CN114397380A - Method for determining concentration of western-style gliptin in blood plasma by liquid chromatography-mass spectrometry - Google Patents
Method for determining concentration of western-style gliptin in blood plasma by liquid chromatography-mass spectrometry Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a method for determining the concentration of western gliptin in plasma by liquid chromatography-mass spectrometry, which comprises the following steps of plasma sample pretreatment and sample determination: with K2EDTA as an anticoagulant, and sitagliptin-d 4 as an internal standard; adding 50 mu L of sample into a hole of a 96-hole plate, adding internal standard sitagliptin-d 4 working solution, uniformly mixing, adding methanol, closing a plate, uniformly mixing, and centrifuging the sample at 4 ℃ for 10 ℃; taking 100 mu L of centrifuged supernatant to another 96-well collection plate, adding 300 mu L of 50% methanol, and mixing uniformly; centrifuging the sample at 4 ℃ to obtain a test sample; then injecting 1 mu L of test sample into an efficient liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of sitagliptin and internal standard sitagliptin-d 4 in the sample, and calculating the chromatographic peak according to the chromatographic peaksSitagliptin concentration in a plasma sample; the detection method provided by the invention has the advantages that the sitagliptin-d 4 is used as an internal standard, the precipitator is added for protein precipitation, the diluent is added into the supernatant, the supernatant is pretreated, the supernatant is separated by a chromatographic column, and the detection is carried out by a mass spectrum detector, so that the detection speed is high, the precision is high, and the sensitivity is excellent.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a method for determining concentration of western gliptin in blood plasma by liquid chromatography-mass spectrometry.
Background
Sitagliptin (SGT), a dipeptidyl peptidase 4(DPP-4) inhibitor, improves glycemic control in type 2 diabetic patients by increasing the levels of active incretin. The incretins, including glucagon-like polypeptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are released throughout the day from the intestinal tract and rise in levels after meals. Sitagliptin is able to prevent DPP-4 from hydrolyzing gut insulinotropic hormone, thereby increasing plasma concentrations of GLP-1 and GIP in active form. By increasing the active incretin level, sitagliptin is able to increase insulin release and decrease pancreatic glucagon levels in a glucose-dependent manner. For type 2 diabetic patients with hyperglycemia, the above-described changes in insulin and pancreatic glucagon levels can lower glycated hemoglobin A1c (HbA1c) and lower fasting blood glucose and meal glucose levels.
The existing analysis methods for detecting sitagliptin in human plasma in the prior art are different, non-isotopic internal standards are mostly adopted, and the consistency of elution modes of an object to be detected and the internal standards cannot be ensured. In order to meet the requirement of clinical biological sample analysis, a simpler, reliable and time-saving method for determining the concentration of sitagliptin in human plasma needs to be developed.
Disclosure of Invention
The invention aims to provide a method for determining concentration of sitagliptin in plasma by liquid chromatography-mass spectrometry, which takes sitagliptin-d 4 as an internal standard, adds a precipitator to carry out protein precipitation, takes supernatant fluid and adds a diluent, and after pretreatment, the supernatant fluid is separated by a chromatographic column and is detected by a mass spectrometer, thus having high detection speed, high precision and good sensitivity.
The technical scheme of the invention is a method for determining the concentration of the sitagliptin in plasma by liquid chromatography-mass spectrometry, which is characterized by comprising the following steps of:
(1) pretreatment of a plasma sample:
with K2EDTA as an anticoagulant, and sitagliptin-d 4 as an internal standard; adding 50 mu L of sample into a hole of a 96-hole plate, adding 50 mu L of internal standard sitagliptin-d 4 working solution with the concentration of 100.000ng/mL, uniformly mixing, adding 300 mu L of methanol into each sample hole, closing the plate, uniformly mixing for 10min, and centrifuging the sample for 10min at 2623g at 4 ℃; taking 100 mu L of centrifuged supernatant to another 96-well collection plate, adding 300 mu L of 50% methanol, sealing the plate, and mixing uniformly; centrifuging the sample at 2623g for 10min at 4 ℃ to obtain a test sample;
(2) and (3) sample measurement:
injecting 1 mu L of test sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of sitagliptin and internal standard sitagliptin-d 4 in the sample, and calculating the sitagliptin concentration in the plasma sample according to the chromatographic peaks;
the liquid chromatography conditions were:
a chromatographic column: welch Ultimate XB-C18The column specification is 2.1 multiplied by 100mm, 5 μm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume percentage of water/formic acid is 100/0.1; mobile phase B: methanol; strong washing liquid: the volume percentage of water/methanol/formic acid is 20/80/0.1; plunger cleaning solution: 90/10% water/methanol by volume; the temperature of the autosampler is 4 ℃; gradient elution with flow rate of 0.5mL/min, sample size of 1 μ L, and analysis time of 4 min; sitagliptin and sitagliptin-d 4 expected retention time was about 1.55 min;
the mass spectrum conditions are as follows:
the ion source is an electrospray ion source, the temperature of an ion transmission pipe is 325 ℃, the temperature of steam is 350 ℃, and the residence time is 200 ms. The collision voltages of sitagliptin and sitagliptin-d 4 are respectively 18V and 19V, and positive ion mode detection is carried out; the scanning mode is multiple reaction monitoring.
Preferably, the gradient elution procedure is:
preferably, in the step (2), the concentration of sitagliptin in the plasma sample is calculated by adopting an internal standard method and substituting a standard curve equation into a peak area ratio of sitagliptin and internal standard sitagliptin-d 4.
Preferably, the establishment of the standard curve equation comprises the following steps:
putting 190 mu L of blank plasma in a polypropylene tube, respectively adding 10 mu L of sitagliptin working solution with the concentration of 40.00, 80.000, 200.000, 1000.000, 4000.000, 8000.000, 16000.000 and 20000.000ng/mL in the form of working solution, respectively mixing uniformly, respectively adding 50 mu L of standard sample 1, standard sample 2, standard sample 3, standard sample 4, standard sample 5, standard sample 6, standard sample 7, standard sample 8 and a zero-concentration sample into 50 mu L of 100.000ng/mL internal standard sitagliptin-d 4 solution, adding 50 mu L of methanol aqueous solution with the volume fraction of 50% into the double blank samples, mixing uniformly, adding 300 mu L of methanol into each sample hole, closing the plates, mixing uniformly for 10min, and centrifuging the samples for 10min at 4 ℃ and 2623 g; taking 100 mu L of centrifuged supernatant to another 96-well collection plate, adding 300 mu L of 50% methanol, sealing the plate, and mixing for 10 min; centrifuging the sample at 2623g for 10min at 4 ℃ to obtain a standard sample to be subjected to sample injection;
and respectively injecting 1 mu L of standard sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of sitagliptin and internal standard sitagliptin-d 4 in the sample, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of sitagliptin in the plasma sample.
Preferably: the establishment of quality control comprises the following steps:
preparing quality control sample working solution with sitagliptin concentration of 40.000, 120.000, 1200.000, 6000.000 and 15000.000 mug/mL. Putting 380 mu L of blank plasma in a polypropylene tube, respectively adding 20 mu L of quality control working solution, uniformly mixing, respectively taking 50 mu L of quality control sample, adding 50 mu L of 100.000ng/mL internal standard sitagliptin-d 4 solution, uniformly mixing, adding 300 mu L of methanol into each sample hole, closing the plate, uniformly mixing for 10min, and centrifuging the sample at 2623g for 10min at 4 ℃; taking 100 mu L of centrifuged supernatant to another 96-well collection plate, adding 300 mu L of 50% methanol, sealing the plate, and mixing for 10 min; centrifuging the sample at 2623g for 10min at 4 ℃ to obtain a quality control standard sample;
and (3) respectively injecting 1 mu L of standard sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of sitagliptin and internal standard sitagliptin-d 4 in the quality control sample, and calculating the determination concentration of the quality control sample according to the standard curve.
The method for determining the concentration of the sitagliptin in the blood plasma by liquid chromatography-mass spectrometry has the beneficial effects that:
1. an isotope internal standard is used to ensure the elution consistency of the object to be detected and the internal standard;
2. the pretreatment method is simple and convenient and is suitable for high-throughput measurement;
3. the specificity is strong: under the chromatographic conditions used in the experiment, the retention time of sitagliptin is about 1.55min, and the retention time of internal standard sitagliptin-d 4 is about 1.55 min. The peak shapes of sitagliptin and internal standard sitagliptin-d 4 are good, the measurement is not obviously interfered by a miscellaneous peak, and the base line is stable;
4. the method is rapid, accurate and highly specific, and provides a basis for determining the blood concentration of sitagliptin. The linear range of the plasma standard curve of the method is 2.000-1000.000 ng/mL.
Drawings
FIG. 1 is a product ion scanning mass spectrum of sitagliptin in a LC-MS/MS detection method for human plasma;
FIG. 2 is a product ion scanning mass spectrum of sitagliptin-d 4 in the LC-MS/MS detection method for human plasma;
FIG. 3 is a LC-MS/MS graph of human blank plasma;
FIG. 4 is a LC-MS/MS graph of human blank plasma supplemented with sitagliptin and sitagliptin-d 4;
FIG. 5 is a standard graph of sitagliptin in human plasma measured by LC-MS/MS method.
Detailed Description
In order to facilitate the understanding of the technical solutions of the present invention for those skilled in the art, the technical solutions of the present invention will be further described with reference to the drawings attached to the specification.
A method for determining the concentration of sitagliptin in plasma by liquid chromatography-mass spectrometry, also called LC-MS/MS method, uses sitagliptin-d 4 as an internal standard, adds a precipitator to carry out protein precipitation, takes supernatant fluid and adds a diluent, after pretreatment, the supernatant fluid is separated by a chromatographic column and is detected by a mass spectrometer. The method specifically comprises the following steps:
(1) pretreatment of a plasma sample:
with K2EDTA as an anticoagulant, and sitagliptin-d 4 as an internal standard; adding 50 mu L of sample into a hole of a 96-hole plate, adding 50 mu L of internal standard sitagliptin-d 4 working solution with the concentration of 100.000ng/mL, uniformly mixing, adding 300 mu L of methanol into each sample hole, closing the plate, uniformly mixing for 10min, and centrifuging the sample for 10min at 2623g at 4 ℃; taking 100 mu L of centrifuged supernatant to another 96-well collection plate, adding 300 mu L of 50% methanol (containing 0.5% formic acid), sealing the plate, and mixing uniformly; the sample was centrifuged at 2623g for 10min at 4 ℃ to obtain a test sample to be injected.
(2) And (3) sample measurement:
injecting 1 mu L of test sample into an HPLC tandem mass spectrometer, detecting chromatographic peaks of sitagliptin and internal standard sitagliptin-d 4 in the sample, and calculating the sitagliptin concentration in the plasma sample according to the chromatographic peaks.
The liquid chromatography conditions were:
a chromatographic column: welch Ultimate XB-C18The column specification is 2.1 multiplied by 100mm, 5 μm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume percentage of water/formic acid is 100/0.1; mobile phase B: methanol; strong washing liquid: the volume percentage of water/methanol/formic acid is 20/80/0.1; plunger cleaning solution: 90/10% water/methanol by volume; the temperature of the autosampler is 4 ℃; gradient elution with flow rate of 0.5mL/min, sample size of 1 μ L, and analysis time of 4 min; sitagliptin and sitagliptin-d 4 expected retention times were around 1.55 min.
The mass spectrum conditions are as follows:
the ion source is an electrospray ion source, the temperature of an ion transmission pipe is 325 ℃, the temperature of steam is 350 ℃, and the residence time is 200 ms. The collision voltages of sitagliptin and sitagliptin-d 4 are respectively 18V and 19V, and positive ion mode detection is carried out; the scanning mode is multiple reaction monitoring. The ion reactions for quantitative analysis were: sitagliptin: m/z 408.1 → 235.1 and sitagliptin-d 4: m/z 412.2 → 239.1.
The procedure for the gradient elution described above was:
in the step (2), an internal standard method is adopted, and the concentration of sitagliptin in the plasma sample is calculated by substituting the peak area ratio of sitagliptin and the internal standard sitagliptin-d 4 into a standard curve equation.
In the technical scheme, the establishment of the standard curve equation comprises the following steps:
putting 190 mu L of blank plasma in a polypropylene tube, respectively adding 10 mu L of sitagliptin working solution with the concentration of 40.00, 80.000, 200.000, 1000.000, 4000.000, 8000.000, 16000.000 and 20000.000ng/mL in the form of working solution, respectively mixing uniformly, respectively adding 50 mu L of standard sample 1, standard sample 2, standard sample 3, standard sample 4, standard sample 5, standard sample 6, standard sample 7, standard sample 8 and a zero-concentration sample into 50 mu L of 100.000ng/mL internal standard sitagliptin-d 4 solution, adding 50 mu L of methanol aqueous solution with the volume fraction of 50% into the double blank samples, mixing uniformly, adding 300 mu L of methanol into each sample hole, closing the plates, mixing uniformly for 10min, and centrifuging the samples for 10min at 4 ℃ and 2623 g; collecting supernatant 100 μ L after centrifugation, adding into another 96-well collection plate, adding 300 μ L50% methanol (containing 0.5% formic acid), sealing, and mixing for 10 min; the sample was centrifuged at 2623g for 10min at 4 ℃ to obtain a standard sample to be injected.
And respectively injecting 1 mu L of standard sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of sitagliptin and internal standard sitagliptin-d 4 in the sample, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of sitagliptin in the plasma sample.
In the technical scheme: the establishment of quality control comprises the following steps:
preparing quality control sample working solution with sitagliptin concentration of 40.000, 120.000, 1200.000, 6000.000 and 15000.000 mug/mL. Putting 380 mu L of blank plasma in a polypropylene tube, respectively adding 20 mu L of quality control working solution, uniformly mixing, respectively taking 50 mu L of quality control sample, adding 50 mu L of 100.000ng/mL internal standard sitagliptin-d 4 solution, uniformly mixing, adding 300 mu L of methanol into each sample hole, closing the plate, uniformly mixing for 10min, and centrifuging the sample at 2623g for 10min at 4 ℃; collecting supernatant 100 μ L after centrifugation, adding into another 96-well collection plate, adding 300 μ L50% methanol (containing 0.5% formic acid), sealing, and mixing for 10 min; centrifuging the sample at 2623g for 10min at 4 deg.C to obtain quality control standard sample for sample injection.
And (3) respectively injecting 1 mu L of standard sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of sitagliptin and internal standard sitagliptin-d 4 in the quality control sample, and calculating the determination concentration of the quality control sample according to the standard curve.
In order to further understand the method for determining the concentration of the sitagliptin in the plasma by liquid chromatography-mass spectrometry in the technical scheme, a group of specific experimental processes are disclosed below.
First, experimental material and analytical equipment
Sitagliptin calcium (analyte): european Pharmacopoeia or the same, higher-grade standard. Western grid
Gliptin-d 4 sodium salt (internal standard): TLC Pharmaceutical Standards Ltd or the same, higher grade standard. The reagents used are shown in Table 1 below and the analytical equipment used is shown in Table 2 below.
TABLE 2 details of reagents
TABLE 2 details of the devices used
Liquid condition:
1. conditions of liquid chromatography
A chromatographic column: welch Ultimate XB-C18The column size was 2.1X 100mm, 5 μm.
Temperature of the chromatographic column: at 40 ℃.
Mobile phase A: the volume percentage of water/formic acid was 100/0.1. Mobile phase B: methanol.
Strong washing liquid: the volume percentage of water/methanol/formic acid was 20/80/0.1.
Plunger cleaning solution: the water/methanol volume percentage was 90/10. The autosampler temperature was 4 ℃.
Gradient elution, flow rate of 0.5mL/min, sample size of 1 μ L, analysis time of 4 min.
Sitagliptin and sitagliptin-d 4 expected retention times were around 1.55 min.
Specific gradient elution procedures are shown in table 3;
TABLE 3 gradient elution procedure
2. Mass spectrum conditions:
the ion source is an electrospray ion source, the temperature of an ion transmission pipe is 325 ℃, the temperature of steam is 350 ℃, and the residence time is 200 ms. The collision voltages of sitagliptin and sitagliptin-d 4 are respectively 18V and 19V, and positive ion mode detection is carried out; the scanning mode is multiple reaction monitoring.
The ion reactions for quantitative analysis were: sitagliptin: m/z 408.1 → 235.1 and sitagliptin-d 4: m/z 412.2 → 239.1.
Second, the experimental procedure
1. Preparation of sitagliptin standard curve working solution
Preparing a sitagliptin standard curve working solution: accurately weighing a proper amount of sitagliptin standard substance, and dissolving the sitagliptin standard substance in 80% methanol after correcting the mass correction coefficient to obtain a stock solution with the final concentration of 1.000 mg/mL. And sequentially diluting with 50% methanol to prepare a sitagliptin working solution, wherein the specific dilution concentration is shown in the following table 4.
TABLE 4 preparation concentration of sitagliptin working solution
SS denotes stock solution, WS denotes working solution. The sitagliptin standard curve working solution is prepared in a brown glass bottle and stored at the temperature of-20 ℃, and the volume can be increased or decreased according to the requirement.
2. Preparation of sitagliptin quality control working solution
Preparing a sitagliptin working solution: accurately weighing a proper amount of sitagliptin standard substance, dissolving the sitagliptin standard substance in 80% methanol after correcting the mass correction coefficient to obtain a stock solution with the final concentration of 1.000mg/mL, and sequentially diluting with 50% methanol to prepare a sitagliptin working solution, wherein the specific dilution concentration is shown in the following table 5.
TABLE 5 preparation concentration of sitagliptin quality control working solution
SS denotes stock solution, WS denotes working solution. The sitagliptin working solution is prepared in a brown glass bottle and stored at the temperature of-20 ℃, and the volume can be increased or reduced according to the requirement.
3. Preparation of internal standard working solution of sitagliptin-d 4
Preparation of a working solution of sitagliptin-d 4 internal standard: taking one standard sitagliptin-d 4, correcting by a mass correction coefficient, and dissolving the standard sitagliptin-d 4 into 80% methanol to obtain a stock solution with the final concentration of 0.500 mg/mL. And then diluting with 50% volume fraction methanol aqueous solution to prepare 20.000ng/mL internal standard sitagliptin-d 4 working solution, wherein the specific dilution concentration is shown in the following table 6.
TABLE 6 preparation concentrations of sitagliptin-d 4 working solution
The internal standard working solution of sitagliptin-d 4 is prepared in a brown glass bottle and stored at the temperature of-20 ℃, and the volume can be increased or decreased proportionally according to the needs.
4. Linear experiment
Unfreezing blank plasma at room temperature; transferring 8 parts of 190 mu L blank plasma into a polypropylene tube (each standard curve sample), respectively and precisely adding 10 mu L of sitagliptin working solution with different concentrations to prepare each sample, uniformly mixing to prepare drug-containing plasma with different concentrations, and operating according to 'plasma sample pretreatment'. And calculating the ratio Y (Y is As/Ai) of the peak area As of sitagliptin and the peak area Ai of the internal standard sitagliptin-d 4, and performing regression calculation on the blood concentration X by using the peak area ratio Y. The mean ratio Y is used for carrying out regression calculation on the blood concentration X, and the lowest quantitative limit of the blood concentration of sitagliptin measured by the method is as follows: 2.000 ng/mL. The standard curve parameters of sitagliptin in human plasma measured by the LC-MS/MS method are shown in Table 7.
TABLE 7 Standard Curve (ng/mL) of sitagliptin in human plasma measured by LC-MS/MS method
5. Accuracy and precision
Unfreezing blank plasma at room temperature; a blank plasma with proper volume is transferred into a polypropylene tube, and sitagliptin quality control working solution is added to prepare 5 drug-containing plasma quality control samples (LLOQ, LQC, M1QC, M2QC and HQC) with different concentrations and a follower standard curve, and the operation is carried out according to the 'plasma sample pretreatment'. And in at least two days, completing in at least three analysis batches, calculating the ratio Y of the sitagliptin peak area As to the internal standard sitagliptin-d 4 peak area Ai, substituting the ratio Y into the standard curve of the current day to obtain the measured concentration, calculating the precision between the batches according to the measured concentration, wherein the ratio of the measured concentration to the added concentration is the accuracy, and the result is shown in the table 8. The result shows that the precision and accuracy of the sitagliptin plasma sample in batches and among batches are less than +/-15 percent and meet the requirements.
As required for each assay batch, a sufficient volume was selected to be dispensed into labeled polypropylene tubes and stored at-70 ℃. The volume may be scaled up or down as desired.
TABLE 8 LC-MS/MS method for determining the accuracy and precision of the plasma midwifery
6. Matrix effect
Six different blank plasma samples are respectively from different healthy human bodies, and the six different blank plasma samples are prepared and analyzed in the same analysis batch according to the sample preparation steps to evaluate the interference of the different blank plasma on the sitagliptin analyte and the internal standard sitagliptin-d 4. Wherein the CV percent of the internal standard normalized matrix factor is less than or equal to 1.8 percent. See table 9 for details.
In table 9, the area peak area is considered zero when "no significant peak can be integrated (or no peak)" or "the retention time of the peak area does not match the retention time of the analyte or internal standard in the sample".
TABLE 9 matrix Effect of blank healthy human plasma from six different sources
As can be seen from table 9, blank plasma from different human bodies did not interfere with the results of sitagliptin detection. Therefore, the method can be used for detecting the concentrations of sitagliptin in the plasma of different human bodies.
7. Dilution reliability
The sample preparation method for inspecting the reliability of 2 times of sample dilution comprises the following steps: taking a sitagliptin stock solution with the concentration of 1.000mg/mL, and taking 50% methanol as a diluent to prepare a working solution with the sitagliptin concentration of 30000.000 ng/mL.
Unfreezing blank plasma at room temperature, transferring 380 mu L of blank plasma to a polypropylene tube, adding 20 mu L of the working solution to prepare a drug-containing plasma dilution quality control sample (the concentration is 1500.000ng/mL), uniformly mixing by vortex to prepare a sample higher than the upper limit of quantification, then taking 50 mu L of the sample, adding 50 mu L of the blank plasma in a new polypropylene tube, and diluting by 2 times to obtain a diluted 2-fold sample (the concentration is 750.000 ng/mL). The "plasma sample pretreatment" procedure was followed, with 6 replicates, and the results are shown in Table 10. The accuracy deviation is-0.6% -5.0%, which shows that the dilution reliability meets the acceptance standard.
TABLE 10 dilution reliability examination results
8. Recovery rate
The extraction recovery rate is determined by comparing the analyte/internal standard response in the treated spiked sample with the response value of the spiked analyte/internal standard in the treated blank matrix. Samples of 3 concentrations of LQC, M2QC, HQC were prepared, with 6 replicates per concentration level. Standards were accepted for the analyte of each concentration level of sample and internal standards for all concentration levels: the peak area of the normal extracted sample, the peak area of the sample after the substrate is extracted, and the CV percent of the recovery rate of the sample at each concentration level are 1.3 to 2.8 percent. Sitagliptin and sitagliptin-d 4 both met the acceptance criteria and the results are shown in tables 11 and 12.
TABLE 11 recovery rate of extraction of analyte
Table 12 internal standard recovery
9. Stability of
(1) Freeze thaw stability
The preparation method of the sample is the same as that of the quality control sample, the sample is respectively placed in a refrigerator at the temperature of-70 ℃ or a refrigerator at the temperature of-20 ℃ for storage after the preparation is finished, then the sample is taken out and placed for melting under the condition of room temperature, each freezing and thawing is carried out for 5 times, each group is parallel to 6 parts, then 1 mu L of the test sample is taken and injected into the high performance liquid chromatography tandem mass spectrometer according to the operation of 'plasma sample pretreatment', and the result is shown in tables 13 and 14.
The deviation between the measured concentration and the standard value of the high/low concentration sample is in the range of-8.9 to-0.1 percent under the condition of 5 times of freezing and thawing at-70 ℃, and the deviation between the measured concentration and the standard value of the high/low concentration sample is in the range of-13.7 to-3.8 percent under the condition of 5 times of freezing and thawing at-20 ℃, which indicates that the sample is stable after 5 times of freezing and thawing treatment in a refrigerator at-70 ℃ or-20 ℃.
TABLE 13-70 ℃ stability test results of 5 times of freezing and thawing at room temperature
TABLE 14-20 ℃ stability test results of 5 times of freezing and thawing at room temperature
(2) Long term stability
The preparation method of the sample is the same as that of the quality control sample, the sample is respectively placed in a refrigerator with the temperature of-70 ℃ for storage for 46 days after the preparation is finished, then the sample is taken out and placed at room temperature for melting, 6 parts of each group are parallel, then 1 mu L of the test sample is taken and injected into the high performance liquid chromatography tandem mass spectrometer according to the operation of 'pretreatment of plasma samples', and the result is shown in Table 15.
After the high/low concentration samples were stored at-70 ℃ for 46 days, the average deviation of accuracy between the measured concentration and the indicated value was in the range of-4.8% to 3.9%, indicating that the samples were stable when stored in a refrigerator at-70 ℃ for 46 days.
TABLE 15-70 ℃ long-term storage stability test results
10. Human plasma sample detection
Samples thawed at room temperature or newly formulated samples were vortexed and mixed. Adding 50 mu L of sample (standard curve, quality control sample, system applicability sample or biological sample to be detected, and adding 50 mu L of blank matrix sample for double blank sample or zero sample) into the hole of a 96-well plate; for a double blank sample or a ULOQ Without IS sample, 50 mu L of 50% methanol solution IS added, 50 mu L of internal standard working solution (100.000ng/mL) IS added for other samples, 300 mu L of methanol IS added into each sample hole after uniform mixing, a sealing plate IS sealed, uniform mixing IS carried out for 10min, the sample IS centrifuged at 4 ℃ for 10min, 100 mu L of supernatant after centrifugation IS taken to another 96-hole collecting plate, 300 mu L of 50% methanol (containing 0.5% formic acid) IS added, the sealing plate IS sealed, the sample IS centrifuged at 2623 ℃ for 10min after uniform mixing for 10min, and the sample IS ready to be injected. The above processes are all carried out under room temperature yellow light.
Four, small knot
In conclusion, the invention provides a simple and convenient method for determining the concentration of the sitagliptin in the blood plasma by the pretreatment method, adopts simple protein precipitation treatment, and is suitable for conventional determination; from the analysis, under the chromatographic conditions adopted in the experiment, the retention time of sitagliptin is about 1.55min, the retention time of internal standard sitagliptin-d 4 is about 1.55min, the peak shapes of the sitagliptin and the internal standard sitagliptin-d 4 are good, the measurement is free of obvious peak interference, and the base line is stable. The linear range of the plasma standard curve of the method is 2.000 ng/mL-1000.000 ng/mL, and the precision CV% in batch and between batches are both less than +/-15%; the method has the advantages of high specificity, good stability, convenience, controllability and capability of accurately measuring the concentration of sitagliptin in blood plasma; meanwhile, the method is accurate and good in reproducibility, and provides a basis for the blood concentration determination of sitagliptin.
Technical solution of the invention is described above with reference to the accompanying drawings, it is obvious that the specific implementation of the invention is not limited by the above-mentioned manner, and it is within the scope of the invention to adopt various insubstantial modifications of the inventive method concept and technical solution, or to apply the inventive concept and technical solution to other occasions without modification.
Claims (5)
1. A method for determining the concentration of sitagliptin in plasma by liquid chromatography-mass spectrometry is characterized by comprising the following steps:
(1) pretreatment of a plasma sample:
with K2EDTA as an anticoagulant, and sitagliptin-d 4 as an internal standard; add 50. mu.L of sample to wells of 96-well plate and add 50. mu.LMixing the internal standard sitagliptin-d 4 working solution with the L concentration of 100.000ng/mL, adding 300 mu L of methanol into each sample hole after uniformly mixing, closing a plate, uniformly mixing for 10min, and centrifuging the sample for 10min at 2623g at 4 ℃; taking 100 mu L of centrifuged supernatant to another 96-well collection plate, adding 300 mu L of 50% methanol, sealing the plate, and mixing uniformly; centrifuging the sample at 2623g for 10min at 4 ℃ to obtain a test sample to be subjected to sample injection;
(2) and (3) sample measurement:
injecting 1 mu L of test sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of sitagliptin and internal standard sitagliptin-d 4 in the sample, and calculating the sitagliptin concentration in the plasma sample according to the chromatographic peaks;
the liquid chromatography conditions were:
a chromatographic column: welch Ultimate XB-C18The column specification is 2.1 multiplied by 100mm, 5 μm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume percentage of water/formic acid is 100/0.1; mobile phase B: methanol; strong washing liquid: the volume percentage of water/methanol/formic acid is 20/80/0.1; plunger cleaning solution: 90/10% water/methanol by volume; the temperature of the autosampler is 4 ℃; gradient elution with flow rate of 0.5mL/min, sample size of 1 μ L, and analysis time of 4 min; sitagliptin and sitagliptin-d 4 expected retention time was about 1.55 min;
the mass spectrum conditions are as follows:
the ion source is an electrospray ion source, the temperature of an ion transmission pipe is 325 ℃, the temperature of steam is 350 ℃, and the residence time is 200 ms. The collision voltages of sitagliptin and sitagliptin-d 4 are respectively 18V and 19V, and positive ion mode detection is carried out; the scanning mode is multiple reaction monitoring.
2. The method for determining the concentration of sitagliptin in plasma in combination liquid chromatography mass spectrometry according to claim 1, wherein the gradient elution procedure is as follows:
3. the method for determining the concentration of sitagliptin in plasma by liquid chromatography mass spectrometry as claimed in claim 1, wherein in the step (2), the concentration of sitagliptin in the plasma sample is calculated by adopting an internal standard method and substituting the peak area ratio of sitagliptin and internal standard sitagliptin-d 4 into a standard curve equation.
4. The method for determining the concentration of sitagliptin in plasma in combination liquid chromatography mass spectrometry as claimed in claim 1, wherein the establishment of the standard curve equation comprises the following steps:
putting 190 mu L of blank plasma in a polypropylene tube, respectively adding 10 mu L of sitagliptin working solution with the concentration of 40.00, 80.000, 200.000, 1000.000, 4000.000, 8000.000, 16000.000 and 20000.000ng/mL in the form of working solution, respectively mixing uniformly, respectively adding 50 mu L of standard sample 1, standard sample 2, standard sample 3, standard sample 4, standard sample 5, standard sample 6, standard sample 7, standard sample 8 and a zero-concentration sample into 50 mu L of 100.000ng/mL internal standard sitagliptin-d 4 solution, adding 50 mu L of methanol aqueous solution with the volume fraction of 50% into the double blank samples, mixing uniformly, adding 300 mu L of methanol into each sample hole, closing the plates, mixing uniformly for 10min, and centrifuging the samples for 10min at 4 ℃ and 2623 g; taking 100 mu L of centrifuged supernatant to another 96-well collection plate, adding 300 mu L of 50% methanol, sealing the plate, and mixing for 10 min; centrifuging the sample at 2623g for 10min at 4 ℃ to obtain a standard sample to be subjected to sample injection;
and respectively injecting 1 mu L of standard sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of sitagliptin and internal standard sitagliptin-d 4 in the sample, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of sitagliptin in the plasma sample.
5. The method for determining the concentration of sitagliptin in plasma in combination of liquid chromatography mass spectrometry according to claim 1, wherein the concentration of sitagliptin in plasma is determined by the following steps: the establishment of quality control comprises the following steps:
preparing quality control sample working solution with sitagliptin concentration of 40.000, 120.000, 1200.000, 6000.000 and 15000.000 mug/mL. Putting 380 mu L of blank plasma in a polypropylene tube, respectively adding 20 mu L of quality control working solution, uniformly mixing, respectively taking 50 mu L of quality control sample, adding 50 mu L of 100.000ng/mL internal standard sitagliptin-d 4 solution, uniformly mixing, adding 300 mu L of methanol into each sample hole, closing the plate, uniformly mixing for 10min, and centrifuging the sample at 2623g for 10min at 4 ℃; taking 100 mu L of centrifuged supernatant to another 96-well collection plate, adding 300 mu L of 50% methanol, sealing the plate, and mixing for 10 min; centrifuging the sample at 4 deg.C and 2623g for 10min to obtain quality control standard sample to be injected;
and (3) respectively injecting 1 mu L of standard sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of sitagliptin and internal standard sitagliptin-d 4 in the quality control sample, and calculating the determination concentration of the quality control sample according to the standard curve.
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| GABRIEL ONN KIT LOH ET AL.: "Simple and rapid LC-MS/MS method for determination of sitagliptin in human plasma and application to bioequivalence study" * |
| 刘珊珊 等: "LC-MS/MS法分析人血浆中西格列汀浓度及其应用研究" * |
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