CN102384954B - Method for determining concentration of lovastatin and hydroxyl lovastatin acid in human plasma - Google Patents
Method for determining concentration of lovastatin and hydroxyl lovastatin acid in human plasma Download PDFInfo
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Abstract
The invention discloses a method for determining concentration of lovastatin and hydroxyl lovastatin acid in K2ETDA human plasma (containing formic acid) by LC/MS/MS method. Through the test, the result shows that the concentration determining method disclosed in the invention is suitable for determining lovastatin and hydroxyl lovastatin acid with a concentration in a range of 0.5-48 ng/mL.
Description
Technical field
The present invention relates to a kind of method measuring the concentration of Lovastatin and hydroxyl lovastatin acid in human plasma, particularly a kind of liquid chromatography-mass spectrography (LC/MS/MS method), for measuring containing formic acid ethylenediamine tetraacetic acid anticoagulate plasma (ETDA-K
2human plasma) in the method for concentration of Lovastatin and hydroxyl lovastatin acid.
Background technology
In current mensuration blood plasma, the method for Lovastatin and lovastatin acid mainly contains HPLC/GC/MS, HPLC/MS, HPLC/MS/MS, RP-HPLC and solid phase extraction etc.HPLC/GC/MS and HPLC/MS method measures the blood plasma large usage quantity needed, and minimal detectable concentration high (being generally 0.36-2.5 μ g/L).The preprocess method of HPLC/MS/MS method be centrifugal after get upper liquid direct injected, the impurity in blood plasma easily retains on a column, and cause post voltage rise high, the lost of life, minimal detectable concentration is also higher, is generally 0.97 μ g/L.RP-HPLC method sample preparation is complicated, finding speed is slow, can not meet the needs of actual detection.Solid extraction method step is many, and cost is high, and minimal detectable concentration is generally 0.5 μ g/L, is unfavorable for a large amount of sample tests.This law adopts LC/MS/MS method to measure containing formic acid ethylenediamine tetraacetic acid anticoagulate plasma (ETDA-K
2human plasma) in the method for Lovastatin and hydroxyl Lovastatin acid concentration there is not yet bibliographical information, this law has the advantages such as blood plasma consumption is few, good stability, minimal detectable concentration are little, and be stable for the sample of standing storage, therefore may be used for the detection of a large amount of plasma sample.
Summary of the invention
The object of the invention is to open one and measure ETDA-K
2the method of the concentration of Lovastatin and hydroxyl lovastatin acid in human plasma.
The present invention is achieved by the following scheme:
Method for measurement of concentration of the present invention comprises the steps:
A. solution is equipped with:
(1) preparation of blank medium: the blank K of preparation pH 4 ± 0.1
2eTDA human plasma
Pipette 166.7 μ L 96% formic acid to the blank ETDA-K of 50mL
2(EDTAP dipotassium ethylene diamine tetraacetate) human plasma, and fully mix;
(2) preparation of pin liquid is washed: methyl alcohol: water=50: 50
Pipette 500mL water and 500mL methyl alcohol in container; Abundant mixing, room temperature is deposited;
The preparation of (3) 0.1% formic acid solutions (preparing with methyl alcohol)
Pipette the formic acid of 100 μ L and 100ml methyl alcohol in reagent bottle; Abundant mixing, room temperature is deposited;
(4) mobile phase A: the preparation of 0.5% aqueous formic acid
Pipette 1000mL water and 5mL formic acid in reagent bottle; Abundant mixing, ultrasonic 5min, room temperature is deposited;
(5) Mobile phase B: the preparation of 0.5% formic acid methanol solution
Pipette 1000mL methyl alcohol and 5mL formic acid in reagent bottle; Abundant mixing, ultrasonic 5min, room temperature is deposited;
The preparation of methanol/water (50: the 50) solution of (6) 0.5% formic acid
Pipette 500mL methyl alcohol, 500mL water and 5mL formic acid, to reagent bottle, fully mix, ultrasonic 5min, and room temperature is deposited;
(7) preparation of standard reserving solution
Lovastatin standard reserving solution: accurately take 5mg Lovastatin in 5mL volumetric flask, dissolve with 0.1% formic acid solution and be diluted to scale, fully mixing, obtain the Lovastatin standard reserving solution that concentration is 1mg/ml, in-20 DEG C of storages;
Hydroxyl lovastatin acid standard reserving solution: accurately take 5mg hydroxyl lovastatin acid sodium in 5mL volumetric flask, dissolve with 0.1% formic acid solution and be diluted to scale, abundant mixing, obtains the hydroxyl lovastatin acid standard reserving solution that concentration is 1mg/ml, in-20 DEG C of storages;
Standard reserving solution 1: pipette 0.1mL Lovastatin standard reserving solution and 0.1mL hydroxyl lovastatin acid standard reserving solution in 10mL volumetric flask, scale is diluted to 0.1% formic acid methanol solution, obtain the hybrid standard storing solution of the hydroxyl lovastatin acid of Lovastatin that concentration is 10 μ g/ml and 10 μ g/ml, abundant mixing, obtain standard reserving solution 1, in-20 DEG C of storages;
(8) preparation of typical curve standard working solution
Typical curve standard working solution: prepare with 0.1% formic acid methanol solution dilution standard storing solution 1, as shown in table 1; Fully mix and be incorporated in-20 DEG C of storages;
The preparation of table 1 typical curve standard working solution
(9) preparation of storing solution
Lovastatin storing solution: accurately take 5mg Lovastatin in 5mL volumetric flask, dissolve with 0.1% formic acid solution and be diluted to scale, namely abundant mixing obtains the Lovastatin storing solution that concentration is 1mg/ml; In-20 DEG C of storages;
Hydroxyl lovastatin acid storing solution: accurately take 5mg hydroxyl lovastatin acid sodium in 5mL volumetric flask, dissolve with 0.1% formic acid solution and be diluted to scale, namely abundant mixing obtains the hydroxyl lovastatin acid storing solution that concentration is 1mg/ml, in-20 DEG C of storages;
Storing solution 1: pipette 0.1mL Lovastatin storing solution and 0.1mL hydroxyl lovastatin acid storing solution in 10mL volumetric flask, scale is diluted to 0.1% formic acid methanol solution, obtain the mixing storing solution of the hydroxyl lovastatin acid of Lovastatin that concentration is 10 μ g/ml and 10 μ g/ml, abundant mixing, obtain storing solution 1, in-20 DEG C of storages;
(10) preparation of sample working solution: prepare, shown in table 2 with K2ETDA human plasma dilution storing solution 1; In-20 DEG C of storages;
The preparation of table 2 sample working solution
Note: QCH: enriched sample
QCM: middle concentration samples
QCL: low concentration sample
(11) preparation of inner mark solution
Hydroxyl simvastatin acid sodium storing solution: accurately take 5mg hydroxyl simvastatin acid sodium to 25mL volumetric flask, dissolve with 0.1% formic acid solution and be diluted to scale, namely abundant mixing obtains the hydroxyl simvastatin acid sodium storing solution that final concentration is 200 μ g/ml, in-20 DEG C of storages;
Simvastatin storing solution: accurately take 5mg Simvastatin to 25mL volumetric flask, dissolve with 0.1% formic acid solution and be diluted to scale, namely abundant mixing obtains the Simvastatin storing solution that concentration is 200 μ g/ml, in-20 DEG C of storages;
Interior mark application of sample solution: pipette hydroxyl simvastatin acid sodium storing solution that 20 μ L concentration are 200 μ g/ml and 20 μ L concentration are that the Simvastatin storing solution of 200 μ g/ml is in 20mL volumetric flask, scale is diluted to 0.1% formic acid solution, abundant mixing, obtain interior mark application of sample solution, in-20 DEG C of storages;
B. extract
(1) as standard working solution prepared by table 3:
The preparation of table 3 standard working solution
(2) by standard working solution, storing solution 1, testing sample, and blank ETDA-K
2human plasma respectively in ice-water bath eddy oscillating thaw;
(3) draw 500 μ L blood plasma (containing two blank, blank and sample) and respectively put a glass test tube, add 0.1% formic acid solution of 50 μ L;
(4) two blank adds 0.1% formic acid solution of 10 μ L, adds respectively in 10 μ L and mark application of sample solution in other sample, eddy current 15 seconds;
(5) add 2mL Mobile phase B, rear abundant eddy current mixing added a cover by test tube;
(6) with 3500rpm centrifugation 10 minutes, another glass test tube transferred to by supernatant, often props up and in vitro adds 2mL mobile phase A, fully mix;
(7) SPE plate is first rinsed 2 times by 1000 μ L Mobile phase B, then rinse 2 times by 1000 μ L mobile phase A, low vacuum is drained;
(8) solution of step (6) is moved to SPE plate, allow sample liquid slowly by packed bed, low vacuum is transferred to maximum vacuum again to ensure that all supernatants are drained completely after each cell becomes dry;
(9) rinse each cell twice by 1000 μ L mobile phase A respectively, drain 1 minute, then use the formic acid methyl alcohol of 1000 μ L 0.5%: water=50: the solution of 50 rinses each cell 3 times, and low vacuum drains 15 seconds;
(10) one piece of 2.2mL deep hole 96 hole dash receiver is placed in below SPE plate, each aperture uses 1000 μ L Mobile phase B wash-outs respectively, and eluent nitrogen dries up under 35 DEG C of conditions;
(11) dried residue 200 μ L Mobile phase B is dissolved, eddy current 30 seconds; To be greater than the centrifugation 10 minutes of 3200rpm, supernatant is transferred to HPLC sample injection bottle;
Wherein, above-mentioned steps (1), to (5), except eddy current mixing, all operates in ice-water bath;
C. data analysis
Retention time and peak area are determined by data acquisition and analysis software (Version 1.4.1); With peak area ratio, standard working curve is done to concentration, obtains following equation of linear regression, in order to calculate the concentration of analyte in solution to be measured:
y＝ax+b
Wherein: y=analyte and interior target peak area ratio
The slope of a=respective standard working curve
The intercept of b=respective standard working curve
The concentration (ng/mL) of x=analyte
(employing weighted least-squares method)
Accompanying drawing explanation
Fig. 1: the representative collection of illustrative plates of the two blank medium of Lovastatin;
Fig. 2: the representative collection of illustrative plates of the two blank medium of lovastatin acid;
Fig. 3: the blank medium of Lovastatin represents collection of illustrative plates;
Fig. 4: the blank medium of lovastatin acid represents collection of illustrative plates;
Fig. 5: the representative collection of illustrative plates of Lovastatin lower bound calibration standard solution;
Fig. 6: the representative collection of illustrative plates of lovastatin acid lower bound calibration standard solution;
Fig. 7: Lovastatin represents linear graph;
Fig. 8: lovastatin acid represents linear graph;
Fig. 9: Lovastatin mean blood plasma concentration-time curve;
Figure 10: lovastatin acid mean blood plasma concentration-time curve.
The invention discloses a kind of LC/MS/MS of employing method and measure K
2the method of the concentration of Lovastatin and hydroxyl lovastatin acid in ETDA human plasma (containing formic acid).Experiment employing 500 μ L sample, the quantitative limit of Lovastatin and hydroxyl lovastatin acid is 0.5ng/mL.Prove that the analyst coverage of this method to Lovastatin and hydroxyl lovastatin acid is all 0.5-48ng/mL through experiment.The sample that analytical concentration exceeds upper limit of quantification can use K
2eTDA human plasma is diluted in appropriate concentration range (dilution gfactor=20).It 6 hours is all stable that Lovastatin and hydroxyl lovastatin acid are placed in human plasma under 3 Frozen-thawed cycled and ice-water condition.It is stable that middle sample is placed after 48 hours at 4 DEG C.It is also stable that Lovastatin and hydroxyl lovastatin acid are stored in human plasma 42 days respectively under-20 DEG C of conditions.It 18,30,16,16 and 30 days is also stable for Lovastatin storing solution (1mg/mL), hydroxyl lovastatin acid storing solution (1mg/mL), Lovastatin storing solution (10 μ g/mL), hydroxyl lovastatin acid storing solution (10 μ g/mL) and hydroxyl simvastatin acid storing solution being placed respectively under-20 DEG C of conditions.It is also stable that hydroxyl lovastatin acid (25ng/mL), Simvastatin and hydroxyl simvastatin acid standard loaded sample are placed 23 days, 27 days and 2 months respectively under-20 DEG C of conditions.Result shows that method for measurement of concentration of the present invention is applicable to the Lovastatin of concentration within the scope of 0.5-48ng/mL and the mensuration of hydroxyl lovastatin acid.
Following experimental example and embodiment are used for further illustrating but are not limited to the present invention.
Following experimental example 1-8 is for measuring K according to method described in the embodiment of the present invention 1
2the concentration of Lovastatin and hydroxyl lovastatin acid in ETDA human plasma, uses Simvastatin and simvastatin acid to be the concentration that interior mapping determines Lovastatin and hydroxyl lovastatin acid respectively.In following experiments, confirmatory assessment is carried out to the method, evaluated the concentration whether it is applicable to measure Lovastatin and hydroxyl lovastatin acid in K2ETDA human plasma (containing formic acid).Result of study shows that the method meets requirements completely, can be used for biological study.All data are all satisfied the criteria.
Experimental example 1: the selectivity experiment of method for measurement of concentration of the present invention
The contrast blood plasma (n=6) from 6 parts of different batches is collected with the inventive method analysis, 1 part of contrast blood plasma adds interior mark (0ng/mL sample, n=1), 1 part adds containing interior target lower limit of quantitation (Low Limit ofQuantitation, LLOQ) sample (n=1).Analysis result is in table 4 and table 5.
The research of table 4 Lovastatin selectivity
* note: use normal data
The selectivity research of table 5 lovastatin acid
* note: use normal data
Result shows at Lovastatin and hydroxyl lovastatin acid and does not have obvious baseline interference near target retention time (Rt) in them: at the retention time place of analysis purpose thing without remarkable Interference Peaks (being less than 20% of LLOQ).And at the retention time place of internal standard compound matter without remarkable Interference Peaks (being less than 5% of interior mark signal).Accompanying drawing 1,2,3,4,5 and 6 is shown in respectively by the representative collection of illustrative plates of two blank medium, blank medium and calibration standard lower limit (0.5ng/mL Lovastatin and hydroxyl lovastatin acid).The retention time of Lovastatin and hydroxyl lovastatin acid is about 1.36min and 1.33min respectively.
The sensitivity experiment of experimental example 2 method for measurement of concentration of the present invention
The lower limit of quantitation (LLOQ) of Lovastatin and hydroxyl lovastatin acid is set as 0.5ng/mL.In order to confirm sensitivity, have detected the sample that 6 parts add the analysis purpose thing of LLOQ concentration level, calculating measured concentration according to corresponding calibration curve.Lovastatin the results are shown in Table 6, hydroxyl lovastatin acid the results are shown in Table 7:
Table 6 Lovastatin lower limit of quantitation sample result
Table 7 hydroxyl lovastatin acid lower limit of quantitation sample result
Note: * exceptional value, deletes in statistics
Result proves, this method all reaches the requirement of bioanalysis research at the preci-sion and accuracy of LLOQ: accuracy (%Nom) is within the scope of (100 ± 20) %, and the coefficient of variation (%CV) is not more than 20%.Data show, this method sensitivity is enough to measure Lovastatin and the hydroxyl lovastatin acid that concentration is 0.5ng/mL.
Experimental example 3 method for measurement of concentration calibration curve of the present invention normal concentration is tested
The measured concentration of the standard solution of every bar calibration curve of Lovastatin and hydroxyl lovastatin acid is respectively in table 8 and table 9.
The measured concentration of the calibration standard solution of table 8 Lovastatin
The measured concentration of the calibration standard solution of table 9 hydroxyl lovastatin acid
Result shows, this method meet bioanalytical method checking research requirement: measured concentration do not exceed theoretical concentration ± 15%, and the measured concentration of LLOQ concentration point do not exceed theoretical concentration ± 20%; At least 75% non-zero concentration point (comprising LLOQ and ULOQ) must reach above-mentioned standard.
Experimental example 4 method for measurement of concentration Linear Experiment of the present invention
The linear of this method is evaluated in Lovastatin and hydroxyl lovastatin acid 0.5-48ng/mL concentration range.Linear regression (weighted least-squares method) is most suitable for reflecting K
2lovastatin and the concentration of hydroxyl lovastatin acid and the relation of response in ETDA human plasma (containing formic acid).The calibration curve parameter of Lovastatin and hydroxyl lovastatin acid is respectively in table 10 and table 11, and the representative linear graph of Lovastatin and hydroxyl lovastatin acid is shown in accompanying drawing 7 and accompanying drawing 8 respectively:
The calibration curve parameter of table 10 hydroxyl lovastatin acid
The calibration curve parameter of table 11 hydroxyl lovastatin acid
Result: all linear graph of Lovastatin all has good correlation (R), is greater than 0.997.The correlativity (R) of the every bar linear graph of hydroxyl lovastatin acid is all greater than 0.993.
Experimental example 5 method for measurement of concentration of the present invention is in a few days tested with day to day precision
Select 3 variable concentrations levels (Lovastatin of 1.5ng/ml, 15ng/ml, 36ng/ml and hydroxyl lovastatin acid) respectively, investigate the inventive method in a few days and day to day precision.The statistics of Lovastatin and hydroxyl lovastatin acid sample is respectively in table 12 and table 13.
Table 12 Lovastatin in a few days and accuracy in the daytime and precision
Table 13 hydroxyl lovastatin acid in a few days and accuracy in the daytime and precision
Result shows the requirement in a few days meeting bioanalysis research with day to day precision of method for measurement of concentration of the present invention: accuracy (%Nom) is within the scope of (100 ± 15) %, and the coefficient of variation (%CV) is not more than 15%.
The experiment of the experimental example 6 method for measurement of concentration recovery of the present invention
The recovery of Lovastatin and hydroxyl lovastatin acid is measured with two (QC) concentration levels (1.5ng/ml, 36ng/ml) respectively.Each condition prepares 3 parts of Duplicate Samples respectively, compares with the mean value of result.The recovery of Lovastatin variable concentrations level the results are shown in Table 14, is respectively 74.3% and 76.4%.The recovery of hydroxyl lovastatin acid variable concentrations level the results are shown in Table 15, is respectively 72.5% and 65.8%.
The table 14 Lovastatin recovery
Table 15 hydroxyl Lovastatin acid recovering rate
Respectively containing adding Simvastatin respectively in the Lovastatin of 1.5ng/ml and 36ng/ml and the sample of hydroxyl lovastatin acid and hydroxyl simvastatin acid reaches 200ng/mL to concentration, to mark the recovery of Simvastatin and hydroxyl simvastatin acid in this measured in solution.Each condition prepares 3 parts of parallel sample respectively, compares with the mean value of result.The recovery of Simvastatin variable concentrations level the results are shown in Table 16 and table 17, is respectively 85.5% and 81.6%.The recovery of hydroxyl lovastatin acid variable concentrations level the results are shown in Table 18 and table 19, is respectively 85.0% and 96.2%.
Table 16 adds the recovery of the Simvastatin (interior mark) of Lovastatin (1.5ng/ml)
Table 17 adds the recovery of the Simvastatin (interior mark) of Lovastatin (36ng/ml)
Table 18 adds the recovery of the hydroxyl simvastatin acid (interior mark) of hydroxyl lovastatin acid (1.5ng/ml)
Table 19 adds the recovery of the hydroxyl simvastatin acid (interior mark) of hydroxyl lovastatin acid (36ng/ml)
The experiment of experimental example 7 method for measurement of concentration dilution of the present invention integrality
When analyzing sample, just must dilute sample when analytical concentration exceeds upper limit of quantification.In order to ensure the analytic signal of the dilution of enriched sample in analyst coverage and acceptable deviation limits, in pooled plasma, add the Lovastatin and hydroxyl lovastatin acid (Lovastatin of 500mg/mL and hydroxyl lovastatin acid) that exceed the inventive method analyst coverage concentration respectively.By sample pooled plasma being diluted 20 times of 6 parts of Duplicate Samples obtained respectively containing 25ng/mL Lovastatin and hydroxyl lovastatin acid.Sample after dilution is analyzed with Lovastatin and hydroxyl lovastatin acid standards calibration curve respectively.Measured concentration is multiplied by corresponding dilution gfactor (20) and obtains corrected concentrations.Result shows that dilution Integrity Assessment meets the requirement of bioanalysis research: the accuracy (%Nom) of corrected concentrations is within the scope of (100 ± 15) %, and the coefficient of variation (%CV) is less than 15%.The dilution integrity test result of Lovastatin and hydroxyl lovastatin acid is acceptable, respectively in table 20 and table 21.
The dilution integrality result of study of table 20 Lovastatin
The dilution integrality result of study of table 21 hydroxyl lovastatin acid
The stability experiment of experimental example 8 method for measurement of concentration of the present invention
1, short-term stability (ice-water)
The Lovastatin of two concentration (1.5ng/mL and 36ng/mL) and hydroxyl lovastatin acid sample are placed under frozen water condition 6 hours to investigate their short-term stabilities in K2ETDA human plasma (containing formic acid), adopt the new calibration curve formulated to carry out cubage.Lovastatin the results are shown in Table 22, hydroxyl lovastatin acid the results are shown in Table 23:
K after 6 hours is placed under table 22 frozen water condition
2the short-term stability result of Lovastatin in ETDA human plasma (containing formic acid)
K after 6 hours is placed under table 23 frozen water condition
2the short-term stability result of hydroxyl lovastatin acid in ETDA human plasma (containing formic acid)
It is stable that Lovastatin in result reference blood plasma and hydroxyl lovastatin acid place 6 hours under ice-water condition.
2, long-time stability
The sample of two concentration (Lovastatin of 1.5ng/ml, 36ng/ml and hydroxyl lovastatin acid, n=6) is placed under-20 DEG C of general chilling temperatures 14 days and investigate K in 42 days
2the long-time stability under-20 DEG C of conditions of storage of Lovastatin and hydroxyl lovastatin acid in ETDA human plasma (containing formic acid), carry out cubage with freshly prepd calibration curve, Lovastatin the results are shown in Table 24 and table 25, hydroxyl lovastatin acid the results are shown in Table 26 and table 27:
The K of 14 days placed by table 24 under-20 DEG C of freezing conditions
2the long-time stability result of Lovastatin in EDTA human plasma (containing formic acid)
The K of 42 days placed by table 25 under-20 DEG C of freezing conditions
2the long-time stability result of Lovastatin in EDTA human plasma (containing formic acid)
The K of 14 days placed by table 26 under-20 DEG C of freezing conditions
2the long-time stability result of hydroxyl lovastatin acid in EDTA human plasma (containing formic acid)
The K of 42 days placed by table 27 under-20 DEG C of freezing conditions
2the long-time stability result of hydroxyl lovastatin acid in EDTA human plasma (containing formic acid)
Lovastatin in result reference blood plasma and hydroxyl lovastatin acid were stable in 42 days.
3, freeze-thaw stability
(stability after Frozen-thawed cycled of Lovastatin and hydroxyl lovastatin acid in K2ETDA human plasma (containing formic acid) investigated by the Lovastatin of 1.5ng/ml, 36ng/ml and the sample of hydroxyl lovastatin acid, and each concentration establishes 6 parallel sample to use two concentration.It is stable that Lovastatin in result reference blood plasma and hydroxyl lovastatin acid operate through 3 freeze thawing, in table 28 and table 29:
The freeze-thaw stability result of table 28 Lovastatin
The freeze-thaw stability result of table 29 hydroxyl lovastatin acid
4, middle sample (Processed Sample) stability
Middle sample after rough handling is placed at 4 DEG C after 48 hours with freshly prepd calibration curve analysis and investigation K again
2the stability of the middle sample of Lovastatin and hydroxyl lovastatin acid in ETDA human plasma (containing formic acid).The results are shown in Table 30 and table 31:
Table 30 middle sample places the Lovastatin stability result of 48 hours under 4 DEG C of conditions
Table 31 middle sample places the hydroxyl lovastatin acid stability result of 48 hours under 4 DEG C of conditions
Result proves that Lovastatin and hydroxyl lovastatin acid middle sample are stable.
5, storing solution stability
Lovastatin storing solution (1mg/ml), hydroxyl lovastatin acid storing solution (1mg/ml), Lovastatin storing solution (10 μ g/ml), hydroxyl lovastatin acid storing solution (10 μ g/ml) and hydroxyl simvastatin acid storing solution are placed under-20 DEG C of conditions the stability under investigating storing solution freezing conditions in 18,30,16,16 and 30 days respectively, the results are shown in Table 32, table 33, table 34, table 35, table 36:
The stability result of 18 days is placed under table 32 Lovastatin storing solution (1mg/ml)-20 DEG C of conditions
The stability result of 30 days is placed under table 33 hydroxyl lovastatin acid storing solution (1mg/ml)-20 DEG C of conditions
The stability result of 16 days is placed under table 34 Lovastatin storing solution (10 μ g/ml)-20 DEG C of conditions
The stability result of 16 days is placed under table 35 hydroxyl lovastatin acid storing solution (10 μ g/ml)-20 DEG C of conditions
The stability result of 30 days is placed under table 36 hydroxyl simvastatin acid storing solution-20 DEG C of conditions
It is stable that result proves that Lovastatin storing solution (1mg/ml), hydroxyl lovastatin acid storing solution (1mg/ml), Lovastatin storing solution (10 μ g/ml), hydroxyl lovastatin acid storing solution (10 μ g/ml) and hydroxyl simvastatin acid storing solution place 18,30,16,16 and 30 days respectively under-20 DEG C of conditions.
6, standard loaded sample (Standard Spike) stability
Hydroxyl lovastatin acid (25ng/mL), Simvastatin and hydroxyl simvastatin acid standard loaded sample are placed 23 days, 27 days and 2 months respectively under-20 DEG C of conditions.Compare with hydroxyl simvastatin acid with the hydroxyl lovastatin acid newly prepared, Simvastatin respectively.Table 37 is stability result that Lovastatin standard loaded sample is placed respectively 23 days under-20 DEG C of conditions, table 38 is stability result that hydroxyl lovastatin acid standard loaded sample is placed respectively 27 days under-20 DEG C of conditions, and table 39 is stability result that hydroxyl simvastatin acid standard loaded sample is placed respectively 2 months under-20 DEG C of conditions.
The stability result of 23 days is placed under table 37 hydroxyl lovastatin acid standard loaded sample-20 DEG C of conditions
The stability result of 27 days is placed under table 38 Simvastatin standard loaded sample-20 DEG C of conditions
The stability result of 2 months is placed under table 39 hydroxyl simvastatin acid standard loaded sample-20 DEG C of conditions
Following embodiment all can realize effect described in above-mentioned experimental example.
Embodiment
Embodiment 1: method for measurement of concentration of the present invention
One, facilities and equipments:
LC/MS/MS, Shimadzu AP15000 type phase autoclave pump and automatic sampler;
Data acquisition and analysis software (Version 1.4.1);
Hydro-extractor, 3500 turns;
Chromatographic column: Atlantis
tMdC18,3 μm, 3.0 × 20mm;
Analytical balance, minimum weighing is to 0.00001g;
Pure water system: ELGA Classic UV (> 18.0M Ω);
Solid-Phase Extraction (SPE) 96 orifice plate (cleanert C896-well, Agela);
2.2ml 96 orifice plate.
Two, reagent and material:
Hydroxyl lovastatin acid sodium standard items
Hydroxyl simvastatin acid sodium (interior mark) standard items
Lovastatin standard items
Simvastatin (interior mark) standard items
Methyl alcohol chromatographically pure
96% formic acid chromatographically pure
Water >=18.0M Ω
Blank human plasma anticoagulation K2ETDA
Three, instrument condition:
(1) high-efficient liquid phase chromatogram condition:
Mobile phase A: 0.5% aqueous formic acid
Mobile phase B: 0.5% formic acid methanol solution
Flow velocity: 1.0mL/min
Sampling volume: 10 μ L
Wash pin liquid: methanol/water (50: 50)
Auto injection actuator temperature: 4 DEG C
Column oven: room temperature
Data acquisition time: 7.0min
Chromatographic column: Atlantis
tMdC18,3 μm, 3.0 × 20mm
Gradient program (Lovastatin and Simvastatin):
The gradient elution time is respectively 0,1,3,3.1,5,5.1,7min, the number percent of mobile phase A used, B is respectively 40% and 60%, 5% and 95%, 5% and 95%, 0 and 100%, 0 and 100%, 40% and 60%, 40% and 60%.
Gradient program (lovastatin acid and simvastatin acid):
The gradient elution time is respectively 0,1,5,5.1,7min, the number percent of mobile phase A used, B is respectively 40% and 60%, 5% and 95%, 5% and 95%, 40% and 60%, 40% and 60%.
(2) MS/MS condition
Lovastatin:
Polarity is positive ion, and the m/z of parent ion is 405.4, and the m/z of daughter ion is 199.1, and sweep time is 150msec, and time out is 5msec, and retention time is ~ 1.36min.
Simvastatin (interior mark):
Polarity is positive ion, and the m/z of parent ion is 419.5, and the m/z of daughter ion is 199.3, and sweep time is 100msec, and time out is 5msec, and retention time is ~ 1.42min.
Lovastatin acid:
Polarity is negative ion, and the m/z of parent ion is 421.6, and the m/z of daughter ion is 319.3, and sweep time is 100msec, and time out is 5msec, and retention time is ~ 1.33min.
Simvastatin acid:
Polarity is negative ion, and the m/z of parent ion is 435.4, and the m/z of daughter ion is 319.4, and sweep time is 100msec, and time out is 5msec, and retention time is ~ 1.38min.
Four, implementation step:
A. solution is equipped with:
(1) preparation of blank medium: the blank K of preparation pH 4 ± 0.1
2eTDA human plasma
Pipette 166.7 μ L 96% formic acid to the blank ETDA-K of 50mL
2human plasma, and fully mix;
(2) preparation of pin liquid is washed: methyl alcohol: water=50: 50
Pipette 500mL water and 500mL methyl alcohol in container; Abundant mixing, room temperature is deposited;
The preparation of (3) 0.1% formic acid solutions (preparing with methyl alcohol)
Pipette the formic acid of 100 μ L and 100ml methyl alcohol in reagent bottle; Abundant mixing, room temperature is deposited;
(4) mobile phase A: the preparation of 0.5% aqueous formic acid
Pipette 1000mL water and 5mL formic acid in reagent bottle; Abundant mixing, ultrasonic 5min, room temperature is deposited;
(5) Mobile phase B: the preparation of 0.5% formic acid methanol solution
Pipette 1000mL methyl alcohol and 5mL formic acid in reagent bottle; Abundant mixing, ultrasonic 5min, room temperature is deposited;
The preparation of methanol/water (50: the 50) solution of (6) 0.5% formic acid
Pipette 500mL methyl alcohol, 500mL water and 5mL formic acid, to reagent bottle, fully mix, ultrasonic 5min, and room temperature is deposited;
(7) preparation of standard reserving solution
Lovastatin standard reserving solution: accurately take 5mg Lovastatin in 5mL volumetric flask, dissolve with 0.1% formic acid solution and be diluted to scale, fully mixing, obtain the Lovastatin standard reserving solution that concentration is 1mg/ml, in-20 DEG C of storages;
Hydroxyl lovastatin acid standard reserving solution: accurately take 5mg hydroxyl lovastatin acid sodium in 5mL volumetric flask, dissolve with 0.1% formic acid solution and be diluted to scale, abundant mixing, obtains the hydroxyl lovastatin acid standard reserving solution that concentration is 1mg/ml, in-20 DEG C of storages;
Standard reserving solution 1: pipette 0.1mL Lovastatin standard reserving solution and 0.1mL hydroxyl lovastatin acid standard reserving solution in 10mL volumetric flask, scale is diluted to 0.1% formic acid methanol solution, obtain the hybrid standard storing solution of the hydroxyl lovastatin acid of Lovastatin that concentration is 10 μ g/ml and 10 μ g/ml, abundant mixing, obtain standard reserving solution 1, in-20 DEG C of storages;
(8) preparation of typical curve standard working solution
Typical curve standard working solution: prepare with 0.1% formic acid methanol solution dilution standard storing solution 1, as shown in table 1; Fully mix and be incorporated in=20 DEG C of storages;
The preparation of table 1 typical curve standard working solution
(9) preparation of storing solution
Lovastatin storing solution: accurately take 5mg Lovastatin in 5mL volumetric flask, dissolve with 0.1% formic acid solution and be diluted to scale, namely abundant mixing obtains the Lovastatin storing solution that concentration is 1mg/ml; In-20 DEG C of storages;
Hydroxyl lovastatin acid storing solution: accurately take 5mg hydroxyl lovastatin acid sodium in 5mL volumetric flask, dissolve with 0.1% formic acid solution and be diluted to scale, namely abundant mixing obtains the hydroxyl lovastatin acid storing solution that concentration is 1mg/ml, in-20 DEG C of storages;
Storing solution 1: pipette 0.1mL Lovastatin storing solution and 0.1mL hydroxyl lovastatin acid storing solution in 10mL volumetric flask, scale is diluted to 0.1% formic acid methanol solution, obtain the mixing storing solution of the hydroxyl lovastatin acid of Lovastatin that concentration is 10 μ g/ml and 10 μ g/ml, abundant mixing, obtain storing solution 1, in-20 DEG C of storages;
(10) preparation of sample working solution: use K
2eTDA human plasma dilution storing solution 1 is prepared, shown in table 2; In-20 DEG C of storages;
The preparation of table 2 sample working solution
Note: QCH: enriched sample
QCM: middle concentration samples
QCL: low concentration sample
(11) preparation of inner mark solution
Hydroxyl simvastatin acid sodium storing solution: accurately take 5mg hydroxyl simvastatin acid sodium to 25mL volumetric flask, dissolve with 0.1% formic acid solution and be diluted to scale, namely abundant mixing obtains the hydroxyl simvastatin acid sodium storing solution that final concentration is 200 μ g/ml, in-20 DEG C of storages;
Simvastatin storing solution: accurately take 5mg Simvastatin to 25mL volumetric flask, dissolve with 0.1% formic acid solution and be diluted to scale, namely abundant mixing obtains the Simvastatin storing solution that concentration is 200 μ g/ml, in-20 DEG C of storages;
Interior mark application of sample solution: pipette hydroxyl simvastatin acid sodium storing solution that 20 μ L concentration are 200 μ g/ml and 20 μ L concentration are that the Simvastatin storing solution of 200 μ g/ml is in 20mL volumetric flask, scale is diluted to 0.1% formic acid solution, abundant mixing, obtain interior mark application of sample solution, in-20 DEG C of storages;
B. extract
(1) as standard working solution prepared by table 3:
The preparation of table 3 standard working solution
(2) by standard working solution, storing solution 1, testing sample, and blank ETDA-K
2human plasma respectively in ice-water bath eddy oscillating thaw;
(3) draw 500 μ L blood plasma (containing two blank, blank and sample) and respectively put a glass test tube, add 0.1% formic acid solution of 50 μ L;
(4) two blank adds 0.1% formic acid solution of 10 μ L, adds respectively in 10 μ L and mark application of sample solution in other sample, eddy current 15 seconds;
(5) add 2mL Mobile phase B, rear abundant eddy current mixing added a cover by test tube;
(6) with 3500rpm centrifugation 10 minutes, another glass test tube transferred to by supernatant, often props up and in vitro adds 2mL mobile phase A, fully mix;
(7) SPE plate is first rinsed 2 times by 1000 μ L Mobile phase B, then rinse 2 times by 1000 μ L mobile phase A, low vacuum is drained;
(8) solution of step (6) is moved to SPE plate, allow sample liquid slowly by packed bed, low vacuum is transferred to maximum vacuum again to ensure that all supernatants are drained completely after each cell becomes dry;
(9) rinse each cell twice by 1000 μ L mobile phase A respectively, drain 1 minute, then use the formic acid methyl alcohol of 1000 μ L 0.5%: water=50: the solution of 50 rinses each cell 3 times, and low vacuum drains 15 seconds;
(10) one piece of 2.2mL deep hole 96 hole dash receiver is placed in below SPE plate, each aperture uses 1000 μ L Mobile phase B wash-outs respectively, and eluent nitrogen dries up under 35 DEG C of conditions;
(11) dried residue 200 μ L Mobile phase B is dissolved, eddy current 30 seconds; To be greater than the centrifugation 10 minutes of 3200rpm, supernatant is transferred to HPLC sample injection bottle;
Wherein, above-mentioned steps (1), to (5), except eddy current mixing, all operates in ice-water bath;
C. data analysis
Retention time and peak area are determined by data acquisition and analysis software (Version 1.4.1); With peak area ratio, standard working curve is done to concentration, obtains following equation of linear regression, in order to calculate the concentration of analyte in solution to be measured:
y＝ax+b
Wherein: y=analyte and interior target peak area ratio
The slope of a=respective standard working curve
The intercept of b=respective standard working curve
The concentration (ng/mL) of x=analyte
(employing weighted least-squares method)
Five, result of study
1. statin
The pharmacokinetic parameter of Lovastatin and statistics are respectively in table 40 and accompanying drawing 9.
Single oral dose 1200mg XZK capsule or 20mg
lovastatin pharmacokinetic parameter shown in table 40.
The mean value (standard deviation) of table 40 Lovastatin pharmacokinetic parameter
Pharmacokinetic parameter C
max, AUC
0-t, and AUC
0-∞(ANOVA) of variance analysis the results are shown in Table 40.The Lovastatin systemic exposure of 1200mg XZK test group and 20mg lovastatin capsule is suitable.But absorbability, the cmax value of XZK capsule are all much higher.
2. lovastatin acid
Lovastatin acid pharmacokinetic parameter and statistics see accompanying drawing 10 and table 41 respectively.
Single oral dose 1200mg XZK capsule or 20mg
lovastatin acid pharmacokinetic parameter shown in table 41.
The mean value (standard deviation) of table 41 lovastatin acid pharmacokinetic parameter
Pharmacokinetic parameter C
max, AUC
0-t, and AUC
0-∞variance analysis (ANOVA) the results are shown in Table 41.Result shows: the absorption rate of 1200mg XZK test group lovastatin acid or uptake is all remarkable in 20mg lovastatin capsule control group.
Claims (1)
1. measure a method for the concentration of Lovastatin and hydroxyl lovastatin acid in human plasma, it is characterized in that the method comprises the steps:
a. solution is equipped with:
(1) preparation of blank medium: the blank K of preparation pH 4 ± 0.1
2eTDA human plasma
Pipette the blank K of 166.7 μ L 96% formic acid to 50 mL
2eTDA(EDTAP dipotassium ethylene diamine tetraacetate) human plasma, and fully mix;
(2) preparation of pin liquid is washed: methyl alcohol: water=50:50
Pipette 500 mL water and 500 mL methyl alcohol in container; Abundant mixing, room temperature is deposited;
The preparation of (3) 0.1% formic acid solutions (preparing with methyl alcohol)
Pipette the formic acid of 100 μ L and 100 ml methyl alcohol in reagent bottle; Abundant mixing, room temperature is deposited;
(4) mobile phase A: the preparation of 0.5% aqueous formic acid
Pipette 1000 mL water and 5 mL formic acid in reagent bottle; Abundant mixing, ultrasonic 5 min, room temperature is deposited;
(5) Mobile phase B: the preparation of 0.5% formic acid methanol solution
Pipette 1000 mL methyl alcohol and 5 mL formic acid in reagent bottle; Abundant mixing, ultrasonic 5 min, room temperature is deposited;
(6) by methyl alcohol 500ml with water 500ml dose volume than the solution being 50:50, then add 5ml formic acid wherein
Pipette 500 mL methyl alcohol, 500 mL water and 5 mL formic acid, to reagent bottle, fully mix, ultrasonic 5 min, and room temperature is deposited;
(7) preparation of standard reserving solution
lovastatin standard reserving solution: accurately take in 5 mg Lovastatin to 5 mL volumetric flasks, dissolve with 0.1% formic acid solution and be diluted to scale, fully mixing, obtain the Lovastatin standard reserving solution that concentration is 1 mg/ml, in-20 DEG C of storages;
hydroxyl lovastatin acid standard reserving solution: accurately take in 5 mg hydroxyl lovastatin acid sodium to 5 mL volumetric flasks, dissolve with 0.1% formic acid solution and be diluted to scale, fully mixing, obtaining the hydroxyl lovastatin acid standard reserving solution that concentration is 1 mg/ml, in-20 DEG C of storages;
standard reserving solution 1:pipette 0.1mL Lovastatin standard reserving solution and 0.1mL hydroxyl lovastatin acid standard reserving solution in 10mL volumetric flask, scale is diluted to 0.1% formic acid methanol solution, obtain the hybrid standard storing solution of the hydroxyl lovastatin acid of Lovastatin that concentration is 10 μ g/ml and 10 μ g/ml, abundant mixing, obtain standard reserving solution 1, in-20 DEG C of storages;
(8) preparation of typical curve standard working solution
Typical curve standard working solution: prepare with 0.1% formic acid methanol solution dilution standard storing solution 1, as shown in table 1; Fully mix and be incorporated in-20 DEG C of storages;
The preparation of table 1 typical curve standard working solution
(9) preparation of storing solution
lovastatin storing solution: accurately take in 5 mg Lovastatin to 5 mL volumetric flasks, dissolve and be diluted to scale with 0.1% formic acid solution, namely abundant mixing obtains the Lovastatin storing solution that concentration is 1 mg/ml; In-20 DEG C of storages;
hydroxyl lovastatin acid storing solution:accurately take in 5 mg hydroxyl lovastatin acid sodium to 5 mL volumetric flasks, dissolve with 0.1% formic acid solution and be diluted to scale, namely abundant mixing obtains the hydroxyl lovastatin acid storing solution that concentration is 1 mg/ml, in-20 DEG C of storages;
storing solution 1:pipette 0.1mL Lovastatin storing solution and 0.1mL hydroxyl lovastatin acid storing solution in 10mL volumetric flask, scale is diluted to 0.1% formic acid methanol solution, obtain the mixing storing solution of the hydroxyl lovastatin acid of Lovastatin that concentration is 10 μ g/ml and 10 μ g/ml, abundant mixing, obtain storing solution 1, in-20 DEG C of storages;
(10) preparation of sample working solution: use K
2eTDA human plasma dilution storing solution 1 is prepared, shown in table 2; In-20 DEG C of storages;
The preparation of table 2 sample working solution
Note: QCH: enriched sample
QCM: middle concentration samples
QCL: low concentration sample
(11) preparation of inner mark solution
hydroxyl simvastatin acid sodium storing solution: accurately take 5 mg hydroxyl simvastatin acid sodium to 25 mL volumetric flasks, dissolve and be diluted to scale with 0.1% formic acid solution, namely abundant mixing obtains the hydroxyl simvastatin acid sodium storing solution that final concentration is 200 μ g/ml, in-20 DEG C of storages;
simvastatin storing solution: accurately take 5 mg Simvastatin to 25 mL volumetric flasks, dissolve and be diluted to scale with 0.1% formic acid solution, namely abundant mixing obtains the Simvastatin storing solution that concentration is 200 μ g/ml, in-20 DEG C of storages;
interior mark application of sample solution:pipette hydroxyl simvastatin acid sodium storing solution that 20 μ L concentration are 200 μ g/ml and 20 μ L concentration are that the Simvastatin storing solution of 200 μ g/ml is in 20mL volumetric flask, scale is diluted to 0.1% formic acid solution, abundant mixing, obtains interior mark application of sample solution, in-20 DEG C of storages;
b. extract
(1) as standard working solution prepared by table 3:
The preparation of table 3 standard working solution
(2) by standard working solution, storing solution 1, testing sample, and blank K
2eTDA human plasma eddy oscillating in ice-water bath thaws;
(3) draw 500 μ L blood plasma, comprise containing two blank, blank and sample, respectively puts a glass test tube, adds 0.1% formic acid solution of 50 μ L;
(4) two blank adds 0.1% formic acid solution of 10 μ L, adds respectively in 10 μ L and mark application of sample solution in other sample, eddy current 15 seconds;
(5) add 2 mL Mobile phase B, rear abundant eddy current mixing added a cover by test tube;
(6) with 3500 rpm centrifugation 10 minutes, another glass test tube transferred to by supernatant, often props up and in vitro adds 2 mL mobile phase A, fully mix;
(7) SPE plate is first rinsed 2 times by 1000 μ L Mobile phase B, then rinse 2 times by 1000 μ L mobile phase A, low vacuum is drained;
(8) solution of step (6) is moved to SPE plate, allow sample liquid slowly by packed bed, low vacuum is transferred to maximum vacuum again to ensure that all supernatants are drained completely after each cell becomes dry;
(9) rinse each cell twice by 1000 μ L mobile phase A respectively, drain 1 minute, then use the formic acid methyl alcohol of 1000 μ L0.5%: the solution of water=50:50 rinses each cell 3 times, and low vacuum drains 15 seconds;
(10) one piece of 2.2 mL deep hole 96 hole dash receiver is placed in below SPE plate, each aperture uses 1000 μ L Mobile phase B wash-outs respectively, and eluent nitrogen dries up under 35 DEG C of conditions;
(11) dried residue 200 μ L Mobile phase B is dissolved, eddy current 30 seconds; To be greater than the centrifugation 10 minutes of 3200 rpm, supernatant is transferred to HPLC sample injection bottle;
Wherein, above-mentioned steps (1), to (5), except eddy current mixing, all operates in ice-water bath;
c. data analysis
Retention time and peak area are determined by data acquisition and analysis software (Version 1.4.1); With peak area ratio, standard working curve is done to concentration, obtains following equation of linear regression, in order to calculate the concentration of analyte in solution to be measured:
y = ax + b
Wherein: y=analyte and interior target peak area ratio
The slope of a=respective standard working curve
The intercept of b=respective standard working curve
The concentration (ng/mL) of x=analyte
Adopt weighted least-squares method.
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| KR20020005862A (en) * | 2000-07-10 | 2002-01-18 | 박호군 | Method for the determination of lovastatin acid in plasma |
| CN1987449A (en) * | 2005-12-19 | 2007-06-27 | 北京维信学知科技发展有限公司 | Method for detecting quality of blood fat recovery capsule |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20020005862A (en) * | 2000-07-10 | 2002-01-18 | 박호군 | Method for the determination of lovastatin acid in plasma |
| CN1987449A (en) * | 2005-12-19 | 2007-06-27 | 北京维信学知科技发展有限公司 | Method for detecting quality of blood fat recovery capsule |
Non-Patent Citations (3)
| Title |
|---|
| Application of a novel ultra-low elution volume 96-well solid-phase extraction method to the LC/MS/MS determination of simvastatin and simvastatin acid in human plasma;Amy Y. Yang et al;《Journal of Pharmaceutical and Biomedical Analysis》;20050731;第38卷;521-527 * |
| Microsample Determination of Lovastatin and its Hydroxy Acid Metabolite in Mouse and Rat Plasma by Liquid Chromatography/Ionspray Tandem Mass Spectrometry;Yunhui Wu et al;《JOURNAL OF MASS SPECTROMETRY》;19970430;第32卷;379-387 * |
| 洛伐他汀检测方法研究进展;文镜等;《北京联合大学学报(自然科学版)》;20030930;第17卷(第3期);70-74 * |
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