A method for simultaneously determining 18 illegally added antiviral drugs

Abstract

The present invention belongs to the technical field of analysis and detection, and specifically relates to a method for simultaneously determining 18 kinds of illegally added antiviral drugs. The method uses high performance liquid chromatography-tandem mass spectrometry to separate and determine the sample to be tested after ultrasonic treatment with a methanol-acetonitrile solution. It has high accuracy, stability and sensitivity, and can be qualitative and quantitative at the same time; and it does not require complex sample pretreatment of the sample, the instrument use cost is low, and the analysis speed is fast. The analysis and detection of 18 antiviral drugs with significant structural differences in a sample can be completed within 20 minutes, which greatly shortens the analysis time and greatly improves the detection efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and more specifically, relates to a method for simultaneously determining 18 kinds of illegally added antiviral drugs. Background Art

[0002] As a traditional beverage deeply loved by the people of Lingnan, herbal tea was selected as one of the first batch of national intangible cultural heritage recognized by the State Council in 2006 and is one of the representatives of Guangdong traditional Chinese medicine culture. However, there are constant reports of illegal addition of chemical drugs in herbal tea and Chinese medicine preparations, and the number and types of illegal additions are increasing. If taken with other drugs without knowing it, it is easy to aggravate adverse drug reactions or cause drug interactions, which poses a great safety hazard. In recent years, under the influence of viral infection, the illegal addition of antiviral drugs in herbal tea and Chinese medicine preparations has spread rapidly. However, the current research focuses on the detection of illegal addition of antipyretic analgesics, antihistamines, antitussives and expectorants, antibacterials, and glucocorticoids in herbal tea and Chinese medicine preparations. For example, Wang Jing disclosed an LC-MS / MS detection method for the illegal addition of 18 antipyretic analgesics and adrenocortical hormones in herbal tea (Wang Jing. Research on LC-MS / MS detection method of 18 illegal chemical drugs added to herbal tea [D]. Guangxi University of Chinese Medicine, 2013.). Therefore, there is still a lack of detection methods for the illegal addition of antiviral drugs into herbal teas and Chinese medicine preparations.

[0003] According to chemical structure, antiviral drugs can be mainly divided into open-ring nucleosides (such as acyclovir, famciclovir, ganciclovir, etc.), non-open-ring nucleosides (such as ribavirin, etc.), tricyclic amines (such as amantadine, rimantadine, etc.), peptide analogs (such as ritonavir), neuraminic acid analogs (such as oseltamivir, zanamivir, peramivir, etc.). It can be seen that there are many types of antiviral drugs, with great structural differences, and the hydrophilicity and polarity of the compounds are widely distributed. It is difficult to achieve simultaneous retention and separation of multiple types of drugs for detection in a common reversed-phase chromatography system. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art, such as the lack of a detection method for antiviral drugs illegally added to herbal teas and traditional Chinese medicine preparations, the large variety of antiviral compounds and their large structural differences, and the difficulty in achieving simultaneous retention and separation of multiple types of drugs in a chromatographic system. A method for simultaneously determining 18 types of illegally added antiviral drugs in herbal teas and traditional Chinese medicine preparations is provided.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] A method for simultaneously determining 18 illegally added antiviral drugs using high performance liquid chromatography-tandem mass spectrometry, specifically comprising the following steps:

[0007] S1. Preparation of standard solution: Dissolve each standard substance with a solvent, make up to volume, mix and dilute to form a mixed standard solution, adjust the concentration according to needs, store and wait for testing;

[0008] S2. Preparation of test solution: Take the sample to be tested and use the methanol-acetonitrile solution for ultrasonic extraction, pass through the membrane, and wait for testing;

[0009] S3, the high performance liquid chromatography: non-bonded silica gel type HILIC column, column temperature 35 ~ 40 ℃, flow rate 0.2 ~ 0.4mL / min; containing 0.1% acetic acid in 10mmol ammonium acetate solution as mobile phase A, 0.1% formic acid acetonitrile as mobile phase B, according to the following elution program for gradient elution: 0 ~ 0.5min, 3% mobile phase A; 0.5 ~ 2.0min, 3 ~ 5% mobile phase A; 2.0 ~ 4.0min, 5 ~ 10% mobile phase A; 4.0 ~ 9.0min, 10 ~ 20% mobile phase A; 9.0 ~ 14.0min, 20% mobile phase A; 14.0 ~ 14.1min, 20 ~ 3% mobile phase A; 14.1 ~ 18.0min, 3% mobile phase A;

[0010] S4. The mass spectrometry conditions are as follows: the ion source is an electrospray ion source; the electrospray voltage is 5000-5500 V for positive ion mode detection; the ion source temperature is 500-550° C.; the nebulizing gas pressure is 45-55 psi; the curtain gas pressure is 35-45 psi; the collision gas pressure is 8-10 psi, and multiple reaction monitoring is adopted.

[0011] Furthermore, the 18 illegally added antiviral drugs are morpholino hydrochloride, amantadine hydrochloride, rimantadine hydrochloride, penciclovir, oseltamivir phosphate, peramivir trihydrate, zanamivir, ribavirin, arbidol, favipiravir, mabaloxavir, lopinavir, ritonavir, laninamivir, acyclovir, ganciclovir, famciclovir, and valacyclovir hydrochloride.

[0012] Furthermore, in step S1, when the standard substance is morpholinoguanidine hydrochloride, amantadine hydrochloride, rimantadine hydrochloride, oseltamivir phosphate, peramivir trihydrate, ribavirin, arbidol, favipiravir, mabaloxavir, lopinavir, famciclovir, or valacyclovir hydrochloride, the reagent is methanol; when the standard substance is ritonavir, penciclovir, acyclovir, or ganciclovir, it is first dissolved in dimethyl sulfoxide and then fixed to volume with methanol; when the standard substance is zanamivir or laninamivir, the solvent is water.

[0013] Furthermore, the limit of quantification of amantadine and famciclovir is 0.0125 mg / kg; the limit of quantification of rimantadine and mabaloxavir is 0.025 mg / kg; the limit of quantification of morphine is 0.05 mg / kg; the limit of quantification of ribavirin and oseltamivir is 0.125 mg / kg; the limit of quantification of acyclovir and valacyclovir is 0.25 mg / kg; the limit of quantification of ganciclovir, penciclovir, arbidol, peramivir and lopinavir is 0.5 mg / kg; the limit of quantification of favipiravir, ritonavir, laninamivir and zanamivir is 5.0 mg / kg.

[0014] Furthermore, the detection limit of favipiravir, ritonavir, laninamivir and zanamivir is 2.0 mg / kg; the detection limit of rimantadine and mabaloxavir is 0.01 mg / kg; the detection limit of ganciclovir, penciclovir, arbidol, peramivir and lopinavir is 0.2 mg / kg; the detection limit of ribavirin and oseltamivir is 0.05 mg / kg; the detection limit of amantadine and famciclovir is 0.005 mg / kg; the detection limit of acyclovir and valacyclovir is 0.1 mg / kg; the detection limit of morphine is 0.02 mg / kg.

[0015] Furthermore, in step S2, the sample to be tested is herbal tea or a Chinese medicine preparation. Preferably, the Chinese medicine preparation is a tablet, injection, capsule, oral liquid, granule or other preparation made with Chinese medicine as the main active ingredient, such as cold medicine, antiviral medicine, heat-clearing and detoxifying medicine, etc.

[0016] Furthermore, in step S2, the volume ratio of methanol to acetonitrile in the methanol-acetonitrile solution is (20-30):(70-80).

[0017] Furthermore, in step S2, the ultrasonic extraction time is 25 to 35 minutes.

[0018] Furthermore, in step S3, the non-bonded silica gel type HILIC column is a Waters ACQUITY BEH HILIC column or an Agilent RRHD HILIC Plus column. Preferably, the specifications of the Waters ACQUITY BEH HILIC column are 2.1 mm × 100 mm, 1.7 μm; the specifications of the Agilent RRHD HILIC Plus column are 2.1 mm × 100 mm, 1.8 μm. It was found in the experiment that the retention and separation effects of the non-bonded silica gel type HILIC column are better.

[0019] Furthermore, the absolute difference between two independent determination results in the repeatability verification of the method shall not exceed 15% of the arithmetic mean.

[0020] Furthermore, the average recovery rate of the standard product is 70-115%.

[0021] The present invention has the following beneficial effects:

[0022] The present invention provides a method for simultaneously determining 18 kinds of illegally added antiviral drugs. After the sample to be tested is ultrasonically treated with a methanol-acetonitrile solution, it is separated and determined by a high performance liquid chromatography-tandem mass spectrometry method. The method has high accuracy, stability and sensitivity, and can simultaneously perform qualitative and quantitative analysis. Moreover, it does not require complex sample pretreatment, the instrument use cost is low, and the analysis speed is fast. The analysis and detection of 18 antiviral drugs with significant structural differences in one sample can be completed within 20 minutes, which greatly shortens the analysis time and greatly improves the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The liquid chromatography-tandem mass spectrometry chromatogram of the mixed standard working solution in Example 1 is shown.

[0024] Figure 2 The chromatograms of ribavirin, acyclovir, ganciclovir and penciclovir when detected with different mobile phases in Example 2 are shown.

[0025] Figure 3 The chromatograms of mabaloxavir, lopinavir, ritonavir, arbidol, peramivir, laninamivir and zanamivir when detected with different mobile phases in Example 2 are shown.

[0026] Figure 4 This is the chromatogram detected by the Poreshell HILIC-5OH chromatographic column in Example 2.

[0027] Figure 5 This is the chromatogram detected by the Poreshell HILIC-Z chromatographic column in Example 2.

[0028] Figure 6 This is the chromatogram detected by the Accucore Urea-HILIC column in Example 2.

[0029] Figure 7 The chromatogram detected by the ACQUITY BEH HILIC column in Example 2. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0031] Among them, acetonitrile (CH 3 CN), methanol (CH 3 OH), dimethyl sulfoxide (C 2 H 6 OS) were all chromatographically pure, formic acid (HCOOH), glacial acetic acid (CH 3 COOH), ammonium acetate (CH 3 COONH 4 ) were all analytically pure.

[0032] Morphoguanidine hydrochloride, amantadine hydrochloride, rimantadine hydrochloride, penciclovir, oseltamivir phosphate, peramivir trihydrate, zanamivir, ribavirin, arbidol, favipiravir, mabaloxavir, lopinavir, ritonavir, laninamivir, acyclovir, ganciclovir, famciclovir, and valacyclovir hydrochloride were used as standard substances with a purity of ≥99.0%.

[0033] Solution configuration:

[0034] (1) Methanol-acetonitrile solution (v / v, 25:75): add 250 mL of methanol to 750 mL of acetonitrile and mix well.

[0035] (2) Blank matrix solution: Weigh 1 g (accurate to 0.01 g) of a blank sample with a similar matrix composition and without the target compound and place it in a 50-mL volumetric flask. Add about 40 mL of methanol-acetonitrile solution (v / v, 25:75). Vortex for 30 s, perform ultrasonic extraction for 30 min, cool to room temperature, make up to volume with methanol-acetonitrile solution (v / v, 25:75), shake well, filter through a microporous filter membrane (0.22 μm, nylon membrane) into a sample bottle, and set aside.

[0036] (3) Standard stock solution: Accurately weigh an appropriate amount of each standard substance (accurate to 0.1 mg) to prepare a standard stock solution with a concentration of 1 mg / mL; among them, morphine hydrochloride, amantadine hydrochloride, rimantadine hydrochloride, oseltamivir phosphate, peramivir trihydrate, ribavirin, arbidol, favipiravir, mabaloxavir, lopinavir, famciclovir, and valacyclovir hydrochloride are dissolved in methanol and then diluted to volume; ritonavir, penciclovir, acyclovir, and ganciclovir are dissolved in a small amount of dimethyl sulfoxide and then diluted to volume with methanol; zanamivir and laninamivir are dissolved in water and then diluted to volume; the above stock solutions are stored at -18°C.

[0037] (4) Mixed standard intermediate solution: Accurately pipette an appropriate amount of the standard stock solution and dilute it step by step to the required concentration using methanol-acetonitrile solution (v / v, 25:75) and prepare it before use.

[0038] (5) Mixed standard matrix matching working series solution: Accurately pipette an appropriate amount of the mixed standard intermediate solution and dilute it with the blank matrix solution to form an appropriate mixed standard matrix matching working series solution, which should be prepared before use.

[0039] (6) Mixed standard working series solution: Accurately pipette an appropriate amount of the mixed standard intermediate solution and dilute it with methanol-acetonitrile solution (v / v, 25:75) to form an appropriate mixed standard series solution, which should be prepared before use.

[0040] (7) Test solution: Weigh 1 g of the sample to be tested (accurate to 0.01 g) and place it in a 50-mL volumetric flask. Add about 40 mL of methanol-acetonitrile solution (v / v, 25:75). Vortex for 30 s, perform ultrasonic extraction for 30 min, cool to room temperature, make up to the mark with methanol-acetonitrile solution (v / v, 25:75), shake well, filter through a microporous filter (0.22 μm, nylon membrane) into a sample bottle, and wait for testing.

[0041] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0042] Example 1 A method for simultaneously determining 18 kinds of illegally added antiviral drugs in herbal tea

[0043] 1. Testing conditions:

[0044] (1) Liquid chromatography conditions

[0045] Chromatographic column: Waters ACQUITY BEH HILIC (2.1 mm × 100 mm, 1.7 μm), column temperature: 40 °C, injection volume: 2 μL;

[0046] Mobile phase A: 10 mmol / L ammonium acetate solution (containing 0.1% acetic acid), mobile phase B: 0.1% formic acid acetonitrile, flow rate: 0.3 mL / min; gradient elution was performed, and the elution program was shown in Table 1.

[0047] Table 1 Gradient elution program

[0048]

[0049]

[0050] (2) Mass spectrometry conditions

[0051] Ion source: electrospray ion source, ion source temperature: 550°C, electrospray voltage: positive ion mode detection, electrospray voltage is 5500V, nebulizer gas pressure: 50psi, curtain gas pressure: 40psi, collision gas pressure: 9psi, detection method: multiple reaction monitoring (MRM), monitoring conditions see Table 2.

[0052] Table 2 Multiple reaction monitoring (MRM) conditions

[0053] Serial number Compound Precursor ion (m / z) Product ion (m / z) Declustering voltage (V) Collision Energy(eV) 1 Favipiravir 158.0 <![CDATA[140.9 * ,112.9]]> 70 18，25 2 Mabaloxavir 572.3 <![CDATA[246.8 * ,214.0]]> 79 35，70 3 Ritonavir 721.4 <![CDATA[296.2 * ,268.0]]> 140 24，40 4 Lopinavir 629.4 <![CDATA[447.3 * ,429.3]]> 120 21，31 5 Ribavirin 245.0 <![CDATA[113.1 * ,133.1]]> 79 17，16 6 Famciclovir 322.1 <![CDATA[136.1 * ,280.1]]> 130 42，29 7 Acyclovir 226.1 <![CDATA[152.0 * ,134.9]]> 100 19，38 8 Ganciclovir 256.2 <![CDATA[152.0 * ,135.0]]> 77 20，44 9 Penciclovir 254.0 <![CDATA[152.0 * ,134.7]]> 140 26，48 10 Abidol 478.3 <![CDATA[433.0 * ,389.0]]> 60 28，32 11 Rimantadine 180.1 <![CDATA[163.1 * ,81.0]]> 75 21，33 12 Amantadine 152.2 <![CDATA[135.1 * ,107.1]]> 100 21，34 13 Oseltamivir 313.1 <![CDATA[166.0 * ,207.9]]> 50 29，21 14 Morphoguanidine 172.2 <![CDATA[113.1 * ,88.1]]> 93 28，29 15 Peramivir 329.3 <![CDATA[270.1 * ,100.0]]> 110 28，39 16 Laninamivir 347.1 <![CDATA[60.1 * ,121.0]]> 108 38，40 17 Valacyclovir 325.2 <![CDATA[152.0 * ,146.0]]> 84 27，24 18 Zanamivir 333.1 <![CDATA[60.0 * ,121.0]]> 124 37，37

[0054] Note: * indicates quantitative ion.

[0055] 2. Qualitative analysis:

[0056] According to the above detection conditions, the test solution and the mixed standard matrix matching working solution series are measured. If the retention time of the mass chromatographic peak in the test solution is consistent with that of the mixed standard matrix matching working series solution, the relative abundance of the ion pair is consistent with that of the mixed standard matrix matching working series solution with equivalent concentration, and the relative abundance deviation does not exceed the provisions of Table 3, then it can be judged that the corresponding target compound exists in the test solution. The HPLC-MS / MS chromatogram of the mixed standard matrix matching working series solution can be found in Figure 1 The peak numbers marked in the figure correspond to those in Table 2.

[0057] Table 3 Maximum allowable deviation of relative ion abundance for qualitative confirmation

[0058] Relative ion abundance / % >50 >20-50 >10-20 ≤10 Allowable relative deviation / % ±20 ±25 ±30 ±50

[0059] 3. Quantitative analysis:

[0060] According to the above detection conditions, the chromatograms of the test solution and the mixed standard matrix matching working series solution were measured, the peak areas were recorded, and the external standard method was used for quantification. The concentration of the target substance in the test solution was obtained based on the standard curve fitted by the mixed standard matrix matching working series solution.

[0061] The content X of the target substance in the test sample is calculated as follows:

[0062]

[0063] Where:

[0064] X: the content of each target substance in the test sample, in milligrams per kilogram (mg / kg);

[0065] c: The concentration of each target substance in the test solution read from the standard curve, in nanograms per milliliter (ng / mL);

[0066] V: final volume of the test solution, in milliliters (mL);

[0067] M: mass of the test sample, in grams (g);

[0068] F: dilution factor.

[0069] The calculation result is expressed as the arithmetic mean of two independent determination results obtained under repeatability conditions, and the result is rounded to three significant figures.

[0070] Note: The blank value should be deducted from the calculated results.

[0071] 4. Test results:

[0072] 54 batches of herbal tea samples were tested under the above conditions. The test results are shown in Table 4.

[0073] Table 4 Herbal tea test results

[0074]

[0075]

[0076]

[0077] As can be seen from the table, 15 batches of herbal tea or herbal tea products were found to contain adamantane and morpholino, while no other ingredients were detected.

[0078] Example 2 Detection Method Condition Comparison and Optimization

[0079] 1. Comparison and optimization of chromatographic conditions

[0080] (1) Comparison and optimization of mobile phase

[0081] The detection conditions of reference example 1 are different in that acetonitrile is used as mobile phase B without adding formic acid, and the mixed standard working series solution is measured. Figures 2-3 ,As can be seen from the figure, when acetonitrile does not contain formic acid, the peaks of compounds such as ribavirin, arbidol, and acyclovir are tailing, and the responses of compounds such as lopinavir, ritonavir, zanamivir, and laninamivir are poor.

[0082] The detection conditions of Reference Example 1 are different in that no buffer salt (sodium acetate) is added to mobile phase A or different buffer salts and different buffer salt concentrations (5mmol / L ammonium acetate (containing 0.1% acetic acid), 10mmol / L ammonium acetate (containing 0.1% acetic acid), 5mmol / L ammonium formate (containing 0.1% formic acid), 10mmol / L ammonium formate (containing 0.1% formic acid)) are used to measure the mixed standard working series solution. The results show that after adding buffer salts to the mobile phase, the ionic strength of each compound is adjusted, and the peak shape and separation are significantly improved; when the buffer salt concentration is 10mmol / L, the retention of each compound is increased, and the separation effect is better; when 10mmol / L ammonium acetate (containing 0.1% acetic acid) is used, compounds such as valacyclovir, penciclovir, and lopinavir have a higher response.

[0083] (2) Comparison and optimization of chromatographic columns

[0084] The stationary phases of hydrophilic interaction chromatography are becoming increasingly diverse. Currently, the most commonly used ones are unbonded silica / hybrid silica, zwitterionic, amide, amino, cyano, diol, and sugar bonded phases. However, different types of HILIC columns have different effects on compounds.

[0085] The detection conditions of Reference Example 1 were different in that ACQUITY BEH HILIC (2.1 mm × 100 mm, 1.7 μm), Poreshell HILIC-Z (2.1 mm × 100 mm, 2.7 μm), Poreshell HILIC-5OH (2.1 mm × 100 mm, 2.7 μm), and Accucore Urea-HILIC (2.1 mm × 100 mm, 2.6 μm) chromatographic columns were used to measure the mixed standard working series solution.

[0086] Results Figures 4 to 7 As can be seen from the figure, Accucore Urea-HILIC has poor retention for strongly basic compounds (such as morpholinoguanidine, amantadine, rimantadine, ritonavir, lopinavir), arbidol, and famciclovir. This may be because the polarity of the stationary phase is weakened after the silica gel is bonded to urea, which is not suitable for the retention and separation of strongly basic antiviral compounds. The peak shape and retention factor of ritonavir, lopinavir, famciclovir, arbidol, etc. on Poreshell HILIC-5OH and Poreshell HILIC-Z are poor. When using non-bonded silica HILIC columns such as Agilent RRHD HILIC Plus and ACQUITY BEH HILIC, the retention, peak shape and separation are all good. Especially when using ACQUITY BEH HILIC columns, the chromatographic retention and separation effects of each compound are excellent, and a better peak shape and lower column pressure can be obtained.

[0087] 2. Comparison and optimization of pretreatment conditions

[0088] The detection conditions of Reference Example 1 were different in that acetonitrile, methanol-acetonitrile solution (v / v, 25:75) and methanol-acetonitrile solution (v / v, 50:50) were used as extraction solvents for the test samples, and the recovery and precision were measured at the LOQ level, 2 times the LOQ level, and 10 times the LOQ level of blank sample spiked solutions.

[0089] The results showed that when acetonitrile was used as the extraction solvent, the extraction rates of zanamivir, laninamivir and peramivir decreased significantly. This may be because the octanol-water partition coefficients Log P of these compounds were small (-4.13, -3.06 and -1.37, respectively), they were highly hydrophilic and had low solubility in acetonitrile. After the sample extract was treated with methanol-acetonitrile solution (v / v, 50:50), the peak shapes of ritonavir, lopinavir and mabaloxavir were split, showing a large solvent effect. When methanol-acetonitrile solution (v / v, 25:75) was used as the extraction solvent, the peak shapes and extraction rates of each compound were relatively ideal.

[0090] 3. Comparison and optimization of quantitative methods

[0091] In the mass spectrometry quantitative method, the matrix effect is one of the key factors affecting the accuracy. The matrix effect coefficient is used to evaluate the influence of the matrix effect on the quantitative results. The mixed standard working series solution and the mixed standard matrix matching working series solution were used respectively, and the standard curve was fitted by the external standard method, and the detection conditions of reference Example 1 were used for detection. The matrix effect coefficient ME = (slope of the matrix matching standard curve / slope of the solvent standard curve-1) × 100% was used for evaluation. A negative value indicates the presence of an inhibitory effect, a positive value indicates the presence of an enhancement effect, and the larger the absolute value, the stronger the matrix effect. When |ME|>50%, it indicates that there is a serious matrix effect; when 20% <|ME|≤50%, it indicates that there is a moderate degree of matrix effect; when |ME|≤20%, it can be considered that there is no matrix effect. The results are shown in Table 5.

[0092] Table 5 Compound matrix effect

[0093]

[0094]

[0095] As can be seen from the table, most of the 18 target compounds showed a matrix enhancement effect, and the matrix effect coefficient ME in herbal tea was -7% to 59%, indicating that some target compounds had serious matrix effects and needed to use blank matrix solution to prepare standard curves for quantitative correction.

[0096] Example 3 Methodological Investigation

[0097] 1. Linear relationship, method detection limit and method quantification limit

[0098] A mixed standard matrix matching working series solution was taken and analyzed and detected according to the detection conditions of Example 1. The mass concentration of each compound was used as the abscissa (x, μg / L) and the corresponding chromatographic peak area was used as the ordinate (y). Linear regression analysis was performed, and the method detection limit (LOD) and quantification limit (LOQ) were determined at 3 times and 10 times the signal-to-noise ratio. The results are shown in Table 6.

[0099] Table 6 Linear relationships, detection limits and quantification limits of 18 target compounds

[0100]

[0101]

[0102] It can be seen from the table that each target compound has good linearity within the corresponding concentration range, with a correlation coefficient of 0.9980-0.9999, a detection limit of 0.005-2.0 mg / kg, a quantification limit of 0.0125-5.0 mg / kg, and good sensitivity.

[0103] 2. Recovery and precision

[0104] A blank matrix solution was selected, and spike recovery was performed at three levels (LOQ, 2 times LOQ, and 10 times LOQ), and 6 samples were prepared for each level. The accuracy and precision of the method were investigated with reference to the detection conditions of Example 1. The solution preparation method was as follows:

[0105] (1) The 18 target compounds were divided into three groups according to their corresponding levels; Group A: amantadine, famciclovir, rimantadine, mabaloxavir, and morphine; Group B: oseltamivir, ribavirin, valacyclovir, acyclovir, arbidol, penciclovir, ganciclovir, lopinavir, and peramivir; Group C: ritonavir, favipiravir, zanamivir, and laninamivir.

[0106] Take an appropriate amount of the standard stock solution of each compound in Group A to prepare a mixed solution of Group A. The concentrations of the compounds are 1.25 μg / ml, 1.25 μg / ml, 2.5 μg / ml, 2.5 μg / ml, and 5 μg / ml, respectively.

[0107] Take an appropriate amount of the standard stock solution of each compound in group B to prepare a mixed solution of group B. The concentrations of each compound are: 12.5μg / ml, 12.5μg / ml, 25μg / ml, 25μg / ml, 50μg / ml, 50μg / ml, 50μg / ml, 50μg / ml, 50μg / ml, and 50μg / ml, respectively.

[0108] (2) Preparation of spiked solution: Take 1 ml of the mixed solution of group A and group B, and 0.5 ml of the standard stock solution of each compound in group C, and dilute them to volume with methanol-acetonitrile solution (v / v, 25:75) in the same 50 ml volumetric flask. Mix well to obtain the spiked solution.

[0109] (3) LOQ level: Take 1 g of blank sample in a 50-ml volumetric flask, add 0.5 ml of spiked solution, dilute to about 40 ml with methanol-acetonitrile solution (v / v, 25:75), extract by ultrasonic wave for 30 min, cool to room temperature, make up to the mark with methanol-acetonitrile solution (v / v, 25:75), shake well, and perform detection according to the detection conditions of Example 1.

[0110] (4) 2 times LOQ level: Take 1 g of blank sample in a 50 ml volumetric flask, add 1.0 ml of spiked solution, dilute to about 40 ml with methanol-acetonitrile solution (v / v, 25:75), extract by ultrasonic for 30 min, cool to room temperature, dilute to scale with methanol-acetonitrile solution (v / v, 25:75), shake well, and perform detection according to the detection conditions of Example 1

[0111] (5) 10 times LOQ level: Take 1 g of blank sample in a 50-ml volumetric flask, add 5.0 ml of spiked solution, dilute to about 40 ml with methanol-acetonitrile solution (v / v, 25:75), extract by ultrasonic wave for 30 min, cool to room temperature, make up to scale with methanol-acetonitrile solution (v / v, 25:75), shake well, and perform detection according to the detection conditions of Example 1.

[0112] See Table 7 for the results.

[0113] Table 7 Average recovery and relative standard deviation of target compounds in herbal tea samples (n=6)

[0114]

[0115] As can be seen from the table, the average recovery rate of each target compound is 70.3% to 115%, and the repeatability RSD (n=6) is 1.1% to 11%, indicating that the method has high accuracy and good repeatability and can meet the analysis requirements.

[0116] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for simultaneously determining 18 illegally added antiviral drugs, characterized in that: The determination was performed by high performance liquid chromatography-tandem mass spectrometry, which specifically included the following steps: S1. Preparation of standard solution: Dissolve each standard substance with a solvent, make up to volume, mix and dilute to form a mixed standard solution, adjust the concentration according to needs, store and wait for testing; S2. Preparation of test solution: Take the sample to be tested and use the methanol-acetonitrile solution for ultrasonic extraction, pass through the membrane, and wait for testing; S3, the high performance liquid chromatography: non-bonded silica gel type HILIC column, column temperature 35~40℃, flow rate 0.2~0.4 mL / min; using 10 mmol ammonium acetate solution containing 0.1% acetic acid as mobile phase A, 0.1% formic acid acetonitrile as mobile phase B, gradient elution according to the following elution program: 0~0.5 min, 3% mobile phase A; 0.5~2.0 min, 3~5% mobile phase A; 2.0~4.0min, 5~10% mobile phase A; 4.0~9.0 min, 10~20% mobile phase A; 9.0~14.0 min, 20% mobile phase A; 14.0~14.1min, 20~3% mobile phase A; 14.1~18.0 min, 3% mobile phase A; S4. Mass spectrometry conditions: the ion source is an electrospray ion source; the electrospray voltage is 5000-5500 V in positive ion mode; the ion source temperature is 500-550 °C; the nebulizer gas pressure is 45-55 psi; the curtain gas pressure is 35-45 psi; the collision gas pressure is 8-10 psi, and multiple reaction monitoring is used; the 18 illegally added antiviral drugs are morpholino hydrochloride, amantadine hydrochloride, rimantadine hydrochloride, penciclovir, oseltamivir phosphate, peramivir trihydrate, zanamivir, ribavirin, arbidol, favipiravir, mabaloxavir, lopinavir, ritonavir, laninamivir, acyclovir, ganciclovir, famciclovir and valacyclovir hydrochloride; The sample to be tested is herbal tea or traditional Chinese medicine preparation; The volume ratio of methanol to acetonitrile in the methanol-acetonitrile solution is (20-30):(70-80).

2. The method according to claim 1, characterized in that: In step S1, when the standard substance is morpholinoguanidine hydrochloride, amantadine hydrochloride, rimantadine hydrochloride, oseltamivir phosphate, peramivir trihydrate, ribavirin, arbidol, favipiravir, mabaloxavir, lopinavir, famciclovir, or valacyclovir hydrochloride, the solvent is methanol; when the standard substance is ritonavir, penciclovir, acyclovir, or ganciclovir, it is first dissolved in dimethyl sulfoxide and then fixed to volume with methanol; when the standard substance is zanamivir or laninamivir, the solvent is water.

3. The method according to claim 1, characterized in that: The limit of quantification of amantadine and famciclovir is 0.0125 mg / kg; the limit of quantification of rimantadine and mabaloxavir is 0.025 mg / kg; the limit of quantification of morphine is 0.05 mg / kg; the limit of quantification of ribavirin and oseltamivir is 0.125 mg / kg; the limit of quantification of acyclovir and valacyclovir is 0.25 mg / kg; the limit of quantification of ganciclovir, penciclovir, arbidol, peramivir and lopinavir is 0.5 mg / kg; the limit of quantification of favipiravir, ritonavir, laninamivir and zanamivir is 5.0 mg / kg.

4. The method according to claim 1, characterized in that: The detection limit of favipiravir, ritonavir, laninamivir and zanamivir is 2.0 mg / kg; the detection limit of rimantadine and mabaloxavir is 0.01 mg / kg; the detection limit of ganciclovir, penciclovir, arbidol, peramivir and lopinavir is 0.2 mg / kg; the detection limit of ribavirin and oseltamivir is 0.05 mg / kg; the detection limit of amantadine and famciclovir is 0.005 mg / kg; the detection limit of acyclovir and valacyclovir is 0.1 mg / kg; the detection limit of morphine is 0.02 mg / kg.

5. The method according to claim 1, characterized in that: In step S3, the non-bonded silica gel type HILIC chromatographic column is a Waters ACQUITY BEH HILIC chromatographic column or an Agilent RRHD HILIC Plus chromatographic column.

6. The method according to any one of claims 1 to 5, characterized in that: The absolute difference between two independent measurement results in the repeatability verification of the method shall not exceed 15% of the arithmetic mean.

7. The method according to any one of claims 1 to 5, characterized in that: The average recovery of the standards was 70-115%.

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