CN106918675B - The detection method of Risperidone and paliperidone in a kind of blood plasma - Google Patents
The detection method of Risperidone and paliperidone in a kind of blood plasma Download PDFInfo
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- 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 discloses the detection methods of Risperidone and 9 hydroxyrisperidones in a kind of blood plasma, include the following steps：(1) plasma sample is subjected to pre-treatment, obtains plasma sample sample solution；(2) the plasma sample sample solution obtained to step (1) with high performance liquid chromatography is measured, and analyzes the measurement result of plasma sample sample solution.The present invention is using the Risperidone and 9 hydroxyrisperidones in liquid-liquid extraction method extraction blood plasma, it is detected using C4 chromatographic columns, optimize extraction conditions and the chromatographic condition of sample detection, the extraction yield of Risperidone and 9 hydroxyrisperidones improves, endogenous substance in blood plasma is eliminated simultaneously to interfere, the Risperidone and 9 hydroxyrisperidones in blood plasma can be easily and fast detected, specificity, accuracy, the accuracy of method are good.
Description
Technical field
The present invention relates to the detection method of a kind of Risperidone and paliperidone, in especially a kind of blood plasma Risperidone and
The detection method of paliperidone.
Background technology
Risperidone, chemical name are 3- [2- [4- (fluoro- 1, the 2- benzos isoxazole -3- bases of 6-) -1- piperidyls] ethyl] -6,
7,8,9- tetrahydrochysene -2- methyl -4H- pyridos [1,2- α] pyrimidin-4-ones, are benzo Isoxazole derivatives, are the anti-essences of a new generation
Refreshing disease medicine.It is partly metabolized as paliperidone in vivo, there is same pharmacological activity.Risperidone is a kind of unique
The selective monoaminergic antagonist of matter, it with 5-hydroxytryptamines can 5-HT2 receptors and dopamine D2 receptors have it is very high affine
Power.Risperidone can also be combined with adrenergic receptor, and in lower affinity and H1-histaminergic receptors and α 2- kidneys
Adrenoceptor combines.Risperidone is not combined with cholinergic recepter.Risperidone is strong D2 antagonists, can improve spirit point
The positive symptom of disease is split, but motor function caused by it inhibits and catalepsy will be fewer than classical antipsychotic drug,
The possibility that extrapyramidal side effect occurs can be reduced to the balance of 5-hydroxytryptamines and the dopamine antagonist effect of cental system,
And its therapeutic effect is expanded into schizoid negative symptoms and affective symptom.
Risperidone and paliperidone are extracted from blood plasma at present there are mainly two types of method, solid phase extraction (SPE) and
Liquid-liquid extraction method.SPE approach is higher using import solid phase extraction column cost, is found when liquid-liquid extraction method is extracted using ether
Ether can be such that some components in EP tube materials dissolve out, consistent with Risperidone retention time, interfere the measurement of Risperidone；Use two
When chloromethanes extracts the active constituent in blood plasma, because dichloromethane density is larger, dichloromethane is relatively tired at solution bottom, separation
It is difficult；By in the Risperidone and paliperidone back extraction to acid aqueous phase solution in organic phase, aqueous phase solution uses pH2.2
When phosphate buffer, it is found that the extraction yield of paliperidone is relatively low, it is also necessary to reduce the pH value of solution；Use C18 reverse phases
When chromatographic column carries out plasma sample detection, the endogenous substance in blood plasma has more interference.
Invention content
Based on this, profit training in a kind of blood plasma is provided it is an object of the invention to overcome in place of above-mentioned the deficiencies in the prior art
The detection method of ketone and paliperidone.This method is at low cost, can facilitate Risperidone and 9- in detection human or animal's blood plasma
Hydroxyrisperidone, method is reproducible, and accuracy is high, and operability is strong.
To achieve the above object, the technical solution used in the present invention is：Risperidone and the training of 9- hydroxyl profits in a kind of blood plasma
The detection method of ketone, includes the following steps：
(1) plasma sample is subjected to pre-treatment, obtains plasma sample sample solution；
(2) the plasma sample sample solution obtained to step (1) with high performance liquid chromatography is measured, and analyzes blood plasma sample
The measurement result of product sample solution；
Wherein, high-efficient liquid phase chromatogram condition is：
A) chromatographic column：C4 columns, 4.6mm × 250mm, 5 μm of grain size；
B) mobile phase be potassium dihydrogen phosphate and acetonitrile mixture, wherein the potassium dihydrogen phosphate with it is described
The volume ratio of acetonitrile is 65:35~85:15；Flow velocity is 0.5~1.5mL/min；Detection wavelength is 278nm.
Potassium dihydrogen phosphate and acetonitrile ratio are too high, and Risperidone, paliperidone and internal standard retention time extend, and increase and divide
The time is analysed, potassium dihydrogen phosphate and acetonitrile ratio are very little, and paliperidone appearance is very fast, cannot effectively divide with component in blood plasma
From present inventor attempts by a large amount of, finds potassium dihydrogen phosphate and the acetonitrile mixing of above-mentioned volume ratio, can be effective
Component in separated plasma, and can suitable control analysis time.
It is highly preferred that the volume ratio of the potassium dihydrogen phosphate and the acetonitrile is 77:23.The di(2-ethylhexyl)phosphate of this volume ratio
Hydrogen potassium solution and acetonitrile mixing are used as mobile phase, can guarantee that the separation for making component in blood plasma while controlling analysis time is imitated
Fruit is more preferable.
Preferably, the sample size of high performance liquid chromatography is 100 μ L；Column temperature is 35 DEG C.
The present invention is detected using C4 chromatographic columns, and compared with C18 chromatographic columns, it is dry to reduce endogenous substance in plasma
It disturbs.Compared with using SPE solid-phase extraction columns, cost is greatly saved, while using conventional H PLC detections, avoiding the limit of instrument
System.
Preferably, in the step (1), the pretreatment process of the plasma sample is：
It is 12 or more that the pH value that strong base solution adjusts acidity to solution is added in plasma sample, and ethyl acetate is then added
It extracted, extracted, obtain ethyl acetate layer；Ethyl acetate layer is extracted with hydrochloric acid solution, is extracted, lower water is obtained
Phase dries up lower layer's water phase；It is redissolved with hydrochloric acid solution, obtains plasma sample sample solution.
Due to of high cost using SPE approach processing plasma sample, the present invention is using in liquid-liquid extraction method extraction blood plasma
Risperidone and paliperidone optimize extraction conditions and the chromatographic condition of sample detection, Risperidone and paliperidone
Extraction yield improve, while eliminating in blood plasma endogenous substance and interfering, can easily and fast detect the Risperidone and 9- hydroxyls in blood plasma
Base Risperidone, specificity, accuracy, the accuracy of method are good.
Preferably, in the step (1), before the plasma sample is handled before progress, it is additionally added internal standard compound.
It is highly preferred that the internal standard compound is Clozapine, the w/v of the Clozapine and the plasma sample is 0.1
0.1 μ g Clozapines are added in i.e. every milliliter of plasma sample in μ g/ml.
Preferably, the volume ratio of the ethyl acetate and the plasma sample is 4:1.
Risperidone and paliperidone therein are extracted with ethyl acetate vortex oscillation extraction, compared with using ether,
Avoid the interference that leachable component measures Risperidone in EP, PP pipe (centrifuge tube) material, Risperidone and paliperidone
Extraction yield 80% or more, the ethyl acetate of above-mentioned volume ratio, more to the effect of extracting of Risperidone and paliperidone
It is good.
Preferably, in the described hydrochloric acid solution for being extracted, being extracted to ethyl acetate layer, a concentration of 0.5mol/L of hydrochloric acid
The volume ratio of~5mol/L, the hydrochloric acid solution and the ethyl acetate is 1:100~1:20.
It is highly preferred that in the hydrochloric acid solution for being extracted, being extracted to ethyl acetate layer, a concentration of 2mol/ of hydrochloric acid
The volume ratio of L, the hydrochloric acid solution and the ethyl acetate is 3：80.Inventors be surprised to learn that the salt of above-mentioned concentration and volume
Acid can extract Risperidone in ethyl acetate and paliperidone in HCl solution, effectively remove wherein fat-soluble object
Matter reduces interference, improves extraction effect.
Preferably, in the hydrochloric acid solution redissolved to lower layer's water phase, a concentration of 0.1mol/L of hydrochloric acid.
It is highly preferred that the volume ratio of the hydrochloric acid solution and the plasma sample is 3：20.
Preferably, the strong base solution is sodium hydroxide solution, and sodium hydroxide is a concentration of in the sodium hydroxide solution
2mol/L。
It is highly preferred that the volume ratio of the sodium hydroxide solution and the plasma sample is 1:1.
Preferably, the pH value of the potassium dihydrogen phosphate is 2.0~6.0.
Present inventor attempts to find by a large amount of, the pH of potassium dihydrogen phosphate outside the above range when, Li Pei
Ketone and paliperidone and inner mark solution retention time are obviously delayed, and sample analysis time is elongated, wherein Risperidone and interior
Mark peak broadens, and trails, and causes integral difficult.
It is highly preferred that the pH value of the potassium dihydrogen phosphate is 4.0.Present inventor has been surprisingly found that, works as di(2-ethylhexyl)phosphate
When the pH value of hydrogen potassium solution is 4.0, peak type is preferable, noiseless with component in blood plasma, and each component can efficiently separate, and chromatographic condition is most
It is excellent.
Preferably, a concentration of 0.05mol/L of the potassium dihydrogen phosphate.When the concentration of potassium dihydrogen phosphate is less than
When 0.05mol/L, component separating degree does not reach requirement and (is less than 1.5) in paliperidone and blood plasma, when potassium dihydrogen phosphate is molten
When the concentration of liquid is more than 0.05mol/L, separating degree is not obviously improved compared with the concentration of 0.05mol/L, therefore potassium dihydrogen phosphate
The concentration of solution is selected as 0.05mol/L.
Preferably, in the plasma sample, the concentration of Risperidone and paliperidone is respectively 5~100ng/ml.
Compared with the existing technology, beneficial effects of the present invention are：
The present invention using liquid-liquid extraction method extraction blood plasma in Risperidone and paliperidone, using C4 chromatographic columns into
Row detection, optimizes extraction conditions and the chromatographic condition of sample detection, and the extraction yield of Risperidone and paliperidone improves, together
When eliminate in blood plasma endogenous substance and interfere, can easily and fast detect the Risperidone and paliperidone in blood plasma, method
Specificity, accuracy, accuracy are good.
Description of the drawings
Fig. 1 is the liquid chromatogram of plasma sample of the present invention；
Fig. 2 is the liquid chromatogram of blank plasma of the present invention；
Fig. 3 is the liquid chromatogram extracted using anhydrous ether after plasma sample；
Fig. 4 is the liquid chromatogram tested plasma sample using C18 chromatographic columns；
The peak area ratio and the graph of relation containing concentration that Fig. 5 is paliperidone and Clozapine；
The peak area ratio and the graph of relation containing concentration that Fig. 6 is Risperidone and Clozapine.
Specific implementation mode
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.Wherein, the instrument in embodiment is：1260 high performance liquid chromatograph VWD detectors of Agilent, chromatographic column are
Shiseido proteonavi 4.6mm × 250mm, C4 analysis of protein chromatographic column (aperture)；Reagent in embodiment
For：Potassium dihydrogen phosphate analyzes pure Tianjin great Mao chemical reagent factories；The acetonitrile chromatographically pure U.S. world；Risperidone, Clozapine reference substance
It is purchased from Chinese food and drug assay research institute；Paliperidone reference substance is purchased from USP pharmacopeia.
Embodiment 1
A kind of embodiment of the detection method of Risperidone and paliperidone in blood plasma of the present invention, the present embodiment institute
The method of stating includes the following steps：
(1) plasma sample is subjected to pre-treatment：In plasma sample, according to the volume of sodium hydroxide solution and plasma sample
Than being 1:1, the sodium hydroxide solution that naoh concentration is 2mol/L is added, the pH value for adjusting acidity to solution is 12 or more,
Then it is 4 according to the volume ratio of ethyl acetate and plasma sample:1, be added ethyl acetate, vortex oscillation 5min, 10000rpm from
Heart 10min is extracted, is extracted, obtains ethyl acetate layer；It is 1 according to the volume ratio of hydrochloric acid solution and ethyl acetate：100, it uses
The hydrochloric acid solution of a concentration of 0.5mol/L of hydrochloric acid extracts ethyl acetate layer, is extracted, vortex oscillation 5min, stands, obtains
To lower layer's water phase, lower layer's water phase is dried up；It is 3 according to the volume ratio of hydrochloric acid solution and plasma sample：20, it is a concentration of with hydrochloric acid
The hydrochloric acid solution of 0.1mol/L redissolves, and obtains plasma sample sample solution.
(2) the plasma sample sample solution obtained to step (1) with high performance liquid chromatography is measured, and analyzes blood plasma sample
The measurement result of product sample solution；
Wherein, high-efficient liquid phase chromatogram condition is：
Chromatographic column is Shiseido proteonavi 4.6mm × 250mm, C4 analysis of protein chromatographic column (aperture
5 μm of grain size)；The potassium dihydrogen phosphate (pH value of potassium dihydrogen phosphate is 2.0) and acetonitrile that mobile phase is 0.05mol/L are pressed
It is 65 according to volume ratio:35 mixed solutions mixed, flow velocity 0.5mL/min；Detection wavelength is 278nm；Sample size is 100
μL；Column temperature is 35 DEG C.
Embodiment 2
A kind of embodiment of the detection method of Risperidone and paliperidone in blood plasma of the present invention, the present embodiment institute
The method of stating includes the following steps：
(1) plasma sample is subjected to pre-treatment：In plasma sample, according to 0.1 μ g chlorine is added in every milliliter of plasma sample
The flat ratio of nitrogen is added Clozapine and is uniformly mixed；It is 1 according to the volume ratio of sodium hydroxide solution and plasma sample:1, hydrogen is added
The sodium hydroxide solution of a concentration of 2mol/L of sodium oxide molybdena, the pH value for adjusting acidity to solution is 12 or more, then according to acetic acid second
The volume ratio of ester and plasma sample is 4:1, ethyl acetate is added, vortex oscillation 5min, 10000rpm centrifugation 10min is extracted
It takes, extract, obtain ethyl acetate layer；It is 3 according to the volume ratio of hydrochloric acid solution and ethyl acetate：80, it is a concentration of with hydrochloric acid
The hydrochloric acid solution of 2mol/L extracts ethyl acetate layer, is extracted, vortex oscillation 5min, stand, obtain lower layer's water phase, will under
Layer water phase drying；It is 3 according to the volume ratio of hydrochloric acid solution and plasma sample：20, with the hydrochloric acid of a concentration of 0.1mol/L of hydrochloric acid
Solution redissolves, and obtains plasma sample sample solution.
(2) the plasma sample sample solution obtained to step (1) with high performance liquid chromatography is measured, and analyzes blood plasma sample
The measurement result of product sample solution；
Wherein, high-efficient liquid phase chromatogram condition is：
Chromatographic column is Shiseido proteonavi 4.6mm × 250mm, C4 analysis of protein chromatographic column (aperture
5 μm of grain size)；The potassium dihydrogen phosphate (pH value of potassium dihydrogen phosphate is 4.0) and acetonitrile that mobile phase is 0.05mol/L are pressed
It is 77 according to volume ratio:23 mixed solutions mixed, flow velocity 1.5mL/min；Detection wavelength is 278nm；Sample size is 100
μL；Column temperature is 35 DEG C.
Embodiment 3
A kind of embodiment of the detection method of Risperidone and paliperidone in blood plasma of the present invention, the present embodiment institute
The method of stating includes the following steps：
(1) plasma sample is subjected to pre-treatment：In plasma sample, according to the volume of sodium hydroxide solution and plasma sample
Than being 1:1, the sodium hydroxide solution that naoh concentration is 2mol/L is added, the pH value for adjusting acidity to solution is 12 or more,
Then it is 4 according to the volume ratio of ethyl acetate and plasma sample:1, be added ethyl acetate, vortex oscillation 5min, 10000rpm from
Heart 10min is extracted, is extracted, obtains ethyl acetate layer；It is 1 according to the volume ratio of hydrochloric acid solution and ethyl acetate：20, it uses
The hydrochloric acid solution of a concentration of 5mol/L of hydrochloric acid extracts ethyl acetate layer, is extracted, vortex oscillation 5min, stands, obtains
Lower layer's water phase dries up lower layer's water phase；It is 3 according to the volume ratio of hydrochloric acid solution and plasma sample：20, it is a concentration of with hydrochloric acid
The hydrochloric acid solution of 0.1mol/L redissolves, and obtains plasma sample sample solution.
(2) the plasma sample sample solution obtained to step (1) with high performance liquid chromatography is measured, and analyzes blood plasma sample
The measurement result of product sample solution；
Wherein, high-efficient liquid phase chromatogram condition is：
Chromatographic column is Shiseido proteonavi 4.6mm × 250mm, C4 analysis of protein chromatographic column (aperture
5 μm of grain size)；The potassium dihydrogen phosphate (pH value of potassium dihydrogen phosphate is 6.0) and acetonitrile that mobile phase is 0.05mol/L are pressed
It is 85 according to volume ratio:15 mixed solutions mixed, flow velocity 0.5mL/min；Detection wavelength is 278nm；Sample size is 100
μL；Column temperature is 35 DEG C.
Embodiment 4
The present embodiment to the detection result of the detection method of Risperidone and paliperidone in blood plasma of the present invention into
Verification is gone.
Blank plasma pre-treatment：It takes in blank plasma 1ml to 10ml EP pipes, adds the Clozapine solution of 100 μ L1 μ g/ml,
Mixing, is added the sodium hydroxide solution of 1ml 2mol/L, and 4ml ethyl acetate, vortex oscillation 5min, 10000rpm is added in mixing
10min is centrifuged, (HCl solution for being previously added 150 μ L 2mol/L in EP pipes), whirlpool are taken in upper layer ethyl acetate to 5ml EP pipes
Whirlpool vibrates 5min, stands, discards upper layer ethyl acetate, and lower layer's water phase is dried up with nitrogen evaporator, multiple with the HCl of 150 μ L 0.1mol/L
It is molten, it is detected into liquid phase.
Plasma sample pre-treatment：It is the plasma sample of 50ng/ml, pre-treatment to take Risperidone, paliperidone concentration respectively
The step of operation is with blank plasma pre-treatment.
Chromatographic column is Shiseido proteonavi 4.6mm × 250mm, C4 analysis of protein chromatographic column (aperture)；
The potassium dihydrogen phosphate and acetonitrile that mobile phase is 0.05mol/L are 77 according to volume ratio:23 mixed solutions mixed, stream
Speed is 1mL/min；Detection wavelength is 278nm；Sample size is 100 μ L；Column temperature is 35 DEG C.
Testing result：In liquid chromatogram as depicted in figs. 1 and 2, abscissa is retention time, and ordinate is that signal is strong
Degree, wherein in liquid chromatogram Fig. 1 of plasma sample, the retention time of paliperidone is 6.716min, the reservation of Risperidone
Time is 8.466min, and the retention time of Clozapine is 14.113min, by the control with Fig. 2 blank plasma liquid chromatograms,
As can be seen that plasma sample is noiseless at Risperidone, paliperidone, Clozapine appearance, Risperidone and the training of 9- hydroxyl profits
Ketone separation is good.
Embodiment 5
The present embodiment by the ethyl acetate in embodiment 4 other than being changed to anhydrous ether, and other methods step is the same as implementation
Example 4 carries out the detection of Risperidone and paliperidone, Clozapine in blank plasma, plasma sample.
Testing result：Plasma sample has impurity peaks, disturbed specimen to measure at paliperidone, as shown in Fig. 3；It is logical
It crosses and is compared with the detection result in embodiment 4, further illustrate the present invention the progressive of organic solvent selection ethyl acetate.
Embodiment 6
The present embodiment by C4 by the chromatographic column in embodiment 4 in addition to being changed to Féraud door Gemini, Agilent ZORBAX
Outside SB-C18, Luna chromatographic column, other methods step carried out to Risperidone in blank plasma, plasma sample with embodiment 4 and
The detection of paliperidone.
Testing result：Plasma sample is at paliperidone, other substances are not kept completely separate in blood plasma, with 9- hydroxyl profits
Training ketone has an overlapping, the measurement of disturbed specimen, as shown in Fig. 4.This also further illustrates in assay method of the present invention and selects C4
The progressive of chromatographic column.
Embodiment 7
The concentration for the hydrochloric acid solution that the present embodiment extracts organic solvent layer by Experimental Research, extracts trains profit
The effect of extracting of ketone, paliperidone, Clozapine.
Blank plasma and plasma sample are prepared with embodiment 4, prepare Risperidone respectively, paliperidone concentration is respectively
The plasma sample of 5ng/ml, 20ng/ml, 50ng/ml.Risperidone in ethyl acetate and paliperidone are being transferred to water phase
When, aqueous phase solution uses the phosphate-buffered of the 2mol/L HCl solutions of same amount, 0.1mol/LHCl solution, pH2.2 respectively
Liquid, compares the extraction yield of Risperidone, paliperidone, Clozapine, and experimental result is as shown in table 1：
Effect of extracting of the hydrochloric acid solution of 1 various concentration of table to Risperidone, paliperidone, Clozapine
From table 1 it follows that using the HCl solution of 2mol/L, Risperidone, paliperidone, Clozapine all have
Higher extraction yield.
Embodiment 8
The present embodiment is to the pH of potassium dihydrogen phosphate in mobile phase to the extraction of Risperidone, paliperidone, Clozapine
It takes the influence of effect to carry out correlation to probe into.
Research method：Prepare blank plasma and plasma sample according to 4 method of embodiment, respectively use pH3.0, pH4.0,
The potassium dihydrogen phosphate and acetontrile mobile phase of pH5.0 utilizes method detection Risperidone in the same manner as in Example 4,9-
Hydroxyrisperidone, Clozapine.
Testing result：When preparing mobile phase using pH3.0 potassium dihydrogen phosphates, the retention time of three kinds of substances carries
Before, it causes paliperidone not to be kept completely separate with component in blood plasma, interferes the detection of paliperidone；Use pH5.0 phosphorus
When acid dihydride potassium solution prepares mobile phase, the retention time of three kinds of substances is obviously delayed, and chromatographic peak obviously broadens, and hangover is made
It is difficult at integral；When preparing mobile phase using pH4.0 potassium dihydrogen phosphates, peak type is good, noiseless with component in blood plasma, respectively
Component can efficiently separate, and chromatographic condition is optimal.
Embodiment 9
The present embodiment has carried out detection method of the present invention exclusive according to biological sample analysis guide for method principle
Property, standard curve (linear, range), accuracy, precision, detection limit, quantitative limit etc. verified.
Specificity is analyzed：The blank plasma of 6 Different Individuals is determined respectively, the experimental results showed that 6 Different Individuals
Blank plasma is noiseless to the measurement of active constituent and internal standard (Clozapine).
Standard curve：It is prepared for Risperidone according to detection method of the present invention, the concentration of paliperidone is respectively
The plasma sample of 5ng/ml~100ng/ml has simultaneously carried out liquid chromatographic detection, has studied Risperidone, paliperidone and chlorine
The flat peak area ratio of nitrogen and containing the relationship between concentration, specific data result is as shown in table 2, table 3, Fig. 5, Fig. 6.
The peak area data of 2 paliperidone of table and Clozapine
| Ng/ml containing concentration | 9-hydroxy-risperidone peak area | Internal standard peak area | 9-hydroxy-risperidone/internal standard |
| 4.99 | 59577 | 1391354 | 0.04 |
| 9.99 | 108205 | 1375150 | 0.08 |
| 19.98 | 233245 | 1261585 | 0.18 |
| 29.97 | 367512 | 1374800 | 0.27 |
| 39.96 | 529705 | 1409348 | 0.38 |
| 49.95 | 615856 | 1385397 | 0.44 |
| 74.92 | 977967 | 1465415 | 0.67 |
| 99.90 | 1316276 | 1387427 | 0.95 |
The peak area data of 3 Risperidone of table and Clozapine
| Concentration ng/ml | Risperidone peak area | Internal standard peak area | Risperidone/internal standard |
| 5.02 | 78582 | 1391354 | 0.06 |
| 10.03 | 130657 | 1375150 | 0.10 |
| 20.07 | 256343 | 1261585 | 0.20 |
| 30.10 | 416114 | 1374800 | 0.30 |
| 40.14 | 563317 | 1409348 | 0.40 |
| 50.17 | 661362 | 1385397 | 0.48 |
| 75.26 | 1043563 | 1465415 | 0.71 |
| 100.35 | 1340619 | 1387427 | 0.97 |
From the data in table 2, table 3 can be seen that the concentration in Risperidone, paliperidone be respectively 5ng/ml~
In the plasma sample concentration range of 100ng/ml, the peak area related coefficient of Risperidone, paliperidone and Clozapine is put down
Square R2It is all higher than 0.99；It can be seen that in above-mentioned drug containing concentration range from Fig. 5, Fig. 6, Risperidone, paliperidone and chlorine
The flat peak area ratio of nitrogen is in good linear relationship with concentration is contained.
Accuracy, repeatability are probed into：Risperidone is prepared respectively, the concentration of paliperidone is respectively 10ng/ml, 20ng/
As basic, normal, high drug containing concentrations Plasma sample, parallel 5 samples of each concentration calculate each concentration samples by ml, 50ng/ml
The rate of recovery and RSD, specific data are as shown in table 4.
The rate of recovery and RSD data of 4 sample of table
From the data in table 4 can be seen that the sample recovery rate of high, medium and low three concentration 85%~115% can
Receive in range, RSD is respectively less than 15%, illustrates that the accuracy of detection method of the present invention and repeatability are good.
Detection limit, quantitative limit：The plasma sample of various concentration is prepared, record signal-to-noise ratio 10 is quantitative limit concentration, signal-to-noise ratio 3
Concentration is limited for detection, experiment obtains：The detection of Risperidone and paliperidone is limited to 1ng/ml, is quantitatively limited to 5ng/ml.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than is protected to the present invention
The limitation of range is protected, although being explained in detail to the present invention with reference to preferred embodiment, those skilled in the art should
Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And range.
Claims (7)
1. the detection method of Risperidone and paliperidone in a kind of blood plasma, which is characterized in that include the following steps：
(1) plasma sample is subjected to pre-treatment, obtains plasma sample sample solution；
(2) the plasma sample sample solution obtained to step (1) with high performance liquid chromatography is measured, and is analyzed on plasma sample
The measurement result of sample liquid；
Wherein, high-efficient liquid phase chromatogram condition is：
A) chromatographic column：C4 columns, 4.6mm × 250mm, 5 μm of grain size；
B) mobile phase is the mixture of potassium dihydrogen phosphate and acetonitrile, wherein the potassium dihydrogen phosphate and the acetonitrile
Volume ratio be 65:35~85:15；Flow velocity is 0.5~1.5mL/min；Detection wavelength is 278nm；
In the step (1), the pretreatment process of the plasma sample is：
It is 12 or more that the pH value that strong base solution adjusts acidity to solution is added in plasma sample, and ethyl acetate is then added and carries out
Extraction, extracting, obtain ethyl acetate layer；Ethyl acetate layer is extracted with hydrochloric acid solution, is extracted, lower layer's water phase is obtained, it will
Lower layer's water phase drying；It is redissolved with hydrochloric acid solution, obtains plasma sample sample solution；
In the hydrochloric acid solution for being extracted, being extracted to ethyl acetate layer, a concentration of 0.5mol/L~5mol/L of hydrochloric acid, institute
The volume ratio for stating hydrochloric acid solution and the ethyl acetate is 1:100~1:20；
The pH value of the potassium dihydrogen phosphate is 2.0~6.0.
2. the detection method of Risperidone and paliperidone in blood plasma as described in claim 1, which is characterized in that the step
(1) in, before the plasma sample is handled before progress, it is additionally added internal standard compound.
3. the detection method of Risperidone and paliperidone in blood plasma as claimed in claim 2, which is characterized in that the internal standard
Object is Clozapine, and the w/v of the Clozapine and the plasma sample is 0.1 μ g/ml.
4. the detection method of Risperidone and paliperidone in blood plasma as described in claim 1, which is characterized in that the acetic acid
The volume ratio of ethyl ester and the plasma sample is 4:1.
5. the detection method of Risperidone and paliperidone in blood plasma as described in claim 1, which is characterized in that the highly basic
Solution is sodium hydroxide solution, a concentration of 2mol/L of sodium hydroxide in the sodium hydroxide solution.
6. the detection method of Risperidone and paliperidone in blood plasma as described in claim 1, which is characterized in that the phosphoric acid
A concentration of 0.05mol/L of dihydro potassium solution.
7. the detection method of Risperidone and paliperidone, feature exist in the blood plasma as described in any one of claim 1~6
In in the plasma sample, the concentration of Risperidone and paliperidone is respectively 5~100ng/ml.
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