Improved precision in oxytetracycline detection via fluorescence spectrometry impacted by pH

Improved precision in oxytetracycline detection via fluorescence spectrometry impacted by pH | 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 Improved precision in oxytetracycline detection via fluorescence spectrometry impacted by pH Xiaodian Huang, Dong Yang, Liang Song, Yongcan Jiang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4780110/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Sep, 2024 Read the published version in Journal of Fluorescence → Version 1 posted 7 You are reading this latest preprint version Abstract Accurate quantification of antibiotics in environmental samples is typically challenging due to the low antibiotic concentrations and the complexity of environmental matrices. This paper presents a fluorescence spectrometry method for determining oxytetracycline under alkaline conditions. The ionic distribution of the oxytetracycline solution was analyzed based on its dissociation constant. The dimethylamino group played a crucial role in this method, as it promotes intramolecular charge transfer in the electronic excited state through its electron-donating capability with a lone electron pair. The presented method is straightforward, cost-effective, and holds potential for analyzing oxytetracycline in water sample after further investigation. Fluorescence Oxytetracycline Antibiotic Seawater Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Over the past few decades, research on environmental contaminants has expanded beyond conventional pollutants to pharmaceuticals and personal care products such as antibiotics, which are widely used in human and veterinary medicine as a preventive or curative treatment for bacterial infections or as growth promoters [ 1 , 2 ]. Due to the incomplete elimination during wastewater treatment, antibiotics in trace amounts enter the environment and exert selective pressure on microorganisms, leading to the enrichment of antibiotic resistance genes (ARGs). Bacteria carrying ARGs upon entering a healthy organism tend to proliferate, increasing in number and forming a resistance module against antibiotics within the host. Consequently, the therapeutic efficacy of antibiotics is diminished [ 3 ]. Previously, antibiotics and ARGs have been frequently reported in rivers [ 4 , 5 ], lakes [ 6 ], groundwater [ 7 ], and estuarine and coastal environments [ 8 ]. Nevertheless, quantifying antibiotics accurately in environmental samples is typically challenging due to the low concentrations of antibiotic and the complexity of environmental matrices [ 9 ]. Currently, the method for detecting antibiotics primarily involves the utilization of Liquid Chromatography-Mass Spectrometry (LC-MS) in conjunction with appropriate pretreatment techniques, such as solid-phase extraction and dispersive liquid-liquid microextraction [ 10 ]. The widespread adoption of this method is limited by the high cost of analysis and complex procedures. Therefore, it is preferable to employ screening analytical techniques that streamline sample separation and cleaning procedures, while also minimizing the volume of organic solvents required [ 11 ]. The Fluorescence Spectrometry (FS) technique is widely recognized as a rapid and cost-effective method for investigating the composition, concentration, distribution, and dynamics of organic matter derived from various sources in diverse aquatic environments. [ 12 ]. However, the direct detection precision of antibiotics by FS is inadequate. For instance, León-Aguirre et al [ 11 ] carried out the detection of three antibiotics, namely enrofloxacin, oxytetracycline (OTC) and sulfamethoxazole, using FS. The Limit Of Detection (LOD) for these antibiotics were 0.133 mg/L, 0.149 mg/L and 2.532 mg/L, respectively. Thus the majority of spectrophotometric methodologies employed for the determination of antibiotics, such as Tetracyclines (TCs), are based on complexation [ 13 ]. Li et al. [ 14 ] synthesized a polynuclear lanthanide metal-organic framework featuring high selectivity and sensitivity towards TC, manifesting LOD of 8 µg/L. Jiang et al. (2022) utilized terbium doped CdTe QDs to form a Schiff base complex structure with norfloxacin, thereby enhancing the emission at 645 nm and achieving a LOD of 1.92 µg/L within 2 seconds. The present study has developed a FS method for the determination of OTC, which is based on the alkaline-induced dissociation properties of OTC. The OTC belongs to the group of TCs, which are commonly employed as therapeutic antibacterial agents in both human and veterinary medicine, as well as being used for prophylaxis and growth promotion in livestock production [ 16 ]. The molecular structure of OTC is depicted in Fig. 1 . The presence of the tricarbonyl group (highlighted in red dash frame), the phenolic-diketone group (highlighted in blue dash frame) and the dimethylamino group (highlighted in green dash frame) contribute to the formation of four OTC dissociation species (as depicted in Fig. 2 ) in solution depending on the pH level [ 17 ]. The four forms demonstrate distinct fluorescence characteristics, which serve as the foundation of this study. 2. Material and methods The OTC (oxytetracycline hydrochloride, CAS: 2058-46-0, > 97.0%) with a chromatographical purity of over 97% was procured from J&K Scientific (China) and utilized without any additional purification. Other chemicals, excluding the solvent using for LC-MS, were all of analytical grade. The seawater utilized in the case study was collected from Xiao Deng island, Xiamen, Fujian province, China. The dissociation constant experiments were conducted using an ultraviolet spectrophotometer (Agilent Cary 60, USA). The fluorescence experiments were performed utilizing a fluorospectro photometer (Agilent Cary Eclipse, USA). Additionally, a LC system (Agilent 1260, USA) equipped with mass spectrometry (Agilent 6460, USA) was employed for the comparative experiments. The pH of the OTC solution (1 mg/L) was adjusted using 1 mmol/L HCl and NaOH in order to conduct experiments on dissociation constant and fluorescence spectra. Standard stock solutions of OTC were prepared by dissolving 1 mg of OTC in 100 mL of deionized water, resulting in a concentration of 10 mg/L. A series of standard solutions with varying concentrations were obtained by diluting the stock solutions with deionized water. Subsequently, the pH was adjusted by adding 1 µL NaOH (1 mmol/L) per 1 mL of standard solutions, which were then sequentially analyzed using the corresponding instrument. 3. Results and discussion 3.1 Dissociation constant of oxytetracycline As shown in Fig. 2 , OTC exhibits four distinct species in solution based on pH, namely OTCH + , OTC ± , OTC − and OTC 2− . To gain a deeper understanding of its distribution in solution, the acid dissociation constants (pKa) of OTC were initially investigated using UV-visible spectrometry. The absorption spectra of OTC at multiple pH conditions (Fig. 3 a) revealed significant changes in the maximum absorption peak as pH increased. Specifically, at pH = 2.14, the absorption peak at 270 nm reached its highest intensity before gradually decreasing and shifting towards 280 nm. Additionally, while the absorption peak at 355 nm initially increased with increasing pH values, it shifted towards 375 nm when the pH exceeded 8.1. The absorbance and pH of the solutions were used to determine the pKa values by fitting the data to the following equation [ 18 ]: $$\:\begin{array}{c}{\text{ϵ}}_{\text{total}}\text{=}{\text{φ}}_{{\text{OTCH}}^{\text{+}}}\text{∙}{\text{ϵ}}_{{\text{OTCH}}^{\text{+}}}\text{+}{\text{φ}}_{{\text{OTC}}^{\text{±}}}\text{∙}{\text{ϵ}}_{{\text{OTC}}^{\text{±}}}\text{+}{\text{φ}}_{{\text{OTC}}^{\text{\--}}}\text{∙}{\text{ϵ}}_{{\text{OTC}}^{\text{\--}}}\text{+}{\text{φ}}_{{\text{OTC}}^{\text{2\--}}}\text{∙}{\text{ϵ}}_{{\text{OTC}}^{\text{2\--}}}\#\left(\text{1}\right)\end{array}$$ Where \(\:{\text{ϵ}}_{\text{X}}\) and \(\:{\text{φ}}_{\text{X}}\) represent the absorbance and proportion of X (X = OTCH + , OTC ± , OTC − and OTC 2− ). According to the definition of pKa, the proportion of OTCH + , OTC ± , OTC − and OTC 2− can be expressed as follows: $$\:\begin{array}{c}\left\{\begin{array}{c}{\text{φ}}_{{\text{OTCH}}^{\text{+}}}\text{=}\frac{\text{1}}{{\text{1+10}}^{\text{pH\--}{\text{pKa}}_{\text{1}}}\text{+}{\text{10}}^{\text{2∙pH\--}{\text{pKa}}_{\text{1}}\text{\--}{\text{pKa}}_{\text{2}}}{\text{+10}}^{\text{3∙pH\--}{\text{pKa}}_{\text{1}}\text{\--}{\text{pKa}}_{\text{2}}\text{\--}{\text{pKa}}_{\text{3}}}}\\\:{\text{φ}}_{{\text{OTC}}^{\text{±}}}\text{=}\frac{{\text{10}}^{\text{pH\--}{\text{pKa}}_{\text{1}}}}{{\text{1+10}}^{\text{pH\--}{\text{pKa}}_{\text{1}}}\text{+}{\text{10}}^{\text{2∙pH\--}{\text{pKa}}_{\text{1}}\text{\--}{\text{pKa}}_{\text{2}}}{\text{+10}}^{\text{3∙pH\--}{\text{pKa}}_{\text{1}}\text{\--}{\text{pKa}}_{\text{2}}\text{\--}{\text{pKa}}_{\text{3}}}}\\\:{\text{φ}}_{{\text{OTC}}^{\text{\--}}}\text{=}\frac{{\text{10}}^{\text{2∙pH\--}{\text{pKa}}_{\text{1}}\text{\--}{\text{pKa}}_{\text{2}}}}{{\text{1+10}}^{\text{pH\--}{\text{pKa}}_{\text{1}}}\text{+}{\text{10}}^{\text{2∙pH\--}{\text{pKa}}_{\text{1}}\text{\--}{\text{pKa}}_{\text{2}}}{\text{+10}}^{\text{3∙pH\--}{\text{pKa}}_{\text{1}}\text{\--}{\text{pKa}}_{\text{2}}\text{\--}{\text{pKa}}_{\text{3}}}}\\\:{\text{φ}}_{{\text{OTC}}^{\text{2\--\:}}}\text{=}\frac{{\text{10}}^{\text{3∙pH\--}{\text{pKa}}_{\text{1}}\text{\--}{\text{pKa}}_{\text{2}}\text{\--}{\text{pKa}}_{\text{3}}}}{{\text{1+10}}^{\text{pH\--}{\text{pKa}}_{\text{1}}}\text{+}{\text{10}}^{\text{2∙pH\--}{\text{pKa}}_{\text{1}}\text{\--}{\text{pKa}}_{\text{2}}}{\text{+10}}^{\text{3∙pH\--}{\text{pKa}}_{\text{1}}\text{\--}{\text{pKa}}_{\text{2}}\text{\--}{\text{pKa}}_{\text{3}}}}\end{array}\right.\#\left(\text{2}\right)\end{array}$$ By combining the Eqs. (1) and (2), the relationship between the total absorbance and pH can be derived as follows: $$\:\begin{array}{c}{\text{ϵ}}_{\text{total}}\text{=}\frac{{{\text{ϵ}}_{{\text{OTCH}}^{\text{+}}}\text{+}{\text{ϵ}}_{{\text{OTC}}^{\text{±}}}\text{∙10}}^{\text{pH\--}{\text{pKa}}_{\text{1}}}\text{+}{{\text{ϵ}}_{{\text{OTC}}^{\text{\--}}}\text{∙10}}^{\text{2∙pH\--}{\text{pKa}}_{\text{1}}\text{\--}{\text{pKa}}_{\text{2}}}{\text{+}{\text{ϵ}}_{{\text{OTC}}^{\text{2\--}}}\text{∙10}}^{\text{3∙pH\--}{\text{pKa}}_{\text{1}}\text{\--}{\text{pKa}}_{\text{2}}\text{\--}{\text{pKa}}_{\text{3}}}}{{\text{1+10}}^{\text{pH\--}{\text{pKa}}_{\text{1}}}\text{+}{\text{10}}^{\text{2∙pH\--}{\text{pKa}}_{\text{1}}\text{\--}{\text{pKa}}_{\text{2}}}{\text{+10}}^{\text{3∙pH\--}{\text{pKa}}_{\text{1}}\text{\--}{\text{pKa}}_{\text{2}}\text{\--}{\text{pKa}}_{\text{3}}}}\#\left(\text{3}\right)\end{array}$$ By fitting the data at 270 nm and 375 nm with the Eq. (3) (Fig. 3 b), the pKa values of OTC were determined to be 2.99 (pKa 1 ), 7.70 (pKa 2 ) and 8.55 (pKa 3 ). These results are consistent with a previous study conducted using potentiometric titration [ 19 ]. By substituting these pKa values into Eq. (2), the pH-dependent variations of OTCH + , OTC ± , OTC − and OTC 2− can be obtained, as illustrated in Fig. 4 . The concentration of OTCH + decreases as pH increases, eventually disappearing when pH exceeds 6. Conversely, at pH above 7, OTC 2− starts to appear in solution with its concentration increasing alongside rising pH levels. OTC − is only present within the pH range of 5 to11, reaching a maximum concentration that does not exceed 60%. OTC ± exhibits a broader pH range of 0–10 and is predominantly present in solution under neutral condition. 3.2 Fluorescence spectra under varying pH Based on the pKa study of OTC, the fluorescence spectrums (as shown in Fig. 5 ) have been detected in pH of 1.97, 4.83, 8.09 and 10.55, confirming the predominant presence of OTCH + , OTC ± , OTC − and OTC 2− in the respective solutions. An intense fluorescence response was observed at excitation wavelength (Ex) of 375 nm and emission wavelength (Em) of 515 nm in the pH 10.55 spectrum, which differed significantly from the others. Figure 6 (a) illustrates the intensity at pH of 1.97, 4.83, 8.09 and 10.55 under the same Ex value (375 nm). The maximum peaks at pH of 1.97 and 4.83 appeared at Em = 570 nm, while the peaks at pH of 8.09 and 10.55 were observed at Em = 515 nm, indicating that the intense fluorescence was attributed to OTC 2− . The correlation between the variation trend of fluorescence intensity at Ex = 375 nm and Em = 515 nm with respect to OTC 2− proportion is demonstrated in Fig. 6 (b), thereby substantiating this perspective. The primary distinction between OTC 2− and other forms of OTC in solution lies in the presence of the neutral dimethylamino group. Ma et al (2012) reported a noticeable quenching of fluorescence in dansyl acid as the pH gradually decreased from 7 to 2. Park et al (2007) observed a decrease in emission band around 432 nm upon lowering the pH of the solution containing 7-(dimethylamino)-2-fluorenesulfonate. All these compounds contain a dimethylamino group connected to a hexatomic ring, forming a D(Donor)−π − A(Acceptor) system [ 21 ].The dimethylamino group herein acted as an electron-donor due to its pronounced electron-donating capability through a lone electron pair [ 22 ], thereby facilitating intramolecular charge transfer (ICT) in the electronic excited state [ 23 ]. However, the interaction between the lone pair of electrons and H + had a detrimental effect on the ion electron-donating ability of the dimethylamino group, leading to fluorescence quenching caused by ICT [ 24 , 25 ]. 3.3 Calibration and validation of the method Based on the excellent fluorescence property of OTC under alkaline conditions, a series of standard OTC solutions (20-1000 µg/L) were adjusted to a pH above 10. Subsequently, standard solutions were sequentially analyzed by FS at Ex = 375 nm and Em = 515 nm. By measuring the fluorescence intensity of standard solutions, a linear relationship between the fluorescence intensity and the concentrations of OTC in solution was obtained as depicted in Fig. 7 . Taking into account the standard deviation of the signal-to-noise ratio, the limit of determination (LOD) for OTC was calculated to be 1.57 µg/L. In comparison, direct detection of OTC by FS yielded a LOD of 149 µg/L [ 11 ], which is nearly 100 times higher than that achieved in this study. Although there still exists a gap when compared to the common method involving HPLC-MS/MS coupled with solid phase extraction [ 26 ], this method can be employed in specific scenarios due to its rapidity and cost-effectiveness. The precision of this method was assessed by triplicate analysis of the samples, yielding relative standard deviations (RSDs) below 7.01%. To verify the accuracy of the method, a comparative experiment employing LC-MS/MS was conducted. The results of the comparison are depicted in Fig. 8 . Deviations between the results obtained from FS and LC-MS/MS fell within a range of 15%, indicating the suitability of the presented method for quantifying OTC. (pH = 10; Ex = 375 nm; Em = 515 nm) 3.4 Case application in seawater As antibiotics are prevalent in estuarine and coastal environments due to substantial terrestrial input, aquaculture effluent, and sewage discharge [ 8 ], the presented method was applied to the seawater sample. OTC with known concentrations were added to seawater samples, and Table 1 presents the recovery results. The observed recovery ranged from 55.15–67.63%. The low recovery may be attributed to the presence of metal ions in seawater, which have a strong tendency to interact with TCs and form metal complexes [ 27 ]. Given the complex composition of seawater, further experiments investigating the influence of metal ions are still necessary for accurate detection of OTC in seawater samples; however, it should be noted that even after correction, this presented method can still be utilized. Table 1 Recovery of OTC in seawater Sample Added (µg/L) Measured (µg/L) Recovery (%) 1 10 6.76 ± 0.17 67.63 2 20 11.03 ± 1.21 55.15 3 50 29.49 ± 0.85 58.97 4 100 55.80 ± 2.72 55.80 4. Conclusions The present study describes the development of a FS method for the determination of OTC, which is based on the ICT emission exhibited by the dimethylamino group under alkaline conditions. The developed method demonstrates excellent precision and accuracy. Furthermore, it offers simplicity and cost-effectiveness, thereby exhibiting potential for analyzing OTC in water samples following further investigations. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding This research was supported by Science and Technology Project of Huadong Engineering (Fujian) Corporation (KY2023-FJ-02-01). Author Contribution Huang wrote the main manuscript text , other authors offer revisions from their own perspectives. All authors reviewed the manuscript Data availability All data generated or analyzed during this study are included in this published article. References Zhou Y, Li W, Kumar V, Necibi MC, Mu Y-J, Shi C, Chaurasia D, Chauhan S, Chaturvedi P, Sillanpää M, Zhang Z, Awasthi MK, Sirohi R (2022) Synthetic organic antibiotics residues as emerging contaminants waste-to-resources processing for a circular economy in China: Challenges and perspective. Environ Res 211:113075. https://doi.org/10.1016/j.envres.2022.113075 Bu Q, Wang B, Huang J, Deng S, Yu G (2013) Pharmaceuticals and personal care products in the aquatic environment in China: a review. J Hazard Mater 262:189–211. https://doi.org/10.1016/j.jhazmat.2013.08.040 Leonard AFC, Zhang L, Balfour AJ, Garside R, Gaze WH (2015) Human recreational exposure to antibiotic resistant bacteria in coastal bathing waters. Environ Int 82:92–100. https://doi.org/10.1016/j.envint.2015.02.013 Chen L, Li H, Liu Y, Cui Y, Li Y, Yang Z (2020) Distribution, residue level, sources, and phase partition of antibiotics in surface sediments from the inland river: a case study of the Xiangjiang River, south-central China. Environ Sci Pollut Res Int 27:2273–2286. https://doi.org/10.1007/s11356-019-06833-0 Feng L, Cheng Y, Zhang Y, Li Z, Yu Y, Feng L, Zhang S, Xu L (2020) Distribution and human health risk assessment of antibiotic residues in large-scale drinking water sources in Chongqing area of the Yangtze River. Environ Res 185:109386. https://doi.org/10.1016/j.envres.2020.109386 Yang Y, Song W, Lin H, Wang W, Du L, Xing W (2018) Antibiotics and antibiotic resistance genes in global lakes: A review and meta-analysis. Environ Int 116:60–73. https://doi.org/10.1016/j.envint.2018.04.011 Zainab SM, Junaid M, Xu N, Malik RN (2020) Antibiotics and antibiotic resistant genes (ARGs) in groundwater: A global review on dissemination, sources, interactions, environmental and human health risks. Water Res 187:116455. https://doi.org/10.1016/j.watres.2020.116455 Zheng D, Yin G, Liu M, Chen C, Jiang Y, Hou L, Zheng Y (2021) A systematic review of antibiotics and antibiotic resistance genes in estuarine and coastal environments. Sci Total Environ 777:146009. https://doi.org/10.1016/j.scitotenv.2021.146009 Yi X, Bayen S, Kelly BC, Li X, Zhou Z (2015) Improved detection of multiple environmental antibiotics through an optimized sample extraction strategy in liquid chromatography-mass spectrometry analysis. Anal Bioanal Chem 407:9071–9083. https://doi.org/10.1007/s00216-015-9074-7 Liang N, Huang P, Hou X, Li Z, Tao L, Zhao L (2016) Solid-phase extraction in combination with dispersive liquid-liquid microextraction and ultra-high performance liquid chromatography-tandem mass spectrometry analysis: the ultra-trace determination of 10 antibiotics in water samples. Anal Bioanal Chem 408:1701–1713. https://doi.org/10.1007/s00216-015-9284-z León-Aguirre K, Hernández-Núñez E, González-Sánchez A, Méndez-Novelo R, Ponce-Caballero C, Giácoman-Vallejos G (2019) A Rapid and Green Method for the Determination of Veterinary Pharmaceuticals in Swine Wastewater by Fluorescence Spectrophotometry. Bull Environ Contam Toxicol 103:610–616. https://doi.org/10.1007/s00128-019-02701-2 Hudson N, Baker A, Reynolds D (2007) Fluorescence analysis of dissolved organic matter in natural, waste and polluted waters—a review. River Res Applic 23:631–649. https://doi.org/10.1002/rra.1005 Leichtweis J, Vieira Y, Welter N, Silvestri S, Dotto GL, Carissimi E (2022) A review of the occurrence, disposal, determination, toxicity and remediation technologies of the tetracycline antibiotic. Process Saf Environ Prot 160:25–40. https://doi.org/10.1016/j.psep.2022.01.085 Li R, Wang W, El-Sayed E-SM, Su K, He P, Yuan D (2021) Ratiometric fluorescence detection of tetracycline antibiotic based on a polynuclear lanthanide metal–organic framework. Sens Actuators B 330:129314. https://doi.org/10.1016/j.snb.2020.129314 Jiang R, Lin D, Zhang Q, Li L, Yang L (2022) Multiplex chroma-response based fluorescent smartphone sensing platform for rapid and visual quantitative determination of antibiotic residues. Sens Actuators B 350:130902. https://doi.org/10.1016/j.snb.2021.130902 Bi X, Xu B, Lin Y-L, Hu C-Y, Ye T, Qin C (2013) Monochloramination of Oxytetracycline: Kinetics, Mechanisms, Pathways, and Disinfection By-Products Formation, CLEAN - Soil, Air, Water n/a-n/a . https://doi.org/10.1002/clen.201200344 Jin X, Xu H, Qiu S, Jia M, Wang F, Zhang A, Jiang X (2017) Direct photolysis of oxytetracycline: Influence of initial concentration, pH and temperature. J Photochem Photobiol A 332:224–231. https://doi.org/10.1016/j.jphotochem.2016.08.032 Boreen AL, Arnold WA, McNeill K (2004) Photochemical Fate of Sulfa Drugs in the Aquatic Environment: Sulfa Drugs Containing Five-Membered Heterocyclic Groups. Environ Sci Technol 38:3933–3940. https://doi.org/10.1021/es0353053 Qiang Z, Adams C (2004) Potentiometric determination of acid dissociation constants (pKa) for human and veterinary antibiotics. Water Res 38:2874–2890. https://doi.org/10.1016/j.watres.2004.03.017 Park KK, Park JW, Hamilton AD (2007) Solvent and pH Effects on the Fluorescence of 7-(Dimethylamino)-2-Fluorenesulfonate. J Fluoresc 17:361–369. https://doi.org/10.1007/s10895-007-0193-1 Yang D, Liu P, Bai T, Kong J (2020) -Dimethyl-Substituted Boron Ketoiminates for Multicolor Fluorescent Initiators and Polymers. Macromolecules 53:3339–3348. https://doi.org/10.1021/acs.macromol.9b02633 Chen C, Fang C (2023) Fluorescence Modulation by Amines: Mechanistic Insights into Twisted Intramolecular Charge Transfer (TICT) and Beyond. Chemosensors 11:87. https://doi.org/10.3390/chemosensors11020087 Yuan L, Lin W, Zheng K, He L, Huang W (2012) Far-red to near infrared analyte-responsive fluorescent probes based on organic fluorophore platforms for fluorescence imaging. Chem Soc Rev 42:622–661. https://doi.org/10.1039/C2CS35313J Ma L, Cao W, Liu J, Deng D, Wu Y, Yan Y, Yang L (2012) A highly selective and sensitive fluorescence dual-responsive pH probe in water. Sens Actuators B 169:243–247. https://doi.org/10.1016/j.snb.2012.04.076 Saha SK, Purkayastha P, Das AB (2008) Photophysical characterization and effect of pH on the twisted intramolecular charge transfer fluorescence of trans-2-[4-(dimethylamino)styryl]benzothiazole. J Photochem Photobiol A 195:368–377. https://doi.org/10.1016/j.jphotochem.2007.11.004 Zhang X, Zhang T, Dong L, Shi S, Yang W, Zhang L, Zhou L, Huang Y (2012) Determination of Antibiotics in Hospital Wastewater Using HPLC-MS /MS Coupled with Solid Phase Extraction. J Instrumental Anal 31:453–458 Pulicharla R, Hegde K, Brar SK, Surampalli RY (2017) Tetracyclines metal complexation: Significance and fate of mutual existence in the environment. Environ Pollut 221:1–14. https://doi.org/10.1016/j.envpol.2016.12.017 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 26 Sep, 2024 Read the published version in Journal of Fluorescence → Version 1 posted Editorial decision: Revision requested 11 Aug, 2024 Reviews received at journal 09 Aug, 2024 Reviewers agreed at journal 08 Aug, 2024 Reviewers invited by journal 08 Aug, 2024 Editor assigned by journal 25 Jul, 2024 Submission checks completed at journal 25 Jul, 2024 First submitted to journal 22 Jul, 2024 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4780110","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":338740684,"identity":"a11048ac-233a-4314-b79a-54696dc229bf","order_by":0,"name":"Xiaodian Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYBACAyjN2C8BZxOrZeYMkrVsuEGsFnOJ5GcPvzAclt18u3njgx8MdnK6DQS0WM5IMzeWYUgz3nbnWLFhD0OysdkBQg67kWAmLcFgk7jtRo6ZNAPDgcRthLWkfwNqkUjcPCPH/DeRWnLMJD8AbdkgkWPGTJyWM2/KgO5JM55xI61YsseAGL8cT98m+QMYYv0zkjd++FFhJ0dQCwgw8/6Dm0CEchBg/EGkwlEwCkbBKBihAACicED4GKPRkQAAAABJRU5ErkJggg==","orcid":"","institution":"Power China Huadong Engineering Corporation Ltd","correspondingAuthor":true,"prefix":"","firstName":"Xiaodian","middleName":"","lastName":"Huang","suffix":""},{"id":338740687,"identity":"5c510fd3-137a-41c6-970c-021f85578497","order_by":1,"name":"Dong Yang","email":"","orcid":"","institution":"Power China Huadong Engineering Corporation Ltd","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"","lastName":"Yang","suffix":""},{"id":338740689,"identity":"45ce0d07-a104-4f6d-8944-712139a82671","order_by":2,"name":"Liang Song","email":"","orcid":"","institution":"Power China Huadong Engineering Corporation Ltd","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Song","suffix":""},{"id":338740691,"identity":"322b8357-aadc-4c2e-9d38-05709b3df082","order_by":3,"name":"Yongcan Jiang","email":"","orcid":"","institution":"Power China Huadong Engineering Corporation Ltd","correspondingAuthor":false,"prefix":"","firstName":"Yongcan","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2024-07-22 07:59:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4780110/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4780110/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10895-024-03941-0","type":"published","date":"2024-09-26T15:58:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62981790,"identity":"afe43f66-64fb-42b4-88b4-24bd195103aa","added_by":"auto","created_at":"2024-08-21 17:44:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23077,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular structure of OTC\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4780110/v1/58be05f24eaaf88e18d56cff.png"},{"id":62981791,"identity":"88d869c6-4d7b-44f1-80cb-b6f310308434","added_by":"auto","created_at":"2024-08-21 17:44:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":60503,"visible":true,"origin":"","legend":"\u003cp\u003eStructures and ionization equilibriums of OTC\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4780110/v1/73d6c17e8ab9d4f503ebdf38.png"},{"id":62982188,"identity":"73f4f0ac-cf7e-4eff-bcee-2966952184c5","added_by":"auto","created_at":"2024-08-21 17:52:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31810,"visible":true,"origin":"","legend":"\u003cp\u003eThe absorption spectra of OTC under various pH conditions (a) and the corresponding fitting results (b)\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4780110/v1/2942838e914a5365d656c8c3.png"},{"id":62981792,"identity":"64ab60c7-afd9-4ce8-a5cf-7c1fbf62673b","added_by":"auto","created_at":"2024-08-21 17:44:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9266,"visible":true,"origin":"","legend":"\u003cp\u003eThe pH-dependent variation of OTCH\u003csup\u003e+\u003c/sup\u003e, OTC\u003csup\u003e±\u003c/sup\u003e, OTC\u003csup\u003e-\u003c/sup\u003e and OTC\u003csup\u003e2-\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4780110/v1/e67200860aaf067666764566.png"},{"id":62981796,"identity":"210d352c-19f2-46a5-9131-9aacc2aa944e","added_by":"auto","created_at":"2024-08-21 17:44:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":457387,"visible":true,"origin":"","legend":"\u003cp\u003eThe fluorescence spectra of OTC at pH of 1.97, 4.83, 8.09 and 10.55\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4780110/v1/7bee7d83deaaeb0a3da50258.png"},{"id":62981793,"identity":"5e35d4dc-8df2-4984-86aa-3d4f5e117362","added_by":"auto","created_at":"2024-08-21 17:44:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":30657,"visible":true,"origin":"","legend":"\u003cp\u003eThe fluorescence spectra (a) and the trend of intensity variation (b) of OTC at various pH with Ex=375 nm\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4780110/v1/270cec494e16f225d0c6d09d.png"},{"id":62981794,"identity":"683d0cc6-effd-4acd-8f67-e6acf97c16c0","added_by":"auto","created_at":"2024-08-21 17:44:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":31347,"visible":true,"origin":"","legend":"\u003cp\u003eThe standard curve of OTC detected by FS\u003c/p\u003e\n\u003cp\u003e(pH=10; Ex=375 nm; Em=515 nm)\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-4780110/v1/1ce020a842d7632505dc97b3.png"},{"id":62981797,"identity":"1fb0a08b-e864-451f-9a4a-4f3832ff451e","added_by":"auto","created_at":"2024-08-21 17:44:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":23636,"visible":true,"origin":"","legend":"\u003cp\u003eThe comparative experimental results between FS and LC-MS/MS\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-4780110/v1/411294c309b3f2c371304d9a.png"},{"id":65627337,"identity":"d224da7a-fe24-4768-a0d0-8a678bc4fc12","added_by":"auto","created_at":"2024-09-30 16:15:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":803814,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4780110/v1/f02aa7c1-9421-4a81-aca9-e5eee8654776.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Improved precision in oxytetracycline detection via fluorescence spectrometry impacted by pH","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOver the past few decades, research on environmental contaminants has expanded beyond conventional pollutants to pharmaceuticals and personal care products such as antibiotics, which are widely used in human and veterinary medicine as a preventive or curative treatment for bacterial infections or as growth promoters [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Due to the incomplete elimination during wastewater treatment, antibiotics in trace amounts enter the environment and exert selective pressure on microorganisms, leading to the enrichment of antibiotic resistance genes (ARGs). Bacteria carrying ARGs upon entering a healthy organism tend to proliferate, increasing in number and forming a resistance module against antibiotics within the host. Consequently, the therapeutic efficacy of antibiotics is diminished [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePreviously, antibiotics and ARGs have been frequently reported in rivers [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], lakes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], groundwater [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and estuarine and coastal environments [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Nevertheless, quantifying antibiotics accurately in environmental samples is typically challenging due to the low concentrations of antibiotic and the complexity of environmental matrices [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Currently, the method for detecting antibiotics primarily involves the utilization of Liquid Chromatography-Mass Spectrometry (LC-MS) in conjunction with appropriate pretreatment techniques, such as solid-phase extraction and dispersive liquid-liquid microextraction [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The widespread adoption of this method is limited by the high cost of analysis and complex procedures. Therefore, it is preferable to employ screening analytical techniques that streamline sample separation and cleaning procedures, while also minimizing the volume of organic solvents required [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Fluorescence Spectrometry (FS) technique is widely recognized as a rapid and cost-effective method for investigating the composition, concentration, distribution, and dynamics of organic matter derived from various sources in diverse aquatic environments. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the direct detection precision of antibiotics by FS is inadequate. For instance, Le\u0026oacute;n-Aguirre et al [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] carried out the detection of three antibiotics, namely enrofloxacin, oxytetracycline (OTC) and sulfamethoxazole, using FS. The Limit Of Detection (LOD) for these antibiotics were 0.133 mg/L, 0.149 mg/L and 2.532 mg/L, respectively. Thus the majority of spectrophotometric methodologies employed for the determination of antibiotics, such as Tetracyclines (TCs), are based on complexation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Li et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] synthesized a polynuclear lanthanide metal-organic framework featuring high selectivity and sensitivity towards TC, manifesting LOD of 8 \u0026micro;g/L. Jiang et al. (2022) utilized terbium doped CdTe QDs to form a Schiff base complex structure with norfloxacin, thereby enhancing the emission at 645 nm and achieving a LOD of 1.92 \u0026micro;g/L within 2 seconds.\u003c/p\u003e \u003cp\u003eThe present study has developed a FS method for the determination of OTC, which is based on the alkaline-induced dissociation properties of OTC. The OTC belongs to the group of TCs, which are commonly employed as therapeutic antibacterial agents in both human and veterinary medicine, as well as being used for prophylaxis and growth promotion in livestock production [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The molecular structure of OTC is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The presence of the tricarbonyl group (highlighted in red dash frame), the phenolic-diketone group (highlighted in blue dash frame) and the dimethylamino group (highlighted in green dash frame) contribute to the formation of four OTC dissociation species (as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) in solution depending on the pH level [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The four forms demonstrate distinct fluorescence characteristics, which serve as the foundation of this study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cp\u003eThe OTC (oxytetracycline hydrochloride, CAS: 2058-46-0, \u0026gt;\u0026thinsp;97.0%) with a chromatographical purity of over 97% was procured from J\u0026amp;K Scientific (China) and utilized without any additional purification. Other chemicals, excluding the solvent using for LC-MS, were all of analytical grade. The seawater utilized in the case study was collected from Xiao Deng island, Xiamen, Fujian province, China.\u003c/p\u003e \u003cp\u003eThe dissociation constant experiments were conducted using an ultraviolet spectrophotometer (Agilent Cary 60, USA). The fluorescence experiments were performed utilizing a fluorospectro photometer (Agilent Cary Eclipse, USA). Additionally, a LC system (Agilent 1260, USA) equipped with mass spectrometry (Agilent 6460, USA) was employed for the comparative experiments.\u003c/p\u003e \u003cp\u003eThe pH of the OTC solution (1 mg/L) was adjusted using 1 mmol/L HCl and NaOH in order to conduct experiments on dissociation constant and fluorescence spectra. Standard stock solutions of OTC were prepared by dissolving 1 mg of OTC in 100 mL of deionized water, resulting in a concentration of 10 mg/L. A series of standard solutions with varying concentrations were obtained by diluting the stock solutions with deionized water. Subsequently, the pH was adjusted by adding 1 \u0026micro;L NaOH (1 mmol/L) per 1 mL of standard solutions, which were then sequentially analyzed using the corresponding instrument.\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Dissociation constant of oxytetracycline\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, OTC exhibits four distinct species in solution based on pH, namely OTCH\u003csup\u003e+\u003c/sup\u003e, OTC\u003csup\u003e\u0026plusmn;\u003c/sup\u003e, OTC\u003csup\u003e\u0026minus;\u003c/sup\u003e and OTC\u003csup\u003e2\u0026minus;\u003c/sup\u003e. To gain a deeper understanding of its distribution in solution, the acid dissociation constants (pKa) of OTC were initially investigated using UV-visible spectrometry. The absorption spectra of OTC at multiple pH conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) revealed significant changes in the maximum absorption peak as pH increased. Specifically, at pH\u0026thinsp;=\u0026thinsp;2.14, the absorption peak at 270 nm reached its highest intensity before gradually decreasing and shifting towards 280 nm. Additionally, while the absorption peak at 355 nm initially increased with increasing pH values, it shifted towards 375 nm when the pH exceeded 8.1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe absorbance and pH of the solutions were used to determine the pKa values by fitting the data to the following equation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\begin{array}{c}{\\text{ϵ}}_{\\text{total}}\\text{=}{\\text{\u0026phi;}}_{{\\text{OTCH}}^{\\text{+}}}\\text{∙}{\\text{ϵ}}_{{\\text{OTCH}}^{\\text{+}}}\\text{+}{\\text{\u0026phi;}}_{{\\text{OTC}}^{\\text{\u0026plusmn;}}}\\text{∙}{\\text{ϵ}}_{{\\text{OTC}}^{\\text{\u0026plusmn;}}}\\text{+}{\\text{\u0026phi;}}_{{\\text{OTC}}^{\\text{\\--}}}\\text{∙}{\\text{ϵ}}_{{\\text{OTC}}^{\\text{\\--}}}\\text{+}{\\text{\u0026phi;}}_{{\\text{OTC}}^{\\text{2\\--}}}\\text{∙}{\\text{ϵ}}_{{\\text{OTC}}^{\\text{2\\--}}}\\#\\left(\\text{1}\\right)\\end{array}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{ϵ}}_{\\text{X}}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{\u0026phi;}}_{\\text{X}}\\)\u003c/span\u003e\u003c/span\u003e represent the absorbance and proportion of X (X\u0026thinsp;=\u0026thinsp;OTCH\u003csup\u003e+\u003c/sup\u003e, OTC\u003csup\u003e\u0026plusmn;\u003c/sup\u003e, OTC\u003csup\u003e\u0026minus;\u003c/sup\u003e and OTC\u003csup\u003e2\u0026minus;\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eAccording to the definition of pKa, the proportion of OTCH\u003csup\u003e+\u003c/sup\u003e, OTC\u003csup\u003e\u0026plusmn;\u003c/sup\u003e, OTC\u003csup\u003e\u0026minus;\u003c/sup\u003e and OTC\u003csup\u003e2\u0026minus;\u003c/sup\u003e can be expressed as follows:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\begin{array}{c}\\left\\{\\begin{array}{c}{\\text{\u0026phi;}}_{{\\text{OTCH}}^{\\text{+}}}\\text{=}\\frac{\\text{1}}{{\\text{1+10}}^{\\text{pH\\--}{\\text{pKa}}_{\\text{1}}}\\text{+}{\\text{10}}^{\\text{2∙pH\\--}{\\text{pKa}}_{\\text{1}}\\text{\\--}{\\text{pKa}}_{\\text{2}}}{\\text{+10}}^{\\text{3∙pH\\--}{\\text{pKa}}_{\\text{1}}\\text{\\--}{\\text{pKa}}_{\\text{2}}\\text{\\--}{\\text{pKa}}_{\\text{3}}}}\\\\\\:{\\text{\u0026phi;}}_{{\\text{OTC}}^{\\text{\u0026plusmn;}}}\\text{=}\\frac{{\\text{10}}^{\\text{pH\\--}{\\text{pKa}}_{\\text{1}}}}{{\\text{1+10}}^{\\text{pH\\--}{\\text{pKa}}_{\\text{1}}}\\text{+}{\\text{10}}^{\\text{2∙pH\\--}{\\text{pKa}}_{\\text{1}}\\text{\\--}{\\text{pKa}}_{\\text{2}}}{\\text{+10}}^{\\text{3∙pH\\--}{\\text{pKa}}_{\\text{1}}\\text{\\--}{\\text{pKa}}_{\\text{2}}\\text{\\--}{\\text{pKa}}_{\\text{3}}}}\\\\\\:{\\text{\u0026phi;}}_{{\\text{OTC}}^{\\text{\\--}}}\\text{=}\\frac{{\\text{10}}^{\\text{2∙pH\\--}{\\text{pKa}}_{\\text{1}}\\text{\\--}{\\text{pKa}}_{\\text{2}}}}{{\\text{1+10}}^{\\text{pH\\--}{\\text{pKa}}_{\\text{1}}}\\text{+}{\\text{10}}^{\\text{2∙pH\\--}{\\text{pKa}}_{\\text{1}}\\text{\\--}{\\text{pKa}}_{\\text{2}}}{\\text{+10}}^{\\text{3∙pH\\--}{\\text{pKa}}_{\\text{1}}\\text{\\--}{\\text{pKa}}_{\\text{2}}\\text{\\--}{\\text{pKa}}_{\\text{3}}}}\\\\\\:{\\text{\u0026phi;}}_{{\\text{OTC}}^{\\text{2\\--\\:}}}\\text{=}\\frac{{\\text{10}}^{\\text{3∙pH\\--}{\\text{pKa}}_{\\text{1}}\\text{\\--}{\\text{pKa}}_{\\text{2}}\\text{\\--}{\\text{pKa}}_{\\text{3}}}}{{\\text{1+10}}^{\\text{pH\\--}{\\text{pKa}}_{\\text{1}}}\\text{+}{\\text{10}}^{\\text{2∙pH\\--}{\\text{pKa}}_{\\text{1}}\\text{\\--}{\\text{pKa}}_{\\text{2}}}{\\text{+10}}^{\\text{3∙pH\\--}{\\text{pKa}}_{\\text{1}}\\text{\\--}{\\text{pKa}}_{\\text{2}}\\text{\\--}{\\text{pKa}}_{\\text{3}}}}\\end{array}\\right.\\#\\left(\\text{2}\\right)\\end{array}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eBy combining the Eqs.\u0026nbsp;(1) and (2), the relationship between the total absorbance and pH can be derived as follows:\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:\\begin{array}{c}{\\text{ϵ}}_{\\text{total}}\\text{=}\\frac{{{\\text{ϵ}}_{{\\text{OTCH}}^{\\text{+}}}\\text{+}{\\text{ϵ}}_{{\\text{OTC}}^{\\text{\u0026plusmn;}}}\\text{∙10}}^{\\text{pH\\--}{\\text{pKa}}_{\\text{1}}}\\text{+}{{\\text{ϵ}}_{{\\text{OTC}}^{\\text{\\--}}}\\text{∙10}}^{\\text{2∙pH\\--}{\\text{pKa}}_{\\text{1}}\\text{\\--}{\\text{pKa}}_{\\text{2}}}{\\text{+}{\\text{ϵ}}_{{\\text{OTC}}^{\\text{2\\--}}}\\text{∙10}}^{\\text{3∙pH\\--}{\\text{pKa}}_{\\text{1}}\\text{\\--}{\\text{pKa}}_{\\text{2}}\\text{\\--}{\\text{pKa}}_{\\text{3}}}}{{\\text{1+10}}^{\\text{pH\\--}{\\text{pKa}}_{\\text{1}}}\\text{+}{\\text{10}}^{\\text{2∙pH\\--}{\\text{pKa}}_{\\text{1}}\\text{\\--}{\\text{pKa}}_{\\text{2}}}{\\text{+10}}^{\\text{3∙pH\\--}{\\text{pKa}}_{\\text{1}}\\text{\\--}{\\text{pKa}}_{\\text{2}}\\text{\\--}{\\text{pKa}}_{\\text{3}}}}\\#\\left(\\text{3}\\right)\\end{array}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eBy fitting the data at 270 nm and 375 nm with the Eq.\u0026nbsp;(3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), the pKa values of OTC were determined to be 2.99 (pKa\u003csub\u003e1\u003c/sub\u003e), 7.70 (pKa\u003csub\u003e2\u003c/sub\u003e) and 8.55 (pKa\u003csub\u003e3\u003c/sub\u003e). These results are consistent with a previous study conducted using potentiometric titration [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. By substituting these pKa values into Eq.\u0026nbsp;(2), the pH-dependent variations of OTCH\u003csup\u003e+\u003c/sup\u003e, OTC\u003csup\u003e\u0026plusmn;\u003c/sup\u003e, OTC\u003csup\u003e\u0026minus;\u003c/sup\u003e and OTC\u003csup\u003e2\u0026minus;\u003c/sup\u003e can be obtained, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The concentration of OTCH\u003csup\u003e+\u003c/sup\u003e decreases as pH increases, eventually disappearing when pH exceeds 6. Conversely, at pH above 7, OTC\u003csup\u003e2\u0026minus;\u003c/sup\u003e starts to appear in solution with its concentration increasing alongside rising pH levels. OTC\u003csup\u003e\u0026minus;\u003c/sup\u003e is only present within the pH range of 5 to11, reaching a maximum concentration that does not exceed 60%. OTC\u003csup\u003e\u0026plusmn;\u003c/sup\u003e exhibits a broader pH range of 0\u0026ndash;10 and is predominantly present in solution under neutral condition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Fluorescence spectra under varying pH\u003c/h2\u003e \u003cp\u003eBased on the pKa study of OTC, the fluorescence spectrums (as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) have been detected in pH of 1.97, 4.83, 8.09 and 10.55, confirming the predominant presence of OTCH\u003csup\u003e+\u003c/sup\u003e, OTC\u003csup\u003e\u0026plusmn;\u003c/sup\u003e, OTC\u003csup\u003e\u0026minus;\u003c/sup\u003e and OTC\u003csup\u003e2\u0026minus;\u003c/sup\u003e in the respective solutions. An intense fluorescence response was observed at excitation wavelength (Ex) of 375 nm and emission wavelength (Em) of 515 nm in the pH 10.55 spectrum, which differed significantly from the others. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (a) illustrates the intensity at pH of 1.97, 4.83, 8.09 and 10.55 under the same Ex value (375 nm). The maximum peaks at pH of 1.97 and 4.83 appeared at Em\u0026thinsp;=\u0026thinsp;570 nm, while the peaks at pH of 8.09 and 10.55 were observed at Em\u0026thinsp;=\u0026thinsp;515 nm, indicating that the intense fluorescence was attributed to OTC\u003csup\u003e2\u0026minus;\u003c/sup\u003e. The correlation between the variation trend of fluorescence intensity at Ex\u0026thinsp;=\u0026thinsp;375 nm and Em\u0026thinsp;=\u0026thinsp;515 nm with respect to OTC\u003csup\u003e2\u0026minus;\u003c/sup\u003e proportion is demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (b), thereby substantiating this perspective.\u003c/p\u003e \u003cp\u003eThe primary distinction between OTC\u003csup\u003e2\u0026minus;\u003c/sup\u003e and other forms of OTC in solution lies in the presence of the neutral dimethylamino group. Ma et al (2012) reported a noticeable quenching of fluorescence in dansyl acid as the pH gradually decreased from 7 to 2. Park et al (2007) observed a decrease in emission band around 432 nm upon lowering the pH of the solution containing 7-(dimethylamino)-2-fluorenesulfonate. All these compounds contain a dimethylamino group connected to a hexatomic ring, forming a D(Donor)\u0026minus;π\u0026thinsp;\u0026minus;\u0026thinsp;A(Acceptor) system [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].The dimethylamino group herein acted as an electron-donor due to its pronounced electron-donating capability through a lone electron pair [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], thereby facilitating intramolecular charge transfer (ICT) in the electronic excited state [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, the interaction between the lone pair of electrons and H\u003csup\u003e+\u003c/sup\u003e had a detrimental effect on the ion electron-donating ability of the dimethylamino group, leading to fluorescence quenching caused by ICT [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Calibration and validation of the method\u003c/h2\u003e \u003cp\u003eBased on the excellent fluorescence property of OTC under alkaline conditions, a series of standard OTC solutions (20-1000 \u0026micro;g/L) were adjusted to a pH above 10. Subsequently, standard solutions were sequentially analyzed by FS at Ex\u0026thinsp;=\u0026thinsp;375 nm and Em\u0026thinsp;=\u0026thinsp;515 nm. By measuring the fluorescence intensity of standard solutions, a linear relationship between the fluorescence intensity and the concentrations of OTC in solution was obtained as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Taking into account the standard deviation of the signal-to-noise ratio, the limit of determination (LOD) for OTC was calculated to be 1.57 \u0026micro;g/L. In comparison, direct detection of OTC by FS yielded a LOD of 149 \u0026micro;g/L [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], which is nearly 100 times higher than that achieved in this study. Although there still exists a gap when compared to the common method involving HPLC-MS/MS coupled with solid phase extraction [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], this method can be employed in specific scenarios due to its rapidity and cost-effectiveness.\u003c/p\u003e \u003cp\u003eThe precision of this method was assessed by triplicate analysis of the samples, yielding relative standard deviations (RSDs) below 7.01%. To verify the accuracy of the method, a comparative experiment employing LC-MS/MS was conducted. The results of the comparison are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Deviations between the results obtained from FS and LC-MS/MS fell within a range of 15%, indicating the suitability of the presented method for quantifying OTC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(pH\u0026thinsp;=\u0026thinsp;10; Ex\u0026thinsp;=\u0026thinsp;375 nm; Em\u0026thinsp;=\u0026thinsp;515 nm)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Case application in seawater\u003c/h2\u003e \u003cp\u003eAs antibiotics are prevalent in estuarine and coastal environments due to substantial terrestrial input, aquaculture effluent, and sewage discharge [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the presented method was applied to the seawater sample. OTC with known concentrations were added to seawater samples, and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the recovery results. The observed recovery ranged from 55.15\u0026ndash;67.63%. The low recovery may be attributed to the presence of metal ions in seawater, which have a strong tendency to interact with TCs and form metal complexes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Given the complex composition of seawater, further experiments investigating the influence of metal ions are still necessary for accurate detection of OTC in seawater samples; however, it should be noted that even after correction, this presented method can still be utilized.\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\u003eRecovery of OTC in seawater\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdded\u003c/p\u003e \u003cp\u003e(\u0026micro;g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMeasured\u003c/p\u003e \u003cp\u003e(\u0026micro;g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRecovery\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\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=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e67.63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e29.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e58.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e55.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55.80\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"},{"header":"4. Conclusions","content":"\u003cp\u003eThe present study describes the development of a FS method for the determination of OTC, which is based on the ICT emission exhibited by the dimethylamino group under alkaline conditions. The developed method demonstrates excellent precision and accuracy. Furthermore, it offers simplicity and cost-effectiveness, thereby exhibiting potential for analyzing OTC in water samples following further investigations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by Science and Technology Project of Huadong Engineering (Fujian) Corporation (KY2023-FJ-02-01).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHuang wrote the main manuscript text , other authors offer revisions from their own perspectives. All authors reviewed the manuscript\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhou Y, Li W, Kumar V, Necibi MC, Mu Y-J, Shi C, Chaurasia D, Chauhan S, Chaturvedi P, Sillanp\u0026auml;\u0026auml; M, Zhang Z, Awasthi MK, Sirohi R (2022) Synthetic organic antibiotics residues as emerging contaminants waste-to-resources processing for a circular economy in China: Challenges and perspective. Environ Res 211:113075. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envres.2022.113075\u003c/span\u003e\u003cspan address=\"10.1016/j.envres.2022.113075\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBu Q, Wang B, Huang J, Deng S, Yu G (2013) Pharmaceuticals and personal care products in the aquatic environment in China: a review. J Hazard Mater 262:189\u0026ndash;211. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jhazmat.2013.08.040\u003c/span\u003e\u003cspan address=\"10.1016/j.jhazmat.2013.08.040\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeonard AFC, Zhang L, Balfour AJ, Garside R, Gaze WH (2015) Human recreational exposure to antibiotic resistant bacteria in coastal bathing waters. Environ Int 82:92\u0026ndash;100. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envint.2015.02.013\u003c/span\u003e\u003cspan address=\"10.1016/j.envint.2015.02.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen L, Li H, Liu Y, Cui Y, Li Y, Yang Z (2020) Distribution, residue level, sources, and phase partition of antibiotics in surface sediments from the inland river: a case study of the Xiangjiang River, south-central China. Environ Sci Pollut Res Int 27:2273\u0026ndash;2286. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11356-019-06833-0\u003c/span\u003e\u003cspan address=\"10.1007/s11356-019-06833-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng L, Cheng Y, Zhang Y, Li Z, Yu Y, Feng L, Zhang S, Xu L (2020) Distribution and human health risk assessment of antibiotic residues in large-scale drinking water sources in Chongqing area of the Yangtze River. Environ Res 185:109386. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envres.2020.109386\u003c/span\u003e\u003cspan address=\"10.1016/j.envres.2020.109386\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Song W, Lin H, Wang W, Du L, Xing W (2018) Antibiotics and antibiotic resistance genes in global lakes: A review and meta-analysis. Environ Int 116:60\u0026ndash;73. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envint.2018.04.011\u003c/span\u003e\u003cspan address=\"10.1016/j.envint.2018.04.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZainab SM, Junaid M, Xu N, Malik RN (2020) Antibiotics and antibiotic resistant genes (ARGs) in groundwater: A global review on dissemination, sources, interactions, environmental and human health risks. Water Res 187:116455. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.watres.2020.116455\u003c/span\u003e\u003cspan address=\"10.1016/j.watres.2020.116455\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng D, Yin G, Liu M, Chen C, Jiang Y, Hou L, Zheng Y (2021) A systematic review of antibiotics and antibiotic resistance genes in estuarine and coastal environments. Sci Total Environ 777:146009. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2021.146009\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2021.146009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi X, Bayen S, Kelly BC, Li X, Zhou Z (2015) Improved detection of multiple environmental antibiotics through an optimized sample extraction strategy in liquid chromatography-mass spectrometry analysis. Anal Bioanal Chem 407:9071\u0026ndash;9083. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00216-015-9074-7\u003c/span\u003e\u003cspan address=\"10.1007/s00216-015-9074-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang N, Huang P, Hou X, Li Z, Tao L, Zhao L (2016) Solid-phase extraction in combination with dispersive liquid-liquid microextraction and ultra-high performance liquid chromatography-tandem mass spectrometry analysis: the ultra-trace determination of 10 antibiotics in water samples. Anal Bioanal Chem 408:1701\u0026ndash;1713. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00216-015-9284-z\u003c/span\u003e\u003cspan address=\"10.1007/s00216-015-9284-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe\u0026oacute;n-Aguirre K, Hern\u0026aacute;ndez-N\u0026uacute;\u0026ntilde;ez E, Gonz\u0026aacute;lez-S\u0026aacute;nchez A, M\u0026eacute;ndez-Novelo R, Ponce-Caballero C, Gi\u0026aacute;coman-Vallejos G (2019) A Rapid and Green Method for the Determination of Veterinary Pharmaceuticals in Swine Wastewater by Fluorescence Spectrophotometry. Bull Environ Contam Toxicol 103:610\u0026ndash;616. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00128-019-02701-2\u003c/span\u003e\u003cspan address=\"10.1007/s00128-019-02701-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHudson N, Baker A, Reynolds D (2007) Fluorescence analysis of dissolved organic matter in natural, waste and polluted waters\u0026mdash;a review. River Res Applic 23:631\u0026ndash;649. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/rra.1005\u003c/span\u003e\u003cspan address=\"10.1002/rra.1005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeichtweis J, Vieira Y, Welter N, Silvestri S, Dotto GL, Carissimi E (2022) A review of the occurrence, disposal, determination, toxicity and remediation technologies of the tetracycline antibiotic. Process Saf Environ Prot 160:25\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.psep.2022.01.085\u003c/span\u003e\u003cspan address=\"10.1016/j.psep.2022.01.085\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi R, Wang W, El-Sayed E-SM, Su K, He P, Yuan D (2021) Ratiometric fluorescence detection of tetracycline antibiotic based on a polynuclear lanthanide metal\u0026ndash;organic framework. Sens Actuators B 330:129314. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.snb.2020.129314\u003c/span\u003e\u003cspan address=\"10.1016/j.snb.2020.129314\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang R, Lin D, Zhang Q, Li L, Yang L (2022) Multiplex chroma-response based fluorescent smartphone sensing platform for rapid and visual quantitative determination of antibiotic residues. Sens Actuators B 350:130902. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.snb.2021.130902\u003c/span\u003e\u003cspan address=\"10.1016/j.snb.2021.130902\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBi X, Xu B, Lin Y-L, Hu C-Y, Ye T, Qin C (2013) Monochloramination of Oxytetracycline: Kinetics, Mechanisms, Pathways, and Disinfection By-Products Formation, CLEAN - Soil, Air, Water \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003en/a-n/a\u003c/span\u003e\u003cspan address=\"http://n/a-n/a\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/clen.201200344\u003c/span\u003e\u003cspan address=\"10.1002/clen.201200344\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin X, Xu H, Qiu S, Jia M, Wang F, Zhang A, Jiang X (2017) Direct photolysis of oxytetracycline: Influence of initial concentration, pH and temperature. J Photochem Photobiol A 332:224\u0026ndash;231. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jphotochem.2016.08.032\u003c/span\u003e\u003cspan address=\"10.1016/j.jphotochem.2016.08.032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoreen AL, Arnold WA, McNeill K (2004) Photochemical Fate of Sulfa Drugs in the Aquatic Environment: Sulfa Drugs Containing Five-Membered Heterocyclic Groups. Environ Sci Technol 38:3933\u0026ndash;3940. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/es0353053\u003c/span\u003e\u003cspan address=\"10.1021/es0353053\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiang Z, Adams C (2004) Potentiometric determination of acid dissociation constants (pKa) for human and veterinary antibiotics. Water Res 38:2874\u0026ndash;2890. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.watres.2004.03.017\u003c/span\u003e\u003cspan address=\"10.1016/j.watres.2004.03.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark KK, Park JW, Hamilton AD (2007) Solvent and pH Effects on the Fluorescence of 7-(Dimethylamino)-2-Fluorenesulfonate. J Fluoresc 17:361\u0026ndash;369. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10895-007-0193-1\u003c/span\u003e\u003cspan address=\"10.1007/s10895-007-0193-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang D, Liu P, Bai T, Kong J (2020) -Dimethyl-Substituted Boron Ketoiminates for Multicolor Fluorescent Initiators and Polymers. Macromolecules 53:3339\u0026ndash;3348. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.macromol.9b02633\u003c/span\u003e\u003cspan address=\"10.1021/acs.macromol.9b02633\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen C, Fang C (2023) Fluorescence Modulation by Amines: Mechanistic Insights into Twisted Intramolecular Charge Transfer (TICT) and Beyond. Chemosensors 11:87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/chemosensors11020087\u003c/span\u003e\u003cspan address=\"10.3390/chemosensors11020087\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan L, Lin W, Zheng K, He L, Huang W (2012) Far-red to near infrared analyte-responsive fluorescent probes based on organic fluorophore platforms for fluorescence imaging. Chem Soc Rev 42:622\u0026ndash;661. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/C2CS35313J\u003c/span\u003e\u003cspan address=\"10.1039/C2CS35313J\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa L, Cao W, Liu J, Deng D, Wu Y, Yan Y, Yang L (2012) A highly selective and sensitive fluorescence dual-responsive pH probe in water. Sens Actuators B 169:243\u0026ndash;247. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.snb.2012.04.076\u003c/span\u003e\u003cspan address=\"10.1016/j.snb.2012.04.076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaha SK, Purkayastha P, Das AB (2008) Photophysical characterization and effect of pH on the twisted intramolecular charge transfer fluorescence of trans-2-[4-(dimethylamino)styryl]benzothiazole. J Photochem Photobiol A 195:368\u0026ndash;377. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jphotochem.2007.11.004\u003c/span\u003e\u003cspan address=\"10.1016/j.jphotochem.2007.11.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Zhang T, Dong L, Shi S, Yang W, Zhang L, Zhou L, Huang Y (2012) Determination of Antibiotics in Hospital Wastewater Using HPLC-MS /MS Coupled with Solid Phase Extraction. J Instrumental Anal 31:453\u0026ndash;458\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePulicharla R, Hegde K, Brar SK, Surampalli RY (2017) Tetracyclines metal complexation: Significance and fate of mutual existence in the environment. Environ Pollut 221:1\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envpol.2016.12.017\u003c/span\u003e\u003cspan address=\"10.1016/j.envpol.2016.12.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Fluorescence, Oxytetracycline, Antibiotic, Seawater","lastPublishedDoi":"10.21203/rs.3.rs-4780110/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4780110/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAccurate quantification of antibiotics in environmental samples is typically challenging due to the low antibiotic concentrations and the complexity of environmental matrices. This paper presents a fluorescence spectrometry method for determining oxytetracycline under alkaline conditions. The ionic distribution of the oxytetracycline solution was analyzed based on its dissociation constant. The dimethylamino group played a crucial role in this method, as it promotes intramolecular charge transfer in the electronic excited state through its electron-donating capability with a lone electron pair. The presented method is straightforward, cost-effective, and holds potential for analyzing oxytetracycline in water sample after further investigation.\u003c/p\u003e","manuscriptTitle":"Improved precision in oxytetracycline detection via fluorescence spectrometry impacted by pH","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-21 17:44:00","doi":"10.21203/rs.3.rs-4780110/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-11T14:11:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-09T04:06:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162101535795069284611203044942657232327","date":"2024-08-09T03:05:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-08T12:06:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-25T17:28:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-25T17:26:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2024-07-22T07:57:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3a5bba79-05f4-4fd7-844f-0caf7ff82721","owner":[],"postedDate":"August 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:06:34+00:00","versionOfRecord":{"articleIdentity":"rs-4780110","link":"https://doi.org/10.1007/s10895-024-03941-0","journal":{"identity":"journal-of-fluorescence","isVorOnly":false,"title":"Journal of Fluorescence"},"publishedOn":"2024-09-26 15:58:00","publishedOnDateReadable":"September 26th, 2024"},"versionCreatedAt":"2024-08-21 17:44:00","video":"","vorDoi":"10.1007/s10895-024-03941-0","vorDoiUrl":"https://doi.org/10.1007/s10895-024-03941-0","workflowStages":[]},"version":"v1","identity":"rs-4780110","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4780110","identity":"rs-4780110","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}