Method for instantly detecting concentration of antihypertensive drug in plasma based on miniature mass spectrometer

Abstract

The invention discloses a method for instantly detecting the concentration of an antihypertensive drug in plasma based on a miniature mass spectrometer, the antihypertensive drug is an amlodipine and benazepril compound drug, and the detection method comprises the following steps: S1, adding a standard substance into a blank sample to obtain a standard plasma sample with a series of concentrations, processing and analyzing the standard plasma sample according to the steps S2 and S3 to obtain a standard curve; s2, adding an acetonitrile-water solution into a to-be-detected plasma sample, extracting with diethyl ether, and centrifuging to take an upper-layer organic phase; drying the upper-layer organic phase by blowing nitrogen, and dissolving residues by using an acetonitrile-water solution to obtain a blood sample; s3, the blood sample is injected in a paper capillary spraying mode, and scanning is performed in two sequences in a positive ion scanning mode; and S4, substituting the detection result of the blood sample into the standard curve to obtain the content of amlodipine and benazepril antihypertensive drugs in the blood sample. The method has the characteristic that the amlodipine and the benazepril can be effectively and quickly detected at one time at the same time.

Description

Technical Field

[0001] This invention relates to a method for detecting drug concentration in blood, and particularly to a method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer. Background Technology

[0002] Hypertension is one of the most common and serious diseases worldwide, and it can trigger many other diseases, such as coronary heart disease, cerebral infarction, and kidney disease. It can cause damage to target organs such as the heart, brain, and kidneys, making it a "silent killer" of cardiovascular and cerebrovascular diseases. Currently, the main treatment for hypertension is long-term oral antihypertensive medication.

[0003] Amlodipine is a dihydropyridine calcium channel blocker (CCB). It inhibits the influx of extracellular calcium ions by blocking slow calcium channels on the cell membranes of myocardial and vascular smooth muscle cells, thereby reducing the intracellular calcium ion concentration and inhibiting the contraction of myocardial and vascular smooth muscle cells. This leads to the dilation of coronary arteries and peripheral arterioles, thus reducing afterload.

[0004] Benazepril is a non-sulfhydryl angiotensin-converting enzyme inhibitor. In the liver, it is rapidly hydrolyzed by esterases into its active metabolite, benazeprilat. Benazeprilat competitively inhibits the binding of angiotensin I to angiotensin-converting enzyme, blocking the conversion of angiotensin I to angiotensin II, reducing various angiotensin I-mediated effects, and also inhibiting the degradation of bradykinin, thus reducing vascular resistance and producing a hypotensive effect without causing compensatory fluid retention. It can also reduce ventricular afterload without increasing heart rate, and improve left ventricular hypertrophy and glucose tolerance in diabetes.

[0005] Because hypertensive patients need to take medication long-term, prolonged use of a single antihypertensive drug can easily lead to drug tolerance. Combination therapy, especially using antihypertensive drugs with different mechanisms of action, can complement each other, enhancing the antihypertensive effect, and reducing the risk of adverse reactions. For example, the combination of amlodipine and benazepril can more effectively control blood pressure, and because the two drugs have different effects on serum potassium, it can help maintain the patient's serum potassium level within the normal range, reducing the risk of adverse reactions and edema caused by abnormal potassium levels.

[0006] However, due to individual differences, the efficacy and side effects of the same dosage of medication can vary greatly among different individuals. Furthermore, the drug concentration can differ even when the same dosage is used by different patients with the same disease. Therefore, individualized medication regimens based on blood drug concentration monitoring are necessary to improve therapeutic efficacy, minimize adverse reactions, and achieve safe, effective, and rational drug use in clinical practice.

[0007] Currently, blood drug concentration detection typically employs techniques such as liquid chromatography, gas chromatography, and mass spectrometry, which offer advantages such as high selectivity, high separation, and high sensitivity. However, these techniques require large laboratory equipment and specialized technical personnel, causing numerous inconveniences for routine testing.

[0008] Although existing technologies exist for determining the concentrations of amlodipine and benazepril separately, there is no method for simultaneously determining the concentrations of amlodipine and benazepril. Furthermore, amlodipine has a lower ionization sensitivity than benazepril, resulting in a significant difference in their ionization sensitivities. Additionally, the polarities of amlodipine and benazepril differ considerably. Therefore, it is difficult to employ a single method to simultaneously detect amlodipine and benazepril, reduce ionization competition and interference between them, ensure a sufficiently low limit of quantitation, and avoid excessively high response from benazepril that could lead to response saturation at high concentrations. Summary of the Invention

[0009] The purpose of this invention is to provide a method for the real-time detection of antihypertensive drug concentrations in plasma based on a miniature mass spectrometer. This invention features the ability to simultaneously and effectively detect amlodipine and benazepril in a single step.

[0010] The technical solution of this invention: a method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer, wherein the antihypertensive drug is a combination drug of amlodipine and benazepril, and the detection method includes the following steps:

[0011] S1. Construction of Standard Curve:

[0012] Add standard substances containing amlodipine and benazepril to blank samples to obtain a series of standard plasma samples of different concentrations. After processing the standard plasma samples according to step S2, they are analyzed by mass spectrometry according to the conditions of step S3 to obtain a standard curve.

[0013] S2. Plasma sample processing:

[0014] a. Add acetonitrile-water solution to the plasma sample to be tested and mix well. Then add diethyl ether to extract and centrifuge to collect the upper organic phase.

[0015] b. Dry the upper organic phase with nitrogen gas, and dissolve the dried residue with acetonitrile-water solution to obtain a blood sample;

[0016] S3, Blood Sample Testing:

[0017] The blood sample was injected into the mass spectrometer using the paper capillary spray method, and the positive ion scanning mode was used to scan the sample in two sequences.

[0018] S4. Blood Sample Analysis:

[0019] The blood sample test results were substituted into the standard curve, and linear regression was performed using the concentration corresponding to the peak area to obtain the content of amlodipine and benazepril antihypertensive drugs in the blood sample.

[0020] In the aforementioned method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer, in step S1, the standard substance is a mixed solution of amlodipine and benazepril dissolved in acetonitrile, with a concentration of 100 μg·mL. -1 The blank sample is a plasma sample that, after processing in step S2, did not show any amlodipine or benazepril detected by portable mass spectrometry.

[0021] In the aforementioned method for real-time detection of antihypertensive drug concentrations in plasma based on a miniature mass spectrometer, in step S1, the serial concentrations of amlodipine and benazepril in the standard plasma sample are 50 ng / mL. -1 100 ng·mL -1 250 ng / mL -1 500 ng·mL -1 1 μg·mL -1 2 μg·mL -1 5 μg·mL -1 .

[0022] In the aforementioned method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer, in step S2, the volume ratio of acetonitrile to aqueous solution is 4:6; the centrifugation speed is 4000–8 r / min; and the centrifugation time is 8–12 min.

[0023] In the aforementioned method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer, in step S3, the precursor ion of amlodipine is m / z 409, and the daughter ions are m / z 238 and m / z 294; the precursor ion of benazepril is m / z 425, and the daughter ions are m / z 351 and m / z 190.

[0024] In the aforementioned method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer, in step S3, the ionization voltage of amlodipine is 3900V, the ISO1 energy of amlodipine is 10V, the ISO2 energy of amlodipine is 5V, and the CID energy of amlodipine is 1.5V.

[0025] In the aforementioned method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer, in step S3, the ionization voltage of benazepril is 4200V, the ISO1 energy of benazepril is 8V, the ISO2 energy of benazepril is 4V, and the CID energy of benazepril is 2.0V.

[0026] In the aforementioned method for real-time detection of antihypertensive drug concentrations in plasma based on a miniature mass spectrometer, the detection limit for amlodipine is 0.3 μg·mL. -1 The limit of quantification for amlodipine is 1 μg / mL. -1 .

[0027] In the aforementioned method for real-time detection of antihypertensive drug concentrations in plasma based on a miniature mass spectrometer, the detection limit for benazepril is 15 μg·L⁻¹. -1 The limit of quantification for benazepril is 50 μg·L⁻¹. -1 .

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] This invention establishes a point-of-care testing (POCT) method for the antihypertensive drugs amlodipine and benazepril based on a miniature mass spectrometer (mMS). A paper-capillary spray (PCS) ionization source is used to achieve rapid detection of these two antihypertensive drugs within 30 seconds. Both amlodipine and benazepril are ionized in positive ion mode using mMS. The fractional ions from secondary mass spectrometry were used to analyze simultaneously injected amlodipine and benazepril. The ion pairs for amlodipine were determined to be m / z 409>m / z 238 and m / z 409>m / z 294; the ion pairs for benazepril were m / z 425>m / z 351 and m / z 425>m / z 190. This effectively reduces ionization competition, attenuation, and interference between the precursor ions of the two drugs, enabling simultaneous and effective detection of both amlodipine and benazepril. This method ensures a sufficiently low limit of quantitation while avoiding excessively high response from benazepril that could lead to response saturation at high concentrations. It features strong qualitative capabilities, high detection sensitivity, and the ability to perform real-time dynamic monitoring.

[0030] Further optimization of instrument parameters was performed: the ionization voltages of amlodipine and benazepril were set to 3900V and 4200V, respectively; the ISO1 energies were 10V and 8V, respectively; the ISO2 energies were 5V and 4V, respectively; and the CID energies of amlodipine and benazepril were 1.5V and 2.0V, respectively. This resulted in the maximum corresponding intensity of the precursor ions for amlodipine and benazepril, while the intensity of impurity peaks was essentially zero.

[0031] The method was validated, and the limits of detection for amlodipine and benazepril were 0.3 μg·mL, respectively. -1 and 15 μg·L -1 The instrument's quantitation limit is 1 μg·mL. -1 and 50 μg·L -1 The intra-day precision RSDs for amlodipine and benazepril were 7.9%–9.1% and 7.0%–8.8%, respectively, while the inter-day precisions were 9.8%–12.6% and 6.9%–8.6%, respectively. Furthermore, the recoveries of amlodipine and benazepril ranged from 86.3% to 94.7%, with relative deviations (RSDs) of 6.2% to 9.5%.

[0032] Therefore, this invention has important clinical significance for monitoring blood drug concentration during hypertension medication, predicting drug efficacy, and customizing personalized medication regimens. Attached Figure Description

[0033] Figure 1 a is the secondary mass spectrum of amlodipine;

[0034] Figure 1 b is the secondary mass spectrum of benazepril. Detailed Implementation

[0035] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0036] Example:

[0037] Instrument used: Miniπ micro-mass spectrometer. This micro-mass spectrometer (mMS) has full scan capability from m / z 50 to m / z 1000 in positive ion mode, negative ion mode, ion trap CID, neutral loss scan, and precursor ion scan, with a scan rate of 20000 Da / s. During sample analysis, ions generated in the ambient ionization source enter the micro-mass spectrometer via DAPI. DAPI is momentarily turned on by a pulsed voltage for ion introduction and then turned off for the remainder of each scan cycle for ion manipulation and mass analysis.

[0038] The miniature mass spectrometer features a collision-induced dissociation (CID) mass ratio (q) of 3.5, a CID voltage of 0.5–3 V, and a linear ion trap radio frequency (RF) of 1033 kHz. Resonant jetting was performed at a scan rate of 1000 Da / s and an AC frequency of 376 kHz. Instrument control and data acquisition were performed using SpecMS software.

[0039] Antihypertensive drugs: Amlodipine and benazepril combination therapy.

[0040] Clinical subject selection:

[0041] Six healthy adult subjects (half male and half female) were selected, all in good health (no significant clinical symptoms or clinically significant abnormal laboratory results); another six were selected as hypertensive patients whose daily medication was amlodipine and benazepril capsules. All subjects were non-smokers, had no history of alcohol or drug allergies, and had not participated in other clinical trials within the month prior to this trial.

[0042] Blood samples from healthy adult subjects were collected at any time. For hypertensive patients, venous blood was collected in heparin tubes 2 hours after taking amlodipine and benazepril compound capsules. The collected blood samples were immediately centrifuged, and the obtained plasma samples were frozen at -80°C.

[0043] A method for real-time detection of antihypertensive drug concentrations in plasma based on a miniature mass spectrometer includes the following steps:

[0044] S1. Construction of Standard Curve:

[0045] Standard substances were added to blank samples to obtain a series of concentrations (the series concentrations of amlodipine and benazepril were 50 ng / mL). -1 100 ng·mL -1 250 ng·mL -1 500 ng·mL -1 1 μg·mL -1 2 μg·mL -1 5 μg·mL -1 The standard blood sample was processed according to step S2, and then analyzed by mass spectrometry according to the conditions in step S3. A linear regression was performed with the peak area of ​​the target analyte in the standard blood sample as the ordinate and the concentration of the standard blood sample as the abscissa to obtain the standard curve.

[0046] The standard substance was a mixed solution of amlodipine and benazepril dissolved in acetonitrile, with a concentration of 100 μg·mL⁻¹. -1 .

[0047] Blank samples are plasma samples from healthy adult subjects that, after processing in step S2, did not show any detection of amlodipine or benazepril by portable mass spectrometry.

[0048] S2. Plasma sample processing:

[0049] Plasma samples of 500 μL were collected from 6 hypertensive patients who were taking compound amlodipine and benazepril capsules orally. 100 μL of acetonitrile-water (40:60, v / v) solution was added and the mixture was vortexed for 1 min to mix. Then, 3 mL of ether was added and the mixture was extracted by vortexing for 3 min. After centrifugation for 10 min (6000 r / min), the upper organic phase was collected and dried under nitrogen at 40 °C. The residue after drying was dissolved in 100 μL of acetonitrile-water (40:60, v / v) solution to obtain blood samples.

[0050] This procedure purifies the blood sample to remove most of the proteins and lipids, thereby reducing matrix interference.

[0051] S3, Blood Sample Testing:

[0052] Take 5 μL of blood sample and put it into the paper capillary spray (PCS) kit. Place the PCS kit in a laboratory desiccator and dry for about 30 seconds. Then, add 100 μL of methanol to the PCS kit to elute the sample. Finally, put the PCS kit into a miniature mass spectrometer for MS / MS secondary mass spectrometry analysis.

[0053] The mass spectrometry conditions for the mass spectrometry analysis were as follows: positive ion scanning mode was used, and two injection sequences were set up for each blood sample, performing two scans on the same sample. The ionization voltages of amlodipine and benazepril were set to 3900V and 4200V, respectively, resulting in the strongest signals; the ISO1 energies of amlodipine and benazepril were set to 10V and 8V, respectively, achieving the highest signal intensity; the ISO2 energies of amlodipine and benazepril were set to 5V and 4V, respectively, with the impurity peak intensities essentially zero. These first-stage scanning parameters maximized the precursor ion response of amlodipine and benazepril.

[0054] The parent ion of amlodipine is m / z 409, and the daughter ions are m / z 238 and m / z 294.

[0055] The parent ion of benazepril is m / z 425, and the daughter ions are m / z 351 and m / z 190.

[0056] The collision-induced dissociation (CID) energy of amlodipine is 1.5 V. The daughter ions m / z 238 and m / z 294 have the strongest and most stable responses, at 6.7e5 and 4.5e5, respectively. The coefficient of variation for daughter ion m / z 238 is 9.0%, and that for daughter ion m / z 294 is 19.1%.

[0057] The collision-induced dissociation (CID) energy of benazepril is 2.0 V, with the strongest and most stable responses at m / z 351 and m / z 190, at 4.5e6 and 5.5e5, respectively.

[0058] The ion pairs for amlodipine are m / z 409-m / z 238 and m / z 409-m / z 294; the ion pairs for benazepril are m / z 425-m / z 351 and m / z 425-m / z 190.

[0059] S4. Blood Sample Analysis:

[0060] The results of blood sample analysis using a miniature mass spectrometer were substituted into a standard curve, and linear regression was performed using the concentration corresponding to the peak area to obtain the content of amlodipine and benazepril antihypertensive drugs in the blood sample.

[0061] S5. Analysis of test results:

[0062] Amlodipine and benazepril molecules were successfully detected in plasma samples taken 2 hours after oral administration of the medication. Quantitative analysis using a standard curve revealed limits of detection of 0.3 μg / mL for both amlodipine and benazepril. -1 and 15 μg·L -1 RSD < 15%.

[0063] The detection results are relatively stable, therefore this method can be used for point-of-care testing (POCT) of blood concentrations of amlodipine and benazepril in hypertensive patients.

[0064] Method verification:

[0065] Following the above method, amlodipine and benazepril were injected together for MS / MS analysis. Amlodipine was injected at a concentration of 50 ppb, and benazepril at a concentration of 10 ppb. The results are as follows. Figure 1 As shown.

[0066] from Figure 1 As can be seen, the spectra of amlodipine and benazepril under secondary mass spectrometry were relatively clean, and no interference from impurities was observed. Furthermore, the target compounds amlodipine and benazepril could be qualitatively identified using a multiple reaction method where one precursor ion corresponds to one daughter ion.

[0067] Benazepril in the range of 50–5000 μg·L -1 Good linear correlation within the range (r) 2 =0.9987), amlodipine in the range of 1–100 μg·mL -1 Good linear correlation within the range (r) 2=0.9925). Based on the signal-to-noise ratio (SNR) of qualitative ions ≥3 and the SNR of quantitative ions ≥10, the limits of detection (LOD) for amlodipine and benazepril were calculated to be 0.3 μg·mL⁻¹. -1 and 15 μg·L -1 The instrument's limit of quantitation (LOQ) was 1 μg·mL. -1 and 50 μg·L -1 The results showed that mMS had good sensitivity to benazepril, while amlodipine had relatively low ionization efficiency, which was further reduced under the influence of whole blood matrix.

[0068] Amlodipine at 1 μg·mL -1 5 μg·mL -1 and 25 μg·mL -1 At a concentration of 50 μg·L⁻¹, benazepril... -1 200 μg·L -1 and 1000 μg·L -1 The precision of the method was assessed using mixed standard solutions of varying concentrations. For each concentration, six consecutive injections were performed, and the intra-day precision (RSD) for amlodipine and benazepril was calculated to be 7.9%–9.1% and 7.0%–8.8%, respectively. For three days, six consecutive injections were performed daily, and the inter-day precision for amlodipine and benazepril was calculated to be 9.8%–12.6% and 6.9%–8.6%, respectively. Furthermore, the recoveries of amlodipine were 86.3%–90.6% with relative deviations (RSDs) of 6.2%–9.5%, and the recoveries of benazepril were 87.3%–94.7% with relative deviations (RSDs) of 6.4%–8.7%. See Table 1 for details. These findings demonstrate the high accuracy and reliability of the method.

[0069] Table 1. Linearity, Limit of Detection, and Limit of Quantitation for the Two Drugs

[0070]

[0071] Therefore, this method can detect the concentrations of amlodipine and benazepril in plasma in a single step, with good linearity. It reduces ionization competition, attenuation, and interference between the two, ensuring that the analytical method has a sufficiently low limit of quantitation while avoiding the possibility of response saturation at high concentration points due to excessively high benazepril response.

Claims

1. A method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer, characterized in that: The antihypertensive drug is a combination of amlodipine and benazepril, and the detection method includes the following steps: S1. Construction of Standard Curve: Add standard substances containing amlodipine and benazepril to blank samples to obtain a series of standard plasma samples of different concentrations. After processing the standard plasma samples according to step S2, they are analyzed by mass spectrometry according to the conditions of step S3 to obtain a standard curve. S2. Plasma sample processing: a. Add acetonitrile-water solution to the plasma sample to be tested and mix well. Then add diethyl ether to extract and centrifuge to collect the upper organic phase. b. Dry the upper organic phase with nitrogen gas, and dissolve the residue with acetonitrile-water solution to obtain a blood sample; S3, Blood Sample Testing: The blood sample was injected into the mass spectrometer using the paper capillary spray method, and the positive ion scanning mode was used to scan the sample in two sequences. S4. Blood Sample Analysis: The blood sample test results were substituted into the standard curve, and linear regression was performed using the concentration corresponding to the peak area to obtain the content of amlodipine and benazepril antihypertensive drugs in the blood sample.

2. The method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer according to claim 1, characterized in that: In step S1, the standard substance is a mixed solution of amlodipine and benazepril dissolved in acetonitrile, and the concentration of the mixed solution is 100 μg·mL -1 ; the blank sample is a blood plasma sample in which amlodipine and benazepril are not detected by portable mass spectrometry after step S2.

3. The method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer according to claim 1, characterized in that: In step S1, the series concentrations of amlodipine and benazepril in the standard plasma sample are 50 ng·mL -1 , 100 ng·mL -1 , 250 ng·mL -1 , 500 ng·mL -1 , 1 μg·mL -1 , 2 μg·mL -1 , 5 μg·mL -1 .

4. The method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer according to claim 1, characterized in that: In step S2, the volume ratio of acetonitrile to aqueous solution is 4:6; the centrifugation speed is 4000–8 r / min; and the centrifugation time is 8–12 min.

5. The method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer according to claim 1, characterized in that: In step S3, the precursor ion of amlodipine is m / z 409, and the daughter ions are m / z 238 and m / z 294; the precursor ion of benazepril is m / z 425, and the daughter ions are m / z 351 and m / z 190.

6. The method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer according to claim 1, characterized in that: In step S3, the ionization voltage of amlodipine is 3900V, the ISO1 energy of amlodipine is 10V, the ISO2 energy of amlodipine is 5V, and the CID energy of amlodipine is 1.5V.

7. The method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer according to claim 1, characterized in that: In step S3, the ionization voltage of benazepril is 4200V, the ISO1 energy of benazepril is 8V, the ISO2 energy of benazepril is 4V, and the CID energy of benazepril is 2.0V.

8. The method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer according to claim 1, characterized in that: The detection limit for amlodipine was 0.3 μg·mL. -1 The limit of quantification for amlodipine is 1 μg / mL. -1 .

9. The method for real-time detection of antihypertensive drug concentration in plasma based on a miniature mass spectrometer according to claim 1, characterized in that: The detection limit for benazepril was 15 μg·L⁻¹. -1 The limit of quantification for benazepril is 50 μg·L⁻¹. -1 .

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