Separation and identification reagent and method for ofloxacin isomer

Abstract

The invention relates to a reagent and a method for separation and identification of ofloxacin isomers, and relates to the technical field of analysis and test technologies and drug analysis, and the method comprises the following steps: mixing an isomer R / S ofloxacin sample with the reagent for separation and identification of ofloxacin isomers to prepare a mixed solution of R / S-OFLX-Vanco or R / S-OFLX-GM-metal ions; introducing the mixed solution into an ion source to generate compound ions, wherein the compound ions are monovalent positive ions of R / S-OFLX-Vanco or R / S-OFLX-GM-metal ions; and controlling the experimental device to measure the ion mobility spectrometry of the compound ions so as to obtain the enantiomeric structure information of R / S ofloxacin. The method has the effect of efficiently separating and quantitatively analyzing the enantiomer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of analytical test technology and pharmaceutical analysis, and particularly relates to a separation and identification reagent and method for ofloxacin isomers. BACKGROUND

[0002] Ofloxacin (OFLX) is a third-generation quinolone antibacterial drug developed and synthesized by the Japanese First Pharmaceutical Corporation in the 1980s. It has strong bactericidal effect on gram-negative bacilli, good oral absorption, wide distribution in the body, and few adverse reactions. It has good efficacy for various infections. Ofloxacin contains a chiral carbon atom in the molecule, and there are two enantiomers, namely, S-ofloxacin (S-OFLX) and R-ofloxacin (R-OFLX). The commonly used in clinical is the racemic mixture (i.e., 1:1 ratio of S-OFLX and R-OFLX), but in recent years, it has been found that the pharmacological effects of the enantiomers are significantly different. S-OFLX is found to have higher antibacterial activity and better pharmacokinetic properties. Experimental results show that the antibacterial activity of S-OFLX is 8-126 times that of R-OFLX, and R-OFLX also exhibits central nervous system toxicity, cardiovascular toxicity, and liver toxicity. Therefore, research on the enantiomeric separation technology of ofloxacin is of great significance for improving drug efficacy, reducing adverse reactions, and optimizing clinical medication schemes.

[0003] Common analysis methods for ofloxacin enantiomers include high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Although HPLC is widely used, it usually requires a relatively long separation time and a relatively expensive chiral column. CE requires complex sample preparation steps and a relatively long migration time in some studies.

[0004] In view of the above related technologies, in the experimental process of analyzing ofloxacin enantiomers, the required time is long whether in separation time or migration time; therefore, the problem of being unable to efficiently, quickly and sensitively obtain the analysis results of ofloxacin enantiomers arises. SUMMARY

[0005] In order to efficiently, quickly and sensitively obtain the analysis results of ofloxacin enantiomers, the present application provides a separation and identification reagent and method for ofloxacin isomers.

[0006] In a first aspect, the present application provides a separation and identification reagent for ofloxacin isomers, which adopts the following technical solution:

[0007] A separation and identification reagent for ofloxacin isomers, comprising a mixed solution for analyzing ofloxacin isomers, the mixed solution comprising a mixture of vancomycin or a metal ion-containing compound and gentamicin C1a.

[0008] By adopting the technical scheme, the ofloxacin isomer molecules form ternary / binary complexes with structural differences by mixing the solvent with vancomycin or gentamicin C1a and the metal ion-containing compound to form a special separation and identification reagent, thereby providing a structural basis for subsequent IM-MS analysis, and realizing efficient, rapid and sensitive separation and accurate identification of ofloxacin isomers.

[0009] Optionally, the molecular formula of gentamicin C1a is C 19 H 39 N5O7, and the molecular weight is 449.54, the molecular formula of vancomycin is C 66 H 75 Cl2N9O 24 , and the molecular weight is 1449.25.

[0010] By adopting the technical scheme, the molecular formula of the aminoglycoside and the corresponding molecular weight are limited, so that the aminoglycoside is fixed, the situation of selecting the aminoglycoside incorrectly when preparing the reagent is avoided, and the efficiency and accuracy of reagent preparation are improved.

[0011] Optionally, the metal ion-containing compound is any one of a metal ion-containing salt or a metal ion-containing soluble base.

[0012] By adopting the technical scheme, the metal ion-containing compound is limited to a metal ion-containing salt or a metal ion-containing soluble base, so that the selection range of the metal ion-containing compound is increased, the situation that the difficulty of obtaining the metal ion-containing compound is increased due to the less selection of the metal ion-containing compound is avoided, and the flexibility of selection of the metal ion-containing compound is improved.

[0013] Optionally, the mixed solution for mixing vancomycin or the metal ion-containing compound with gentamicin C1a is one of pure water or a mixture of pure water and methanol, ethanol, acetonitrile.

[0014] By adopting the technical scheme, the solvent is limited, so that the preparation system of the mixed solution is more stable and controllable, the problems of insufficient solubility, stratification or precipitation of solutes due to unstable solvents are avoided, the stability of the target ion signal in subsequent mass spectrometric detection is ensured, and the repeatability and reliability of experimental operation are improved.

[0015] The experimental reagent can adapt to different operation scenarios and cope with various experimental scenarios, thereby maximizing the flexibility and experimental adaptability of the reagent configuration.

[0016] In a second aspect, the present application provides a method for separating and identifying ofloxacin isomers, which adopts the following technical solution:

[0017] A method for separating and identifying ofloxacin isomers, comprising:

[0018] Step 1: mixing the isomer R / S ofloxacin sample with one of the separating and identifying reagents for ofloxacin isomers as described in any one of the above to prepare an R / S-OFLX-Vanco or R / S-OFLX-GM-metal ion mixed solution;

[0019] Step 2: passing the mixed solution into an ion source to generate complex ions, which are monovalent positive ions of R / S-OFLX-Vanco or R / S-OFLX-GM-metal ions;

[0020] Step 3: controlling the experimental device to measure the ion mobility spectrum of the complex ions to obtain the enantiomeric structure information of the R / S ofloxacin.

[0021] By adopting the above technical solution, by mixing the R / S ofloxacin sample with the special separating and identifying reagent to form a specific complex, generating characteristic ions after the ion source, and using ion mobility spectrum to obtain the isomer structure information, the limitations of low separation efficiency and poor structure differentiation of isomers in the traditional method are avoided, the efficient separation and accurate identification of ofloxacin drug isomer molecules are realized, and the specificity and reliability of isomer analysis are improved.

[0022] Optionally, the ion source type is one of an electrospray ionization ion source, a laser-assisted desorption ionization ion source, and a desorption electrospray ionization ion source.

[0023] By adopting the above technical solution, by providing multiple ion source type options, the characteristics of different chemicals in the mixed solution can be adapted to avoid the limitations of a single ion source for generating specific complex ions, and the compatibility and ionization efficiency of the method for different experimental scenarios are improved.

[0024] Optionally, the method for preparing the mixed solution comprises:

[0025] Step 10: pouring the solvent corresponding to the mixed solution into a mixed container containing R / S ofloxacin and ultrasonically dissolving to obtain an R / S ofloxacin mother liquor;

[0026] Step 11: pouring the solvent corresponding to the mixed solution into a mixed container containing vancomycin or gentamicin C1a and a compound containing metal ions and ultrasonically dissolving to obtain a mixed solution, which is a vancomycin mother liquor or a GM-metal ion compound mother liquor;

[0027] Step 12: Pour the R / S ofloxacin mother liquor and the mixed solution into a mixing container according to the preset volume ratio, mix them, and control the vortex oscillation of the mixing container to obtain the composite.

[0028] By adopting the above technical solution, the R / S ofloxacin mother liquor and the GM-metal ion / vancomycin mother liquor are prepared by stepwise ultrasonic dissolution, and then the composite is formed by vortex oscillation mixing according to the preset ratio, which avoids the problems of insufficient dissolution and uneven composite formation caused by direct mixing, ensures efficient combination of each component, and improves the repeatability and operation convenience of the mixed solution preparation.

[0029] Optionally, the method further includes a further method for preparing the mixed solution, which comprises:

[0030] Step 13: Obtain the mixing components;

[0031] Step 14: Find the corresponding adjustment data based on the mixing components;

[0032] Step 15: Adjust the mixing components according to the adjustment data;

[0033] Step 16: When a preset adjustment completion signal is received, execute steps 10 to 12.

[0034] By adopting the above technical solution, the mixing components are adjusted according to the corresponding adjustment data after obtaining the mixing components, and then the subsequent preparation steps are executed after the adjustment is completed, which improves the dissolution rate and dissolution completeness of R / S ofloxacin, vancomycin, and GM-metal ion compounds in the solvent.

[0035] Optionally, the method further includes a method for simulating the concentration of the mixed solution, which comprises:

[0036] Step 120: Obtain the required concentration and the mixing amount corresponding to the mixing components;

[0037] Step 121: Obtain the mixing device based on the mixing amount, and the mixing device comprises a mixing container;

[0038] Step 122: Obtain the simulated mixed concentration based on the mixing amount and the mixing device by using a preset simulation calculation method;

[0039] Step 123: When the simulated mixed concentration is consistent with the required concentration, output the mixing amount as the accurate mixing amount;

[0040] Step 124: When the simulated mixed concentration is inconsistent with the required concentration, obtain the simulated increase amount based on the mixing amount and the simulated mixed concentration;

[0041] Step 125: Obtain the accurate mixing amount output based on the simulated increase amount and the mixing amount.

[0042] By adopting the technical scheme, the accurate mixing amount is determined by acquiring the required concentration and the to-be-mixed amount, and combining the mixing device to simulate the mixed concentration, so that the precise prediction and determination of the mixed solution concentration before preparation are realized, and the reliability and efficiency of the reagent preparation are improved.

[0043] Optionally, the method further comprises pouring the simulated added amount into the mixing container, and the method comprises the following steps:

[0044] Step 1240: acquiring the mixed concentration;

[0045] Step 1241: when the mixed concentration reaches the required concentration, steps 2 to 3 are executed;

[0046] Step 1242: when the mixed concentration does not reach the required concentration, and the mixing device is a preset flow guide device, the corresponding supplementary mixing amount of the to-be-mixed component is calculated according to the required concentration and the mixed concentration;

[0047] Step 1243: when the supplementary mixing amount is consistent with the simulated added amount, the simulated added amount is poured into the mixing container as the supplementary mixing amount;

[0048] Step 1244: when the supplementary mixing amount is inconsistent with the simulated added amount, the simulated added amount is updated according to the supplementary mixing amount and poured into the mixing container;

[0049] Step 1245: if the mixing device is a preset wall-following device, the mixing container is closed and the mixing container is controlled to execute a preset complete dissolution scheme.

[0050] By adopting the technical scheme, by analyzing whether the concentration meets the standard, whether the test added amount needs to be added is determined according to the device when the concentration does not meet the standard, so that the concentration deviation and the insufficient dissolution problem caused by the mixed concentration not meeting the standard are avoided, the mixed solution concentration is ensured to be stable and meet the requirements, and the adaptability of the reagent preparation to different mixing devices and the reliability of the mixed solution preparation are improved.

[0051] In summary, the present application has at least one of the following beneficial technical effects:

[0052] 1. The present application realizes the rapid and accurate identification and separation of the enantiomeric structure of R / S ofloxacin isomer molecules, and the present application innovatively designs R / S-OFLX-Vanco or R / S-OFLX-GM-metal ion chiral separation reagent to form a ternary complex with R / S ofloxacin with structural differences, generates a monovalent positive ion of R / S-OFLX-Vanco or R / S-OFLX-GM-metal ion by using an ion source, and combines ion mobility-mass spectrometry technology, so that the baseline separation of R / S ofloxacin isomer molecules can be realized without relying on chemical derivatization and complex processing, the time-consuming pain points of the prior art are solved, the sensitivity and accuracy of the analysis are ensured, and the present application can be used for the accurate separation and identification of OFLX isomer molecules in different samples.

[0053] 2. The present application avoids the error caused by inaccurate reagent preparation by performing simulation test on the mixed solution, judging whether the mixing amount needs to be increased to reach the required concentration according to the simulation result, and improving the reliability and accuracy of the analysis result. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 is a schematic diagram of the vancomycin and gentamicin C1a structure in the embodiments of the present application;

[0055] Figure 2 is a schematic diagram of the chemical structure of ofloxacin in the embodiments of the present application;

[0056] Figure 3 is a schematic diagram of the TIMS-MS analysis mass spectrum and ion mobility spectrum of R / S ofloxacin in the positive ion mode in the embodiments of the present application;

[0057] Figure 4 is a schematic diagram of the mass spectrum and ion mobility spectrum of the binary complex formed by mixing R / S ofloxacin and chiral reagent Vanco in the embodiments of the present application;

[0058] Figure 5 is a schematic diagram of the mass spectrum and ion mobility spectrum of the ternary complex and quaternary complex formed by mixing R / S ofloxacin, chiral reagent GM and metal ions in the embodiments of the present application;

[0059] Figure 6 is a flowchart of a method for ofloxacin isomers in the embodiments of the present application;

[0060] Figure 7 is a flowchart of a method for simulating the concentration of a mixed solution in the embodiments of the present application. DETAILED DESCRIPTION

[0061] The present application will be further described in detail below in combination with the drawings and embodiments.

[0062] The present application discloses a reagent and method for ofloxacin isomer. Referring to Figures 1 to 4 , a reagent for ofloxacin isomer, comprising a mixed solution of vancomycin or metal ion-containing compound mixed with solvent and gentamicin C1a, wherein, vancomycin can be abbreviated as Vanco, and its molecular formula is C 66 H 75 Cl2N9O 24 , and the corresponding molecular weight is 1449.25; gentamicin C1a can be abbreviated as GM, and its molecular formula is C 19 H 39 N5O7, and the corresponding molecular weight is 449.54. The structures of vancomycin and gentamicin C1a are shown in Figure 1 , wherein: a is the structural diagram of vancomycin, and b is the structural diagram of gentamicin. The metal ion-containing compound can be any metal ion salt or metal ion-containing soluble base. The solvent includes pure water or a mixed solution of water and liquid such as methanol, ethanol, acetonitrile, etc. In the mixed solution of GM-metal ion-containing compound, the weight ratio of GM-metal ion-containing compound can be any ratio.

[0063] Referring to Figure 2 and Figure 3 , Figure 2 , the structural diagrams of isomers R / S ofloxacin are shown in, wherein: a is the structural diagram of S-OFLX, i.e. levofloxacin, and b is the structural diagram of R-OFLX, i.e. dextrofloxacin, and R / S ofloxacin can also be referred to as R / S-Ofloxacin (OFLX), and its molecular formula is C 18 H 20 FN3O4, and the corresponding molecular weight is 361.37. Figure 3 , the mass spectrum and ion mobility spectrum of R / S ofloxacin in positive ion mode are shown in, wherein: a is the mass spectrum of [R / S-OFLX+H] + , and b is the ion mobility spectrum of [R / S-OFLX+Vanco+H] + . As shown in Figure 3 , S-OFLX and R-OFLX are first analyzed by TIMS-MS in positive ion mode without adding any additive. TIMS-MS refers to ion mobility mass spectrometry. As shown in Figure 3 , the mass spectrum of a is shown, wherein the abscissa is mass-to-charge ratio (m / z), and the ordinate is relative intensity. When [R / S-OFLX+H] + is detected, the peak with the strongest relative intensity appears at a mass-to-charge ratio of 362.15, and [R / S-OFLX+H] +This is a positive ion formed when ofloxacin molecules combine with protons during mass spectrometry ionization. For example... Figure 3 The ion mobility diagram for b is shown below, where the horizontal axis represents ion mobility (1 / K0) in V·s / cm. 2 The vertical axis represents relative intensity. [S - OFLX + H] was observed. + and [R-OFLX+H] + The presence of a consistent mobility peak and an Rp-p value of 0 indicates that simple mass spectrometry and mobility spectrometry cannot distinguish between ofloxacin enantiomers, and TIMS cannot directly identify these isomers. Here, Rp-p represents the inter-peak resolution.

[0064] Reference Figure 4 , Figure 4 This is a schematic diagram of the mass spectrum and ion mobility spectra of the binary complex formed by mixing R / S ofloxacin with the chiral reagent Vanco, where: a represents [R / S-OFLX+Vanco+H] + A schematic diagram of the mass spectrum, where b represents [R / S-OFLX+Vanco+H]. + A schematic diagram of the ion mobility spectra. To distinguish the S / R-OFLX enantiomers, they were mixed with the chiral reagent Vanco, forming related binary complexes, indicating a strong non-covalent interaction between Vanco and OFLX. Figure 4 As shown in figure a, the mass spectrometry of the Vanco and OFLX mixed solution reveals the presence of the corresponding binary complex [R / S-OFLX+Vanco+H]. + (m / z = 1811.58). The mobilities of these binary complexes were compared, such as... Figure 4 As shown in b, [S-OFLX+Vanco+H] + and [R-OFLX+Vanco+H] + The mobility was 1.994 V·s / cm. 2 and 1.951 V·s / cm 2 The peak separation resolution is 1.943.

[0065] Reference Figure 5 , Figure 5 Schematic diagrams of mass spectra and ion mobility spectra of ternary and quaternary complexes formed by mixing R / S ofloxacin with chiral reagent GM and metal ions, where: a is a schematic diagram of the mass spectrum of the mixture of R / S-OFLX and GM, b is a schematic diagram of the mass spectrum of R / S-OFLX and GM, Li + A schematic diagram of the mixed mass spectrum, where c represents [R / S-OFLX+GM+H]. + A schematic diagram of the ion mobility spectrum, where d represents [R / S-OFLX+2GM+H]. +schematic diagram of ion mobility spectrometry of the mixture of GM and OFLX enantiomers, e is [R / S-OFLX+GM+Li] + schematic diagram of ion mobility spectrometry of the mixture of GM and OFLX enantiomers, f is [R / S-OFLX+2GM+Li] + schematic diagram of ion mobility spectrometry of the mixture of GM and OFLX enantiomers. GM is non-covalently combined with OFLX enantiomers. As shown in Figure 5 a, m / z in the mass spectrum is 811.43, 1172.57 and 1260.72 respectively, corresponding to ions [R / S-OFLX+GM+H] + ion, [2R / S-OFLX+GM+H] + ion and [R / S-OFLX+2GM+H] + ion, which indicates that for the mixture of GM and OFLX enantiomers, the formation of enantiomeric or diastereomeric hetero-trimer is inevitable, which may complicate the mobility.

[0066] ion mobility spectrometry of the enantiomeric binary complex after protonation is shown in Figure 5 c. Binary complex ions [S-OFLX+GM+H] + and [R-OFLX+GM+H] + have no obvious difference in mobility, which indicates that the structural difference of R / S-OFLX enantiomers is not large after being combined with one GM. As shown in Figure 5 d, ternary complex ions [S-OFLX+2GM+H] + and [R-OFLX+2GM+H] + formed by S-OFLX and R-OFLX respectively have obvious difference in mobility, wherein 1 / K0 value is 1.675 V·s / cm 2 and 1.657 V·s / cm 2 , and Rp-p is 1.058. The results show that the ternary complex formed with GM can provide greater structural difference. Since the ion mobility depends on shape separation, compared with the binary complex, the ternary complex can bring greater improvement in discrimination. Such results show that multiple combinations between the ligand and the target are needed to make the discrimination large enough.

[0067] Further, metal ion Li + is also added to the mixture of OFLX and GM, and is detected by using TIMS-MS. As shown in Figure 5 b, R / S-OFLX enantiomers form ternary complex ions [R / S-OFLX+GM+Li] + and quaternary complex ions [R / S-OFLX+2GM+Li] + with GM and Li + respectively. As shown in Figure 5e and Figure 5 As shown in FIG. 2, compared with the resolution of the ternary complex (Rp-p is 0.636), the resolution of the quaternary complex is greatly improved, reaching 3.34, and baseline separation has been achieved.

[0068] Based on the same inventive concept, the embodiment of the present application provides a method for ofloxacin isomers.

[0069] Referring to Figure 6 A method for ofloxacin isomers, comprising:

[0070] Step 1: mixing the isomer R / S ofloxacin sample with a separation and identification reagent for ofloxacin isomers to prepare an R / S-OFLX-Vanco or R / S-OFLX-GM-metal ion mixed solution.

[0071] Here, the mixing method is to mix the mother liquor prepared from the R / S ofloxacin sample with the analysis reagent by vortex oscillation. Here, the pouring method can be directly poured slowly along the wall of the container, glass rod drainage or micro-volume pipetting equipment. Here, the weight ratio of GM-metal ion can be any ratio, which can be adjusted according to experimental requirements, for example: 1:1, 1:10, 10:1, etc.

[0072] Step 2: passing the mixed solution into the ion source to generate complex ions.

[0073] The complex ion is a monovalent positive ion of R / S-OFLX-Vanco or R / S-OFLX-GM-metal ion. Here, the complex ion is specifically [S-OFLX+Vanco+H] + and [R-OFLX+Vanco+H] + Here, the passing method is to use a micro-volume injection pump to inject the mixed solution into the ion source at a constant flow rate.

[0074] Step 3: controlling the experimental device to measure the ion mobility spectrum of the complex ion to obtain the enantiomeric structure information of R / S ofloxacin.

[0075] Here, the measurement-related data of the measurement analysis method is that the ion source voltage is 2.5-4.5 kV, the sample flow rate is 1-5 μLmin-1; the atomizer pressure is 0.3 bar; and the drying gas is 3 Lmin-1at 200 °C.

[0076] The experimental device here is an ion mobility spectrometry-mass spectrometry (IM-MS) instrument. The ion mobility spectrometry function is to drive the ions to migrate in the drift tube under the action of an electric field, and the mobility is directly related to the collision cross section (CCS) of the ions, because the collision cross section is determined by the spatial structure of the ions. Because the spatial conformation of the ternary complex formed by the R / S ofloxacin isomers is different, the collision cross section is different, so there is a difference in the drift time in the ion mobility spectrometry. At the same time, the peak separation degree (Rp-p) can quantify the separation effect between the isomers, and judge whether baseline separation can be achieved.

[0077] The function of mass spectrometry is to confirm the chemical composition of ions through the mass-to-charge ratio, to verify that the detected is the ternary complex of R / S-OFLX-Vanco or R / S-OFLX-GM-metal ion, not an impurity ion, and to provide verification at the composition level for the separation result of ion mobility spectrometry.

[0078] Finally, by combining the drift time, collision cross section, and peak separation degree, the R / S ofloxacin enantiomers can be accurately distinguished from the ion mobility spectrometry, and the isomer structure information can be identified.

[0079] Among them, the ion source type is one of electrospray ionization ion source, laser-assisted desorption ionization ion source, and desorption electrospray ionization ion source. The ion source here is not limited to the above types of ion sources, and can be selected according to experimental needs or existing ion sources in the laboratory.

[0080] The method for preparing the mixed solution includes:

[0081] Step 10: Pour the solvent corresponding to the mixed solution into the mixed container containing R / S ofloxacin and perform ultrasonic dissolution to obtain the R / S ofloxacin mother liquor.

[0082] The solvent here is pure water or a mixture of pure water and methanol, ethanol, acetonitrile, etc., which can be selected according to experimental needs.

[0083] Ultrasonic dissolution refers to a method of using the vibrational energy of ultrasonic waves to accelerate the dissolution of solid samples in solvents. The ultrasonic dissolution here is performed by using a device contained in the laboratory, placing a container containing the solid sample and the corresponding solvent in the device, and dissolving by ultrasonic vibration.

[0084] The experimental data of ultrasonic dissolution here can be adjusted according to experimental requirements, for example: if the solute is vancomycin (solid powder, medium solubility), 300-500W power and 5-10 minutes may be needed, if it is a salt containing metal ions (such as lithium chloride, easy to dissolve), 200-300W and 2-5 minutes can be used, if a high concentration mother liquor (such as 10mmol / L) is needed, it may need to be ultrasonic dissolved in several times (5 minutes each time, interval 2 minutes); for low concentration mother liquor (such as 1mmol / L), single ultrasonic dissolution can be used. Ultrasonic dissolution device, such as ultrasonic cleaner, probe type ultrasonic cell disruptor.

[0085] Here, the pouring method is to first put R / S ofloxacin into the mixing container, then pour the corresponding solvent into the mixing container according to the required amount by directly pouring along the wall of the container, glass rod drainage or micro-volume pipetting equipment.

[0086] The R / S ofloxacin mother liquor refers to a mixed liquid containing solvent and R / S ofloxacin. Here, the way to get it is to ultrasonic dissolve the solvent poured into the mixing container with R / S ofloxacin until completely dissolved to obtain the R / S ofloxacin mother liquor.

[0087] Step 11: Pour the corresponding solvent of the mixed solution into the mixing container containing vancomycin or gentamicin C1a and metal ion containing compound and ultrasonic dissolve to obtain a mixed solution, which is vancomycin mother liquor or GM-metal ion containing compound mother liquor.

[0088] Vancomycin mother liquor refers to a mixed liquid containing solvent and vancomycin. GM-metal ion containing compound mother liquor refers to a mixed liquid containing solvent and GM-metal ion containing compound. The way to get it, the pouring method and the ultrasonic dissolution method are consistent with the method introduced in step 10, which is not repeated here.

[0089] Step 12: Pour the R / S ofloxacin mother liquor and the mixed solution into the mixing container according to the preset volume ratio, mix and control the vortex oscillation of the mixing container to obtain the composite.

[0090] Volume ratio refers to the ratio of the volume of R / S ofloxacin mother liquor to the mixed solution. It is obtained by the laboratory staff according to the concentration of the mixed solution required by the experiment, for example: 1:1. Complex refers to a mixed solution containing solvent, R / S ofloxacin, vancomycin or gentamicin C1a and metal ion-containing compounds. Here it is obtained by sequentially pouring R / S ofloxacin mother liquor and vancomycin mother liquor or GM-metal ion-containing compound mother liquor into a mixing container, and then mixing by vortexing. Here vortexing refers to a method of using the high-speed rotation of a vortexing device to generate centrifugal force to drive the liquid to form a vortex, thereby accelerating the mixing of liquid and solid samples or the uniform mixing of liquids. Here the vortexing method is to use the device contained in the laboratory to place the container containing the mixed substances on the oscillation platform of the device, and mix the substances in the container by high-speed rotation to form a vortex. The experimental data of vortexing can be adjusted according to experimental needs, for example: low speed (800-1500 rpm) is suitable for small amount of sample (such as 1-5 mL of mixed solution), high speed (2000-3000 rpm) is suitable for larger volume (such as 10-20 mL) of mixed solution, if the mixed solution is easy to produce bubbles (such as mixed solution containing ethanol), intermittent oscillation (oscillation for 5 seconds, pause for 2 seconds, repeat 3-5 times) can be used. Vortexing devices include, for example, benchtop vortexers and handheld vortexers.

[0091] Further methods for preparing a mixed solution are also included, which comprise:

[0092] Step 13: Obtain the mixed ingredients.

[0093] The mixed ingredients refer to the solute and solvent components to be mixed. Here the method of obtaining is that the workers in the field input the solute and solvent components during the experiment into the system to obtain them.

[0094] Step 14: Find the corresponding adjustment data based on the mixed ingredients.

[0095] The adjustment data refers to the relevant data that need to be adjusted for the mixed ingredients. Here the method of finding is that different mixed ingredients correspond to different optimal mixing data, and the system outputs the data difference between the optimal mixing data and the current data as the adjustment data. Here the optimal mixing data is obtained by workers in the field according to their own experience and network search and then input into the database, and the system finds the corresponding optimal mixing data in the database and then calculates the adjustment data after receiving the mixed ingredients. Here the current data is obtained by the original device of the experimental device.

[0096] Step 15: Adjust the mixed ingredients according to the adjustment data.

[0097] The adjustment mode is that the system controls the adjustment device in the experimental device to adjust according to the adjustment data. The data is adjusted to the optimal mixing data, the mixing of the solvent and the solute is accelerated, and the efficiency of the experiment is improved.

[0098] Step 16: When the preset adjustment completion signal is received, steps 10 to 12 are executed.

[0099] The adjustment completion signal refers to a signal that the data has been adjusted to the optimal mixing data. The receiving mode is that the system outputs the adjustment completion signal and receives when the current data is the optimal mixing data.

[0100] When the preset adjustment completion signal is received, it means that the conditions for preparing the mixed solution at this time are the optimal conditions, and at this time, the mixed solution can be quickly prepared, so steps 10 to 12 are executed.

[0101] Reference Figure 7 The method also includes simulating the concentration of the mixed solution, and the method includes:

[0102] Step 120: Obtain the required concentration and the corresponding mixed amount of the to-be-mixed components.

[0103] The required concentration refers to the concentration of the mixed solution required. The mixed amount refers to the amount of various components to be mixed. The obtaining mode is that the experimental personnel input the required concentration and the mixed amount into the experimental device.

[0104] Step 121: Obtain the mixing device based on the mixed amount.

[0105] The mixing device refers to a device for mixing solute and solvent into mixed liquid. The mixing device includes a mixing container. The obtaining mode is that different mixed amounts correspond to different mixing devices, and the working personnel in the field input the mixing devices corresponding to the commonly used mixed amounts into the system according to their own experience.

[0106] Step 122: Obtain the simulated mixing concentration based on the mixed amount and the mixing device by using a preset simulation calculation method.

[0107] The simulation calculation method refers to a method for calculating the concentration of the mixed solution simulated by the system. The simulated mixing concentration refers to the concentration of the mixed solution simulated by the system. The obtaining method is that different mixing devices have different simulated mixing concentrations according to different mixed amounts, and the working personnel in the field input the mixing concentrations corresponding to the mixed amounts obtained from multiple experimental data into the database as the average values of the multiple experimental data.

[0108] Step 123: When the simulated mixing concentration is consistent with the required concentration, output the mixed amount as the accurate mixed amount.

[0109] The accurate mixing amount refers to the amount of the mixed component after mixing, which can reach the required concentration of the solution.

[0110] When the simulated mixing concentration is consistent with the required concentration, it means that the solvent and solute are mixed to obtain the required reagent, and no additional mixed component needs to be added, so the to-be-mixed amount is output as the accurate mixing amount.

[0111] Step 124: When the simulated mixing concentration is inconsistent with the required concentration, the simulated increase amount is obtained based on the to-be-mixed amount and the simulated mixing concentration.

[0112] The simulated increase amount refers to the amount of the mixed component that needs to be additionally added through simulation. Here, the required concentration is subtracted from the simulated mixing concentration to obtain a concentration difference, and then the concentration difference is multiplied by the ratio of the simulated mixing concentration to the to-be-mixed amount to obtain the simulated increase amount.

[0113] When the simulated mixing concentration is inconsistent with the required concentration, it means that the content of the solute or solvent in the solution is not enough, and needs to be additionally increased, so the simulated increase amount is obtained based on the required concentration, the simulated mixing concentration, and the to-be-mixed amount.

[0114] Step 125: The accurate mixing amount is output based on the simulated increase amount and the to-be-mixed amount.

[0115] Here, the simulated increase amount is added to the to-be-mixed amount to obtain the accurate mixing amount.

[0116] The method further includes pouring the simulated increase amount into the mixing container, and the method includes:

[0117] Step 1240: Obtain the mixing concentration.

[0118] The mixing concentration refers to the concentration of the solution containing the solvent, R / S ofloxacin, vancomycin or gentamicin C1a, and the compound containing metal ions. Here, the mixing concentration is obtained by using existing devices in the laboratory, such as ultraviolet-visible spectrophotometer, high-performance liquid chromatograph, etc.

[0119] Step 1241: When the mixing concentration reaches the required concentration, steps 2 to 3 are performed.

[0120] When the mixing concentration reaches the required concentration, it means that the concentration of the mixed solution meets the experimental requirements, so steps 2 to 3 are performed.

[0121] Step 1242: When the mixing concentration does not reach the required concentration, and the mixing device is a preset drainage device, the to-be-mixed component corresponding to the supplementary mixing amount is calculated according to the required concentration and the mixing concentration.

[0122] Draining device refers to the device that can make the solution flow into the mixed liquid in the mixing container through the draining rod and other draining objects. For example: glass rod, beaker with draining nozzle, separating funnel, etc.

[0123] Supplemental mixing amount refers to the amount of solvent, solute or solution that needs to be supplemented into the mixing container. Here the calculation method is consistent with step 124, which will not be repeated here.

[0124] Step 1243: When the supplemental mixing amount is consistent with the simulated increase amount, pour the simulated increase amount into the mixing container as the supplemental mixing amount.

[0125] Here the pouring method is to slowly flow into the container through the corresponding draining device in step 1242.

[0126] When the supplemental mixing amount is consistent with the simulated increase amount, it means that the simulation result is accurate and does not need to be updated, so the simulated increase amount is poured into the mixing container as the supplemental mixing amount.

[0127] Step 1244: When the supplemental mixing amount is not consistent with the simulated increase amount, update the simulated increase amount according to the supplemental mixing amount and pour it into the mixing container.

[0128] Here the update method is to update the simulated increase amount as the supplemental mixing amount.

[0129] When the supplemental mixing amount is not consistent with the simulated increase amount, it means that the simulated increase amount is not accurate. In order to avoid the problem of insufficient mixing concentration caused by the inaccurate simulated increase amount, update the simulated increase amount according to the supplemental mixing amount and pour it into the mixing container.

[0130] Step 1245: If the mixing device is a preset wall-following device, then close the mixing container and control the mixing container to execute a preset complete dissolution scheme.

[0131] Wall-following device refers to the device that makes the solution flow into the mixed liquid in the container along the container wall. For example: pipette, dropper, beaker directly poured into, etc.

[0132] Complete dissolution scheme refers to the method of completely dissolving the solute or mixed solution in the mixing container.

[0133] If the mixing device is a preset wall-following device, since adding solution along the wall may cause some solution to remain on the container wall and not completely dissolve into the mixed solution, it is necessary to close the mixing container and control the mixing container to execute a preset complete dissolution scheme, so that the solution or solvent hanging on the container wall can be fully mixed with the mixed solution.

[0134] The above merely describes the preferred embodiments of the present application, and the protection scope of the present application is not limited to the above-described embodiments. Any technical solution falling within the concept of the present application shall fall within the protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, some improvements and refinements without departing from the principles of the present application shall also be considered as falling within the protection scope of the present application.

Claims

1. A reagent for the separation and identification of ofloxacin isomers, characterized in that: Includes a mixed solution for analyzing ofloxacin isomers, said mixed solution comprising a mixture of vancomycin or a metal ion-containing compound with gentamicin C1a containing a solvent.

2. The reagent for the separation and identification of ofloxacin isomers according to claim 1, characterized in that: The molecular formula of gentamicin C1a is C 19 H 39 N5O7 has a molecular weight of 449.54, and vancomycin has the molecular formula C. 66 H 75 Cl2N9O 24 Its molecular weight is 1449.

25.

3. The reagent for the separation and identification of ofloxacin isomers according to claim 1, characterized in that: The compound containing metal ions is either a salt containing metal ions or a soluble base containing metal ions.

4. The reagent for the separation and identification of ofloxacin isomers according to claim 1, characterized in that: The solvent used to mix vancomycin or a compound containing metal ions with gentamicin C1a is pure water, or a mixture of pure water with methanol, ethanol, or acetonitrile.

5. A method for the separation and identification of ofloxacin isomers, characterized in that, include: Step 1: Mix the R / S ofloxacin isomer sample with a reagent for the separation and identification of ofloxacin isomers as described in any one of claims 1 to 4 to prepare a mixed solution of R / S-OFLX-Vanco or R / S-OFLX-GM-metal ions. Step 2: Pass the mixed solution into an ion source to generate complex ions, wherein the complex ions are monovalent positive ions of R / S-OFLX-Vanco or R / S-OFLX-GM-metal ions; Step 3: Control the experimental setup to measure the ion mobility spectrum of the complex ions to obtain enantiomeric structural information of R / S ofloxacin.

6. The method for separating and identifying ofloxacin isomers according to claim 5, characterized in that: The ion source type is one of the following: electrospray ionization source, laser-assisted desorption ionization source, or desorption electrospray ionization source.

7. The method for separating and identifying ofloxacin isomers according to claim 5, characterized in that, Methods for preparing mixed solutions include: Step 10: Pour the solvent corresponding to the mixed solution into a mixing container containing R / S ofloxacin and sonicate to dissolve it to obtain R / S ofloxacin stock solution; Step 11: Pour the solvent corresponding to the mixed solution into a mixing container containing vancomycin or gentamicin C1a and a compound containing metal ions and sonicate to dissolve it to obtain a mixed solution, wherein the mixed solution is a vancomycin stock solution or a GM-metal ion compound stock solution; Step 12: Pour the R / S ofloxacin mother liquor and the mixed solution into a mixing container according to the preset volume ratio, and control the mixing container to vortex and oscillate to obtain the complex.

8. The method for separating and identifying ofloxacin isomers according to claim 7, characterized in that, It also includes a further method for preparing a mixed solution, the method comprising: Step 13: Obtain the components to be mixed; Step 14: Find the corresponding adjustment data based on the components to be mixed; Step 15: Adjust the components to be mixed based on the adjustment data; Step 16: Upon receiving the preset adjustment completion signal, execute steps 10 to 12.

9. A method for separating and identifying ofloxacin isomers according to claim 7, characterized in that, It also includes methods for simulating the concentration of mixed solutions, which include: Step 120: Obtain the required concentration and the corresponding amount of the component to be mixed; Step 121: Obtain a mixing device based on the amount to be mixed, the mixing device including a mixing container; Step 122: Based on the amount to be mixed and the mixing device, obtain the simulated mixing concentration using a preset simulation calculation method; Step 123: When the simulated mixing concentration matches the required concentration, output the amount to be mixed as the accurate mixing amount; Step 124: When the simulated mixing concentration is inconsistent with the required concentration, obtain the simulated increase based on the amount to be mixed and the simulated mixing concentration; Step 125: Obtain the accurate mixing amount output based on the simulated increase and the amount to be mixed.

10. A method for separating and identifying ofloxacin isomers according to claim 9, characterized in that, It also includes a method for pouring the simulated increase into a mixing container, the method comprising: Step 1240: Obtain the mixed concentration; Step 1241: When the mixed concentration reaches the required concentration, proceed to steps 2 and 3; Step 1242: When the mixing concentration does not reach the required concentration and the mixing device is a preset diversion device, calculate the supplementary mixing amount corresponding to the component to be mixed based on the required concentration and the mixing concentration; Step 1243: When the amount of additional mixing matches the amount of simulated increase, pour the simulated increase into the mixing container as the additional mixing amount; Step 1244: When the amount of additional mixing is inconsistent with the simulated increase, update the simulated increase based on the amount of additional mixing and pour it into the mixing container; Step 1245: If the mixing device is a preset wall-mounted device, then close the mixing container and control the mixing container to execute the preset complete dissolution scheme.

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