Comparison of Quadrupole Linear Ion Trap Mass Spectrometry and Quadrupole Electrostatic Field Orbital Trap Mass Spectrometry for Confirmation of Positive Results of Norfloxacin in Freshwater Shrimp

Comparison of Quadrupole Linear Ion Trap Mass Spectrometry and Quadrupole Electrostatic Field Orbital Trap Mass Spectrometry for Confirmation of Positive Results of Norfloxacin in Freshwater Shrimp | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparison of Quadrupole Linear Ion Trap Mass Spectrometry and Quadrupole Electrostatic Field Orbital Trap Mass Spectrometry for Confirmation of Positive Results of Norfloxacin in Freshwater Shrimp Lian Wang, Chunying Luo, Xin Ning, Shu Zhang, Wenqian Hou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7044135/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract To investigate the confirmation method of positive results for the determination of norfloxacin in freshwater shrimp by liquid chromatography quadrupole tandem mass spectrometry using multi reactive ion monitoring mode. Liquid chromatography quadrupole tandem mass spectrometry was used to detect norfloxacin standard solution, initial positive samples, and samples with added norfloxacin standard concentration at the detection limit level of the original method by changing the gradient conditions of the mobile phase and monitoring the number of ions. Liquid chromatography quadrupole linear ion trap tandem mass spectrometry was performed using enhanced ion scanning mode, and liquid chromatography quadrupole electrostatic field orbital trap tandem mass spectrometry was performed using full MS-dd-MS 2 collection mode. The results showed that all three methods were able to qualitatively detect the standard solution of norfloxacin and the sample with added norfloxacin standard. The positive sample detected for the first time was not detected. The conclusion is that there are false positive results in the original liquid chromatography quadrupole tandem mass spectrometry method. The three established methods for confirming positive results in liquid chromatography quadrupole tandem mass spectrometry provide strong technical support for food safety supervision. quadrupole linear ion trap tandem mass spectrometry Quadrupole electrostatic field orbital trap tandem mass spectrometry Norfloxacin False positive Veterinary drug residue Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Quinolones (QNs) are a class of artificially synthesized antibiotics that have been produced for 4 generations and over 20 species. The antibacterial spectrum of these drugs has gradually evolved from a single anti-Gram negative bacterium to a broad-spectrum anti-Gram positive bacterium, anaerobic bacterium, mycobacteria, Legionella, mycoplasma, and Chlamydia (Cardoso O et al. 2025). Due to its low price and strong antibacterial activity, it has been widely used in the aquaculture industry since the 1980s. Due to the abuse of quinolone antibiotics, the problem of drug residues has become increasingly serious. The residue of quinolone antibiotics in aquatic products is one of the important items for food safety risk assessment ( Suryoprabowo et al. 2025 ; Suryoprabowo S et al. 2023). There are 11 quinolone antibiotics involved in the monitoring of aquatic products in the " National Food Pollution and Harmful Factors Risk Monitoring Work Manual" (China National Center for Food Safety Risk Assessment 2023 ) (hereinafter referred to as the manual), and norfloxacin is one of the banned antibiotics. Freshwater shrimp are rich in nutrients and have many metabolites in their bodies. The sample matrix interferes greatly with the detection method in the manual - liquid chromatography tandem mass spectrometry (LC-MS/MS) (Carmen et al. 2023 ; Shen K et al. 2022 ), which can easily affect the qualitative judgment of norfloxacin. In the initial detection, 60 out of 86 freshwater shrimp samples were found to contain norfloxacin. This study selected representative initial positive samples and used optimized quadrupole mass spectrometry (QQQ), quadrupole and trap tandem mass spectrometry (Qtrap), enhanced product ion scanning (EPI), and quadrupole electrostatic field orbital trap tandem mass spectrometry (Q Exactive) full MS dd-MS 2 for confirmation detection. Different mass spectrometers and specific scanning modes were used, suitable for laboratories at different levels, to confirm norfloxacin in freshwater shrimp for food safety supervision. Testing provides multiple solutions with wide application value and significant social value. 1 Materials and Methods 1.1 Instruments and reagents Ultimate DGP-3600 High Performance Liquid Chromatography (Thermo Fisher Scientific, USA) - Qtrap3200 Tandem Mass Spectrometer (AB Sciex, USA), Ultimate3000RS High Performance Liquid Chromatography - Q Exactive Plus Tandem Mass Spectrometer (Thermo Fisher Scientific, USA), Vortex Genius 3 Vortex Mixer (IKA, Germany), GX-274 Fully Automatic Solid Phase Extraction Instrument (Gilson, USA), MPEva Quantitative Concentrator (Rui Lai Bo Instrument Guangzhou Co., Ltd.). The concentrations of norfloxacin (purchased from Beijing Tanmo Quality Inspection Technology Co., Ltd.) and norfloxacin D5 isotope standard reserve solution (A Chemtek, USA) are both 1.0 mg/mL. Citric acid, disodium hydrogen phosphate, glacial acetic acid, sodium hydroxide, and disodium ethylenediaminetetraacetic acid are analytical purities, while formic acid and acetonitrile (LC-MS grade, Dikma, USA) are used. HLB solid-phase extraction column (200 mg, 6 mL, Waters, USA) is used, and ultrapure water is used for the experiment. Mcllvaine buffer solution: Mix 1000 mL of 0.1 mol/L citric acid solution with 625 mL of 0.2 mol/L disodium hydrogen phosphate solution, and adjust the pH to 4.0 ± 0.05 with hydrochloric acid or sodium hydroxide. 0.1mol/L EDTA McCllvaine buffer solution: Weigh 60.5 g of disodium ethylenediaminetetraacetic acid and place it in 1625 mL of Mcllvaine buffer solution. Shake to dissolve. 1.2 Method 1.2.1 Preparation of Matrix Standard Solution Accurately draw 800 µ L of norfloxacin standard reserve solution into a 10 mL volumetric flask, dilute with methanol to the mark, and obtain a standard application solution with a concentration of 8.0 µg/mL; Accurately draw 1.25 mL of 8.0 µ g/mL standard application solution into a 10mL volumetric flask, dilute with methanol to the mark, and obtain a standard working solution of 1.0 µg/mL. Draw 1mL of norfloxacin D 5 isotope standard solution into a 10mL volumetric flask, dilute with methanol to the mark, and obtain an internal standard working solution with a concentration of 10.0 µg/mL. Accurately extract a certain amount of 1.0 µg/mL standard working solution and internal standard working solution to prepare standard solutions with concentrations of 1.0, 2.5, 5.0, 20, 50, and 100 ng/mL, and an internal standard concentration of 20 ng/mL. Dilute and mix the blank sample matrix extraction solution for testing. 1.2.2 Samples and Pretreatment Accurately weigh 2.00 g of the edible portion of freshwater shrimp that tested positive for norfloxacin for the first time, and place it in a 50 mL plastic centrifuge tube. Add 20 µL of internal standard application solution and 8 mL of 0.1 mol/L EDTA Mcllvaine buffer solution, mix with a vortex at 1000 r/min for 1 minute, extract with ultrasound for 10 minutes, and centrifuge at 10000 r/min for 5 minutes (temperature below 5℃). Repeat the extraction twice and collect the supernatant. Filter the supernatant using fast filter paper. The filtrate was passed through an HLB solid-phase extraction column (200 mg, 6 mL, activated with 6 mL of methanol and equilibrated with 6 mL of water before use) at a rate of 2–3 mL/min. The filtrate was discarded, washed with 2 mL of 5% methanol aqueous solution, and the eluent was discarded. Finally, the eluent was washed with 6 mL of methanol and collected. The eluent was washed in a 40℃ water bath, purged to dryness with a nitrogen blower, dissolved in 1 mL of 0.1% (v/v) formic acid water solution, and vortexed at 1000 r/min for 1 minute for LC-MS/MS determination. 1.3 Initial detection by quadrupole tandem mass spectrometry (QQQ) 1.3.1 Chromatographic conditions Chromatography column: Waters XSelect ® HSS T3 column (2.1 × 100mm, 2.5 µ m), column temperature: 40 ℃; Injection volume: 10 µL; Flow rate: 400 µ L/min; Mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid aqueous solution; The conditions for gradient elution of mobile phase are shown in Table 1 . Table 1 Gradient elution conditions for QQQ detection of norfloxacin positive freshwater shrimp Time/min Mobile phase A/% Mobile phase B/% 0.0 10 90 2.5 22 78 4.7 22 78 5.2 80 20 7.5 80 20 7.6 95 5 8.0 95 5 8.1 10 90 10.1 10 90 1.3.2 Mass spectrometry conditions The ionization mode is ESI + (electric spray positive ionization source), the curtain gas pressure (CUR) is 172 kPa, the collision gas pressure (CAD) is 34.5 kPa, the ion source voltage is 5500 V, the ion source temperature is 550 ℃, the atomization gas pressure is 172 kPa, and the auxiliary heating pressure is 172 kPa. The scanning mode is Multi Reactive Ion Monitoring (MRM), with a Dwell Time of 50ms for each sub ion. The ion conditions are shown in Table 2 . Table 2 MRM conditions for norfloxacin and norfloxacin D 5 chemical compound Primary ion ( m/z ) Product ion ( m/z ) Cluster voltage/V Collision energy/V Norfloxacin 320.2 233.3* 57 28 302.1 57 32 Norfloxacin D5 325.2 307.4* 57 32 238.2 57 32 Note: * represents quantitative ions 1.4 Quadrupole tandem mass spectrometry (QQQ) confirmation detection 1.4.1 Chromatographic conditions Same as 1.3.1, confirm that the gradient changes of the detected mobile phase are adjusted according to Table 3 . Table 3 QQQ confirms the elution conditions of the mobile phase gradient for detection Time/min A/% B/% 0.0 10 90 5.0 22 78 5.1 10 90 6.6 10 90 1.4.2 Mass spectrometry conditions Same as 1.3.2, the residence time of each sub ion is 100ms, and the MRM conditions are shown in Table 4 . Table 4 Confirm the MRM conditions for detecting norfloxacin and norfloxacin D5 chemical compound Primary ion (m/z) Product ion (m/z) Cluster voltage/V Collision energy/V Norfloxacin 320.2 302.1 47 28 233.3 57 32 276.2 57 23 Norfloxacin D5 325.2 307.4 57 32 1.5 Quadrupole Linear Ion Trap Tandem Mass Spectrometry (Qtrap) Confirmation Detection 1.5.1 Chromatographic conditions Same as 1.3.1 1.5.2 Mass spectrometry conditions The ionization mode is ESI + (electric spray positive ionization source), the curtain gas pressure (CUR) is 172 kPa, the collision gas pressure (CAD) is 75.9 kPa, the ion source voltage is 5500 V, the ion source temperature is 550℃, the atomizing gas pressure is 345 kPa, the auxiliary heating pressure is 345 kPa, the declustering voltage (DP) is 50 v, the inlet voltage (EP) is 5.0 v, the collision energy (CE) is 35v ± 15v, the scanning mode is enhancer ion scanning (EPI), and the scanning speed (Scan Rate) is 1000 Da/s. 1.6 Quadrupole electrostatic field orbit trap tandem mass spectrometry (Q Exactive) confirmation detection 1.6.1 Chromatographic conditions Same as 1.3.1 1.6.2 Mass spectrometry conditions The ionization mode is H-ESI + , the sheath gas pressure is 345 kPa, the collision gas pressure is 75.9 kPa, the spray voltage is 3500 V, the capillary temperature is 350℃, the heating rod temperature is 425℃, and the auxiliary gas pressure is 86.2 kPa. The scanning mode is full MS dd-MS 2 , with full scanning part: micro scan 1, resolution of 70000, AGC of 3 × 10 6 , allowing maximum ion implantation time of 100ms; dd-MS2 part, micro scan 1, resolution of 17500, AGC of 3 × 10 5 , allowing maximum ion implantation time of 50 ms, cycle number of 10, MSX count of 1, TopN of 10, isolation window of 0.4 m/z, collision energy of 25v, 50v, 75v; DD settings section: The AGC is set to 500, with a trigger threshold of 1 × 10 4 . 2 Results 2.1 Initial QQQ Test Results According to the instrument conditions in section 1.3.1 , a standard solution with a norfloxacin content of 5 ng/mL and a norfloxacin D 5 isotope internal standard content of 20 ng/mL (hereinafter referred to as the standard solution) was prepared for blank matrix. The extraction ion current (XIC) diagrams of norfloxacin isotope internal standard, norfloxacin quantitative ion, and norfloxacin qualitative ion in the standard solution are shown in Fig. 1 (a, b, c). The retention time of the three ions is the same, and the intensity ratio of norfloxacin quantitative ion to qualitative ion is 2.20. The d, e, and f in Fig. 1 are the XIC plots of norfloxacin isotope internal standard, norfloxacin quantitative ion, and norfloxacin qualitative ion in the sample solution, respectively. The retention times of the three ions are the same, and the intensity ratio of norfloxacin quantitative ion to qualitative ion is 2.22. In the initial detection, QQQ detected one parent ion and two product ions of the same compound, and the abundance ratio of the two product ions was similar to that of the standard substance. According to the EU 96/23/EC resolution (European Commission, 2002 ), it can be considered that the sample solution contains norfloxacin (low resolution mass spectrometer has one parent ion and two product ions, with 4 identification points). As this compound is a prohibited substance, confirmation testing is required to determine whether there are endogenous (freshwater shrimp samples) or exogenous (reagents, consumables, or instrument tubing) co flowing interferents, resulting in false positive results. According to the standard curve preparation and sample pretreatment in method 1.2, three methods, QQQ (changing instrument conditions), QTrap, and Q Exactive, were used for confirmation testing. 2.2 QQQ Confirmation test results Confirm the detection by changing the gradient conditions of the mobile phase (see Table 3 ) to increase the separation of the analyte from possible endogenous and exogenous interferences; Increase the number of standard solution product ion monitoring (Table 4 ). Qualitative analysis was conducted by confirming the extracted sub ion signals and abundance ratios at the retention time of the analyte in the standard solution and sample solution during testing (Wei D et al. 2020 ). Measure the above standard solution and sample solution according to the conditions of 1.3.2, and the corresponding XIC diagram is shown in Fig. 2 . From the XIC plots (b ~ d) of the standard solution, it can be seen that three characteristic ion pairs were detected at m/z 320.2/302.1, 320.2/233.3, and 320.2/276.2 when the retention time of norfloxacin was 4.91 minutes; From the XIC plots (f ~ h) of the sample solution, it can be seen that no corresponding signals were detected in the XIC plots of the three characteristic ions. It can be concluded that the sample solution does not contain the analyte. The experiment further verified that after adding 0.5 ng/g (limit of quantification) of norfloxacin to the sample for extraction and testing under confirmed experimental conditions, the results showed that all three ions detected signals at retention times of 4.91min for m/z 320.2/302.1, 320.2/233.3, and 320.2/276.2, indicating that the sample solution does not contain norfloxacin. 2.3 Qtrap Confirmation Test Results Qtrap is a hybrid mass spectrometer consisting of a quadrupole rod and a linear ion trap in series. It has all scanning modes of triple quadrupole rod series mass spectrometry, as well as linear ion trap mass spectrometry function. It can scan all the sub ions of the parent ion selected by the primary screening after collision fragmentation. As it is a full scan of all the product ions of the analyte, it greatly increases the qualitative ability (Xu F et al. 2020). In this study, the enhancer ion scanning (EPI) function of a linear ion trap was utilized, using MS/MS full scan-EPI mode. The first mass analyzer (Q1) performed a full scan of m/z in the range of 50–330, selecting ions with m/z of 320.0 for fragmentation in the collision cell (Q2). The second mass analyzer was a linear ion trap (LIT), where fragment ions were aggregated for mass spectrometry scanning. The results are shown in Fig. 3 (a ~ d), where a is the total ion chromatogram (TIC) of the standard solution, the peak at a retention time of 2.93 minutes is norfloxacin, and b is the fragment ion scan of the ion formed in LIT. It can be seen from this that the stronger ones are m/z 320.2, m/z 302.1, m/z 276.2, m/z 256.1, m/z 233.2, m/z 120.1, m/z 110.0, etc. c is the TIC image of the sample solution, and at a retention time of 2.93 minutes, there is also a high intensity m/z 320.0 signal. d is the scan image of the fragmented ions formed by the ion in LIT, and the stronger ones are m/z 320.2, m/z 302.2, m/z 276.2, m/z 248.2, m/z 231.2, m/z 110.0, etc. There is a significant difference in the ratio of fragment ion types and abundances between d and b. Using b plot to establish a spectral library, the matching degree between d plot and spectral library is only 28% (usually considered to be greater than 90% for detection), indicating that the sample solution does not contain norfloxacin. The experiment further verified that after adding 0.5 ng/g (quantification limit) of norfloxacin to the sample for extraction, the matching degree was greater than 90% when tested under confirmed experimental conditions. According to the breaking law of chemical bonds, the fragment ions of norfloxacin were analyzed, as shown in Fig. 4 , and the production pathways of fragment ions such as m/z 256, m/z 233, and m/z 120 were speculated. However, the ions with m/z 248.2, m/z 231.2, and m/z 110.0 in the d diagram cannot be reasonably inferred from the fragmentation rules, further illustrating the difference between the norfloxacin ions in the standard solution and the ion of m/z 320 in the sample solution. It also suggests that when detecting norfloxacin in freshwater shrimp, when the ions with m/z 320.2, m/z 302.2, and m/z 276.2 appear at the same time, further confirmation of other characteristic ions is still needed for qualitative analysis. 2.4 Q Exactive Confirmation test results The mass analyzer of the Q Exactive mass spectrometer consists of a quadrupole and an electrostatic field orbital ion trap (Orbitrap). Voltage is applied to four electrode rods to form an electric field, allowing the measured ions to become resonant ions and pass smoothly through the quadrupole. The Orbitrap separates ions of different m/z by the difference in their frequency of motion in the z direction, achieving high-resolution function (Cai R et al. 2024 ; Shi Y et al. 2023 ). In the experiment, the full MS/dd-MS 2 scanning mode was used to first collect the full scan spectrum of the sample solution, and automatically trigger the secondary mass spectrometry of the parent ions with higher intensity in the full scan spectrum according to the set parameters. Due to the high-resolution mass analysis of fragment ions by Orbitrap, the method has good qualitative ability. The experimental results are shown in Fig. 5 (a ~ d). Figure a is the XIC diagram of the standard solution, where ions with m/z ranging from 320.13730 to 320.14370 were extracted. The retention time of norfloxacin was 3.06 minutes. Figure b is the secondary fragment ion diagram obtained by passing the ions extracted from the first stage near this retention time through a high-energy collision cell; The XIC diagram of the sample solution is shown in Figure C. It can be seen that in Figure A, no ions with m/z values of 320.13730-320.14370 were extracted near the retention time of norfloxacin. This is because high-resolution mass spectrometry increased the mass accuracy (relative to QQQ, the extracted m/z is 320.2), and there was no peak observed in both the standard solution and the sample solution at 3.09 minutes in the XIC diagram during QQQ detection. Further examination of the secondary fragment plot d at a retention time of 3.06 minutes in the sample solution reveals that the fragment ions in plot d and plot b are different, indicating the absence of norfloxacin in the sample solution. The experiment further verified that after adding 0.5 ng/g (limit of quantification) of norfloxacin to the sample for extraction, a secondary fragment ion map was obtained by testing under confirmed experimental conditions near the retention time of norfloxacin from the first stage extracted ions after passing through a high-energy collision cell. 3 Discussions This study used three detection methods to confirm the detection of norfloxacin positive samples in freshwater shrimp by liquid chromatography tandem mass spectrometry, and analyzed and speculated on fragment ions. Changing the gradient elution conditions of the mobile phase and increasing the number of product ion monitoring is a common way to confirm positive detection results in daily testing. However, this method has certain limitations. If the gradient change is not significant or the chromatographic behavior of the test substance is not significantly affected by changes in the mobile phase conditions, it will create the illusion of confirming the detection again as positive. The EPI function of Qtrap, as secondary mass analysis is a full scan of the analyte ion, theoretically greatly increases the information of secondary fragment ions and enhances qualitative ability. However, if encountering compounds with simple results and less secondary fragment information, this function has little significance for qualitative ability. Both the first and second level quality analysis of Q Exactive can utilize the high-resolution function of Orbitrap, and the precise mass number effectively ensures qualitative ability and avoids false positives. It is the most reliable method among the three positive confirmation methods. However, compared with the serial quadrupole, high-resolution instruments are relatively expensive and less popular than QQQ, which limits their use. Therefore, each of the three confirmation detection methods has its own advantages and disadvantages, and is suitable for different instrument configurations and laboratory levels. Overall, all three methods in this study can be used for confirming the positive results of quinolone (such as norfloxacin) detection in freshwater shrimp using traditional triple quadrupole tandem mass spectrometry. To a certain extent, they can all eliminate false positive results and qualitatively confirm positive results. They are suitable for laboratories under different conditions and have promotional value, providing strong technical support for food safety supervision. Declarations Competing Interest declaration: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution Wang wrote the main manuscript text, Luo and Ning prepared figures1-5, Zhang and Hou prepared Table1-4. All authors reviewed the manuscript. References Cardoso O, Donato MM, Henriques SC, Ramos F (2025) Fluoroquinolone Residues in Piglet Viscera and Their Impact on Intestinal Microbiota Resistance: A One Health Approach. J Microorganisms. 13: 1389–1399 Suryoprabowo S, Nasyiruddin RL, Wang ZX, Hendriko A, Tristanto NA (2025) A comprehensive review on the pretreatment and detection methods of fluoroquinolones in food and environment. J FOOD COMPOS ANAL 140:107179 Anamika S, Kirty P, Singh BD, Avinash T, Vikas N (2023) A review on Api-products: current scenario of potential contaminants and their food safety concerns. 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Brussels Wei D, Guo M, Zhang J (2020) Determination of 10 Fluoroquinolone Residues in Aquatic Products by Accelerated Solvent Extraction Magnetic Solid Phase Extraction High Performance Liquid Chromatography Tandem Mass. Spectrom J Chromatogr Chin 38:1413–1422 Xu F, Liu F, Wang C, Wei Y (2018) Use of phenyl/tetrazolyl-functionalized magnetic microspheres and stable isotope labeled internal standards for significant reduction of matrix effect in determination of nine fluoroquinolones by liquid chromatography-quadrupole linear ion trap mass spectrometry. J Anal Bioanal Chem 410:1709–1724 Cai R, Liu J, Wang X, An T, Zhang L (2024) Identification of daurisoline metabolites in rats via the UHPLC-Q-exactive orbitrap mass spectrometer. J Pharm Biomed Anal .252:116482‌ Shi Y, Zhang Y, He Q, Zhang L, Jin M (2023) Rapid determination of 48 stimulant residues in milk and dairy products by high-performance liquid chromatography coupled with quadrupole/Orbitrap high-resolution mass spectrometry. J Chem Pap 77:5237–5258 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 21 Aug, 2025 Reviews received at journal 05 Aug, 2025 Reviews received at journal 04 Aug, 2025 Reviews received at journal 01 Aug, 2025 Reviewers agreed at journal 30 Jul, 2025 Reviews received at journal 29 Jul, 2025 Reviewers agreed at journal 24 Jul, 2025 Reviewers agreed at journal 24 Jul, 2025 Reviewers agreed at journal 24 Jul, 2025 Reviewers agreed at journal 24 Jul, 2025 Reviewers invited by journal 24 Jul, 2025 Editor assigned by journal 11 Jul, 2025 Submission checks completed at journal 11 Jul, 2025 First submitted to journal 04 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7044135","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":490608364,"identity":"4746ad9d-28c0-4c7f-ba5f-9b4bd93325fa","order_by":0,"name":"Lian 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Prevention","correspondingAuthor":false,"prefix":"","firstName":"Wenqian","middleName":"","lastName":"Hou","suffix":""}],"badges":[],"createdAt":"2025-07-04 07:38:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7044135/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7044135/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87762228,"identity":"af32184e-3060-4056-a405-4846c0be376f","added_by":"auto","created_at":"2025-07-28 17:08:46","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":200594,"visible":true,"origin":"","legend":"\u003cp\u003ea XIC of norfloxacin D5 in standard solution Figure 1-b XIC of quantitative ions of norfloxacin in standard solution Figure 1-c XIC of qualitative ions of norfloxacin in standard solution Figure 1-d XIC of norfloxacin D5 in sample solution Figure 1-e XIC of quantitative ions of norfloxacin in sample solution Figure 1-f XIC of qualitative ions of norfloxacin in sample solution\u003c/p\u003e\n\u003cp\u003eExtraction ion chromatogram of norfloxacin and internal standard in standard solution and sample solution during initial inspection of QQQ\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7044135/v1/513aba2d2f2bb1456549fa15.jpeg"},{"id":87762216,"identity":"eed934e0-a618-4056-8d57-7a65aec85dee","added_by":"auto","created_at":"2025-07-28 17:08:46","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":265781,"visible":true,"origin":"","legend":"\u003cp\u003ea XIC of norfloxacin D5 in standard solution, Figure 2-b XIC of norfloxacin quantitative ions in standard solution, Figure 2-c XIC of norfloxacin qualitative ion 1 in standard solution, Figure 2-d XIC of norfloxacin qualitative ion 2 in standard solution, Figure 2-e XIC of norfloxacin D5 in sample solution, Figure 2-f XIC of norfloxacin quantitative ions in sample solution, Figure 2-g XIC of norfloxacin qualitative ion 1 in sample solution, Figure 2-h XIC of norfloxacin qualitative ion 2 in sample solution\u003c/p\u003e\n\u003cp\u003eIonic chromatogram of norfloxacin and internal standard extraction from standard solution and sample solution during QQQ confirmation testing\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7044135/v1/3f1fedd9cf5726c306509be0.jpeg"},{"id":87762218,"identity":"e9f781d8-8164-41c4-9278-dab5ae95691d","added_by":"auto","created_at":"2025-07-28 17:08:46","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":276787,"visible":true,"origin":"","legend":"\u003cp\u003eTIC diagram of 3-a standard solution, EPI diagram of 3-b standard solution, TIC diagram of 3-c sample solution, and EPI diagram of 3d sample solution\u003c/p\u003e\n\u003cp\u003eTIC and EPI plots of standard solution and sample solution during EPI detection\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7044135/v1/078be1b4c20a228919bcbaca.jpeg"},{"id":87762491,"identity":"7b5e5fd1-7e2a-4ac9-8d4a-752fe1525383","added_by":"auto","created_at":"2025-07-28 17:16:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":47757,"visible":true,"origin":"","legend":"\u003cp\u003eCracking of characteristic fragment ions of norfloxacin\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7044135/v1/4a29980123aab3312a990a0f.jpg"},{"id":87762222,"identity":"7626cd53-b5e0-4084-bae6-a9e1dbd656c6","added_by":"auto","created_at":"2025-07-28 17:08:46","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":315533,"visible":true,"origin":"","legend":"\u003cp\u003ea XIC of standard solution Figure 5-b Secondary fragment ion diagram of standard solution Figure 5-c XIC of sample solution Figure 5-d Secondary fragment ion diagram of sample solution\u003c/p\u003e\n\u003cp\u003eXIC diagram and secondary fragment ion diagram of standard solution and sample solution during Q Exactive detection\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7044135/v1/637990ac4c5e459ec1290d21.jpeg"},{"id":87764213,"identity":"5f2142da-c18d-48b8-bc0c-757eb16eb306","added_by":"auto","created_at":"2025-07-28 17:40:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2020194,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7044135/v1/499959e1-988a-4b51-9c50-653d7aa3c677.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparison of Quadrupole Linear Ion Trap Mass Spectrometry and Quadrupole Electrostatic Field Orbital Trap Mass Spectrometry for Confirmation of Positive Results of Norfloxacin in Freshwater Shrimp","fulltext":[{"header":"Introduction","content":"\u003cp\u003eQuinolones (QNs) are a class of artificially synthesized antibiotics that have been produced for 4 generations and over 20 species. The antibacterial spectrum of these drugs has gradually evolved from a single anti-Gram negative bacterium to a broad-spectrum anti-Gram positive bacterium, anaerobic bacterium, mycobacteria, Legionella, mycoplasma, and Chlamydia (Cardoso O et al. 2025). Due to its low price and strong antibacterial activity, it has been widely used in the aquaculture industry since the 1980s. Due to the abuse of quinolone antibiotics, the problem of drug residues has become increasingly serious. The residue of quinolone antibiotics in aquatic products is one of the important items for food safety risk assessment ( Suryoprabowo et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Suryoprabowo S et al. 2023).\u003c/p\u003e\u003cp\u003eThere are 11 quinolone antibiotics involved in the monitoring of aquatic products in the \" National Food Pollution and Harmful Factors Risk Monitoring Work Manual\" (China National Center for Food Safety Risk Assessment \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) (hereinafter referred to as the manual), and norfloxacin is one of the banned antibiotics. Freshwater shrimp are rich in nutrients and have many metabolites in their bodies. The sample matrix interferes greatly with the detection method in the manual - liquid chromatography tandem mass spectrometry (LC-MS/MS) (Carmen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shen K et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), which can easily affect the qualitative judgment of norfloxacin. In the initial detection, 60 out of 86 freshwater shrimp samples were found to contain norfloxacin. This study selected representative initial positive samples and used optimized quadrupole mass spectrometry (QQQ), quadrupole and trap tandem mass spectrometry (Qtrap), enhanced product ion scanning (EPI), and quadrupole electrostatic field orbital trap tandem mass spectrometry (Q Exactive) full MS dd-MS\u003csup\u003e2\u003c/sup\u003e for confirmation detection. Different mass spectrometers and specific scanning modes were used, suitable for laboratories at different levels, to confirm norfloxacin in freshwater shrimp for food safety supervision. Testing provides multiple solutions with wide application value and significant social value.\u003c/p\u003e"},{"header":"1 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e1.1 Instruments and reagents\u003c/h2\u003e\u003cp\u003eUltimate DGP-3600 High Performance Liquid Chromatography (Thermo Fisher Scientific, USA) - Qtrap3200 Tandem Mass Spectrometer (AB Sciex, USA), Ultimate3000RS High Performance Liquid Chromatography - Q Exactive Plus Tandem Mass Spectrometer (Thermo Fisher Scientific, USA), Vortex Genius 3 Vortex Mixer (IKA, Germany), GX-274 Fully Automatic Solid Phase Extraction Instrument (Gilson, USA), MPEva Quantitative Concentrator (Rui Lai Bo Instrument Guangzhou Co., Ltd.).\u003c/p\u003e\u003cp\u003eThe concentrations of norfloxacin (purchased from Beijing Tanmo Quality Inspection Technology Co., Ltd.) and norfloxacin D5 isotope standard reserve solution (A Chemtek, USA) are both 1.0 mg/mL. Citric acid, disodium hydrogen phosphate, glacial acetic acid, sodium hydroxide, and disodium ethylenediaminetetraacetic acid are analytical purities, while formic acid and acetonitrile (LC-MS grade, Dikma, USA) are used. HLB solid-phase extraction column (200 mg, 6 mL, Waters, USA) is used, and ultrapure water is used for the experiment.\u003c/p\u003e\u003cp\u003eMcllvaine buffer solution: Mix 1000 mL of 0.1 mol/L citric acid solution with 625 mL of 0.2 mol/L disodium hydrogen phosphate solution, and adjust the pH to 4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 with hydrochloric acid or sodium hydroxide.\u003c/p\u003e\u003cp\u003e0.1mol/L EDTA McCllvaine buffer solution: Weigh 60.5 g of disodium ethylenediaminetetraacetic acid and place it in 1625 mL of Mcllvaine buffer solution. Shake to dissolve.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e1.2 Method\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e1.2.1 Preparation of Matrix Standard Solution\u003c/h2\u003e\u003cp\u003eAccurately draw 800 \u0026micro; L of norfloxacin standard reserve solution into a 10 mL volumetric flask, dilute with methanol to the mark, and obtain a standard application solution with a concentration of 8.0 \u0026micro;g/mL; Accurately draw 1.25 mL of 8.0 \u0026micro; g/mL standard application solution into a 10mL volumetric flask, dilute with methanol to the mark, and obtain a standard working solution of 1.0 \u0026micro;g/mL. Draw 1mL of norfloxacin D\u003csub\u003e5\u003c/sub\u003e isotope standard solution into a 10mL volumetric flask, dilute with methanol to the mark, and obtain an internal standard working solution with a concentration of 10.0 \u0026micro;g/mL.\u003c/p\u003e\u003cp\u003eAccurately extract a certain amount of 1.0 \u0026micro;g/mL standard working solution and internal standard working solution to prepare standard solutions with concentrations of 1.0, 2.5, 5.0, 20, 50, and 100 ng/mL, and an internal standard concentration of 20 ng/mL. Dilute and mix the blank sample matrix extraction solution for testing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e1.2.2 Samples and Pretreatment\u003c/h2\u003e\u003cp\u003eAccurately weigh 2.00 g of the edible portion of freshwater shrimp that tested positive for norfloxacin for the first time, and place it in a 50 mL plastic centrifuge tube. Add 20 \u0026micro;L of internal standard application solution and 8 mL of 0.1 mol/L EDTA Mcllvaine buffer solution, mix with a vortex at 1000 r/min for 1 minute, extract with ultrasound for 10 minutes, and centrifuge at 10000 r/min for 5 minutes (temperature below 5℃). Repeat the extraction twice and collect the supernatant. Filter the supernatant using fast filter paper. The filtrate was passed through an HLB solid-phase extraction column (200 mg, 6 mL, activated with 6 mL of methanol and equilibrated with 6 mL of water before use) at a rate of 2\u0026ndash;3 mL/min. The filtrate was discarded, washed with 2 mL of 5% methanol aqueous solution, and the eluent was discarded. Finally, the eluent was washed with 6 mL of methanol and collected. The eluent was washed in a 40℃ water bath, purged to dryness with a nitrogen blower, dissolved in 1 mL of 0.1% (v/v) formic acid water solution, and vortexed at 1000 r/min for 1 minute for LC-MS/MS determination.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e1.3 Initial detection by quadrupole tandem mass spectrometry (QQQ)\u003c/h2\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e1.3.1 Chromatographic conditions\u003c/h2\u003e\u003cp\u003eChromatography column: Waters XSelect \u0026reg; HSS T3 column (2.1 \u0026times; 100mm, 2.5 \u0026micro; m), column temperature: 40 ℃; Injection volume: 10 \u0026micro;L; Flow rate: 400 \u0026micro; L/min; Mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid aqueous solution; The conditions for gradient elution of mobile phase are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGradient elution conditions for QQQ detection of norfloxacin positive freshwater shrimp\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime/min\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMobile phase A/%\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMobile phase B/%\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e1.3.2 Mass spectrometry conditions\u003c/h2\u003e\u003cp\u003eThe ionization mode is ESI\u003csup\u003e+\u003c/sup\u003e(electric spray positive ionization source), the curtain gas pressure (CUR) is 172 kPa, the collision gas pressure (CAD) is 34.5 kPa, the ion source voltage is 5500 V, the ion source temperature is 550 ℃, the atomization gas pressure is 172 kPa, and the auxiliary heating pressure is 172 kPa. The scanning mode is Multi Reactive Ion Monitoring (MRM), with a Dwell Time of 50ms for each sub ion. The ion conditions are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMRM conditions for norfloxacin and norfloxacin D\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003echemical compound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimary ion (\u003cem\u003em/z\u003c/em\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eProduct ion (\u003cem\u003em/z\u003c/em\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCluster voltage/V\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCollision energy/V\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNorfloxacin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e320.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e233.3*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e302.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNorfloxacin D5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e325.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e307.4*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e238.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: * represents quantitative ions\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e1.4 Quadrupole tandem mass spectrometry (QQQ) confirmation detection\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e1.4.1 Chromatographic conditions\u003c/h2\u003e\u003cp\u003eSame as 1.3.1, confirm that the gradient changes of the detected mobile phase are adjusted according to Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eQQQ confirms the elution conditions of the mobile phase gradient for detection\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime/min\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eA/%\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eB/%\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e1.4.2 Mass spectrometry conditions\u003c/h2\u003e\u003cp\u003eSame as 1.3.2, the residence time of each sub ion is 100ms, and the MRM conditions are shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eConfirm the MRM conditions for detecting norfloxacin and norfloxacin D5\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003echemical compound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimary ion (m/z)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eProduct ion (m/z)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCluster voltage/V\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCollision energy/V\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eNorfloxacin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e320.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e302.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e233.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e276.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNorfloxacin D5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e325.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e307.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e1.5 Quadrupole Linear Ion Trap Tandem Mass Spectrometry (Qtrap) Confirmation Detection\u003c/h2\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e1.5.1 Chromatographic conditions\u003c/h2\u003e\u003cp\u003eSame as 1.3.1\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e1.5.2 Mass spectrometry conditions\u003c/h2\u003e\u003cp\u003eThe ionization mode is ESI\u003csup\u003e+\u003c/sup\u003e(electric spray positive ionization source), the curtain gas pressure (CUR) is 172 kPa, the collision gas pressure (CAD) is 75.9 kPa, the ion source voltage is 5500 V, the ion source temperature is 550℃, the atomizing gas pressure is 345 kPa, the auxiliary heating pressure is 345 kPa, the declustering voltage (DP) is 50 v, the inlet voltage (EP) is 5.0 v, the collision energy (CE) is 35v\u0026thinsp;\u0026plusmn;\u0026thinsp;15v, the scanning mode is enhancer ion scanning (EPI), and the scanning speed (Scan Rate) is 1000 Da/s.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e1.6 Quadrupole electrostatic field orbit trap tandem mass spectrometry (Q Exactive) confirmation detection\u003c/h2\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e1.6.1 Chromatographic conditions\u003c/h2\u003e\u003cp\u003eSame as 1.3.1\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e1.6.2 Mass spectrometry conditions\u003c/h2\u003e\u003cp\u003eThe ionization mode is H-ESI\u003csup\u003e+\u003c/sup\u003e, the sheath gas pressure is 345 kPa, the collision gas pressure is 75.9 kPa, the spray voltage is 3500 V, the capillary temperature is 350℃, the heating rod temperature is 425℃, and the auxiliary gas pressure is 86.2 kPa. The scanning mode is full MS dd-MS\u003csup\u003e2\u003c/sup\u003e, with full scanning part: micro scan 1, resolution of 70000, AGC of 3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e, allowing maximum ion implantation time of 100ms; dd-MS2 part, micro scan 1, resolution of 17500, AGC of 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e, allowing maximum ion implantation time of 50 ms, cycle number of 10, MSX count of 1, TopN of 10, isolation window of 0.4 m/z, collision energy of 25v, 50v, 75v; DD settings section: The AGC is set to 500, with a trigger threshold of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"2 Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Initial QQQ Test Results\u003c/h2\u003e\u003cp\u003eAccording to the instrument conditions in section \u003cspan refid=\"Sec8\" class=\"InternalRef\"\u003e1.3.1\u003c/span\u003e, a standard solution with a norfloxacin content of 5 ng/mL and a norfloxacin D\u003csub\u003e5\u003c/sub\u003e isotope internal standard content of 20 ng/mL (hereinafter referred to as the standard solution) was prepared for blank matrix. The extraction ion current (XIC) diagrams of norfloxacin isotope internal standard, norfloxacin quantitative ion, and norfloxacin qualitative ion in the standard solution are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e (a, b, c). The retention time of the three ions is the same, and the intensity ratio of norfloxacin quantitative ion to qualitative ion is 2.20. The d, e, and f in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e are the XIC plots of norfloxacin isotope internal standard, norfloxacin quantitative ion, and norfloxacin qualitative ion in the sample solution, respectively. The retention times of the three ions are the same, and the intensity ratio of norfloxacin quantitative ion to qualitative ion is 2.22.\u003c/p\u003e\u003cp\u003eIn the initial detection, QQQ detected one parent ion and two product ions of the same compound, and the abundance ratio of the two product ions was similar to that of the standard substance. According to the EU 96/23/EC resolution (European Commission, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), it can be considered that the sample solution contains norfloxacin (low resolution mass spectrometer has one parent ion and two product ions, with 4 identification points). As this compound is a prohibited substance, confirmation testing is required to determine whether there are endogenous (freshwater shrimp samples) or exogenous (reagents, consumables, or instrument tubing) co flowing interferents, resulting in false positive results. According to the standard curve preparation and sample pretreatment in method 1.2, three methods, QQQ (changing instrument conditions), QTrap, and Q Exactive, were used for confirmation testing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e2.2 QQQ Confirmation test results\u003c/h2\u003e\u003cp\u003eConfirm the detection by changing the gradient conditions of the mobile phase (see Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) to increase the separation of the analyte from possible endogenous and exogenous interferences; Increase the number of standard solution product ion monitoring (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Qualitative analysis was conducted by confirming the extracted sub ion signals and abundance ratios at the retention time of the analyte in\u003c/p\u003e\u003cp\u003ethe standard solution and sample solution during testing (Wei D et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Measure the above standard solution and sample solution according to the conditions of 1.3.2, and the corresponding XIC diagram is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e. From the XIC plots (b\u0026thinsp;~\u0026thinsp;d) of the standard solution, it can be seen that three characteristic ion pairs were detected at \u003cem\u003em/z\u003c/em\u003e 320.2/302.1, 320.2/233.3, and 320.2/276.2 when the retention time of norfloxacin was 4.91 minutes; From the XIC plots (f\u0026thinsp;~\u0026thinsp;h) of the sample solution, it can be seen that no corresponding signals were detected in the XIC plots of the three characteristic ions. It can be concluded that the sample solution does not contain the analyte. The experiment further verified that after adding 0.5 ng/g (limit of quantification) of norfloxacin to the sample for extraction and testing under confirmed experimental conditions, the results showed that all three ions detected signals at retention times of 4.91min for \u003cem\u003em/z\u003c/em\u003e 320.2/302.1, 320.2/233.3, and 320.2/276.2, indicating that the sample solution does not contain norfloxacin.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Qtrap Confirmation Test Results\u003c/h2\u003e\u003cp\u003eQtrap is a hybrid mass spectrometer consisting of a quadrupole rod and a linear ion trap in series. It\u003c/p\u003e\u003cp\u003ehas all scanning modes of triple quadrupole rod series mass spectrometry, as well as linear ion trap mass spectrometry function. It can scan all the sub ions of the parent ion selected by the primary screening after collision fragmentation. As it is a full scan of all the product ions of the analyte, it greatly increases the qualitative ability (Xu F et al. 2020). In this study, the enhancer ion scanning (EPI) function of a linear ion trap was utilized, using MS/MS full scan-EPI mode. The first mass analyzer (Q1) performed a full scan of m/z in the range of 50\u0026ndash;330, selecting ions with m/z of 320.0 for fragmentation in the collision cell (Q2). The second mass analyzer was a linear ion trap (LIT), where fragment ions were aggregated for mass spectrometry scanning. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e (a\u0026thinsp;~\u0026thinsp;d), where a is the total ion chromatogram (TIC) of the standard solution, the peak at a retention time of 2.93 minutes is norfloxacin, and b is the fragment ion scan of the ion formed in LIT. It can be seen from this that the stronger ones are m/z 320.2, m/z 302.1, m/z 276.2, m/z 256.1, m/z 233.2, m/z 120.1, m/z 110.0, etc. c is the TIC image of the sample solution, and at a retention time of 2.93 minutes, there is also a high intensity m/z 320.0 signal. d is the scan image of the fragmented ions formed by the ion in LIT, and the stronger ones are m/z 320.2, m/z 302.2, m/z 276.2, m/z 248.2, m/z 231.2, m/z 110.0, etc. There is a significant difference in the ratio of fragment ion types and abundances between d and b. Using b plot to establish a spectral library, the matching degree between d plot and spectral library is only 28% (usually considered to be greater than 90% for detection), indicating that the sample solution does not contain norfloxacin. The experiment further verified that after adding 0.5 ng/g (quantification limit) of norfloxacin to the sample for extraction, the matching degree was greater than 90% when tested under confirmed experimental conditions.\u003c/p\u003e\u003cp\u003eAccording to the breaking law of chemical bonds, the fragment ions of norfloxacin were analyzed, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e, and the production pathways of fragment ions such as m/z 256, m/z 233, and m/z 120 were speculated. However, the ions with m/z 248.2, m/z 231.2, and m/z 110.0 in the d diagram cannot be reasonably inferred from the fragmentation rules, further illustrating the difference between the norfloxacin ions in the standard solution and the ion of m/z 320 in the sample solution. It also suggests that when detecting norfloxacin in freshwater shrimp, when the ions with m/z 320.2, m/z 302.2, and m/z 276.2 appear at the same time, further confirmation of other characteristic ions is still needed for qualitative analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Q Exactive Confirmation test results\u003c/h2\u003e\u003cp\u003eThe mass analyzer of the Q Exactive mass spectrometer consists of a quadrupole and an electrostatic\u003c/p\u003e\u003cp\u003efield orbital ion trap (Orbitrap). Voltage is applied to four electrode rods to form an electric field, allowing the measured ions to become resonant ions and pass smoothly through the quadrupole. The Orbitrap separates ions of different m/z by the difference in their frequency of motion in the z direction, achieving high-resolution function (Cai R et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Shi Y et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the experiment, the full MS/dd-MS\u003csup\u003e2\u003c/sup\u003e scanning mode was used to first collect the full scan spectrum of the sample solution, and automatically trigger the secondary mass spectrometry of the parent ions with higher intensity in the full scan spectrum according to the set parameters. Due to the high-resolution mass analysis of fragment ions by Orbitrap, the method has good qualitative ability. The experimental results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003e (a\u0026thinsp;~\u0026thinsp;d). Figure a is the XIC diagram of the standard solution, where ions with m/z ranging from 320.13730 to 320.14370 were extracted. The retention time of norfloxacin was 3.06 minutes. Figure b is the secondary fragment ion diagram obtained by passing the ions extracted from the first stage near this retention time through a high-energy collision cell; The XIC diagram of the sample solution is shown in Figure C. It can be seen that in Figure A, no ions with m/z values of 320.13730-320.14370 were extracted near the retention time of norfloxacin. This is because high-resolution mass spectrometry increased the mass accuracy (relative to QQQ, the extracted m/z is 320.2), and there was no peak observed in both the standard solution and the sample solution at 3.09 minutes in the XIC diagram during QQQ detection. Further examination of the secondary fragment plot d at a retention time of 3.06 minutes in the sample solution reveals that the fragment ions in plot d and plot b are different, indicating the absence of norfloxacin in the sample solution. The experiment further verified that after adding 0.5 ng/g (limit of quantification) of norfloxacin to the sample for extraction, a secondary fragment ion map was obtained by testing under confirmed experimental conditions near the retention time of norfloxacin from the first stage extracted ions after passing through a high-energy collision cell.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Discussions","content":"\u003cp\u003eThis study used three detection methods to confirm the detection of norfloxacin positive samples in freshwater shrimp by liquid chromatography tandem mass spectrometry, and analyzed and speculated on fragment ions. Changing the gradient elution conditions of the mobile phase and increasing the number of product ion monitoring is a common way to confirm positive detection results in daily testing. However, this method has certain limitations. If the gradient change is not significant or the chromatographic behavior of the test substance is not significantly affected by changes in the mobile phase conditions, it will create the illusion of confirming the detection again as positive. The EPI function of Qtrap, as secondary mass analysis is a full scan of the analyte ion, theoretically greatly increases the information of secondary fragment ions and enhances qualitative ability. However, if encountering compounds with simple results and less secondary fragment information, this function has little significance for qualitative ability. Both the first and second level quality analysis of Q Exactive can utilize the high-resolution function of Orbitrap, and the precise mass number effectively ensures qualitative ability and avoids false positives. It is the most reliable method among the three positive confirmation methods. However, compared with the serial quadrupole, high-resolution instruments are relatively expensive and less popular than QQQ, which limits their use. Therefore, each of the three confirmation detection methods has its own advantages and disadvantages, and is suitable for different instrument configurations and laboratory levels. Overall, all three methods in this study can be used for confirming the positive results of quinolone (such as norfloxacin) detection in freshwater shrimp using traditional triple quadrupole tandem mass spectrometry. To a certain extent, they can all eliminate false positive results and qualitatively confirm positive results. They are suitable for laboratories under different conditions and have promotional value, providing strong technical support for food safety supervision.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eCompeting Interest declaration: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWang wrote the main manuscript text, Luo and Ning prepared figures1-5, Zhang and Hou prepared Table1-4. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCardoso O,\u0026ensp;Donato MM,\u0026ensp;Henriques SC, Ramos F (2025) Fluoroquinolone Residues in Piglet Viscera and Their Impact on Intestinal Microbiota Resistance: A One Health Approach. J Microorganisms. 13: 1389\u0026ndash;1399\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSuryoprabowo S, Nasyiruddin RL, Wang ZX, Hendriko A, Tristanto NA (2025) A comprehensive review on the pretreatment and detection methods of fluoroquinolones in food and environment. J FOOD COMPOS ANAL 140:107179\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnamika S, Kirty P, Singh BD, Avinash T, Vikas N (2023) A review on Api-products: current scenario of potential contaminants and their food safety concerns. J Food Control 145:109499\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChina National Center for Food Safety Risk Assessment (2023) National Food Safety Risk Assessment Center 2024 National Food Pollution and Harmful Factors Risk Monitoring Work Manual. Medium Volume, Beijing\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarmen M, Juan LS, Julia M, Irene A, Esteban A (2023) Automatised on-line SPE-chiral LC-MS/MS method for the enantiomeric determination of main fluoroquinolones and their metabolites in environmental water samples. J Microchemical 185:108217\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShen K, Zou X, Wang J (2022) Simultaneous determination of the four key fluoroquinolones and two antipsychotics in fish and shrimp by LC-MS/MS. J FOOD ADDIT CONTAM A 39:678\u0026ndash;686\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEuropean Commission (2002) Implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (2002/657/EC). Brussels\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei D, Guo M, Zhang J (2020) Determination of 10 Fluoroquinolone Residues in Aquatic Products by Accelerated Solvent Extraction Magnetic Solid Phase Extraction High Performance Liquid Chromatography Tandem Mass. Spectrom J Chromatogr Chin 38:1413\u0026ndash;1422\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu F, Liu F, Wang C, Wei Y (2018) Use of phenyl/tetrazolyl-functionalized magnetic microspheres and stable isotope labeled internal standards for significant reduction of matrix effect in determination of nine fluoroquinolones by liquid chromatography-quadrupole linear ion trap mass spectrometry. J Anal Bioanal Chem 410:1709\u0026ndash;1724\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCai R, Liu J, Wang X, An T, Zhang L (2024) Identification of daurisoline metabolites in rats via the UHPLC-Q-exactive orbitrap mass spectrometer. J Pharm Biomed Anal .252:116482\u0026zwnj;\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi Y, \u0026ensp;Zhang Y, He Q, Zhang L, Jin M (2023) Rapid determination of 48 stimulant residues in milk and dairy products by high-performance liquid chromatography coupled with quadrupole/Orbitrap high-resolution mass spectrometry. J Chem Pap 77:5237\u0026ndash;5258\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-analytical-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Analytical Methods](https://www.springer.com/journal/12161)","snPcode":"12161","submissionUrl":"https://submission.nature.com/new-submission/12161/3","title":"Food Analytical Methods","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"quadrupole linear ion trap tandem mass spectrometry, Quadrupole electrostatic field orbital trap tandem mass spectrometry, Norfloxacin, False positive, Veterinary drug residue","lastPublishedDoi":"10.21203/rs.3.rs-7044135/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7044135/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo investigate the confirmation method of positive results for the determination of norfloxacin in freshwater shrimp by liquid chromatography quadrupole tandem mass spectrometry using multi reactive ion monitoring mode. Liquid chromatography quadrupole tandem mass spectrometry was used to detect norfloxacin standard solution, initial positive samples, and samples with added norfloxacin standard concentration at the detection limit level of the original method by changing the gradient conditions of the mobile phase and monitoring the number of ions. Liquid chromatography quadrupole linear ion trap tandem mass spectrometry was performed using enhanced ion scanning mode, and liquid chromatography quadrupole electrostatic field orbital trap tandem mass spectrometry was performed using full MS-dd-MS\u003csup\u003e2\u003c/sup\u003e collection mode. The results showed that all three methods were able to qualitatively detect the standard solution of norfloxacin and the sample with added norfloxacin standard. The positive sample detected for the first time was not detected. The conclusion is that there are false positive results in the original liquid chromatography quadrupole tandem mass spectrometry method. The three established methods for confirming positive results in liquid chromatography quadrupole tandem mass spectrometry provide strong technical support for food safety supervision.\u003c/p\u003e","manuscriptTitle":"Comparison of Quadrupole Linear Ion Trap Mass Spectrometry and Quadrupole Electrostatic Field Orbital Trap Mass Spectrometry for Confirmation of Positive Results of Norfloxacin in Freshwater Shrimp","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 17:08:41","doi":"10.21203/rs.3.rs-7044135/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-21T08:57:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-05T09:37:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-04T15:35:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-01T19:31:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154137250628457507655755610354071935266","date":"2025-07-30T11:15:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-29T14:45:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"246226435610484847907029277601546001455","date":"2025-07-25T03:29:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321610620424837905190752961116969117448","date":"2025-07-24T11:29:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"190194988935132722321965268502873229506","date":"2025-07-24T11:04:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"335883571332155607550577167028226658830","date":"2025-07-24T08:40:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-24T08:17:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-11T05:30:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-11T05:30:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food Analytical Methods","date":"2025-07-04T07:22:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-analytical-methods","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food Analytical Methods](https://www.springer.com/journal/12161)","snPcode":"12161","submissionUrl":"https://submission.nature.com/new-submission/12161/3","title":"Food Analytical Methods","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3ddd03bb-5f8e-404c-9199-762f576c1266","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-14T10:23:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-28 17:08:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7044135","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7044135","identity":"rs-7044135","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}