Method for separating dextrofloxacin and levofloxacin based on high performance liquid chromatography-differential ion mobility mass spectrometry

Abstract

The invention belongs to the technical field of analysis and detection, and provides a method for separating dextrofloxacin and levofloxacin based on high performance liquid chromatography-differential ion mobility mass spectrometry. According to the method for analyzing the ofloxacin isomeride based on the differential ion mobility mass spectrometry, by optimizing parameters such as a modifier, separation voltage and compensation voltage, efficient mobility separation of dextrorotatory / levorotatory ofloxacin is achieved for the first time, the problem of isomeride identification of a traditional chromatography method is solved, and the method is suitable for large-scale popularization and application. The background noise is obviously reduced; and the detection sensitivity is improved. The method is compatible with an existing chromatography-mass spectrometry system, an electrospray ion source and multiple reaction monitoring parameters do not need to be adjusted after a DMS function is started, operation is easy and convenient, high-value consumables such as a chiral chromatographic column are omitted, the detection time is greatly shortened, a solution which is high in sensitivity, low in cost and easy to popularize is provided for chiral drug analysis, and the method is suitable for rapid screening and conventional quality inspection scenes.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of analytical detection, and particularly relates to a method for separating dextrofloxacin and levofloxacin based on high performance liquid chromatography-differential ion mobility mass spectrometry. BACKGROUND

[0002] Ofloxacin is a racemic mixture composed of two enantiomers, levofloxacin (L-form, levofloxacin) and dextrofloxacin (D-form). The levofloxacin has antibacterial activity, and the dextrofloxacin has almost no antibacterial activity. Levofloxacin is a pure levorotatory isomer (L-form) of ofloxacin, which retains only the active levorotatory form and removes the inactive dextrofloxacin, thereby significantly improving the efficacy and drug efficiency.

[0003] Currently, the analysis techniques for determining dextrofloxacin and levofloxacin mainly include high performance liquid chromatography and liquid chromatography-triple quadrupole mass spectrometry. However, the analysis method cannot separate the levorotatory isomer and the dextrofloxacin isomer of ofloxacin. The current chiral separation methods for ofloxacin mainly include separation by using a chiral mobile phase or a derivatization method. The chiral chromatographic column has high cost, and the applicability of different chiral columns has large differences, and the universality is low. Some chiral columns are sensitive to the pH, ionic strength or organic phase ratio of the mobile phase, and the conditions may need to be strictly controlled. SUMMARY

[0004] To solve the above technical problems, the application provides a method for separating dextrofloxacin and levofloxacin based on high performance liquid chromatography-differential ion mobility mass spectrometry.

[0005] The technical scheme of the application is as follows: The method for separating dextrofloxacin and levofloxacin based on high performance liquid chromatography-differential ion mobility mass spectrometry comprises the following steps: (1) Sample pretreatment: taking a sample to be tested, diluting or preparing a solution, filtering, and then detecting; (2) Adjusting the chromatographic conditions: The chromatographic column is a Phenomenex Kintex F5 column with a specification of 100*3.0 mm and a particle size of 2.6 μm. The mobile phase A is 0.1%-0.5% formic acid water, and the mobile phase B is 0.1%-0.5% formic acid acetonitrile. The flow rate is 0.3-1.0 mL / min. The gradient elution program is as follows: 0~2 min, 5% B; 2~5 min, 5%-95% B; 5~8 min, 95% B; 8~8.1 min, 95%-5% B; 8.1~10 min, 5% B. (3) Adjusting the mass spectrometry conditions: The ion source heating temperature is 400-600 DEG C, and the detection mode is a multi-reaction selection ion monitoring mode; positive ion detection; The spray voltage is 4500-6500 V; the atomization gas pressure is 300-400 kPa; the auxiliary gas pressure is 300-400 kPa; the curtain gas pressure is 200-210 kPa; the differential ion mobility temperature is 120-170 DEG C. The differential ion mobility modifier is isopropanol. (4) Injection detection: injection amount: 1 muL.

[0006] Preferably, in (1), the sample pretreatment process is as follows: taking the sample to be tested, diluting or preparing a solution by using 50% methanol aqueous solution, filtering through a 0.22 mu m polytetrafluoroethylene (PTFE) or regenerated cellulose (RC) filter membrane, discarding the initial filtrate, and taking 1.0 mL of the filtered sample into an injection vial; wherein the 50% methanol aqueous solution contains 0.1% formic acid.

[0007] Preferably, in (2), the mobile phase A is 0.2% formic acid water, and the mobile phase B is 0.2% formic acid acetonitrile. The flow rate is 0.6 mL / min.

[0008] Preferably, in (3), the ion source heating temperature is 500 DEG C. The spray voltage is 5500 V; the atomization gas pressure is 345 kPa; the auxiliary gas pressure is 345 kPa; the curtain gas pressure is 207 kPa; the differential ion mobility temperature is 150 DEG C.

[0009] The present application has the following advantages and effects relative to the prior art: (1) The ion mobility difference mass spectrometry (DMS) technology of the method easily realizes the separation of the isomers dextrofloxacin and levofloxacin, solves the problem of distinguishing dextrofloxacin and levofloxacin, realizes the accurate qualitative and quantitative determination of levofloxacin, and also reduces the background noise and improves the sensitivity; (2) The method realizes the mobility separation of the isomers dextrofloxacin and levofloxacin which are difficult to separate by chromatography by optimizing the modifier, separation voltage and compensation voltage and other conditions, solves the problem of distinguishing dextrofloxacin and levofloxacin, and realizes the accurate qualitative and quantitative determination of levofloxacin; (3) After the differential ion mobility mass spectrometry function is turned on, the chromatographic conditions, electrospray ion source and multi-reaction monitoring parameters do not need to be changed, which is simple and easy to operate, reduces the consumable cost of, for example, a chiral chromatographic column, has a short running time and high detection efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1Mobility separation profile of ofloxacin with two configurations after introducing modifier isopropanol; Figure 2 Mobility separation profile of ofloxacin without modifier; Figure 3 Mobility separation profile of ofloxacin with modifier methanol at low flow rate; Figure 4 Mobility separation profile of ofloxacin with modifier isopropanol at low flow rate; Figure 5 Mobility separation profile of ofloxacin with modifier acetonitrile at low flow rate; Figure 6 Mobility separation profile of ofloxacin with modifier isopropanol at high flow rate; Figure 7 Optimization of mobility pool temperature, wherein A is the mobility separation profile of ofloxacin with two configurations at low temperature 150℃, B is the mobility separation profile of ofloxacin with two configurations at medium temperature 225℃, C is the mobility separation profile of ofloxacin with two configurations at high temperature 300℃; Figure 8 Optimization of separation voltage SV value; Figure 9 Optimization of separation voltage SV value; Figure 10 Multiple reaction monitoring chromatogram of ofloxacin without opening differential ion mobility; Figure 11 Multiple reaction monitoring chromatogram of dextro ofloxacin at CoV =-23.7V and levo ofloxacin at CoV =-8.4V after opening differential ion mobility; Figure 12 Standard curve in Example 3; Figure 13 Chromatogram of detecting fatty acid isomers by differential ion mobility mass spectrometry in Comparative Example 2. DETAILED DESCRIPTION

[0011] In order to make the person skilled in the art better understand the present application, the present application will be further described in conjunction with specific embodiments.

[0012] 1.1 Instruments, reagents and materials Triple Quad 6500 + Triple Quad 6500, methanol, water, isopropanol, formic acid, acetonitrile, ofloxacin and levofloxacin were purchased from Shanghai Anpu Experimental Technology Co., Ltd., and the purity was greater than 98.0%.

[0013] 1.2 Preparation of standard solution The standard solution was prepared by dissolving the standard substance in methanol to a concentration of 1000 μg / mL and stored at -20 ℃. The mixed standard solution was prepared by dissolving the standard substance in 50% methanol water (0.1% formic acid) to a concentration of 1 μg / mL, and the required concentration was prepared by using 50% methanol water (0.1% formic acid) before use.

[0014] Example 1 Method for separating levofloxacin and ofloxacin based on high performance liquid chromatography-differential ion mobility mass spectrometry: (1) Chromatographic conditions Chromatographic column: Phenomenex Kintex F5 (100 x 3.0 mm, 2.6 μm, USA); Mobile phase A: 0.2% formic acid water, mobile phase B: 0.2% formic acid acetonitrile; Flow rate: 0.6 mL / min; Gradient elution program: 0~2 min, 5% B; 2~5 min, 5~95% B; 5~8 min, 95% B; 8~8.1 min, 95~5% B; 8.1~10 min, 5% B. Injection volume: 1 μL.

[0015] (2) Mass spectrometry conditions The electrospray ionization (ESI) source was used, the ion source heating temperature was 500 ℃, and the detection mode was multiple reaction ion monitoring (MRM) mode. Positive ion detection; spray voltage: 5500 V; nebulization gas pressure: 345 kPa (50 psi); auxiliary gas pressure: 345 kPa (50 psi); curtain gas pressure: 207 kPa (30 psi); differential ion mobility temperature: 150 ℃; differential ion mobility modifier: isopropanol. The optimized differential ion mobility mass spectrometry parameters of levofloxacin and ofloxacin are shown in Table 1.

[0016] Table 1 Differential ion mobility mass spectrometry parameters of levofloxacin and ofloxacin compounds Compound Parent ion (m / z) Daughter ion (m / z) De-clustering voltage DP / V Collision energy CE / eV Compensation voltage CoV / V Separation voltage SV / V Dextro ofloxacin 362.3 318 50 26 -23.7 3700 Levo ofloxacin 362.3 318 50 26 -8.4 3700

[0017] Example 2 Optimization of differential ion mobility mass spectrometry parameters In this example, the two compounds were separated by differential ion mobility mass spectrometry. After turning on the differential ion mobility mass spectrometry function, the electrospray ion source and multiple reaction monitoring parameters did not need to be changed, and the differential ion mobility mass spectrometry related parameters were optimized by directly injecting 500 ng / mL of the mixed standard solution. The specific optimization process is as follows: 2.1 Optimization of modifier (1) Screening of different modifiers The conditions of the modifying agent were optimized in detail during the experiment, and the addition of no modifying agent and different modifying agents (methanol, acetonitrile, isopropanol) was compared, and the results showed (as shown in Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 indicated) that when no modifying agent, methanol and acetonitrile were added as the modifying agent, the dextro-ofloxacin and the levofloxacin were not effectively separated, when isopropanol was used as the modifying agent, the dextro-ofloxacin and the levofloxacin were effectively separated, and therefore, the experiment finally selected isopropanol as the modifying agent. Therefore, by pumping the isopropanol modifying agent to optimize the coefficient of variation (CoV), the mobility separation diagram (as shown in Figure 1 ) showed that there were obvious peak values.

[0018] (2) Screening of isopropanol modifying agent flow rate The expression of the flow rate of the modifying agent involved in each embodiment of the present application is 325 μL / min for the low flow rate and 500 μL / min for the high flow rate, unless otherwise specified.

[0019] As shown in the spectrum in Figure 4 、 Figure 6 , since the increase of the flow rate of the modifying agent will lead to a decrease in response and separation can be achieved at a low flow rate, the low flow rate (325 μL / min) of isopropanol is selected, and the next parameter adjustment is carried out.

[0020] 2.2 Optimization of the mobility cell temperature (DT) The separation degree and sensitivity of the two isomers were improved by optimizing different DTs. The results showed (as shown in Figure 7 ) that with the increase of DT, the sensitivity gradually increased, but the separation degree became worse and worse.

[0021] Table 2. Detection information of target substances at different mobility cell temperatures Tailing pool temperature 150℃ 225℃ 300℃ Levo ofloxacin (CoV value) -8.5 V -9 V -11.5 V Dextro ofloxacin (CoV value) -23.8 V -22.5 V -11.5 V

[0022] Therefore, 150℃ is selected as the mobility cell temperature, which not only ensures good separation degree, but also has high sensitivity.

[0023] 2.3 Confirmation of the corresponding CoV values of dextro-ofloxacin and levofloxacin During the experiment, a single standard needle pump was used for sampling to confirm the CoV values of dextro-ofloxacin and levofloxacin. When a single standard dextro-ofloxacin (500 ng / mL) needle pump was used for sampling, the corresponding CoV value was -23.7V; when a single standard levofloxacin (500 ng / mL) needle pump was used for sampling, the corresponding CoV value was -8.4V. 2.4 Optimization of separation voltage SV Based on the obtained CoV value, the SV value is optimized to achieve the best sensitivity (as shown in Figure 8 , Figure 9 The SV is continuously changed from 3000 V to 4100 V in 5 V steps, and the sensitivity of levofloxacin (CoV = -23.7) and ofloxacin (CoV = -8.4) is optimal at SV = 3700 V, so the separation voltage SV value is selected as 3700 V.

[0024] Analysis performance verification of the method of Example 3 A levofloxacin standard solution is prepared, and determination is performed under the analysis conditions determined in Example 1, with the mass concentration (ng / mL) of each substance as the abscissa X and the corresponding peak area as the ordinate Y, to draw a standard curve (as shown in Figure 12 ). The standard curve has a good linearity in the range of 1 ng / mL to 25 ng / mL, and the correlation coefficient r is greater than 0.999.

[0025] The levofloxacin content in a certain ofloxacin ear drop (purchased from a pharmacy) is analyzed. The drug solution is diluted 300000 times with 50% methanol water (0.1% formic acid) and directly injected. Significant levofloxacin and ofloxacin chromatograms can be seen, and the levofloxacin concentration in the sample is quantified as 5.9 ng / mL (after dilution 300000 times) by using the levofloxacin standard curve.

[0026] Comparative Example 1 Compared with the existing method, i.e., the conventional LCMS detection method, when the ion mobility technology is not used, the levofloxacin content cannot be accurately quantified (as shown in Figure 10 ) because the chromatographic separation cannot be achieved and the monitoring ion pair information of levofloxacin and ofloxacin is completely the same. After using the differential ion mobility mass spectrometry technology, the multiple reaction monitoring chromatograms of ofloxacin at a compensation voltage of -23.7 V and levofloxacin at a compensation voltage of -8.4 V are shown in Figure 11 , and the two channels do not interfere with each other, so that the separation and qualitative and quantitative analysis of ofloxacin and levofloxacin can be achieved.

[0027] Comparative Example 2 The differential ion mobility mass spectrometry technology is tried to be used to separate two isomers of fatty acids C18:2 (Z9, Z12) and C18:2 (Z9, E11), but still cannot distinguish between the two (as shown in Figure 13 , and the CoV of the two compounds is -8.8 V without difference). It is shown that the differential ion mobility mass spectrometry technology is not applicable to the separation of all isomers.

[0028] Figure 13The CoV of the two compounds, medium fatty acid C18:2(Z9,Z12) and C18:2(Z9,E11), is -8.8 V after introducing isopropanol modifier when optimizing CoV using differential ion mobility mass spectrometry technology.

[0029] In summary, the present application realizes the efficient separation of dextrofloxacin and levofloxacin for the first time by DMS mobility technology, solves the isomer identification problem that is difficult to distinguish by traditional chromatography, and provides a precise qualitative and quantitative analysis method for levofloxacin.

[0030] The above is only a preferred embodiment of the present application, and does not limit the patent scope of the present application, and any equivalent changes and modifications made within the scope of the present application shall still fall within the scope of the present application.

Claims

1. A method for separating dextrorotatory ofloxacin and levorotatory ofloxacin based on high performance liquid chromatography-differential molecular mobility mass spectrometry, characterized in that, The steps include the following: (1) Sample pretreatment: Take the ofloxacin sample to be tested, dilute or prepare a solution, filter it, and then test it; (2) Adjust chromatographic conditions: Column: Phenomenex Kintex F5, 100×3.0 mm, 2.6 μm; Mobile phase A is 0.1%-0.5% formic acid in water, and mobile phase B is 0.1%-0.5% formic acid in acetonitrile; Flow rate: 0.3-1.0 mL / min; Gradient elution program: 0-2 min, 5% B; 2-5 min, 5%-95% B; 5-8 min, 95% B; 8~8.1 min, 95%~5% B; 8.1~10min, 5% B; (3) Adjust mass spectrometry conditions: An electrospray ionization source is used, with an ion source heating temperature of 400-600 ℃. The detection method is multiple reaction selection ion monitoring mode; positive ion detection is employed. Spray voltage: 4500-6500 V; atomizing gas pressure: 300-400 kPa; auxiliary gas pressure: 300-400 kPa; curtain gas pressure: 200-210 kPa; differential ion mobility temperature: 120-170℃. Differential ion mobility modifier: isopropanol; the pumping flow rate of the differential ion mobility modifier is 300-400 μL / min; (4) Sample injection and detection: Injection volume: 1 μL.

2. The method as described in claim 1, characterized in that, (1) The sample pretreatment process is as follows: take the sample to be tested, dilute or prepare a solution using 50% methanol aqueous solution, filter with a 0.22 μm PTFE or RC filter membrane, discard the initial filtrate, and take 1.0 mL of the filtered sample into a vial; wherein the 50% methanol aqueous solution contains 0.1% formic acid.

3. The method as described in claim 1, characterized in that, (2) In this context, mobile phase A is 0.2% formic acid water, and mobile phase B is 0.2% formic acid acetonitrile; Flow rate: 0.6 mL / min.

4. The method as described in claim 1, characterized in that, In (3), the heating temperature of the ion source is 500 °C; The spray voltage is 5500 V; the atomizing gas pressure is 345 kPa; the auxiliary gas pressure is 345 kPa; the curtain gas pressure is 207 kPa; the differential ion mobility temperature is 150℃. The pumping flow rate of the differential molecular mobility modifier was 325 μL / min.

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