Method for simultaneously detecting florfenicol and florfenicol amine and application thereof

Abstract

This application discloses a method for the simultaneous detection of florfenicol and florfenicolamine. After spiked with florfenicol-D3 and florfenicolamine-D3, the sample is derivatized using phenyl isothiocyanate. Quantitative analysis is then performed using an internal standard method combined with ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-QTS-MS / MS). This method enables the simultaneous detection of florfenicol and florfenicolamine in negative ion mode, achieving detection limits of 0.5 μg / kg and quantitation limits of 1.0 μg / kg for both. The method offers rapid analysis and high sensitivity, providing a novel technical means for the detection of florfenicol and its metabolite florfenicolamine.

Description

Technical Field

[0001] This application relates to the field of chromatographic detection technology, and in particular to a method for the simultaneous detection of florfenicol and florfenicolamine and its application. Background Technology

[0002] Florfenicol (FF) belongs to the amide alcohol class of broad-spectrum antibacterial drugs. It has a strong inhibitory effect on most Gram-positive and Gram-negative bacteria, as well as mycoplasma, in animals. Compared with other amide alcohol drugs (chloramphenicol and thiamphenicol), FF has strong antibacterial ability and low toxicity, and is currently commonly used in the livestock, poultry, and aquaculture industries for the treatment of bacterial diseases.

[0003] Existing research indicates that FF possesses certain embryotoxicity and hematologic toxicity. Its residues in animal-derived foods may induce bacterial resistance and cause dysbiosis in the human gut microbiota. The main metabolite of FF in animals is florfenicol (FFA), which lacks antibacterial activity and has a long residual time. GB 31650—2019, "Maximum Residue Limits for Veterinary Drugs in Food," calculates the maximum residue limit (MRL) for this type of substance in animal-derived foods based on the sum of the contents of FF and its metabolite FFA. To scientifically regulate drug use in animal husbandry and strictly control withdrawal periods, my country and the EU, among other countries, have stipulated the MRLs for FF and FFA in animal tissues (muscle, skin, liver, and kidneys, etc.), and prohibit the use of FF in cattle or sheep during lactation. Therefore, to meet relevant standards and regulations, it is necessary to establish a rapid, accurate, and quantitative detection method for simultaneously determining FF and its metabolite FFA.

[0004] Currently, commonly used detection methods for FF and FFA mainly include high-performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography-mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS), and immunoassay (IA). Among these, immunoassay is simple to operate and highly sensitive, but when measuring tissue samples, it often requires converting FFA to FF first, and it cannot be quantified separately, leading to false positives or false negatives. Among the many chromatographic methods mentioned above, LC-MS / MS remains the primary technical means relied upon for administrative supervision due to its high selectivity and fast separation speed.

[0005] To strengthen the monitoring of florfenicol and its metabolite florfenicol (FFA) residues, my country has successively promulgated national food safety standards GB 31658.5-2022 "Determination of Florfenicol and Florfenicolamine Residues in Animal-Derived Foods by Liquid Chromatography-Tandem Mass Spectrometry" and GB 31658.20-2022 "Determination of Amide Alcohol Drugs and Their Metabolites Residues in Animal-Derived Foods by Liquid Chromatography-Tandem Mass Spectrometry." However, due to differences in molecular structure, the national standard method requires simultaneous switching between positive and negative ion electrospray ionization (ESI) modes for FFA and FF data acquisition. Furthermore, because FFA molecules are highly polar, they are not easily retained on ordinary C18 columns, resulting in poor peak shape and susceptibility to interference from impurities in quantitative detection results. To improve the chromatographic behavior of FFA, some researchers have attempted to use functional C18 columns, such as the Eclipse Plus C18 column suitable for separating basic compounds and the Kinetex F5 column suitable for separating highly polar compounds. However, when mass spectrometry collects both positive and negative ions simultaneously, it is difficult to achieve optimal response values ​​for both FF and FFA simultaneously by adding acid, alkali, or ammonium acetate to the aqueous mobile phase.

[0006] Therefore, there is a need for a method that can simultaneously determine florfenicol and its metabolite florfenicolamine, providing a new technical means to ensure food safety. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and provide a method for simultaneously detecting florfenicol and florfenicolamine, and its application.

[0008] The primary objective of this invention is to provide a sample pretreatment method for the simultaneous detection of florfenicol and florfenicolamine.

[0009] A second objective of this invention is to provide a method for simultaneously detecting florfenicol and florfenicolamine.

[0010] A third objective of this invention is to provide the application of the above method in the simultaneous detection of florfenicol and florfenicolamine.

[0011] To achieve the above objectives, the present invention is implemented through the following solution: A sample pretreatment method for simultaneous detection of florfenicol and florfenicolamine, specifically comprising: Florfenicol-D3 and florfenicolamine-D3 were added to the test sample at a final concentration of 2–10 μg / L as internal standards. The spiked test sample was extracted with ammoniated acetonitrile and purified by solid-phase extraction column. The purified solution was collected, mixed with derivatization reagent, and fully derivatized to obtain the derivatized test sample. The derivatizing reagent is phenyl isothiocyanate.

[0012] The derivatization reaction involves reacting florfenicol and florfenicol-D3 in the purified liquid to produce florfenicol derivatives and florfenicol-D3 derivatives.

[0013] Preferably, the sample to be tested is milk.

[0014] Preferably, extraction is performed using ammoniated acetonitrile with a final volume concentration of 2%.

[0015] More preferably, the extraction is vortex extraction.

[0016] Preferably, the solid phase extraction column is an Oasis PRiME HLB solid phase extraction column.

[0017] Preferably, the purification solution and the derivatizing reagent are mixed in a volume ratio of 3-7:7-3, and the concentration of the derivatizing reagent is 75-125 mM.

[0018] More preferably, the purification solution and the derivatization reagent are mixed evenly at a volume ratio of 1:1.

[0019] More preferably, the concentration of the derivatizing reagent is 100 mM.

[0020] More preferably, the purification solution is obtained by drying the effluent after purification by the solid phase extraction column and then resolving it in a Na2CO3-NaHCO3 buffer solution; the concentration of the Na2CO3-NaHCO3 buffer solution is 20 mM and the pH is 9.0.

[0021] Preferably, the derivatization reaction is carried out at 20–40°C in the dark for 5–100 min.

[0022] More preferably, the derivatization reaction is carried out at 25°C in the dark for 60 minutes.

[0023] Preferably, after the derivatization reaction has been fully carried out, the supernatant is collected by solid-liquid separation of the article after the derivatization reaction to obtain the derivatized test sample.

[0024] More preferably, the solid-liquid separation specifically involves centrifuging the fully reacted material at 12,000 r / min, filtering it using a filter membrane, and collecting the supernatant.

[0025] This invention claims protection for a method for simultaneously detecting florfenicol and florfenicolamine, comprising the following steps: S1. Process the sample to be tested according to any of the sample pretreatment methods described above to obtain the derivatized sample to be tested; S2. The derivatized test sample obtained in step S1 was quantitatively analyzed by using the internal standard method combined with ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry to obtain the content of florfenicol and florfenicolamine in the test sample.

[0026] Before performing ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-QFS / Q-MS), the method of this invention first uses phenyl isothiocyanate (PITC) as a derivatizing reagent to derivatize florfenicol in the sample to be tested, as shown in the sample pretreatment method above. This ensures that florfenicol (FF) and florfenicol (FFA) can be detected simultaneously in negative ion mode during the detection process. However, when the derivatizing reagent is adjusted to phenyl 4-chloroisothiocyanate and phenyl 3,5-dichloroisothiocyanate, the florfenicol derivative florfenicol (PFFA) cannot be effectively derivatized, and the simultaneous detection of FF and FFA in one ion mode cannot be achieved after derivatization.

[0027] Preferably, in step S2, when performing quantitative analysis using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry, the chromatographic conditions are as follows: using a C18 column, controlling the column temperature at 25–45°C, the injection volume at 2–10 μL, the flow rate at 0.2–0.4 mL / min, mobile phase A being methanol or acetonitrile, and mobile phase B being water; The elution program was as follows: 0–1 min, 10% mobile phase A; 1–4 min, 10%–90% mobile phase A; 4–5.5 min, 90% mobile phase A; 5.5–6 min, 90%–10% mobile phase A; 6–7 min, 10% mobile phase A.

[0028] More preferably, the mobile phase A is methanol.

[0029] More preferably, the chromatographic conditions are controlled as follows: column temperature 35°C, injection volume 10 μL, and flow rate 0.3 mL / min.

[0030] Preferably, in step S2, when performing quantitative analysis using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry, the mass spectrometry conditions are as follows: ESI negative ion mode scanning, capillary voltage of 0.5–3.0 kV, ion source temperature of 100–150 °C, cone voltage of 20–80 V, cone gas flow rate of 20–100 L / h, desolvation gas temperature of 300–800 °C, desolvation gas flow rate of 600–1000 L / h, scanning range m / z of 100–600 Da, low collision energy channel voltage of 4 eV, and high collision energy channel voltage of 10–45 eV.

[0031] More preferably, the mass spectrometry conditions are as follows: ESI negative ion mode scanning, capillary voltage of 1.0 kV, ion source temperature of 120℃, cone voltage of 40 V, cone gas flow rate of 50 L / h, desolvation gas temperature of 550℃, and desolvation gas flow rate of 900 L / h.

[0032] Preferably, in the quantitative analysis described in step S2, when quantifying florfenicol, florfenicol-D3 is used as an internal standard, and the content of florfenicol in the sample to be tested is calculated by combining the florfenicol quantitative analysis formula; The quantitative analysis formula for florfenicol is obtained by combining the results of ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry of samples with known florfenicol content, with the florfenicol content in the sample as the abscissa and the ratio of the peak area of ​​florfenicol in the sample to the peak area of ​​florfenicol-D3 as the ordinate. When quantifying florfenicol, florfenicol-D3 is used as an internal standard, and the content of florfenicol in the sample is calculated by combining the quantitative analysis formula of florfenicol. The quantitative analysis formula for florfenicol is obtained by combining the results of ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry of samples with known florfenicol content. The formula uses the florfenicol content in the sample as the x-axis and the ratio of the peak area of ​​florfenicol to the peak area of ​​florfenicol-D3 as the y-axis.

[0033] More preferably, the quantitative analysis formula for florfenicol is shown in Formula I; Formula I: y = 0.551656x - 0.501299; Where y is the ratio of the peak area of ​​florfenicol to the peak area of ​​florfenicol-D3 in the sample to be tested, and x is the content of florfenicol in the sample to be tested. The quantitative analysis formula for florfenicol is shown in Formula II; Formula II: y' = 0.515312x' - 0.471481; Where y' is the ratio of the peak area of ​​the florfenicol derivative to the peak area of ​​the florfenicol-D3 derivative in the sample to be tested, and x' is the content of florfenicol in the sample to be tested.

[0034] More preferably, the retention times and mass spectrometry acquisition parameters of florfenicol, florfenicol-D3, florfenicol amine derivatives, and florfenicol amine-D3 derivatives in the sample to be tested are as follows:

[0035] The present invention also claims protection for the use of any of the methods described above in the simultaneous detection of florfenicol and florfenicolamine.

[0036] Preferably, the simultaneous detection of florfenicol and florfenicolamine is performed simultaneously in a milk sample.

[0037] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for the simultaneous detection of florfenicol and florfenicolamine. After spiked with florfenicol-D3 and florfenicolamine-D3, the sample is derivatized using phenyl isothiocyanate. Quantitative analysis is then performed using an internal standard method combined with ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-QTS-MS / MS). This method enables the simultaneous detection of florfenicol and florfenicolamine in negative ion mode, achieving detection limits of 0.5 μg / kg and quantitation limits of 1.0 μg / kg for both. The method offers rapid analysis and high sensitivity, providing a novel technical means for the detection of florfenicol and its metabolite florfenicolamine. Attached Figure Description

[0038] Figure 1 This is a diagram of the reaction process in Example 1 where the FFA derivatization reaction is converted into PFFA; Figure 2 The first-order mass spectrometry full scan results are shown in the negative ion mode in Example 2; a is the first-order mass spectrometry full scan results when 4-chloroisothiocyanate phenyl ester solution is used as the derivatization stock solution; b is the first-order mass spectrometry full scan results when 3,5-dichloroisothiocyanate phenyl ester solution is used as the derivatization stock solution. Figure 3 The images shown are full-scan mass spectrometry results from Example 2 when the detection was performed according to the method shown in Example 1; a is the first-stage mass spectrometry result; b is the second-stage mass spectrometry result. Figure 4 This is the chromatogram of the detection using methanol as mobile phase A in Example 2; Figure 5 This is the chromatogram of acetonitrile as mobile phase A in Example 2; Figure 6The figures show the peak areas of PFFA and PFFA-D3 during the optimization of derivatization reaction conditions in Example 2; a) peak areas of PFFA and PFFA-D3 in UHPLC-QT-MS after treatment with different concentrations of PITC derivatization solution; b) peak areas of PFFA and PFFA-D3 in UHPLC-QT-MS after different derivatization reaction times; c) peak areas of PFFA and PFFA-D3 in UHPLC-QT-MS after treatment with different concentrations of carbonate buffer; and d) peak areas of PFFA and PFFA-D3 in UHPLC-QT-MS under different volume ratios of derivatization treatment. Figure 7 The chromatograms are for LOD and LOQ detection in blank milk samples spiked with 0.5 μg / kg and 1.0 μg / kg, respectively, in Example 3. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0040] Example 1: A method for simultaneous detection of florfenicol and florfenicolamine in milk samples. I. Instruments and Reagents Instruments: Xevo G2-XS Q-TOF quadrupole-time-of-flight mass spectrometer (Waters Corporation, USA), Multifige X4R Pro benchtop high-speed centrifuge (Thermo Fisher Scientific, USA), 24-position solid-phase extraction apparatus (Thermo Fisher Scientific, USA), N-EVAP-111 nitrogen blower (Organomation Corporation, USA), MS3 vortex mixer (IKA GmbH, Germany).

[0041] Reagents: Florfenicol standard solution (FF, 100 mg / L, Tanmo Quality Inspection Technology Co., Ltd., A24030320), Florfenicol amide standard solution (FFA, 100 mg / L, Shanghai Anpu Technology Co., Ltd., 2417034), Florfenicol-D3 internal standard (FF-D3, 100 mg / L, Shanghai Anpu Technology Co., Ltd., 2355491), Florfenicol amide-D3 internal standard (FFA-D3, 100 mg / L, Shanghai Anpu) Technology Co., Ltd., 2577023), phenyl isothiocyanate (PITC, purity > 99%, Shanghai Maclean Biochemical Technology Co., Ltd., C16562553), phenyl 4-chloroisothiocyanate (Shanghai Aladdin Biochemical Technology Co., Ltd., purity > 98%, B2327636%), phenyl 3,5-dichloroisothiocyanate (Shanghai Aladdin Biochemical Technology Co., Ltd., purity > 99%, G23072182327636%), Oasis PRiME HLB solid phase extraction column (3mL / 60mg, Waters Corporation, USA, 01349245A), leucine enkephalin correction solution (200μg / L, Waters Corporation, USA); Methanol and acetonitrile were LC-MS grade reagents purchased from Merck, Germany; formic acid and ammonia were chromatographic grade reagents purchased from Thermo Fisher Scientific, USA.

[0042] II. Preparation of Standard Solutions Mixed standard working solution: Mix florfenicol standard solution and florfenicolamine standard solution and dilute with methanol to obtain mixed standard working solution (the final concentration of FF and FFA is 1.0 mg / L or 0.1 mg / L).

[0043] Mixed internal standard working solution: The florfenicol-D3 internal standard and the florfenicolamine-D3 internal standard were mixed and diluted with methanol to obtain the mixed internal standard working solution (the final concentration of both FF-D3 and FFA-D3 was 100 μg / L).

[0044] Derivatization stock solution: Place 2.4 mL of phenyl isothiocyanate in a 100 mL volumetric flask and dilute to the mark with acetonitrile to obtain a 200 mM derivatization stock solution (PITC solution).

[0045] III. Method for simultaneous detection of florfenicol and florfenicolamine in milk samples 1. Experimental Methods (1) Sample pretreatment: Take 1 g of milk sample (sample to be tested) and place it in a polytetrafluoroethylene centrifuge tube. Then add 50 μL of mixed internal standard working solution (the final concentration of FF-D3 and FFA-D3 is 100 μg / L). Vortex mix to obtain spiked milk sample. Then add 5 mL of ammoniated acetonitrile (2%, v / v). Vortex (using MS3 vortex mixer) for 5 min. Centrifuge at 4000 r / min at 4℃ for 5 min to obtain supernatant 1 and residue 1. Add residue 1 to 5 mL of ammoniated acetonitrile (2%, v / v). Vortex extract for 5 min. Centrifuge at 4000 r / min at 4℃ for 5 min. Collect supernatant 2 and combine it with supernatant 1 to obtain extract.

[0046] (2) Sample derivatization: Then, 10 mL of acetonitrile-saturated n-hexane was added to the extract obtained in (1), vortexed for 2 min, centrifuged at 4000 r / min for 5 min, and 5 mL of the lower layer liquid was transferred to an Oasis PRiME HLB solid phase extraction column for extraction and the eluent was collected. The eluent was dried under nitrogen in a 45℃ water bath and then redissolved in 500 μL of Na2CO3-NaHCO3 solution (20 mM, carbonate buffer, pH=9.0) to obtain 500 μL of purification solution. 500 μL of purification solution and 500 μL of derivatization solution (100 mM PITC solution) were mixed evenly and derivatized in the dark for 60 min. Then, the mixture was centrifuged at 12000 r / min, filtered through a 0.22 μm filter membrane and the supernatant was collected to obtain the derivatized milk sample. The derivatization reaction involves reacting florfenicolamine (FFA) and florfenicolamine-D3 (FFA-D3) to produce florfenicolamine derivatives (PFFA) and florfenicolamine-D3 derivatives (PFFA-D3). The reaction process for the derivatization of FFA to PFFA is as follows: Figure 1 As shown.

[0047] (3) Chromatographic mass spectrometry detection: The derivatized milk sample obtained in (2) was subjected to ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry using a Xevo G2-XS Q-TOF quadrupole-time-of-flight mass spectrometer (Waters Corporation, USA) to quantitatively analyze florfenicol and florfenicolamine in the milk sample; During the ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry process, the chromatographic conditions were as follows: using Phenomenex Kinetex C 18The column (100 mm × 2.1 mm, 2.6 μm) was controlled at 35 °C, the injection volume was 10 μL, the flow rate was 0.3 mL / min, the mobile phase A was methanol, and the mobile phase B was water; the elution program was: 0–1 min, 10% mobile phase A; 1–4 min, 10%–90% mobile phase A; 4–5.5 min, 90% mobile phase A; 5.5–6 min, 90%–10% mobile phase A; 6–7 min, 10% mobile phase A. During ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-TOF), the mass spectrometry conditions were as follows: ESI negative ion mode scanning, capillary voltage of 1.0 kV, ion source temperature of 120℃, cone voltage of 40 V, cone gas flow rate of 50 L / h, desolvation gas temperature of 550℃, desolvation gas flow rate of 900 L / h, scan range of m / z of 100–600 Da, low collision energy channel voltage of 4 eV, and high collision energy channel voltage of 10–45 eV. Q-TOF was calibrated in real time using leucine-enkephalin correction solution (200 μg / L, m / z 554.2615). Qualitative confirmation required successful matching of the precursor ion and a characteristic fragment ion, with a mass accuracy error of less than 5.0 × 10⁻⁶. -6 The retention time deviation is less than 0.2 min; for quantification, the chromatographic peak of the target analyte is integrated by the precise mass number of the primary precursor ion, the mass deviation is set to 50 mDa, and the internal standard is used for calibration and quantitative analysis.

[0048] Table 1 shows the retention times of the target analytes and the mass spectrometry acquisition parameters during ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry. Table 1 Target analyte retention time and mass spectrometry acquisition parameters

[0049] Based on the results of ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry, the florfenicol content (μg / kg) in the milk sample was calculated using Formula I, and the florfenicol amine content (μg / kg) in the milk sample was calculated using Formula II. Formula I: y = 0.551656x - 0.501299; Where y is the ratio of the peak area of ​​florfenicol (FF) to the peak area of ​​florfenicol-D3 (FF-D3) during ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry, and x is the content of florfenicol in the sample to be tested (μg / kg). Formula II: y' = 0.515312x' - 0.471481; Where y' is the ratio of the peak area of ​​florfenicolamine derivative (PFFA) to the peak area of ​​florfenicolamine-D3 derivative (PFFA-D3) during ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry, and x' is the content of florfenicolamine in the sample to be tested (μg / kg).

[0050] Example 2: Investigation of a method for simultaneous detection of florfenicol and florfenicolamine in milk samples I. Investigation of Derivatizing Reagents 1. Experimental Methods As shown in "III. Method for Simultaneous Detection of Florfenicol and Florfenicolamine in Milk Samples" in Example 1, Yantang Pure Milk (batch number: 20251030A) was used as the test sample. Florfenicol and florfenicolamine-D3 with a final concentration of 5 μg / L and 5 μg / L respectively were added to the test sample and then detected. The mass spectra during the ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry process were recorded.

[0051] Next, the derivatization stock solution in the sample derivatization process of "III. Method for Simultaneous Detection of Florfenicol and Florfenicol Amine in Milk Samples" in Example 1 was replaced sequentially with 200 mM phenyl 4-chloroisothiocyanate solution and 200 mM phenyl 3,5-dichloroisothiocyanate solution, while the rest remained unchanged. The mass spectra during the ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry process were recorded.

[0052] 2. Experimental Results The full scan results of primary mass spectrometry in negative ion mode are shown in the figure below. Figure 2 As shown, Figure 2 In the image, 'a' represents the first-order full-scan mass spectrometry result using phenyl 4-chloroisothiocyanate solution as the derivatization stock solution. Figure 2 Figure b shows the first-order full-scan mass spectrometry result when phenyl 3,5-dichloroisothiocyanate solution was used as the derivatization stock solution. The results show that after replacing the derivatization stock solution (PITC solution) with equal concentrations of phenyl 4-chloroisothiocyanate and phenyl 3,5-dichloroisothiocyanate, no florfenicolamine derivative (PFFA, theoretically...) was detected in the mass spectrum. m / z The generation of 381.1083 indicates that phenyl 4-chloroisothiocyanate and phenyl 3,5-dichloroisothiocyanate cannot effectively derivatize florfenicol, thus failing to achieve simultaneous detection of florfenicol and florfenicol in a single ion mode.

[0053] The mass spectrometry full scan result when the detection was performed according to the method shown in Example 1 is shown in the figure below. Figure 3 As shown, Figure 3 In the image, 'a' represents the result of a first-order mass spectrometry test. Figure 3b in the figure represents the result of a secondary mass spectrometry test; the results show that when using PITC solution as the derivatization stock solution (i.e., according to the method shown in Example 1), the measured mass-to-charge ratio of the PFFA precursor ion is [value missing]. m / z 381.1083 ( Figure 3 (a) In the middle, a series of isotope peaks near this peak m / z 382.1127 and m / z 383 and 1064 are consistent with the theoretical molecular weight of the compound (PFFA); by applying collision energies of 10–45 eV to the quasi-molecular ion peak of PFFA, fragment ions were obtained, and secondary mass spectrometry showed that ( Figure 3 (b) In negative electron mode, PFFA and FF exhibit similar mass spectrometry fragmentation patterns, with fragment ion 361.0997 fragmenting in [MH-HF]. - The fragment ion at 185.0458 is [CH3SO2-C6H4-CHOH]. - This indicates that PITC can effectively derivatize florfenicol, thereby enabling the simultaneous detection of florfenicol and florfenicol in a single ion mode.

[0054] II. Mobile Phase Optimization 1. Experimental Methods As shown in "III. Method for Simultaneous Detection of Florfenicol and Florfenicolamine in Milk Samples" in Example 1, Yantang Pure Milk (batch number: 20251030A) was used as the test sample. Florfenicol and florfenicolamine-D3 with a final concentration of 5 μg / L and 5 μg / L respectively were added to the test sample for detection, and the chromatograms during the ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry process were recorded.

[0055] Next, the mobile phase A in "III. Method for Simultaneous Detection of Florfenicol and Florfenicolamine in Milk Samples" of Example 1 was changed to acetonitrile and methanol, with the rest remaining unchanged. The chromatograms of the ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry process were recorded.

[0056] 2. Experimental Results The chromatogram when methanol is used as mobile phase A is as follows: Figure 4 As shown, the chromatogram when acetonitrile is used as mobile phase A for detection is as follows: Figure 5 As shown; the results indicate that when simultaneously detecting florfenicol and florfenicolamine using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry, methanol is used as the mobile phase A ( Figure 4 When water was used as mobile phase B, the separation efficiency of the target analytes (florfenicol, florfenicol-D3, florfenicol amine derivatives, and florfenicol amine-D3 derivatives) was higher, and the response value was significantly higher than when acetonitrile was used as mobile phase A. Figure 5 ).

[0057] III. Optimization of Derivatization Reaction Conditions 1. Experimental Methods As shown in "III. Method for Simultaneous Detection of Florfenicol and Florfenicolamine in Milk Samples" in Example 1, Yantang Pure Milk (batch number: 20251030A) was used as the test sample. Florfenicol and florfenicolamine-D3 with a final concentration of 5 μg / L and 5 μg / L respectively were added to the test sample for detection. The peak areas of derivatives PFFA and PFFA-D3 during the ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry process were recorded.

[0058] Next, the concentration of PITC derivatization solution in the sample derivatization process of "III. Method for simultaneous detection of florfenicol and florfenicolamine in milk samples" in Example 1 was replaced with 10, 20, 50, 100 and 200 mM, and the peak areas of derivatives PFFA and PFFA-D3 in the ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry process after treatment with PITC derivatization solution of different concentrations were recorded.

[0059] The derivatization reaction time in the sample derivatization process of "III. Method for Simultaneous Detection of Florfenicol and Florfenicolamine in Milk Samples" in Example 1 was adjusted to 5, 20, 40, 60, 80 and 100 min in sequence, and the peak areas of derivatives PFFA and PFFA-D3 in the ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry process at different derivatization reaction times were recorded.

[0060] In Example 1, "III. Method for Simultaneous Detection of Florfenicol and Florfenicolamine in Milk Samples", the concentration of carbonate buffer during sample derivatization was adjusted to 10, 20, 50, and 100 mM, and the peak areas of derivatives PFFA and PFFA-D3 were recorded during ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry after treatment with different concentrations of carbonate buffer.

[0061] Replace the volume ratio of the purification solution and the derivatization solution in the sample derivatization process of "III. Method for Simultaneous Detection of Florfenicol and Florfenicol Amine in Milk Samples" in Example 1 with 3:7, 4:6, 5:5, 6:4 and 7:3, and record the peak areas of derivatives PFFA and PFFA-D3 in the ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry process under different volume ratio derivatization processes.

[0062] 2. Experimental Results The peak area diagrams of PFFA and PFFA-D3 during the optimization of derivatization reaction conditions are shown below. Figure 6 As shown, Figure 6In the figure, 'a' represents the peak areas of derivatives PFFA and PFFA-D3 during ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-QT-MS / MS) after treatment with PITC derivatized solutions of different concentrations. Figure 6 In the figure, b represents the peak area of ​​derivatives PFFA and PFFA-D3 during ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry processes with different derivatization reaction times. Figure 6 In the figure, 'c' represents the peak areas of the derivatives PFFA and PFFA-D3 during ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry after treatment with different concentrations of carbonate buffer. Figure 6 In this context, d represents the peak areas of derivatives PFFA and PFFA-D3 during ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry under different volume ratios of derivatization treatment.

[0063] The results showed that: (1) As the concentration of PITC derivatizing solution increased, the mass spectrometry response of the derivatives continued to increase. The peak area of ​​PFFA and PFFA-D3 reached its maximum after the concentration of PITC derivatizing solution was increased to 100 mM. Further increasing the concentration of derivatizing agent had no significant effect. Therefore, the concentration of PITC derivatizing solution was selected to be 100 mM during the sample derivatization process.

[0064] (2) PITC derivatization solution can rapidly derivatize florfenicol. Obvious derivatives are generated when the reaction time is 5 min, but the peak area response value of the derivatives is low at this time. As the reaction time increases (5-60 min), the peak areas of PFFA and PFFA-D3 continue to increase. When the reaction time is 60 min, the peak areas of the derivatives (PFFA and PFFA-D3) reach their peak values. Further increasing the reaction time will reduce the peak area of ​​the derivatives to varying degrees. Therefore, the derivatization reaction time is 60 min during the sample derivatization process.

[0065] (3) During the sample derivatization process of the method shown in Example 1, the reaction system can proceed efficiently at lower carbonate buffer concentrations (10 mM and 20 mM), and the peak areas of the derivatives (PFFA and PFFA-D3) are high. As the carbonate buffer concentration increases, the derivatization reaction efficiency shows a significant decreasing trend. When the carbonate buffer concentration is increased to 100 mM, the peak area of ​​the derivative PFFA is only about one-third of that at 20 mM. In order to ensure the buffering capacity of the buffer, the optimal carbonate buffer concentration is 20 mM during the sample derivatization process.

[0066] (4) When the volume ratio of the purification solution to the derivatizing solution in the sample derivatization process is 6:4 and 7:3, the reaction efficiency is reduced due to the relatively insufficient content of the derivatizing solution in the system, resulting in a smaller amount of derivatives and a smaller corresponding chromatographic peak area. Under the condition that the volume ratio of the purification solution to the derivatizing solution in the sample derivatization process is 3:7 and 4:6, although the peak area of ​​the derivative is higher, the proportion of organic phase in the system increases because the solvent of the derivatizing reagent is acetonitrile, which may affect the solubility or stability of the derivative, resulting in a wider and asymmetrical chromatographic peak shape, poor peak area repeatability, and large deviation. In contrast, when the volume ratio of the purification solution to the derivatizing reagent in the sample derivatization process is 5:5, the chromatographic peak shape of the derivative is sharp and symmetrical, the peak area is larger, and the reproducibility is good. Therefore, in the sample derivatization process, the volume ratio of the purification solution to the derivatizing solution is 5:5 as the optimal derivatization conditions.

[0067] Example 3: Methodological validation of a method for the simultaneous detection of florfenicol and florfenicolamine in milk samples. I. Linear range and detection limit investigation 1. Experimental Methods Matrix-matched standard working solutions: Prepare matrix-matched standard working solutions of florfenicol and florfenicolamine with mass concentrations of 0.5, 1, 2, 5, 10, 20 and 50 μg / L respectively.

[0068] In each matrix-matched standard working solution of different concentrations, the mixed internal standard working solution from Example 1 was added to a final concentration of 5 μg / L to obtain matrix-matched standard working solutions of different concentrations after spiking.

[0069] Next, using the spiked matrix-matched standard working solutions at various concentrations as the test samples, the detection was carried out according to Example 1, "III. Method for Simultaneous Detection of Florfenicol and Florfenicolamine in Milk Samples," and the mass spectra of the spiked matrix-matched standard working solutions at various concentrations were recorded. Then, a florfenicol standard curve was plotted (Formula I) with the mass concentration of florfenicol in the spiked matrix-matched working solutions at various concentrations as the abscissa and the ratio of the peak area of ​​florfenicol to the peak area of ​​florfenicol-D3 in the mass spectrum as the ordinate. A florfenicol standard curve was plotted (Formula II) with the mass concentration of florfenicol in the spiked matrix-matched working solutions at various concentrations as the abscissa and the ratio of the peak area of ​​florfenicolamine derivative to the peak area of ​​florfenicolamine-D3 derivative in the mass spectrum as the ordinate.

[0070] The target analytes (florfenicol and florfenicolamine) at concentrations of 0.2–1.0 μg / kg were added to blank milk samples (milk samples that did not contain FF and FFA according to GB 31658.20-2022). Blank milk samples with different concentrations of florfenicol and florfenicolamine were used as test samples. The detection was carried out according to "III. Method for Simultaneous Detection of Florfenicol and Florfenicolamine in Milk Samples" in Example 1. The florfenicol and florfenicolamine were quantitatively detected by combining the florfenicol standard curve (Formula I) and the florfenicolamine standard curve (Formula II). The limit of detection (LOD) was determined by a signal-to-noise ratio (S / N) ≥ 3, and the limit of quantitation (LOQ) was determined by a signal-to-noise ratio (S / N) ≥ 10. The limits of detection and quantitation of florfenicol and florfenicolamine in milk samples by the method shown in Example 1 were obtained.

[0071] 2. Experimental Results The limits of detection and limits of quantitation for florfenicol and florfenicolamine in milk samples as shown in Example 1 are listed in Table 2; the chromatograms of LOD and LOQ after adding 0.5 μg / L to blank milk samples are shown in Table 2. Figure 7 As shown.

[0072] Table 2. Limits of detection and limits of quantitation for florfenicol and florfenicolamine.

[0073] In Formula I, y is the ratio of the peak area of ​​florfenicol (FF) to the peak area of ​​florfenicol-D3 (FF-D3) during ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-QQMS), and x is the concentration of florfenicol in the sample (μg / L). In Formula II, y' is the ratio of the peak area of ​​florfenicolamine derivative (PFFA) to the peak area of ​​florfenicolamine-D3 derivative (PFFA-D3) during UHPLC-QQMS, and x' is the concentration of florfenicolamine in the sample (μg / L).

[0074] The results showed that the method described in Example 1 exhibited good linearity for the detection of florfenicol and florfenicol amine in milk samples within the range of 0.5–50 μg / L, with a correlation coefficient (r) of [missing value]. 2The values ​​of florfenicol and florfenicolamine in milk samples were all greater than 0.995. When the content of florfenicol and florfenicolamine in the milk sample was 0.5 μg / kg, the signal-to-noise ratios (S / N) of florfenicol and florfenicolamine were 5.14 and 4.56, respectively, with both S / N greater than 3. Therefore, 0.5 μg / kg can be used as the method detection limit (LOD) of the method shown in Example 1. When the content of florfenicol and florfenicolamine in the milk sample was 1.0 μg / kg, the S / N of florfenicol and florfenicolamine were 12.82 and 14.31, respectively, with both S / N greater than 10. Therefore, 1.0 μg / kg can be used as the method quantitation limit (LOQ) of the method shown in Example 1. The units of LOD and LOQ were converted to internationally accepted units according to the formula X=C×V / m, where X is the converted unit, C is the concentration calculated according to the standard curve (x or x'), V is the volumetric volume (1 mL), and m is the weight of the blank milk (1 g).

[0075] II. Recovery Rate and Precision Study 1. Experimental Methods Add the mixed internal standard working solution shown in Example 1 to blank milk samples (milk samples that do not contain FF and FFA according to GB 31658.20-2022) to prepare milk samples for recovery testing with FF and FF final concentrations of 1.0 μg / kg, 2.0 μg / kg, 5.0 μg / kg and 20.0 μg / kg respectively.

[0076] Next, milk samples with different concentrations of recovery rate were used as test samples and tested according to Example 1, "III. Method for simultaneous detection of florfenicol and florfenicolamine in milk samples". The FF content and FFA content in each recovery rate milk sample were calculated by combining Formula I and Formula II.

[0077] Five replicates were set up for the recovery rate test of milk samples at each concentration (to calculate the relative standard deviation of intraday recovery rate), and the tests were repeated continuously for 5 days (to calculate the relative standard deviation of interday recovery rate). The average recovery rate of each milk sample was then calculated.

[0078] 2. Experimental Results The results of the recovery rate and precision test are shown in Table 3.

[0079] Table 3 Results of Recovery and Precision Testing

[0080] The results showed that when using the method described in Example 1 to detect florfenicol and florfenicolamine in milk samples containing different concentrations, the average recovery rates were between 89.4% and 103.5% and between 85.3% and 108.2%, respectively, and the relative standard deviations of the intra-day and inter-day recovery rates were both less than 15%. This indicates that the method described in Example 1 has high accuracy and precision in detecting florfenicol and florfenicolamine in milk.

[0081] Example 4: Application of a method for simultaneous detection of florfenicol and florfenicolamine in milk samples I. Experimental Methods Following the method described in "III. Simultaneous Detection of Florfenicol and Florfenicolamine in Milk Samples" in Example 1, the following milk samples were tested: Yili Golden Classic Organic Pure Milk (batch numbers: 20260217 C9, 20260221 A2, 20260223 A2, 20260305 C4, 202600406 C4), Yantang Pure Milk (batch numbers: 20251030A, 20260105B, 20260112B, 20260116B, 20260217A), Sanyuan Pure Milk (batch numbers: 20260107 B6, 202600127B6, 20260208 B6, 20260209 X, 20260218 X), and Bright Dairy Pure Milk (batch number: 20251027). Y03, 20260122 Y03, 20260204 T14, 20260211 Y03, 20260311 F03) were used as test samples and the detection status of florfenicol and florfenicolamine in the test samples was recorded.

[0082] II. Experimental Results For 20 commercially available milk samples, the method described in "III. Method for Simultaneous Detection of Florfenicol and Florfenicol in Milk Samples" in Example 1 was used, but florfenicol and florfenicol were not detected qualitatively in any of them.

[0083] This indicates that none of the 20 commercially available milk samples contained florfenicol or florfenicolamine, and therefore met the requirements.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A sample pretreatment method for simultaneous detection of florfenicol and florfenicol amine, characterized by, Specifically: Florfenicol-D3 and florfenicolamine-D3 were added to the test sample at a final concentration of 2–10 μg / L as internal standards to obtain spiked test samples. The spiked test samples were then extracted with ammoniated acetonitrile and purified using a solid-phase extraction column. The purified solution was collected, mixed evenly with derivatization reagent, and fully derivatized to obtain derivatized test samples. The derivatizing reagent is phenyl isothiocyanate.

2. The sample preparation method of claim 1, wherein, The sample to be tested was milk.

3. The sample preparation method of claim 1, wherein The derivatization reaction is carried out at 20–40°C in the dark for 5–100 min.

4. A method for simultaneous detection of florfenicol and florfenicolamine, characterized in that, Includes the following steps: S1. The sample to be tested is processed according to any one of the sample pretreatment methods of claims 1 to 3 to obtain a derivatized sample to be tested; S2. The derivatized test sample obtained in step S1 was quantitatively analyzed by using the internal standard method combined with ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry to obtain the content of florfenicol and florfenicolamine in the test sample.

5. The method according to claim 4, characterized in that, When performing quantitative analysis using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry as described in step S2, the chromatographic conditions are as follows: using a C18 column, controlling the column temperature at 25–45℃, the injection volume at 2–10 μL, the flow rate at 0.2–0.4 mL / min, mobile phase A being methanol or acetonitrile, and mobile phase B being water; The elution program was as follows: 0–1 min, 10% mobile phase A; 1–4 min, 10%–90% mobile phase A; 4–5.5 min, 90% mobile phase A; 5.5–6 min, 90%–10% mobile phase A; 6–7 min, 10% mobile phase A.

6. The method according to claim 5, characterized in that, The mobile phase A is methanol.

7. The method according to claim 5, characterized in that, The chromatographic conditions were controlled as follows: column temperature 35℃, injection volume 10 μL, and flow rate 0.3 mL / min.

8. The method according to claim 4, characterized in that, When performing quantitative analysis using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-QTSMS) as described in step S2, the mass spectrometry conditions are as follows: ESI negative ion mode scanning, capillary voltage of 0.5–3.0 kV, ion source temperature of 100–150 °C, cone voltage of 20–80 V, cone gas flow rate of 20–100 L / h, desolvation gas temperature of 300–800 °C, desolvation gas flow rate of 600–1000 L / h, scanning range of m / z of 100–600 Da, low collision energy channel voltage of 4 eV, and high collision energy channel voltage of 10–45 eV.

9. The method according to claim 4, characterized in that, In the quantitative analysis described in step S2, when quantifying florfenicol, florfenicol-D3 is used as an internal standard, and the content of florfenicol in the sample is calculated by combining the florfenicol quantitative analysis formula. The quantitative analysis formula for florfenicol is obtained by combining the results of ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry of samples with known florfenicol content, with the florfenicol content in the sample as the abscissa and the ratio of the peak area of ​​florfenicol in the sample to the peak area of ​​florfenicol-D3 as the ordinate. When quantifying florfenicol, florfenicol-D3 is used as an internal standard, and the content of florfenicol in the sample is calculated by combining the quantitative analysis formula of florfenicol. The quantitative analysis formula for florfenicol is obtained by combining the results of ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry of samples with known florfenicol content. The formula uses the florfenicol content in the sample as the x-axis and the ratio of the peak area of ​​florfenicol to the peak area of ​​florfenicol-D3 as the y-axis.

10. The application of the method according to any one of claims 4 to 9 in the simultaneous detection of florfenicol and florfenicolamine.

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