CN114755335B - Quantitative detection method for pyrimidine derivative 1D228 enantiomer in biological sample - Google Patents
Quantitative detection method for pyrimidine derivative 1D228 enantiomer in biological sample Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention relates to the technical field of analysis and detection, in particular to a quantitative detection method for pyrimidine derivative 1D228 enantiomer in a biological sample. According to the quantitative detection method provided by the invention, chiral separation of R-1D228 and S-1D228 in a biological sample is realized through ultra-efficient phase-combining chromatography tandem mass spectrometry detection, and the problems that the sensitivity of an HPLC method is insufficient and the quantitative detection cannot be realized due to incompatibility of normal phase solvents in a LC-MS/MS (liquid chromatography-mass spectrometry) using chiral columns are overcome; meanwhile, the detection method provided by the invention has high sensitivity and good precision and accuracy, and meets the requirements of enantiomer detection in pyrimidine derivatives in preclinical and clinical biological samples.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a quantitative detection method for pyrimidine derivative 1D228 enantiomer in a biological sample.
Background
1D228 (structural formula is shown as follows) is a novel pyrimidine derivative for targeted treatment of lung cancer and liver cancer, has good anti-tumor activity and in vivo stability, and the anti-tumor activity of an enantiomer S-1D228 of 1D228 is obviously superior to that of R-1D228, so whether the optical activity conversion of S-1D228 occurs in vivo is a key research that must be examined in the development of preclinical and clinical studies of chiral medicines, and therefore, a detection method for quantitatively measuring the enantiomer R-1D228 and S-1D228 of the novel pyrimidine derivative in a sample is required to be established.
The quantitative analysis of the medicine in the biological sample has the characteristics of small sampling amount, low medicine concentration, more interference substances, large individual difference and the like, and the current common detection method for the enantiomers R-1D228 and S-1D228 of the novel pyrimidine derivative 1D228 is High Performance Liquid Chromatography (HPLC), but the sensitivity of the HPLC method does not meet the requirement of quantitative detection of the medicine in the biological sample. The most commonly used analytical method in biological analysis is high performance liquid chromatography tandem mass spectrometry (LC-MS/MS), but the chiral chromatographic columns capable of separating R-1D228 and S-1D228 in liquid phase conditions by LC-MS/MS are normal phase columns, normal phase solvents such as normal hexane and isopropanol must be used, and normal phase solvents are not compatible with mass spectrometry, so quantitative detection of enantiomers of novel pyrimidine derivatives in biological samples cannot be performed by LC-MS/MS.
Disclosure of Invention
In view of the above, the present invention aims to provide a quantitative detection method for pyrimidine derivative 1D228 enantiomer in biological samples, which has high sensitivity and can realize quantitative detection for pyrimidine derivative 1D228 enantiomer in biological samples.
In order to achieve the above object, the present invention provides a quantitative detection method for pyrimidine derivative 1D228 enantiomer in biological sample, comprising the steps of:
performing ultra-efficient synthetic phase chromatography tandem mass spectrometry detection on a sample liquid to be detected to obtain a detection result of a pyrimidine derivative 1D228 enantiomer;
the detection conditions of the ultra-high performance phase-combining chromatography comprise: the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is CO 2 The mobile phase B is a methanol-ammonium formate-formic acid system;
the flow rate of the mobile phase system is 1.0mL/min;
the elution mode is gradient elution; the gradient elution procedure was:
0.0 to 1.0min: the volume percentage of the mobile phase B is 60%;
1.0 to 5.0min: the volume percentage of the mobile phase B is increased from 60% to 80% at a constant speed;
5.0 to 8.0min: the volume percentage of the mobile phase B is 80%;
8.0 to 10.0min: the volume percentage of the mobile phase B is reduced from 80% to 60% at a constant speed; the method comprises the steps of carrying out a first treatment on the surface of the
The detection conditions of the mass spectrum include: the ion source is an electrospray ion source; the detection mode is multi-reaction ion monitoring; the scanning mode is a positive ion mode; the capillary voltage is 2.0kV; taper hole voltage: 20V; taper hole air flow rate: 10L/h; desolventizing temperature: 500 ℃; desolventizing flow rate: 1000L/h;
the enantiomer of the pyrimidine derivative 1D228 is R-1D228 and S-1D228;
the pyrimidine derivative 1D228 has the structural formula:
preferably, the conditions of the ultra-high performance phase-combining chromatographic conditions further include: the conditions for ultra-high performance phase-combining chromatography detection also include: the chromatographic column is AcquityUPCC Trefoil CEL chromatographic column, acquity UPCC Trefoil CEL chromatographic column or ACQUITY UPCC Torus Diol chromatographic column; the column temperature is 25-40 ℃; the sample injection amount is 1-7.5 mu L; the back pressure of the system is 800-2000 psi.
Preferably, the molar concentration of ammonium formate in the mobile phase B is 5-15 mmol/L; the volume concentration of the formic acid in the mobile phase B is 0.05-0.2%.
Preferably, the CO 2 The purity of the product is more than or equal to 99.9 percent.
Preferably, the method for obtaining the sample liquid to be tested comprises the following steps:
and mixing the biological sample to be detected, the internal standard substance and the protein precipitant, and centrifuging to obtain the sample liquid to be detected.
Preferably, the biological sample to be tested comprises plasma, tissue homogenate, urine, bile or faecal homogenate.
Preferably, the volume ratio of the biological sample to be detected to the protein precipitant is 1:2-3.
Preferably, the internal standard is tolbutamide.
Preferably, the rotation speed of the centrifugation is 10000-14000 rpm, and the centrifugation time is 5-20 min.
Preferably, the temperature of the centrifugation is 4 to 6 ℃.
Preferably, the protein precipitant is methanol and/or acetonitrile.
The invention provides a quantitative detection method of pyrimidine derivative 1D228 enantiomer in a biological sample, which comprises the following steps: performing ultra-efficient synthetic phase chromatography tandem mass spectrometry detection on a sample liquid to be detected to obtain a detection result of a pyrimidine derivative 1D228 enantiomer; the ultra-high performance phase-combining chromatography tandem mass spectrometry detection comprises ultra-high performance phase-combining chromatography detection and mass spectrometry detection; the conditions for ultra-high performance phase-combining chromatography detection include: the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is CO 2 The mobile phase B is a methanol-ammonium formate-formic acid system; the flow rate of the mobile phase system is 1.0mL/min; the elution mode is gradient elution; the gradient elution procedure was: 0.0 to 1.0min: the volume percentage of the mobile phase B is 60%;1.0 to 5.0min: the volume percentage of the mobile phase B is increased from 60% to 80% at a constant speed; 5.0 to 8.0min: the volume percentage of the mobile phase B is 80%;8.0 to 10.0min: the volume percentage of the mobile phase B is reduced from 80% at a constant speedTo 60%; the conditions for mass spectrometry detection include: the ion source is an electrospray ion source; the detection mode is multi-reaction ion monitoring; the scanning mode is a positive ion mode; the capillary voltage is 2.0kV; taper hole voltage: 20V; taper hole air flow rate: 10L/h; desolventizing temperature: 500 ℃; desolventizing flow rate: 1000L/h;
the enantiomer of the pyrimidine derivative 1D228 is R-1D228 and S-1D228.
According to the invention, chiral separation of R-1D228 and S-1D228 in a biological sample is realized through ultra-efficient phase-combining chromatography tandem mass spectrometry detection, and the problems that the sensitivity of an HPLC method is insufficient and quantitative detection cannot be realized due to incompatibility of normal phase solvents and mass spectrometry by using a chiral column by LC-MS/MS are overcome; meanwhile, the detection method has high sensitivity and good precision and accuracy, and meets the requirements of enantiomer detection in pyrimidine derivatives in preclinical and clinical biological samples.
Drawings
FIG. 1 is a graph of a linear representation of the concentration of R-1D 228;
FIG. 2 is a graph showing a linear representation of the concentration of S-1D228;
FIG. 3 is a chromatogram obtained under selective detection conditions for a blank plasma sample;
FIG. 4 is a chromatogram obtained under selective assay conditions of a blank plasma sample supplemented with a minimum quantitative concentration of pyrimidine derivative enantiomer and an internal standard working fluid;
FIG. 5 is a graph of mean plasma concentration versus time for S-1D288 concentrations in Beagle canine plasma for each subject group.
Detailed Description
The invention provides a quantitative detection method of pyrimidine derivative 1D228 enantiomer in a biological sample, which comprises the following steps:
performing ultra-efficient synthetic phase chromatography tandem mass spectrometry (UPCC-MS/MS) detection on a sample liquid to be detected to obtain a detection result of pyrimidine derivative enantiomer;
the pyrimidine derivative 1D228 has the structural formula:
in the present invention, unless otherwise specified, the reagents used are commercially available products well known to those skilled in the art.
In the present invention, the method for obtaining the sample liquid to be tested preferably includes the following steps.
And mixing the biological sample to be detected, the internal standard substance and the protein precipitant, and centrifuging to obtain the sample liquid to be detected.
In the invention, the biological sample to be tested preferably comprises plasma, tissue homogenate, urine, bile or faecal homogenate; the plasma in the embodiments of the present invention is preferably Beagle canine plasma. In the present invention, the internal standard is preferably tolbutamide. In the present invention, the protein precipitant is preferably methanol and/or acetonitrile, and more preferably acetonitrile. In the invention, the volume ratio of the biological sample to be detected to the protein precipitant is preferably 1:2-3, and more preferably 1:3.
In the present invention, the rotational speed of the centrifugation is preferably 10000 to 14000rpm, more preferably 13000 to 14000rpm; the time for the centrifugation is preferably 5 to 20 minutes, more preferably 10 to 15 minutes. In the present invention, the number of times of centrifugation is preferably 2 times; in the invention, supernatant obtained by centrifugation is used as a sample liquid to be detected.
After obtaining the sample liquid to be detected, the invention carries out ultra-efficient synthetic phase chromatography tandem mass spectrometry detection on the sample liquid to be detected to obtain the detection result of pyrimidine derivative enantiomer.
In the invention, the ultra-high performance synthetic phase chromatography tandem mass spectrometry detection comprises ultra-high performance synthetic phase chromatography detection and mass spectrometry detection.
In the invention, the conditions for ultra-high performance synthetic phase chromatography detection include: the mobile phase system comprises a mobile phase A and a mobile phase B. In the invention, the mobile phase A is CO 2 The CO 2 The purity of (C) is preferably not less than 99.9%. In the present invention, the mobile phase B is a methanol-ammonium formate-formic acid system; the concentration of ammonium formate in the mobile phase B is preferably 5-15 mmol/L, and the volume concentration of formic acid is preferably 0.05-0.2%; the methanol is preferably chromatographic grade methanol.
In the present invention, the flow rate of the mobile phase system is 1.0mL/min.
In the invention, the elution mode is gradient elution; the gradient elution procedure was:
0.0 to 1.0min: the volume percentage of the mobile phase B is 60%;
1.0 to 5.0min: the volume percentage of the mobile phase B is increased from 60% to 80% at a constant speed;
5.0 to 8.0min: the volume percentage of the mobile phase B is 80%;
8.0 to 10.0min: the volume percentage of the mobile phase B is reduced from 80% to 60% at a constant speed.
In the present invention, the conditions for the ultra-high performance synthetic phase chromatography detection preferably further include: the column temperature is preferably 20 to 40 ℃, and more preferably 35 ℃; the sample amount is preferably 1 to 7.5. Mu.L, more preferably 5. Mu.L; the back pressure of the system is preferably 800-2000 psi, more preferably 1500psi; the chromatographic column is preferably Acquity UPCC Trefoil CEL chromatographic column, acquityUPCC Trefoil CEL chromatographic column or ACQUITY UPCC Torus Diol chromatographic column, and further preferably Acquity UPCC Trefoil CEL chromatographic column; the size of the Acquity UPCC Trefoil CEL column is preferably (3.0X105 mm 2.7 μm), the compensation solution is preferably a methanolic formate solution with a volume concentration of 0.1%, and the flow rate of the compensation solution is preferably 0.1-0.5 mL/min, and more preferably 0.4mL/min.
In the present invention, the conditions for mass spectrometry include: the ion source is an electrospray ion source; the detection mode is multi-reaction ion monitoring; the scanning mode is a positive ion mode; the capillary voltage is 2.0kV; taper hole voltage: 20V; taper hole air flow rate: 10L/h; desolventizing temperature: 500 ℃; desolventizing flow rate: 1000L/h.
In the present invention, the detected ion pairs, collision energy, and cone hole voltages for R-1D228 and S-1D228, as well as for the internal standard, are shown in Table 1.
Table 1 Mass Spectrometry parameters for R-1D228 and S-1D228 and tosituric acid
| Compounds of formula (I) | Parent ion (m/z) | Sub-ions (m/z) | Crash energy (V) | Taper hole voltage (V) |
| R-1D228 | 508.36 | 111.95 | 30 | 40 |
| S-1D228 | 508.36 | 111.95 | 30 | 40 |
| Toluene sulfobutyl urea | 466 | 344 | 20 | 17 |
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In an embodiment of the present invention, the specifications of the specific instrument, reagent and sample to be tested are as follows:
the instrument is specifically as follows: the model of the ultra-high performance combined chromatography mass spectrometer (UPCC-MS/MS) is preferably UPC2-XEVO TQD, UPC2-XEVO TQS or XEVO TQ-XS, and more preferably UPC2-XEVO TQD.
The conditions for ultra-high performance synthetic phase chromatography tandem mass spectrometry detection are as follows:
conditions for ultra-high performance phase-combining chromatography detection include: the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is CO 2 The purity is more than or equal to 99.9 percent, and the mobile phase B is a methanol-ammonium formate-formic acid system; the molar concentration of ammonium formate in the methanol-ammonium formate-formic acid system is 5 to 15mmol/L, more preferably 10mmol/L; the volume concentration of formic acid is 0.05 to 0.2%, more preferably 0.1%; the chromatographic column is Acquity UPCC Trefoil CEL2 chromatographic column; the column temperature is 35 ℃; the sample injection amount is 5 mu L; the system back pressure is 1500psi; the flow rate of the mobile phase system is 1.0mL/min;
the elution mode is gradient elution; the gradient elution procedure was:
0.0 to 1.0min: the volume percentage of the mobile phase B is 60%;
1.0 to 5.0min: the volume percentage of the mobile phase B is increased from 60% to 80% at a constant speed;
5.0 to 8.0min: the volume percentage of the mobile phase B is 80%;
8.0 to 10.0min: the volume percentage of the mobile phase B is reduced from 80% to 60% at a constant speed;
the conditions for mass spectrometry include: the ion source is an electrospray ion source; the detection mode is multi-reaction ion monitoring; the scanning mode is a positive ion mode; the capillary voltage is 2.0kV; taper hole voltage: 20V; taper hole air flow rate: 10L/h; desolventizing temperature: 500 ℃; desolventizing flow rate: 1000L/h.
Example 1
1. Preparation of sample and working fluid
1. Preparation of standard Curve sample
(1) Preparation of stock solution:
preparing R-1D228 and S-1D228 reference substance stock solutions: after dissolving 10mg of R-1D228 and S-1D228 with a small amount of ethanol, diluting the solution to a volume of 10mL with 50% methanol, and storing the solution in a brown volumetric flask to obtain control stock solutions of R-1D228 and S-1D228 with concentrations of 1 mg/mL.
Preparing tolbutamide internal standard stock solution: 10mg of tolbutamide is dissolved by using a small amount of methanol, diluted by using 50% methanol with volume concentration to a volume of 10mL brown volumetric flask, and prepared into tolbutamide internal standard stock solution with concentration of 1mg/mL, and the stock solution is refrigerated and stored.
(2) Preparation of Mixed Linear samples
A proper amount of R-1D228 and S-1D228 reference stock solutions are respectively removed by a pipette, and are prepared into mixed linear samples of R-1D228 and S-1D228 with serial concentrations of 1,2, 5, 10, 20, 50, 100, 200 and 500ng/mL by using blank plasma.
(3) Preparing an internal standard working solution: an appropriate amount of tolbutamide internal standard stock solution is respectively removed by a pipette, and 50% methanol is used for preparing internal standard working solutions with series concentration of 1000 ng/mL.
2. Preparation of mixed quality control sample and working solution
(1) Preparing a mixed quality control sample: a proper amount of R-1D228 and S-1D228 reference substance stock solutions are respectively removed by a pipette, and are prepared into mixed quality control samples of R-1D228 and S-1D228 with serial concentrations of 1, 3, 30 and 375ng/mL by using blank plasma.
(2) Preparing a mixed quality control working solution: and respectively transferring a proper amount of R-1D228 and S-1D228 reference substance stock solution by using a pipette, and preparing a series of mixed quality control working solutions of R-1D228 and S-1D228 with concentration of 30 and 750ng/mL by using 50% methanol.
2. Obtaining a sample fluid to be tested
(1) Plasma sample pretreatment is known: and precisely transferring 50 mu L of mixed linear sample, adding 5 mu L of internal standard working solution (1000 ng/mL), then adding 150 mu L of acetonitrile, swirling for 30s, centrifuging for 10min at 14000r/min, controlling the temperature at 4 ℃, taking supernatant, centrifuging for 10min at 14000r/min again, controlling the temperature at 4 ℃, and taking supernatant.
(2) Pretreatment of unknown plasma samples: accurately removing 50 mu L of unknown plasma sample, adding 5 mu L of internal standard working solution (1000 ng/mL), adding 150 mu L of acetonitrile, swirling for 30s, centrifuging for 10min at 14000r/min, controlling the temperature at 4 ℃, collecting supernatant, centrifuging for 10min at 14000r/min again, controlling the temperature at 4 ℃, and collecting supernatant.
3. Linear regression equation
And (3) taking 50 mu L of mixed linear samples (1, 2, 5, 10, 20, 50, 100, 200 and 500 ng/mL) of R-1D228 and S-1D228, operating according to the operation steps of 'two, (1) known plasma sample pretreatment', and then carrying out sample injection analysis according to the conditions of ultra-high performance liquid chromatography tandem mass spectrometry detection. And (3) carrying out linear regression by taking the concentration of the mixed linear sample of R-1D228 and S-1D228 as an abscissa and the ratio Y of the peak area of R-1D228 and S-1D228 to the area of toluene sulfonic acid Ding Niaofeng as an ordinate, so as to obtain a linear regression equation, wherein the results are shown in figures 1 and 2. FIGS. 1 and 2 are linear representations of the concentration of R-1D228 and S-1D228, respectively. As can be seen from FIGS. 1-2, the R-1D228 and S-1D228 standard curves have linear ranges of 1-500 ng/mL, and the linearity R is greater than 0.99, and the linearity is good.
4. Methodological validation test
1. Selective detection
(1) Selecting 50 mu L of each blank plasma sample from different sources, adding 150 mu L of acetonitrile, swirling for 30s, centrifuging for 10min at 14000r/min, controlling the temperature at 4 ℃, taking supernatant, centrifuging for 10min at 14000r/min again, controlling the temperature at 4 ℃, taking supernatant, and then carrying out sample injection analysis according to the conditions of ultra-high performance liquid chromatography-tandem mass spectrometry detection to obtain a blank plasma sample chromatogram, as shown in figure 3.
(2) Pyrimidine derivative enantiomers (R-1D 228 and S-1D 228) with the lowest quantitative concentration (LLOQ, 1 ng/mL) and 5 mu L of internal standard working solution are added into blank plasma, then 150 mu L of acetonitrile is added, the mixture is vortexed for 30S,14000R/min for centrifugation for 10min at 4 ℃, the temperature is controlled, the supernatant is taken, the mixture is centrifuged for 10min again at 14000R/min, the temperature is controlled at 4 ℃, the supernatant is taken, and then the corresponding chromatogram is obtained according to the sample injection analysis under the condition of the ultra-high performance liquid chromatography tandem mass spectrometry detection, and the corresponding chromatogram is shown in figure 4.
It is required that the peak area of the target compound in the blank sample should not exceed 20% of the peak area of the LLOQ (1 ng/mL) sample and be less than 5% of the internal standard response, and it is acceptable that the peak area of the target compound in the blank sample should not exceed 20% of the peak area of the LLOQ sample and be less than 5% of the internal standard response, as shown in FIGS. 3 to 4, and thus meet the standard.
2. Quantitative lower limit precision and accuracy verification
Three mixed quality control samples with the concentrations of 3, 30 and 375ng/mL (6 samples with each concentration of each batch) are detected according to the detection conditions of the ultra-high performance phase-combining chromatography-tandem mass spectrometry, and the precision and the accuracy between batches are obtained by data of three analysis batches, and the detection results are shown in tables 2-3.
And verifying the precision and accuracy of the lower limit of the quantification, wherein the accuracy (relative error: RE%) is required to be less than or equal to 20%, and the precision (variation coefficient: CV%) is less than or equal to 20%. )
TABLE 2 results of verification of precision and accuracy of UPCC-MS/MS determination of R-1D228 in plasma
TABLE 3 results of verification of precision and accuracy of UPCC-MS/MS determination of S-1D228 in plasma
From tables 2 to 3, it can be seen that: UPCC-MS/MS determines that the inter-batch CV% of the lower limit of the quantification of R-1D228 in plasma is 12.67% and the inter-batch RE% is-5.02%; UPCC-MS/MS measured that the CV% between batches was 7.91% and the RE% between batches was 0.79% at the lower limit of the quantification of S-1D228 in plasma.
3. Residual verification
After injection of the upper limit of quantification (500 ng/mL), 2 blank samples were set to investigate the residue. The residue in the blank sample after the upper limit of quantification does not exceed 20% of the lower limit of quantification and does not exceed 5% of the response of the internal standard. The residue meets the acceptance criteria.
4. Matrix effect validation
Taking 45 mu L of blank plasma of 6 animals respectively, adding 150 mu L of acetonitrile, swirling for 30s, centrifuging for 10min at 14000r/min, controlling the temperature at 4 ℃, taking supernatant, centrifuging for 10min at 14000r/min again, controlling the temperature at 4 ℃, taking supernatant, adding 5 mu L of low-concentration and high-concentration (3, 375 ng/mL) mixed quality control working solution and 5 mu L of internal standard working solution into the obtained supernatant, swirling and uniformly mixing, and carrying out sample injection analysis.
The blank plasma was replaced with 50% methanol by volume concentration and was subjected to sample injection analysis according to the blank plasma procedure. Calculating matrix factors of R-1D228, S-1D228 and an internal standard by detecting the area ratio of the obtained target compound to the internal standard peak; the internal standard normalized matrix factor is calculated by dividing the matrix factor of the analyte by the matrix factor of the internal standard, and the overall coefficient of variation of the calculated value must not be greater than 15%, i.e., meet the standard.
The results of the matrix effect are shown in tables 4 and 5, and from tables 4 to 5, it can be seen that: the average value of the peak area ratio of the target compound R-1D228 of the R-1D228 in the blank plasma measured by UPCC-MS/MS is 118.50 percent and 77.95 percent respectively, the average value of the peak area ratio of the internal standard tolbutamide is 90.39 percent and 89.84 percent respectively, the average value of the internal standard normalized matrix factors is 1.31 and 0.87 respectively, the overall variation coefficient is 6.70 percent and 6.75 percent respectively, and the standard meets the acceptance standard; the peak area ratio average values of the target compound R-1D228 of the S-1D228 under low concentration and high concentration are 91.882 percent and 93.52 percent respectively, the peak area ratio average values of the internal standard tolbutamide are 90.39 percent and 89.84 percent respectively, the average values of the internal standard normalized matrix factors are 1.02 and 1.04 respectively, and the overall variation coefficients are 5.52 percent and 6.32 percent respectively, so that the compound meets the acceptance standard.
TABLE 4 results of validation of the matrix effect of measuring the concentration of R-1D228 in plasma by UPCC-MS/MS
TABLE 5 results of validation of the effect of the matrix on the concentration of S-1D228 in plasma by UPCC-MS/MS
5. Recovery verification
Taking 50 mu L of mixed quality control samples of 3ng/mL and 375ng/mL, adding 5 mu L of internal standard working solution (1000 ng/mL), then adding 150 mu L of acetonitrile, swirling for 30s, centrifuging for 10min at 14000r/min, controlling the temperature at 4 ℃, taking supernatant, centrifuging for 10min at 14000r/min again, controlling the temperature at 4 ℃, taking supernatant, and then carrying out sample injection analysis according to the conditions of ultra-high performance liquid chromatography-tandem mass spectrometry detection, wherein each concentration is 6 samples.
And (3) taking 45 mu L of blank plasma, carrying out treatment according to the operation of pretreatment of the second (1) known plasma sample to obtain supernatant, adding 5 mu L of mixed quality control working solution of 3ng/mL and 375ng/mL and 5 mu L of internal standard working solution into the supernatant, mixing uniformly by vortex, and carrying out sample injection analysis. Recovery was calculated as the ratio of peak area for each concentration for both treatments and the recovery data are shown in tables 6-7. As can be seen from tables 6 to 7: UPCC-MS/MS determines that the average recovery rate of R-1D228 in the plasma is 93.68 percent and 102.87 percent at the concentration of 3 and 375ng/mL respectively; the average recovery rate of S-1D228 at the concentration of 3ng/mL and 375ng/mL is 98.78 percent and 99.13 percent respectively; the average recovery rate of the internal standard tolbutamide at the concentration of 3ng/mL and 375ng/mL is 97.01% and 98.14%, respectively.
TABLE 6UPCC-MS/MS determination of concentration extraction recovery of R-1D228 in Beagle canine plasma Table
TABLE 7UPCC-MS/MS determination of concentration extraction recovery of S-1D228 in Beagle canine plasma Table
7. Stability verification
Standing for 24 hours at the temperature of the automatic sampler for stability: taking 50 mu L of a mixed quality control sample (QC) with the concentration of 3ng/mL and 375ng/mL, adding 5 mu L of an internal standard working solution (1000 ng/mL), then adding 150 mu L of acetonitrile, centrifuging for 10min at 14000r/min, controlling the temperature at 4 ℃, taking supernatant, centrifuging for 10min at 14000r/min again, controlling the temperature at 4 ℃, taking supernatant, preparing 6 samples at each concentration, placing the samples at the temperature of an automatic sampler for 24 hours, measuring the concentration of the samples, and evaluating the placement stability of the samples at the temperature of the automatic sampler according to the relative error (RE%) of the measured concentration of the samples and the marked concentration, wherein the test results of the stability are shown in tables 8-9. The measured concentration is compared with the indicated concentration, and the mean value of each concentration is within + -15% of the indicated concentration. From tables 8 to 9, it can be seen that: the relative error (RE%) between the detected concentration and the marked concentration of the quality control sample with low concentration and high concentration R-1D228 in Beagle dog plasma measured by UPCC-MS/MS is 2.62 percent and 2.84 percent respectively after the sample is placed for 24 hours at the temperature of a sample injector; the relative error (RE%) between the detected concentration and the marked concentration of the low-concentration and high-concentration quality control samples of S-1D228 after being placed for 24 hours at the temperature of the sample injector is 9.47% and 3.55% respectively. The method is in accordance with quantitative detection analysis of chiral drugs S-1D228 and R-1D228 in plasma.
TABLE 8UPCC-MS/MS determination of R-1D228 concentration in plasma stability validation results after 24h of standing at the temperature of the injector
TABLE 9UPCC-MS/MS stability verification results table when placed at 24h at the temperature of the sample injector for measuring S-1D228 concentration in plasma
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Example 2: single gastric lavage pharmacokinetic test of pyrimidine derivatives (S-1D 228) on Beagle dogs
The method comprises the steps of 18 Beagle dogs, dividing the Beagle dogs into 3 groups at equal intervals, respectively taking low, medium and high doses of test substances, respectively taking 6 animals in each group, respectively taking 1,2 and 4mg/kg of the test substances, taking the test substances by single gastric lavage, taking blood 0h before and 0.25, 0.5, 1,2, 4, 6, 8, 12 and 24h after the administration of the test substances, taking blood from four limbs veins alternately, taking blood with a volume of about 1.0mL, adding heparin sodium for anticoagulation, centrifuging the blood taking tube at a temperature of 20 ℃ for 15min by using 2000g of relative centrifugal force, and taking upper blood plasma. The samples are stored in a refrigerator at the temperature of minus 80 ℃, operated according to the operation steps of 'two, (2) pretreatment of unknown plasma samples', and then the concentrations of the enantiomers of the novel pyrimidine derivatives in the Beagle canine plasma are detected by sample injection analysis according to the conditions of ultra-efficient phase-combination chromatography tandem mass spectrometry detection, wherein fig. 5 is a graph of average blood concentration-time of the S-1D288 concentration in the Beagle canine plasma of each test group, and the R-1D288 concentration in the Beagle canine plasma of each test group at each time point is lower than the quantitative lower limit (1 ng/mL), so that the graph of average blood concentration-time cannot be drawn. As can be seen from fig. 5: the chiral pyrimidine derivative S-1D288 is singly and intravenously administrated to the Beagle dogs, and the S-1D288 does not generate optical rotation conversion in the Beagle dogs.
Claims (10)
1. A method for quantitatively detecting the enantiomer of pyrimidine derivative 1D228 in a biological sample, comprising the steps of:
performing ultra-efficient synthetic phase chromatography tandem mass spectrometry detection on a sample liquid to be detected to obtain a detection result of a pyrimidine derivative 1D228 enantiomer;
ultra-efficient phase-combining chromatographic detection stripThe piece includes: the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is CO 2 The mobile phase B is a methanol-ammonium formate-formic acid system;
the flow rate of the mobile phase system is 1.0mL/min;
the elution mode is gradient elution; the gradient elution procedure was:
0.0 to 1.0min: the volume percentage of the mobile phase B is 60%;
1.0 to 5.0min: the volume percentage of the mobile phase B is increased from 60% to 80% at a constant speed;
5.0 to 8.0min: the volume percentage of the mobile phase B is 80%;
8.0 to 10.0min: the volume percentage of the mobile phase B is reduced from 80% to 60% at a constant speed;
the detection conditions of the mass spectrum include: the ion source is an electrospray ion source; the detection mode is multi-reaction ion monitoring; the scanning mode is a positive ion mode; the capillary voltage is 2.0kV; taper hole voltage: 20V; taper hole air flow rate: 10L/h; desolventizing temperature: 500 ℃; desolventizing flow rate: 1000L/h;
the enantiomer of the pyrimidine derivative 1D228 is R-1D228 and S-1D228;
the pyrimidine derivative 1D228 has the structural formula:
2. the method according to claim 1, wherein the conditions for detecting the ultra-high performance co-phase chromatography further comprise: the chromatographic column is Acquity UPCC Trefoil CEL chromatographic column, acquity UPCC Trefoil CEL chromatographic column or ACQUITY UPCC Torus Diol chromatographic column; the column temperature is 25-40 ℃; the sample injection amount is 1-7.5 mu L; the back pressure of the system is 800-2000 psi.
3. The method according to claim 1, wherein the molar concentration of ammonium formate in the mobile phase B is 5-15 mmol/L and the volume concentration of formic acid is 0.05-0.2%.
4. The method of claim 1, wherein the CO 2 The purity of the product is more than or equal to 99.9 percent.
5. The method according to claim 1, wherein the method for obtaining the sample liquid to be tested comprises the steps of:
and mixing the biological sample to be detected, the internal standard substance and the protein precipitant, and centrifuging to obtain the sample liquid to be detected.
6. The method of claim 5, wherein the biological sample comprises plasma, tissue homogenate, urine, bile, or stool homogenate.
7. The method according to claim 5, wherein the internal standard is tolbutamide.
8. The method according to claim 5, wherein the rotational speed of the centrifugation is 10000 to 14000rpm and the time of the centrifugation is 5 to 20 minutes.
9. The method according to claim 5 or 8, wherein the temperature of the centrifugation is 4 to 6 ℃.
10. The method according to claim 5, wherein the protein precipitant is methanol and/or acetonitrile.
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