CN114235985A - Method for detecting guanfacine in human plasma by using HPLC-MS/MS - Google Patents
Method for detecting guanfacine in human plasma by using HPLC-MS/MS 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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- 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
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Abstract
The invention relates to a method for detecting guanfacine in human plasma by HPLC-MS/MS combination, which comprises the following steps: (1) pretreating a human plasma sample; (2) performing liquid chromatography-mass spectrometry detection, and performing gradient elution by using a mobile phase A and a mobile phase B as a mixed mobile phase, wherein: the mobile phase A is acetonitrile; the mobile phase B is 2-20 mM ammonium formate aqueous solution; (3) determination of guanfacine concentration in human plasma. According to the method, the isotope internal standard is used as an internal standard substance, the ACE C18-PFP is used as a chromatographic column for gradient elution, the isotope internal standard and the substance to be detected have the same retention time, chemical properties and matrix effect, and the reproducibility and accuracy for measuring the concentration of guanfacine in plasma are good. The method of the invention can be used to evaluate the bioequivalence of guanfacine.
Description
Technical Field
The invention belongs to the technical field of biological analysis, and particularly relates to a method for detecting guanfacine in human plasma by using HPLC-MS/MS in a combined manner.
Background
The guanfacine hydrochloride sustained-release tablet is a selective alpha-2A adrenoceptor agonist developed by the U.S. fire pharmaceutical company, is approved by the U.S. FDA to be on the market in 9, 2 and 9 months in 2009, and is the first alpha-2A receptor agonist approved for treating ADHD. The FDA has approved Guanfacine hydrochloride sustained release tablets (Guanfacine, intuiv) for the treatment of Attention Deficit and Hyperactivity Disorder (ADHD) in children and adolescents aged 6-17. The document shows that the guanfacine hydrochloride sustained release tablet can directly act on prefrontal cortex receptors in the prefrontal cortex area of the brain, and postsynaptic alpha-2A receptors can enhance memory, reduce interference, improve attention, enhance behavior inhibition capability and control impulsive behaviors after being stimulated.
At present, a method for measuring the content of guanfacine by adopting liquid chromatography-mass spectrometry is reported in domestic literature. For example, the document LC-MS/MS method for measuring the mass concentration of guanfacine in Beagle dog plasma (korea, shenyang university of pharmacy, 2014) discloses a method for detecting guanfacine in Beagle dog plasma, but the pretreatment of plasma samples adopts a liquid-liquid extraction method, which is complicated in steps and long in time, and the plasma dosage is more than 500 μ L, so that the method is not suitable for analyzing samples in large scale. And in the detection method, the non-deuterated internal standard granisetron hydrochloride is used as the internal standard, so that the matrix effect of the analyte and the matrix effect of the internal standard cannot be controlled to be the same, and the difference between the detection result of the sample and the true value can be caused.
In order to meet the requirement of clinical large-batch sample analysis and evaluation on the biological equivalence of the medicine, a simpler, reliable, rapid and high-flux sample pretreatment method and a method for detecting the concentration of guanfacine in human plasma need to be developed.
Disclosure of Invention
The invention aims to provide a method for detecting the concentration of guanfacine in human plasma, which has the advantages of small sample amount, good reproducibility, high sensitivity, high analysis speed, small matrix effect influence and higher extraction recovery rate, on the basis of the prior art.
The technical scheme of the invention is as follows:
a method for detecting guanfacine in human plasma by HPLC-MS/MS combination comprises the following steps: (1) human plasma samples are pretreated; (2) performing liquid chromatography-mass spectrometry detection, and performing gradient elution by using a mobile phase A and a mobile phase B as a mixed mobile phase, wherein the mobile phase A is acetonitrile; the mobile phase B is 2-20 mM ammonium formate aqueous solution; (3) determination of guanfacine concentration in human plasma. Wherein, the human plasma sample is pretreated by adopting a protein precipitation method, and a precipitator is methanol; the chromatographic column is ACE C18-PFP; the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 30:70 to 40:60 at a constant speed within 0-2.5 minutes; in 2.5-2.8 minutes, the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 40:60 to 90:10 at a constant speed; the volume ratio of the mobile phase A to the mobile phase B is 90:10 within 2.8-3.8 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 90:10 to 30:70 at a constant speed within 3.8-4.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is 30:70 within 4.0-5.5 minutes; in the gradient elution process, the strong needle washing liquid is a methanol-acetonitrile-isopropanol mixed solution. The specific gradient elution procedure is shown in table 1:
table 1: guanfacine liquid chromatography gradient
For the invention, other gradient elution processes are adopted, the separation effect is poor, the analyte in the residual sample after the upper limit sample is quantified is easy to generate residue, the accuracy of sample determination after the high-concentration sample is influenced, and the residue is accumulated and eluted at the peak position due to the high initial water phase ratio.
When the method adopts HPLC-MS/MS combination to detect guanfacine in human plasma, in a preferred scheme, in the gradient elution process, the weak needle washing solution is a methanol-water mixed solution, in order to better implement the method, the volume ratio of methanol to water in the weak needle washing solution is 1-9: 9-1, and under the condition that the effect of the method is not influenced, in a further preferred scheme, the volume ratio of methanol to water in the weak needle washing solution is 1: 1. In the experimental process, it is found that by using the commonly used strong needle washing liquid, such as methanol, acetonitrile, methanol-ethanol, methanol-isopropanol and methanol-acetonitrile-isopropanol, as the strong needle washing liquid, in addition to methanol-acetonitrile-isopropanol, other solvents as the strong needle washing liquid have higher analyte residues after high concentration samples, which affects accurate determination of the samples after the high concentration samples. For the invention, the strong needle washing liquid is a methanol-acetonitrile-isopropanol mixed solution, residues can be effectively removed, and the detection result is accurate and reliable, in a preferable scheme, the volume ratio of methanol to acetonitrile to isopropanol in the strong needle washing liquid is 1-2: 2-3: 1, and in a further preferable scheme, the volume ratio of methanol to acetonitrile to isopropanol in the strong needle washing liquid is 1:2:1 under the condition of not influencing the effect of the invention.
When the invention adopts HPLC-MS/MS combination to detect guanfacine in human plasma, in a preferred scheme, the mobile phase B is 10mM ammonium formate aqueous solution. In order to improve the chromatographic separation selectivity, it may be considered to adjust the polarity of the mobile phase. In the mobile phase B, the total volume of the ammonium formate aqueous solution is 100 percent as a reference, and the mobile phase B contains 0.05 to 0.20 percent of formic acid in volume ratio; in a preferable embodiment, the mobile phase B contains 0.1% by volume of formic acid based on 100% by volume of the total ammonium formate solution, i.e., the mobile phase B is 0.1% formic acid to 10mM ammonium formate solution.
According to the invention, when guanfacine in human plasma is detected by adopting HPLC-MS/MS combination, ACE3C18-PFP is used as a chromatographic column, and furthermore, the length of the chromatographic column is 75mm, the diameter of the chromatographic column is 4.6mm, and the particle size of a filler is 3 μm, namely ACE3C18-PFP (75 multiplied by 4.6mm,3 μm).
In chromatography, the choice of the chromatographic column is important and the requirements for the chromatographic column: high column efficiency, good selectivity, high analysis speed and the like. The invention adopts the mobile phase mentioned above to carry out gradient elution, takes ACE C18-PFP as a chromatographic column, and endogenous substances do not interfere the determination of samples under the coordination of other conditions, and the invention has good reproducibility, high sensitivity, fast analysis speed and small influence of matrix effect. And other similar chromatographic columns are adopted, for example, the chromatographic column is an active ZORBAX XDB-Phenyl, the separation effect is poor, the chromatographic peak response is low, the peak is widened, and the tailing phenomenon is generated.
When the internal standard method is adopted, the selection of the internal standard substance is very important work. The ideal internal standard should be capable of being added to the sample in an accurate, known amount, and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be sufficiently separated from the components of the sample. When the method adopts HPLC-MS/MS combination to detect the guanfacine in the human plasma, the guanfacine-13C-15N 3 is used as an internal standard, the isotope internal standard is used as an internal standard substance, the isotope internal standard and the substance to be detected have the same retention time, chemical property and matrix effect, and the reproducibility and accuracy of measuring the guanfacine concentration in the plasma are better.
In the step (1), the human plasma sample is pretreated by a protein precipitation method, and methanol is used as a precipitator. The method for pretreating the human plasma sample by adopting the protein precipitation method can avoid the complicated and time-consuming liquid-liquid extraction process and simultaneously obtain the surprisingly high recovery rate. The invention adopts a protein precipitation method to pretreat human plasma samples, takes ACE3C18-PFP as a chromatographic column, and under the coordination of other conditions, the total extraction recovery rate of guanfacine is 100.69%. And other similar precipitants such as acetonitrile are adopted, so that the total extraction recovery rate is only about 70%, the viscosity of the acetonitrile is low, and the acetonitrile is easy to drip and splash in the pretreatment process, so that the sample is polluted.
In one embodiment, in step (1), the pretreatment of the human plasma sample comprises: adding internal standard working solution and a precipitator into a human plasma sample, taking supernate after vortex and centrifugation, and mixing the supernate with a diluent to obtain a sample to be detected; wherein the internal standard in the internal standard working solution is guanfacine-13C-15N 3. The diluent is 10mM ammonium formate solution, and the volume ratio of formic acid is 0.1-0.15% based on the total volume of the 10mM ammonium formate solution as 100%.
The detection method of the invention also comprises the steps of preparing an internal standard working solution: weighing a guanfacine-13C-15N 3 reference substance, dissolving the reference substance by using acetonitrile to obtain a guanfacine-13C-15N 3 stock solution with the concentration of 200 mu g/mL, and diluting the stock solution by using a mixed solution of methanol and water with the volume ratio of 50:50 to obtain an internal standard working solution with the concentration of 10.0ng/mL guanfacine-13C-15N 3.
In a more preferred embodiment, in step (1), the pretreatment of the human plasma sample comprises: and (3) taking 100 mu L of human plasma sample, adding 50 mu L of internal standard working solution and 350 mu L of methanol, performing vortex and centrifugation, taking 150 mu L of supernatant, and mixing with 100 mu L of diluent to obtain the sample to be detected. Wherein the diluent is 10mM ammonium formate solution, and the volume ratio of the ammonium formate solution is 0.1% based on 100% of the total volume of the 10mM ammonium formate solution, and the ammonium formate solution can be simplified into 10mM ammonium formate solution (containing 0.1% formic acid).
The invention adopts a protein precipitation method for pretreatment of a human plasma sample, wherein the conditions of vortex and centrifugation are as follows: vortex for 10min and centrifuge at 4000rpm/min for 5min at 4 ℃.
In the invention, during the chromatographic detection, a sample to be detected is placed in an automatic sample injector for HPLC-MS/MS analysis, the sample injection volume is 10 mu L, and the temperature of the sample injector is 4 ℃.
The detection method of the invention, the step (2) of adopting the liquid chromatography-mass spectrometry combined detection comprises the following steps: the detailed chromatographic conditions of the invention are as follows: using ACE C18-PFP (75 × 4.6mm,3 μm) as chromatographic column, and performing gradient elution according to the above-mentioned elution process, wherein the column temperature is 30-45 deg.C, preferably 40 deg.C; the flow rate is 0.5 to 1.0mL/min, preferably 0.8 mL/min.
The mass spectrometry conditions of the present invention include: with electrospray ion source, positive ions are abundantReaction monitoring and scanning, wherein the spraying voltage is 5500V, and the ion source temperature is 550 ℃; guanfacine, [ M + H ]]+M/z 246.1 → 60.1, DP value 90V, CE value 24V; guanfacine-13C-15N 3, [ M + H ]]+M/z 250.0 → 159.1, DP 80V, CE 20V.
The detection method comprises the following steps of (3) determining the concentration of guanfacine in human plasma: preparing the plasma to be detected according to the sample pretreatment method in the step (1), detecting according to the liquid chromatography-mass spectrometry in the step (2), recording the peak area corresponding to guanfacine, and determining the peak area ratio of guanfacine to the internal standard according to the weight coefficient w ═ 1/x2And performing linear regression, wherein the formula is expressed as y ═ ax + b, and calculating to obtain the concentration of guanfacine in the blood plasma to be detected.
The detection method of the invention can be used for clinical pharmacokinetic sample monitoring. The step of calculating the clinical pharmacokinetic parameters comprises: pharmacokinetic parameters were calculated with DAS3.2.8, including: cmax、Tmax、t1/2、AUC0-tAnd simultaneously calculating the average number and the standard deviation of each parameter.
By adopting the technical scheme of the invention, the advantages are as follows:
(1) the method takes the isotope internal standard as the internal standard substance, the isotope internal standard and the substance to be detected have the same retention time, chemical property and matrix effect, and the reproducibility and accuracy of the determination of the concentration of guanfacine in plasma are better.
(2) The detection method of the invention adopts the methanol-acetonitrile-isopropanol mixed solution as the strong needle washing solution (for example, the volume ratio is 1:2:1) in the gradient elution process, and can effectively remove residues, so that the detection result is accurate and reliable.
(3) The sample size used in the method is only 100 mu L, the sample size used is small, and the method is suitable for detecting large-batch plasma samples.
(4) The detection method provided by the invention selects a specific chromatographic column and a specific mobile phase, optimizes the elution time and the proportion of the mobile phase in the gradient elution process, has the advantages of good reproducibility, high sensitivity, high analysis speed, small matrix effect influence, high recovery rate and the like, is verified by all methods including specificity, accuracy, precision, matrix effect, extraction recovery rate and stability, and can be reliably used for evaluating the bioequivalence of guanfacine in a human body.
Drawings
FIG. 1 is a guanfacine ion scan;
FIG. 2 is a graph of a guanfacine-13C-15N 3 daughter ion scan;
FIG. 3 is a specific chromatogram for determining guanfacine in plasma by LC-MS/MS method;
wherein, fig. 3-1 to fig. 3-6 are blank plasma chromatograms of 6 different individuals, in each of fig. 3-1 to fig. 3-6, the left chromatogram is guanfacine, and the right chromatogram is guanfacine-13C-15N 3;
FIG. 4 is a mixed blank plasma chromatogram;
wherein, the left chromatogram is guanfacine, and the right chromatogram is guanfacine-13C-15N 3;
FIG. 5 is a quantitative lower limit sample chromatogram;
wherein, the left chromatogram is guanfacine, and the right chromatogram is guanfacine-13C-15N 3;
FIG. 6 is a chromatogram of guanfacine in comparative example 2;
wherein the left chromatogram is the chromatogram of guanfacine in a quantitative lower limit sample of which the chromatographic column is of active ZORBAX XDB-Phenyl (75 multiplied by 4.6mm,3.5 mu m), and the right chromatogram is the chromatogram of guanfacine in a quantitative lower limit sample of which the chromatographic column is of ACE C18-PFP (75 multiplied by 4.6mm,3 mu m);
FIG. 7 is a chromatogram of guanfacine in comparative example 3;
wherein, the left chromatogram is the chromatogram of guanfacine in the residual sample of which the strong needle washing liquid is methanol and acetonitrile (1:1, v/v), and the right chromatogram is the chromatogram of guanfacine in the residual sample of which the strong needle washing liquid is methanol and acetonitrile and isopropanol (1:2:1, v/v/v).
FIG. 8 is a chromatogram of guanfacine in comparative example 4;
wherein, the left chromatogram is the chromatogram of the upper limit sample guanfacine, and the right chromatogram is the chromatogram of the residual sample guanfacine.
Detailed Description
The detection method of the present invention is further illustrated by the following examples in conjunction with the drawings, but the present invention is not limited to these examples.
Materials and methods
1. Instruments and reagents
High phase liquid chromatography (Shimadzu LC-30AD series); mass Spectrometry (API 4000, Applied Biosystems/Sciex); water purifiers (MilliDirectQ, Millipore); microbalance (XP6, METTLER TOLEDO); centrifuge (heraeus muitifiduge X1R, ThermoFisher); an oscillator (LPD2500, LE PARD).
Methanol (Merck, HPLC grade), acetonitrile (Merck, HPLC grade), water (lab homemade/minister, ultrapure/distilled water), formic acid (Aladdin, HPLC grade), ammonium formate (Aladdin, HPLC grade), 10M ammonium formate solution (BioUltra, HPLC grade), isopropanol (national pharmaceutical group chemicals limited, HPLC grade). Blank plasma was derived from healthy subjects. Guanfacine hydrochloride (TLC, batch No.: 1169-089A1), guanfacine-13C-15N 3(TRC, batch No.: 6-THT-103-3)
2. Liquid condition
Liquid phase conditions: a chromatographic column: ACE C18-PFP (75X 4.6mm,3 μm); column temperature: 40 ℃; sample injector temperature: 4 ℃; the mobile phase A is acetonitrile; mobile phase B was 0.1% formic acid-10 mM aqueous ammonium formate solution (containing 0.1% formic acid by volume based on 100% total volume of 10mM aqueous ammonium formate solution); the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 30:70 to 40:60 at a constant speed within 0-2.5 minutes; in 2.5-2.8 minutes, the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 40:60 to 90:10 at a constant speed; the volume ratio of the mobile phase A to the mobile phase B is 90:10 within 2.8-3.8 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 90:10 to 30:70 at a constant speed within 3.8-4.0 minutes; the volume ratio of mobile phase A to mobile phase B is 30:70 in 4.0-5.5 minutes. The flow rate is 0.8 mL/min; the needle washing mode comprises the following steps: rinse Pump the Port; weak needle washing liquid: methanol water (1:1, v/v); washing volume: 1000 μ L; strong needle washing liquid: methanol acetonitrile isopropanol (1:2:1, v/v/v).
Mass spectrum conditions: ion detection mode: multiple Reaction Monitoring (MRM); an ionization mode: pneumatically assisted electrospray ionization (ESI); ion polarity: positive ion (Pos)An ive); detecting an object: guanfacine, [ M + H ]]+M/z 246.1 → 60.1, DP value 90V, CE value 24V; guanfacine-13C-15N 3, [ M + H ]]+M/z 250.0 → 159.1, DP 80V, CE 20V. Mass spectrum parameters: IonSpray Voltage: 5500V; TEM: at 550 ℃. Ion scans of specific guanfacine and guanfacine-13C-15N 3 are shown in FIGS. 1 and 2.
3. Preparation of standard solution
Preparing a guanfacine working solution: accurately weighing two parts of guanfacine reference substances, correcting the reference substances by using a mass correction coefficient, dissolving the reference substances by using methanol to obtain two parts of guanfacine stock solutions with the concentration of 200 mu g/mL, and storing the stock solutions in a refrigerator at the temperature of 20 ℃ below zero. After the stock solution is qualified, precisely measuring one part of guanfacine stock solution, diluting the guanfacine stock solution with methanol to water (50:50, v/v), preparing a series of guanfacine standard curve sample working solutions with the concentrations of 200, 180, 100, 40.0, 18.0, 6.00, 2.00 and 1.00ng/mL, precisely measuring the other part of guanfacine stock solution, diluting the other part of guanfacine stock solution with methanol to water (50:50, v/v), and preparing QC working solutions with the concentrations of 150, 20.0 and 3.00 ng/mL.
Preparing a guanfacine-13C-15N 3 working solution: accurately weighing a guanfacine-13C-15N 3 reference substance, correcting the reference substance by a mass correction coefficient, dissolving the reference substance by using methanol to obtain a guanfacine-13C-15N 3 stock solution with the concentration of 200 mu g/mL, and storing the stock solution in a refrigerator at the temperature of-20 ℃. A certain amount of guanfacine-13C-15N 3 stock solution is precisely measured and diluted by methanol and water (50:50, v/v) to prepare internal standard working solution with the concentration of 10.0 ng/mL.
4. Preparation of standard curve sample and quality control sample
For the standard curve samples and quality control samples at each concentration level, the formulation procedure is exemplified as follows: adding 10 mu L of corresponding working solution into 190 mu L of blank plasma, mixing uniformly, and preparing the volume which can be properly adjusted according to actual conditions, and sequentially preparing a standard curve sample containing guanfacine with the concentration of 0.0500, 0.100, 0.300, 0.900, 2.00, 5.00, 9.00 and 10.00ng/mL and a quality control sample with the concentration of 0.0500ng/mL (LLOQ QC), 0.150ng/mL (LQC), 1.00ng/ML (MQC) and 7.50ng/mL (HQC).
5. Sample pretreatment
Adding 100 mu L of samples (biological samples to be detected, standard curve samples and quality control samples) into a 2.0mL 96-well plate; for both the double blank samples and the blank samples, 100. mu.L of blank matrix was added. Add 50. mu.L of solvent methanol water (1:1, v/v) to the double blank samples, add 50. mu.L of internal standard working solution to all wells except the double blank samples, add 350. mu.L of precipitant methanol, vortex the 96-well plate at 2000rpm/min for 10min, continue to centrifuge the 96-well plate at 4 ℃ for 5min at 4000 rpm/min. 150 μ L of the supernatant was added to a clean 96-well plate, and 100 μ L of 10mM ammonium formate solution (containing 0.1% formic acid) was added and mixed well and placed in a sample room or a refrigerator at the same temperature to be tested.
6. Methodology review content
And (3) carrying out methodology verification on the detection method according to a verification guiding principle of the quantitative analysis method of the biological sample in Chinese pharmacopoeia of 2015 edition to ensure the accuracy, repeatability and stability of detection. The verification comprises the following steps: specificity, standard curve, precision and accuracy, matrix effect, extraction recovery rate, stability.
Results and discussion
1. Specificity
Under the chromatographic conditions adopted in the test, the retention time of guanfacine is about 2.16min, as shown in figure 5; the retention time of the internal standard (guanfacine-13C-15N 3) is about 2.16min, as shown in FIG. 4; respectively taking 100 mu L of blank plasma from 6 different sources, and obtaining blank plasma sample chromatograms according to sample pretreatment operation except for adding no internal standard, such as figures 3-1-3-6; the chromatogram of the lower limit sample is shown in FIG. 5; the results show that endogenous substances in the plasma do not affect the detection of guanfacine, and meanwhile, the internal standard does not affect the detection of guanfacine.
2. Accuracy and precision test
Preparing quality control samples with the concentration of guanfacine of 0.0500ng/mL, 0.150ng/mL, 1.00ng/mL and 7.50ng/mL respectively, preparing 6 samples for each concentration, preparing two standard curves (obtained by regression of two sets of standard curve samples), calculating the ratio f of a guanfacine peak area As of guanfacine to an internal standard peak area Ai of the guanfacine, marking the ratio f As f, substituting f into the standard curve on the day to obtain the average value and the accuracy of the actually measured concentration and calculate the precision and the accuracy in batch, and obtaining the result shown in Table 2. The results show that: except for the lowest limit of quantitation (LLOQ), the precision RSD of the quality control samples in the guanfacine batch is less than 15%, the accuracy RE in the batch is at least 67% and not more than +/-15%, and the deviation of the quality control samples with at least 50% of each concentration level from the theoretical value is not more than +/-15%. The precision RSD of the quality control samples in the batch of the minimum quantitative limit guanfacine samples is less than 20%, the accuracy RE in the batch is at least 67% and is not more than +/-20%, and the deviation of the quality control samples with the quantity of at least 50% of each concentration level and the theoretical value of the quality control samples is not more than +/-20%.
TABLE 2 precision and accuracy of in-batch and inter-batch sample testing
3. Matrix effect investigation
Preparing a matrix sample: using 6 batches of blank plasma from different donors, preparing nine repeated double blank samples from each batch of blank plasma, obtaining a blank matrix extracting solution according to the sample pretreatment operation, and adding a certain amount of analyte and internal standard after extraction so as to lead the final concentration of the blank matrix extracting solution to be consistent with the sample injection concentration of low, medium and high concentration quality control samples respectively (each concentration level, three repetitions).
Preparing a solution sample: and (3) replacing blank plasma with pure water to carry out a pretreatment step, and then diluting the working solution to prepare a corresponding concentration so that the concentration of the added 100 mu L blank matrix extracting solution or 100 mu L pure water extracting solution is consistent with the concentration of the pre-treated low, medium and high concentration quality control samples. And 3 replicate samples per concentration.
The result shows that the total matrix effect factor of the guanfacine matrix effect (calculated by peak area ratio) is 0.94-1.04, the precision is less than 2.3%, and the accurate quantification of guanfacine is not influenced by the plasma matrix. The results of the matrix effect data are shown in table 3.
TABLE 3 matrix Effect
4. Investigation of extraction recovery
Preparing a matrix sample: using plasma mixed by 6 batches of blank plasma of different donors to prepare 9 repeated double blank samples, obtaining blank plasma extract according to sample pretreatment operation, adding a certain amount of analyte and internal standard after extraction so as to lead the final concentration to be consistent with the sample injection concentration of low, medium and high concentration quality control samples respectively (each concentration level, 3 repetitions).
Preparing a quality control sample: and (3) taking quality control samples with low, medium and high concentrations to process according to a sample processing method, and preparing 6 parts of each concentration level.
And evaluating the recovery rate by comparing the response value of the analyte or the internal standard in a single quality control sample with the average value of the response values of the double blank samples added with the analyte and the internal standard after extraction.
The accepted criteria for recovery are: the precision of recovery should be within 15.0% for each concentration level and all concentration levels. The extraction recovery rate (calculated by peak area ratio) of guanfacine is 100.69%, wherein the extraction recovery rates of the low, medium and high concentrations are 103.93%, 97.91% and 100.22% respectively. The results are shown in Table 4.
TABLE 4 recovery rate of extraction
5. Stability survey
Sample stability after treatment: after the analysis batch for inspecting precision and accuracy is subjected to sample injection analysis for the first time, the sample is placed in an automatic sample injector (4 ℃) for 137h, a newly prepared standard curve sample and an analyzed sample are subjected to sample injection analysis, a chromatogram is recorded, the result is shown in table 5, and the sample injection solution after the guanfacine plasma sample is treated is placed in the sample injector for 137h, so that the stability is good, and the requirement of biological sample analysis is met.
Stability at room temperature: the prepared quality control samples with low and high concentration levels have guanfacine concentrations of 0.150ng/mL and 7.50ng/mL respectively, after the samples with each concentration level are uniformly mixed, the samples are placed at room temperature for 24 hours, LC-MS/MS analysis is carried out, chromatogram is recorded, and the results are shown in Table 6, and the stability of the plasma samples is good after the plasma samples are placed at room temperature for 24 hours.
Freeze-thaw stability: samples with the concentration of guanfacine of 0.150ng/mL and 7.50ng/mL are prepared in a fresh manner and are respectively put into a refrigerator with the temperature of 80 ℃ below zero for 5 times of freeze-thaw cycle. The acceptance criteria were: the average measured value of the stability sample and the% RE of the theoretical value of the stability sample should not exceed +/-15.0%, the% RSD of the measured value of the stability sample at each concentration level should be less than or equal to 15.0%, the result is shown in Table 7, and the stability of the sample is good after 5 freeze-thaw cycles at-80 ℃.
Long-term stability: samples with the concentration of guanfacine of 0.150ng/mL and 7.50ng/mL are prepared and put into a refrigerator with the temperature of 80 ℃ below zero respectively for freezing for 41 days and then are detected. The acceptance criteria were: the average measured value of the stability sample and the% RE of the theoretical value of the stability sample should not exceed +/-15.0%, the% RSD of the measured value of the stability sample at each concentration level should be less than or equal to 15.0%, the result is shown in Table 8, and the stability is good when the sample is frozen and stored for 41 days at minus 80 ℃.
TABLE 5 post-preparation sample stability
TABLE 6 stability during pretreatment of biological samples (stability at room temperature)
TABLE 7 Freeze thaw stability
TABLE 8 Long term stability
The method establishes an HPLC-MS/MS determination method of guanfacine in blood plasma, has good specificity, ensures that endogenous substances in the blood plasma do not interfere with the determination of a sample, has a standard curve linear range of 0.0500-10.0 ng/mL and has good linear relation: the precision of the detection results of the quality control samples with high concentration (7.50ng/mL), medium concentration (1.00ng/mL) and low concentration (0.150ng/mL) in batches and between batches is less than 15.0 percent; the precision of the detection result of the quality control sample (0.0500ng/mL) with the lower limit of quantification is less than 20.0 percent in batch and between batches. The total matrix effect factor of guanfacine is 0.94-1.04, the precision is less than 2.3%, and the plasma matrix does not influence the accurate quantification of guanfacine. The extraction recovery of guanfacine was 100.69%. The stability of the guanfacine plasma is good after the guanfacine plasma is placed at room temperature for 24 hours; the stability is good after 5 cycles of freezing/unfreezing; after the plasma sample is treated, the plasma sample is placed in an autosampler at 4 ℃ for 137h, and the stability is good; the guanfacine plasma sample has good stability after being placed at the temperature of minus 80 ℃ for 41 days, and meets the analysis requirement of biological samples.
In conclusion, the method for determining the concentration of guanfacine in human plasma by the HPLC-MS/MS method established by the method meets the relevant requirements in the 'biological sample quantitative analysis method verification guide principle' in the 2020 pharmacopoeia, and can be used for the analysis and detection of plasma samples in clinical tests.
Comparative example 1
The sample pretreatment process and mass spectrometry conditions were the same as in example 1.
Liquid phase conditions: a chromatographic column: ACE C18-PFP (75X 4.6mm,3 μm); column temperature: 40 ℃; sample injector temperature: 4 ℃; the mobile phase A is acetonitrile; the mobile phase B is 0.1% formic acid-10 mM ammonium acetate aqueous solution (containing 0.1% formic acid by volume based on 100% of the total volume of the 10mM ammonium acetate aqueous solution); the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 30:70 to 40:60 at a constant speed within 0-2.5 minutes; in 2.5-2.8 minutes, the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 40:60 to 90:10 at a constant speed; the volume ratio of the mobile phase A to the mobile phase B is 90:10 within 2.8-3.8 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 90:10 to 30:70 at a constant speed within 3.8-4.0 minutes; the volume ratio of mobile phase A to mobile phase B is 30:70 in 4.0-5.5 minutes. The flow rate is 0.8 mL/min; the needle washing mode comprises the following steps: rinse Pump the Port; weak needle washing liquid: methanol water (1:1, v/v); washing volume: 1000 μ L; strong needle washing liquid: methanol acetonitrile isopropanol (1:2:1, v/v/v).
Mass spectrum conditions: ion detection mode: multiple Reaction Monitoring (MRM); an ionization mode: pneumatically assisted electrospray ionization (ESI); ion polarity: positive ions (Positive); detecting an object: guanfacine, [ M + H ]]+M/z 246.1 → 60.1, DP value 90V, CE value 24V; guanfacine-13C-15N 3, [ M + H ]]+M/z 250.0 → 159.1, DP 80V, CE 20V. Mass spectrum parameters: IonSpray Voltage: 5500V; TEM: at 550 ℃.
As a result, it was found that: ammonium formate in the mobile phase B is adjusted to ammonium acetate, the chromatographic peak response is basically unchanged, the baseline drift is too high, and the accuracy of sample determination is influenced.
Comparative example 2
The sample pretreatment process and mass spectrometry conditions were the same as in example 1.
Liquid phase conditions: a chromatographic column: agent ZORBAX XDB-Phenyl (75X 4.6mm,3.5 μm); column temperature: 40 ℃; sample injector temperature: 4 ℃; the mobile phase A is acetonitrile; mobile phase B was 0.1% formic acid-10 mM aqueous ammonium formate solution (containing 0.1% formic acid by volume based on 100% total volume of 10mM aqueous ammonium formate solution); the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 30:70 to 40:60 at a constant speed within 0-2.5 minutes; in 2.5-2.8 minutes, the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 40:60 to 90:10 at a constant speed; the volume ratio of the mobile phase A to the mobile phase B is 90:10 within 2.8-3.8 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 90:10 to 30:70 at a constant speed within 3.8-4.0 minutes; the volume ratio of mobile phase A to mobile phase B is 30:70 in 4.0-5.5 minutes. The flow rate is 0.8 mL/min; the needle washing mode comprises the following steps: rinse Pump the Port; weak needle washing liquid: methanol water (1:1, v/v); washing volume: 1000 μ L; strong needle washing liquid: methanol acetonitrile isopropanol (1:2:1, v/v/v).
Mass spectrum conditions: ion detection mode: multiple Reaction Monitoring (MRM); an ionization mode: pneumatically assisted electrospray ionization (ESI); ion polarity: positive ions (Positive); detecting an object: guanfacine, [ M + H ]]+M/z 246.1 → 60.1, DP value 90V, CE value 24V; guanfacine-13C-15N 3, [ M + H ]]+M/z 250.0 → 159.1, DP 80V, CE 20V. Mass spectrum parameters: IonSpray Voltage: 5500V; TEM: at 550 ℃.
As a result, it was found that: as shown in fig. 6, changing the column type, the chromatographic peak response becomes low, the peak broadens and tailing phenomenon occurs.
Comparative example 3
The sample pretreatment process and mass spectrometry conditions were the same as in example 1.
Liquid phase conditions: a chromatographic column: ACE3C18-PFP (75X 4.6mm,3 μm); column temperature: 40 ℃; sample injector temperature: 4 ℃; the mobile phase A is acetonitrile; mobile phase B was 0.1% formic acid-10 mM aqueous ammonium formate solution (containing 0.1% formic acid by volume based on 100% total volume of 10mM aqueous ammonium formate solution); the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 30:70 to 40:60 at a constant speed within 0-2.5 minutes; in 2.5-2.8 minutes, the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 40:60 to 90:10 at a constant speed; the volume ratio of the mobile phase A to the mobile phase B is 90:10 within 2.8-3.8 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 90:10 to 30:70 at a constant speed within 3.8-4.0 minutes; the volume ratio of mobile phase A to mobile phase B is 30:70 in 4.0-5.5 minutes. The flow rate is 0.8 mL/min; the needle washing mode comprises the following steps: rinse Pump the Port; weak needle washing liquid: methanol water (1:1, v/v); washing volume: 1000 μ L; strong needle washing liquid: methanol acetonitrile (1:1, v/v).
Mass spectrum conditions: ion detection mode: multiple Reaction Monitoring (MRM); an ionization mode: pneumatically assisted electrospray ionization (ESI); ion polarity: positive ions (Positive); detecting an object: guanfacine, [ M + H ]]+M/z 246.1 → 60.1, DP value 90V, CE value 24V; guanfacine-13C-15N 3, [ M + H ]]+M/z 250.0 → 159.1, DP 80V, CE 20V. Mass spectrum parameters: IonSpray Voltage: 5500V; TEM: at 550 ℃.
As a result, it was found that: as shown in fig. 7, when the strong needle wash is adjusted, the analyte is likely to remain in the sample remaining after the upper limit sample is quantified, and the accuracy of the sample measurement after the high concentration sample is affected.
Comparative example 4
The sample pretreatment process and mass spectrometry conditions were the same as in example 1.
A chromatographic column: ACE C18-PFP (75X 4.6mm,3 μm); column temperature: 40 ℃; sample injector temperature: 4 ℃; the mobile phase A is acetonitrile; mobile phase B was 0.1% formic acid-10 mM aqueous ammonium formate solution (containing 0.1% formic acid by volume based on 100% total volume of 10mM aqueous ammonium formate solution); the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 10:90 to 30:70 at a constant speed within 0-1.2 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 30:70 to 40:60 at a constant speed within 1.2-2.5 minutes; in 2.5-2.8 minutes, the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 40:60 to 90:10 at a constant speed; the volume ratio of the mobile phase A to the mobile phase B is 90:10 within 2.8-3.8 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 90:10 to 30:70 at a constant speed within 3.8-4.0 minutes; the volume ratio of mobile phase A to mobile phase B is 30:70 in 4.0-5.5 minutes. The flow rate is 0.8 mL/min; the needle washing mode comprises the following steps: rinse Pump the Port; weak needle washing liquid: methanol water (1:1, v/v); washing volume: 1000 μ L; strong needle washing liquid: methanol acetonitrile isopropanol (1:2:1, v/v/v).
As a result, it was found that: as shown in fig. 8, when the gradient elution process is adjusted, the analyte in the residual sample after the upper limit sample is quantified is likely to remain, which affects the accuracy of the sample measurement after the high concentration sample, and the possible reason for the occurrence of the residue is that the initial water phase ratio is relatively high, which causes the residue to accumulate and be eluted at the peak position.
Comparative example 5
Sample pretreatment: adding 100 mu L of samples (biological samples to be detected, standard curve samples and quality control samples) into a 2.0mL 96-well plate; for both the double blank samples and the blank samples, 100. mu.L of blank matrix was added. Add 50. mu.L of solvent methanol water (1:1, v/v) to the double blank samples, add 50. mu.L of internal standard working solution to all wells except the double blank samples, add 350. mu.L of precipitant acetonitrile, vortex the 96-well plate at 2000rpm/min for 10min, continue to centrifuge the 96-well plate at 4 ℃ for 5min at 4000 rpm/min. 150 μ L of the supernatant was added to a clean 96-well plate, and 100 μ L of 10mM ammonium formate solution (containing 0.1% formic acid) was added and mixed well and placed in a sample room or a refrigerator at the same temperature to be tested.
The liquid phase conditions and mass spectrometry conditions were the same as in example 1.
As a result, it was found that: the precipitant is changed into acetonitrile, the total extraction recovery rate is only about 70%, the acetonitrile viscosity is low, and the acetonitrile is easy to drip and splash in the pretreatment process, so that the sample pollution is caused.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for detecting guanfacine in human plasma by HPLC-MS/MS is characterized by comprising the following steps:
(1) human plasma samples are pretreated; (2) performing liquid chromatography-mass spectrometry detection, and performing gradient elution by using a mobile phase A and a mobile phase B as a mixed mobile phase, wherein the mobile phase A is acetonitrile; the mobile phase B is 2-20 mM ammonium formate aqueous solution; (3) measuring the concentration of guanfacine in human plasma;
wherein, the human plasma sample is pretreated by adopting a protein precipitation method, and a precipitator is methanol; the chromatographic column is ACE C18-PFP; the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 30:70 to 40:60 at a constant speed within 0-2.5 minutes; in 2.5-2.8 minutes, the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 40:60 to 90:10 at a constant speed; the volume ratio of the mobile phase A to the mobile phase B is 90:10 within 2.8-3.8 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 90:10 to 30:70 at a constant speed within 3.8-4.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is 30:70 within 4.0-5.5 minutes; in the gradient elution process, the strong needle washing liquid is a methanol-acetonitrile-isopropanol mixed solution.
2. The method for detecting guanfacine in human plasma by HPLC-MS/MS combined use according to claim 1, wherein in the gradient elution process, the volume ratio of methanol to acetonitrile to isopropanol in the strong needle washing solution is 1-2: 2-3: 1, preferably the volume ratio of methanol to acetonitrile to isopropanol in the strong needle washing solution is 1:2: 1; the weak needle washing liquid is a methanol-water mixed solution, preferably, the volume ratio of methanol to water in the weak needle washing liquid is 1-9: 9-1, and more preferably, the volume ratio of methanol to water in the weak needle washing liquid is 1: 1.
3. The method for detecting guanfacine in human plasma by HPLC-MS/MS combined use according to claim 1, wherein the mobile phase B is 10mM ammonium formate aqueous solution; preferably, the mobile phase B contains 0.05-0.20% by volume of formic acid based on 100% of the total volume of the ammonium formate aqueous solution; more preferably, in mobile phase B, formic acid is contained in a volume ratio of 0.1% based on 100% of the total volume of the aqueous ammonium formate solution.
4. The method for detecting guanfacine in human plasma by HPLC-MS/MS combined use according to claim 1, wherein the liquid chromatography conditions comprise: the length of the chromatographic column is 75mm, the diameter is 4.6mm, and the particle size of the filler is 3 mu m; the column temperature is 30-45 ℃, and preferably 40 ℃; the flow rate is 0.5 to 1.0mL/min, preferably 0.8 mL/min.
5. The method for detecting guanfacine in human plasma by HPLC-MS/MS combined use according to claim 1, wherein in the step (1), the pretreatment of the human plasma sample comprises: adding internal standard working solution and a precipitator into a human plasma sample, taking supernate after vortex and centrifugation, and mixing the supernate with a diluent to obtain a sample to be detected; the internal standard in the internal standard working solution is guanfacine-13C-15N 3; the diluent is 10mM ammonium formate solution, and the diluent contains 0.1-0.15% of formic acid in volume ratio based on 100% of the total volume of the 10mM ammonium formate solution.
6. The method for detecting guanfacine in human plasma by HPLC-MS/MS combined use according to claim 5, wherein in step (1), the internal standard working solution is prepared as follows: weighing a guanfacine-13C-15N 3 reference substance, dissolving the reference substance by using acetonitrile to obtain a guanfacine-13C-15N 3 stock solution with the concentration of 200 mu g/mL, and diluting the stock solution by using a mixed solution of methanol and water with the volume ratio of 50:50 to obtain an internal standard working solution with the concentration of 10.0ng/mL guanfacine-13C-15N 3.
7. The method for detecting guanfacine in human plasma by HPLC-MS/MS combined use according to claim 6, wherein in the step (1), the pretreatment of the human plasma sample comprises: taking 100 mu L of human plasma sample, adding 50 mu L of internal standard working solution and 350 mu L of methanol, taking 150 mu L of supernatant after vortex and centrifugation, and mixing with 100 mu L of diluent to obtain a sample to be detected; the diluent is 10mM ammonium formate solution, and contains 0.1% formic acid by volume based on 100% of the total volume of the 10mM ammonium formate solution.
8. The method for detecting guanfacine in human plasma by HPLC-MS/MS combined use according to claim 6 or 7, wherein the conditions of vortexing and centrifugation are as follows: vortexing for 10min, and centrifuging at 4000rpm/min for 5min at 4 deg.C; and (3) placing the sample to be detected in an automatic sample injector, and performing HPLC-MS/MS analysis, wherein the sample injection volume is 10 mu L, and the sample injector temperature is 4 ℃.
9. The method for detecting guanfacine in human plasma by HPLC-MS/MS combination according to claim 1, wherein the mass spectrum conditions comprise: adopting an electrospray ion source, monitoring and scanning positive ion multiple reaction, wherein the spraying voltage is 5500V, and the ion source temperature is 550 ℃; guanfacine, [ M + H ]]+M/z 246.1 → 60.1, DP value 90V, CE value 24V; guanfacine-13C-15N 3, [ M + H ]]+M/z 250.0 → 159.1, DP 80V, CE 20V.
10. The method for detecting guanfacine in human plasma by HPLC-MS/MS combined use according to claim 1, wherein the method is applicable to clinical pharmacokinetic sample monitoring.
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| CN116735761A (en) * | 2023-08-16 | 2023-09-12 | 四川省药品检验研究院(四川省医疗器械检测中心) | UPLC-MS/MS detection method of brivaracetam in human plasma |
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