CN114397379A - Method for determining concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry - Google Patents
Method for determining concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry Download PDFInfo
- Publication number
- CN114397379A CN114397379A CN202111538151.9A CN202111538151A CN114397379A CN 114397379 A CN114397379 A CN 114397379A CN 202111538151 A CN202111538151 A CN 202111538151A CN 114397379 A CN114397379 A CN 114397379A
- Authority
- CN
- China
- Prior art keywords
- ornidazole
- sample
- concentration
- plasma
- standard
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- G01N30/02—Column chromatography
-
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- 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
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
-
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
Abstract
The invention discloses a method for determining the concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry, which uses EDTA-K2As an anticoagulant, taking ornidazole-d 5 as an internal standard; adding 50 mu L of plasma sample to be detected into a hole of a 96-well plate; then 50 mul of internal standard ornidazole-d 5 working solution with the concentration of 1.000 mug/mL is added; after mixing uniformly, adding 300 mu L of methanol into each sample hole, sealing the plate, mixing uniformly, and centrifuging; taking 50 mu L of centrifuged supernatant to another 96-well collection plate, adding 200 mu L of 50% methanol into each sample well, sealing the plates, uniformly mixing, and centrifuging to obtain a test sample; injecting 1 mu L of test sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of ornidazole and an internal standard ornidazole-d 5 in the test sample, and calculating according to the chromatographic peaksThe concentration of ornidazole in the blood plasma sample to be tested; the LC-MS/MS detection method provided by the invention is characterized in that ornidazole-d 5 is used as an internal standard, a precipitator is added for protein precipitation, a diluent is added into a supernatant, after pretreatment, separation is carried out through a chromatographic column, and a mass spectrum detector is used for detection, so that the concentration of ornidazole in human plasma is obtained.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a method for determining the concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry.
Background
Ornidazole (Ornidazole, ORZ) also known as alpha chloromethyl-2-methyl-5-nitroimidazole-1-ethanol is an anti-anaerobe and anti-protozoan drug which is taken orally, has good curative effect and low toxic and side effect, has good curative effect on trichomonas, amoeba, giardia and anaerobe infection, is superior to metronidazole and is slightly superior to tinidazole. It is clinically used for infectious diseases caused by anaerobes or protozoa. Is mainly used for preventing postoperative anaerobic bacteria infection, and treating infection caused by sensitive anaerobic bacteria, such as septicemia, meningitis, peritonitis, postoperative wound infection, puerperal sepsis, septic abortion and endometritis. The blood concentration reaches the peak value within 2 hours after the medicine is orally taken, the elimination half-life period is about 14 hours, and the plasma protein binding rate is less than 15 percent. Are widely distributed in human tissues and fluids, including cerebrospinal fluid. Ornidazole is metabolized in the liver, excreted in the urine mainly as conjugates and metabolites, and excreted in small amounts in the feces. A single oral dose has been reported to eliminate 85% in 5 days, 63% in urine and 22% in feces. Biliary excretion is about 4.1% of the elimination of oxmetazole and its metabolites.
At present, the prior art has few analysis methods for detecting ornidazole in human plasma, mostly adopts non-isotopic internal standards, and cannot ensure the consistency of the elution mode of an object to be detected and the internal standards. In order to meet the requirements of clinical biological sample analysis, a simpler, reliable and time-saving method for determining the concentration of ornidazole in human plasma needs to be developed.
Disclosure of Invention
The invention aims to provide a method for determining concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry, which comprises the steps of taking ornidazole-d 5 as an internal standard, adding a precipitator for protein precipitation, taking supernate, adding a diluent, pretreating, separating by a chromatographic column, and detecting by a mass spectrometer to obtain the concentration of ornidazole in human blood plasma, wherein the linear range of a standard curve of the blood plasma obtained by the method is 50.000 ng/mL-20000.000 ng/mL, and the precision CV% in and among batches is less than +/-15%.
The technical scheme of the invention is a method for determining the concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry, which comprises the following steps:
(1) pretreatment of a plasma sample to be detected:
under room temperature yellow light conditions, with EDTA-K2As an anticoagulant, taking ornidazole-d 5 as an internal standard; adding 50 mu L of plasma sample to be detected into a hole of a 96-hole plate; then 50 mul of internal standard oxpoconazole-d 5 working solution with the concentration of 1.000 mug/mL is added; after mixing uniformly, adding 300 mu L of methanol into each sample hole, closing a plate, mixing uniformly for 10min, and centrifuging the sample at 2623g for 10min at 4 ℃;
taking 50 mu L of centrifuged supernatant to another 96-well collection plate, adding 200 mu L of 50% methanol into each sample well, sealing the plates, 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 under the condition of yellow light at room temperature, detecting chromatographic peaks of ornidazole in the test sample and an internal standard ornidazole-d 5, and calculating the concentration of ornidazole in a plasma sample to be detected according to the chromatographic peaks; and (3) adopting an internal standard method, and substituting the peak area ratio of the ornidazole and the internal standard ornidazole-d 5 into a standard curve equation to calculate the concentration of the ornidazole in the plasma sample to be detected.
Preferably, the liquid chromatography conditions of the high performance liquid chromatography tandem mass spectrometer are as follows:
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: pure acetonitrile; needle washing liquid: the volume percentage of water/methanol/formic acid is 20/80/0.1; 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; ornidazole and Ornidazole-d 5 expected retention time was about 1.79min；
The mass spectrum conditions of the high performance liquid chromatography tandem mass spectrometer are as follows:
the ion source is an electrospray ion source, the spray voltage is 5500.00V, the atomization temperature is 500 ℃, the air curtain gas is 20psi, the atomization gas is 30psi, the auxiliary gas is 35psi, and the collision gas is 8; collision voltage of ornidazole and ornidazole-d 5 is 23V, and positive ion detection is carried out; the scanning mode is multiple reaction monitoring.
Preferably, the procedure for gradient elution is:
preferably, the establishment of the standard curve equation comprises the following steps:
putting 190 mu L of blank plasma into a polypropylene tube, adding 10 mu L of ornidazole working solutions with the concentrations of 1.000 mu g/mL, 2.000 mu g/mL, 5.000 mu g/mL, 20.000 mu g/mL, 80.000 mu g/mL, 160.000 mu g/mL, 320.000 mu g/mL and 400.000 mu g/mL respectively in the form of working solution, mixing uniformly, 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 zero-concentration sample into 50 mu L of 1.000 mu g/mL of internal standard ornidazole-d 5 solution, adding 50 mu L of 50% methanol aqueous solution with volume fraction into the double blank samples, mixing uniformly, adding 300 mu L of methanol into each sample hole, sealing the plate, mixing uniformly for 10min, and centrifuging the samples at 4 ℃ for 2623g for 10 min; taking 50 μ L of centrifuged supernatant to another 96-well collection plate, adding 200 μ L of 50% methanol, sealing the plate, and mixing for 10 min; centrifuging the sample at 2623g for 10min at 4 ℃ to obtain a labeled sample;
and respectively injecting 1 mu L of standard sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of the ornidazole and the internal standard ornidazole-d 5 in the sample, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of the ornidazole in the plasma sample to be detected.
Preferably, in the determination of the plasma sample to be determined, the determination of the quality control sample concentration of the 96-well plate kit is carried out by the following steps:
preparing quality control sample working solution with the concentration of ornidazole being 1.000 mug/mL, 3.000 mug/mL, 15.000 mug/mL, 150.000 mug/mL and 300.000 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 1.000 mu g/mL internal standard ornidazole-d 5 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 4 ℃ and 2623g for 10 min; taking 50 μ L of centrifuged supernatant to another 96-well collection plate, adding 200 μ L of 50% methanol, sealing the plate, and mixing for 10 min; centrifuging the sample at 2623g for 10min at 4 deg.C to obtain quality control sample;
and (3) respectively injecting 1 mu L of quality control sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of ornidazole and an internal standard ornidazole-d 5 in the quality control sample, and calculating the concentration of the quality control sample according to a standard curve.
The method for determining the concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry has the following beneficial effects:
(1) the method is characterized in that ornidazole-d 5 is used as an internal standard, and an isotope internal standard is used to ensure the elution consistency of the substance to be detected and the internal standard;
(2) the pretreatment method of the blood sample to be detected is simple and convenient, and is suitable for high-throughput measurement;
(3) the specificity is strong: under the chromatographic conditions used in the technical scheme, the retention time of ornidazole is about 1.79min, and the retention time of the internal standard ornidazole-d 5 is about 1.79 min. The ornidazole and the internal standard ornidazole-d 5 have good peak shapes, no mixed peak obviously interferes the measurement, and the base line is stable;
(4) the method is rapid, accurate and highly specific, and provides a basis for the blood concentration determination of ornidazole; the linear range of the plasma standard curve of the method is 50.000-20000.000 ng/mL.
Drawings
Fig. 1 is a product ion scanning mass spectrogram of ornidazole in the method for determining concentration of ornidazole in blood plasma by LC-MS in the technical scheme;
FIG. 2 is a scanning mass spectrum of product ions of ornidazole-d 5 in the method for determining concentration of ornidazole in blood plasma by LC-MS;
FIG. 3 is a LC-MS/MS graph of human blank plasma;
FIG. 4 is a LC-MS/MS graph of human blank plasma with added ornidazole and ornidazole-d 5;
FIG. 5 is a standard curve diagram of ornidazole in human plasma measured by LC-MS/MS detection 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 now be further described with reference to the drawings attached to the specification.
The invention discloses a method for determining the concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry, which is also called LC-MS/MS detection method of the concentration of ornidazole in human blood plasma, and is called LC-MS/MS detection method for short, and comprises the following steps:
(1) pretreatment of a plasma sample to be detected:
under room temperature yellow light conditions, with EDTA-K2As an anticoagulant, taking ornidazole-d 5 as an internal standard; adding 50 mu L of plasma sample to be detected into a hole of a 96-hole plate; then 50 mul of internal standard oxpoconazole-d 5 working solution with the concentration of 1.000 mug/mL is added; after mixing uniformly, adding 300 mu L of methanol into each sample hole, closing a plate, mixing uniformly for 10min, and centrifuging the sample at 2623g for 10min at 4 ℃;
taking 50 mu L of centrifuged supernatant to another 96-well collection plate, adding 200 mu L of 50% methanol into each sample well, sealing the plates, 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 under the condition of yellow light at room temperature, detecting chromatographic peaks of ornidazole in the test sample and an internal standard ornidazole-d 5, and calculating the concentration of ornidazole in a plasma sample to be detected according to the chromatographic peaks; and (3) adopting an internal standard method, and substituting the peak area ratio of the ornidazole and the internal standard ornidazole-d 5 into a standard curve equation to calculate the concentration of the ornidazole in the plasma sample to be detected.
In the above (2), the liquid chromatography conditions of the hplc-tandem mass spectrometer are:
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: pure acetonitrile; needle washing liquid: the volume percentage of water/methanol/formic acid is 20/80/0.1; 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; ornidazole and ornidazole-d 5 are expected to remain for approximately 1.79 min.
The mass spectrum conditions of the high performance liquid chromatography tandem mass spectrometer are as follows:
the ion source is an electrospray ion source, the spray voltage is 5500.00V, the atomization temperature is 500 ℃, the air curtain gas is 20psi, the atomization gas is 30psi, the auxiliary gas is 35psi, and the collision gas is 8; collision voltage of ornidazole and ornidazole-d 5 is 23V, and positive ion detection is carried out; the scanning mode is multiple reaction monitoring.
In the liquid chromatography conditions of the high performance liquid chromatography tandem mass spectrometer, the gradient elution procedure is as follows:
in the above (2), the establishment of the standard curve equation includes the steps of:
putting 190 mu L of blank plasma into a polypropylene tube, adding 10 mu L of ornidazole working solutions with the concentrations of 1.000 mu g/mL, 2.000 mu g/mL, 5.000 mu g/mL, 20.000 mu g/mL, 80.000 mu g/mL, 160.000 mu g/mL, 320.000 mu g/mL and 400.000 mu g/mL respectively in the form of working solution, mixing uniformly, 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 zero-concentration sample into 50 mu L of 1.000 mu g/mL of internal standard ornidazole-d 5 solution, adding 50 mu L of 50% methanol aqueous solution with volume fraction into the double blank samples, mixing uniformly, adding 300 mu L of methanol into each sample hole, sealing the plate, mixing uniformly for 10min, and centrifuging the samples at 4 ℃ for 2623g for 10 min; taking 50 μ L of centrifuged supernatant to another 96-well collection plate, adding 200 μ L of 50% methanol, sealing the plate, and mixing for 10 min; the sample was centrifuged at 2623g for 10min at 4 ℃ to obtain the labeled sample.
And then, respectively injecting 1 mu L of standard sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of the ornidazole and the internal standard ornidazole-d 5 in the sample, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of the ornidazole in the plasma sample to be detected.
In the above (2), in the measurement of a plasma sample to be measured, it is necessary to measure the quality control sample concentration of a 96-well plate kit by a method comprising:
preparing quality control sample working solution with the concentration of ornidazole being 1.000 mug/mL, 3.000 mug/mL, 15.000 mug/mL, 150.000 mug/mL and 300.000 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 1.000 mu g/mL internal standard ornidazole-d 5 solution, uniformly mixing, adding 300 mu L of methanol into each sample hole, closing the plate, uniformly mixing for 10min, centrifuging the sample at 4 ℃, 2623g for 10min, taking 50 mu L of centrifuged supernatant into another 96-hole collection plate, adding 200 mu L of 50% methanol, closing the plate, uniformly mixing for 10min, centrifuging the sample at 4 ℃, 2623g for 10min, and obtaining the quality control sample.
And then, respectively injecting 1 mu L of quality control samples into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of ornidazole and an internal standard ornidazole-d 5 in the quality control samples, and calculating the concentration of the quality control samples according to a standard curve.
Based on the technical scheme, the following method for determining the concentration of ornidazole in blood plasma by LC-MS discloses specific experimental steps. The experimental process is carried out under room temperature yellow light condition.
First, experimental material and analytical equipment
Calcium ornidazole (analyte): china food and drug testing research institute or the same and higher-grade standard;
ornidazole-d 5 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 1: details of reagents
| Solvent/reagent name | Rank of | Manufacturer of the product |
| Methanol | Chromatographic grade | Sigma |
| Formic acid | Chromatographic grade | Aladdin |
| Acetonitrile | Chromatographic grade | Sigma |
| Ultrapure water | NA | Preparation in the laboratory |
Table 2: details of the equipment used
Second, liquid condition
1. Conditions of liquid chromatography
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: pure acetonitrile;
needle washing liquid: the volume percentage of water/methanol/formic acid is 20/80/0.1;
autosampler temperature: 4 ℃;
elution procedure: gradient elution with flow rate of 0.5mL/min, sample size of 1 μ L, and analysis time of 4 min; ornidazole and ornidazole-d 5 are expected to remain for approximately 1.79 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 spraying voltage is 5500.00V, the atomizing temperature is 500 ℃, the air curtain gas is 20psi, the atomizing gas is 30psi, the auxiliary gas is 35psi, and the collision gas is 8. Collision voltage of ornidazole and ornidazole-d 5 is 23V, and positive ion detection is carried out; the scanning mode is multiple reaction monitoring. The ionic reactions used for the quantitative analysis were respectively: ornidazole: m/z 220.1 → 128.0 and ornidazole-d 5: m/z 225.1 → 128.0.
Third, the experimental process
1. Preparation of ornidazole standard curve working solution
Preparing an ornidazole standard curve working solution: accurately weighing a proper amount of ornidazole standard substance, correcting by a mass correction coefficient, and dissolving the ornidazole standard substance in pure methanol to obtain a stock solution with the final concentration of 1.000 mg/mL. Then sequentially diluting with 50% methanol to prepare an ornidazole working solution, wherein the specific dilution concentration is shown in the following table 4:
table 4: concentration of Ornidazole working solution
SS denotes stock solution, WS denotes working solution. The ornidazole 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 proportion as required.
2. Preparation of ornidazole quality control working solution
Preparing an ornidazole working solution: accurately weighing a proper amount of ornidazole standard substance, after correcting the mass correction coefficient, dissolving the ornidazole standard substance in pure methanol to obtain a stock solution with the final concentration of 1.000mg/mL, and sequentially diluting with 50% methanol to prepare an ornidazole working solution, wherein the specific dilution concentration is shown in the following table 5:
table 5: ornidazole quality control working solution preparation concentration
SS denotes stock solution, WS denotes working solution. The ornidazole 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.
3. Preparation of working solution of ornidazole-d 5 internal standard
Preparing an ornidazole-d 5 internal standard working solution: and taking one ornidazole-d 5 standard product, correcting the mass correction coefficient, and dissolving the ornidazole-d 5 standard product in pure methanol to obtain a stock solution with the final concentration of 0.500 mg/mL. Then methanol aqueous solution with volume fraction of 50% is dissolved and diluted to prepare 1.000 mu g/mL internal standard ornidazole-d 5 working solution, and the specific dilution concentration is shown in the following table 6:
table 6: ornidazole-d 3 working solution preparation concentration
The ornidazole-d 5 is abbreviated as d5PIT, and the ornidazole-d 5 internal standard 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 proportion as required.
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), precisely adding 10 mu L of ornidazole working solution with different concentrations to prepare each sample, mixing uniformly to prepare drug-containing plasma with different concentrations, and carrying out pretreatment on the plasma samples. And calculating the ratio Y (Y is As/Ai) of the area As of the ornidazole peak and the area Ai of the ornidazole-d 5 peak, and performing regression calculation on the blood concentration X by using the area ratio Y. The average ratio Y is used for carrying out regression calculation on the blood concentration X, and the lowest quantitative limit of the blood concentration of the ornidazole measured by the method is as follows: 50.000 ng/mL. The standard curve parameters of oxpoconazole in human plasma determined by LC-MS/MS method are shown in Table 7:
table 7: standard curve (ng/mL) of ornidazole in human plasma measured by LC-MS/MS method
5. Accuracy and precision
Unfreezing blank plasma at room temperature; the appropriate volume of blank plasma was transferred to polypropylene tubes, and 5 drug-containing plasma quality control samples (LLOQ, LQC, M1QC, M2QC, HQC) of different concentrations and a follow-up standard curve were prepared by adding ornidazole quality control working solution, and the procedure was followed as "plasma sample pretreatment". And (3) in at least two days, completing in at least three analysis batches, calculating the ratio Y of the peak area As of the ornidazole and the peak area Ai of the internal standard ornidazole-d 5, substituting the ratio Y into a standard curve on the same day to obtain the measured concentration, calculating the precision between batches according to the measured concentration, and determining the ratio of the measured concentration to the added concentration As the accuracy, wherein the result is shown in a table 8. The result shows that the precision and accuracy of the ornidazole plasma sample in batches and between 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 precision and accuracy of ornidazole in plasma
6. Matrix effect
Six different blank plasma samples are respectively from different healthy human bodies, and are prepared and analyzed in the same analysis batch according to the sample preparation steps to evaluate the interference of the different blank plasmas on the ornidazole analyte and the internal standard ornidazole-d 5, and the specific data of the interference are shown in a table 9. Wherein the CV percent of the internal standard normalized matrix factor is less than or equal to 3.1 percent.
Table 9: interference data comparison table of blank healthy human plasma from six different sources
Note: 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".
As can be seen from table 9, blank plasma from different human bodies did not interfere with the detection result of ornidazole. Therefore, the method can be used for detecting the concentration of ornidazole 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 ornidazole stock solution with the concentration of 1.000mg/mL, and taking 50% methanol as a diluent to prepare working solution with the concentration of 600.000 mug/mL of ornidazole.
Thawing 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 (with the concentration of 30000.000ng/mL), vortexing and mixing to prepare a sample higher than the upper limit of quantitation, and then taking 50. mu.L of the sample, adding 50. mu.L of the sample into a new polypropylene tube, and diluting 2 times to obtain a diluted 2-fold sample (with the concentration of 15000.000 ng/mL). The "plasma sample pretreatment" procedure was followed, with 6 replicates, and the results are shown in Table 10. The deviation in accuracy was 2.9%, indicating that dilution reliability meets the acceptance criteria.
Table 10: dilution reliability investigation results
8. Recovery rate
The extraction recovery is determined by comparing the analyte/internal standard response in the treated spiked sample with the analyte/internal standard response in the treated blank matrix. Samples of 3 concentration levels 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 sample extracted normally, the peak area of the sample after the matrix is extracted, and CV percent of the recovery rate of the sample at each concentration level are less than or equal to 4.7 percent. Both ornidazole and ornidazole-d 5 met the acceptance criteria and the results are shown in tables 11 and 12.
Table 11: extraction recovery rate of substance to be tested
Table 12: recovery of internal standard
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 a high-efficiency 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 of the measured concentration and the standard value of the high/low concentration sample is in the range of-12.1% -1.4% under the condition of 5 times of freezing and thawing at-70 ℃, and the deviation of the measured concentration and the standard value of the high/low concentration sample is in the range of-11.4% -2.7% 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: stability test results of 5 times of freezing and thawing between-70 ℃ and room temperature
Table 14: stability test results of 5 times of freezing and thawing between-20 ℃ and 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 placed in a refrigerator with the temperature of-70 ℃ for storage for 52 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 test sample is taken and injected into a high performance liquid chromatography tandem mass spectrometer according to the operation of 'plasma sample pretreatment', and the result is shown in Table 15.
After the high/low concentration samples were stored at-70 ℃ for 52 days, the average deviation of accuracy between the measured concentration and the indicated value was in the range of-5.4% to 3.9%, indicating that the samples were stable when stored in a refrigerator at-70 ℃ for 52 days.
Table 15: long term storage stability at-70 ℃ 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 the double blank sample or ULOQ Without IS sample, add 50 μ L50% methanol solution, for other samples add 50 μ L internal standard working solution (1.000 μ g/mL), vortex and mix; adding 300 mu L of methanol into each sample hole, sealing the plate, and uniformly mixing for 10 min; centrifuging the sample at 2623g for 10min at 4 ℃; taking 50 μ L of centrifuged supernatant to another 96-well collection plate, adding 200 μ L of 50% methanol, sealing the plate, and mixing for 10 min; the sample was centrifuged at 2623g for 10min at 4 ℃ for injection.
Four, small knot
In conclusion, the invention provides a simple and convenient method for determining the concentration of ornidazole in blood plasma by a pretreatment method, adopts simple protein precipitation treatment, and is suitable for conventional determination; from the above analysis, under the chromatographic conditions adopted in the experiment, the retention time of ornidazole is about 1.79min, the retention time of the internal standard ornidazole-d 5 is about 1.79min, the peaks of ornidazole and the internal standard ornidazole-d 5 are good, no obvious interference of mixed peaks is generated in measurement, and the baseline is stable. The linear range of the plasma standard curve of the method is 50.000 ng/mL-20000.000 ng/mL, and the precision CV% in batch and between batches are both less than +/-15%; the method has higher specificity and good stability, is convenient and controllable, and can accurately determine the concentration of ornidazole in blood plasma; meanwhile, the method is accurate and has good reproducibility, and a basis is provided for the blood concentration determination of ornidazole.
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 concept and technical solution, or to apply the inventive concept and technical solution to other occasions without any modification.
Claims (5)
1. A method for determining the concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry is characterized by comprising the following steps: under the condition of yellow light at room temperature,
(1) pretreatment of a plasma sample to be detected:
under room temperature yellow light conditions, with EDTA-K2As an anticoagulant, taking ornidazole-d 5 as an internal standard; adding 50 mu L of plasma sample to be detected into a hole of a 96-well plate; then 50 mul of internal standard ornidazole-d 5 working solution with the concentration of 1.000 mug/mL is added; after mixing evenly, 300 mu L of methanol is added into each sample hole,sealing the plates, mixing uniformly for 10min, and centrifuging the sample at 2623g for 10min at 4 ℃;
taking 50 mu L of centrifuged supernatant to another 96-well collection plate, adding 200 mu L of 50% methanol into each sample well, sealing the plates, and mixing uniformly; centrifuging the sample at 2623g for 10min at 4 ℃ to obtain a test sample;
(2) and (3) sample measurement:
under the condition of yellow light at room temperature, injecting 1 mu L of test sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of ornidazole and an internal standard ornidazole-d 5 in the test sample, and calculating the concentration of ornidazole in a plasma sample to be detected according to the chromatographic peaks; and (3) adopting an internal standard method, and substituting the peak area ratio of the ornidazole and the internal standard ornidazole-d 5 into a standard curve equation to calculate the concentration of the ornidazole in the plasma sample to be detected.
2. The method for determining the concentration of ornidazole in plasma by LC-MS as claimed in claim 1,
the liquid chromatogram conditions of the high performance liquid chromatogram tandem mass spectrometer are as follows:
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: pure acetonitrile; needle washing liquid: the volume percentage of water/methanol/formic acid is 20/80/0.1; 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; ornidazole and ornidazole-d 5 expected retention time was about 1.79 min;
the mass spectrum conditions of the high performance liquid chromatography tandem mass spectrometer are as follows:
the ion source is an electrospray ion source, the spray voltage is 5500.00V, the atomization temperature is 500 ℃, the air curtain gas is 20psi, the atomization gas is 30psi, the auxiliary gas is 35psi, and the collision gas is 8; collision voltage of ornidazole and ornidazole-d 5 is 23V, and positive ion detection is carried out; the scanning mode is multiple reaction monitoring.
3. The method for determining the concentration of ornidazole in plasma by LC-MS as claimed in claim 2, wherein the gradient elution procedure is as follows:
4. the method for determining the concentration of ornidazole in plasma by LC-MS as claimed in claim 1, wherein the establishment of the standard curve equation includes the following steps:
putting 190 mu L of blank plasma into a polypropylene tube, adding 10 mu L of ornidazole working solutions with the concentrations of 1.000 mu g/mL, 2.000 mu g/mL, 5.000 mu g/mL, 20.000 mu g/mL, 80.000 mu g/mL, 160.000 mu g/mL, 320.000 mu g/mL and 400.000 mu g/mL respectively in the form of working solution, mixing uniformly, 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 zero-concentration sample into 50 mu L of 1.000 mu g/mL of internal standard ornidazole-d 5 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 a plate and mixing uniformly for 10min, and centrifuging the samples for 3g 26210 min at 4 ℃; taking 50 μ L of centrifuged supernatant to another 96-well collection plate, adding 200 μ L of 50% methanol, sealing the plate, and mixing for 10 min; centrifuging the sample at 2623g for 10min at 4 ℃ to obtain a labeled sample;
and respectively injecting 1 mu L of standard sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of ornidazole and an internal standard ornidazole-d 5 in the sample, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of the ornidazole in the plasma sample to be detected.
5. The method for determining the concentration of ornidazole in plasma by LC-MS (liquid chromatography-Mass spectrometer) as claimed in claim 1, wherein in the determination of the plasma sample to be determined, the concentration of the quality control sample of a 96-well plate kit needs to be determined by the following steps:
preparing quality control sample working solution with the concentration of ornidazole being 1.000 mug/mL, 3.000 mug/mL, 15.000 mug/mL, 150.000 mug/mL and 300.000 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 1.000 mu g/mL internal standard ornidazole-d 5 solution, uniformly mixing, adding 300 mu L of methanol into each sample hole, sealing the plate, uniformly mixing for 10min, and centrifuging the sample at 2623g for 10min at 4 ℃; taking 50 μ L of centrifuged supernatant to another 96-well collection plate, adding 200 μ L of 50% methanol, sealing the plate, and mixing for 10 min; centrifuging the sample at 2623g for 10min at 4 deg.C to obtain quality control sample;
and (3) respectively injecting 1 mu L of quality control sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of ornidazole and an internal standard ornidazole-d 5 in the quality control sample, and calculating the concentration of the quality control sample according to a standard curve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111538151.9A CN114397379A (en) | 2021-12-15 | 2021-12-15 | Method for determining concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111538151.9A CN114397379A (en) | 2021-12-15 | 2021-12-15 | Method for determining concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114397379A true CN114397379A (en) | 2022-04-26 |
Family
ID=81226603
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111538151.9A Pending CN114397379A (en) | 2021-12-15 | 2021-12-15 | Method for determining concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114397379A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116124927A (en) * | 2022-12-23 | 2023-05-16 | 上海嘉葆药银医药科技有限公司 | Method for measuring concentration of JDB0131 and metabolite thereof in blood plasma |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103048401A (en) * | 2012-12-07 | 2013-04-17 | 中国检验检疫科学研究院 | Determining method for 15 kinds of forbidden nitro imidazoles antibiotics in cosmetics |
| CN109507342A (en) * | 2019-01-21 | 2019-03-22 | 中国检验检疫科学研究院 | A kind of remaining method of nitroimidazoles medicine in measurement blood sample |
-
2021
- 2021-12-15 CN CN202111538151.9A patent/CN114397379A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103048401A (en) * | 2012-12-07 | 2013-04-17 | 中国检验检疫科学研究院 | Determining method for 15 kinds of forbidden nitro imidazoles antibiotics in cosmetics |
| CN109507342A (en) * | 2019-01-21 | 2019-03-22 | 中国检验检疫科学研究院 | A kind of remaining method of nitroimidazoles medicine in measurement blood sample |
Non-Patent Citations (5)
| Title |
|---|
| MARK CRONLY ET AL.: "Development and validation of a rapid method for the determination and confirmation of 10 nitroimidazoles in animal plasma using liquid chromatography tandem mass spectrometry" * |
| 孟茜;郑荣;王柯;: "高效液相色谱-串联质谱法测定化妆品中16种抗真菌类和硝基咪唑类化合物" * |
| 尚彬如;陈义强;张丽英;王宗义;: "液相色谱-串联质谱测定饲料中7种硝基咪唑类药物" * |
| 杜江波等: "人血浆中奥硝唑及其主要代谢物的定性与定量分析" * |
| 杨万兴,向明凤: "人体血浆中奥硝唑的HPLC测定" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116124927A (en) * | 2022-12-23 | 2023-05-16 | 上海嘉葆药银医药科技有限公司 | Method for measuring concentration of JDB0131 and metabolite thereof in blood plasma |
| CN116124927B (en) * | 2022-12-23 | 2023-08-22 | 上海嘉葆药银医药科技有限公司 | Method for measuring concentration of JDB0131 and metabolite thereof in blood plasma |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dai et al. | Determination of serum uric acid using high-performance liquid chromatography (HPLC)/isotope dilution mass spectrometry (ID-MS) as a candidate reference method | |
| WO2023005687A1 (en) | Metabolic spectrum detection kit, and usage method and application thereof | |
| Berlin et al. | Micromethod for the determination of diphenylhydantoin in plasma and cerebrospinal fluid—a comparison between a gas chromatographic and a spectrophotometric method | |
| CN108072712B (en) | Quantitative analysis method for blood concentration of new compound WSJ-557 in SD rat plasma | |
| CN115902048A (en) | Method for detecting water-soluble vitamins in serum by methyl derivatization-high performance liquid chromatography tandem mass spectrometry | |
| CN111458417B (en) | Method and kit for combined detection of multiple antibiotics in sample to be detected | |
| CN110865137A (en) | Method and kit for detecting aldosterone in blood plasma | |
| CN114397379A (en) | Method for determining concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry | |
| CN112697935B (en) | Method for simultaneous determination of perindopril and perindopril A concentration in human plasma | |
| CN114544796B (en) | Method for measuring settop alcohol in plasma by liquid phase mass spectrometry | |
| CN115420812B (en) | Method for detecting calcic acid in urine and application thereof | |
| CN115389684A (en) | Method for simultaneously detecting oxalic acid, citric acid and cystine in 24h urine | |
| CN115656400A (en) | Method for detecting 11-dehydrothromboxane B in urine 2 Liquid chromatography-tandem mass spectrometry method and kit | |
| Eckfeldt et al. | Determination of serum cholesterol by isotope dilution mass spectrometry with a benchtop capillary gas chromatograph/mass spectrometer: comparison with the national reference system's definitive and reference methods | |
| CN114384170A (en) | Method for determining concentration of amlodipine in blood plasma by liquid chromatography-mass spectrometry | |
| CN110007023B (en) | High-resolution mass spectrum screening method for sulfonamides in fish body and analysis method for interaction of sulfonamides and protein macromolecules | |
| CN113109493A (en) | Method for measuring rifampicin in plasma by high performance liquid chromatography-mass spectrometry | |
| CN114563504B (en) | Method and kit for determining content of free aldosterone in blood plasma | |
| CN114384171A (en) | Method for determining concentration of lacosamide in blood plasma by liquid chromatography-mass spectrometry | |
| CN113341023B (en) | Serum diaminopimelic acid detection kit based on liquid chromatography-mass spectrometry, detection method and application | |
| CN112986452B (en) | Method for determining tandospirone concentration in human plasma | |
| CN116087373B (en) | Detection method and pretreatment method for folic acid and 5-methyltetrahydrofolate in erythrocytes | |
| WO2023024727A1 (en) | Method for treating blood plasma sample containing clopidogrel oxide, and measurement method | |
| CN114397380A (en) | Method for determining concentration of western-style gliptin in blood plasma by liquid chromatography-mass spectrometry | |
| CN114397378A (en) | Method for determining pitavastatin concentration in blood plasma by liquid chromatography-mass spectrometry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |