Method for simultaneously determining ritocaine and prilocaine in plasma based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology

Abstract

The invention provides a method for simultaneously determining ritocaine and prilocaine in blood plasma based on a liquid chromatography-tandem mass spectrometry (LC-MSMS) technology. The specific operation of the method comprises the following steps: adding an internal standard solution and an organic solvent into a plasma sample, and ensuring full extraction of lidocaine and prilocaine through vortex mixing. And separating by using a centrifugal technology, and collecting supernate as a pretreated sample to be detected. An Eclipse Plus C18 chromatographic column is adopted for isocratic elution, effective separation of lidocaine and prilocaine is achieved, and then a sample is subjected to mass spectrometric detection. A standard curve is drawn based on the mass spectrum peak area ratio, a regression equation is obtained, and the concentration of lidocaine and prilocaine in the sample to be detected is obtained through calculation. The method has the characteristics of simplicity and convenience in pretreatment operation, low plasma consumption, short analysis time and high sensitivity, and is suitable for detection of lidocaine and prilocaine in plasma and clinical pharmacokinetic research of the lidocaine cream.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for simultaneously determining the concentrations of lidocaine and prilocaine in plasma based on liquid chromatography-tandem mass spectrometry (LC-MS / MS). Background Technology

[0002] Lidocaine and prilocaine are both amide-type local anesthetics. They stabilize nerve cell membranes by inhibiting the ion flux required for nerve impulse initiation and conduction, thereby producing a local anesthetic effect.

[0003] Lidocaine and prilocaine cream is a compound cream prepared by mixing lidocaine and prilocaine in a 1:1 weight ratio. By releasing lidocaine and prilocaine into the epidermis and dermis through the cream, the synergistic effect of the compound is stronger, resulting in a more potent local anesthetic effect, thus providing analgesia for various minor superficial skin surgeries. This product successfully solves the problem of other drugs being unable to safely and effectively penetrate intact skin and achieve prolonged analgesia. Using this product can significantly reduce injection pain and unnecessary risks associated with general anesthesia in children, playing an irreplaceable role in alleviating injection pain in children.

[0004] To advance the clinical application of lidocaine cream, it is necessary to monitor the concentration levels of lidocaine and prilocaine in human plasma to obtain pharmacokinetic characteristics and then evaluate the bioequivalence of the original drug and generic drugs.

[0005] Due to the skin barrier, the amount of topical preparations absorbed through the skin is relatively limited, resulting in extremely low drug exposure in the bloodstream. This places extremely high demands on the sensitivity of analytical methods. Furthermore, since this product is a compound preparation, ideally, the concentrations of both drug components in human plasma should be accurately measured simultaneously. However, there are few reported analytical methods for the simultaneous determination of lidocaine and prilocaine in human plasma; most methods measure lidocaine or prilocaine separately. Only Yadlapalli et al. (2019) used LC-MS / MS to simultaneously determine lidocaine and prilocaine in human plasma, achieving a limit of quantification of 0.100 ng / ml for both. However, this method requires a large plasma volume (200 μL per sample), limiting its application in clinical studies with limited sample sizes, especially in pediatric samples. Additionally, the solid-phase extraction (SPE) method for sample pretreatment is costly and has low throughput, making it unsuitable for the rapid and economical analysis of large-scale samples in clinical studies.

[0006] Given the shortcomings of existing technologies for the simultaneous determination of lidocaine and prilocaine in terms of throughput, cost, and sample applicability, there is a need to develop an analytical method that is simple to perform pretreatment, highly sensitive, and low in cost, so as to be suitable for the analysis of large numbers of samples in clinical research. Summary of the Invention

[0007] To fill the gap in the prior art, the present invention aims to provide an analytical method for determining the concentrations of lidocaine and prilocaine in plasma based on LC-MS / MS technology.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] On one hand, the present invention provides a method for determining the concentrations of lidocaine and prilocaine in plasma based on LC-MS / MS technology, which includes the following steps:

[0010] Internal standard solution and organic solvent were added to the plasma sample, and lidocaine and prilocaine were fully extracted by vortex mixing. Separation was performed using centrifugation, and the supernatant was collected as the pretreated sample for testing.

[0011] Isocratic elution was performed using an Eclipse Plus C18 column to achieve effective separation of lidocaine and prilocaine. The samples were then analyzed by mass spectrometry. A standard curve was plotted based on the peak area ratio, and a regression equation was obtained to calculate the concentrations of lidocaine and prilocaine in the sample.

[0012] In the above method, the internal standard solution is a lidocaine and prilocaine structural analog or a stable isotope-labeled lidocaine and prilocaine. Preferably, the internal standard solution is lidocaine-d6 and prilocaine-d7. More preferably, the concentration of the internal standard solution is 3.00 ng / mL.

[0013] In the above method, the organic solution is methanol or acetonitrile or a combination of both. Preferably, the organic solvent is acetonitrile. More preferably, the proportion of the organic solvent added is 3-5 times the volume of the blood plasma.

[0014] In the above method, preferably, the centrifugation time is 10 min, the centrifugation temperature is 4℃, and the centrifugation speed is 3900 rpm.

[0015] In the above method, preferably, the chromatographic column used for liquid chromatography separation is an Eclipse Plus C18 column, 3.5 μm, 4.6 × 100 mm; the mobile phase used is: phase A: aqueous solution containing 5 mM ammonium acetate, phase B: acetonitrile.

[0016] In the above method, preferably, the liquid chromatography separation and elution conditions are as follows:

[0017] Isocratic elution: Phase A: Phase B = 25%: 75%

[0018] Washing time: 3.00 min;

[0019] Flow rate: 0.700 mL / min;

[0020] Injection volume: 5.00 μL;

[0021] Autosampler temperature: 4℃;

[0022] Column temperature: 35℃.

[0023] In the above method, preferably, the mass spectrometry conditions for mass spectrometry detection are as follows:

[0024] Ion source: Electrospray ionization (ESI);

[0025] Injection voltage: 5000V;

[0026] Gas spray (Gas1): 50 psi;

[0027] Auxiliary gas (Gas2): 50 psi;

[0028] Detection method: positive ions;

[0029] Ion source temperature: 500℃;

[0030] Collision-induced dissociation (CAD): 6;

[0031] Curtain gas: 35 psi;

[0032] Dwell time: 80ms.

[0033] In the above method, preferably, mass spectrometry detection uses quantitative analysis ion pairs, wherein the quantitative analysis ion pairs are:

[0034] Lidocaine m / z 235.2→86.3, collision energy (CE) 24eV, declustering voltage (DP) 60V;

[0035] Priligycaine m / z 221.1→86.1, collision energy (CE) 14eV, declustering voltage (DP) 60V;

[0036] Lidocaine -d6 m / z 241.3→86.1, collision energy (CE) 24eV, declustering voltage (DP) 60V;

[0037] Priligycaine - d7 m / z 228.1→86.1, collision energy (CE) 14eV, declustering voltage (DP) 60V.

[0038] In the above method, preferably, the standard curve is specifically prepared as follows:

[0039] Using the theoretical concentration of the sample to be tested as the abscissa and the peak area ratio of the sample to the internal standard as the ordinate, a linear regression equation was obtained by regression analysis.

[0040] On the other hand, the present invention also provides the application of the above-described method in analyzing the concentrations of lidocaine and prilocaine in plasma samples; the lidocaine-prilocaine cream is used for various needle punctures and superficial surgical anesthesia of the skin and mucous membranes, which can greatly improve the pain of patients during medical treatment.

[0041] The beneficial effects of this invention are:

[0042] (1) Significant improvement over existing technologies: Currently, only one paper presents a complete methodological study on the simultaneous determination of lidocaine and prilocaine in human plasma using LC-MS / MS technology. However, this method has limitations such as large plasma volume (200 μL) and high pretreatment costs (solid phase extraction, SPE method). This invention establishes for the first time a more economical and micro-sample LC-MS / MS method for the simultaneous detection of two components. The methodological validation indicators all meet the requirements of the specifications, representing a significant optimization and improvement over existing technical solutions.

[0043] (2) The method has wide applicability, especially for special populations: The method of this invention only requires 50 μL of plasma, which significantly reduces the sample consumption. This feature is particularly suitable for pediatric subjects with limited sample size, thus expanding the clinical application scope of the method.

[0044] (3) Outstanding advantages in analytical efficiency and throughput: The pretreatment steps of the method of this invention are extremely simple, requiring only one extraction step for direct sample injection, which significantly improves operational efficiency; the extracted samples have good stability, making them easy to preserve and retest. At the same time, the analytical method is fast and efficient, with an analysis time of only 3 minutes for a single sample, which can fully meet the needs of rapid detection of large-scale, high-throughput biological samples in studies such as bioequivalence.

[0045] (4) High sensitivity, effectively promoting the research and development process: The method of this invention has higher analytical sensitivity, with the limits of quantitation for lidocaine and prilocaine reaching 0.100 ng / mL and 0.050 ng / mL, respectively. This high sensitivity characteristic can ensure the accurate determination of extremely low blood drug concentrations after topical administration, thereby providing reliable data support for the bioequivalence evaluation of the generic drug.

[0046] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0047] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0048] Figure 1 MRM chromatograms of lidocaine (A) and prilocaine (C) and internal standards lidocaine-d6 (B) and prilocaine-d7 (D) in blank plasma samples;

[0049] Figure 2 MRM chromatograms of lidocaine (A) and prilocaine (C) and internal standards lidocaine-d6 (B) and prilocaine-d7 (D) in samples with lower limits of quantitation;

[0050] Figure 3 MRM chromatograms of lidocaine (A) and prilocaine (C) and internal standards lidocaine-d6 (B) and prilocaine-d7 (D) in plasma samples from healthy subjects after topical application of 15g lidocaine cream;

[0051] Figure 4 Plasma drug concentration-time curve after topical application of 15g of liprocaine cream to one healthy subject (approximately 100cm² of application area). 2 Remove the cream after 4 hours of application. Detailed Implementation

[0052] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The processes, conditions, reagents, experimental methods, etc., for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any special limitations.

[0053] Example

[0054] The rapid analytical method for determining the concentration of lidocaine and prilocaine in plasma provided by this invention can generally be divided into three parts: extraction method (i.e., pretreatment method), liquid chromatography method, and mass spectrometry method. This invention addresses the shortcomings of existing technologies by establishing an analytical method from these three aspects.

[0055] I. Preprocessing:

[0056] This invention uses only 50.0 μL of plasma, suitable for bioanalytical work involving small sample volumes in clinical research. Regarding the extraction method, this invention employs protein precipitation, which exhibits high recoveries for lidocaine and prilocaine, and is simple and rapid to operate, eliminating the need for costly SPE concentration steps and significantly shortening extraction time. Furthermore, this method, when used with 96-well plates, is ideally suited for the high-throughput sample pretreatment needs of clinical research.

[0057] The specific pretreatment steps are as follows:

[0058] 1. Add 50.0 μL of plasma sample, 50.0 μL of internal standard solution (lidocaine-d6 and prilocaine-d7 concentrations of 3.00 / 3.00 ng / mL) and 150 μL of acetonitrile to a 96-well plate;

[0059] 2. Vortex mix and centrifuge for 10 min (4℃, 3900 rpm);

[0060] 3. Take 100 μL of supernatant into another clean 96-well plate, add 100 μL of ultrapure water and mix well;

[0061] 4. The injection volume is 5.0 μL.

[0062] II. Chromatographic Analysis:

[0063] Chromatography was used to separate the sample into analytes using liquid chromatography with an Eclipse Plus C18 column and isocratic elution [25% phase A: 75% phase B]. Mobile phase A was an aqueous solution containing 5 mM ammonium acetate, and mobile phase B was acetonitrile.

[0064] Lidocaine and prilocaine have small molecular weights, making them susceptible to interference from other endogenous substances in plasma during assay. Furthermore, the limited transdermal absorption of topical formulations results in extremely low blood drug concentrations, placing stringent demands on the sensitivity of analytical methods. Therefore, this invention employs an Eclipse Plus C18 column for chromatographic separation, which exhibits excellent retention characteristics for both the target analyte and the internal standard. In addition, ammonium acetate is added to the mobile phase as a modifier, which helps optimize peak shape and improve peak symmetry. Based on these advantages, this method achieves high analytical throughput with a chromatographic run time of only 3 minutes, making it highly suitable for large-scale sample analysis in clinical research.

[0065] III. Mass Spectrometry Analysis:

[0066] An electrospray ionization source was used with positive ion detection. The spray voltage was 5000V, gas 1 (Gas1) was 50psi, gas 2 (Gas2) was 50psi, curtain gas was 35psi, the ion source temperature was 500℃, collision-induced dissociation was 6, and the residence time was 80ms. For lidocaine quantitative analysis, the ion pair m / z was 235.2→86.3, the collision energy (CE) was 24eV, and the declustering voltage (DP) was 60V. For prilocaine quantitative analysis, the ion pair m / z was 221.1→86.1, the collision energy (CE) was 14eV, and the declustering voltage (DP) was 60V. For internal standard lidocaine-d6 quantitative analysis, the ion pair m / z was 241.3→86.1, the collision energy (CE) was 24eV, and the declustering voltage (DP) was 60V. For internal standard prilocaine-d7 quantitative analysis, the ion pair m / z was... 228.1→86.1, collision energy (CE) 14eV, declustering voltage (DP) 60V.

[0067] The present invention will now be described in detail through specific embodiments:

[0068] Example 1:

[0069] Abbreviation Explanation:

[0070]

[0071] 1. Materials

[0072] 1.1 Instruments

[0073] Chromatograph: LC-30AD rapid liquid chromatography system, Shimadzu Corporation, Japan.

[0074] Mass spectrometer: Model 5500 triple quadrupole tandem mass spectrometer, equipped with an electrospray ionization source (Turbo Ion Spray), Sciex Corporation, USA.

[0075] Data processing was performed using Analyst (version 1.6.3), manufactured by Sciex, Canada.

[0076] Centrifuge: 5810R multi-functional benchtop centrifuge, Eppendorf GmbH, Germany.

[0077] Analytical balance: CPA225D analytical balance, Beijing Sartorius Instruments Co., Ltd.

[0078] 1.2 Reference Standards and Reagents

[0079] Lidocaine (99.7% purity) was purchased from the National Institutes for Food and Drug Control, while prilocaine (99.1% purity) and internal standard lidocaine-d6 (96.1% purity) were purchased from Beijing Manhag Biotechnology Co., Ltd. Prilocaine-d7 (97.5% purity) was purchased from TLC Corporation (Canada). Methanol (HPLC grade) was purchased from Sigma-Aldrich (USA). Formic acid (HPLC grade) was purchased from TCI Corporation. Ammonium acetate (HPLC grade) was purchased from ROE Corporation. Deionized water (18.2 mΩ, TOC ≤ 50 ppb) was prepared using a Milli-Q ultrapure water system.

[0080] 2 Methods

[0081] 2.1 Preparation of solutions and samples

[0082] Standard series samples: Accurately weigh an appropriate amount of the reference standard, dissolve it in methanol and dilute to volume to prepare stock solutions with lidocaine and prilocaine concentrations of approximately 1.00 mg / mL. Accurately pipette an appropriate amount of each stock solution and dilute with acetonitrile:water (50:50, v / v) to obtain working solutions. Dilute with blank human plasma to obtain a mixed standard series sample, with lidocaine and prilocaine concentration ranges of 0.100–50.0 ng / mL and 0.050–25.0 ng / mL, respectively.

[0083] Quality control samples: Four mixed quality control samples of lidocaine and prilocaine at four concentration levels were prepared using a method similar to that used for the standard series samples. The low quality control (LQC) concentration was 0.250 / 0.125 ng / mL, the accessorial middle quality control (AMQC) concentration was 3.50 / 1.75 ng / mL, the medium quality control (MQC) concentration was 15.0 / 7.50 ng / mL, and the high quality control (HQC) concentration was 40.0 / 20.0 ng / mL.

[0084] Internal standard solutions: Accurately weigh lidocaine-d6 and prilocaine-d7 reference standards, dissolve and dilute to volume with methanol to prepare internal standard stock solutions with a concentration of approximately 200 μg / mL. Accurately pipette an appropriate amount of the above internal standard stock solution and dilute with acetonitrile:water (50:50, v / v) to obtain internal standard solutions with lidocaine-d6 and prilocaine-d7 concentrations of 3.00 ng / mL and 3.00 ng / mL, respectively.

[0085] 2.2 Plasma Sample Processing

[0086]

[0087] 2.3 Chromatographic and Mass Spectrometric Conditions Chromatographic conditions:

[0088]

[0089] Mass spectrometry conditions:

[0090]

[0091]

[0092] 3. Verification of the method in this embodiment

[0093] The LC-MS / MS method validation provided in this embodiment for simultaneously testing lidocaine and prilocaine includes selectivity, standard curve and limit of quantitation, precision and accuracy, matrix effect, recovery, residue and stability, etc., which meet the requirements of ICH M10 guidance.

[0094] [Selectivity]

[0095] Evaluation was conducted using six blank plasma and LLOQ samples from different sources. The requirement was that at the retention time of each analyte and internal standard, the peak area of ​​the co-eluting interfering substances was less than 20% (analyte) and 5% (internal standard) of the LLOQ peak area.

[0096] [Standard curve and lower limit of quantitation]

[0097] Plotting the theoretical concentration of the analyte on the x-axis (x) and the peak area ratio of the analyte to the internal standard on the y-axis, a weighted (W = 1 / x²) least squares linear regression is performed, and the resulting equation is the standard curve. The standard curve is acceptable if the backcalculated concentration accuracy is within ±15.0% for at least 75% of the standard points (and at least 6 concentration levels) (within ±20.0% for the lower limit of quantitation).

[0098] Accuracy and precision

[0099] For each analytical batch, at least six samples of quality control samples at four different concentrations should be measured. The intra-batch and inter-batch accuracy at the limit of quantitation should be within ±20.0%, and the precision within 20% to be acceptable. For the remaining concentration levels of quality control samples, the intra-batch and inter-batch accuracy should be within ±15%, and the precision within 15.0%.

[0100] Recovery rate

[0101] The recovery rate was calculated by comparing the peak area ratio of the two treatment methods before and after extraction with the addition of quality control sample working solution.

[0102] Matrix effect

[0103] Using six blank plasma samples from different sources, two quality control plasma samples with low and high concentrations were prepared. Three samples were analyzed for each concentration. The average accuracy of the measured values ​​for each quality control sample per source per concentration was within ±15.0% of the labeled value.

[0104] [Residue]

[0105] After the ULOQ sample analysis was completed, the blank plasma sample was analyzed immediately. By comparing the chromatographic peak areas at the retention times of the analyte and internal standard in the blank plasma sample and the LLOQ sample, the accuracy of the analysis was assessed.

[0106]

stability

[0107] To investigate the stability of the analyte in plasma samples, plasma samples at LQC and HQC concentration levels were placed under different temperatures and environments, for a total of four placement conditions:

[0108] 1) Short-term stability: After being placed at room temperature for 18 hours,

[0109] 2) Place in the autosampler at 4℃ for 123 hours.

[0110] 3) It undergoes four freeze-thaw cycles (from -80°C to room temperature).

[0111] 4) Store at -80℃ for 223 days.

[0112] A sample is considered stable if the accuracy (RE%) of the stability measurement at each concentration level under all storage conditions does not exceed ±15.0%.

[0113] 4 Results of method verification in this embodiment

[0114] Method selectivity

[0115] The retention times of lidocaine and internal standard lidocaine on day 6 were approximately 2.53 min and 2.50 min, respectively; the retention times of prilocaine and internal standard prilocaine on day 7 were approximately 2.19 min and 2.17 min, respectively. Figure 1 and Figure 2 As shown, no interference was observed at any retention time.

[0116] Standard curve and lower limit of quantitation

[0117] The linear ranges for lidocaine and prilocaine in plasma samples from clinical studies were 0.100–50.0 ng / mL and 0.050–25.0 ng / mL, respectively. The typical linear regression equation for the standard curve of the analytes is as follows:

[0118] Lidocaine: y = 0.298x - 0.0000458 (r) 2=0.9992)

[0119] Priligy: y = 0.364x - 0.000851(r) 2 =0.9980)

[0120] The lower limit of quantitation (LDQ) concentrations in the samples were 0.100 ng / mL for lidocaine and 0.050 ng / mL for prilocaine. The intra- and inter-batch accuracy of the LDQ for lidocaine was between 2.5% and 5.5%, with intra- and inter-batch precision within 5.0%; the intra- and inter-batch accuracy of the LDQ for prilocaine was between 1.8% and 12.1%, with intra- and inter-batch precision within 6.5%; both met the acceptance criteria.

[0121] Precision and accuracy of the method

[0122] The intra-assay and inter-assay accuracy of lidocaine was between 0.8% and 8.9%, and the intra-assay and inter-assay precision was within 3.5%; the intra-assay and inter-assay accuracy of prilocaine was between 0.8% and 7.1%, and the intra-assay and inter-assay precision was within 4.0%; both met the acceptance criteria, and the results are shown in Table 1.

[0123] Table 1: Precision and accuracy of determination of lidocaine and prilocaine in human plasma

[0124]

[0125]

[0126] Treatment recovery rate

[0127] At LQC, MQC, and HQC concentration levels, the extraction recoveries of lidocaine were 101.5%, 98.7%, and 101.6%, respectively, while those of prilocaine were 99.1%, 98.2%, and 99.7%, respectively; the recoveries of the internal standards lidocaine-d6 and prilocaine-d7 were 100.1% and 99.3%, respectively. These results meet the requirements for analytical detection.

[0128] Matrix effect

[0129] The average accuracy (RE) of lidocaine and prilocaine determinations from quality control samples of different sources ranged from -9.1% to -0.8% of the labeled values. These results indicate that matrix effects do not interfere with the accuracy of analyte analysis.

[0130] Residue

[0131] Immediately after ULOQ sample analysis, blank plasma samples were analyzed. The peak areas at the retention times of both the analyte and internal standard in the blank plasma samples were less than 20% of the peak area of ​​the analyte in the LLOQ sample and less than 5% of the peak area of ​​the internal standard. This indicates that there are no residual effects under this method.

[0132] Plasma stability study

[0133] The results of the plasma stability test are shown in Table 2. The results indicate that lidocaine and prilocaine are stable under the various test conditions.

[0134] Table 2. Stability of lidocaine and prilocaine in human plasma under different conditions.

[0135]

[0136]

[0137] Example 2: Application of the method in Example 1 in human pharmacokinetic studies

[0138] Example 2: Application of the method in Example 1 in human pharmacokinetic studies The validated method was used to analyze lidocaine and prilocaine in human plasma to evaluate their pharmacokinetic characteristics. One healthy subject applied 15g of lidocaine cream topically to the anterior thigh, covered with an occlusive dressing, covering an area of ​​approximately 100 cm². 2 After applying the medication for 4 hours, wipe off the cream. See the plasma drug concentration-time curve below. Figure 4 The detection method has high sensitivity and can fully depict the pharmacokinetic characteristics of lidocaine and prilocaine. In addition, the method has good reproducibility and high accuracy.

[0139] Specific embodiments have been used to illustrate the principles and implementation methods of this invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of this invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this invention. Therefore, the content of this specification should not be construed as a limitation of this invention.

Claims

1. A method for simultaneous determination of lidocaine and prilocaine concentrations in plasma based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) technique, comprising the following steps: adding internal standard solution and organic solvent to plasma sample and mixing by vortex to ensure sufficient extraction of lidocaine and prilocaine. Separation by centrifugation, collecting supernatant as pretreated sample to be tested. Effective separation of lidocaine and prilocaine is achieved by isocratic elution using Eclipse Plus C18 column, followed by mass spectrometry detection. Standard curve is drawn based on mass spectrometry peak area ratio to obtain regression equation for calculating lidocaine and prilocaine concentrations in sample to be tested.

2. The method of claim 1, wherein, The internal standard solution is a structural analogue of lidocaine and prilocaine or stable isotope labeled lidocaine and prilocaine, preferably, the internal standard solution is lidocaine-d6 and prilocaine-d7, further preferably, the concentration of the internal standard solution is 3.00 ng / mL.

3. The method of claim 1, wherein, The organic solution is methanol or acetonitrile or a combination of the two, preferably, the organic solvent is acetonitrile, further preferably, the proportion of the organic solvent added is 3-5 times the volume of plasma.

4. The method of claim 1, preferably, the centrifugation time is 10 min, the centrifugation temperature is 4℃, and the centrifugation speed is 3900 rpm.

5. The method of claim 1, wherein, The chromatographic column used for liquid chromatography separation is: Eclipse Plus C18 column, 3.5 μm, 4.6 x 100 mm; the mobile phase used is: phase A: 5 mM ammonium acetate in water, phase B: acetonitrile.

6. The method of claim 5, wherein, The elution conditions for liquid chromatography separation are: Isocratic elution: phase A: phase B = 25%:75% Elution time: 3.00 min; Flow rate: 0.700 mL / min; Injection volume: 5.00 μL; Automatic injector temperature: 4℃; Column temperature: 35℃.

7. The method of claim 1, wherein, The mass spectrometry conditions for mass spectrometry detection are: Ion source: electrospray ion source (ESI); Spray voltage: 5000 V; Spray gas (Gas 1): 50 psi; Auxiliary gas (Gas 2): 50 psi; Detection mode: positive ion; Ion source temperature: 500℃; Collision-induced dissociation (CAD): 6; Curtain gas: 35 psi; Residence time: 80 ms.

8. The method of claim 1 or 6, wherein, The quantitative analysis ion pairs used for mass spectrometry detection are: Lidocaine m / z 235.2→86.3, collision energy (CE) 24 eV, de-clustering voltage (DP) 60 V; Prilocaine m / z 221.1→86.1, collision energy (CE) 14 eV, de-clustering voltage (DP) 60 V; Lidocaine-d6 m / z 241.3→86.1, collision energy (CE) 24 eV, de-clustering voltage (DP) 60 V; Prilocaine-d7 m / z 228.1→86.1, collision energy (CE) 14 eV, de-clustering voltage (DP) 60 V.

9. The method of claim 1, wherein, The standard curve is prepared as follows: The peak area ratio of the sample to the internal standard was taken as the vertical coordinate, and the theoretical concentration of the sample was taken as the horizontal coordinate, and a linear regression equation was obtained by regression analysis.

10. Use of the method according to any one of claims 1 to 9 for the analysis of lidocaine and prilocaine concentrations in plasma samples; the lidoprocaine cream for the anaesthesia of all kinds of needle puncture and skin, mucous membrane superficial surgical operation, can greatly improve the pain of patients during the medical process.

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