CN109541103B

CN109541103B - Method for measuring aminoglycoside drug residue in animal derived food

Info

Publication number: CN109541103B
Application number: CN201811391769.5A
Authority: CN
Inventors: 严丽娟; 徐敦明; 林立毅; 吴敏; 张洁; 赖国银; 林建忠; 张志刚; 张峰
Original assignee: Xiamen Customs Technical Center
Current assignee: Xiamen Customs Technical Center
Priority date: 2018-11-21
Filing date: 2018-11-21
Publication date: 2021-07-30
Anticipated expiration: 2038-11-21
Also published as: CN109541103A

Abstract

The invention relates to a method for determining residual aminoglycoside drugs in animal derived food, which comprises the steps of extracting the residual aminoglycoside drugs in a sample by adopting a phosphate buffer solution, enriching and purifying a SupelMIP SPE-amino glycosides solid phase extraction column, separating a SIELC Obelisc chromatographic column and a Hypercarb chromatographic column, and determining by high performance liquid chromatography-tandem mass spectrometry. The method for determining the residual aminoglycoside drug in the animal-derived food can simultaneously detect the residual situation of 16 aminoglycoside drugs in the animal-derived food. Compared with the reversed phase chromatography for analyzing the medicines, the method does not add ion pair reagent in the whole detection process, and has the advantage of not polluting a mass spectrometer; compared with hydrophilic interaction chromatography, the method has the advantages of high sensitivity, good peak pattern, strong response and various analyzed medicines.

Description

Method for measuring aminoglycoside drug residue in animal derived food

Technical Field

The invention relates to a detection technology of aminoglycoside Antibiotics (AGs), in particular to a method for determining aminoglycoside drug residue in animal derived food.

Background

Aminoglycoside Antibiotics (AGs) are a class of antibiotics containing aminoglycoside bonds, have a broad antibacterial spectrum, have strong antibacterial activity against aerobic gram-negative bacilli and pseudomonas aeruginosa, and currently have dozens of natural antibiotics or semi-synthetic varieties. The natural aminoglycoside antibiotics are produced by streptomyces, micromonospora and bacteria, and comprise streptomycin, kanamycin, tobramycin, spectinomycin, neomycin, gentamicin, sisomicin, astemicin and the like; semi-synthetic aminoglycoside antibiotics include amikacin, netilmicin, and the like. Because of its high efficiency and low price, aminoglycoside antibiotics are one of the most common antibiotics used in the world today, and are widely used in the breeding industry, and are also commonly added into feed to be used as animal growth promoters. Side effects of AGs are severe ototoxicity and nephrotoxicity, blocking neuromuscular transmission, and impairment of the intestinal tract affecting their absorptive function. In addition, the compounds have strong affinity with tissues, so that the residual time is long, and potential harm is brought to consumers if the consumers eat animal food with excessive AGs residues. The european union clearly stipulates that aminoglycoside antibiotics are prohibited from being used as growth-promoting additives, and the FDA in the united states pays much attention to the residues of the drugs in bee products due to the toxic and side effects and the easy generation of drug resistance of the drugs. In recent years, China also strengthens the monitoring of the residue of the medicines in bee products. Because some beekeepers unscientially use aminoglycoside medicines to prevent and treat bee larvae diseases, the residue of the aminoglycoside medicines in bee products exceeds the standard, and therefore, the monitoring of the residue of the aminoglycoside medicines in the bee products has very important significance.

The invention patent CN106053638B discloses a method for detecting the residual quantity of aminoglycoside antibiotics in animal-derived food, which is suitable for detecting 6 antibiotics such as neomycin, spectinomycin, dihydrostreptomycin, streptomycin, gentamicin, apramycin and the like, but is not suitable for detecting other 10 aminoglycoside antibiotics such as paromomycin, ribostamycin and the like. In addition, the method adopts a hydrophilic interaction chromatographic column to analyze 6 antibiotics such as neomycin, spectinomycin, dihydrostreptomycin, streptomycin, gentamicin, apramycin and the like, the lower detection limit of the method is 100ug/kg, the sensitivity is low, and the actual monitoring requirement cannot be met.

Disclosure of Invention

The aminoglycoside antibiotic is formed by combining aminosugar and alkaline 1, 3-diaminoinositol by glycosidic bond, and the 1, 3-diaminoinositol is a basic polycyclocyclohexanol structure, so that the aminoglycoside antibiotic has the characteristics of alkalinity and strong polarity. Currently, in conventional reverse phase chromatography-mass spectrometry, an ion pair reagent, such as heptafluorobutyric acid, is usually added to a mobile phase or a sample to promote the ionization of a drug, so that the target substance is retained on a column by detecting through an ion pair chromatographic mode. Alternatively, the HILIC mode can be used to improve the chromatographic behavior by adding high concentrations of salts (such as ammonium formate) to the mobile phase, as described in patent application CN 108519456A. However, the influence of ions on the reagent on the mass spectrum detector is great, which may cause ion suppression, ion source contamination, and the like. Therefore, the separation and detection of the drugs are one of the challenges faced by detection workers at present.

The invention aims to overcome the defects that the detection types of aminoglycoside antibiotics in the existing detection method are limited, and the residue situation of at least 10 aminoglycoside antibiotics can not be obtained at one time, and provides a method for measuring aminoglycoside drug residue in animal derived food. Compared with a reversed phase chromatography (an ion pair reagent needs to be added in a mobile phase), the method has the advantages of no mass spectrometer pollution, and compared with a hydrophilic interaction chromatography, the method has the advantages of high sensitivity, good peak pattern, strong response and multiple types of analyzed medicines.

The invention selects 16 aminoglycoside drugs as research objects, adopts liquid chromatography tandem mass spectrometry to carry out simultaneous detection, and adopts SIELC Obelisc under the condition of not adding ion pair reagent in the whole process^TMThe R and Hyercarb chromatographic columns have good retention and separation effects on aminoglycoside drugs, can be used for separating and detecting 16 aminoglycoside antibiotics, and have the advantages of simplicity, rapidness and strong adaptability.

The specific scheme is as follows:

a method for determining aminoglycoside drug residue in animal derived food comprises the following steps:

step 1: extracting residual aminoglycoside drug in sample

Putting the sample into a plastic centrifuge tube, adding phosphate buffer solution, covering, whirling, centrifuging, and taking supernatant for later use;

taking another negative sample without aminoglycoside drugs, putting the negative sample into a plastic centrifuge tube, adding phosphate buffer solution, covering, whirling, centrifuging, and taking supernatant as blank matrix solution;

step 2: enrichment and purification of solid phase extraction column

Activating a SupelMIPSPE-amino-ligands solid phase extraction column by using methanol and phosphate buffer solution in sequence before use; passing the supernatant obtained by the sample through a solid phase extraction column, leaching with water and ammonia water in sequence, and then vacuum-pumping to dry; then leaching by using a mixed solution of acetonitrile and water, and then performing vacuum pumping; then, leaching by using a mixed solution of dichloromethane and methanol, and then, vacuumizing and drying; finally, eluting the mixture of acetonitrile containing formic acid and water into a plastic centrifuge tube, filtering the eluent, placing the filtered eluent into a plastic sample bottle, and marking the solution as a solution S for later use;

adding known amount of aminoglycoside drug into the blank matrix solution to prepare a matrix matching mixed standard solution;

and step 3: high performance liquid chromatography-tandem mass spectrometry

And (3) respectively separating the aminoglycoside medicines in the solution S and the matrix matching mixed standard solution by adopting an SIELC ObeliscR chromatographic column and a Hypercarb chromatographic column, determining by adopting a tandem mass spectrum, drawing a standard working curve according to the determination result of the matrix matching mixed standard solution, and calculating the content of the aminoglycoside medicines to be determined in the sample.

Further, the bee product is honey or royal jelly.

Further, the aminoglycoside drug is composed of a compound M and a compound N, wherein the compound M is at least one of streptomycin, dihydrostreptomycin or spectinomycin, and the compound N is at least one of neomycin, apramycin, tobramycin, gentamicin, kanamycin, hygromycin B, micronomicin, paromomycin, ribostamycin, amikacin, sisomicin, etimicin or netilmicin.

Further, the SIELC ObeliscR column in step 3 is suitable for separating compound M from the sample, and the Hypercarb column in step 3 is suitable for separating compound N from the sample.

Further, step 1 comprises: weighing 2g of honey sample or 1g of royal jelly sample into a 10mL plastic centrifuge tube, adding 5mL of 50mM phosphate buffer solution, adjusting the pH value of the phosphate buffer solution to 8.0, covering, whirling for 1min, centrifuging at 9000r/min for 5min, and taking the supernatant to pass through a column.

Further, step 2 comprises: SupelMIPSPE-amino-ligands solid phase extraction cartridge was activated with 1mL methanol, 1mL 50mM phosphate buffer (pH8.0) in that order before use. Taking all the supernatant fluid to pass through a column, and controlling the flow rate of the sample to be less than 1 mL/min; leaching with 3mL of water and 1mL of 0.1% ammonia water in sequence, then vacuum-drying for 5min, leaching with 1mL of acetonitrile/water solution, then vacuum-drying for 10s, leaching with 1mL of dichloromethane/methanol solution, and then vacuum-drying for 10s, wherein the flow rate of the whole leaching process is controlled to be less than 1 mL/min; finally, eluting the mixture into a 5mL plastic centrifuge tube by using 1.0mL acetonitrile/water solution containing 5 volume percent of formic acid, and controlling the elution flow rate to be less than 0.5 mL/min; the eluent is filtered by a 0.22 mu m filter membrane to a 2mL plastic sample bottle for the determination of a liquid chromatogram-tandem mass spectrometer.

Further, step 2 comprises: taking another blank matrix solution, and adding a known amount of aminoglycoside drug to ensure that: streptomycin, dihydrostreptomycin, tobramycin, hygromycin B, ribostamycin and sisomicin are respectively 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL and 100ng/mL, spectinomycin, neomycin, apramycin, gentamycin, kanamycin, micronomicin, paromomycin, amikacin, etimicin and netilmicin are respectively 20ng/mL, 50ng/mL, 100ng/mL, 250ng/mL and 500ng/mL, and are used as matrix matching mixed standard solutions.

Further, the chromatographic conditions for analyzing streptomycin, dihydrostreptomycin and spectinomycin in step 3 are as follows: a chromatographic column: SIELC Obelisc^TMR column (2.1 mm. times.100 mm,5 μm); flow rate: 0.4 mL/min; column temperature: 35 ℃; the sample injection amount is 10 mu L; mobile phase: a was 0.1 vol% formic acid solution and B was acetonitrile, and the elution was performed in the following gradient:

time in minutes	A, volume%	B, volume%
			0	5	95
2	5	95
			5	35	65
8	95	5
			14	95	5
14.1	5	95
			20	5	95

In step 3, the chromatographic conditions for neomycin, apramycin, tobramycin, gentamicin, kanamycin, hygromycin B, micronomicin, paromomycin, ribostamycin, amikacin, sisomicin, etimicin and netilmicin are analyzed as follows: a chromatographic column: hypercarb chromatography column (2.1 mm. times.150 mm,5 μm); flow rate: 0.35 mL/min; column temperature: 35 ℃; the sample injection amount is 20 mu L; mobile phase: a is 2.5% ammonia water solution, B is acetonitrile, and the gradient elution is carried out by the following steps:

time in minutes	A, volume%	B, volume%
			0	95	5
0.5	95	5
			9.0	5	95
9.1	95	5
			13.0	95	5

Further, the mass spectrum conditions in step 3 are as follows: ESI positive ion mode; monitoring multiple reactions; electrospray voltage: 5500.0V; ion source temperature: 550 ℃; atomizing gas pressure: 50.00 Psi; auxiliary gas pressure: 50.00 Psi; air curtain pressure: 35.00 Psi; collision gas pressure: medium; inlet voltage (EP): 10V; outlet voltage (CXP): 10V; residence time (Dwell): 20 ms; the ion pair, the de-clustering voltage (DP), and the Collision Energy (CE) were monitored as follows:

wherein is the quantitative ion pair.

Has the advantages that: the method for determining aminoglycoside drug residue in animal derived food can simultaneously detect the residue of 16 aminoglycosides in animal derived food, and the determination limit of streptomycin, dihydrostreptomycin, tobramycin, hygromycin B, ribostamycin and sisomicin in honey is 5 mug/kg; the determination lower limit of spectinomycin, neomycin, apramycin, gentamicin, kanamycin, micronomicin, paromomycin, amikacin, etimicin and netilmicin is 25 mug/kg; the lower limit of the determination of the streptomycin, dihydrostreptomycin, tobramycin, hygromycin B, ribostamycin and sisomicin in the royal jelly is 10 mug/kg; the determination limit of spectinomycin, neomycin, apramycin, gentamycin, kanamycin, micronomicin, paromomycin, amikacin, etimicin and netilmicin is 50 mug/kg.

Drawings

In order to illustrate the technical solution of the present invention more clearly, the drawings will be briefly described below, and it is apparent that the drawings in the following description relate only to some embodiments of the present invention and are not intended to limit the present invention.

FIG. 1 is a streptomycin standard spectrum provided in example 1 of the present invention;

FIG. 2 is a spectrum of a dihydrostreptomycin standard provided in example 1 of the present invention;

FIG. 3 is a spectinomycin standard spectrum provided in example 1 of the present invention;

FIG. 4 is a neomycin standard spectrum as provided in example 1 of the present invention;

FIG. 5 is an apramycin standard spectrum provided in example 1 of the present invention;

FIG. 6 is a spectrum of a tobramycin standard according to example 1 of the present invention;

fig. 7 is a spectrum of a standard spectrum of gentamicin provided in example 1 of the present invention;

FIG. 8 is a spectrum of kanamycin standard provided in example 1 of the present invention;

FIG. 9 is a spectrum of hygromycin B standard provided in example 1 of the present invention;

FIG. 10 is a micronomicin standard spectrum provided in example 1 of the present invention;

FIG. 11 is a spectrum of paromomycin standard according to example 1 of the present invention;

FIG. 12 is a ribostamycin standard spectrum provided in example 1 of the present invention;

FIG. 13 is an amikacin standard spectrum provided in example 1 of the present invention;

FIG. 14 is a sisomicin standard spectrum provided in accordance with an embodiment 1 of the present invention;

FIG. 15 is a graph of a standard spectrum of etimicin according to one embodiment 1 of the present invention;

fig. 16 is a spectrum of the netilmicin standard provided in example 1 of the present invention.

Detailed Description

The definitions of some of the terms used in the present invention are given below, and other non-mentioned terms have definitions and meanings known in the art:

the 16 aminoglycoside drugs selected by the invention comprise: streptomycin, dihydrostreptomycin, spectinomycin, neomycin, apramycin, tobramycin, gentamycin, kanamycin, hygromycin B, micronomicin, paromomycin, ribostamin, amikacin, sisomicin, etimicin, and netilmicin. The aminoglycoside antibiotic is formed by combining aminosugar and alkaline 1, 3-diaminoinositol by glycosidic bond, and the 1, 3-diaminoinositol is a basic polycyclocyclohexanol structure, so that the aminoglycoside antibiotic has the characteristics of alkalinity and strong polarity. The common reverse phase solid phase extraction cartridge is not easy to be reserved, so the invention selects the Oasis WCX solid phase extraction cartridge with weak cation exchange performance and the SupelMIPSPE-amino-ligands solid phase extraction cartridge with molecular imprinting function. The supel MIP solid phase extraction column is composed of highly cross-linked polymers, introducing selectivity during MIP synthesis, wherein a template molecule designed to mimic an analyte directs the formation of a specific cavity or imprint that is spatially and chemically complementary to the analyte of interest, allowing the extraction of a single target analyte or a class of structurally related analytes with extremely high selectivity. The extraction selectivity is obviously improved, so that the background noise is effectively reduced, and the lower detection limit is obtained. Experimental results show that the recovery rate of the 16 aminoglycoside drugs in the WCX extraction column is in the range of 40-90%, the recovery rate range of the MIP extraction column is over 85%, the selectivity of the molecular imprinting MIP column is better, and the sample purification effect is better. Therefore, SupelMIPSPE-amino-glycosides solid phase extraction column is finally selected to purify the animal-derived food.

The SupelMIPSPE-amino-glycosides solid phase extraction column has a set of recommended activation, elution and elution methods, the recommended eluent is acetonitrile/water (20: 80) solution of 1mL 1% formic acid containing 5mM heptafluorobutyric acid, the experiment optimizes the eluent, ion pair reagent heptafluorobutyric acid is not added, the formic acid concentration is increased, the recovery rates of the target compounds when the eluent contains 1%, 2%, 5% and 10% formic acid are compared, the experiment shows that when the formic acid content in the eluent is 1% or 2%, only part of aminoglycoside medicines can be eluted, the recovery rate is below 40%, and when the formic acid content in the eluent is more than or equal to 5%, 16 aminoglycoside medicines can be eluted, the recovery rates are more than 85%, so the invention selects acetonitrile/water (20: 80) solution of 5% formic acid as the elution solution of the MIP solid phase extraction column.

In the selection of the chromatographic column, the applicant considers that the polarity of the compound to be detected is stronger, the compound is not retained by using the common C18 column, and the compound is usually required to be added with an ion pair reagent in a mobile phase to obtain better retention. Under the condition of not adding ions to a reagent, most of the prior literature methods adopt a HILIC (hydrophilic interaction chromatography) column for separation, so that the inventor firstly selects Waters Antithiac, PhenomenexHILIC, Merck ZIC-Hilic and other hydrophilic interaction chromatography columns, tests are respectively carried out according to the conditions of the reference literature, only partial aminoglycoside compounds can generate peaks, but the sensitivity and the peak type are poor, and the accurate qualitative and quantitative determination of the compounds is influenced.

Through repeated experiments, the SIELC ObeliscR chromatographic column and the Hypercarb chromatographic column are both suitable for separating polar compounds, and the analysis of aminoglycosides by the two chromatographic columns is attempted. By optimizing the conditions of the mobile phase, the SIELC Obelisc R chromatographic column can have better retention and sensitivity on analysis of streptomycin, dihydrostreptomycin and spectinomycin, while the Hypercarb chromatographic column can have better retention and sensitivity on analysis of neomycin, apramycin, tobramycin, gentamycin, kanamycin, hygromycin B, micronomicin, paromomycin, ribostamycin, amikacin, sisomicin, etimicin and netilmicin. The two columns are combined for use, so that the analysis requirements of 16 aminoglycoside drugs can be met.

Since aminoglycosides have a pKa greater than 7 and are basic compounds, when the pH of the buffer is less than the pKa, the compounds exist in ionic form, and when the pH of the buffer is greater than the pKa, the compounds exist in neutral molecular form. The SIELC ObeliscR chromatographic column has a mixed ion exchange action mechanism, 0.1 percent of formic acid and acetonitrile are selected as a mobile phase, an ion exchange action mechanism exists between an analyte and a chromatographic column stationary phase, namely, under an acidic condition, an alkaline substance is in a cation state, the adsorption capacity on the stationary phase is stronger, the retention is stronger, and finally streptomycin, dihydrostreptomycin and spectinomycin can obtain more satisfactory retention and peak patterns on the SIELC ObeliscR chromatographic column. The Hypercarb chromatographic column is made of porous graphitized carbon, and is different from a chromatographic column made of a common silica gel material in that the chromatographic column has super-strong pH stability and can tolerate a mobile phase with a pH range of 0-14, so that ammonia water is used as the mobile phase in the method, and the pH value of the mobile phase is adjusted to gradually convert the aminoglycoside drug from an ionic state to a molecular state, thereby obtaining satisfactory retention. In the experiment, ammonia water with different concentrations of 0.1%, 1%, 2.5% and 5% is selected as a mobile phase, and the results show that 13 aminoglycoside medicaments such as neomycin, apramycin, tobramycin, gentamicin, kanamycin, hygromycin B, micronomicin, paromomycin, ribostamycin, amikacin, sisomicin, etimicin, netilmicin and the like can obtain more satisfactory retention and peak patterns when the mobile phase is 2.5% ammonia water.

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. In the following examples, "%" refers to volume percent, unless otherwise specified.

The main reagents used in the examples are as follows: methanol, acetonitrile, formic acid, ammonia water: are all chromatographically pure; dichloromethane, dipotassium hydrogen phosphate, phosphoric acid: are all analytically pure; the water is first-grade water specified in GB/T6682. 100mM phosphate buffer (pH 8.0): weighing 17.4g of dipotassium hydrogen phosphate, adding water to dissolve, fixing the volume to 1000mL, uniformly mixing, and adjusting the pH value to 8.0 by using phosphoric acid. 50mM phosphate buffer (pH 8.0): 100mM phosphate buffer (pH8.0) was diluted one time with water and mixed well. Standard substance: streptomycin (CAS 3810-74-0), dihydrostreptomycin (CAS 5490-27-7), spectinomycin (CAS64058-48-6), neomycin (CAS 1405-10-3), apramycin (CAS 65710-07-8), tobramycin (CAS32986-56-4), gentamycin (CAS 1405-41-0), kanamycin (CAS 25389-94-0), hygromycin B (CAS31282-04-9), micronomicin (CAS 66803-19-8), paromomycin (CAS 1263-89-4), ribostamycin (CAS53797-35-6), amikacin (CAS 37517-28-5), sisomicin (CAS 32385-11-8), etimicin (CAS59711-96-5), Netilmicin (CAS 56391-57-2). The standard substance needs to be converted according to the molecular formula and the purity.

Standard stock solution (100. mu.g/mL): accurately weighing appropriate amount of standard substances, respectively, preparing into standard stock solutions of 100 μ g/mL with water, and storing in refrigerator at 4 deg.C. Mix standard stock solution (1. mu.g/mL): 1.00mL of standard stock solution is accurately weighed into a 100mL volumetric flask, diluted to the scale with methanol and stored in a refrigerator at 4 ℃. Matrix standard working solution: the mixed standard stock solution is diluted to a proper concentration by a blank matrix solution and is prepared immediately before use.

The main instruments and equipment used in the examples are as follows: high performance liquid chromatography-tandem mass spectrometer: an electrical distribution spray ionization (ESI) source; balance: sensory amounts 0.1mg and 0.01 g; a high-speed centrifuge: the rotating speed is more than 9000 r/min; a constant-temperature water bath kettle; a vortex oscillator; a pH meter; a solid phase extraction device; SupelMIP amino-glycosides solid phase extraction cartridge (50mg/3 mL); 0.22 μm filter.

Example 1

Sample preparation

Taking 500g of a representative honey sample, forcibly stirring the sample without crystallization uniformly, plugging a sample bottle cap with the sample with crystallization, placing the sample bottle cap in a water bath at the temperature of not more than 60 ℃ for warming, stirring uniformly after the sample is completely melted, and rapidly cooling to room temperature. Care should be taken to prevent evaporation of moisture upon thawing. The prepared sample is divided into two parts, and the two parts are respectively filled into sample bottles, sealed and marked with marks. One was used as a test sample and the other was stored at-18 ℃ or below.

Taking 500g of a representative royal jelly sample, unfreezing at room temperature, stirring uniformly after the sample is completely melted, dividing the sample into two parts, respectively filling the two parts into sample bottles, sealing and marking. One was used as a test sample and the other was stored at-18 ℃ or below.

During the sample preparation operation, contamination of the sample or changes in the content of residues should be prevented. Glassware was avoided throughout the analysis process.

In the present embodiment, the honey sample and the royal jelly sample are taken as examples, and do not limit the application range of the present invention, and the present invention is also applicable to other animal-derived foods, such as meat, aquatic products, eggs, milk and the like, and the extraction is performed according to the existing sample pretreatment method or the sample preparation method of the bee product when in use.

The method for determining the residue condition of the aminoglycoside drug in the sample comprises the following steps:

step 1: extracting residual aminoglycoside drug in sample

Weighing 2g Mel sample or 1g Lac Regis Apis sample (accurate to 0.01g) into 10mL plastic centrifuge tube, adding 5mL50mM phosphate buffer (pH8.0), covering, vortexing for 1min, centrifuging at 9000r/min for 5min, and collecting supernatant to be passed through column.

A blank matrix solution was additionally prepared: weighing 2g of negative honey sample or 1g (accurate to 0.01g) of royal jelly sample, adding 5mL of 50mM phosphate buffer (pH8.0) into a 10mL plastic centrifuge tube, covering, vortexing for 1min, centrifuging at 9000r/min for 5min, and collecting supernatant to pass through a column.

Step 2: enrichment and purification of solid phase extraction column

SupelMIPSPE-amino glycosides solid phase extraction cartridge was activated with 1mL methanol, 1mL 50mM phosphate buffer (pH8.0) in that order before use; taking all the supernatant fluid to pass through a column, and controlling the flow rate of the sample to be less than 1 mL/min; leaching with 3mL of water and 1mL of 0.1% ammonia water in sequence, then vacuum-drying for 5min, leaching with 1mL of acetonitrile/water solution, then vacuum-drying for 10s, leaching with 1mL of dichloromethane/methanol solution, and then vacuum-drying for 10s, wherein the flow rate of the whole leaching process is controlled to be less than 1 mL/min; finally, eluting the mixture into a 5mL plastic centrifuge tube by using 1.0mL acetonitrile/water solution containing 5 volume percent of formic acid, and controlling the elution flow rate to be less than 0.5 mL/min; the eluent is filtered by a 0.22 mu m filter membrane to a 2mL plastic sample bottle for the determination of a liquid chromatogram-tandem mass spectrometer.

And step 3: high performance liquid chromatography-tandem mass spectrometry

1.1 liquid chromatography conditions 1 (for streptomycin, dihydrostreptomycin and spectinomycin)

a) A chromatographic column: SIELC ObeliscR column (2.1 mm. times.100 mm,5 μm);

b) mobile phase: a was 0.1% formic acid solution and B was acetonitrile, and the elution was performed in gradient, the procedure of which is shown in the following table.

TABLE 1 gradient elution procedure

c) Flow rate: 0.4 mL/min;

d) column temperature: 35 ℃;

e) the sample injection amount is 10 mu L.

1.2 liquid chromatography Condition 2 (for neomycin, apramycin, tobramycin, gentamicin, kanamycin, hygromycin B, micronomicin, paromomycin, ribostamycin, amikacin, sisomicin, etimicin, and netilmicin)

a) A chromatographic column: hypercarb chromatography column (2.1 mm. times.150 mm,5 μm);

b) mobile phase: a is 2.5% ammonia water solution, B is acetonitrile, gradient elution, elution procedure is shown in the following table.

TABLE 2 gradient elution procedure

Time(min)	A(％)	B(％)
			0	95	5
0.5	95	5
			9.0	5	95
9.1	95	5
			13.0	95	5

c) Flow rate: 0.35 mL/min;

d) column temperature: 35 ℃;

e) the sample injection amount is 20 mu L.

1.3 Mass Spectrometry conditions:

a) an ion source: electrospray Source (ESI), positive ion mode;

b) and (3) monitoring mode: multiple Reaction Monitoring (MRM);

c) ESI positive ion mode; multiple Reaction Monitoring (MRM);

d) electrospray voltage (IS): 5500.0V; ion source Temperature (TEM): 550 ℃;

e) atomization gas pressure (GS 1): 50.00 Psi; assist gas pressure (GS 2): 50.00 Psi; air curtain pressure (CUR): 35.00 Psi; collision gas pressure (CAD): medium;

f) inlet voltage (EP): 10V; outlet voltage (CXP): 10V; residence time (Dwell): 20 ms; the ion pair, the de-clustering voltage (DP), and the Collision Energy (CE) were monitored as shown in Table 3.

Ion pair and declustering voltage and collision energy for monitoring 316 aminoglycoside drugs in table

(is a quantitative ion pair)

And respectively adding a proper amount of mixed standard stock solution into the blank matrix solution to prepare a matrix matching mixed standard solution series, preparing a standard curve according to the conditions of the steps 1.1-1.3, and quantifying the residual medicine in the sample by using the standard working curve.

Qualitative determination

1 parent ion and more than 2 daughter ions are selected for each component to be tested, and the deviation of the retention time of a substance to be tested in a sample and the retention time of a standard calibration solution with corresponding concentration in a matrix matching mixed standard solution is within +/-2.5 percent under the same experimental conditions; and comparing the relative abundance of each component qualitative ion in the sample spectrogram with the relative abundance of the corresponding qualitative ion in the matrix matching mixed standard solution spectrogram with the approximate concentration, wherein the deviation does not exceed the range specified in table 4, and determining that the corresponding object to be detected exists in the sample.

TABLE 4 maximum permissible deviation of relative ion abundance in qualitative confirmation

Relative ion abundance	＞50％	More than 20% to 50%	More than 10% to 20%	≦10％
					Maximum deviation allowed	±20％	±25％	±30％	±50％

Quantitative determination

Respectively injecting samples by using mixed matrix standard working solutions, taking the peak area ratio of an analysis compound as a vertical coordinate and the concentration ratio of the analysis compound as a horizontal coordinate as a standard working curve, and quantifying the samples by using the standard working curve, wherein the response values of the 16 aminoglycoside medicines in the standard working solution and the solution to be detected are within the linear response range of the instrument. The parent and daughter ions of the 16 aminoglycoside are shown in Table 3.

In the case of blank test, the measurement was carried out in the same manner as described above except that no sample was added.

Result calculation and presentation

The residual quantity of the aminoglycoside drug in the sample is calculated by a data processing system or calculated according to the formula (1):

in the formula:

x is the residue of the component to be measured in micrograms per kilogram (mug/kg) of the sample;

c-concentration of the measured component solution in nanograms per milliliter (ng/mL) from the standard working curve;

v is the final volumetric volume of the sample solution in milliliters (mL);

m-the mass of the final sample represented by the sample solution in grams (g).

Blank values are subtracted from the calculation result, and standard spectrograms of the 16 aminoglycoside drugs are shown in the attached drawing of the specification, wherein, a streptomycin standard spectrogram is shown in figure 1; FIG. 2 is a spectrum of a dihydrostreptomycin standard; FIG. 3 is a spectinomycin standard spectrum; FIG. 4 is a neomycin standard spectrum; FIG. 5 is an apramycin standard spectrum; FIG. 6 is a tobramycin standard spectrum; FIG. 7 is a spectrum of a gentamicin standard; FIG. 8 is a chart of kanamycin standards; FIG. 9 is a hygromycin B standard spectrum; FIG. 10 is a micronomicin standard spectrum; FIG. 11 is a paromomycin standard spectrum; FIG. 12 is a ribostamycin standard spectrum; FIG. 13 is an amikacin standard spectrum; FIG. 14 is a sisomicin standard spectrum; FIG. 15 is an etimicin standard spectrum; figure 16 is a netilmicin standard spectrum. As can be seen from the figure, under the condition that the ion pair reagent is not added, the method can better separate 16 aminoglycoside drugs, and has the advantages of high detection sensitivity, good peak pattern and strong response.

Example 2 Linear Range and Linear equation

Referring to the method of example 1, a blank honey base solution was taken and a known amount of aminoglycoside was added such that: streptomycin, dihydrostreptomycin, tobramycin, hygromycin B, ribostamycin and sisomicin are respectively 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL and 100ng/mL, spectinomycin, neomycin, apramycin, gentamycin, kanamycin, micronomicin, paromomycin, amikacin, etimicin and netilmicin are respectively 20ng/mL, 50ng/mL, 100ng/mL, 250ng/mL and 500ng/mL, and the streptomycin, dihydrostreptomycin, tobramycin B, ribostamycin and sisomicin are used as matrix matching mixed standard solutions to perform liquid chromatography determination, and response peak areas (Y axis) of quantitative ions are plotted against corresponding mass concentrations (X axis and ng/mL), and the results are shown in Table 5. The result shows that 16 aminoglycoside drugs have good linear relation between the response peak area of the quantitative ions and the sample concentration in the corresponding linear range.

TABLE 5 Linear equation and correlation coefficient for aminoglycoside drugs

Example 3 determination of lower limits of determination

A series of working standard solutions of 16 aminoglycoside drug mixed matrixes with lower mass concentration are selected, the working standard solutions are processed according to the method in the embodiment 1, the analytical detection is carried out under the chromatographic condition, and the determination lower limit is calculated.

The determination finds that the determination limit of the method for streptomycin, dihydrostreptomycin, tobramycin, hygromycin B, ribostamycin and sisomicin in honey is 5 mug/kg; the determination limit of spectinomycin, neomycin, apramycin, gentamycin, kanamycin, micronomicin, paromomycin, amikacin, etimicin and netilmicin is 25 mug/kg. The method has the determination lower limit of 10 mug/kg for the streptomycin, dihydrostreptomycin, tobramycin, hygromycin B, ribostamycin and sisomicin in royal jelly; the determination limit of spectinomycin, neomycin, apramycin, gentamycin, kanamycin, micronomicin, paromomycin, amikacin, etimicin and netilmicin is 50 mug/kg.

Example 4 recovery of addition and precision experiments

Weighing 2.0g of blank honey sample and 1.0g of blank royal jelly sample respectively, adding 1, 2 and 10 times of mixed standard working solution of aminoglycoside drugs for determining low limit, and preparing 6 parallel samples at each level. The operation is carried out according to the measuring steps in the laboratory method, the quantification is carried out by an external standard method, and the adding recovery rate of each sample is calculated.

The adding recovery rate of streptomycin, dihydrostreptomycin, tobramycin, hygromycin B, ribostamycin and sisomicin in honey is as follows:

when the addition level is 5 mug/kg, the recovery rate ranges from 73.3 to 97.7 percent;

when the addition level is 10 mug/kg, the recovery rate ranges from 74.1 to 96.6 percent;

when the addition level is 50 mug/kg, the recovery rate ranges from 72.6 to 99.4 percent.

The conditions of the addition recovery rates of spectinomycin, neomycin, apramycin, gentamycin, kanamycin, micronomicin, paromomycin, amikacin, etimicin and netilmicin in honey are as follows:

when the addition level is 25 mug/kg, the recovery rate ranges from 72.5 to 96.3 percent;

when the addition level is 50 mug/kg, the recovery rate ranges from 72.7 to 98.8 percent;

the recovery ranged from 73.4 to 95.5% at an add-on level of 250. mu.g/kg.

The adding recovery rate of the royal jelly streptomycin, dihydrostreptomycin, tobramycin, hygromycin B, ribostamycin and sisomicin is as follows:

when the addition level is 10 mug/kg, the recovery rate ranges from 70.5 to 94.2 percent;

when the addition level is 20 mug/kg, the recovery rate ranges from 73.3 to 97.9 percent;

when the addition level is 100 mug/kg, the recovery rate ranges from 72.6 to 96.4 percent.

The conditions of the addition recovery rate of spectinomycin, neomycin, apramycin, gentamycin, kanamycin, micronomicin, paromomycin, amikacin, etimicin and netilmicin in the royal jelly are as follows:

when the addition level is 50 mug/kg, the recovery rate ranges from 71.5 to 92.6 percent;

when the addition level is 100 mug/kg, the recovery rate ranges from 72.7 to 90.3 percent;

when the addition level is 500. mu.g/kg, the recovery rate ranges from 70.8 to 94.1%.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Claims

1. A method for determining aminoglycoside drug residue in animal derived food comprises the following steps:

step 1: extracting residual aminoglycoside drug in sample

Putting the sample into a plastic centrifuge tube, adding phosphate buffer solution, whirling, centrifuging, and taking supernatant for later use;

taking another negative sample without aminoglycoside drugs, putting the negative sample into a plastic centrifuge tube, adding phosphate buffer solution, whirling, centrifuging, and taking supernatant as blank matrix solution;

step 2: enrichment and purification of solid phase extraction column

Activating a SupelMIPSPE-amino-ligands solid phase extraction column by using methanol and phosphate buffer solution in sequence before use; passing the supernatant obtained by the sample through a solid phase extraction column, leaching with water and ammonia water in sequence, and then vacuum-pumping to dry; then leaching by using a mixed solution of acetonitrile and water, and then performing vacuum pumping; then, leaching by using a mixed solution of dichloromethane and methanol, and then, vacuumizing and drying; finally, eluting the mixture of acetonitrile containing formic acid and water into a plastic centrifuge tube, filtering the eluent, placing the filtered eluent into a plastic sample bottle, and marking the solution as a solution S for later use; the mixed solution of the acetonitrile containing formic acid and water is an acetonitrile/water solution containing 5 volume percent of formic acid;

and step 3: high performance liquid chromatography-tandem mass spectrometry

Respectively separating aminoglycoside drugs in the solution S and the matrix matching mixed standard solution by using a SIELC ObeliscR chromatographic column and a Hypercarb chromatographic column, determining by using a tandem mass spectrum, drawing a standard working curve according to the determination result of the matrix matching mixed standard solution, and calculating the content of the aminoglycoside drugs to be determined in the sample;

the chromatographic conditions for analyzing streptomycin, dihydrostreptomycin and spectinomycin in the step 3 are as follows: a chromatographic column: SIELC ObeliscR column, 2.1mm X100 mm,5 μm; flow rate: 0.4 mL/min; column temperature: 35 ℃; sample introduction amount: 10 mu L of the solution; mobile phase: a was 0.1 vol% formic acid solution and B was acetonitrile, and the elution was performed in the following gradient:

In step 3, the chromatographic conditions for neomycin, apramycin, tobramycin, gentamicin, kanamycin, hygromycin B, micronomicin, paromomycin, ribostamycin, amikacin, sisomicin, etimicin and netilmicin are analyzed as follows: a chromatographic column: hypercarb column, 2.1mm x 150mm,5 μm; flow rate: 0.35 mL/min; column temperature: 35 ℃; sample introduction amount: 20 mu L of the solution; mobile phase: a is 2.5 volume percent ammonia water solution, B is acetonitrile, and the elution procedure is as follows:

The mass spectrum conditions in step 3 are as follows: ESI positive ion mode; monitoring multiple reactions; electrospray voltage: 5500.0V; ion source temperature: 550 ℃; atomizing gas pressure: 50.00 Psi; auxiliary gas pressure: 50.00 Psi; air curtain pressure: 35.00 Psi; collision gas pressure: medium; inlet voltage: 10V; outlet voltage: 10V; retention time: 20 ms; the ion pair, declustering voltage, and collision energy were monitored as follows:

wherein is a quantitative ion pair;

the animal-derived food is honey or royal jelly, and the aminoglycoside drug is composed of a compound M and a compound N, wherein the compound M is streptomycin, dihydrostreptomycin and spectinomycin, and the compound N is neomycin, apramycin, tobramycin, gentamicin, kanamycin, hygromycin B, micronomicin, paromomycin, ribostamycin, amikacin, sisomicin, etimicin and netilmicin; the SIELC ObeliscR column in step 3 is suitable for separating compound M from the sample, and the Hypercarb column in step 3 is suitable for separating compound N from the sample.

2. The method according to claim 1, wherein the method comprises the steps of: the step 1 comprises the following steps: weighing 2g of honey sample or 1g of royal jelly sample into a 10mL plastic centrifuge tube, adding 5mL of 50mM phosphate buffer solution, adjusting the pH value of the phosphate buffer solution to 8.0, covering, whirling for 1min, centrifuging for 5min at 9000r/min, and taking the supernatant to pass through a column.

3. The method according to claim 1, wherein the method comprises the steps of: the step 2 comprises the following steps: SupelMIPSPE-amino-ligands solid phase extraction column was activated with 1mL methanol, 1mL 50mM phosphate buffer solution at pH 8.0; taking all the supernatant fluid to pass through a column, and controlling the flow rate of the sample to be less than 1 mL/min; leaching with 3mL of water and 1mL of 0.1% ammonia water in sequence, then vacuum-drying for 5min, leaching with 1mL of acetonitrile/water solution, then vacuum-drying for 10s, leaching with 1mL of dichloromethane/methanol solution, and then vacuum-drying for 10s, wherein the flow rate of the whole leaching process is controlled to be less than 1 mL/min; finally, eluting the mixture into a 5mL plastic centrifuge tube by using 1.0mL acetonitrile/water solution containing 5 volume percent of formic acid, and controlling the elution flow rate to be less than 0.5 mL/min; the eluent is filtered by a 0.22 mu m filter membrane to a 2mL plastic sample bottle for the determination of a liquid chromatogram-tandem mass spectrometer.

4. The method according to claim 1, wherein the method comprises the steps of: the step 2 comprises the following steps: taking another blank matrix solution, and adding a known amount of aminoglycoside drug to ensure that: streptomycin, dihydrostreptomycin, tobramycin, hygromycin B, ribostamycin and sisomicin are respectively 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL and 100ng/mL, spectinomycin, neomycin, apramycin, gentamycin, kanamycin, micronomicin, paromomycin, amikacin, etimicin and netilmicin are respectively 20ng/mL, 50ng/mL, 100ng/mL, 250ng/mL and 500ng/mL, and are used as matrix matching mixed standard solutions.