A method for determining the residual amount of eight antiviral drugs in bee products

Abstract

The application discloses a method for determining the residual amount of eight antiviral drugs in bee products. It relates to the technical field of drug residue determination. The method for determining the residual amount of drugs in the bee products comprises the following steps: S1, extracting the eight antiviral drugs in the bee products by using an acidic acetonitrile mixed extraction liquid to obtain an extraction liquid; S2, purifying the extraction liquid to obtain a purified extraction liquid; S3, determining the residual amount of the eight antiviral drugs in the purified extraction liquid by using an SB-Aq C 18 As a chromatographic column, the purified extraction liquid in step S2 is detected by using a high performance liquid chromatography tandem mass spectrometry method, wherein the mobile phase comprises at least one of formic acid, methanol and ammonium acetate. By using the method, the residual amount of eight antiviral drugs including amantadine, rimantadine, memantine, ribavirin, moroxydine, acyclovir, oseltamivir and ganciclovir in the bee products can be simultaneously determined qualitatively and quantitatively, and the eight drugs have very low detection limits, excellent accuracy and precision.

Description

Technical Field

[0001] This invention relates to the field of drug residue determination technology, and in particular to a method for determining the residue levels of eight antiviral drugs in bee products. Background Technology

[0002] When diseases and infections occur in bee colonies, beekeepers often use antiviral drugs for treatment, such as amantadine, rimantadine, memantine, ribavirin, morpholine guanidine, acyclovir, oseltamivir, and ganciclovir. These drugs are added to bee feed or drinking water to help bees fight viral infections or treat infected bees. To prevent the spread of disease within the colony, beekeepers may also use antiviral drugs around the hive and bees. However, these drugs may leave residues in bee products.

[0003] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is of great significance for the detection of antiviral drug residues in bee products. Detecting these antiviral drug residues in bee products is crucial for ensuring food safety and monitoring the impact of drug residues on bee product quality. However, currently, there is no suitable LC-MS / MS method that can simultaneously and stably detect residues of eight antiviral drugs in bee products: amantadine, rimantadine, memantine, ribavirin, morpholine guanidine, acyclovir, oseltamivir, and ganciclovir. Firstly, bee product samples are complex, containing abundant protein and lipid components, which poses challenges to the extraction and purification of drug residues. Secondly, highly sensitive and selective methods are needed to ensure accurate determination of these antiviral drug residues even at low levels. Furthermore, the different drugs have varying chemical properties, requiring the targeted selection of appropriate chromatographic columns and mass spectrometry conditions to achieve simultaneous detection and quantification of multiple drugs.

[0004] Therefore, there is an urgent need to develop a highly sensitive and selective assay method to simultaneously determine the residues of eight antiviral drugs in peak products. Summary of the Invention

[0005] The technical problem to be solved by this invention is:

[0006] A method for determining drug residues in bee products is provided.

[0007] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows:

[0008] A method for determining drug residues in bee products includes the following steps:

[0009] S1 uses an acidic acetonitrile mixed extraction solution to extract amantadine, rimantadine, memantine, ribavirin, morpholine guanidine, acyclovir, oseltamivir and ganciclovir from bee products to obtain an extract;

[0010] S2 uses at least one of QuERChERS, Prime HLB and EMR-enhanced degreasing dispersion SPE to purify the extract, obtaining a purified extract;

[0011] S3 with SB-Aq C 18 As a chromatographic column, high performance liquid chromatography-tandem mass spectrometry is used to detect the purified extract in step S2, wherein the mobile phase includes at least one of formic acid, methanol and ammonium acetate.

[0012] According to embodiments of the present invention, one of the technical solutions has at least one of the following advantages or beneficial effects:

[0013] Using the method of this invention, it is possible to simultaneously determine the residues of eight antiviral drugs in bee products, including amantadine, rimantadine, memantine, ribavirin, morpholine guanidine, acyclovir, oseltamivir, and ganciclovir. The method has extremely low detection limits for all eight drugs and excellent accuracy and precision.

[0014] Among the eight drugs, ribavirin and moroxydine are highly polar substances, readily soluble in water and slightly soluble in methanol; while the other substances are low to medium polar compounds, slightly soluble in water and readily soluble in methanol; furthermore, ribavirin has a structure similar to sugar. This invention uses an acidic acetonitrile mixed extraction solution, which ensures the extraction of the eight antiviral drugs, and the acidic acetonitrile mixed extraction solution effectively removes sugar from honey, yielding satisfactory chromatograms.

[0015] Among them, since the eight compounds contain amino groups and are alkaline, and ribavirin and moroxydine are highly polar and difficult to elute or have low recovery rates, in order to avoid the cumbersome process of stepwise elution or eluting ribavirin and moroxydine separately, this invention selects at least one of QuERChERS, Prime HLB and EMR enhanced defatted dispersion SPE to purify the extract, ensuring that the eight antiviral drugs are purified at the same time and a good recovery rate is obtained;

[0016] Because ribavirin and morpholine guanidine are highly polar, a high proportion of aqueous phase is required for chromatographic separation. The other six substances are moderately or weakly polar drugs, requiring a high proportion of organic phase for separation. Furthermore, rimantadine and memantine are isomers with similar relative molecular mass and chemical properties. Therefore, selecting a suitable chromatographic column is crucial for the separation of these eight substances. Conventional columns such as HILIC columns are difficult to separate rimantadine and memantine, and graphitized carbon columns are unstable for analyzing samples with complex matrices. This invention utilizes the SB-Aq C... 18 The chromatographic column is a C18 surface-modified reversed-phase column that can withstand 100% aqueous phase, enabling the retention of ribavirin and morpholine guanidine as well as the separation of amantadine and memantine.

[0017] Commonly used mobile phases typically contain acetonitrile, but acetonitrile has a strong elution ability, which can lead to imperfect separation of rimantadine and memantine. Therefore, the mobile phase of this invention includes at least one of formic acid, methanol, and ammonium acetate.

[0018] According to one embodiment of the present invention, the acidic acetonitrile mixed extract includes a formic acid-acetonitrile mixed extract, and the mass percentage of formic acid in the acidic acetonitrile mixed extract is 1-1.5%.

[0019] According to one embodiment of the present invention, when 1-1.5% formic acid acetonitrile is added to bee products, a layering phenomenon will occur, with the upper clear liquid being an acetonitrile system and the lower layer being sugar.

[0020] According to one embodiment of the present invention, when the bee product exhibits stratification after extraction, if the upper organic layer is dried with nitrogen and then reconstituted with the lower aqueous phase layer, it will be found that a large number of endogenous substances contained in the bee product, such as uridine, have structures similar to ribavirin and cannot be separated, resulting in disordered peak shapes that cannot be analyzed.

[0021] According to one embodiment of the present invention, the acidic acetonitrile mixed extract further contains methanol and / or water, and the acidic acetonitrile mixed extract comprises acetonitrile:methanol in a mass ratio of 7-7.5:3-2.5. After extracting eight substances from bee products, analysis of the lower aqueous phase after filtration revealed high levels of ribavirin and morpholine guanidine, as well as a small amount of ganciclovir. This indicates that although the acidic acetonitrile mixed extract can effectively remove sugars from bee products and obtain satisfactory chromatograms, its extraction efficiency is not high for substances that are more soluble in water, such as ribavirin, morpholine guanidine, and ganciclovir. Therefore, the present invention adds different proportions of water, methanol, and other solvents to the acidic acetonitrile mixed extract to improve the extraction efficiency. Studies have found that adding a low proportion of methanol to the acidic acetonitrile mixed extract significantly improves the extraction efficiency of eight antiviral drugs.

[0022] According to one embodiment of the present invention, the acidic acetonitrile mixed extract comprises acetonitrile:methanol:water in a mass ratio of 7-7.5:2-2.5:1.

[0023] According to one embodiment of the present invention, when the mobile phase of the present invention does not contain ammonium acetate, the peak shape of morpholine guanidine will split when the concentration of the standard is too high.

[0024] According to one embodiment of the present invention, either QuERChERS or Prime HLB can be used to simultaneously purify eight antiviral drugs in bee products, achieving good recovery rates. Furthermore, the purification effects of the two methods on honey samples are comparable.

[0025] According to one embodiment of the present invention, in step S2, royal jelly in bee products is purified using an EMR-enhanced defatting dispersion SPE method; honey in bee products is purified using the QuERChERS method. Because royal jelly has a high protein and lipid content, the extract experiences significant resistance when flowing out through Prime HLB, causing column blockage. Therefore, an EMR-enhanced defatting dispersion SPE method was selected for royal jelly, which removes lipids while ensuring a satisfactory recovery rate, while the honey sample uses the same type of QuERChERS column.

[0026] According to one embodiment of the present invention, the SB-Aq C 18 The chromatographic column has a particle size of 2.7-3 μm, an inner diameter of 2.1-2.5 mm, and a column length of 100-120 mm. Using other chromatographic columns will result in the inability to simultaneously separate the above eight substances. For example, HILIC columns have difficulty separating rimantadine and memantine; graphitized carbon columns exhibit instability in the analysis of honey and royal jelly samples; only SB-Aq C... 18 The chromatographic column can retain ribavirin and morpholine guanidine while separating amantadine and memantine, thus ensuring excellent separation of the eight substances.

[0027] According to one embodiment of the present invention, the mobile phase in step S3 includes ammonium acetate, and the concentration of ammonium acetate is 5-10 mmol / L. An appropriate concentration of ammonium acetate can further improve ionization efficiency, enhance the stability of morpholine guanidine retention, and ensure an increase in ganciclovir response value.

[0028] According to one embodiment of the present invention, a gradient elution procedure is used in the detection process, and the following settings are made:

[0029] Elution time, volume ratio of formic acid, ammonium acetate mixed solution to methanol;

[0030] 0-1.5 min, 100:0;

[0031] 1.5-4.5 min, 70-100: 0-30;

[0032] 4.5-7.5 min, 10-70: 30-90;

[0033] 7.5-8.0 min, 10:90:

[0034] 8.0-8.1 min, 10-100: 0-90;

[0035] 8.1-10 min, 100:0.

[0036] According to one embodiment of the present invention, the liquid chromatography conditions during the detection process include:

[0037] Column temperature: 30-35℃;

[0038] Flow rate: 0.3-0.6 mL / min;

[0039] Injection volume: 10-15 μL.

[0040] According to one embodiment of the present invention, in step S3, during the detection process using high-performance liquid chromatography-tandem mass spectrometry, mass spectrometry analysis is performed in positive ion scanning mode. All eight antiviral drugs contain amino groups, which readily bind to H protons during ionization and thus carry a positive charge; therefore, positive ion scanning mode is selected for mass spectrometry analysis.

[0041] According to one embodiment of the present invention, in step S3, during the detection process using high-performance liquid chromatography-tandem mass spectrometry, the mass spectrometry conditions include:

[0042] Ion source: Electrospray ionization (ESI);

[0043] Capillary voltage: 3500-3600V;

[0044] Atomization temperature (Gas temp): 300-350℃;

[0045] Gas flow rate: 7-10 L / min;

[0046] Nebulizer pressure: 40-50 psi;

[0047] Shealth gas temperature: 350-450℃;

[0048] Sheath gas flow rate: 12-20 L / min.

[0049] According to one embodiment of the present invention, in step S3, during the detection process using high performance liquid chromatography-tandem mass spectrometry, the MRM parameters of various drugs are shown in Table 1.

[0050] Table 1 Select Ion Setting Parameters

[0051]

[0052]

[0053] According to one embodiment of the present invention, the internal standard method is used for quantification of the target analyte. The matrix effect was investigated, and the results showed that the matrix effects of morpholine guanidine, ribavirin, ganciclovir, acyclovir, amantadine, rimantadine, memantine, and oseltamivir were 16.1%, 13.4%, 41.4%, 37.4%, 61.8%, 85.3%, 69.7%, and 92.2%, respectively. It is generally considered that when the matrix effect is <80%, matrix standards or internal standards are needed to offset the matrix effect during quantification. The experimental results show that the matrix effect is severe for some analytes; therefore, the internal standard method used in this invention can significantly offset the matrix effect.

[0054] According to one embodiment of the present invention, the method for determining drug residues in bee products further includes a honey sample pretreatment step:

[0055] Weigh the honey and place it in a centrifuge tube. Add the internal standard working solution and 1-1.5% formic acid acetonitrile methanol solution. Vortex the mixture and shake it at a constant speed. Centrifuge at 4-6℃, and transfer the supernatant to a dispersive solid-phase extraction column. Vortex the column. Centrifuge again at 4-6℃, and transfer the supernatant to a centrifuge tube. Blow the mixture under nitrogen at 40-50℃ until nearly dry. Reconstitute the honey with 1-2 mL of the reconstituted solution and filter it through a 0.22-0.3 μm PES membrane for analysis.

[0056] According to one embodiment of the present invention, the method for determining drug residues in bee products further includes a step of pretreatment of royal jelly samples:

[0057] Weigh royal jelly and place it in a centrifuge tube. Add internal standard working solution and 1-1.5% formic acid acetonitrile methanol solution. Vortex mix on a vortex mixer and shake at a constant speed. Centrifuge at 4-6℃, and transfer the supernatant to an EMR purification package pre-activated with 2-4 mL of water. Vortex again. Centrifuge at 4-6℃, and transfer the supernatant to a centrifuge tube. Blow under nitrogen at 40-50℃ until nearly dry. Reconstitute with 1-2 mL of the reconstituted solution. If turbidity remains after reconstitution, centrifuge at 4-6℃ and filter through a 0.22-0.3 μm PES membrane before analysis.

[0058] According to one embodiment of the present invention, the qualitative determination method for determining drug residues in bee products includes the following steps:

[0059] For each analyte, one parent ion and two daughter ions are selected. Under the same experimental conditions, the retention time of the analyte in the sample deviates from the corresponding retention time in the standard solution by within ±2.5%; and the relative abundance of the two daughter ions of each component in the sample spectrum is consistent with the relative abundance of the standard solution with a similar concentration, with the deviation not exceeding the following specified range. In qualitative confirmation, the maximum permissible deviation of relative ion abundance is as follows: when the relative ion abundance (%) is >50, >20–50, >10–20, and ≤10, the permissible relative deviation (%) is ±20, ±25, ±30, and ±50, respectively.

[0060] According to one embodiment of the present invention, the method for determining drug residues in bee products includes the following quantitative determination steps:

[0061] Take appropriate amounts of the sample solution and the corresponding concentration of the standard working solution, and perform single-point or multi-point calibration, using the internal standard method for quantification. The response values ​​of the drug in both the standard working solution and the sample solution should be within the linear range of the instrument's detection. The standard working solution should be used during the sample injection process to ensure accurate quantification.

[0062] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0063] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0064] Figure 1 This is a chromatogram of the sample measured by the method of the present invention in Example 1.

[0065] Figure 2 This is a chromatogram of the sample measured by the method of the present invention in Example 1.

[0066] Figure 3 The image shows the chromatogram of the sample in Comparative Example 1 after being measured by the method of the present invention.

[0067] Figure 4 The image shows the chromatogram of the sample in Comparative Example 2 after being measured by the method of the present invention.

[0068] Figure 5 The image shows the chromatogram of the sample in Comparative Example 8 after being measured by the method of the present invention.

[0069] Figure 6The image shows the chromatogram of the sample in Comparative Example 9 after being measured by the method of the present invention.

[0070] Figure 7 The image shows the chromatogram of the sample in Comparative Example 10 after being measured by the method of the present invention.

[0071] Figure 8 The chromatogram is shown for the blank honey sample in Comparative Example 12.

[0072] Figure 9 Add chromatograms to the honey blank sample matrix of Comparative Example 12.

[0073] Figure 10 The chromatogram of the blank royal jelly sample in Comparative Example 12 is shown.

[0074] Figure 11 Add chromatograms to the honey blank sample matrix of Comparative Example 12. Detailed Implementation

[0075] The embodiments of the present invention are described in detail below. Throughout the embodiments, the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0076] In the description of this invention, the use of terms such as "first," "second," etc., is for the purpose of distinguishing technical features only and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

[0077] In the description of this invention, it should be understood that the orientation descriptions, such as up, down, etc., are based on the orientation or positional relationship shown in the embodiments, and are only for the purpose of facilitating the description of this invention and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0078] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0079] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0080] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of the present invention.

[0081] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0082] In the examples and comparative examples, all reagents used, unless otherwise specified, were analytical grade reagents, and the water was Grade I water conforming to GB / T6682.

[0083] In the examples and comparative examples, acetonitrile, methanol, formic acid and ammonium acetate were chromatographically pure reagents.

[0084] In the examples and comparative examples, the preparation method of the 1% formic acid acetonitrile methanol solution includes the following steps: 30 mL of methanol is measured, 70 mL of acetonitrile and 1 mL of formic acid are added, and the mixture is stirred well to obtain the 1% formic acid acetonitrile methanol solution. When preparing formic acid acetonitrile methanol solutions of other concentrations, the amount of formic acid added is adjusted accordingly.

[0085] In the examples and comparative examples, the preparation method of 5 mmol / L ammonium acetate solution (containing 0.1% formic acid) includes the following steps: Weigh 0.385 g of ammonium acetate into approximately 800 mL of water, add 1 mL of formic acid, and then dilute with water to 1000 mL. Mix well to obtain a 5 mmol / L ammonium acetate solution (containing 0.1% formic acid). When preparing ammonium acetate solutions (containing 0.1% formic acid) of other concentrations, the amount of ammonium acetate added should be adjusted accordingly.

[0086] In the examples and comparative examples, the preparation method of the reconstituted solution includes the following steps: 95 mL of 5 mmol / L ammonium acetate solution (containing 0.1% formic acid) and 5 mL of methanol are measured, mixed well, and the reconstituted solution is obtained.

[0087] In the examples and comparative examples, the standard substances included: amantadine, rimantadine hydrochloride, memantine hydrochloride, ribavirin, moroxydine hydrochloride, and ganciclovir, all with a content ≥98.0%; oseltamivir and acyclovir with a content ≥92.0%; and the isotopic internal standard solution: amantadine-d... 15 Amantadine-D4, Memantine-D6, Acyclovir-D4, Ganciclovir-D5, Ribavirin- 13 C5, oseltamivir-d3 concentration is 100 μg / mL, morpholine guanidine-d8 concentration is 500 μg / mL.

[0088] In the examples and comparative examples, the preparation method of a single standard stock solution includes the following steps: accurately weigh appropriate amounts of amantadine, rimantadine, memantine, ribavirin, morpholine guanidine, oseltamivir, ganciclovir, and acyclovir standard substances into a 10 mL volumetric flask, and prepare 1 mg / mL standard stock solutions with methanol respectively. Store at -18°C or below, with a shelf life of 3 months.

[0089] In the examples and comparative examples, the preparation method of the mixed standard intermediate solution includes the following steps: accurately measure 1.00 mL of a single standard stock solution and place it in a 100 mL volumetric flask, prepare a mixed standard intermediate solution with a concentration of 10 μg / mL using methanol, store it below -18°C, and have a shelf life of 1 month.

[0090] In the examples and comparative examples, the preparation method of the mixed standard working solution includes the following steps: accurately measure 1.00 mL of the mixed standard intermediate solution and place it in a 100 mL volumetric flask, prepare a mixed standard working solution with a concentration of 100 ng / mL using methanol, store it below 4°C, and keep it for 1 week.

[0091] In the examples and comparative examples, the preparation method of the mixed internal standard working solution includes the following steps: accurately measuring an appropriate amount of adamantane-d 15 Amantadine-D4, Memantine-D6, Acyclovir-D4, Ganciclovir-D5, Ribavirin- 13 C5, oseltamivir-d3, and morpholine guanidine-d8 standard solutions were placed in the same 100 mL volumetric flask and diluted to the mark with methanol to prepare a mixed internal standard working solution with a concentration of 100 ng / mL. It was stored below 4°C and had a shelf life of 3 months.

[0092] In the examples and comparative examples, the dispersion solid-phase extraction column was 15 mL.

[0093] In the examples and comparative examples, the disposable syringes were 1 mL in size.

[0094] In the examples and comparative examples, the sample preparation process includes the following steps: Uncrystallized honey samples are vigorously stirred until homogeneous; for honey samples with crystallization and royal jelly samples, the sample bottle cap is tightly sealed, and the sample is placed in a water bath not exceeding 50°C to warm until completely melted, then stirred thoroughly. Royal jelly is removed from the frozen environment, stirred thoroughly, and then sampled. During the sample preparation process, contamination or changes in residue content should be prevented.

[0095] In the examples and comparative examples, the sample preservation process included the following steps: honey samples were stored at room temperature away from light. Royal jelly samples were stored at -20°C or below away from light.

[0096] In the examples and comparative examples, the pretreatment of honey samples included the following steps: Weigh 2.0 g of honey (accurate to ±0.02 g), place it in a 50 mL plastic centrifuge tube, add 100 μL of internal standard working solution, add 10 mL of 1% formic acid acetonitrile methanol solution, vortex mix on a vortex mixer, and shake at a constant speed for 10 min. Centrifuge at 10000 rpm for 8 min at 4 °C, collect the supernatant into a dispersible solid-phase extraction column, and vortex for 3 min. Centrifuge at 10000 rpm for 5 min at 4 °C, collect the supernatant into a 10 mL centrifuge tube, blow with nitrogen to near dryness at 40 °C, reconstitute with 1 mL of reconstitution solution, and filter through a 0.22 μm PES membrane for analysis.

[0097] In the examples and comparative examples, the pretreatment of royal jelly samples included the following steps: Weigh 1.0 g of royal jelly (accurate to ±0.01 g), place it in a 50 mL plastic centrifuge tube, add 100 μL of internal standard working solution, add 10 mL of 1% formic acid acetonitrile methanol solution, vortex mix on a vortex mixer, and shake at a constant speed for 10 min. Centrifuge at 10000 rpm for 8 min at 4 °C, collect the supernatant into an EMR purification package pre-activated with 2 mL of water, and vortex for 3 min. Centrifuge at 10000 rpm for 5 min at 4 °C, collect the supernatant into a 10 mL centrifuge tube, blow with nitrogen to near dryness at 40 °C, and reconstitute with 1 mL of reconstitution solution. If turbidity is present after reconstitution, centrifuge at 10000 rpm for 5 min at 4 °C and filter through a 0.22 μm PES membrane before analysis.

[0098] In the examples and comparative examples, the residual amount X of antiviral drugs in the samples is expressed as a mass fraction in micrograms per kilogram (μg / kg) and calculated according to formula (1):

[0099]

[0100] Where: X—the residual amount of antiviral drugs in the sample, in micrograms per kilogram (μg / kg);

[0101] C—The concentration of the corresponding antiviral drug in the sample solution, in nanograms per milliliter (ng / mL);

[0102] V—The volume of the solution used to dissolve the sample, in milliliters (mL);

[0103] m – Sample mass, in grams (g).

[0104] The measurement results are expressed as the arithmetic mean of parallel measurements, and are retained to three significant figures.

[0105] In the attached figures, the horizontal axis of each graph represents the acquisition time; ASTW stands for oseltamivir, AXLW for acyclovir, GXLW for ganciclovir, JGWA for amantadine, JGYA for adamantine, LBWL for ribavirin, MJG for memantine, and MLG for morpholine guanidine.

[0106] Example 1

[0107] A method for determining drug residues in bee products includes the following steps:

[0108] S1 uses a mixed extract containing acetonitrile:methanol at a volume ratio of 7:3 and 1% formic acid to extract amantadine, rimantadine, memantine, ribavirin, morpholine guanidine, acyclovir, oseltamivir, and ganciclovir from honey and royal jelly samples to obtain the extract.

[0109] S2 uses EMR-enhanced degreasing dispersing SPE to purify royal jelly extract and QuERChERS columns to purify honey extract.

[0110] S3 with SB-Aq C 18 The product in step S2 was detected using high performance liquid chromatography-tandem mass spectrometry as the chromatographic column, wherein the mobile phase was a 5 mmol / L ammonium acetate solution (containing 0.1% formic acid by mass).

[0111] The liquid chromatography conditions include:

[0112] Chromatographic column: SB-Aq C18 column, particle size 2.7μm, inner diameter 2.1mm, column length 100mm, or equivalent;

[0113] Column temperature: 30℃;

[0114] Flow rate: 0.3 mL / min;

[0115] Injection volume: 10 μL;

[0116] The gradient elution procedure is shown in Table 2.

[0117] Table 2

[0118]

[0119] The mass spectrometry conditions include:

[0120] Ion source: Electrospray ionization (ESI);

[0121] Scanning method: positive ion scan;

[0122] Capillary voltage: 3500V;

[0123] Atomization temperature (Gas temp): 300℃;

[0124] Gas flow rate: 7 L / min;

[0125] Nebulizer pressure: 40 psi;

[0126] Shealth gas temperature: 350℃;

[0127] Shealth gas flow rate: 12 L / min

[0128] Monitoring mode: Multiple Response Monitoring (MRM acquisition mode).

[0129] The MRM parameters for each drug are shown in Table 1.

[0130] Table 1 Select Ion Setting Parameters

[0131]

[0132]

[0133] Example 2

[0134] The difference between Example 2 and Example 1 is that the extraction solution used in step S1 is different. In Example 1, a mixed extraction solution with an acetonitrile:methanol volume ratio of 7:3 and containing 1% formic acid by mass was used; in Example 2, a mixed extraction solution of acetonitrile and formic acid was used, wherein the formic acid by mass was 1% (hereinafter referred to as 1% formic acid acetonitrile).

[0135] Tests showed that adding 1% formic acid acetonitrile to honey samples resulted in stratification, with the upper clear liquid being an acetonitrile system and sugar located in the lower layer; adding 1% formic acid acetonitrile to royal jelly samples caused the sample matrix to denature and clump together.

[0136] Filtering and analyzing the lower aqueous phase of the honey revealed high levels of ribavirin and morpholine guanidine, as well as a small amount of ganciclovir. This indicates that while this extract effectively removes sugars from honey and yields satisfactory chromatograms, its extraction efficiency is low for water-soluble substances such as ribavirin, morpholine guanidine, and ganciclovir.

[0137] Example 3

[0138] The difference between Example 3 and Example 1 is that the extraction solution used in step S1 is different. Specifically, Example 1 uses a mixed extraction solution with an acetonitrile:methanol volume ratio of 7:3 and containing 1% formic acid by mass; Example 3 uses a mixed extraction solution with an acetonitrile:methanol:water volume ratio of 7:2:1 and containing 1% formic acid by mass.

[0139] Tests showed that the extract from Example 3 was able to normally separate eight substances from honey and royal jelly samples.

[0140] Comparative Example 1

[0141] The difference between Comparative Example 1 and Example 1 lies in the extraction solution used in step S1. Example 1 uses a mixed extraction solution with an acetonitrile:methanol volume ratio of 7:3 and containing 1% formic acid by mass; Comparative Example 1 uses an ammonium acetate / acetonitrile extraction solution, wherein the concentration of ammonium acetate is 5 mmol / L.

[0142] The test results are as follows: Figure 3 As can be seen from the figure, the extraction effect of many compounds was poor after extraction with ammonium acetate / acetonitrile.

[0143] Comparative Example 2

[0144] The difference between Comparative Example 1 and Example 1 lies in the extraction solution used in step S1. Example 1 uses a mixed extraction solution with an acetonitrile:methanol volume ratio of 7:3 and containing 1% formic acid by mass; Comparative Example 1 uses a trichloroacetic acid / acetonitrile extraction solution with a trichloroacetic acid concentration of 5 mmol / L.

[0145] The test results are as follows: Figure 4 As can be seen from the figure, although trichloroacetic acid / acetonitrile can extract all eight target substances, it cannot remove the interference of sugar substances. Ribavirin has a similar structure to sugar, and after instrumental analysis, ribavirin and sugar substances form an encapsulation peak, which has a very serious matrix effect. Neither the internal standard method nor the matrix spiking method can eliminate this matrix effect. In addition, the high boiling point of trichloroacetic acid solution is not conducive to mass spectrometry analysis.

[0146] Comparative Example 3

[0147] The difference between Comparative Example 3 and Example 1 lies in the solid-phase extraction column used in step S2. Example 1 used a QuERChERS column to purify the honey extract, while Comparative Example 3 used a PBA solid-phase extraction column.

[0148] Comparative Example 4

[0149] The difference between Comparative Example 4 and Example 1 lies in the solid-phase extraction column used in step S2. Example 1 used a QuERChERS column to purify the honey extract, while Comparative Example 4 used an HLB solid-phase extraction column.

[0150] Comparative Example 5

[0151] The difference between Comparative Example 5 and Example 1 lies in the solid-phase extraction column used in step S2. Example 1 used a QuERChERS column to purify the honey extract, while Comparative Example 5 used an MCX solid-phase extraction column.

[0152] Comparative Example 6

[0153] The difference between Comparative Example 6 and Example 1 lies in the solid-phase extraction column used in step S2. Example 1 used a QuERChERS column to purify the honey extract, while Comparative Example 6 used a MAX solid-phase extraction column.

[0154] Comparative Example 7

[0155] The difference between Comparative Example 7 and Example 1 lies in the solid-phase extraction column used in step S2. Example 1 used a QuERChERS column to purify the honey extract, while Comparative Example 7 used a Hypercard solid-phase extraction column.

[0156] Comparative Example 8

[0157] The difference between Comparative Example 8 and Example 1 is that the chromatographic column used in step S3 is different. Example 1 uses an SB-Aq C18 column, while Comparative Example 8 uses a HILIC column.

[0158] The test results of Comparative Example 8 are as follows Figure 5 As can be seen from the figure, the HILIC column is a hydrophilic column, which has good retention for polar drugs such as ribavirin, but amantadine and memantine are difficult to separate and cannot be accurately quantified.

[0159] Comparative Example 9

[0160] The difference between Comparative Example 9 and Example 1 is that the chromatographic columns used in step S3 are different. Example 1 uses an SB-Aq C18 column, while Comparative Example 9 uses a graphitized carbon column.

[0161] The test results of Comparative Example 9 are as follows Figure 6 As can be seen from the figure, graphitized carbon chromatographic columns, because their packing material is 100% porous graphitized carbon (PGC) without any other bonded phases, are unstable for the analysis of samples with complex matrices and are not suitable for analyzing honey and royal jelly samples.

[0162] Comparative Example 10

[0163] The difference between Comparative Example 10 and Example 1 lies in the choice of mobile phase. In Example 1, the mobile phase was a 5 mmol / L ammonium acetate solution (containing 0.1% formic acid by mass); in Comparative Example 10, the mobile phase was water-acetonitrile (containing 0.1% formic acid by mass).

[0164] The test results of Comparative Example 10 are as follows Figure 7 As can be seen from the figure, in the acetonitrile system, due to the strong elution ability of acetonitrile, the separation effect of amantadine and memantine is not perfect. In addition, since the standards are all diluted with methanol, there is a solvent effect, which broadens the ganciclovir peak and bifurcates the peak shape. Ribavirin and the solvent peak co-elute and cannot be separated. At the same time, the response values ​​of the other drugs are low, which seriously affects the method's detection limit and quantitation limit.

[0165] Comparative Example 11

[0166] The difference between Comparative Example 11 and Example 1 lies in the choice of mobile phase. In Example 1, the mobile phase was a 5 mmol / L ammonium acetate solution (containing 0.1% formic acid by mass); in Comparative Example 11, the mobile phase was water-methanol (containing 0.1% formic acid by mass).

[0167] In the methanol system, amantadine and memantine showed better separation than in the acetonitrile system. Ribavirin and ganciclovir both yielded satisfactory chromatographic peaks. Except for ganciclovir, the response values ​​of all drugs were improved. However, when the concentration of the standard was too high, the peak shape of morpholine guanidine showed bifurcation.

[0168] Comparative Example 12

[0169] Comparative Example 12 is a blank experimental scheme, which differs from Example 1 in that no sample is added to Comparative Example 12.

[0170] Performance testing:

[0171] The samples extracted using different extraction solutions in Examples 1-3 were analyzed and tested, and the test results are shown in Table 3.

[0172] Table 3

[0173]

[0174]

[0175] As can be seen from Table 3, the extracts of Examples 1-3 can all extract eight antiviral drugs from bee products, but the 1% formic acid acetonitrile: methanol (7:3) used in Example 1 has the best extraction efficiency.

[0176] Recovery rate tests were performed on samples from comparative examples 3-7, and the test results are shown in Table 4.

[0177] Table 4

[0178]

[0179] Table 4 shows that, due to the presence of amino groups in the eight compounds, which are basic, most were not retained on the MAX column, and even those that were retained had very low recoveries. HLB, MCX, and PBA retained most substances, but because ribavirin and morpholine guanidine are highly polar, they had strong retention capabilities on ion exchange columns, making them difficult to elute or resulting in low recoveries. After purification with Hypercard, ganciclovir was undetectable, and some substances had low recoveries, making it unsuitable for purifying antiviral drugs. Some literature uses two of these solid-phase extraction columns in series, but this requires stepwise elution or separate elution of ribavirin, making the process complex and unsuitable for large-scale sample processing.

[0180] The test results of the sample measured by the method of the present invention in Example 1 are as follows: Figure 1 ,from Figure 1 As can be seen from the present invention, the method of the present invention can effectively separate eight antiviral drugs.

[0181] The test results of the sample measured by the method of the present invention in Example 2 are as follows: Figure 2 As can be seen from the figure, its extraction efficiency is not high.

[0182] The matrix effect of the sample in Example 1 was tested. The matrix effect refers to the influence of components other than the analyte in the sample on the measured value of the analyte, i.e., the interference of the matrix on the ability of the analytical method to accurately determine the analyte. The matrix effect is calculated as follows:

[0183] Matrix Effect (%) = B / A × 100%

[0184] A: Slope of the solvent standard curve

[0185] B: Slope of the matrix standard curve

[0186] This invention investigated the matrix effect. The results showed that the matrix effects of morpholine guanidine, ribavirin, ganciclovir, acyclovir, amantadine, rimantadine, memantine, and oseltamivir were 16.1%, 13.4%, 41.4%, 37.4%, 61.8%, 85.3%, 69.7%, and 92.2%, respectively. It is generally considered that when the matrix effect is <80%, matrix standards or internal standards are needed to offset the matrix effect. The experimental results show that some analytes exhibit significant matrix effects; therefore, all embodiments of this invention employ internal standard quantification to greatly offset the matrix effect.

[0187] The linear range and detection limit of the sample from Example 1 were tested. The results of the linear range test are shown in Table 5, and the results of the detection limit test are shown in Table 6.

[0188] Accurately measure an appropriate amount of the antiviral drug mixed standard intermediate solution, add 100 μL of isotope internal standard mixed standard working solution, and prepare a series of standard working solutions with concentrations of 0, 0.5, 1, 5, 10, 50, and 100 ng / mL for amantadine, rimantadine, memantine, ribavirin, morpholine guanidine, acyclovir, oseltamivir, and ganciclovir, for liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis. Plot a standard curve with the peak area ratio of the obtained solution to the internal standard as the ordinate and the corresponding standard solution concentration as the abscissa, and calculate the regression equation and correlation coefficient. The results show a good linear relationship within the concentration range of 0-100 ng / mL, with linear correlation coefficients all greater than 0.99, meeting the quantitative requirements.

[0189] Table 5. Standard curve parameters for eight analytes

[0190]

[0191] The limit of detection (LOD) is one of the main technical indicators for drug additive detection. An appropriate amount of mixed standard solution is added to a blank sample, extracted, and then measured. The LOD is determined based on a signal-to-noise ratio of 3, and the LOD is calculated based on a signal-to-noise ratio of 10. Therefore, the method's LOD is obtained.

[0192] Table 6 Qualitative and quantitative limits of honey and royal jelly

[0193]

[0194] As shown in Table 6, the limits of detection (LOD) for amantadine, rimantadine, memantine, morpholine guanidine, oseltamivir, acyclovir, and ganciclovir in honey using this method are 0.2 μg / kg and the limits of quantitation (LOQ) are 0.5 μg / kg; the LOD for ribavirin is 0.5 μg / kg and the LOD is 1 μg / kg.

[0195] As shown in Table 6, the detection limits for amantadine, rimantadine, memantine, oseltamivir, acyclovir, and ganciclovir in royal jelly using this method are 0.2 μg / kg and the quantitation limits are 0.5 μg / kg; the detection limits for morpholine guanidine and ribavirin are 0.5 μg / kg and the quantitation limits are 1 μg / kg.

[0196] The spiked recovery rate and precision of the method in Example 1 were tested. To examine the accuracy and precision of the method, a spiked recovery experiment was conducted to determine these parameters. Following the above processing and measurement methods, blank samples of honey and royal jelly were selected, and three different concentrations (LOQ, 2LOQ, and 10LOQ) were added to each sample. Three replicates were set for each level, and the experiment was repeated for three batches. Appropriate amounts of sample solution and corresponding concentrations of standard working solution were taken for single-point or multi-point calibration, and quantification was performed using the internal standard method. The response values ​​of the drug in both the standard working solution and the sample solution should be within the linear range of the instrument's detection. The standard working solution was interpolated during sample injection to ensure accurate quantification. The recovery rate and intra-batch and inter-batch relative standard deviations for each sample were calculated. The results are shown in Tables 7-8.

[0197] Table 7 Results of the recovery experiment of eight antiviral drugs in honey blank samples.

[0198]

[0199]

[0200]

[0201] As shown in Table 7, the intra-batch relative standard deviations for the eight drugs ranged from 0.37% to 10.75%, and the inter-batch relative standard deviations ranged from 1.6% to 8.4%.

[0202] As shown in Table 7, in honey samples, the recoveries of the eight antiviral drugs ranged from 62.7% to 106.7% when the addition level was LOQ; from 75.5% to 114.5% when the addition level was 2 LOQ; and from 75.0% to 113.0% when the addition level was 10 LOQ.

[0203] Table 8 Results of the recovery experiment of eight antiviral drugs in royal jelly blank samples.

[0204]

[0205]

[0206]

[0207] As shown in Table 8, the intra-batch relative standard deviations for the eight drugs ranged from 0.46% to 11.80%, and the inter-batch relative standard deviations ranged from 2.0% to 10.7%.

[0208] Table 8 shows that in royal jelly samples, the recoveries of the eight antiviral drugs ranged from 62.8% to 103.1% when the addition level was LOQ; from 65.2% to 104.1% when the addition level was 2 LOQ; and from 63.4% to 115.0% when the addition level was 10 LOQ.

[0209] Comparative Example 12 was a blank experimental protocol. To clearly observe and analyze the interference of impurities on the drug, a blank sample matrix addition experiment was performed, and the chromatograms were compared with those of the blank sample. Figure 8-11 As shown, where, Figure 8 The chromatogram of the blank honey sample for Comparative Example 12 is shown below. Figure 9 Add chromatograms (2 ng / g) to the honey blank sample matrix. Figure 10 The chromatogram of a blank royal jelly sample is shown. Figure 11 A chromatogram (5 ng / g) was added to the honey blank sample matrix. The results showed that there was no interference from impurities at the analyte peak positions in the blank sample.

[0210] Actual sample testing: Using the method of this invention, residues of eight antiviral drugs were detected in 28 honey samples and 4 royal jelly samples randomly collected from a bee farm. The results showed that two batches of honey contained morpholine guanidine, with levels of 8.6 ng / g and 5.1 ng / g, respectively; one batch of royal jelly contained amantadine, with a content of 26.1 ng / g. This demonstrates that the method of this invention can be applied to actual testing.

[0211] The above are merely embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for determining drug residues in bee products, characterized in that: Includes the following steps: S1 uses an acidic acetonitrile mixed extract to extract amantadine, rimantadine, memantine, ribavirin, morpholine guanidine, acyclovir, oseltamivir, and ganciclovir from bee products, obtaining an extract; the acidic acetonitrile mixed extract comprises acetonitrile:methanol:water in a mass ratio of 7-7.5:2-2.5:1; the acidic acetonitrile mixed extract includes a formic acid acetonitrile mixed extract, and the mass percentage of formic acid in the acidic acetonitrile mixed extract is 1-1.5%; S2. The extract is purified by using QuERChERS and / or EMR-enhanced defatting dispersion SPE to obtain a purified extract; royal jelly in bee products is purified by EMR-enhanced defatting dispersion SPE; honey in bee products is purified by QuERChERS. S3 with SB-Aq C 18 As a chromatographic column, high performance liquid chromatography-tandem mass spectrometry was used to detect the purified extract in step S2, wherein the mobile phase was 5-10 mmol / L ammonium acetate solution and methanol, and the ammonium acetate solution also contained 0.1% formic acid by mass. The detection process employs a gradient elution procedure, configured as follows: Elution time, volume ratio of formic acid, ammonium acetate mixed solution to methanol; 0-1.5 min, 100:0; 1.5-4.5 min, 100:0-70:30; 4.5-7.5 min, 70:30-10:90; 7.5-8.0 min, 10:90: 8.0-8.1 min, 10:90-100:0; 8.1-10 min, 100:

0.

2. The method according to claim 1, characterized in that: The SB-Aq C 18 The chromatographic column has a particle size of 2.7-3 μm, an inner diameter of 2.1-2.5 mm, and a column length of 100-120 mm.

3. The method according to claim 1, characterized in that: In step S3, during the detection process using high performance liquid chromatography-tandem mass spectrometry, mass spectrometry analysis is performed in positive ion scanning mode.

4. The method according to claim 1, characterized in that: The internal standard method is used to quantify the target analyte.

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