Method for detecting piperazine new psychoactive substances in levofloxacin eye drops

Abstract

The invention provides a method for detecting new piperazine psychoactive substances in levofloxacin eye drops. The detection method comprises the following steps: S1, mixing the piperazine new psychoactive substance with a diluent to prepare mixed solutions with different concentrations as reference substance solutions; s2, detecting each reference substance solution by adopting a liquid chromatograph-mass spectrometer, and carrying out regression analysis on the concentration of each reference substance solution according to the spectrogram peak area of each reference substance solution to obtain a standard curve; step S3, under the same test condition as the step S2, detecting the levofloxacin eye drops by adopting a liquid chromatograph and mass spectrometer to obtain spectrogram peak area detection data of the piperazine new psychoactive substances in the levofloxacin eye drops; and substituting detection data into the standard curve to obtain the concentration of the piperazine new psychoactive substances in the levofloxacin eye drops. The detection method is high in accuracy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis, in particular to a method for detecting piperazine new psychoactive substances in levofloxacin eye drops. BACKGROUND

[0002] The safety of a drug depends not only on its own toxicity, but also on the impurities it contains. Piperazine substances are a class of amphetamines, and their basic chemical structure is a six-membered heterocyclic ring with two nitrogen atoms at positions 1 and 4. Piperazine NPS is a class of NPS with piperazine ring as the parent structure and structural modification at multiple positions. Piperazine NPS can stimulate the release of dopamine, norepinephrine and serotonin by inhibiting the reuptake of these monoamine neurotransmitters, and has a mild excitatory effect on the central nervous system and a part of hallucinogenic effect and dependence.

[0003] Piperazine is an important pharmaceutical intermediate and is widely used. Studies have shown that the potential toxic effects of piperazine drugs can cause damage to human myocardial cells. For example, taking piperazine NPS can cause irritability, shortness of breath, headache, dizziness, anxiety, insomnia, vomiting, chest pain, tachycardia, hypertension and other adverse reactions a few minutes after taking the drug, and can cause hallucinations, disorientation, hearing impairment, persecutory delusion and other symptoms after a period of time, and can cause serious arrhythmia, cardiac arrest, acute renal failure, acute psychosis, rhabdomyolysis, disseminated intravascular coagulation and other symptoms and death. Piperazine NPS simulates the molecular mechanism of amphetamines, stimulates the release of dopamine, norepinephrine and serotonin, and inhibits the reuptake of these monoamine neurotransmitters, and has a mild excitatory effect on the central nervous system and a part of hallucinogenic effect and dependence.

[0004] Levofloxacin is a broad-spectrum antibacterial, strong quinolone drugs, levofloxacin eye drops for light yellow to light yellow or light yellow green clear liquid, can be used for the treatment of most enterobacteriaceae bacteria, some staphylococcus, streptococcus pneumoniae, chlamydia, etc. also have good antibacterial effect, suitable for staphylococcus, streptococcus, pneumococcus, coccobacillus, enterococcus, corynebacterium, pseudomonas, green pyocyanin, hemophilus (influenza hemophilus, conjunctivitis hemophilus (k-wisdom bacillus)), mora (bran) catheter bacteria, mora bacillus, mora-arsenical bacillus, serratia, klebsiella, proteus, acinetobacter, enterobacter, anaerobic bacteria (propionic acid bacillus) caused by the following infectious diseases: blepharitis, hordeolum, dacryocystitis, conjunctivitis, hordeolum, keratitis.

[0005] According to the structure characteristics, synthesis process, degradation pathway and stability of levofloxacin raw material, the impurities generated in the process, the impurities generated in the stability test of the raw material, the impurities possibly generated in the preparation process and the stability test of the preparation, it is found that piperazine new psychoactive substances are the main raw materials and synthesis intermediates of quinolone drugs such as ofloxacin, levofloxacin, levofloxacin hydrochloride, etc. It is necessary to control piperazine new psychoactive substances. The existing analysis methods of piperazine new psychoactive substances include gas chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, capillary electrophoresis and other technologies. However, the existing analysis methods mainly focus on qualitative analysis, and there is no report on the detection of piperazine new psychoactive substances in levofloxacin eye drops. SUMMARY

[0006] The main purpose of the present application is to provide a detection method of piperazine new psychoactive substances in levofloxacin eye drops, to solve the problem that the prior art lacks detection of piperazine new psychoactive substances in levofloxacin eye drops.

[0007] In order to achieve the above-mentioned purpose, according to one aspect of the present application, a detection method of piperazine new psychoactive substances in levofloxacin eye drops is provided, which comprises the following steps: step S1, mixing piperazine new psychoactive substances and diluent to prepare mixed solutions with different concentrations as control solution; step S2, detecting each control solution by liquid chromatography and mass spectrometry, and performing regression analysis on the concentration of each control solution according to the peak area of the spectrum of each control solution to obtain a standard curve; step S3, under the same test conditions as step S2, detecting levofloxacin eye drops by liquid chromatography and mass spectrometry to obtain the peak area detection data of piperazine new psychoactive substances in levofloxacin eye drops; and substituting the detection data into the standard curve to obtain the concentration of piperazine new psychoactive substances in levofloxacin eye drops.

[0008] Further, the packing material of the chromatographic column in the liquid chromatography is octadecylsilane-bonded silica gel; and / or, the inner diameter of the chromatographic column in the liquid chromatography is 1.0-4.6 mm; and / or, the length of the chromatographic column in the liquid chromatography is 150-250 mm; and / or, the particle size of the packing material of the chromatographic column in the liquid chromatography is 1.7-3.5 μm.

[0009] Further, the flow rate of the mobile phase in the liquid chromatography is 0.6-1.0 mL / min; preferably, the mobile phase comprises mobile phase A and mobile phase B; and in the gradient elution procedure of the liquid chromatography, the ratio of mobile phase A and mobile phase B is as follows: when 0≤t1≤4 min, 100% of mobile phase A and 0% of mobile phase B; when 4

[0010] Further, the mobile phase A is selected from aqueous formic acid and / or aqueous acetic acid; and / or, the mobile phase B is selected from methanol and / or acetonitrile; preferably, the mass concentration of the aqueous formic acid is 0.05%-0.5%.

[0011] Further, the temperature of the chromatographic column in the liquid chromatography is 30-40 °C; and / or, the temperature of the sample tray in the liquid chromatography is 15-30 °C; and / or, the sample injection volume in the liquid chromatography is 1-20 μL; and / or, when the sample starts to flow into the liquid chromatography, the time is t2, when 2

[0012] Further, the ion source of the mass spectrometer is an electrospray ion source; in the electrospray ion source, the spray voltage is 5500-6000 V, the atomization temperature is 400-500 °C, the atomization gas pressure is 50-55 Psi, the auxiliary gas pressure is 50-60 Psi, the curtain gas pressure is 50-60 Psi, the collision gas pressure is 7-9 Psi, and the transition residence time is 200-260 ms.

[0013] Further, the monitoring mode of the mass spectrometer is multiple ion reaction monitoring in the positive ion mode.

[0014] Further, the above-mentioned new psychoactive piperazines include 1-nitroso-piperazine, 1-nitroso-4-methyl-piperazine and N-nitrosamine-levofloxacin.

[0015] Further, the mass concentration of 1-nitroso-piperazine in the above-mentioned control solution is not more than 322.848 ng / mL; and / or, the mass concentration of 1-nitroso-4-methyl-piperazine in the control solution is not more than 322.666 ng / mL; and / or, the mass concentration of N-nitrosamine-levofloxacin in the control solution is not more than 1.224 μg / mL.

[0016] Further, the above-mentioned control solution has at least six groups of different concentrations; preferably, the concentration of 1-nitroso-piperazine in the first group of concentrations of the control solution is 16.142 ng / mL, the concentration of 1-nitroso-4-methyl-piperazine is 16.133 ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.061 μg / mL; the concentration of 1-nitroso-piperazine in the second group of concentrations of the control solution is 48.427 ng / mL, the concentration of 1-nitroso-4-methyl-piperazine is 48.400 ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.184 μg / mL; the concentration of 1-nitroso-piperazine in the third group of concentrations of the control solution is 80.712 ng / mL, the concentration of 1-nitroso-4-methyl-piperazine is 80.667 ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.306 μg / mL; the concentration of 1-nitroso-piperazine in the fourth group of concentrations of the control solution is 161.424 ng / mL, the concentration of 1-nitroso-4-methyl-piperazine is 161.333 ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.612 μg / mL; the concentration of 1-nitroso-piperazine in the fifth group of concentrations of the control solution is 242.136 ng / mL, the concentration of 1-nitroso-4-methyl-piperazine is 242.000 ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.918 μg / mL; the concentration of 1-nitroso-piperazine in the sixth group of concentrations of the control solution is 322.848 ng / mL, the concentration of 1-nitroso-4-methyl-piperazine is 322.666 ng / mL, and the concentration of N-nitrosamine-levofloxacin is 1.224 μg / mL.

[0017] By the technical scheme of the present application, the measuring instrument used in the present application is a liquid chromatograph and mass spectrometer, the liquid chromatograph can better separate the components in the levofloxacin eye drops, so that the mass spectrometer can detect the components in the levofloxacin eye drops, thereby obtaining the structure of each component in the levofloxacin eye drops. The external standard method is used to prepare a plurality of concentration gradient control solution for detection, thereby obtaining the linear equation of the peak area and the concentration of the spectrum, and the result of the levofloxacin eye drops after measurement is substituted into the linear equation, so that the content of the piperazine new psychoactive substance in the levofloxacin eye drops can be obtained. The detection method of the present application can accurately quantitatively analyze the piperazine new psychoactive substance in the levofloxacin eye drops, the analysis time is short, the sample amount is small, the detection method has high sensitivity, good specificity and repeatability, is accurate and reliable, and the detection method of the present application has low economic cost, is simple and efficient, is less affected by external environmental factors, can be realized in different laboratories, different liquid chromatographs and mass spectrometers, different analysis test personnel and analysis environmental conditions, has good durability and good applicability. BRIEF DESCRIPTION OF DRAWINGS

[0018] The drawings accompanying the specification of the present application form a part thereof, serve to provide further understanding of the present application, and together with the description of the exemplary embodiments of the present application and the explanation thereof serve to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0019] Figure 1 The LC-MS graph of the first reference solution in Example 1 of the present application is shown;

[0020] Figure 2 The standard curve of 1-nitrosopiperazine in Example 1 of the present application is shown;

[0021] Figure 3 The standard curve of 1-nitrosopiperazine in Example 1 of the present application is shown;

[0022] Figure 4 The standard curve of N-nitrosamine-levofloxacin in Example 1 of the present application is shown. DETAILED DESCRIPTION

[0023] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0024] As analyzed in the background art of the present application, the prior art lacks detection of piperazine new psychoactive substances in levofloxacin eye drops, in order to solve the above problems, the present application provides a detection method for piperazine new psychoactive substances in levofloxacin eye drops.

[0025] In an embodiment of the present application, a method for detecting piperazine-type new psychoactive substances in levofloxacin eye drops is provided, which comprises the following steps: S1, mixing the piperazine-type new psychoactive substances with diluents to prepare mixed solutions with different concentrations as control solutions; S2, detecting the control solutions by using a liquid chromatograph-mass spectrometer, and performing regression analysis on the peak area of the spectrum of each control solution and its concentration to obtain a standard curve; S3, detecting the levofloxacin eye drops under the same test conditions as in step S2 by using the liquid chromatograph-mass spectrometer to obtain the peak area detection data of the piperazine-type new psychoactive substances in the levofloxacin eye drops; and S4, substituting the detection data into the standard curve to obtain the concentration of the piperazine-type new psychoactive substances in the levofloxacin eye drops.

[0026] The measuring instrument used in the present application is a liquid chromatograph-mass spectrometer. The liquid chromatograph can separate the components in the levofloxacin eye drops well, so that the mass spectrometer can detect the components in the levofloxacin eye drops and obtain the structure of each component in the levofloxacin eye drops. The external standard method is used to prepare control solutions with multiple concentration gradients for detection, so as to obtain a linear equation of the peak area and the concentration. The result of the levofloxacin eye drops after measurement is substituted into the linear equation to obtain the content of the piperazine-type new psychoactive substances in the levofloxacin eye drops. The detection method of the present application can accurately quantify and analyze the piperazine-type new psychoactive substances in the levofloxacin eye drops. The analysis time is short, the sample size is small, the detection method has high sensitivity, good specificity and repeatability, and is accurate and reliable. The detection method of the present application has low economic cost, is simple and efficient, is less affected by external environmental factors, can be realized in different laboratories, different liquid chromatograph-mass spectrometers, different analysis test personnel and different analysis environmental conditions, has good durability and good applicability.

[0027] The diluent is selected from any one or more of water, ethanol, acetonitrile and methanol.

[0028] In order to further improve the separation efficiency of each component in the levofloxacin eye drops and improve the detection accuracy, in an embodiment of the present application, the packing material of the chromatographic column in the liquid chromatograph is octadecylsilane bonded silica gel; and / or, the inner diameter of the chromatographic column in the liquid chromatograph is 1.0-4.6 mm; and / or, the length of the chromatographic column in the liquid chromatograph is 150-250 mm; and / or, the particle size of the packing material of the chromatographic column in the liquid chromatograph is 1.7-3.5 μm.

[0029] In an embodiment of the present application, the flow rate of the mobile phase in the liquid chromatography is 0.6-1.0 mL / min; preferably, the mobile phase comprises mobile phase A and mobile phase B; in the gradient elution procedure of the liquid chromatography, the proportion of mobile phase A and mobile phase B is as follows: when 0≤t1≤4 min, 100% of mobile phase A and 0% of mobile phase B; when 4

[0030] Controlling the flow rate of the mobile phase within the above range helps to make the sample stay in the chromatographic column for an appropriate time, thereby helping to improve the separation efficiency of each component while shortening the analysis time. Mobile phase A and B are responsible for enhancing the polarity or non-polarity characteristics of the sample in the liquid chromatography, respectively, and by gradient elution, the polarity environment of the liquid chromatography can be gradually adjusted, thereby optimizing the separation effect of different compounds. The step of 100% of mobile phase A and 0% of mobile phase B helps to quickly introduce the sample into the chromatographic column; the change of gradually reducing mobile phase A to 10% and increasing mobile phase B to 90% helps to promote the effective separation of compounds according to their polarity differences, and finally, mobile phase A returns to 100% and mobile phase B returns to 0%, which helps to clean the chromatographic column, improves the accuracy and repeatability of the next analysis, and is also beneficial to the final elution of strongly retained compounds.

[0031] In order to further improve the separation effect of each component in the levofloxacin eye drops and thereby improve the accuracy of the detection method, in an embodiment of the present application, the above-mentioned mobile phase A is selected from aqueous formic acid solution and / or aqueous acetic acid solution; and / or, mobile phase B is selected from methanol and / or acetonitrile; preferably, the mass concentration of aqueous formic acid solution is 0.05%-0.5%.

[0032] Controlling the mass concentration of aqueous formic acid solution within the above range helps to improve the separation efficiency of each component in the levofloxacin eye drops while maintaining the stability of the sample.

[0033] In an embodiment of the present application, the above-mentioned mobile phase A is aqueous formic acid solution and mobile phase B is methanol.

[0034] The aqueous formic acid solution can effectively improve the solubility of the sample in the reversed-phase liquid chromatography due to its acidic property, especially for the compounds with strong polarity, which helps to improve the retention time and achieve better separation. Methanol as a non-polar mobile phase can accelerate the elution of non-polar or weakly polar compounds, which helps to make the target analyte be detected at a suitable time point. The combination of the two can further improve the separation efficiency of each component in the levofloxacin eye drops.

[0035] In an embodiment of the present application, the temperature of the chromatographic column in the above-mentioned liquid chromatography is 30-40℃; and / or, the temperature of the sample tray in the liquid chromatography is 15-30℃; and / or, the sample injection volume in the liquid chromatography is 1-20μL; and / or, the sample flow channel in the liquid chromatography is connected to the mass spectrometer when 2

[0036] Controlling the temperature of the chromatographic column within the above-mentioned range helps to reduce the chromatographic peak tailing caused by excessive viscosity of the stationary phase while maintaining the stability of the sample, thereby obtaining sharper chromatographic peaks and improving the accuracy of detection. Controlling the temperature of the sample tray in the liquid chromatography within the above-mentioned range helps to slow down the evaporation and denaturation of the sample, maintain the uniformity between samples, and thus improve the accuracy of the detection results. Controlling the sample injection volume in the liquid chromatography within the above-mentioned range helps to improve the sensitivity and accuracy of detection while maintaining the non-overload of the chromatographic column and the detector. Controlling the time when the sample flow channel in the liquid chromatography is connected to the mass spectrometer within the above-mentioned range helps to reduce the entry of non-target substances into the mass spectrometer, reduce background noise and ion suppression effect, and thus improve the detection accuracy of the target analyte.

[0037] In an embodiment of the present application, the ion source of the above-mentioned mass spectrometer is an electrospray ion source; the spray voltage of the electrospray ion source is 5500-6000V, the atomization temperature is 400-500℃, the atomization gas pressure is 50-55Psi, the auxiliary gas pressure is 50-60Psi, the curtain gas pressure is 50-60Psi, the collision gas pressure is 7-9Psi, and the transition residence time is 200-260ms.

[0038] The electrospray ion source can effectively convert the analytes in the liquid chromatography effluent into gaseous ions, achieving good ionization effect even for difficult-to-volatile or thermally unstable compounds. Controlling the spray voltage and atomization temperature within the above range helps to improve the efficiency of the formation of electric liquid droplets, accelerate the evaporation and ionization process of the liquid droplets, thereby helping to improve the sensitivity of mass spectrometric detection. Controlling the atomization gas pressure within the above range helps to form a stable spray, break the liquid droplets into smaller particles, and promote the further evaporation and ionization of the liquid droplets. Controlling the auxiliary gas pressure within the above range helps to maintain the dryness of the atomization area, further promote the evaporation of the liquid droplets, and reduce the interference of pollutants on the mass spectrometric signal. Controlling the curtain gas pressure within the above range helps to protect the ion source from the interference of the external environment, thereby helping to improve the stability and repeatability of the detection. Controlling the collision gas pressure within the above range helps to generate more abundant fragment ion information, helping to identify the compounds and improve the specificity of the detection. Controlling the transition residence time within the above range helps to improve the stability of the mass spectrometric signal.

[0039] In an embodiment of the present application, the monitoring mode of the mass spectrometry is multiple ion reaction monitoring in positive ion mode.

[0040] The multiple ion reaction monitoring mode is a mass spectrometry technique for selectively identifying and quantifying specific compounds by monitoring specific reaction pairs of parent ions to daughter ions, which helps to improve the sensitivity and specificity of the detection.

[0041] In an embodiment of the present application, the above-mentioned new psychoactive substance of piperazine includes 1-nitroso-piperazine, 1-nitroso-4-methyl-piperazine and N-nitrosamine-levofloxacin.

[0042] In an embodiment of the present application, the mass concentration of 1-nitroso-piperazine in the above-mentioned reference solution is not more than 322.848 ng / mL; and / or, the mass concentration of 1-nitroso-4-methyl-piperazine in the reference solution is not more than 322.666 ng / mL; and / or, the mass concentration of N-nitrosamine-levofloxacin in the reference solution is not more than 1.224 μg / mL.

[0043] By setting the upper limit of the concentration, it helps to avoid the formation of accumulation of excessive compounds in the chromatographic column, thereby helping to improve the effectiveness of the separation process and the reliability of the detection result. And it helps to make the linear interval of the standard curve cover the concentration range that the actual sample may exist, thereby helping to improve the accuracy and applicability of the detection method.

[0044] In order to improve the accuracy of the detection result, in an embodiment of the present application, the different concentrations of the above-mentioned control solution have at least six groups; preferably, in the first group of concentrations of the control solution, the concentration of 1-nitroso-piperazine is 16.142 ng / mL, the concentration of 1-nitroso-4-methyl-piperazine is 16.133 ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.061 μg / mL; in the second group of concentrations of the control solution, the concentration of 1-nitroso-piperazine is 48.427 ng / mL, the concentration of 1-nitroso-4-methyl-piperazine is 48.400 ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.184 μg / mL; in the third group of concentrations of the control solution, the concentration of 1-nitroso-piperazine is 80.712 ng / mL, the concentration of 1-nitroso-4-methyl-piperazine is 80.667 ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.306 μg / mL; in the fourth group of concentrations of the control solution, the concentration of 1-nitroso-piperazine is 161.424 ng / mL, the concentration of 1-nitroso-4-methyl-piperazine is 161.333 ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.612 μg / mL; in the fifth group of concentrations of the control solution, the concentration of 1-nitroso-piperazine is 242.136 ng / mL, the concentration of 1-nitroso-4-methyl-piperazine is 242.000 ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.918 μg / mL; in the sixth group of concentrations of the control solution, the concentration of 1-nitroso-piperazine is 322.848 ng / mL, the concentration of 1-nitroso-4-methyl-piperazine is 322.666 ng / mL, and the concentration of N-nitrosamine-levofloxacin is 1.224 μg / mL.

[0045] The beneficial effects of the present application will be further illustrated below in combination with examples.

[0046] Example 1

[0047] Instrument: liquid chromatograph and mass spectrometer, wherein the type of the chromatographic column in the liquid chromatograph is Shim-pack GIST C18-AQ, the filler in the chromatographic column is octadecylsilane bonded silica gel, the inner diameter of the chromatographic column is 4.6 mm, the length of the chromatographic column is 150 mm, and the particle size of the filler is 3 μm;

[0048] The flow rate of the mobile phase is 0.6 mL / min, the mobile phase comprises mobile phase A and mobile phase B, the mobile phase A is 0.1% mass concentration of formic acid aqueous solution, the mobile phase B is methanol, and the gradient elution program of the liquid chromatograph is as follows: in terms of mass percentage, when 0≤t1≤4 min, 100% of the mobile phase A and 0% of the mobile phase B; when 4

[0049] The temperature of the chromatographic column in the liquid chromatograph is 30℃, the temperature of the sample tray is 15℃, the injection amount is 1 μL, and the time is t2 from the start of sample flow into the liquid chromatograph, when 2

[0050] The ion source of the mass spectrometer is an electrospray ion source, the spray voltage in the electrospray ion source is 5500 V, the atomization temperature is 500℃, the atomization gas pressure is 50 Psi, the auxiliary gas pressure is 50 Psi, the curtain gas pressure is 50 Psi, the collision gas pressure is 7 Psi, the residence time of each transition is 200 ms, and the detection mode of the mass spectrometer is multiple ion reaction monitoring in a positive ion mode.

[0051] System suitability and instrument precision determination

[0052] 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine, N-nitrosamine-levofloxacin and water are mixed to prepare a control solution, wherein the concentration of 1-nitrosopiperazine is 162 ng / mL, the concentration of 1-nitroso-4-methylpiperazine is 162 ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.61 μg / mL.

[0053] Six injections of the control solution are continuously taken, and the numbers are first, second, third, fourth, fifth and sixth in sequence, the LC-MS (liquid chromatograph-mass spectrometer) diagram is recorded, the relative standard deviation (RSD) of the peak area and the RSD of the retention time are calculated, the results are shown in Table 1, and the liquid LC-MS diagram of the first injection is shown in Figure 1The liquid phase LC-MS chart of 1-nitrosopiperazine is shown in Figure (a), the retention time is 3.442 min; the liquid phase LC-MS chart of 1-nitroso-4-methylpiperazine is shown in Figure (b), the retention time is 3.684 min; the liquid phase LC-MS chart of N-nitrosamine-levofloxacin is shown in Figure (c), the retention time is 12.245 min, the RSD of the retention time of 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine and N-nitrosamine-levofloxacin is required to be less than or equal to 1.0%, the RSD of the peak area is required to be less than or equal to 10%.

[0054] Table 1

[0055]

[0056]

[0057] As can be seen from the results in Table 1, the RSD of the retention time of 1-nitrosopiperazine in the reference solution is 0.2%, the RSD of the peak area is 3.8%; the RSD of the retention time of 1-nitroso-4-methylpiperazine is 0.3%, the RSD of the peak area is 5.1%; the RSD of the retention time of N-nitrosamine-levofloxacin is 0.1%, the RSD of the peak area is 1.5%. The results meet the requirements, indicating that the system suitability and instrument precision are good.

[0058] Specificity determination

[0059] Preparation of the blank solution: water.

[0060] Preparation of the reference solution: accurately take 1 mL of the mixed reference solution into a 10 mL volumetric flask, dilute to the mark with water, and shake well to prepare a solution containing about 162 ng, 162 ng and 0.61 μg of 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine and N-nitrosamine-levofloxacin per 1 mL.

[0061] Preparation of the test solution: accurately take 125 μL of levofloxacin eye drops into a 1 mL volumetric flask, dilute to the mark with water, and shake well.

[0062] Preparation of the spiked test solution: accurately take 125 μL of levofloxacin eye drops into a 1 mL volumetric flask, add 100 μL of the mixed reference solution, dilute to the mark with water, and shake well.

[0063] The blank solution, control solution, test solution and spiked test solution were determined, and the LC-MS diagram was recorded. The results are shown in Table 2. The retention time of 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine and N-nitrosamine-levofloxacin was investigated. The blank solution should have no interference, and the retention time of 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine and N-nitrosamine-levofloxacin in the test solution and spiked test solution should be consistent with the peak time of the control solution.

[0064] Table 2

[0065]

[0066]

[0067] As can be seen from the results in Table 2, the blank solution has no interference, the retention time of 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine and N-nitrosamine-levofloxacin in the spiked test solution is consistent with the control solution, and 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine and N-nitrosamine-levofloxacin are not detected in the test solution. The results meet the requirements, indicating that the method has good specificity.

[0068] Quantitative limit and detection limit determination

[0069] The control solution was prepared by accurately measuring 1 mL of the mixed control solution in a 10 mL volumetric flask, diluting with water to the mark, and shaking well to prepare a solution containing about 162 ng, 162 ng and 0.61 μg of 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine and N-nitrosamine-levofloxacin per 1 mL.

[0070] 1 mL of the control solution was measured into a 10 mL volumetric flask, diluted with water to the mark, and shaken well as the quantitative limit solution. Six portions were prepared in parallel, numbered as the 1st, 2nd, 3rd, 4th, 5th and 6th. The above quantitative limit solution was measured, and the LC-MS diagram was recorded. The quantitative limit concentration, RSD of retention time and RSD of peak area were calculated. The results of quantitative limit concentration are shown in Table 3, and the RSD of retention time and RSD of peak area are shown in Table 4.

[0071] 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine and N-nitrosamine-levofloxacin were all investigated by signal-to-noise ratio method, with a signal-to-noise ratio (S / N) ≥ 10; the quantitative limit level content should be below the limit; the RSD of retention time in the six quantitative limit solutions should be ≤ 2.0%, and the RSD of peak area should be ≤ 15%.

[0072] Table 3

[0073]

[0074] Table 4

[0075]

[0076]

[0077] From the results of Table 3 and Table 4, the quantification limit level concentration of 1-nitrosopiperazine is 16.142 ng / mL, equivalent to the limit concentration of 9.9%, equivalent to the content of 26.4623 μg / g in the sample, the RSD of the retention time of 6 quantification limit solutions is 0.3%, the RSD of the peak area is 5.0%, and the signal-to-noise ratio is between 140.4 and 236.6; the quantification limit level concentration of 1-nitroso-4-methylpiperazine is 16.133 ng / mL, equivalent to the limit concentration of 9.9%, equivalent to the content of 26.4475 μg / g in the sample, the RSD of the retention time of 6 quantification limit solutions is 0.3%, the RSD of the peak area is 2.6%, and the signal-to-noise ratio is between 140.2 and 171.0; the quantification limit level concentration of N-nitrosamine-levofloxacin is 0.061 μg / mL, equivalent to the limit concentration of 10.0%, equivalent to the content of 100.3475 μg / g in the sample, the RSD of the retention time of 6 quantification limit solutions is 0.1%, the RSD of the peak area is 1.4%, and the signal-to-noise ratio is between 8636.4 and 14054.7, the results meet the requirements, indicating that the method can meet the quantitative detection requirements.

[0078] Limit of detection determination

[0079] Preparation of the control solution: precisely take 1 mL of the mixed control solution into a 10 mL volumetric flask, dilute to the mark with water, and shake well; prepare a solution containing about 162 ng of 1-nitrosopiperazine, 162 ng of 1-nitroso-4-methylpiperazine, and 0.61 μg of N-nitrosamine-levofloxacin per 1 mL.

[0080] Take 50 μL of the mixed control solution into a 10 mL volumetric flask, dilute to the mark with water, and shake well as the limit of detection solution, and prepare 2 portions in parallel. Take the above limit of detection solution for determination, record the LC-MS chart, and calculate the limit of detection concentration of 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine, and N-nitrosamine-levofloxacin, and the results are shown in Table 5. The signal-to-noise ratio method is used to investigate the quantification limit of 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine, and N-nitrosamine-levofloxacin, and the signal-to-noise ratio (S / N) should be greater than or equal to 3; the limit of detection level content should be less than 50% of the quantification limit.

[0081] Table 5

[0082]

[0083] From the results of Table 5, the detection limit level concentration of 1-nitroso-piperazine is 8.071 ng / mL, equivalent to the limit of quantification concentration of 50.0%, equivalent to the content in the sample of 13.2311 μg / g, and the signal-to-noise ratio is 119.9 / 129.2; the detection limit level concentration of 1-nitroso-4-methyl-piperazine is 8.067 ng / mL, equivalent to the limit of quantification concentration of 50.0%, equivalent to the content in the sample of 13.2246 μg / g, and the signal-to-noise ratio is 78.4 / 70.3; the detection limit level concentration of N-nitrosamine-levofloxacin is 0.031 μg / mL, equivalent to the limit of quantification concentration of 50.0%, equivalent to the content in the sample of 50.1738 μg / g, and the signal-to-noise ratio is 4449.4 / 4792.2, which meets the requirements, indicating that the method can meet the detection requirements.

[0084] Linearity

[0085] Linearity refers to the degree of proportionality between the measured response value and the concentration of the measured substance in the sample within the designed range, which is generally represented by a linear regression equation. Six control solution samples with different concentrations are prepared within the range of the limit of quantification concentration to 200% of the limit concentration, and the peak areas of 1-nitroso-piperazine, 1-nitroso-4-methyl-piperazine and N-nitrosamine-levofloxacin are measured respectively. Linear regression analysis is performed with the concentration as the abscissa (X) and the peak area as the ordinate (Y). The correlation coefficient (r) of the regression equation should be greater than or equal to 0.990, and the Y-axis intercept should be within 25% of the 100% response value. Linear regression is performed on the concentrations (X) and corresponding peak areas (Y) of 1-nitroso-piperazine, 1-nitroso-4-methyl-piperazine and N-nitrosamine-levofloxacin in the above-mentioned solutions respectively, and the regression equation, the correlation coefficient r and the intercept are calculated. The results of 1-nitroso-piperazine are shown in Table 6, and the standard curve is shown in Figure 2 , the results of 1-nitroso-4-methyl-piperazine are shown in Table 7, and the standard curve is shown in Figure 3 , and the results of N-nitrosamine-levofloxacin are shown in Table 8, and the standard curve is shown in Figure 4 .

[0086] Table 6

[0087]

[0088] Table 7

[0089]

[0090] Table 8

[0091]

[0092] From the results of Table 6 to Table 8, it can be seen that 1-nitrosopiperazine has a good linear relationship between peak area and concentration in the concentration range of 16.142 ng / mL to 322.848 ng / mL (equivalent to the content of 26.4623 μg / g to 529.2590 μg / g in the sample, equivalent to 9.9% to 198.5% of the limit concentration); 1-nitroso-4-methylpiperazine has a good linear relationship between peak area and concentration in the concentration range of 16.133 ng / mL to 322.666 ng / mL (equivalent to the content of 26.4475 μg / g to 528.9607 μg / g in the sample, equivalent to 9.9% to 198.4% of the limit concentration); N-nitrosamine-levofloxacin has a good linear relationship between peak area and concentration in the concentration range of 0.061 μg / mL to 1.224 μg / mL (equivalent to the content of 100.0000 μg / g to 2006.5574 μg / g in the sample, equivalent to 10.0% to 200.7% of the limit concentration).

[0093] Repeatability determination

[0094] Repeatability refers to the precision of the results obtained by the same analyst under the same conditions. Prepare a 100% level of spiked test solution (precisely take 125 μL of levofloxacin eye drops into a 1 mL volumetric flask, add 100 μL of mixed control solution, dilute to the mark with water, shake well), prepare 6 parallel samples, numbered S-100%-1, S-100%-2, S-100%-3, S-100%-4, S-100%-5, S-100%-6, determine under the same conditions as possible, calculate the content of 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine and N-nitrosamine-levofloxacin, the results are shown in Table 9, the measured amount of each component is required to have RSD≤15%, and the recovery rate is between 80% and 115%.

[0095] Table 9

[0096]

[0097] From the results of Table 9, it can be seen that in the 6 spiked test solutions prepared by the same analyst, the RSD of the measured amount of 1-nitrosopiperazine is 2.0%, and the recovery rate is between 88.9% and 93.5%; the RSD of the measured amount of 1-nitroso-4-methylpiperazine is 2.6%, and the recovery rate is between 95.4% and 102.5%; the RSD of the measured amount of N-nitrosamine-levofloxacin is 0.7%, and the recovery rate is between 100.1% and 101.9%, which meets the requirements, indicating that the method has good repeatability.

[0098] Accuracy determination

[0099] The spiked sample solutions of the limit of quantification and 50%, 100%, 150% limit concentrations were prepared (125 μL of levofloxacin eye drops was accurately measured into a 1 mL flask, 100 μL of the mixed control solution was added, and water was added to constant volume, and shaken well), 3 samples for each level, and the content was determined, and the samples were numbered as S-LOQ-1, S-LOQ-2, S-LOQ-3, S-50%-1, S-50%-2, S-50%-3, S-100%-1, S-100%-2, S-100%-3, S-150%-1, S-150%-2, S-150%-3, and the measured values were compared with the theoretical values, and the recovery rate was calculated, and the results are shown in Table 10. The recovery rate at the limit of quantification level is required to be between 70% and 125%, and the recovery rate RSD is ≤15%; the recovery rate at the 50%-150% level is required to be between 80% and 115%, and the recovery rate RSD and the total recovery rate RSD are ≤10%.

[0100] Table 10

[0101]

[0102]

[0103] From the results in Table 10, the LOQ level recovery rate of 1-nitroso-piperazine was between 92.8% and 110.7%, the recovery rate RSD was 8.8%, the 50%-150% level recovery rate was between 84.6% and 100.0%, the recovery rate RSD of each level was ≤6.6%, and the total recovery rate RSD was 4.8%; the LOQ level recovery rate of 1-nitroso-4-methyl-piperazine was between 93.8% and 104.8%, the recovery rate RSD was 5.7%, the 50%-150% level recovery rate was between 92.8% and 105.1%, the recovery rate RSD of each level was ≤3.8%, and the total recovery rate RSD was 3.7%; the LOQ level recovery rate of N-nitrosamine-levofloxacin was between 103.7% and 106.8%, the recovery rate RSD was 1.6%, the 50%-150% level recovery rate was between 99.6% and 104.7%, the recovery rate RSD of each level was ≤1.4%, and the total recovery rate RSD was 1.7%, which met the requirements, indicating that the method has good accuracy.

[0104] Solution stability determination

[0105] ​The solution was observed over time to provide a basis for the storage period of each solution during detection. Each solution was detected at different times after preparation, wherein the control solution was prepared by accurately measuring 1 mL of the mixed control solution into a 10 mL volumetric flask, diluting to the mark with water, and shaking well; 1 mL of the solution contained about 162 ng of 1-nitrosopiperazine, 162 ng of 1-nitroso-4-methylpiperazine, and 0.61 μg of N-nitrosamine-levofloxacin; the spiked sample solution was prepared by accurately measuring 125 μL of levofloxacin eye drops into a 1 mL volumetric flask, adding 100 μL of the mixed control solution, diluting to the mark with water, and shaking well. The peak area change was observed, and the results are shown in Table 11. The peak area ratio at different time points to that at 0 h was required to be between 80% and 115%.

[0106] Table 11

[0107]

[0108]

[0109] As can be seen from the results in Table 11, the control solution was stored at 10°C for 24.47 h, and the peak area ratio of each substance to that at 0 h was between 100.0% and 109.4%; the spiked sample solution was stored at 10°C for 25.24 h, and the peak area ratio of each substance to that at 0 h was between 95.8% and 103.3%. The results met the requirements, indicating that the control solution was stable when stored at 10°C for 24.47 h, and the spiked sample solution was stable when stored at 10°C for 25.24 h.

[0110] Sample detection

[0111] Three batches of levofloxacin eye drops were detected, numbered as levofloxacin eye drops (P1), levofloxacin eye drops (P2), and levofloxacin eye drops (P3), and the results are shown in Table 12 below.

[0112] Table 12

[0113]

[0114] As can be seen from the results in Table 12, the three batches of levofloxacin eye drops did not contain 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine, and N-nitrosamine-levofloxacin.

[0115] Example 1 is a methodology investigation, according to the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" of Chinese Pharmacopoeia 2020 Edition Volume IV 9101 and "Mass Spectrometry" of Chinese Pharmacopoeia 2020 Edition Volume IV 0431, the inspection and analysis method of 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine and N-nitrosamine-levofloxacin in levofloxacin eye drops is verified. After the system applicability and instrument precision, specificity, limit of quantification and detection limit, linearity, repeatability, range, accuracy and intermediate precision test, all parameters are within the acceptable range; the solution stability test shows that the control solution is stable at 10°C for 24.47h, and the spiked test sample solution is stable at 10°C for 25.24h. The above results show that this method can be used for the inspection of 1-nitrosopiperazine, 1-nitroso-4-methylpiperazine and N-nitrosamine-levofloxacin in levofloxacin eye drops.

[0116] Example 2

[0117] The difference from Example 1 is that the inner diameter of the chromatographic column in liquid chromatography is 1.0mm, the length of the chromatographic column in liquid chromatography is 200mm, and the particle size of the chromatographic column filler in liquid chromatography is 1.7μm.

[0118] Prepare the spiked test sample solution with the limit of quantification concentration of 50%, 100%, 150% limit concentration, 3 for each level, respectively, determine the content, numbered as S-LOQ-1, S-LOQ-2, S-LOQ-3, S-50%-1, S-50%-2, S-50%-3, S-100%-1, S-100%-2, S-100%-3, S-150%-1, S-150%-2, S-150%-3, compare the measured value with the theoretical value, calculate the recovery rate, the results are shown in Table 13.

[0119] Table 13

[0120]

[0121]

[0122] From the results of Table 13, it can be seen that the LOQ level recovery of 1-nitrosopiperazine was between 95% and 105%, the recovery RSD was 0.07%, the 50% to 150% level recoveries were all between 95% and 105%, the RSD of each level recovery was all ≤0.20%, and the total recovery RSD was 0.10%; the LOQ level recovery of 1-nitroso-4-methylpiperazine was between 95% and 105%, the recovery RSD was 0.65%, the 50% to 150% level recoveries were all between 95% and 105%, the RSD of each level recovery was all ≤0.1%, and the total recovery RSD was 0.13%; the LOQ level recovery of N-nitrosamine-levofloxacin was between 95% and 105%, the recovery RSD was 0.50%, the 50% to 150% level recoveries were all between 95% and 105%, the RSD of each level recovery was all ≤2.0%, and the total recovery RSD was 1.54%. From the recoveries and the RSD of the recoveries, the effects of Example 1 and Example 2 were similar.

[0123] Example 3

[0124] The difference from Example 1 was that the inner diameter of the chromatographic column in the liquid chromatograph was 5.0 mm, the length of the chromatographic column in the liquid chromatograph was 300 mm, and the particle size of the packing material of the chromatographic column in the liquid chromatograph was 4.0 μm.

[0125] The limit concentration of the limit concentration of the spiked sample solution was prepared to be 50%, 100%, and 150%, each level was 3 copies, and the content was determined, numbered S-LOQ-1, S-LOQ-2, S-LOQ-3, S-50%-1, S-50%-2, S-50%-3, S-100%-1, S-100%-2, S-100%-3, S-150%-1, S-150%-2, S-150%-3, and the measured value was compared with the theoretical value, the recovery was calculated, and the results are shown in Table 14.

[0126] Table 14

[0127]

[0128]

[0129] From the results of Table 14, it can be seen that the LOQ level recovery rate of 1-nitrosopiperazine was between 92% and 103%, the recovery rate RSD was 5.85%, the 50%-150% level recovery rate was between 95% and 105%, the RSD of each level recovery rate was ≤1.5%, and the total recovery rate RSD was 1.46%; the LOQ level recovery rate of 1-nitroso-4-methylpiperazine was between 80% and 110%, the recovery rate RSD was 13.57%, the 50%-150% level recovery rate was between 95% and 105%, the RSD of each level recovery rate was ≤2.0%, and the total recovery rate RSD was 1.58%; the LOQ level recovery rate of N-nitrosamine-levofloxacin was between 60% and 105%, the recovery rate RSD was 14.14%, the 50%-150% level recovery rate was between 60% and 100%, the RSD of each level recovery rate was ≤15%, and the total recovery rate RSD was 10.30%. From the recovery rate and the RSD of the recovery rate, it can be seen that the effect of Example 1 was obviously better than that of Example 2.

[0130] Example 4

[0131] The difference from Example 1 was that the gradient elution program of the liquid chromatograph was as follows: when the time of gradient elution was t1, 100% of mobile phase A and 0% of mobile phase B when 0≤t1≤5 min; 10% of mobile phase A and 90% of mobile phase B when 5

[0132] The limit concentration of the spiked sample solution was prepared to be 50%, 100%, and 150%, and each level had 3 replicates. The content of each was determined, and the numbers were S-LOQ-1, S-LOQ-2, S-LOQ-3, S-50%-1, S-50%-2, S-50%-3, S-100%-1, S-100%-2, S-100%-3, S-150%-1, S-150%-2, and S-150%-3. The measured value was compared with the theoretical value, the recovery rate was calculated, and the results are shown in Table 15.

[0133] Table 15

[0134]

[0135]

[0136] From the results of Table 15, it can be seen that the LOQ level recovery of 1-nitrosopiperazine was between 95% and 105%, the recovery RSD was 0.59%, the 50% to 150% level recoveries were all between 95% and 105%, the RSD of each level recovery was all ≤1.5%, and the total recovery RSD was 0.95%; the LOQ level recovery of 1-nitroso-4-methylpiperazine was between 95% and 105%, the recovery RSD was 13.57%, the 50% to 150% level recoveries were all between 95% and 105%, the RSD of each level recovery was all ≤2.0%, and the total recovery RSD was 1.51%; the LOQ level recovery of N-nitrosamine-levofloxacin was between 95% and 105%, the recovery RSD was 1.89%, the 50% to 150% level recoveries were all between 95% and 105%, the RSD of each level recovery was all ≤2.0%, and the total recovery RSD was 1.54%. From the recovery and the RSD of the recovery, the effect of Example 4 was worse than that of Example 1.

[0137] Example 5

[0138] The difference from Example 1 was that the temperature of the chromatographic column in the liquid chromatograph was 40°C, and the temperature of the sample tray in the liquid chromatograph was 15°C.

[0139] The limit of quantification concentration was 50%, 100%, and 150%, and the spiked sample solution was prepared, 3 portions for each level, and the content was determined, numbered S-LOQ-1, S-LOQ-2, S-LOQ-3, S-50%-1, S-50%-2, S-50%-3, S-100%-1, S-100%-2, S-100%-3, S-150%-1, S-150%-2, S-150%-3. The measured value was compared with the theoretical value, the recovery was calculated, and the results were shown in Table 16.

[0140] Table 16

[0141]

[0142]

[0143] From the results of Table 16, the LOQ level recovery of 1-nitrosopiperazine was between 95% and 105%, the recovery RSD was 2.68%, the 50% to 150% level recoveries were all between 95% and 105%, the RSD of each level recovery was all ≤0.5%, and the total recovery RSD was 0.18%; the LOQ level recovery of 1-nitroso-4-methylpiperazine was between 95% and 105%, the recovery RSD was 3.04%, the 50% to 150% level recoveries were all between 95% and 105%, the RSD of each level recovery was all ≤0.6%, and the total recovery RSD was 0.37%; the LOQ level recovery of N-nitrosamine-levofloxacin was between 95% and 105%, the recovery RSD was 0.34%, the 50% to 150% level recoveries were all between 95% and 105%, the RSD of each level recovery was all ≤0.9%, and the total recovery RSD was 0.84%. From the recovery and the RSD of the recovery, the effect of Example 5 was not much different from that of Example 1.

[0144] Example 6

[0145] The difference from Example 1 was that the temperature of the chromatographic column in the liquid chromatography was 25°C, and the temperature of the sample tray in the liquid chromatography was 35°C.

[0146] The limit concentration of the limit of quantification was 50%, 100%, and 150%, and the spiked sample solution was prepared, 3 portions for each level, and the content was determined, numbered S-LOQ-1, S-LOQ-2, S-LOQ-3, S-50%-1, S-50%-2, S-50%-3, S-100%-1, S-100%-2, S-100%-3, S-150%-1, S-150%-2, S-150%-3. The measured value was compared with the theoretical value, the recovery was calculated, and the results were shown in Table 17.

[0147] Table 17

[0148]

[0149]

[0150] From the results of Table 17, it can be seen that the LOQ level recovery of 1-nitrosopiperazine was between 95% and 115%, the recovery RSD was 3.21%, the 50% to 150% level recoveries were all between 95% and 105%, the RSD of each level recovery was all ≤3.0%, and the total recovery RSD was 1.28%; the LOQ level recovery of 1-nitroso-4-methylpiperazine was between 95% and 105%, the recovery RSD was 3.87%, the 50% to 150% level recoveries were all between 95% and 105%, the RSD of each level recovery was all ≤3.0%, and the total recovery RSD was 1.54%; the LOQ level recovery of N-nitrosamine-levofloxacin was between 80% and 120%, the recovery RSD was 16.63%, the 50% to 150% level recoveries were all between 80% and 120%, the RSD of each level recovery was all ≤7.0%, and the total recovery RSD was 5.03%. From the recoveries and the RSD of the recoveries, the effect of Example 6 was worse than that of Example 1.

[0151] Example 7

[0152] The difference from Example 1 was that, with the sample inflow liquid chromatography starting timing, the time was t2, when 1<t2≤7min and 12<t2≤15min, the sample flow channel in the liquid chromatography was connected with the mass spectrometer, when 0≤t2≤1min, 7<t2≤12min and 15<t2≤25min, the sample flow channel in the liquid chromatography was connected with the waste liquid pipeline.

[0153] The limit concentration of the standard addition sample solution was prepared to be 50%, 100%, and 150%, and each level had 3 portions, and the content was determined, and the numbers were S-LOQ-1, S-LOQ-2, S-LOQ-3, S-50%-1, S-50%-2, S-50%-3, S-100%-1, S-100%-2, S-100%-3, S-150%-1, S-150%-2, S-150%-3. The measured value was compared with the theoretical value, the recovery was calculated, and the results were shown in Table 18.

[0154] Table 18

[0155]

[0156]

[0157] From the results of Table 18, it can be seen that the LOQ level recovery rate of 1-nitrosopiperazine was between 80% and 120%, the recovery rate RSD was 13.19%, the 50% to 150% level recovery rate was all between 80% and 120%, the RSD of each level recovery rate was all ≤7.0%, and the total recovery rate RSD was 4.44%; the LOQ level recovery rate of 1-nitroso-4-methylpiperazine was between 80% and 120%, the recovery rate RSD was 12.94%, the 50% to 150% level recovery rate was all between 80% and 120%, the RSD of each level recovery rate was all ≤5.0%, and the total recovery rate RSD was 4.13%; the LOQ level recovery rate of N-nitrosamine-levofloxacin was between 80% and 120%, the recovery rate RSD was 1.56%, the 50% to 150% level recovery rate was all between 80% and 120%, the RSD of each level recovery rate was all ≤2.0%, and the total recovery rate RSD was 2.44%. From the recovery rate and the RSD of the recovery rate, the effect of Example 7 was worse than that of Example 1.

[0158] Example 8

[0159] The difference from Example 1 was that the spray voltage in the electrospray ion source was 6000V, the atomization temperature was 400℃, the atomization gas pressure was 55Psi, the auxiliary gas pressure was 60Psi, the curtain gas pressure was 50Psi, the collision gas pressure was 9Psi, and the residence time of each transition was 260ms.

[0160] Prepare the spiked test sample solution with the limit concentration of 50%, 100%, and 150% of the limit concentration, 3 copies for each level, respectively, and determine the content of each, numbered S-LOQ-1, S-LOQ-2, S-LOQ-3, S-50%-1, S-50%-2, S-50%-3, S-100%-1, S-100%-2, S-100%-3, S-150%-1, S-150%-2, S-150%-3. Compare the measured value with the theoretical value, calculate the recovery rate, and the results are shown in Table 19.

[0161] Table 19

[0162]

[0163]

[0164] From the results of Table 19, it can be seen that the LOQ level recovery rate of 1-nitrosopiperazine was between 80% and 120%, the recovery rate RSD was 10.70%, the 50% to 150% level recovery rate was all between 80% and 120%, the RSD of each level recovery rate was all ≤7.0%, and the total recovery rate RSD was 4.83%; the LOQ level recovery rate of 1-nitroso-4-methylpiperazine was between 80% and 120%, the recovery rate RSD was 4.87%, the 50% to 150% level recovery rate was all between 80% and 120%, the RSD of each level recovery rate was all ≤4.0%, and the total recovery rate RSD was 3.61%; the LOQ level recovery rate of N-nitrosamine-levofloxacin was between 80% and 120%, the recovery rate RSD was 1.54%, the 50% to 150% level recovery rate was all between 80% and 120%, the RSD of each level recovery rate was all ≤2.0%, and the total recovery rate RSD was 2.21%; from the recovery rate and the RSD of the recovery rate, the effect of Example 8 was similar to that of Example 1.

[0165] Example 9

[0166] The difference from Example 1 was that the spray voltage in the electrospray ion source was 5300V, the atomization temperature was 520°C, the atomization gas pressure was 58Psi, the auxiliary gas pressure was 45Psi, the curtain gas pressure was 48Psi, the collision gas pressure was 10Psi, and the residence time for each transition was 190ms.

[0167] Prepare the spiked sample solution with the limit concentration of 50%, 100%, and 150% of the limit concentration, 3 portions for each level, respectively, and determine the content of each, numbered S-LOQ-1, S-LOQ-2, S-LOQ-3, S-50%-1, S-50%-2, S-50%-3, S-100%-1, S-100%-2, S-100%-3, S-150%-1, S-150%-2, S-150%-3. Compare the measured value with the theoretical value, calculate the recovery rate, and the results are shown in Table 20.

[0168] Table 20

[0169]

[0170]

[0171] From the results of Table 20, it can be seen that the LOQ level recovery rate of 1-nitrosopiperazine is between 80% and 120%, the recovery rate RSD is 13.12%, the 50% to 150% level recovery rate is all between 80% and 120%, the RSD of each level recovery rate is all ≤7.0%, and the total recovery rate RSD is 4.44%; the LOQ level recovery rate of 1-nitroso-4-methylpiperazine is between 80% and 120%, the recovery rate RSD is 12.17%, the 50% to 150% level recovery rate is all between 80% and 120%, the RSD of each level recovery rate is all ≤5.0%, and the total recovery rate RSD is 4.15%; the LOQ level recovery rate of N-nitrosamine-levofloxacin is between 80% and 120%, the recovery rate RSD is 1.12%, the 50% to 150% level recovery rate is all between 80% and 120%, the RSD of each level recovery rate is all ≤5.0%, and the total recovery rate RSD is 4.35%. From the recovery rate and the RSD of the recovery rate, the effect of Example 9 is worse than that of Example 1.

[0172] Example 10

[0173] The difference from Example 1 is that the instrument is a liquid chromatograph and mass spectrometer combination, wherein the filler in the chromatographic column of the liquid chromatograph is octadecylsilane bonded silica gel, the inner diameter of the chromatographic column is 2.3 mm, the length is 150 mm, and the particle size of the filler is 1.7 μm.

[0174] The flow rate of the mobile phase is 1 mL / min, the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is 0.5% mass concentration of formic acid aqueous solution, the mobile phase B is methanol, and the gradient elution program of the liquid chromatograph is that, in terms of mass percentage, the time of gradient elution is t1, when 0≤t1≤4 min, 100% of the mobile phase A and 0% of the mobile phase B; when 4

[0175] The temperature of the chromatographic column in the liquid chromatograph is 30℃, the temperature of the sample disc is 10℃, the injection amount is 5 μL, and the timing starts when the sample flows into the liquid chromatograph, the time is t2, when 2

[0176] The ion source of the mass spectrometer is an electrospray ion source, the spray voltage of the electrospray ion source is 5500 V, the atomization temperature is 500 DEG C, the atomization gas pressure is 50 Psi, the auxiliary gas pressure is 50 Psi, the gas curtain gas pressure is 50 Psi, the collision gas pressure is 7 Psi, the residence time of each transition is 200 ms, and the detection mode of the mass spectrometer is multiple ion reaction monitoring in a positive ion mode.

[0177] The limit of quantification concentration of 50%, 100%, 150% of the spiked sample solution was prepared, 3 copies of each level, and the content was determined, numbered as S-LOQ-1, S-LOQ-2, S-LOQ-3, S-50%-1, S-50%-2, S-50%-3, S-100%-1, S-100%-2, S-100%-3, S-150%-1, S-150%-2, S-150%-3, the measured value was compared with the theoretical value, and the recovery rate was calculated, and the results are shown in Table 21.

[0178] Table 21

[0179]

[0180]

[0181] It can be seen from the results in Table 21 that the LOQ level recovery rate of 1-nitrosopiperazine is between 80% and 120%, the recovery rate RSD is 8.76%, the recovery rate of 50% to 150% is between 80% and 120%, the recovery rate RSD of each level is ≤7.0%, and the total recovery rate RSD is 4.86%; the LOQ level recovery rate of 1-nitroso-4-methylpiperazine is between 80% and 120%, the recovery rate RSD is 12.94%, the recovery rate of 50% to 150% is between 80% and 120%, the recovery rate RSD of each level is ≤5.0%, and the total recovery rate RSD is 4.13%; the LOQ level recovery rate of N-nitrosamine-levofloxacin is between 80% and 120%, the recovery rate RSD is 1.56%, the recovery rate of 50% to 150% is between 80% and 120%, the recovery rate RSD of each level is ≤2.0%, and the total recovery rate RSD is 2.44%, from the recovery rate and the RSD of the recovery rate, the effect of Example 10 is similar to that of Example 1.

[0182] From the above description, it can be seen that the above-mentioned examples of the present application achieve the following technical effects:

[0183] The measuring instrument used in the present application is a liquid chromatograph and mass spectrometer, the liquid chromatograph can better separate the components in the levofloxacin eye drops, so that the mass spectrometer can detect the components in the levofloxacin eye drops, so that the structure of each component in the levofloxacin eye drops can be obtained. The external standard method is used, a plurality of concentration gradient control solution is prepared for detection, so that the linear equation of the peak area and the concentration is obtained, the result of the levofloxacin eye drops after measurement is substituted into the linear equation, and the content of the piperazine new psychoactive substance in the levofloxacin eye drops can be obtained. The detection method of the present application can accurately and quantitatively analyze the piperazine new psychoactive substance in the levofloxacin eye drops, the analysis time is short, the sample amount is small, the detection method has high sensitivity, good specificity and repeatability, is accurate and reliable, and the detection method of the present application has low economic cost, is simple and efficient, is less affected by external environmental factors, can be realized in different laboratories, different liquid chromatographs and mass spectrometers, different analysis test personnel and analysis environmental conditions, has good durability and good applicability.

[0184] The above is only an embodiment of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting a piperazine-type new psychoactive substance in a levofloxacin eye drop, characterized by, The detection method comprises: S1, mixing piperazine new psychoactive substances and diluents to prepare mixed solutions with different concentrations as control solutions; S2, detecting each control solution by using a liquid chromatograph and mass spectrometer, and performing regression analysis on the peak area of the spectrum of each control solution and its concentration to obtain a standard curve; S3, under the same test conditions as in S2, detecting the levofloxacin eye drops by using the liquid chromatograph and mass spectrometer to obtain the peak area detection data of the spectrum of the piperazine new psychoactive substances in the levofloxacin eye drops; and substituting the detection data into the standard curve to obtain the concentration of the piperazine new psychoactive substances in the levofloxacin eye drops.

2. The detection method according to claim 1, characterized in that, The packing material of the chromatographic column in the liquid chromatograph is octadecylsilane-bonded silica gel; and / or, the inner diameter of the chromatographic column in the liquid chromatograph is 1.0-4.6 mm; and / or, the length of the chromatographic column in the liquid chromatograph is 150-250 mm; and / or, the particle size of the packing material of the chromatographic column in the liquid chromatograph is 1.7-3.5 μm.

3. The detection method according to claim 1 or 2, characterized in that, The flow rate of the mobile phase in the liquid chromatograph is 0.6-1.0 mL / min; preferably, the mobile phase comprises mobile phase A and mobile phase B; In the gradient elution program of the liquid chromatograph, the ratio of the mobile phase A and the mobile phase B is as follows: when 0≤t1≤4 min, 100% of the mobile phase A and 0% of the mobile phase B; when 4 4. The detection method according to claim 3, characterized in that, The mobile phase A is selected from aqueous formic acid and / or aqueous acetic acid; and / or, the mobile phase B is selected from methanol and / or acetonitrile; Preferably, the mass concentration of the aqueous formic acid is 0.05%-0.5%.

5. The detection method according to any one of claims 1 to 4, characterized in that, The temperature of the chromatographic column in the liquid chromatograph is 30-40℃; and / or, the temperature of the sample tray in the liquid chromatograph is 15-30℃; and / or, the sample injection volume in the liquid chromatograph is 1-20 μL; And / or, the sample flow channel in the liquid chromatograph is connected to the mass spectrometer when 2 6. The detection method according to any one of claims 1 to 5, characterized in that, The ion source of the mass spectrometer is an electrospray ion source; the spray voltage of the electrospray ion source is 5500-6000V, the atomization temperature is 400-500℃, the atomization gas pressure is 50-55Psi, the auxiliary gas pressure is 50-60Psi, the gas curtain gas pressure is 50-60Psi, the collision gas pressure is 7-9Psi, and the conversion residence time is 200-260ms.

7. The detection method according to any one of claims 1 to 6, characterized in that, The monitoring mode of the mass spectrometer is multiple ion reaction monitoring in positive ion mode.

8. The detection method according to any one of claims 1 to 7, characterized in that, The piperazine new psychoactive substances include 1-nitroso piperazine, 1-nitroso-4-methyl piperazine and N-nitrosamine-levofloxacin.

9. The detection method according to claim 8, characterized in that, The mass concentration of 1-nitroso piperazine in the control solution is not more than 322.848ng / mL; and / or, the mass concentration of 1-nitroso-4-methyl piperazine in the control solution is not more than 322.666ng / mL; and / or, the mass concentration of N-nitrosamine-levofloxacin in the control solution is not more than 1.224μg / mL.

10. The detection method according to claim 9, characterized in that, There are at least six groups of different concentrations of the control solution; preferably, the concentration of 1-nitroso piperazine in the first group of concentrations of the control solution is 16.142ng / mL, the concentration of 1-nitroso-4-methyl piperazine is 16.133ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.061μg / mL; the concentration of 1-nitroso piperazine in the second group of concentrations of the control solution is 48.427ng / mL, the concentration of 1-nitroso-4-methyl piperazine is 48.400ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.184μg / mL; the concentration of 1-nitroso piperazine in the third group of concentrations of the control solution is 80.712ng / mL, the concentration of 1-nitroso-4-methyl piperazine is 80.667ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.306μg / mL; the concentration of 1-nitroso piperazine in the fourth group of concentrations of the control solution is 161.424ng / mL, the concentration of 1-nitroso-4-methyl piperazine is 161.333ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.612μg / mL; the concentration of 1-nitroso piperazine in the fifth group of concentrations of the control solution is 242.136ng / mL, the concentration of 1-nitroso-4-methyl piperazine is 242.000ng / mL, and the concentration of N-nitrosamine-levofloxacin is 0.918μg / mL; the concentration of 1-nitroso piperazine in the sixth group of concentrations of the control solution is 322.848ng / mL, the concentration of 1-nitroso-4-methyl piperazine is 322.666ng / mL, and the concentration of N-nitrosamine-levofloxacin is 1.224μg / mL.

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