CN109142593A - HPLC-DAD method measures Quetiapine drug concentration in human serum - Google Patents
HPLC-DAD method measures Quetiapine drug concentration in human serum Download PDFInfo
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Abstract
The present invention relates to Quetiapine drug concentrations in the clinical therapeutic drug monitoring technical field more particularly to HPLC-DAD method of mental disease class drug measurement human serum.The present invention provides the methods that Quetiapine concentration in serum after Quetiapine drug is taken in measurement, and the method includes human serum sample preprocessing steps and high performance liquid chromatography (HPLC-DAD) analytical procedure.The present invention establishes that specificity is strong, HPLC-DAD method of high sensitivity and favorable reproducibility.The method for the measurement Quetiapine blood concentration that the present invention establishes for the first time accuracy, precision, specificity, in terms of verification result meet 2015 version Pharmacopoeia of People's Republic of China related guidance principle (9012) to the analysis of biological sample requirement.
Description
Technical field
The present invention relates to the surveys of the clinical therapeutic drug monitoring technical field more particularly to HPLC-DAD method of mental disease class drug
Determine Quetiapine drug concentration in human serum.
Background technique
Quetiapine is a kind of new atypical antipsychotic agents, is various neurotransmitters receptor antagonist in brain, main to make
For 5-HT2、D1、D2And D4Receptor affirms that safety is good, and adverse reaction is few to Stability of Schizophrenia.By gene pleiomorphism
It influencing, there are biggish individual differences for internal metabolic process, and blood concentration and clinical efficacy and adverse reaction are closely related, according to
Blood concentration can guarantee curative effect of medication and reduction adverse reaction to adjust dosage, to realize personalized medicine.So prison
Patient's blood concentration is surveyed to be of great significance in clinical application.
At present both at home and abroad detection human blood in Quetiapine method mainly have HPLC- UV detection method, efficiently
Liquid chromatogram-Diode array detection, High Performance Liquid Chromatography-Electrospray Ionization Tandem Mass method etc..Wherein high performance liquid chromatography matter
Combination method higher cost is composed, therefore detects the method first choice high performance liquid chromatography of Quetiapine concentration in human serum at present.But it is previous
Method is there are complex pretreatment, sensitivity is low, quantitative analysis narrow range, quantified by external standard method easily lead to that result is not accurate enough etc. to ask
Topic.
Summary of the invention
In order to solve the above technical problems, the present invention proposes Quetiapine drug concentration in HPLC-DAD method measurement human serum.
Specific technical solution is as follows:
HPLC-DAD method measure human serum in Quetiapine drug concentration, it the following steps are included:
(1) preparation of standard working solution
(1) preparation of standard working solution:
Quetiapine fumarate standard items 5.57mg accurately is weighed in 15mL centrifuge tube, is dissolved with methanol, is made into Quetiapine
Concentration is the stock solution S1 of 1.0mg/mL, and standard reserving solution S1 is diluted with methanol, is containing 1.0-60 μ g/mL respectively
Series standard working solution is prepared in the range of Quetiapine, is saved in -80 DEG C of refrigerator freezings；
(2) preparation of internal standard working solution
Precision weighs genistein standard items 2.31mg in 5mL centrifuge tube, is dissolved with methanol, and being made into concentration is 1.0mg/
Standard reserving solution ISS1 is diluted by the stock solution ISS1 of mL with methanol, obtains the internal standard working solution that concentration is 10 μ g/mL,
It is saved in -80 DEG C of refrigerator freezings；
(2) preparation of serum sample
(3) blank blood at least 5mL is taken, 10min is centrifuged at centrifugal speed 3500rpm, supernatant is taken to obtain blank serum,
10 μ L of series standard working solution described in step (1) is mixed with the 190 μ L of blank serum in above-mentioned steps respectively
The standard work serum that Quetiapine drug concentration is 50-3000ng/mL；
(4) blood to be measured at least 5mL is taken, 10min is centrifuged at centrifugal speed 3500rpm, supernatant is taken to obtain test serum,
It is spare to before analyzing that above-mentioned serum is placed in the lower preservation of -20 DEG C of freezings；
(3) blood serum sample is handled
(5) internal standard working solution described in 10 μ L steps (2) is pipetted in 1.5mL centrifuge tube with liquid-transfering gun, be then added
Serum described in 200 μ L step (2), the vortex mixed 3min under the revolving speed of 2000rpm；
(6) the methyl tertiary butyl ether(MTBE) extractant that 800 μ L contain 0%-30% n-hexane being pipetted with liquid-transfering gun, step (5) are added
Centrifuge tube in, 3 min of vortex mixed under the revolving speed of 2000rpm, to achieve the purpose that sufficiently to extract, then in 14000rpm
Revolving speed under high speed centrifugation 5min, obtain supernatant；
(7) it takes 750 μ L of supernatant described in step (6) to be placed in another 1.5mL centrifuge tube, uses nitrogen under room temperature
Slowly drying；
(8) water that 100 μ L contain 45% acetonitrile is pipetted with liquid-transfering gun redissolve drying described in liquid addition step (7)
In centrifuge tube, the vortex mixed 3min under the revolving speed of 2000rpm, then the high speed centrifugation 5min under the revolving speed of 14000rpm, is obtained
It is sample to be tested to supernatant；
(4) calibration curve equation
Liquid chromatograph and DAD detector pair are used after serum described in step (3) is pre-processed according to step (3)
Sample to be tested is detected, and the Quetiapine and internal standard chromatogram of above-mentioned sample to be tested is obtained, in above-mentioned Quetiapine and internal standard chromatography
Target to be measured peak area and internal standard compound peak area are obtained in figure, by the ratio of target to be measured peak area and internal standard compound peak area
As the ordinate y of canonical plotting, target to be measured drug concentration and internal standard in step (3) Plays work serum are worked
Abscissa x of the ratio, that is, relative concentration of liquid concentration as canonical plotting, passes through weighted least-squares for the above detection data
Method carries out linear regression, and obtaining calibration curve equation is y=ax+b, and obtains weight coefficient a and b；
(5) detection of unknown sample
It is detected after test serum described in step (4) is pre-processed according to step (3) using liquid chromatograph and DAD
Device detects sample to be tested, the Quetiapine and internal standard chromatogram of above-mentioned sample to be tested is obtained, in above-mentioned Quetiapine and internal standard
Target to be measured peak area and internal standard compound peak area are obtained in chromatogram, by target to be measured peak area and internal standard compound peak area
Ratio y is substituted into the calibration curve equation in above-mentioned steps (four), and object relative concentration in sample to be tested is obtained by calculation
X, internal standard compound working solution concentration be it is known, the Quetiapine drug concentration in the sample in test serum is thus calculated.
It is dissolved to obtain standard reserving solution S1 and standard reserving solution ISS1 respectively with methanol in step (1) and step (2),
It is diluted respectively with methanol, makes each standard working solution and internal standard working solution, extractant described in step (6) is by 1:4
N-hexane and methyl tertiary butyl ether(MTBE) composition extractant, described in step (8) redissolution liquid be made of the acetonitrile and water of 9:11
Redissolution liquid.
Quetiapine contained in target to be measured drug concentration index quasi- work serum in the above-mentioned standard work serum
Concentration, internal standard working solution concentration refers to the concentration of genistein contained in internal standard working solution.
The standard work serum of seven kinds of various concentrations is used in step (4).
The standard work serum of seven kinds of various concentrations be respectively contain 50,100,150,300,500,1000,
The Quetiapine serum of 3000ng/mL concentration.
SPE solid phase extraction column used in the high performance liquid chromatograph is Shiseido MF ph-12cm, and chromatographic column is
Waters SunFire C18。
The column temperature of the high performance liquid chromatograph setting is 40 DEG C.
Mobile phase used in the high performance liquid chromatograph is the water and acetonitrile of the ammonium acetate containing 30mmol, and using ladder
Degree elution, sample volume are 10 μ L.
The content is the content of volume ratio.
Compared with prior art, the present invention has following advantageous effects:
High performance liquid chromatography-DAD detection method established by the present invention is in the side such as accuracy, precision, specificity, stability
Face meet 2015 version Pharmacopoeia of People's Republic of China related guidance principle (9012) to the analysis of biological sample requirement.Especially
, it is noted that the serum sample volume that this measuring method utilizes is 200 μ L, corresponding extractant volume is 800 μ L, is lower than document
The consumption of the volume of report, lesser sample size demand and relatively small number of organic reagent makes the present invention be suitable for inspection
It surveys patient and takes after Quetiapine drug blood concentration in serum, and reduce reagent contamination.
The method that the present invention establishes for the first time can be used in clinical treatment supervising the Quetiapine blood concentration of patient's body
It surveys, provides theoretical foundation for individual administration, reduction toxic side effect.
Detailed description of the invention
Fig. 1 is chromatography typical case's chromatogram after blank serum pretreatment in embodiment 3；
Fig. 2 is chromatography typical case's chromatogram partial enlargement chromatogram after blank serum pretreatment in embodiment 3；
Fig. 3 is chromatography typical case's chromatogram after the work serum pretreatment of 3 Plays of embodiment；Wherein；
Fig. 4 is chromatography typical case's chromatogram partial enlargement chromatogram after the work serum pretreatment of 3 Plays of embodiment；
In figure, 1- genistein；2- Quetiapine.
Specific embodiment
In order to which more clearly the present invention will be described, below by way of specific implementation example to specific implementation of the invention
Mode is described in more detail.However, it should be understood that specific implementation example as described below, which is only used for the present invention, carries out example
Property explanation, carry out any property restriction not for the present invention, wherein material therefor, reagent, instrument and operating condition are only generation
Table, it is not limited to cited situation.Person of ordinary skill in the field can be to this by reading following explanation
The change and improvement for not departing from protection scope defined by the claims in the present invention are made in invention, these changes and improvement are also at
In scope of the present invention.
Embodiment 1
(1) preparation of standard working solution
(1) preparation of standard working solution:
Quetiapine fumarate standard items 5.57mg (being equivalent to Quetiapine quality is 4.83mg) is accurately weighed to be centrifuged in 15mL
Guan Zhong is dissolved with methanol, is made into the stock solution S1 that Quetiapine concentration is 1.0mg/mL, standard reserving solution S1 is carried out with methanol
Dilution, is respectively prepared the Quetiapine series standard working solution containing 1.0,2.0,3.0,6.0,10,20,60 μ g/mL concentration, in-
80 DEG C of refrigerator freezings save；
(2) preparation of internal standard working solution
Precision weighs genistein standard items 2.31mg (being equivalent to genistein quality is 2.30 mg) in 5mL centrifuge tube
In, it is dissolved with methanol, is made into the stock solution ISS1 that concentration is 1.0mg/mL, standard reserving solution ISS1 is diluted with methanol,
The internal standard working solution that concentration is 10 μ g/mL is obtained, is saved in -80 DEG C of refrigerator freezings；
(2) preparation of serum sample
(3) blank blood 5mL is taken, 10min is centrifuged at centrifugal speed 3500rpm, supernatant is taken to obtain blank serum, will be walked
Suddenly the quinoline sulphur that the μ L of series standard working solution 10 described in (1) is mixed with the 190 μ L of blank serum in above-mentioned steps respectively
Flat blood concentration is the standard work serum of 50,100,150,300,500,1000,3000ng/mL；
(4) blood to be measured at least 5mL is taken, 10min is centrifuged at centrifugal speed 3500rpm, supernatant is taken to obtain test serum,
It is spare to before analyzing that above-mentioned serum is placed in the lower preservation of -20 DEG C of freezings；
(3) blood serum sample is handled
(5) internal standard working solution described in 10 μ L steps (2) is pipetted in 1.5mL centrifuge tube with liquid-transfering gun, be then added
Serum described in 200 μ L step (2), the vortex mixed 3min under the revolving speed of 2000rpm；
(6) 800 μ L methyl tertiary butyl ether(MTBE) extractants are pipetted with liquid-transfering gun to be added in the centrifuge tube of step (5),
Vortex mixed 3min under the revolving speed of 2000rpm, to achieve the purpose that sufficiently to extract, then under the revolving speed of 14000rpm high speed from
Heart 5min, obtains supernatant；
(7) it takes 750 μ L of supernatant described in step (6) to be placed in another 1.5mL centrifuge tube, uses nitrogen under room temperature
Slowly drying；
(8) water that 100 μ L contain 45% acetonitrile is pipetted with liquid-transfering gun redissolve drying described in liquid addition step (7)
In centrifuge tube, the vortex mixed 3min under the revolving speed of 2000rpm, then the high speed centrifugation 5min under the revolving speed of 14000rpm, is obtained
It is sample to be tested to supernatant；
Embodiment 2
(1) preparation of standard working solution
(1) preparation of standard working solution:
Quetiapine fumarate standard items 5.57mg (being equivalent to Quetiapine quality is 4.83mg) is accurately weighed to be centrifuged in 15mL
Guan Zhong is dissolved with methanol, is made into the stock solution S1 that Quetiapine concentration is 1.0mg/mL, standard reserving solution S1 is carried out with methanol
Dilution, is respectively prepared the Quetiapine series standard working solution containing 1.0,2.0,3.0,6.0,10,20,60 μ g/mL concentration, in-
80 DEG C of refrigerator freezings save；
(2) preparation of internal standard working solution
Precision weighs genistein standard items 2.31mg (being equivalent to genistein quality is 2.30 mg) in 5mL centrifuge tube
In, it is dissolved with methanol, is made into the stock solution ISS1 that concentration is 1.0mg/mL, standard reserving solution ISS1 is diluted with methanol,
The internal standard working solution that concentration is 10 μ g/mL is obtained, is saved in -80 DEG C of refrigerator freezings；
(2) preparation of serum sample
(3) blank blood 5mL is taken, 10min is centrifuged at centrifugal speed 3500rpm, supernatant is taken to obtain blank serum, will be walked
Suddenly the quinoline sulphur that the μ L of series standard working solution 10 described in (1) is mixed with the 190 μ L of blank serum in above-mentioned steps respectively
Flat blood concentration is the standard work serum of 50,100,150,300,500,1000,3000ng/mL；
(4) blood to be measured at least 5mL is taken, 10min is centrifuged at centrifugal speed 3500rpm, supernatant is taken to obtain test serum,
It is spare to before analyzing that above-mentioned serum is placed in the lower preservation of -20 DEG C of freezings；
(3) blood serum sample is handled
(5) internal standard working solution described in 10 μ L steps (2) is pipetted in 1.5mL centrifuge tube with liquid-transfering gun, be then added
Serum described in 200 μ L step (2), the vortex mixed 3min under the revolving speed of 2000rpm；
(6) with liquid-transfering gun pipette the methyl tertiary butyl ether(MTBE) extractant that 800 μ L contain 10% n-hexane be added step (5) from
In heart pipe, then the vortex mixed 3min under the revolving speed of 2000rpm is turned with achieving the purpose that sufficiently to extract in 14000rpm
The lower high speed centrifugation 5min of speed, obtains supernatant；
(7) it takes 750 μ L of supernatant described in step (6) to be placed in another 1.5mL centrifuge tube, uses nitrogen under room temperature
Slowly drying；
(8) water that 100 μ L contain 45% acetonitrile is pipetted with liquid-transfering gun redissolve drying described in liquid addition step (7)
In centrifuge tube, the vortex mixed 3min under the revolving speed of 2000rpm, then the high speed centrifugation 5min under the revolving speed of 14000rpm, is obtained
It is sample to be tested to supernatant；
Embodiment 3
(1) preparation of standard working solution
(1) preparation of standard working solution:
Quetiapine fumarate standard items 5.57mg (being equivalent to Quetiapine quality is 4.83mg) is accurately weighed to be centrifuged in 15mL
Guan Zhong is dissolved with methanol, is made into the stock solution S1 that Quetiapine concentration is 1.0mg/mL, standard reserving solution S1 is carried out with methanol
Dilution, is respectively prepared the Quetiapine series standard working solution containing 1.0,2.0,3.0,6.0,10,20,60 μ g/mL concentration, in-
80 DEG C of refrigerator freezings save；
(2) preparation of internal standard working solution
Precision weighs genistein standard items 2.31mg (being equivalent to genistein quality is 2.30 mg) in 5mL centrifuge tube
In, it is dissolved with methanol, is made into the stock solution ISS1 that concentration is 1.0mg/mL, standard reserving solution ISS1 is diluted with methanol,
The internal standard working solution that concentration is 10 μ g/mL is obtained, is saved in -80 DEG C of refrigerator freezings；
(2) preparation of serum sample
(3) blank blood 5mL is taken, 10min is centrifuged at centrifugal speed 3500rpm, supernatant is taken to obtain blank serum, will be walked
Suddenly the quinoline sulphur that the μ L of series standard working solution 10 described in (1) is mixed with the 190 μ L of blank serum in above-mentioned steps respectively
Flat blood concentration is the standard work serum of 50,100,150,300,500,1000,3000ng/mL；
(4) blood to be measured at least 5mL is taken, 10min is centrifuged at centrifugal speed 3500rpm, supernatant is taken to obtain test serum,
It is spare to before analyzing that above-mentioned serum is placed in the lower preservation of -20 DEG C of freezings；
(3) blood serum sample is handled
(5) internal standard working solution described in 10 μ L steps (2) is pipetted in 1.5mL centrifuge tube with liquid-transfering gun, be then added
Serum described in 200 μ L step (2), the vortex mixed 3min under the revolving speed of 2000rpm；
(6) with liquid-transfering gun pipette the methyl tertiary butyl ether(MTBE) extractant that 800 μ L contain 20% n-hexane be added step (5) from
In heart pipe, then the vortex mixed 3min under the revolving speed of 2000rpm is turned with achieving the purpose that sufficiently to extract in 14000rpm
The lower high speed centrifugation 5min of speed, obtains supernatant；
(7) it takes 750 μ L of supernatant described in step (6) to be placed in another 1.5mL centrifuge tube, uses nitrogen under room temperature
Slowly drying；
(8) water that 100 μ L contain 45% acetonitrile is pipetted with liquid-transfering gun redissolve drying described in liquid addition step (7)
In centrifuge tube, the vortex mixed 3min under the revolving speed of 2000rpm, then the high speed centrifugation 5min under the revolving speed of 14000rpm, is obtained
It is sample to be tested to supernatant；
(4) calibration curve equation
Liquid chromatograph and DAD detector pair are used after serum described in step (3) is pre-processed according to step (3)
Sample to be tested is detected, and the Quetiapine and internal standard chromatogram of above-mentioned sample to be tested is obtained, in above-mentioned Quetiapine and internal standard chromatography
Target to be measured peak area and internal standard compound peak area are obtained in figure, by the ratio of target to be measured peak area and internal standard compound peak area
As the ordinate y of canonical plotting, target to be measured blood concentration and internal standard in step (3) Plays work serum are worked
Abscissa x of the ratio, that is, relative concentration of liquid concentration as canonical plotting, data are shown in Table 1, by the above detection data by adding
It weighs least square method and carries out linear regression, obtaining calibration curve equation is y=0.0435*x-0.1509；
1 linear relationship related data of table
| Sample introduction title | Determinand peak area | Internal standard peak area | Y value | Determinand blood concentration | Internal standard concentration | X value |
| sd1_01 | 0.0218 | 1.032 | 0.0211 | 50 | 10 | 5 |
| sd2_01 | 0.0456 | 1.009 | 0.0452 | 100 | 10 | 10 |
| sd3_01 | 0.0699 | 1.096 | 0.0638 | 150 | 10 | 15 |
| sd4_01 | 0.137 | 0.968 | 0.142 | 300 | 10 | 30 |
| sd5_01 | 0.232 | 1.100 | 0.211 | 500 | 10 | 50 |
| sd6_01 | 0.461 | 1.025 | 0.450 | 1000 | 10 | 100 |
| sd7_01 | 1.51 | 1.068 | 1.419 | 3000 | 10 | 300 |
(5) detection of unknown sample
It is detected after test serum described in step (4) is pre-processed according to step (3) using liquid chromatograph and DAD
Device detects sample to be tested, the Quetiapine and internal standard chromatogram of above-mentioned sample to be tested is obtained, in above-mentioned Quetiapine and internal standard
Target to be measured peak area and internal standard compound peak area are obtained in chromatogram, by target to be measured peak area and internal standard compound peak area
Ratio y is substituted into the calibration curve equation in above-mentioned steps (four), and object relative concentration in sample to be tested is obtained by calculation
X, internal standard compound working solution concentration be it is known, the Quetiapine drug concentration in the sample in test serum is thus calculated.
(6) liquid chromatographic detection condition
SPE solid phase extraction column used in high performance liquid chromatograph is Shiseido MF ph-12cm, and chromatographic column is
Waters SunFire C18, column temperature are 40 DEG C, and mobile phase is the water and acetonitrile of the ammonium acetate containing 30mmol, and uses gradient
Elution, gradient elution program are shown in Table 2 and table 3, and sample volume is 10 μ L.
If the content in the application is the content of volume ratio without specified otherwise.
Left pump (cleaning pump) gradient elution program of 2 liquid chromatogram of table
| Time(min) | Acetonitrile (%) | Water (30mM ammonium acetate) (%) | Flow velocity (mL/min) |
| 0 | 35 | 65 | 1.0 |
| 11.5 | 35 | 65 | 1.0 |
The right pump of 3 liquid chromatogram of table (analysis pump) gradient elution program
| Time(min) | Acetonitrile (%) | Water (30mM ammonium acetate) (%) | Flow velocity (mL/min) |
| 0 | 60 | 40 | 1.0 |
| 4.2 | 60 | 40 | 1.0 |
| 4.21 | 98 | 2 | 1.0 |
| 10.5 | 98 | 2 | 1.0 |
| 10.51 | 60 | 40 | 1.0 |
| 11.5 | 60 | 40 | 1.0 |
Method validation is as follows:
One, specificity
Blank serum and sample serum are prepared into sample by 3 pretreatment condition of above-described embodiment, then measured by embodiment
Condition analysis, typical chromatogram are shown in Fig. 1-4, the results showed that, blank serum is noiseless to determinand and internal standard, this method specificity
It is relatively strong.
Two, linear and quantitative limit
The standard work serum of each concentration of above-mentioned preparation is prepared into sample by 3 pretreatment condition of embodiment, is then pressed
Embodiment determination condition, concentration measure from high to low, with chromatographic peak area-concentration mapping, obtain standard curve, the results showed that quinoline
Flat the quantifying of sulphur is limited to 50ng/mL, and for Quetiapine in 50ng/mL to 3000ng/mL range, linear relationship is good, related coefficient
R>0.99。
Three, precision and accuracy
Take Quetiapine stock solution be configured to high (QC3), in (QC2), low (QC1) 3 kinds of concentration and lower limit of quantitation (LLOQ) into
The test of row preci-sion and accuracy measures 3 batches, preci-sion and accuracy result difference by the present embodiment method replicate analysis
Such as the following table 4.
The preci-sion and accuracy result of 4 Quetiapine determination of plasma concentration method of table
In summary verification test, the technical indicators such as the specificity, preci-sion and accuracy of the present embodiment meet the requirements,
Method detects Quetiapine drug concentration in serum, and specificity is strong, and reproducibility is good, improves the accuracy of testing result, and this
Embodiment method pre-treatment step is easy, and organic reagent consumption is lower.
Embodiment described above only describe the preferred embodiments of the invention, not to model of the invention
It encloses and is defined, without departing from the spirit of the design of the present invention, those of ordinary skill in the art are to technical side of the invention
The various changes and improvements that case is made should all be fallen into the protection scope that claims of the present invention determines.
Claims (9)
1.HPLC-DAD method measure human serum in Quetiapine drug concentration, it is characterised in that: it the following steps are included:
(1) preparation of standard working solution
(1) preparation of standard working solution:
Quetiapine fumarate standard items 5.57mg accurately is weighed in 15mL centrifuge tube, is dissolved with methanol, is made into Quetiapine concentration
For the stock solution S1 of 1.0mg/mL, standard reserving solution S1 is diluted with methanol, is containing 1.0-60 μ g/mL Quetiapine respectively
In the range of prepare series standard working solution, saved in -80 DEG C of refrigerator freezings；
(2) preparation of internal standard working solution
Precision weighs genistein standard items 2.31mg in 5mL centrifuge tube, is dissolved with methanol, and being made into concentration is 1.0mg/mL's
Standard reserving solution ISS1 is diluted by stock solution ISS1 with methanol, the internal standard working solution that concentration is 10 μ g/mL is obtained, in -80
DEG C refrigerator freezing saves；
(2) preparation of serum sample
(3) blank blood at least 5mL is taken, 10min is centrifuged at centrifugal speed 3500rpm, supernatant is taken to obtain blank serum, will be walked
Suddenly the quinoline sulphur that the μ L of series standard working solution 10 described in (1) is mixed with the 190 μ L of blank serum in above-mentioned steps respectively
The standard work serum that flat drug concentration is 50-3000ng/mL；
(4) blood to be measured at least 5mL is taken, is centrifuged 10min at centrifugal speed 3500rpm, supernatant is taken to obtain test serum, it is above-mentioned
It is spare to before analyzing that serum is placed in the lower preservation of -20 DEG C of freezings；
(3) blood serum sample is handled
(5) internal standard working solution described in 10 μ L steps (2) is pipetted in 1.5mL centrifuge tube with liquid-transfering gun, 200 μ L step is then added
Suddenly serum described in (two), the vortex mixed 3min under the revolving speed of 2000rpm；
(6) with liquid-transfering gun pipette the methyl tertiary butyl ether(MTBE) extractant that 800 μ L contain 0%-30% n-hexane be added step (5) from
In heart pipe, then the vortex mixed 3min under the revolving speed of 2000rpm is turned with achieving the purpose that sufficiently to extract in 14000rpm
The lower high speed centrifugation 5min of speed, obtains supernatant；
(7) it takes 750 μ L of supernatant described in step (6) to be placed in another 1.5mL centrifuge tube, is slowly blown under room temperature with nitrogen
It is dry；
(8) water that 100 μ L contain 45% acetonitrile is pipetted with liquid-transfering gun redissolve the centrifuge tube that drying described in step (7) is added in liquid
In, the vortex mixed 3min under the revolving speed of 2000rpm, then the high speed centrifugation 5min under the revolving speed of 14000rpm, obtains supernatant
Liquid is sample to be tested；
(4) calibration curve equation
Using liquid chromatograph and DAD detector to be measured after serum described in step (3) is pre-processed according to step (3)
Sample is detected, and the Quetiapine and internal standard chromatogram of above-mentioned sample to be tested is obtained, in above-mentioned Quetiapine and internal standard chromatogram
Obtain target to be measured peak area and internal standard compound peak area, using the ratio of target to be measured peak area and internal standard compound peak area as
The ordinate y of canonical plotting is dense by target to be measured drug concentration and internal standard working solution in step (3) Plays work serum
Abscissa x of the ratio, that is, relative concentration of degree as canonical plotting, by the above detection data by weighted least-squares method into
Row linear regression, obtaining calibration curve equation is y=ax+b, and obtains weight coefficient a and b；
(5) detection of unknown sample
Liquid chromatograph and DAD detector pair are used after test serum described in step (4) is pre-processed according to step (3)
Sample to be tested is detected, and the Quetiapine and internal standard chromatogram of above-mentioned sample to be tested is obtained, in above-mentioned Quetiapine and internal standard chromatography
Target to be measured peak area and internal standard compound peak area are obtained in figure, by the ratio of target to be measured peak area and internal standard compound peak area
Y is substituted into the calibration curve equation in above-mentioned steps (four), and object relative concentration x in sample to be tested is obtained by calculation, interior
Mark object working solution concentration be it is known, the Quetiapine drug concentration in the sample in test serum is thus calculated.
2. Quetiapine drug concentration in HPLC-DAD method measurement human serum as described in claim 1, it is characterised in that: in step
(1) and step (2) is dissolved to obtain standard reserving solution S1 and standard reserving solution ISS1 respectively with methanol, is carried out respectively with methanol
Dilution, makes each standard working solution and internal standard working solution, extractant described in step (6) is the n-hexane and methyl by 1:4
The extractant of tertbutyl ether composition, the redissolution liquid that redissolution liquid described in step (8) is made of the acetonitrile and water of 9:11.
3. Quetiapine drug concentration in HPLC-DAD method measurement human serum as claimed in claim 2, it is characterised in that: on described
State the concentration of Quetiapine contained in target to be measured drug concentration index quasi- work serum in standard work serum, internal standard work
Liquid concentration refers to the concentration of genistein contained in internal standard working solution.
4. Quetiapine drug concentration in HPLC-DAD method measurement human serum as claimed in claim 3, it is characterised in that: in step
(4) the standard work serum of seven kinds of various concentrations is used in.
5. Quetiapine drug concentration in HPLC-DAD method measurement human serum as claimed in claim 4, it is characterised in that: described seven
The standard work serum of kind of various concentration be respectively containing 50,100,150,300,500,1000, the quinoline sulphur of 3000ng/mL concentration
Flat serum.
6. Quetiapine drug concentration in HPLC-DAD method measurement human serum as claimed in claim 5, it is characterised in that: the height
SPE solid phase extraction column used in effect liquid phase chromatogram instrument is Shiseido MF ph-12cm, and chromatographic column is Waters SunFire
C18。
7. Quetiapine drug concentration in HPLC-DAD method measurement human serum as claimed in claim 5, it is characterised in that: the height
The column temperature of effect liquid phase chromatogram instrument setting is 40 DEG C.
8. Quetiapine drug concentration in HPLC-DAD method measurement human serum as claimed in claim 5, it is characterised in that: the height
Mobile phase used in effect liquid phase chromatogram instrument is the water and acetonitrile of the ammonium acetate containing 30mmol, and uses gradient elution, sample volume
For 10 μ L.
9. Quetiapine drug concentration in HPLC-DAD method measurement human serum as described in claim 1, it is characterised in that: described to contain
Amount is the content of volume ratio.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109900820A (en) * | 2019-02-22 | 2019-06-18 | 南京海纳医药科技股份有限公司 | A kind of method that HPLCMS-MS combination detects Quetiapine in human plasma |
| CN110763799A (en) * | 2019-11-29 | 2020-02-07 | 吉林和合医学检验有限公司 | Method for simultaneously detecting quetiapine and N-dealkylated quetiapine contents in blood |
| CN112834679A (en) * | 2020-12-31 | 2021-05-25 | 苏州海科医药技术有限公司 | Analysis method for clinically researching concentration of quetiapine in plasma sample |
-
2018
- 2018-11-12 CN CN201811339098.8A patent/CN109142593A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109900820A (en) * | 2019-02-22 | 2019-06-18 | 南京海纳医药科技股份有限公司 | A kind of method that HPLCMS-MS combination detects Quetiapine in human plasma |
| CN110763799A (en) * | 2019-11-29 | 2020-02-07 | 吉林和合医学检验有限公司 | Method for simultaneously detecting quetiapine and N-dealkylated quetiapine contents in blood |
| CN110763799B (en) * | 2019-11-29 | 2022-03-25 | 吉林和合医学检验有限公司 | Method for simultaneously detecting quetiapine and N-dealkylated quetiapine contents in blood |
| CN112834679A (en) * | 2020-12-31 | 2021-05-25 | 苏州海科医药技术有限公司 | Analysis method for clinically researching concentration of quetiapine in plasma sample |
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