Rapid quantitative analysis method for plasma concentration of ropivacaine

Abstract

The invention relates to the field of mass spectrometric detection, and discloses a rapid quantitative analysis method for plasma concentration of ropivacaine, which relates to the field of mass spectrometric detection, and comprises the following steps: preparing a ropivacaine standard curve sample and a quality control sample; carrying out pretreatment on the standard curve sample, the quality control sample and the sample to be detected by adopting a method of solid-phase extraction after protein precipitation; then performing sample injection detection by adopting a mass spectrometry method; and establishing a regression equation according to the standard curve sample, and calculating the concentration of ropivacaine in the to-be-detected sample. According to the invention, the rapid quantitative detection of the concentration of ropivacaine in human plasma / serum / whole blood can be realized, the linear range concentration of ropivacaine is 10-10000 ng / mL, and the concentrations of quality control samples are 3 ng / mL, 400 ng / mL, 4000 ng / mL and 7500 ng / mL. The detection sensitivity is high, the linear range is wide, the accuracy is high, the analysis speed is high, convenience and rapidness are achieved, and support is provided for rapid monitoring of clinical blood concentration and reasonable medication of ropivacaine.

Description

Technical Field

[0001] This invention relates to the field of mass spectrometry detection, and more specifically to a rapid quantitative analysis method for ropivacaine concentration in various biological samples based on mass spectrometry. Background Technology

[0002] Ropivacaine is a relatively new long-acting local anesthetic. Due to its high pKa value and low lipid solubility, ropivacaine exhibits stronger sensory and motor blocking effects, while its toxicity is significantly lower than other long-acting local anesthetics. Therefore, it has been increasingly widely used in anesthesia, postoperative analgesia, and other fields, including local infiltration, peripheral nerve blocks, and epidural anesthesia.

[0003] Ropivacaine's primary mechanism of action is the blocking of sodium-calcium channels, including those in the heart, thus exhibiting cardiotoxicity. Furthermore, during clinical use, ropivacaine can induce the cell division regulator DRP1, leading to mitochondrial dysfunction and resulting in mitochondrial homeostasis imbalance, ultimately inducing neuronal damage. However, the efficacy and toxicity of ropivacaine vary significantly among individuals, necessitating real-time monitoring of blood drug concentrations and timely dosage adjustments to ensure both efficacy and safety during clinical use. Therefore, an accurate, efficient, simple, and rapid method for quantitative analysis of ropivacaine blood drug concentrations is needed. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid quantitative analytical method for detecting the concentration of ropivacaine in serum, plasma, and whole blood.

[0005] This invention provides an analytical procedure for the quantitative analysis of ropivacaine blood concentration in different samples, characterized by comprising the following steps:

[0006] (1) Prepare ropivacaine standard curve samples and quality control samples;

[0007] (2) Pretreatment of whole blood, plasma and serum samples;

[0008] (3) Detection of ropivacaine concentration in samples based on mass spectrometry;

[0009] (4) Experimental data processing and result analysis.

[0010] The method used was mass spectrometry for detection. The chromatographic conditions were as follows: no column was connected; the mobile phase was 0.05-0.5% formic acid acetonitrile solution; the flow rate was 0.1-0.5 mL / min; isocratic elution was used; and the injection volume was 1 μL to 10 μL.

[0011] The mass spectrometry conditions were as follows: ESI ion source; positive ion mode; drying gas temperature: 150-350℃; drying gas flow rate: 5-20 L / min; spray gas flow rate: 10-50 psi; sheath gas temperature: 250-400℃; sheath gas flow rate: 5-20 L / min; spray voltage: 2500-5000 V; capillary temperature: 150-320℃.

[0012] Preferably, the ropivacaine standard curve sample and quality control sample are prepared using ropivacaine standard and blank biological matrix. The linear range concentration of the ropivacaine standard curve sample is 10-10000 ng / mL, and the concentrations of the quality control samples are 3 ng / mL, 400 ng / mL, 4000 ng / mL, and 7500 ng / mL. The blank biological matrix is ​​whole blood, serum, or plasma from healthy humans or animals, stored at -80°C, and thawed naturally at room temperature before use.

[0013] The internal standard used for ropivacaine analysis in the mixed internal standard solution is deuterated ropivacaine, and the concentration of deuterated ropivacaine in the internal standard solution is 200-1000 ng / mL.

[0014] The mass spectrometry conditions were as follows: ESI ion source; positive ion mode; drying gas temperature: 200-300℃; drying gas flow rate: 10-20 L / min; spray gas flow rate: 20-40 psi; sheath gas temperature: 300-400℃; sheath gas flow rate: 10-20 L / min; spray voltage: 3000-5000 V; capillary temperature: 200-320℃.

[0015] The optimal internal standard for ropivacaine analysis is deuterated ropivacaine, with a concentration of 200 ng / mL. Pretreatment of whole blood, plasma, and serum samples: Take 20 μL of the ropivacaine standard curve sample, quality control sample, or plasma / whole blood / serum sample, add 400 μL of internal standard solution, vortex, and then add to a solid-phase extraction column with phospholipid / protein removal function. Collect the filtrate and inject for analysis.

[0016] Mass spectrometry detection: The chromatographic conditions were as follows: no column connection; mobile phase: 0.1% formic acid acetonitrile solution; flow rate: 0.5 mL / min; isocratic elution; injection volume: 1 μL.

[0017] The mass spectrometry conditions were as follows: ESI ion source; positive ion mode; drying gas temperature: 150-350℃; drying gas flow rate: 5-20 L / min; spray gas flow rate: 10-50 psi; sheath gas temperature: 250-400℃; sheath gas flow rate: 5-20 L / min; capillary voltage: 2500-5000 V; nozzle voltage: 200-800 V.

[0018] The optimal mass spectrometry analysis conditions are as follows: ESI ion source; positive ion mode; drying gas temperature: 250℃; drying gas flow rate: 7L / min; spray gas flow rate: 40psi; sheath gas temperature: 350℃; sheath gas flow rate: 12L / min; capillary voltage: 3000V; nozzle voltage: 500V.

[0019] Beneficial technical effects:

[0020] This invention proposes a rapid quantitative analysis method for ropivacaine blood concentration. Utilizing mass spectrometry and developing suitable detection conditions, the method optimizes the selection of mobile phase, pretreatment methods, and mass spectrometry parameters to achieve rapid quantitative detection of ropivacaine concentration in human plasma / serum / whole blood. Method validation shows that the linear concentration range for ropivacaine is 10-10000 ng / mL, exhibiting high sensitivity, high accuracy, and rapid analysis speed, providing convenient and efficient technical support for rapid clinical blood concentration detection of ropivacaine. Attached Figure Description

[0021] Figure 1 This is a typical mass spectrometry MRM image of the ropivacaine standard curve sample in Example 1 of the present invention;

[0022] Figure 2 This is a typical mass spectrometry MRM image of the ropivacaine blank sample in Example 1 of this invention;

[0023] Figure 3 This is a typical mass spectrometry (MRM) image of a real plasma sample containing ropivacaine in Example 1 of this invention;

[0024] Figure 4 This is the standard curve of ropivacaine in human plasma in Example 1 of the present invention. Detailed Implementation

[0025] Example 1

[0026] Rapid quantitative analysis of ropivacaine concentration in human plasma

[0027] I. Solution Preparation

[0028] 1.1 Reference stock solution

[0029] Accurately weigh two portions of ropivacaine hydrochloride reference standard, approximately 22.6 mg each, and place them in separate 10 mL volumetric flasks. Dissolve and dilute to the mark with water to prepare standard curve stock solutions with a concentration of 2 mg / mL. Store at 4°C. One portion is used as the stock solution for preparing the standard curve sample; the other portion is used as the stock solution for preparing the quality control sample.

[0030] 1.2 Standard curve working solution and quality control working solution

[0031] Working solutions for standard curve samples: Take an appropriate amount of ropivacaine reference stock solution, dilute with water, and prepare a series of working solutions for ropivacaine standard curve samples with concentrations of 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL, and 1000 μg / mL.

[0032] Quality control sample working solutions: Take an appropriate amount of ropivacaine reference stock solution, dilute it with water, and prepare a series of ropivacaine quality control sample working solutions with concentrations of 0.3 μg / mL, 40 μg / mL, 400 μg / mL and 750 μg / mL.

[0033] 1.3 Standard curve samples and quality control samples

[0034] Standard curve samples: 10 μL of each of the above standard curve sample working solutions were added to 990 μL of blank biological matrix to obtain a series of standard curve samples with ropivacaine concentrations of 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL, 5000 ng / mL and 10000 ng / mL.

[0035] Quality control samples: 10 μL of each of the above quality control sample working solutions were added to 990 μL of blank biological matrix to obtain quality control samples with ropivacaine concentrations of 3 ng / mL, 400 ng / mL, 4000 ng / mL and 7500 ng / mL.

[0036] The blank biological matrix is ​​healthy human blood plasma, stored at -80℃, and thawed naturally at room temperature before use.

[0037] 1.4 Preparation of internal standard solution

[0038] Accurately weigh an appropriate amount of stable isotope internal standard ropivacaine-D7 and place it in a volumetric flask. Add an appropriate amount of acetonitrile and dissolve it completely to obtain an internal standard stock solution with a ropivacaine-D7 concentration of 500 μg / mL. Take 10 μL of the above internal standard stock solution and add it to 25 mL of acetonitrile containing 1% formic acid to obtain an internal standard solution with a ropivacaine-D7 concentration of 200 ng / mL.

[0039] II. Sample Pretreatment

[0040] Pretreatment of standard curve samples and quality control samples: Take 20 μL of each of the above standard curve samples and quality control samples, add 400 μL of internal standard solution, vortex for 5 min, add to a solid phase extraction column with phospholipid / protein removal function, take the filtrate, and inject it into a mass spectrometer for analysis.

[0041] Sample pretreatment: The sample to be tested is human plasma, which is stored at -80℃ and thawed naturally at room temperature before use. Take 20 μL of the sample to be tested and add it to 400 μL of internal standard solution. Vortex for 5 min and add it to a solid phase extraction column with phospholipid / protein removal function. Take the filtrate and inject it into a mass spectrometer for analysis.

[0042] III. Sample Testing

[0043] The Agilent K6460 liquid chromatography-mass spectrometry (LC-MS / MS) instrument was used for detection.

[0044] Chromatographic conditions: No column connection; mobile phase: acetonitrile solution containing 0.1% formic acid (v / v); flow rate: 0.5 mL / min; injection volume: 10 μL; isocratic elution: elution time: 2 min; injection solution: a 1:1:1 mixture of methanol, isopropanol, and water (v / v / v).

[0045] Mass spectrometry conditions: Ion source: ESI; Scan mode: positive ion mode; Drying gas temperature: 250℃; Drying gas flow rate: 7 L / min; Spray gas flow rate: 40 psi; Sheath gas temperature: 350℃; Sheath gas flow rate: 12 L / min; Capillary voltage: 3000 V; Nozzle voltage: 500 V; Acquisition mode: Multiple reaction monitoring (MRM). MRM scanning parameters for ropivacaine and stable isotope-labeled internal standards are shown in Table 1.

[0046] Table 1 MRM Mode Scanning Parameters

[0047]

[0048]

[0049] IV. Results Analysis

[0050] 4.1 Standard curve and linear range of ropivacaine

[0051] Typical MRM spectra of ropivacaine in the standard curve samples are shown below. Figure 1 Ten standard curve samples were pretreated and injected sequentially from low to high concentrations. The ropivacaine concentration was plotted on the x-axis (X), and the ratio of the ropivacaine peak area to the internal standard peak area was plotted on the y-axis (Y), with a weighting factor of 1 / x. The linear equation and linear correlation coefficient R of the ropivacaine standard curve were calculated. 2 (like Figure 2 (As shown). The linear regression equation for ropivacaine is Y = 7.634169X - 0.007478, and the linear correlation coefficient R0 is... 2 The value was 0.999. This indicates that ropivacaine in human plasma exhibits good linearity within the range of 10-10000 ng / mL.

[0052] 4.2 Precision and accuracy of ropivacaine

[0053] After pretreatment, the quality control samples were analyzed using the method provided in this invention, with quantitative analysis performed using the internal standard method. The precision and accuracy of this method were verified, and detailed results are shown in Table 2. The accuracy ranged from 91.4% to 106%, the intra-batch precision from 0.60% to 3.0%, and the inter-batch precision from 0.10% to 4.7%. This demonstrates that the method exhibits good precision and accuracy.

[0054] Table 2 Precision and Accuracy of Ropivacaine

[0055]

[0056] 4.3 Matrix effect and extraction recovery of ropivacaine

[0057] After pretreatment, quality control samples were analyzed using the method provided in this invention, with quantitative analysis performed using the internal standard method. The matrix effect and extraction recovery of this method were validated, and detailed results are shown in Table 3. The relative matrix effect of ropivacaine after internal standard correction was between 102% and 108%, the RSD of ropivacaine extraction recovery was between 2.6% and 5.6%, and the RSD of internal standard extraction recovery was between 2.4% and 5.5%. This demonstrates that the matrix effect and extraction recovery of this method are good.

[0058] Table 3. Matrix effect and extraction recovery of ropivacaine

[0059]

[0060] 4.4 Detection of Ropivacaine Concentration in Human Plasma Samples

[0061] The samples to be tested were plasma samples collected from four patients using ropivacaine at different time points. Rapid quantitative analysis of the ropivacaine concentration in the samples was performed. The measured plasma concentrations of ropivacaine in each sample are shown in Table 4.

[0062] Table 4. Concentration of ropivacaine in plasma samples from subjects

[0063]

[0064] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A rapid quantitative analysis method for ropivacaine blood concentration, characterized in that... Includes the following steps: Prepare ropivacaine standard curve samples and quality control samples; pretreat the standard curve samples, quality control samples and test samples by protein precipitation followed by solid phase extraction; then use mass spectrometry for sample injection and detection; establish a regression equation based on the standard curve samples to calculate the concentration of ropivacaine in the test samples.

2. The rapid quantitative analysis method for ropivacaine blood concentration according to claim 1, characterized in that, The standard curve samples and quality control samples of ropivacaine were prepared using ropivacaine reference standard and blank biological matrix.

3. The rapid quantitative analysis method for ropivacaine blood concentration according to claim 2, wherein the linear range concentration of ropivacaine is 10-10000 ng / mL, and the quality control sample concentrations are 3 ng / mL, 400 ng / mL, 4000 ng / mL, and 7500 ng / mL.

4. The rapid quantitative analysis method for ropivacaine blood concentration according to claim 2, characterized in that... The blank biological matrix is ​​whole blood, plasma, or serum from a healthy person.

5. The rapid quantitative analysis method for ropivacaine blood concentration according to claim 1, characterized in that, The test sample is whole blood, plasma or serum collected from a person after administration of ropivacaine, stored in a -80°C freezer, and thawed naturally at room temperature before use.

6. The rapid quantitative analysis method for ropivacaine blood concentration according to claim 1, characterized in that, The pretreatment methods include protein precipitation and solid-phase extraction.

7. The rapid quantitative analysis method for ropivacaine blood concentration according to claim 1, characterized in that, The specific operation of the protein precipitation and solid-phase extraction pretreatment method is as follows: Take an appropriate amount of ropivacaine standard curve sample, quality control sample, or test sample, add 10-40 times the volume of precipitant containing internal standard, vortex, transfer to a solid-phase extraction column with phospholipid / protein removal function for extraction, and inject the filtrate for analysis.

8. The rapid quantitative analysis method for ropivacaine blood concentration according to claim 1, characterized in that, The injection volume is 1 μL to 10 μL.

9. The rapid quantitative analysis method for ropivacaine blood concentration according to claim 1, characterized in that, The chromatographic conditions were set as follows: no column connected; mobile phase was acetonitrile solution containing 0.05% to 0.5% formic acid (volume fraction); flow rate was 0.1 mL / min to 0.5 mL / min; isocratic elution; and analysis time was 0.5 to 2 min.

10. The rapid quantitative analysis method for ropivacaine blood concentration according to claim 1, characterized in that, The chromatographic eluent was analyzed by mass spectrometry using an electrospray ionization source in positive ion detection mode. The mass spectrometry parameters were as follows: drying gas temperature: 150℃-350℃; drying gas flow rate: 5L / min-20L / min; spray gas flow rate: 10psi-50psi; sheath gas temperature: 250℃-400℃; sheath gas flow rate: 5L / min-20L / min; spray voltage: 2500V-5000V; capillary temperature: 150℃-320℃.

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