Ropivacaine concentration detection method based on pipette tip

Abstract

The invention discloses a ropivacaine concentration detection method based on a pipette tip. The ropivacaine concentration detection method comprises the following steps: collecting a proper amount of blood sample, preparing a detection sample, treating the detection sample and analyzing components. According to the method disclosed by the invention, the pipette is combined with LC-MS / MS liquid chromatography-tandem mass spectrometry for analysis, and rapid analysis and monitoring of the plasma concentration of ropivacaine are applied and realized; according to the present invention, during the detection, the required blood sample amount is less, only one drop of blood (about 10 [mu] L) is required, the operation is simple, and the whole solid phase extraction process can be rapidly completed only by using the pipette and the suction head filled with the chromatographic filler such as C18; according to the method, a special solid-phase extraction instrument is not needed, and compared with a conventional solid-phase extraction technology, the technical difficulty is low, the operation is simple, the consumed sample size is small, and the detection cost is relatively low. The method has the advantages of convenience and rapidness in detection and high detection precision.

Description

Technical Field

[0001] The invention relates to the field of detection technology, in particular to a method for detecting ropivacaine concentration based on a pipette tip. Background Art

[0002] Ropivacaine is a pure left-handed (S configuration) long-acting amide local anesthetic. Its mechanism of action is the same as other local anesthetics. It blocks sodium ion channels, blocks nerve excitation and conduction, and then produces an anesthetic effect. It is mainly used clinically for regional block, epidural anesthesia, and epidural postoperative or labor analgesia. Ropivacaine has less cardiotoxicity than bupivacaine, and its toxicity is milder than bupivacaine, but it still has certain toxicity to the cardiovascular system. Excessive blood concentration can inhibit myocardial contraction and cardiac conduction. With the widespread use of large doses of ropivacaine in regional nerve blocks, local anesthetic poisoning also occurs from time to time in clinical practice. Local anesthetic poisoning refers to excessive doses of local anesthetics absorbed into the blood within blood vessels or per unit time, or poor systemic nutritional regulation of patients, liver and kidney dysfunction, which causes excessive concentrations of local anesthetics in the blood to cause toxic reactions, mainly manifested as central nervous system toxicity and cardiovascular dysfunction. Once local anesthetic poisoning occurs, it will cause great damage to the patient's body and may even lead to serious consequences such as cardiac arrest.

[0003] At present, the clinical judgment of local anesthetic poisoning is still based on clinical symptoms. For example, mild poisoning: symptoms such as dizziness, polyphagia, chills, etc., when the drug enters the blood vessels by mistake, there are symptoms such as tinnitus, headache, sweating, etc.; moderate poisoning: symptoms such as irritability, nausea and vomiting, involuntary muscle twitching in the limbs, eyeballs and face; severe poisoning: the patient's muscle twitching is full-body rigidity, clonic convulsions and other neurological symptoms. At the same time, due to decreased myocardial contractility, slow heart rate, pale face, cold limbs, and decreased blood pressure, shock symptoms may also occur.

[0004] Complex and changeable clinical situations may lead to deviations in the judgment of local anesthetic poisoning, or even misjudgment resulting in irreversible consequences. Therefore, it is particularly urgent and important to develop a pharmacokinetic analysis method with high sensitivity and accuracy that can simply, quickly and efficiently monitor the blood concentration of ropivacaine in ropivacaine therapeutic drug monitoring (TDM).

[0005] Currently, the commonly used methods for therapeutic drug monitoring are chemical detection, chromatography and immunoassay. Chemical detection has low sensitivity and specificity, and requires a large amount of samples; immunoassay has complicated and cumbersome operation steps, its scope of application is largely limited by the type of kit, and the cost is relatively high.

[0006] Chromatography is currently the most commonly used detection method. Chromatographic separation is often used in conjunction with mass spectrometry. Because of the high sensitivity, specificity and accuracy of chromatography-mass spectrometry (LC-MS and GC-MS), it is widely used in the field of pharmacokinetic analysis and detection of clinical drugs. The composition of blood is relatively complex and needs to undergo sample pretreatment processes such as protein precipitation, solid phase extraction, and liquid-liquid extraction before it can be used for chromatography-mass spectrometry detection.

[0007] Solid-phase extraction (SPE) is currently the most commonly used and effective sample pretreatment method, mainly used for the separation, purification and concentration of biological samples.

[0008] The conventional SPE method has obvious disadvantages: it requires the use of specialized solid phase extraction instruments and solid phase extraction columns, has high sample pretreatment costs, requires a large amount of samples, has complex and cumbersome operation steps, has high technical requirements for operators, and is time-consuming (up to tens of minutes).

[0009] These factors greatly increase the error that the entire operation process may cause to the experimental results, resulting in poor repeatability of the final results. These shortcomings also greatly limit its application in the field of rapid monitoring of therapeutic drugs.

[0010] In summary, there is still no convenient and fast method for detecting the blood concentration of ropivacaine. Summary of the invention

[0011] The purpose of the present invention is to provide a method for detecting the concentration of ropivacaine based on a pipette tip. The present invention uses the pipette tip to detect the concentration of ropivacaine in blood, which has the advantages of convenient and fast detection and high detection accuracy.

[0012] The technical solution of the present invention is a method for detecting ropivacaine concentration based on a pipette tip, comprising the following process:

[0013] A. Collect appropriate amount of blood sample;

[0014] B. Preparation of test samples: Process the collected blood samples to prepare test samples;

[0015] C. Test sample processing: Use a pipette to process the test sample to obtain a sample extract;

[0016] D. Component analysis: Liquid chromatography-mass spectrometry is used to analyze the sample extract to determine whether the blood sample contains ropivacaine and determine its concentration.

[0017] In the aforementioned method for detecting ropivacaine concentration based on a pipette tip, the specific contents of collecting an appropriate amount of blood sample as described in step A are as follows:

[0018] A 50 μL blood sample was collected using a heparin anticoagulation tube.

[0019] In the aforementioned method for detecting ropivacaine concentration based on a pipette tip, the test sample preparation described in step B is specifically as follows:

[0020] Take 10 μL of blood sample and drop it onto sampling filter paper, and dry it at room temperature; the sampling filter paper has been pre-dropped with 10 μL of ropivacaine-d7 internal standard methanol solution and fully dried; the sampling filter paper is Whatman 903.

[0021] In the aforementioned method for detecting ropivacaine concentration based on a pipette tip, the specific steps of the detection sample processing described in step C are as follows:

[0022] C1. Use a puncher to cut a 3 mm circular blood spot from the center of the blood spot of the test sample;

[0023] C2. Fold the circular blood spot in half and place it on the tip filler of the pipette;

[0024] C3. Immerse the tip in 100 μL of methanol and repeat the aspiration-discharge operation 4 times using a pipette.

[0025] C4. The final methanol extract is used as the sample extract to be tested.

[0026] In the aforementioned ropivacaine concentration detection method based on a pipette tip, the pipette is a micro solid phase extraction pipette, and the filler of the pipette tip is 0.5 mg mixed cation exchange reverse phase adsorbent MCX filler.

[0027] In the aforementioned method for detecting ropivacaine concentration based on a pipette tip, the component analysis described in step D comprises the following specific steps:

[0028] D1. Use ultra-high performance liquid chromatography system for chromatographic separation;

[0029] D2. Perform mass spectrometry analysis using a mass spectrometer.

[0030] In the aforementioned method for detecting ropivacaine concentration based on a pipette tip, the ultra-high performance liquid chromatography system is used for chromatographic separation in step D1, and the chromatographic column used in the ultra-high performance liquid chromatography system is Waters AcquityUPLC, and the specific steps are as follows:

[0031] The chromatographic column of the ultra-high performance liquid chromatography system is a Waters C18 reverse phase chromatographic column, HSS T3, 1.8 μm, 100 mm×2.1 mm, and the column temperature of the chromatographic column is 35° C.

[0032] The injection volume of the sample extract was 1 μL, and the temperature of the sample cell was 8°C;

[0033] The mobile phase A of the ultra-high performance liquid chromatography system is 0.1% formic acid aqueous solution, and the mobile phase B is acetonitrile, with a flow rate of 0.4 mL / min;

[0034] The linear gradient elution chromatographic conditions of the ultra-high performance liquid chromatography system are: 0.0 min, 85% A phase; 1.0 min, 85% A phase; 3.0 min, 40% A phase; 4.0 min, 10% A phase; 6.0 min, 10% A phase;

[0035] The elution time of the ultra-high performance liquid chromatography system is 2.33 min.

[0036] In the aforementioned method for detecting ropivacaine concentration based on a pipette tip, the mass spectrometer described in step D2 is used for mass spectrometry analysis, and the mass spectrometer is a Waters TQD triple quadrupole mass spectrometer, and the specific analysis steps are as follows:

[0037] In positive ion mode, the analysis was performed using an ESI electrospray ion source and selected reaction monitoring (SRM) mode.

[0038] The mass spectrometer parameters are: ion source capillary ionization voltage 3.5KV, cone voltage 30V, desolvation temperature 500°C, desolvation gas flow rate 800L / h, cone gas flow rate 50L / h;

[0039] The SRM monitoring channel was m / z 275→m / z 126.

[0040] Compared with the prior art, the present invention uses a pipette gun combined with LC-MS / MS liquid chromatography-tandem mass spectrometry analysis to apply and realize rapid analysis and monitoring of ropivacaine blood concentration;

[0041] The amount of blood sample required for the test in this application is small, only one drop of blood (about 10 μL), and the operation is simple. The entire solid phase extraction process can be quickly completed by using only a pipette gun and a pipette tip filled with chromatographic fillers such as C18;

[0042] The present application does not require a special solid phase extraction instrument. Compared with conventional solid phase extraction technology, the present application has low technical difficulty, simple operation, small sample consumption and relatively low detection cost.

[0043] In summary, the present invention has the advantages of convenient and fast detection and high detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The chemical structure and tandem mass spectrometry fragmentation diagram of ropivacaine of the present invention;

[0045] Figure 2 Schematic diagram of micro-solid phase extraction of a pipette tip in an embodiment of the present invention;

[0046] Figure 3 Schematic diagram of solid phase extraction effect of pipette tips filled with different types of reverse phase adsorbents in the embodiment;

[0047] Figure 4 Schematic diagram of the effect of the number of cycles of micro-solid phase extraction absorption and discharge of the pipette tip in the embodiment;

[0048] Figure 5 Schematic diagram of the effect of the micro-solid phase extraction elution solvent of the pipette tip on the chromatographic peak area of ​​ropivacaine in the embodiment;

[0049] Figure 6 is a representative SRM detection chromatogram of a blood standard sample to which 5 μg / mL ropivacaine was added in the embodiment;

[0050] Figure 7 1 is a monitoring curve of blood ropivacaine concentration of two subjects in the embodiment. DETAILED DESCRIPTION

[0051] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but they are not intended to limit the present invention.

[0052] Embodiment. A method for detecting ropivacaine concentration based on a pipette tip, the principle is as follows Figure 1 and 2 As shown, the following process is included:

[0053] A. Collect appropriate amount of blood sample;

[0054] B. Preparation of test samples: Process the collected blood samples to prepare test samples;

[0055] C. Test sample processing: Use a pipette to process the test sample to obtain a sample extract;

[0056] D. Component analysis: Liquid chromatography-mass spectrometry is used to analyze the sample extract to determine whether the blood sample contains ropivacaine and determine its concentration.

[0057] The specific contents of collecting an appropriate amount of blood sample described in step A are as follows:

[0058] A 50 μL blood sample was collected using a heparin anticoagulation tube.

[0059] The specific contents of the test sample preparation described in step B are as follows:

[0060] Take 10 μL of blood sample and drop it onto sampling filter paper, and dry it at room temperature; the sampling filter paper has been pre-dropped with 10 μL of ropivacaine-d7 internal standard methanol solution and fully dried; the sampling filter paper is Whatman 903.

[0061] The specific steps of the test sample processing described in step C are as follows:

[0062] C1. Use a puncher to cut a 3 mm circular blood spot from the center of the blood spot of the test sample;

[0063] C2. Fold the circular blood spot in half and place it on the tip filler of the pipette;

[0064] C3. Immerse the tip in 100 μL of methanol and repeat the aspiration-discharge operation 4 times using a pipette.

[0065] C4. The final methanol extract is used as the sample extract to be tested.

[0066] The pipette is a micro solid phase extraction pipette, and the filler of the pipette tip is 0.5 mg mixed cation exchange reverse phase adsorbent MCX filler.

[0067] The component analysis described in step D comprises the following specific steps:

[0068] D1. Use ultra-high performance liquid chromatography system for chromatographic separation;

[0069] D2. Perform mass spectrometry analysis using a mass spectrometer.

[0070] The chromatographic separation using an ultra-high performance liquid chromatography system in step D1, wherein the chromatographic column used in the ultra-high performance liquid chromatography system is a Waters Acquity UPLC, and the specific steps are as follows:

[0071] The chromatographic column of the ultra-high performance liquid chromatography system is a Waters C18 reverse phase chromatographic column, HSS T3, 1.8 μm, 100 mm×2.1 mm, and the column temperature of the chromatographic column is 35° C.

[0072] The injection volume of the sample extract was 1 μL, and the temperature of the sample cell was 8°C;

[0073] The mobile phase A of the ultra-high performance liquid chromatography system is 0.1% formic acid aqueous solution, and the mobile phase B is acetonitrile, with a flow rate of 0.4 mL / min;

[0074] The linear gradient elution chromatographic conditions of the ultra-high performance liquid chromatography system are: 0.0 min, 85% A phase; 1.0 min, 85% A phase; 3.0 min, 40% A phase; 4.0 min, 10% A phase; 6.0 min, 10% A phase;

[0075] The elution time of the ultra-high performance liquid chromatography system is 2.33 min.

[0076] The mass spectrometer used in step D2 is a Waters TQD triple quadrupole mass spectrometer for mass spectrometry analysis, and the specific analysis steps are as follows:

[0077] In positive ion mode, the analysis was performed using an ESI electrospray ion source and selected reaction monitoring (SRM) mode.

[0078] The mass spectrometer parameters are: ion source capillary ionization voltage 3.5KV, cone voltage 30V, desolvation temperature 500°C, desolvation gas flow rate 800L / h, cone gas flow rate 50L / h;

[0079] The SRM monitoring channel was m / z 275→m / z 126.

[0080] Experimental verification

[0081] The effectiveness of the technology of the present invention is verified by conducting comparative experiments

[0082] 1. Prepare chemicals and blood samples

[0083] Ropivacaine was purchased from Merck, Germany, and ropivacaine-d7 deuterated internal standard was purchased from MedChemExpress (MCE), USA. Chromatographic grade methanol was purchased from Merck, Germany. Ultrapure water with a resistivity of 18.2 MΩ·cm was from the Milli-Q system, USA. Blank mixed blood samples were obtained from 6 healthy subjects, and ropivacaine blood samples were obtained from 3 patients anesthetized with ropivacaine.

[0084] 2. Preparation of standard samples

[0085] The 1.0 mg / mL ropivacaine methanol stock solution was serially diluted with methanol to obtain working solutions of different concentrations: 1, 5, 10, 50, 100, 500, and 1000 μg / mL. Similarly, the same procedure was followed to prepare specific concentrations of ropivacaine quality control (QC) working solutions at 2, 80, and 600 μg / mL. In addition, the ropivacaine-d7 deuterated internal standard stock solution was also prepared in methanol at a concentration of 1.0 mg / mL and then diluted with methanol to a working solution at a concentration of 1 μg / mL.

[0086] To prepare the blood samples for calibration, 1 mL of blank mixed human blood was accurately measured and 10 μL of ropivacaine working solution of the corresponding concentration was added to obtain blood standard samples with concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 μg / mL. Similarly, QC blood samples with concentrations of 0.02, 0.8 and 6 μg / mL were prepared by adding the corresponding QC working solution to blank mixed human blood according to the same method.

[0087] 3. Blood sample collection and rapid processing of micro-solid phase extraction with pipette tips

[0088] Use a heparin anticoagulant blood collection tube to collect an appropriate amount of blood sample, take 10 μL and drop it on Whatman903 sampling filter paper (mark the dropping position with a pencil, 10 μL of ropivacaine-d7 internal standard methanol solution has been added in advance and fully dried), and wait until the blood on the filter paper is completely dry at room temperature.

[0089] A 3 mm circular blood spot was cut from the center of the blood spot using a hole punch. The blood spot was folded in half and placed on top of the filler of a micro solid phase extraction pipette tip (10-100 μL, USA). The tip was immersed in 100 μL methanol using a handheld pipette. After repeated aspiration-discharge 4 times, ropivacaine and its internal standard in the blood spot were fully enriched and purified, and the final methanol extract was used for subsequent LC-MS analysis.

[0090] 4. Liquid chromatography-mass spectrometry analysis method

[0091] In this experiment, Waters Acquity UPLC ultra-high performance liquid chromatography system was used for chromatographic separation. The chromatographic column used was Waters C18 reverse phase chromatographic column (HSS T3, 1.8μm, 100mm×2.1mm), and the column temperature was set at 35℃. The sample injection volume was 1μL, and the sample cell temperature was set at 8℃. Mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile. The mobile phase flow rate was set to 0.4mL / min, and the linear gradient elution chromatographic conditions were as follows: 0.0 min, 85% A phase; 1.0 min, 85% A phase; 3.0 min, 40% A phase; 4.0 min, 10% A phase; 6.0 min, 10% A phase. Under this chromatographic condition, the elution time of ropivacaine was 2.33min, and the elution time of ropivacaine-d7 internal standard was 2.30min.

[0092] In this experiment, a Waters TQD triple quadrupole mass spectrometer (UK) was used for mass spectrometry analysis. In positive ion mode, an ESI electrospray ion source was used, and analysis and monitoring were performed in the selected reaction monitoring SRM mode. Specific instrument parameters include: ion source capillary ionization voltage 3.5KV, cone voltage 30V, desolvation temperature 500℃, desolvation gas flow rate 800L / h, cone gas flow rate 50L / h.

[0093] According to the secondary fragmentation mass spectra of the standard and internal standard substances, the SRM monitoring channel m / z 275→m / z 126 was finally determined to be used for the monitoring of ropivacaine, and the SRM monitoring channel m / z 282→m / z 133 was used to monitor the ropivacaine-d7 internal standard.

[0094] 5. Clinical drug monitoring of ropivacaine therapy

[0095] Two patients who underwent elective shoulder arthroscopy under general anesthesia combined with interscalene brachial plexus anesthesia were selected. They were aged 18-60 years, regardless of gender, and were in American Society of Anesthesiologists (ASA) grade Ⅰ or Ⅱ.

[0096] Exclusion criteria: history of allergy to local anesthetics, peripheral neuropathy, abnormal liver or kidney function, hypertension, heart disease, hypoproteinemia, history of diabetes, coagulation disorder, platelet count <8×109 / L, morbid obesity (body mass index BMI>30kg / m2), infection at the puncture site, and history of mental illness.

[0097] After entering the room, the forearm vein was opened, and non-invasive blood pressure, transcutaneous pulse oximetry and electrocardiogram monitoring were performed routinely. Fentanyl 0.01 mg and midazolam 1 mg were injected intravenously.

[0098] A portable ultrasound machine (Sonosite, USA) and a high-frequency probe (6-12MHz) were used to locate the interscalene brachial plexus block. 20 ml of 0.375% ropivacaine was injected cumulatively in divided doses near the needle tip reaching the upper and middle trunks of the brachial plexus nerves by the in-plane technique. Ten minutes after the nerve block, intravenous propofol 2 mg / kg, sufentanil 3 μg / kg, and cisatracurium 0.2 mg / kg were induced and endotracheal intubation was performed. Anesthesia was maintained by inhalation of sevoflurane and remifentanil.

[0099] Venous blood was drawn before and at 5, 10, 20, 30, 60, and 120 minutes after nerve block administration, and quickly dripped onto blood collection filter paper to make dry blood paper slides. Blood spots were cut and used for micro-solid phase extraction with pipette tips. Finally, LC-MS was used to determine the plasma ropivacaine concentration.

[0100] 6. Optimization of detection parameters

[0101] 6.1 Selection of fillers for micro-volume solid phase extraction of pipette tips

[0102] As the stationary phase material in the solid phase extraction column, the main function of the filler is to adsorb and separate the target compounds, and it plays a vital role in solid phase extraction technology.

[0103] In addition to the commercial conventional C18 reverse adsorbent pipette tips, this experiment also used laboratory-made micro-solid phase extraction pipette tips filled with hydrophilic-lipophilic balanced water-wettable reverse adsorbent HLB and mixed cation exchange reverse adsorbent MCX. The final results are shown in the figure. Figure 3 As shown, the MCX-filled pipette tip has the best enrichment and purification effect on ropivacaine, and the obtained ropivacaine chromatographic peak signal is the best;

[0104] Therefore, the present application uses a pipette tip filled with 0.5 mg of mixed cation exchange reverse phase adsorbent MCX.

[0105] 6.2 Optimization of micro-solid phase extraction method using pipette tips

[0106] The steps of conventional solid phase extraction include activation, loading, elution and washing. In order to improve the efficiency of sample pretreatment, this experiment integrated the above four steps. By repeatedly aspirating and discharging the pipette tip in the elution solvent, the enrichment and purification of ropivacaine and its deuterated internal standard in the blood spot was achieved.

[0107] In this experiment, a blood standard sample containing 1 μg / mL ropivacaine was used to optimize the number of aspiration-discharge cycles of the pipette tip in the elution solvent.

[0108] like Figure 4 As shown in the figure, with the increase of the number of C18 pipette tip micro-solid phase extraction aspiration-discharge cycles, the relative standard deviation (RSD) of the chromatographic area ratio of ropivacaine and its internal standard substance gradually decreased.

[0109] Therefore, the present application determines the number of aspiration-discharge cycles of micro-solid phase extraction of a pipette tip to be 4 times.

[0110] 6.3 Selection of elution solvents for micro-solid phase extraction using pipette tips

[0111] The main function of the elution solvent in solid phase extraction is to effectively elute the target compounds adsorbed on the solid phase extraction column by selecting a suitable solvent, thereby optimizing the separation effect and improving the detection sensitivity and recovery rate.

[0112] In this experiment, methanol, acetonitrile and isopropanol were used as elution solvents to perform micro-solid phase extraction pretreatment on pipette tips containing 1 μg / mL ropivacaine. The elution solvents were analyzed by liquid chromatography-mass spectrometry. The final results are shown in Figure 5 As shown in the figure, methanol has the best elution effect, the obtained ropivacaine chromatographic peak area is the highest, and no interference peak appears in the chromatogram. The elution effects of acetonitrile and isopropanol are relatively poor, the obtained ropivacaine chromatographic peak area is relatively low, and obvious interference peaks appear in the chromatogram, indicating that the separation effect is also worse than that of methanol elution.

[0113] Therefore, the present application selects methanol as the elution solvent for micro-solid phase extraction of pipette tips.

[0114] 7. Determine the detection limit

[0115] Using blank mixed human blood samples, ropivacaine standard substances were added to prepare blood standard samples containing ropivacaine concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 μg / mL, which were then added to blood collection paper containing ropivacaine-d7 internal standard. After drying, fixed blood spots were cut and fixed. After micro-solid phase extraction with a pipette tip, the eluted solution was used for LC-MS analysis and detection. The peak area ratio of ropivacaine to the internal standard was used as the ordinate (y), and the blood ropivacaine concentration was used as the abscissa. The weighted least squares method (weight 1 / x 2 ) to perform linear regression and establish a standard curve, and the regression equation is y = 2.1947x-0.2449 (r 2 =0.9919), for rapid quantitative detection of ropivacaine in blood. The limit of quantification of the method was determined to be 0.01 μg / mL based on S / N≥10. The limit of detection (LOD) of the method was determined to be 5 ng / mL based on the signal-to-noise ratio S / N≥3.

[0116] Representative SRM monitoring chromatograms are shown in Figure 6 shown.

[0117] 8. Verify accuracy and precision

[0118] In this experiment, blood quality control samples containing 0.02, 0.8 and 6 μg / mL ropivacaine were prepared. After rapid pretreatment with micro-solid phase extraction using a pipette tip, the eluted solution was analyzed and detected by LC-MS. The actual concentration of ropivacaine was calculated using the above linear regression equation, and the accuracy and precision of the detection method were verified and evaluated. It was found that the accuracy and precision were 91.77-110.61% and 9.99-14.60%, respectively, which met the accuracy (85-115%) and precision (≦15%) quantitative detection methodology verification requirements. The final results are shown in the following table.

[0119] Table 1. Accuracy and precision results of ropivacaine quantitative detection

[0120]

[0121] 9. Subject monitoring and verification

[0122] The detection method of the present application was used to monitor the drug concentration in the blood of patients anesthetized with ropivacaine during surgery. Figure 7 As shown, during the 2 hours of therapeutic drug monitoring, the blood ropivacaine concentration of the first subject was maintained between 0.79-1.29 μg / mL, and the blood ropivacaine concentration of the second subject was maintained between 0.65-0.97 μg / mL.

Claims

1. A method for detecting ropivacaine concentration based on a pipette tip, characterized in that , including the following processes: A. Collect appropriate amount of blood sample; B. Preparation of test samples: Process the collected blood samples to prepare test samples; C. Test sample processing: Use a pipette to process the test sample to obtain a sample extract; D. Component analysis: Liquid chromatography-mass spectrometry is used to analyze the sample extract to determine whether the blood sample contains ropivacaine and determine its concentration.

2. A method for detecting ropivacaine concentration based on a pipette tip according to claim 1, characterized in that: The specific contents of collecting an appropriate amount of blood sample described in step A are as follows: A 50 μL blood sample was collected using a heparin anticoagulation tube.

3. A method for detecting ropivacaine concentration based on a pipette tip according to claim 1, characterized in that: The specific contents of the test sample preparation described in step B are as follows: Take 10 μL of blood sample and drop it onto sampling filter paper, and dry it at room temperature; the sampling filter paper has been pre-dropped with 10 μL of ropivacaine-d7 internal standard methanol solution and fully dried; the sampling filter paper is Whatman 903.

4. A method for detecting ropivacaine concentration based on a pipette tip according to claim 1, characterized in that: The specific steps of the test sample processing described in step C are as follows: C1. Use a puncher to cut a 3 mm circular blood spot from the center of the blood spot of the test sample; C2. Fold the circular blood spot in half and place it on the tip filler of the pipette; C3. Immerse the tip in 100 μL of methanol and repeat the aspiration-discharge operation 4 times using a pipette. C4. The final methanol extract is used as the sample extract to be tested.

5. A method for detecting ropivacaine concentration based on a pipette tip according to claim 4: the pipette is a micro solid phase extraction pipette, and the filler of the pipette tip is 0.5 mg mixed cation exchange reverse phase adsorbent MCX filler.

6. A method for detecting ropivacaine concentration based on a pipette tip according to claim 1, characterized in that: The component analysis described in step D comprises the following specific steps: D1. Use ultra-high performance liquid chromatography system for chromatographic separation; D2. Perform mass spectrometry analysis using a mass spectrometer.

7. A method for detecting ropivacaine concentration based on a pipette tip according to claim 6, characterized in that: The chromatographic separation using an ultra-high performance liquid chromatography system in step D1, wherein the chromatographic column used in the ultra-high performance liquid chromatography system is a Waters Acquity UPLC, and the specific steps are as follows: The chromatographic column of the ultra-high performance liquid chromatography system is a Waters C18 reverse phase chromatographic column, HSS T3, 1.8 μm, 100 mm×2.1 mm, and the column temperature of the chromatographic column is 35° C. The injection volume of the sample extract was 1 μL, and the temperature of the sample cell was 8°C; The mobile phase A of the ultra-high performance liquid chromatography system is 0.1% formic acid aqueous solution, and the mobile phase B is acetonitrile, with a flow rate of 0.4 mL / min; The linear gradient elution chromatographic conditions of the ultra-high performance liquid chromatography system are: 0.0 min, 85% phase A; 1.0 min, 85% phase A; 3.0 min, 40% phase A; 4.0 min, 10% phase A; 6.0 min, 10% phase A; The elution time of the ultra-high performance liquid chromatography system is 2.33 min.

8. A method for detecting ropivacaine concentration based on a pipette tip according to claim 6, characterized in that: The mass spectrometer used in step D2 is a Waters TQD triple quadrupole mass spectrometer for mass spectrometry analysis, and the specific analysis steps are as follows: In positive ion mode, the analysis was performed using an ESI electrospray ion source and selected reaction monitoring (SRM) mode. The mass spectrometer parameters are: ion source capillary ionization voltage 3.5KV, cone voltage 30V, desolvation temperature 500°C, desolvation gas flow rate 800L / h, cone gas flow rate 50L / h; The SRM monitoring channel was m / z 275→m / z 126.

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