundation of the standard curve of the uric acid concentration and peak area the following steps are included:
P1, the uric acid stock solution that 2.00mM is prepared using methanol as solvent, with boric acid-borax buffer solution dilution uric acid storage
The uric acid standard solution that standby liquid compound concentration gradient scope is 10.0~1000uM；
P2, uric acid standard solution is taken, the aqueous solution of 0.1% acetic acid of 2 times of volumes: methanol (9:1, v:v) is added, mixing is equal
It is even；
P3, sample introduction measurement, are ordinate by abscissa, peak area of uric acid concentration, are obtained linearly with weighted least-squares method
Regression equation Y=2704.55X-5522.88, r2=0.9916 (X is uric acid concentration, and unit is μM).
Further, in step S4, the uric acid enzyme concentration and the foundation of the standard curve of urea consumption include following
Step:
It is Q1, molten using PBS buffer solution as the uricase standard that solvent compound concentration gradient scope is 0.25~50.0 μ g/mL
Liquid；
Q2, enzyme reaction: the uricase standard of various concentration is added in 1:3 into 1000 μM of uric acid solution respectively by volume
Solution, water-bath 10min add the perchloric acid of 1/2 volume to terminate reaction, immediately ice bath 5min, and then low-temperature and high-speed is centrifuged, and is taken
The aqueous solution of 0.1% acetic acid of 2 times of volumes: methanol (9:1, v:v) is added in supernatant, is uniformly mixed；
Q3, sample introduction measurement, using uric acid consumption as ordinate, uric acid enzyme concentration is abscissa, uses weighted least-squares method
Obtain regression equation y=-2.109x2+81.90x+61.89, r2=0.994 (x is uric acid enzyme concentration, and unit is μ g/mL), wherein
Uric acid consumption (μM)=1000-1.5 × uric acid measures concentration.
Using technical solution provided by the invention, have the following beneficial effects:
1, uricase concentration analysis of the invention has the wider array of range of linearity, and easy to operate, sensitivity and essence
Density height, specificity and favorable reproducibility, the rate of recovery are high, may be implemented quickly and accurately to quantify uricase in plasma sample
Analysis, all can accurate quantitative analysis within the scope of 0.25~20.0 μ g/mL.
2, using specific condition of gradient elution, uric acid retention time is suitable for that good separating effect, baseline is steady, peak shape is good
Well, without hangover situation, accuracy in detection is high, and detection cycle is short, is conducive to quickly detect, and greatly improves detection efficiency.
3, uricase concentration analysis of the invention can be used for being injected intravenously uricase administration toxicity test poison for power
Research is learned, its pharmacokinetics behavior can be completely drawn, is potential risk of the prediction tested material in Human body package and is the later period
Clinical test provides reference.
Detailed description of the invention
Fig. 1 is the uric acid concentration of embodiment 1 and the standard curve of chromatographic peak area；
Fig. 2 is the uric acid enzyme concentration of embodiment 1 and the standard curve of uric acid consumption；
Fig. 3 is the representative chromatogram of uric acid retention time under 2 elution requirement C of embodiment, and uric acid retention time is
2.6min；
Fig. 4 is the representative chromatogram of the blank plasma samples of embodiment 3；
Fig. 5 be embodiment 6 rat intravenous injection uricase after mean drug concentration-time graph semilog in blood plasma
Figure.
Specific embodiment
Below in conjunction with the attached drawing in the present invention, technical solution of the present invention is clearly and completely described, it is clear that
Described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the implementation in the present invention
Example, those of ordinary skill in the art's all other embodiment obtained under the conditions of not making creative work belong to
The scope of protection of the invention.
The method of uricase in 1 HPLC method analyzing rat blood plasma of embodiment
(1) laboratory apparatus and reagent
Laboratory apparatus: chromatographic column: SymmetryC18,4.6 × 250mm, 5.0 μm, WATERS company；
Experiment reagent: methanol, water, acetic acid, uric acid are chromatographic grade.
The uricase that the present invention selects is PEGylated recombination pig-human urate oxidase, hereinafter referred to as uricase.
(2) in HPLC method analyzing rat blood plasma uricase method
A kind of method of uricase in HPLC method analyzing rat blood plasma, comprising the following steps:
S1, sample acquisition: it is centrifuged after acquisition whole blood, obtains plasma sample；
S2, sample pretreatment: the plasma sample of step S1: 1:3 is mixed uric acid solution by volume, water-bath 10min,
The perchloric acid of 1/2 volume is added to terminate reaction, immediately ice bath 5min, then low-temperature and high-speed is centrifuged, and takes supernatant, 2 times of volumes are added
The aqueous solution of 0.1% acetic acid: methanol (9:1, v:v) obtains sample to be tested；In the present embodiment specifically: take 50 μ L step S1's
Plasma sample, being added containing 150 μ L concentration is 1000 μM of uric acid solution, reacts l0min in 37 DEG C of water-baths；Add 100 μ L 1M
Perchloric acid terminates reaction, immediately ice bath 5min；Then with 10000r/min revolving speed, 10min is centrifuged under the conditions of 4 DEG C；Take supernatant
The Mobile phase B solution (aqueous solution containing 0.1% acetic acid: methanol (9:1, v:v)) of 180 μ L is added in 90.0 μ L, sample introduction point after mixing
It analyses (high performance liquid chromatograph), 20 μ L of sampling volume；
S3, HPLC measurement: chromatographic condition are as follows:
Chromatographic column: SymmetryC18；
Column oven temperature: 30 DEG C；
Flow velocity: 1.20mL/min；
Sampling volume: 20.0 μ L；
Detection wavelength: 288nm；
Autosampler cleaning solution: methanol；
Substantially column pressure when chromatographic column equilibrium state: 18.0Mpa (Typical)；
Pressure foot lifting amount: 52mm；
Autosampler cleaning model: before and after sample introduction
Cleaning solution washes needle body product: 200 μ L
Mobile phase B: the aqueous solution containing 0.1% acetic acid: methanol (9:1, v:v)；
Mobile phase C: methanol；
Using gradient elution, the program of gradient elution is shown in Table 1；
The program of 1 gradient elution of table
| Time (min) | Unit | Processing order | It is worth (%) |
| 0.80 | Pump | C Conc. | 5.00 |
| 1.80 | Pump | C Conc. | 10.0 |
| 2.80 | Pump | C Conc. | 10.0 |
| 3.50 | Pump | C Conc. | 15.0 |
| 3.60 | Pump | C Conc. | 5.00 |
| 5.50 | Controller | Stop |
S4, quantitative analysis: the standard curve and uric acid enzyme concentration and urea consumption of uric acid concentration and peak area are established
Standard curve, resulting peak area ratio is detected according to equation of linear regression and sample to be tested and calculates EGCG in plasma sample
Concentration.
The foundation of the standard curve of the uric acid concentration and peak area the following steps are included:
P1, the uric acid stock solution that 2.00mM is prepared using methanol as solvent, with boric acid-borax buffer solution dilution uric acid storage
The uric acid standard solution that standby liquid compound concentration gradient scope is 10.0~1000uM；
The Mobile phase B solution of 180 μ L is added in P2, the 90.0 μ L of uric acid standard solution for taking various concentration to work as, and is uniformly mixed；
P3, sample introduction measurement, using uric acid concentration as abscissa, peak area is ordinate, is obtained linearly with weighted least-squares method
Regression equation Y=2704.55X-5522.88, r2For 0.9916 (X is uric acid concentration, and unit is μM), Fig. 1 is seen.
The foundation of the standard curve of the uric acid enzyme concentration and urea consumption the following steps are included:
It is Q1, molten using PBS buffer solution as the uricase standard that solvent compound concentration gradient scope is 0.25~50.0 μ g/mL
Liquid；
Q2, enzyme reaction: taking 150 μ L to contain 1000 μM of uric acid solution, and the uricase standard that 50.0 μ L various concentrations are added is molten
Liquid, water-bath 10min add the 1M perchloric acid of 100 μ L to terminate reaction, immediately ice bath 5min, and then low-temperature and high-speed is centrifuged, and is taken
The Mobile phase B solution of 180 μ L is added in clear 90.0 μ L, is uniformly mixed；
Q3, sample introduction measurement, specific measurement result is shown in Table 2, and using uric acid consumption as ordinate, uric acid enzyme concentration is horizontal seat
Mark, obtains regression equation y=-2.109x with weighted least-squares method2+ 81.90x+61.89, r2For 0.994 (x is uric acid enzyme concentration,
Unit is μ g/mL), see Fig. 2, wherein uric acid consumption (μM)=1000-1.5 × uric acid measures concentration.
The standard curve base testing result of 2 uric acid enzyme concentration of table and uric acid consumption
| The concentration (μ g/mL) of PEGylated enzyme | Measure uric acid concentration (μM) | Uric acid consumption (μM) |
| 50.0 | 0 | >1000 |
| 20.0 | 97.379 | 854 |
| 10.0 | 213.475 | 680 |
| 5.00 | 384.791 | 423 |
| 2.00 | 541.058 | 188 |
| 1.00 | 589.445 | 116 |
| 0.500 | 594.508 | 108 |
| 0.250 | 587.023 | 119 |
As shown in Figure 1, peak area ratio and uric acid concentration keep good linear relationship, and standard deviation value is small, and controllability is good,
Stability is high, is conducive to improve detection accuracy, and the signal-to-noise ratio that 10 μM of lower limit of quantitation is 22.01, shows the standard for measuring uric acid
Curve is linear good within the scope of 10.0~1000 μM, and detection sensitivity is high.By Fig. 2 and table 2 it is found that uric acid enzyme concentration is 0.25
Within the scope of~20.0 μ g/mL can accurate quantitative analysis, standard deviation value is small, and controllability is good, can be realized the quick, smart of uric acid enzyme concentration
Really detection；Since the uricase of 50.0 μ g/mL is by substrate total overall reaction, it is not involved in calculating.
The optimization of 2 gradient of embodiment
After completing step S2, sample to be tested is divided into 4 groups (A, B, C, D), respectively the sample introduction under different elution requirements
Detection, is evaluated with disengaging time combination peak shape and recovery of standard addition；
Isocratic elution A: mobile phase is the aqueous solution containing 0.1% acetic acid: methanol (9:1, v:v)
Gradient B:
0-0.80min:100% (vt) Mobile phase B；
0.80-1.80min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
1.80-2.80min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
2.80-3.50min:90% (vt) (vt) mobile phase of Mobile phase B+10% C；
3.50-3.60min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
3.60-5.50min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
Gradient C:
0-0.80min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
0.80-1.80min:90% (vt) (vt) mobile phase of Mobile phase B+10% C；
1.80-2.80min:90% (vt) (vt) mobile phase of Mobile phase B+10% C；
2.80-3.50min:85% (vt) (vt) mobile phase of Mobile phase B+15% C；
3.50-3.60min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
3.60-5.50min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
Gradient D:
0-0.80min:90% (vt) (vt) mobile phase of Mobile phase B+10% C；
0.80-1.80min:85% (vt) (vt) mobile phase of Mobile phase B+15% C；
1.80-2.80min:85% (vt) (vt) mobile phase of Mobile phase B+15% C；
2.80-3.50min:80% (vt) (vt) mobile phase of Mobile phase B+20% C；
3.50-3.60min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
3.60-5.50min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
Recovery of standard addition measurement: the blank plasma samples to be free of uricase, preparing spiked levels respectively is 50 μM, 500 μ
The quality-control sample of M is measured after handling according to step S2, and each concentration 3 times parallel, calculates average recovery of standard addition, specifically
It the results are shown in Table 3.
Recovery testu result under the conditions of the different gradients of table 3
At elution requirement A, uric acid appearance time is 5.3min, and uric acid peak corresponding position has impurity peaks, these substances
Retention time it is identical as uric acid, cause the interference to testing uric acid, that is, there is stronger matrix effect, cause elution item
Under part A, recovery of standard addition is extremely high；Therefore, it is optimized using gradient elution program.At gradient condition B~D, urine
Acid is gradually obvious with the differentiation of impurity peaks, and with the increase of organic phase (mobile phase C) content, impurity is eluted in advance, uric acid
Appearance time be gradually shortened, the uric acid of elution requirement B and impurity peaks are distinguished more apparent, but uric acid retention time is still longer,
For 4.2min, reagent waste is caused, is unfavorable for quickly detecting.Under elution requirement D, uric acid appearance time is 1.9min, with solvent
Peak is close, and peak shape partially overlaps, therefore recovery of standard addition is caused to get higher, and is unfavorable for accurately detecting.At elution requirement C, uric acid
Retention time be 2.6min, see Fig. 3, retention time is suitable for being conducive to quickly detect, especially the needs such as hospital high-volume sample
When product examine is surveyed, detection time is short, detection accuracy is high will greatly improve detection efficiency.Can be seen from figure 3, uric acid peak with it is molten
Agent peak and other impurity peaks show bimodal, reach baseline separation, and baseline is steady, peak shape is good, without hangover situation, and separating degree is high,
Therefore, at elution requirement C, eliminate retention time it is short caused by solvent peak and impurity peaks interference, separating effect is better than isocratic
Elution and condition of gradient elution B and D, and recovery of standard addition is high, can be improved the detection limit of instrument.The present invention is removed and is non-specifically said
Bright, elution requirement all uses above-mentioned condition C.
3 system suitability of embodiment
Blank plasma samples are taken, Operations Analyst are carried out by the acquisition of step S1 sample and step S2 sample pretreatment, wherein not
Uric acid solution is added, obtains blank sample representativeness chromatogram and sees Fig. 4, separately take 6 separate sources rat blank plasma samples respectively
(3 are taken a blood sample from female rats, and 3 are taken a blood sample from male rat), 0.02M PBS buffer solution and boric acid-borax buffering are molten
Liquid carries out Operations Analyst according to the acquisition of step S1 sample and step S2 sample pretreatment, to investigate the blank plasma of separate sources
The influence degree that Interference Peaks in sample integrate uric acid, investigation the results are shown in Table 4.
4 system suitability testing result of table
By table 4 and Fig. 4 it is found that the bare substrate of 6 separate sources, 0.02M PBS buffer solution and boric acid-borax buffering
The response of Interference Peaks in solution at uric acid retention time shows rat blank blood lower than 0.3% of uric acid response in sample
The interference that slurry, 0.02M PBS buffer solution and boric acid-borax buffer solution integrate uric acid is negligible, and specificity is preferable.Blank
There is extremely low uric acid peak in plasma sample, this is because itself causing containing uric acid in organism, but content is little, ties to detection
Dried fruit is disturbed negligible.
4 accuracy of embodiment, precision and determination of recovery rates
Substrate uric acid is acquired with bare substrate by step S1 sample respectively and step S2 sample pretreatment carries out operation point
Analysis measures the deviation for being back-calculated concentration and uric acid mark concentration of uric acid, is shown in Table 5.
5 accuracy of table, precision and determination of recovery rates result
As shown in Table 5, the average deviation for being back-calculated concentration and indicating concentration of uric acid is 11.3%, RSD in 6 parallel samples
It is 0.7%, for there are a certain amount of urine in bare substrate the reason of average recovery rate 111.3%, the rate of recovery is more than 100%
Acid increases the concentration of uric acid in system, and blank plasma plus uric acid group are deducted bare substrate background uric acid concentration, are calculated
The bat and the rate of recovery of uric acid are respectively 99.8%, average deviation 1.6%, RSD 0.2%, illustrate the method standard
Really, reliably, stablize, favorable reproducibility.
5 stability test of embodiment
1) (short-term) stability that uricase condition of ice bath is placed in matrix sample
By 1.0 μ g/mL uricase working solutions, place for 24 hours that (estimation of stability quality-control sample) afterwards and newly in condition of ice bath
The uricase working solution (stability control sample) of fresh preparation respectively with substrate uric acid solution, according to step S2 specimen preprocessing
After reason operation, sample introduction is analyzed.The peak area of the peak area of uric acid and uric acid in stability control sample in stability quality-control sample
The deviation theory of ratio is no more than ± 15%, and investigation the results are shown in Table 6.
(short-term) stability test measurement result that 6 uricase ice bath of table is placed
As shown in Table 6, the deviation that uric acid concentration is averagely measured in stability quality-control sample and stability control sample is
0.5%, show that analyte uricase in matrix places at least having good stability for 24 hours in room temperature condition of ice bath.It carries out simultaneously
The assessment of long-time stability experiment, by 1.0 μ g/mL uricase working solutions in -20 DEG C of refrigerator storage 21d (stability Quality Control sample
Product) after, with the uricase working solution (stability control sample) of Fresh respectively with substrate uric acid solution reaction, detection
The deviation for averagely measuring uric acid concentration is 0.6%, and at least stability of 21d is good under the conditions of analyte is stored in -20 DEG C in matrix
It is good.
2) freezing of uricase and thaw cycle (short-term) stability in matrix sample
1.0 μ g/mL uricase working solutions are subjected to freeze thawing, freezing and thaw cycle should freeze at least 12h for the first time
After carry out, recycled each time next, sample all needs to freeze completely, then melt again, in total carry out 5 times freezing and melt
Recycle (estimation of stability sample), with 1.0 μ g/mL uricase working solutions (stability control sample) of Fresh respectively with
Substrate uric acid solution, after step S2 sample pretreatment operation, sample introduction is analyzed, and investigation the results are shown in Table 7.
The freezing and thaw cycle (short-term) stability test measurement result of 7 uricase of table
As shown in Table 7, the deviation that uric acid concentration is averagely measured in estimation of stability sample and stability control sample is
0.4%, show that analyte is in having good stability after at least 5 times freezings and thaw cycle in matrix.
6 uricase pharmacokinetic of embodiment
24 SD rats, are randomly divided into basic, normal, high 3 groups, and every group of male and female each 4, be injected intravenously 0.420 respectively,
0.840, the uricase injection of tri- dosage of 1.68mg/kg, before administration and administration after 10min and 2h, 4h, 8h, 12h,
16h, for 24 hours, 36h, 2d, 3d, 4d, 5d, 6d, 8d, 10d, 14d, 16d acquire plasma sample, detect rat plasma sample in uric acid
Enzyme concentration obtains uricase in the intracorporal Pharmacokinetic Characteristics of rat, corresponding averaged plasma drug concentration time curve half
Logarithmic curve chart is shown in Fig. 5.
6.3 medicine of plasma drug concentration data application WinNonlin of every group of animal is selected into non-compartment model for software simultaneously
It counts moments method and calculates pharmacokinetic parameter, the results are shown in Table 8.
Mean serum pharmacokinetic parameter summary sheet after 8 rat intravenous injection uricase of table
| Group | Low dose group | Middle dose group | High dose group |
| Dosage (mg/kg) | 0.420 | 0.840 | 1.68 |
| t1/2(h) | 117 | 90.1 | 137 |
| Cmax(μg/mL) | 11.5 | 27.9 | 50.3 |
| AUC0-384h(h·μg/mL) | 1007 | 1814 | 4124 |
| AUC0-∞(h·μg/mL) | 1122 | 1922 | 4435 |
| Vd(mL/kg) | 56.7 | 54.2 | 74.9 |
| CL(mL/h/kg) | 0.362 | 0.421 | 0.379 |
As shown in Figure 5, uric acid enzyme concentration gradually decreases as time went on, pharmacokinetics relevant to internal exposed amount
Parameter terminal elimination half-life t1/2Respectively 117,90.1,137h, peak concentration of drug CmaxRespectively 11.5,27.9,50.3 μ g/
ML, area AUC under plasma drug level-time graph0-384hRespectively 1007,1814,4124h μ g/mL, drug absorption
It is good.By Cmax、AUC0-384hLinear fit, the related coefficient (r of linear regression are carried out to dosage2) 0.9 or so, show
C in 0.420~1.68mg/kg dosage rangemax、AUC0-384hIt is related to dosage；This method can be successfully applied to uricase blood medicine
The quantitative analysis of concentration and development pharmacokinetic.
Claims (6)
1. a kind of method of uricase in HPLC method analyzing rat blood plasma, which comprises the following steps:
S1, sample acquisition: it is centrifuged after acquisition whole blood, obtains plasma sample；
S2, sample pretreatment: the plasma sample of step S1: 1:3 is mixed uric acid solution by volume, and water-bath 10min adds 1/
The perchloric acid of 2 volumes terminates reaction, immediately ice bath 5min, and then low-temperature and high-speed is centrifuged, and takes supernatant, obtains sample to be tested；
S3, HPLC measurement: chromatographic condition are as follows:
Chromatographic column: SymmetryC18；
Flow velocity: 1.20mL/min；
Detection wavelength: 288nm；
Mobile phase B: the aqueous solution containing 0.1% acetic acid: methanol (9:1, v:v)；
Mobile phase C: methanol；
Using gradient elution, the program of gradient elution are as follows:
0-0.80min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
0.80-1.80min:90% (vt) (vt) mobile phase of Mobile phase B+10% C；
1.80-2.80min:90% (vt) (vt) mobile phase of Mobile phase B+10% C；
2.80-3.50min:85% (vt) (vt) mobile phase of Mobile phase B+15% C；
3.50-3.60min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
3.60-5.50min:95% (vt) (vt) mobile phase of Mobile phase B+5% C；
S4, quantitative analysis: the standard curve of uric acid concentration and peak area and the mark of uric acid enzyme concentration and urea consumption are established
Directrix curve goes out the concentration of uricase according to regression equation calculation.
2. the method for uricase in HPLC method analyzing rat blood plasma as described in claim 1, which is characterized in that in step S2,
Supernatant is taken, the aqueous solution of 0.1% acetic acid of 2 times of volumes is added: methanol (9:1, v:v) obtains sample to be tested.
3. the method for uricase in HPLC method analyzing rat blood plasma as described in claim 1, which is characterized in that in step S2,
The uric acid solution concentration is 1000 μM.
4. the method for uricase in HPLC method analyzing rat blood plasma as described in claim 1, which is characterized in that in step S2,
The low-temperature and high-speed centrifugal condition is 10000r/min, 4 DEG C.
5. the method for uricase in HPLC method analyzing rat blood plasma as described in claim 1, which is characterized in that in step S4,
The foundation of the standard curve of the uric acid concentration and peak area the following steps are included:
P1, the uric acid stock solution that 2.00mM is prepared using methanol as solvent dilute uric acid stock solution with boric acid-borax buffer solution
Compound concentration gradient scope is the uric acid standard solution of 10.0~1000uM；
P2, uric acid standard solution is taken, the aqueous solution of 0.1% acetic acid of 2 times of volumes: methanol (9:1, v:v) is added, be uniformly mixed；
P3, sample introduction measurement, are ordinate by abscissa, peak area of uric acid concentration, obtain linear regression with weighted least-squares method
Equation Y=2704.55X-5522.88, r2=0.9916 (X is uric acid concentration, and unit is μM).
6. the method for uricase in HPLC method analyzing rat blood plasma as described in claim 1, which is characterized in that in step S4,
The foundation of the standard curve of the uric acid enzyme concentration and urea consumption the following steps are included:
Q1, the uricase standard solution for being 0.25~50.0 μ g/mL as solvent compound concentration gradient scope using PBS buffer solution；
Q2, enzyme reaction: into 1000 μM of uric acid solution, the uricase standard of addition various concentration is molten respectively by 1:3 by volume
Liquid, water-bath 10min add the perchloric acid of 1/2 volume to terminate reaction, immediately ice bath 5min, and then low-temperature and high-speed is centrifuged, and is taken
Clearly, the aqueous solution of 0.1% acetic acid of 2 times of volumes: methanol (9:1, v:v) is added, is uniformly mixed；
Q3, sample introduction measurement, using uric acid consumption as ordinate, uric acid enzyme concentration is abscissa, must be returned with weighted least-squares method
Return equation y=-2.109x2+81.90x+61.89, r2=0.994 (x is uric acid enzyme concentration, and unit is μ g/mL), wherein uric acid
Consumption (μM)=1000-1.5 × uric acid measures concentration.
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