A novel high-conjugated probe of 4-(acridone-10-yl)-phenylethyl chloroformate (ABE-Cl) for rapid detection of amino compounds Using HPLC with Fluorescence detection

A novel high-conjugated probe of 4-(acridone-10-yl)-phenylethyl chloroformate (ABE-Cl) for rapid detection of amino compounds Using HPLC with Fluorescence detection | 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 A novel high-conjugated probe of 4-(acridone-10-yl)-phenylethyl chloroformate (ABE-Cl) for rapid detection of amino compounds Using HPLC with Fluorescence detection Yingying Lou, Huiquan Xiao, Xiaocong Zou, Zenghui Xu, Zhiwei Sun, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5365168/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Jan, 2025 Read the published version in Journal of Fluorescence → Version 1 posted 10 You are reading this latest preprint version Abstract The fluorescence detection of amino compounds and the evaluation of their content in environmental samples are vital, not only for assessing food quality but also for studying soil organic matter. Here, we present the synthesis and application of a novel fluorescent probe, 4-(9-acridone)benzylmethyl carbonochloride (APE-Cl), for detecting amino compounds via a chloromate reaction with fluorescence detection. The complete derivatization reaction of APE-Cl with amino compounds can be accomplished in aqueous acetonitrile within 5 minutes at room temperature, using 0.2 M borate buffer (pH = 9.0). APE-amine derivatives exhibited intense fluorescence with an excitation maximum at λex 254 nm and an emission maximum at λem 418 nm. All derivatives demonstrated high stability, strong fluorescence, and elevated ionization potential under atmospheric pressure chemical ionization (APCI-MS) in positive ion detection mode. The method, combined with gradient elution, provides baseline resolution of common amine derivatives on a reversed-phase C18 column. The LC separation for the derivatized amines shows good reproducibility with aqueous acetonitrile as the mobile phase. The relative standard deviations (n = 6) for each amine derivative are < 3.99%. The detection limits (at a signal-to-noise ratio of 3) per injection ranged from 1.68 to 11.2 femtomole. The established pre-column derivatization method for determining amino compounds in practical samples proved to be satisfactory. HPLC Amino compounds Fluorescence detection Mass spectrometry 4-(9-acridone)benzylmethyl carbonochloride (APE-Cl) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Aliphatic compounds are naturally occurring and widely distributed in nature, including aquatic environments, biological fluids, and industrial waste materials. They typically arise as metabolic products in microorganisms, plants, and animals, with the main pathways for amine formation being the decarboxylation of amino acids, amination of carbonyl compounds, and degradation of nitrogen-containing compounds [ 1 – 4 ]. Aliphatic amines also serve as important raw materials and intermediates in manufacturing, as well as in the chemical and pharmaceutical industries [ 5 ]. Analyzing aliphatic amines in various liquids is crucial due to their toxic or carcinogenic properties [ 3 , 6 – 9 ]. The analysis of amines has traditionally been challenging due to their unique physicochemical properties, such as high polarity, volatility, alkalinity, and water solubility. Gas chromatography is commonly employed to analyze amines using various derivatization reagents [ 10 ]. Other techniques, including enzymatic [ 11 , 12 ] and ion-exchange chromatographic detection [ 13 ], have been reported for the determination of amines in different matrices. However, these methods often face limitations due to low sensitivity. Most aliphatic amines exhibit neither natural UV absorption nor fluorescence. Consequently, chemical derivatization is essential to enhance detection sensitivity and improve selectivity through pre-column or post-column HPLC derivatization methods. Additional techniques used to enhance detection limits include microcolumn LC [ 14 ] and capillary electrophoresis [ 15 ]. Currently, the most popular methods for determining aliphatic amines involve pre-column and post-column derivatization with fluorescence detection. Several common fluorescent derivatizing reagents [ 16 – 27 ] have been fully developed. Despite the popularity of these pre-column methods, numerous reports have highlighted various shortcomings in their application, and to date, no method has been demonstrated to address all these issues. For instance, the o-phthalaldehyde (OPA) method provides greater sensitivity overall, but it is restricted to primary amines. The 2-phthalimidylbenzoyl chloride (PIB-Cl) method offers high sensitivity; however, its derivatives experience slight decomposition during storage [ 28 ]. The 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBDF) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) are more effective derivatization reagents for amino compounds; however, when these reagents are exposed to daylight for 25 minutes in an aqueous methanol solution, approximately 30–50% decomposition occurs [ 29 ]. Recently, 9-fluorenylmethyl chloroformate (FMOC) [ 30 ], 1-(9-fluorenyl)ethyl chloroformate (FLEC) [ 31 ], and 2-(9-anthryl)ethyl chloroformate (AEOC) [ 32 ] reagents have been utilized for the derivatization of amines, amino acids, and peptides for chiral or nonchiral separation in LC or CE. These reagents provide excellent UV absorption and very high sensitivity with laser-induced fluorescence detection; however, current derivatization procedures using FMOC are cumbersome. For effective derivatization, an excess of reagent is required, and the derivatized solution must be extracted with pentane to eliminate the surplus reagent [ 33 , 34 ], as it interferes with the separation of derivatives and negatively impacts column performance. The 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) method is quick and convenient, yielding stable derivatives. Nevertheless, only 10% of the fluorescent intensity in aqueous solution compared to that in pure acetonitrile solution is observed for its derivatives. Consequently, the detection limits for early eluted amine derivatives are typically higher than those for later ones [ 35 ]. Therefore, developing convenient assay methods for highly sensitive monitoring of amino compounds is extremely valuable, not only for water quality assessment but also for soil organic matter research. In our previous studies [ 3 , 36 – 39 ], we described the preparation of several fluorescent probes and their applications for analyzing common amino compounds. The primary objective of this research is to further develop more sensitive fluorescent probes with relatively long emission wavelengths. The results indicated that the synthesized APE-Cl probe molecule can react swiftly with amines to form highly stable derivatives. These derivatives not only exhibit high fluorescence sensitivity but also demonstrate long emission wavelengths. To the best of our knowledge, this is the first report of the APE-Cl fluorescent probe for the determination of amino compounds. In the present work, we investigate the optimal labeling conditions, including derivatization time, reaction temperature, buffer pH, and solvent system. We also determine the linearity, detection limits, and precision of the entire procedure. The detection of trace amounts of amines in various real samples using APE-Cl as the labeling reagent yielded satisfactory results. Experimental Section Apparatus Experiments were conducted using an LC/MSD-Trap-SL electrospray ion trap liquid chromatography/mass spectrometry (1100 Series LC/MSD Trap, a complete LC/MS/MS). All components of the HPLC system were from the HP 1100 series. Derivatives were separated on a BDS-C18 column (200×4.6 mm, 5 µm, Yilite Dalian, China). The HPLC system was operated using HP Chemstation software. The mass spectrometer from Bruker Daltonik (Bremen, Germany) was equipped with atmospheric pressure chemical ionization (APCI). The mass spectrometer system was managed by Esquire-LC NT software, version 4.1. Fluorescence excitation and emission spectra were recorded using an F-7000 fluorescence spectrophotometer (Hitachi). Both excitation and emission bandpass were set to 5 nm. Reagents Acetonitrile and methanol were of chromatographic grade from Luguang Chemical Reagent Co. (Shandong, China). All solvents used for RP-LC were of analytical reagent grade and filtered through a 0.45 micron filtration disk. Acetone, DMF, DMSO and other reagents were also of analytical reagent grade. Methylamine (99%), ethylamine (99%), and diethylamine (99%) as hydrochlorides were sourced from Shanghai Chemical Reagent Co. (Shanghai, China). All other amines used in experiments were of chromatographic grade and purchased from Luguang Chemical Reagent Co. Hyaluronic acid fermentation broth was obtained from Focus Biology Co., Ltd (Qu, Shandong, China). Stinky tofu, yogurt, and shrimp paste were acquired from the local supermarket (Qufu, Shandong, China). Synthesis of 4-(9-acridone)phenylethyl chloroformate (APE-Cl) The synthesis of N-(4-(2-hydroxyethyl)phenyl)acridone (HPA) The synthesis of APE-Cl is illustrated in Fig. 1 . Acridone (2.0 g, 10 mmol), 2-(4-iodophenyl)ethanol (3.0 g, 12 mmol), dipivaloylmethane (DPVM, 2 mL), K 2 CO 3 (2.0 g), and CuI (0.2 g) were combined in 50 mL of DMSO in a 250 mL round-bottom flask. The mixture was rapidly heated to 135℃and stirred at that temperature for 24 h. Upon completion of the reaction, the mixture was cooled to room temperature and filtered to remove insoluble solids (such as CuI and K 2 CO 3 ). Subsequently, 400 mL of water was added with vigorous stirring, and the pH was adjusted to 6.5 using 3M HCl. The precipitated solids were filtered and washed twice with water (2×20 mL) to yield yellow products. The crude product was recrystallized from a mixed solvent of acetonitrile and DMF (1:3, v/v) to obtain 3.8 g of light yellow crystals, with a yield of 84.8%. Mp. 248.3 ℃. Found: C:75.34%, H:5.50%, N:4.75%. Calculated: C: 75.30%, H:5.48%, N:5.85%. 1H NMR (500 MHz, Chloroform ) δ 7.55 (d, J = 70.0 Hz, 14H), 7.45 (s, 1H), 7.23 (d, J = 50.0 Hz, 16H), 7.08 (s, 11H), 7.03 (s, 5H), 3.63 (s, 4H), 2.75 (s, 4H), 1.37 (s, 4H). LC-APCI-MS: m/z 315.4 [M + H] + in positive-ion mode. Preparation of 4-(9-acridone)benzylethyl chloroformate (APE-Cl) To a solution containing 8.5 g of solid phosgene and 100 mL of dichloromethane (0 ℃) in a 500 mL round-bottom flask, a mixture of N-(4-(2-hydroxyethyl)phenyl)acridone (3.0 g) and triethylamine (1.0 g catalyst) in 100 mL of dichloromethane solution was added dropwise over 2 h under electromagnetic stirring. After stirring at 0 ℃ for 4 h, the mixture was allowed to react at ambient temperature for an additional 6 h with electromagnetic stirring. The solution was then concentrated using a rotary evaporator. The residue was extracted three times with acetone; the combined acetone layers were concentrated under vacuum to yield a faint yellow solid. The crude products were recrystallized twice from acetone/acetonitrile (3:1, v/v) to produce yellow crystals.(2.87 g, yield, 80.0%), m.p. 173.5 ℃. Found: C 63.69%, H 4.01%, N 4.64%, Cl 11.75; Calculated: C 63.72%, H 4.11%, N 4.58%, Cl 11.05%; 1 H NMR (500 MHz, DMSO) δ 8.38 (dd, J = 8.0, 1.6 Hz, 2H), 7.72–7.64 (m, 2H), 7.62 (d, J = 8.0 Hz, 2H), 7.45 (dd, J = 31.9, 8.2 Hz, 2H), 7.33 (dd, J = 11.0, 3.9 Hz, 2H), 6.75 (dd, J = 15.2, 8.6 Hz, 2H), 3.90 (dt, J = 123.7, 6.9 Hz, 2H), 3.09–2.91 (m, 2H).;LC-APCI-MS: m/z 377.8 [M + H] + in positive-ion mode. Preparation of APE-butylamine derivative APE-butylamine derivative was synthesized by reacting APE-Cl with an excess of butylamine as follows: To a solution of butylamine (0.146 g, 2.0 mmol) and DMAP (0.122 g, 0.1 mmol) in acetonitrile (50 mL) in a 100 mL round-bottom flask, a solution of APE-Cl (0.307 g, 1.0 mmol) in acetonitrile (50 mL) was added dropwise under electromagnetic stirring. The mixture was rapidly heated to 60 ℃ for 5 minutes and then poured into 300 mL of ice-water, stirring vigorously for 10 minutes; the precipitated solid was collected by filtration, washed with water, and dried at room temperature for 48 hours. The crude product was recrystallized twice from acetonitrile (30 mL) to yield yellow crystals (0.179 g, 52.0%). LC-APCI-MS: m/z 339.0 [M + H] + in positive-ion mode. Preparation of Standard Solutions The APE-Cl reagent solution (approximately 4.0×10 − 3 mol/L) was prepared by dissolving 1.5 mg of APE-Cl in 10 mL of acetonitrile. The concentration of all amine standard solutions (50 ng/mL) was achieved by diluting the stock solutions of each amine (1 mg/mL) with acetonitrile. For long-chain amines (i.e., > C10), the stock solution was prepared by dissolving the long-chain amine in a small amount of DMF and then diluting it with acetonitrile to reach a final concentration of 50 ng/mL APE-butylamine solution (4.0×10 − 3 mol/L) was prepared by dissolving 1.6 mg of APE-butylamine in 10 mL of acetonitrile. The working solution of APE-butylamine for fluorescence spectrometry evaluation was prepared by diluting the stock solution with acetonitrile. Chromatographic Method and MS condition The separation of APE-amine derivatives by HPLC was performed on a reversed-phase BDS-C18 column using a gradient elution. Eluent A consisted of 35% acetonitrile with 20 mM ammonium/formic acid buffer (pH = 3.5), while eluent B was 100% acetonitrile. Gradient elution commenced with 70% B and 30% A, increasing to 100% B over 35 minutes. Before the injection of the next sample, the column needed to be re-equilibrated with mobile phase A for 10 minutes. The flow rate was maintained at 1.0 mL min⁻¹, and the column temperature was set to 30℃. The fluorescence excitation and emission wavelengths were established at Ex = 256 nm and Em = 418 nm, respectively. MS ion source: Dry gas temperature, 350℃; nebulizer pressure, 60.0 psi; dry gas flow, 5.0 L/min; APCI Vap Temp 450℃; corona current (nA) 4000 (pos); capillary voltage 3500 V. Sample preparation procedure Extraction of amines from fermented shrimp sauce To minimize the loss of volatile amines, all collected samples were promptly stored in brown bottles and placed in a freezer at -18 ℃ until used in subsequent experiments. The sample preparation procedure was as follows: 5.0 g of fermented shrimp sauce was homogenized in 25 mL of cooled trichloroacetic acid (TCA) solution (5%, v/v) in a 50 mL centrifuge tube. The centrifuge tube was thoroughly vortexed, then ultrasonicated for 30 min and centrifuged at 6000 r/min for 15 min at 4 ℃. The supernatant was filtered through 0.22 µm syringe filters (Millipore) and stored in a brown glass bottle at -18 ℃ until HPLC analysis. Extraction of amines from stinky tofu To a 50 mL centrifuge tube, 5.0 g of stinky tofu and 25 mL of the cooled trichloroacetic acid solution (5%, v/v) or cooled HClO 4 solution (1.0 M, with 100 mg/L EDTA) were added. After the mixture was vibrated for 3 min, it was ultrasonicated for 30 min and then centrifuged at 6000 r/min for 15 min. The supernatant was filtered through 2 µm Millipore filters. The resulting solution was stored in a brown glass bottle at -18 ℃ until HPLC analysis. Extraction of amines from acidophilus milk To a 10 ml centrifugal tube, yogurt (2 ml), deionized water (2.0 ml), and sodium hydroxide solution (0.5 ml, 4M) were added continuously and vortexed thoroughly for 3 minutes. Subsequently, 20 ml of chloroform was added and sonicated for 10 minutes. The chloroform layer was then transferred to a 50-ml round-bottom flask and adjusted to pH 3.0 with 4 M HCl solution. The solvent was evaporated to dryness using a rotary evaporator. The residue was re-dissolved in 2.0 ml of aqueous acetonitrile (ACN:H 2 O, 4:1, v:v) and stored in a brown glass bottle at -18 ℃ until HPLC analysis. Extraction of amines from hyaluronic acid fermentation fluid. To a solution containing 5 mL of acetonitrile in a 10 mL round-bottom flask, 5 mL of the filtered hyaluronic acid fermentation fluid was added. The contents of the flask were ultrasonicated for 5 minutes. The extraction was performed twice, and the resulting solutions were combined and adjusted to pH = 3.0 using a 4.0 M HCl solution. The combined extracts were then evaporated to dryness at 60 ℃ under reduced pressure. The residue was re-dissolved in 50% aqueous acetonitrile and stored at -18 ℃ until HPLC analysis. Derivatization Procedure To a 2 ml screw-capped test tube, a standard amine mixture (50 µL), borate buffer (200 µL, pH = 9.0), acetonitrile (50 µL), and 100 µL of APE-Cl solution (1.0×10⁻³ mol/L) were successively added. The mixture was allowed to react at room temperature for 10 minutes. After the reaction was complete, a 10 µL volume of the mixture was diluted to 100 µL with acetonitrile. The diluted solution (10 µL) was then injected directly onto the chromatograph (Fig. 1 ). Results and Discussion Stabilities of APE-butylamine Derivative The stabilities of APE-amine derivatives were assessed by analyzing the stored APE-amine solution over a week. As anticipated, when the derivative was stored in pure acetonitrile or methanol, daylight had no impact on its stability. However, the aqueous solution of the derivatives showed slight degradation (approximately 2.6–3.8%) after being exposed to daylight for one week. This indicates that the derivatives were sufficiently stable to be effectively analyzed during the HPLC analysis period. Ultraviolet Absorption of APE-butylamine Acridone and its derivatives represent one of the most significant classes of organic photochromic molecules, characterized by a large molar absorption coefficient. They display intriguing photochromic properties. In previous studies, the 10-phenyl-acridone-2-sulfonyl chloride (PASC) synthesized in our laboratory demonstrated strong UV properties [4] . In this study, APE derivatives exhibited significantly stronger UV and fluorescence properties compared to PASC derivatives. This enhancement can be attributed to the introduction of a benzene group at the N position of the acridone ring via an N-linked mode, which increases the π-conjugated system of the entire APE molecule. Consequently, some acridone derivatives can also function as high-sensitivity UV or fluorescence materials. The maximum ultraviolet absorption responses of APE derivatives are observed at wavelengths of 254 nm, with a molar absorption coefficient (ε) exceeding 7.7×10 4 (L mol -1 cm -1 ). Previous studies indicated that PASC has an absorption maximum at 252 nm in acetonitrile, with a comparatively lower molar absorption coefficient of 3.85×10 4 (L mol -1 cm -1 ). Clearly, due to the enhancement of the molecular conjugation system, UV absorption was significantly improved, with the maximum molar absorption coefficient for the APE molecule being approximately twice that of the PASC molecule. Fluorescence for Representative ABE-butylamine Derivative. Fluorescence spectra of APE derivatives in aqueous acetonitrile were obtained with an excitation maximum at λex 254 nm and an emission maximum at λem 418 nm. The excitation spectra of APE derivatives showed no significant change when varying the polarity of the solvent. An increase in solvent polarity resulted in a slight red shift in the fluorescence emission spectra. The emission intensities in 100% acetonitrile were 2.6 times stronger than those in pure water (Fig. 2 ). The fluorescence intensities in pure acetonitrile showed a significant increase compared to pure water. These results were in excellent agreement with some nitrogen-containing fluorescence derivatives previously reported in our experiments [ 3 , 36 – 39 ]. This can be attributed to the fact that most fluorophores were only partially dissolved in low concentrations of aqueous methanol or acetonitrile. The fluorescence emission exhibited a slight blue shift with the addition of progressively increasing amounts of acetonitrile. A peak blue shift of about 17 nm (from 428 nm to 410 nm) for APE derivatives was observed. Similar results were also noted in methanol. To evaluate the effects of inorganic anions (such as sulfate, nitrate, and phosphate), organic anions (such as citrate), and divalent cations (Ca 2+ , Mg 2+ , Ni 2+ , Co 2+ , Cu 2+ , and Hg 2+ ) on fluorescence emission intensities, aqueous acetonitrile solutions of APE-butylamine (10 mol/L, 80:20, v/v) were utilized. Experiments were conducted by gradually adding increasing amounts of the corresponding anions or divalent cations to the APE-butylamine solution. The addition of progressively increasing amounts of Ca 2+ and Mg 2+ from 1.0 to 100 mol/L to the APE-butylamine solution did not result in a significant change in fluorescence emission intensities. However, the addition of progressively increasing amounts of Ni 2+ and Co 2+ beyond 40 mol/L to the APE-butylamine solution caused a decrease in emission intensities of approximately 4.2–5.3% (the intensity was compared to those obtained in the absence of divalent cations). The addition of progressively increasing amounts of Cu 2+ and Hg 2+ (0–20 mol/L) to the solution did not lead to a significant change in emission intensities. A notable decrease in emission intensities was observed when progressively increasing amounts of Cu 2+ and Hg 2+ beyond 100 mol/L were added, resulting in about a 10% decrease in emission intensities. Typically, the inorganic ions present in environmental samples did not interfere with the measurement results. Effects of Co-solvents on Derivatization Efficiency. In general, acetonitrile or aqueous acetonitrile was selected as the derivatization solvent for amines using chloroformate reagents. Thus, the choice of co-solvents is vital to enhance derivatization yield and improve the solubility of non-water-soluble derivatives. In this study, DMF and methyl sulfoxide (DMSO) were assessed for their ability to boost derivatization yields and enhance the solubility of amine derivatives in aqueous acetonitrile, particularly for some non-water-soluble long-chain amine derivatives. Optimal efficiency was achieved by adding an appropriate amount of DMF or DMSO to the derivatization solution. The yields in acetonitrile solutions containing approximately 10% DMF or DMSO were 20% higher than those in pure acetonitrile. As shown in Table 1 , DMF as a co-solvent exhibited the strongest fluorescence and outperformed other solvents due to its low viscosity, effectively mitigating the issue of precipitation of hydrophobic long-chain amine derivatives (Fig. 3 ). Effects of Temperature, Time and pH on Derivatization APE-Cl exhibits the same chloroformate reaction with primary and secondary amino compounds as BCEOC-Cl, CEOC-Cl, and AEC-Cl, as previously reported [ 21 , 24 , 40 ]. The entire conjugation system of the APE-Cl molecule has been fully enhanced by an N-linked phenyl ring, with an excitation wavelength of λex 252 nm and an emission wavelength of λem 414 nm. The corresponding derivatives displayed higher fluorescence responses. Complete derivatization of APE-Cl with amines could be performed, and the peak heights for all amine derivatives remained constant at 25 ℃ for 5 min. However, the derivatization yields slightly decreased when the temperature exceeded 30 ℃, likely due to the self-hydrolysis of the reagent at relatively high temperatures. When the reaction temperature was below 20 ℃, it took at least 10 minutes to ensure complete derivatization of the long-chain amines. Conversely, prolonged derivatization time also resulted in a slight decrease in signal for all derivatives, probably because the APE-amine derivatives could be hydrolyzed in an alkaline buffer solution environment. Therefore, subsequent derivatization was conducted at 25 ℃ for 5 min, and the solution was immediately neutralized to pH 6.0–6.5 with 36% acetic acid. The fluorescence intensity of APE derivatives increased with the amount of derivatization reagent. A constant fluorescence intensity was achieved with the addition of a 3 to 4-fold molar excess of reagent relative to the total molar amines; further increasing the excess reagent beyond this level had no significant effect on yields. With as little as a 2.0-fold molar excess of derivatization reagent, the derivatization of amines was incomplete, resulting in low detection responses. Additionally, the hydrolysis of the excess APE-Cl produced the corresponding by-product APE-OH [MS, m/z 360.12 [M + H]]; MS/MS: m/z 342.12, m/z 315.12, m/z 285.0, m/z 196.1], and subsequently, the excess ABE-Cl reacted with APE-OH to form the di-substituted derivative APE-O-APE [MS: m/z 656.74 [M + H]]. The presence of APE-OH and (APE)2 did not interfere with the separation of amine derivatives. APE-Cl concentrations ranging from 5.0×10 − 3 to 5.0×10 − 4 mol/L did not significantly alter the derivatization time. When the derivatization solution was neutralized to pH 6.0–6.5 with 36% acetic acid (w/w), the corresponding amine derivatives were found to be stable for more than 48 hours at room temperature. Therefore, the subsequent derivatization was carried out at 25 ℃ for 5 minutes, and the solution was immediately neutralized to pH 6.0–6.5 with 36% acetic acid. Usually, the derivatization reaction was conducted in the presence of basic catalysts; here, borate buffers, carbonate buffers, and phosphate buffers were evaluated. The results indicated that borate buffer was the optimal choice. The effects of pH on the derivatization reaction were then tested with borate buffers (0.2 mol/l) in the pH range of 8.5–10.5. The maximum derivatization yields were achieved in the pH range of 9.0–9.5. Consequently, all subsequent derivatization was performed in this pH range; however, outside this range, particularly in more acidic solutions, decreased responses were observed. At even higher pH values (> 10.0), PAE-Cl molecules were partially hydrolyzed to produce APE-OH forms; therefore, a 0.2 mol/l borate buffer solution at pH 9.0 was selected for all amine derivatization. Linearity and detection limits and for derivatized amines A standard solution containing 50 pmol of the APE-amine derivative was prepared to assess method repeatability. The relative standard deviations (R.S.D; n = 10) of the peak areas and retention times ranged from 0.44–1.19% and 0.023–0.067%, respectively. For precision and accuracy, interday precision for 12 amines was evaluated using three identical shrimp paste samples, with amines spiked at low, medium, and high concentration levels, and analyses were conducted in triplicate. The experimental recoveries ranged from 96.2–105.9%. The detection limit is a crucial parameter for assessing the sensitivity of an analytical method, particularly for analyzing trace amounts of components. Establishing a method with a low detection limit is a prerequisite for ensuring accurate determination. Based on an injection of 1.0 pmol amines, the detection limits (at a signal-to-noise ratio of 3:1) for each amine ranged from 1.68 to 11.2 fmol. Linearities were established over a 1000-fold concentration range for amines, with serial standard solutions analyzed from 1.0×10 − 8 to 1.0×10 − 5 mol/l (corresponding injected amounts of amines from 500 to 5.0 pmol). All amines exhibited linear responses over this range, with correlation coefficients exceeding 0.9994. The linear regression analysis for higher concentrations of amines was not conducted due to significant peak overrun. The linear regression equations are presented in Table 3. Table 1 Linear regression equations, correlation coefficients and detection limits Amine Y = AX + B, X: injected amount (pmol): Y: peak area R 2 Detection limits (fmol) R.S.D.s (%) C1 Y = 46.83X − 2.44 0.9998 11.20 3.48 C2 Y = 71.06X − 0.30 0.9999 3.33 2.70 C3 Y = 93.43X -2.49 0.9998 3.91 3.99 C4 Y = 128.71X − 4.26 0.9997 3.67 3.08 C5 Y = 122.79X − 1.01 0.9994 3.67 3.63 C6 Y = 146.82X − 8.20 0.9997 3.10 2.68 C7 Y = 151.09X − 10.17 0.9997 3.06 2.30 C8 Y = 165.75X − 11.28 0.9997 3.11 2.46 C9 Y = 169.04X − 11.01 0.9997 3.20 2.43 C10 Y = 170.29X − 11.00 0.9997 3.52 2.53 C11 Y = 171.21X − 12.46 0.9999 2.07 2.04 C12 Y = 171.29X − 12.61 0.9999 1.68 2.13 HPLC separation of APE-amine derivatives The separation of the derivatized amine standards could be achieved under slightly acidic conditions (pH from 3.5 to 5.5). When the separation was conducted at pH > 7.0, a noticeable increase in retention time for PAE-amine derivatives was observed, accompanied by slight peak tailing. Subsequently, the separation was performed on a BDS-C18 chromatographic column (200 x 4.6 mm, 5 µm) with gradient elution using aqueous acetonitrile containing 20 mM ammonium/formic acid as the mobile phase. The separation of the blank and sample is illustrated in Fig. 4 (A and B). Sample analysis. The analysis of real samples of hyaluronic acid fermentation fluid (A), shrimp paste (B), stinky tofu (C), and yogurt (D) using fluorescence detection is presented in Fig. 6 (A, B, C and D). Amine compositional data are provided in Table 3. In the sample of hyaluronic acid fermentation fluid (A), fatty amines C 2 , C 5 , and C 6 were found, with amounts of 5.23 ng/L, 9.71 ng/L, and 0.94 ng/L, respectively. Other fatty amines, including C 1 , C 3 -C 4 , and C 7 -C 12 , were not detected or were below detection limits. For shrimp paste samples (B), fatty amines C 2 , C 3 , C 5 , and C 9 were identified, with amounts of 2.54 ng/g, 5.32 ng/g, 4.60 ng/g, and 0.53 ng/g, respectively. Other fatty amines, including C 1 , C 4 , C 6 , C 7 , C 8 , and C 10 -C 12 , were either not detected or below detection limits. For stinky tofu samples (C), high concentrations of fatty amines were detected, except for C 3 and C 7 . The amine amounts were as follows: C 1 (17.98 ng/g), C 2 (57.91 ng/g), C 4 (75.22 ng/g), C 5 (89.33 ng/g), C 6 (1.20 ng/g), C 8 (23.18 ng/g), C 9 (18.58 ng/g), and C 10 (2.26 ng/g). Amines C 3 and C 7 were not detected. Among these fatty amines, C 4 and C 5 exhibited the highest levels. For yogurt samples (D), high concentrations of fatty amines C 1 and C 4 -C 8 were detected, with amounts of 2.07 ng/g, 28.03 ng/g, 3.54 ng/g, 2.89 ng/g, 11.92 ng/g, and 16.71 ng/g, respectively. In all these samples, C 5 amine was detected in relatively high amounts, while C 11 -C 12 were absent. It was clear that the high concentration of amine primarily originated from fermented stinky tofu and yogurt. Generally, the amine levels varied depending on the sample type, with C 4 and C 5 consistently showing relatively high concentrations across all fermented samples. C 3 amine was not detected in any samples except for the shrimp sauce. Table 2 Amine amount in different samples Amines Hyaluronic acid fermentation fluid ( A) Shrimp sauce (B) Stinky tofu (C) Yogurt (D) ng/L ng/g ng/g ng/L C1 No No 17.98 2.07 C2 5.23 2.54 57.91 No C3 No 5.32 No No C4 No No 75.22 28.03 C5 9.71 4.60 89.33 3.54 C6 0.94 No 1.20 2.89 C7 No No No 11.92 C8 No - 23.18 16.71 C9 No 0.53 18.58 No C10 No No 2.26 No C11 No No No No C12 No No No No No: detection or below detection limits. Conclusion Although the analysis of amine compounds is a well-established technique, current liquid chromatography methods still need improvements in analysis speed and sensitivity for a diverse range of samples. In this study, APE-Cl was employed to analyze amines, offering not only high sensitivity but also a simple and convenient derivatization process. The method offers several advantages: (1) the derivatization reaction proceeds quickly and smoothly with a basic catalyst; (2) the reagent and its derivatives remain stable for at least one week during HPLC analysis; (3) the reagent exhibits excellent fluorescence and a high ionization potential in the MS ion chamber.The derivatization reaction of APE-Cl with amines is simple, sensitive, and reproducible. Complete derivatization for higher chain amines (> C12) can be easily achieved under the proposed derivatization conditions. This method has been successfully applied to determine free amines in various real samples with satisfactory results. Further work will focus on improving experimental conditions and investigating trace-level analysis of specific biogenic and aromatic amines in diverse environmental samples. Declarations Ethical Approval Not applicable. Author Contributions Yingying Lou: data collection, purification, methodology, and analysis, writing-original draft. Jinmao You: conceptualization, writing-review, and editing. Huiquan Xiao: methodology. Xiaocong Zou: HPLC-FLD analysis. Zenghui Xu: data curation. Zhiwei Sun, Zan Li, and Zhongyin Ji: conceptualization. All authors reviewed the manuscript. Funding This work was financially supported by the Major Innovation Fund of Shandong Province (No. 2021ZDSYS23) and supported by the National Natural Science Foundation of China (No. 21976105). Data Availability The datasets generated or analyzed for this study are available from reasonable requests to the corresponding author. The results presented in this manuscript have neither been published before nor submitted for publication elsewhere. Conflict of Interest Yingying Lou declares that she has no conflict of interest. Jinmao You declares that he has no conflict of interest. Zhiwei Sun declares that he has no conflict of interest. Zan Li declares that he has no conflict of interest. Zhongyin Ji declares that he has no conflict of interest. References Lu Xxin, Wang et al (2021) Metagenomes of polyaminetransforming bacterioplankton along a nearshore–open ocean transect. Mar Life Sci Technol 4:163–178 Nakano-Fujii S, Kamata T, Kakehashi H et al (2022) Automated simultaneous identification of methamphetamine, precursor compounds to methamphetamine and their metabolites in urine for proof of methamphetamine use. Japanese J Forensic Sci Technol 27:151–160 Bing li L, Ling yun W, Xue guang R et al (2022) Recent advances in fluorescent methods for polyamine detection and the polyamine suppressing strategy in tumor treatment. Biosensors 12:633 Tanifuji R (2022) Skeletal Editing: Recent Progress on Ring-contraction Reactions. 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J Mass Spectrom 56:e4709–e4709 Sarah J, Antoine D, Ema Z et al (2021) An environmentally benign post-polymerization functionalization strategy towards unprecedented poly(vinylamine) polyHIPEs. Polym Chem 12:1155–1164 Watanabe Y, Imai K (1981) High-performance liquid chromatography and sensitive detection of amino acids derivatized with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole. Anal Biochem 116:471–472 Kotaniguchi H, Kawakatsu M, Toyo’oka T, Imai K (1987) Automatic amino acid analysis utilizing 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole. Chromatogr Biomed App 420:141–145 Zheng M, Fu C, Xu H (1993) High-performance liquid chromatographic detection of trace N-nitrosoamines by pre-column derivatization with 4-(2-phthalimidyl) benzoyl chloride. Analyst 118:269–271 Ahnoff M, Grundevik I, Arfwidsson A, Fonselius J, Persson B-A (1981) Derivatization with 4-chloro-7-nitrobenzofurazan for liquid chromatographic determination of hydroxyproline in collagen hydrolysate. Anal Chem 53:485–489 Engstro¨m A, Anderson PE, Jossfsson B, Pfeffer WD (1995) Determination of 2-(9-Anthryl)ethyl chloroformate-labeled amino acids by capillary electrophoresis and liquid chromatography with absorbance or fluorescence detection. Anal Chem 67:3018–3022 Einarsson S, Josefsson B, Moller P, Sanchez D (1987) Separation of amino acid enantiomers and chiral amines using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate and reversed-phase liquid chromatography. Anal Chem 59:1191–1195 Faulkner AJ, Veening H, Becker HD (1991) 2-(9-Anthryl)ethyl chloroformate: a precolumn derivatizing reagent for polyamines determined by liquid chromatography and fluorescence detection. Anal Chem 63:292–296 Dean S, Burgi et al (1992) Improvement in the method of sample stacking for gravity injection in capillary zone electrophoresis. Anal Biochem 202:306–309 Schwer C, Gas B, Lottspeich F, Kenndler E (1993) Computer simulation and experimental evaluation of on-column sample preconcentration in capillary zone electrophoresis by discontinuous buffer systems. Anal Chem 65:2108–2115 Cohen SA, Michaud DP (1993) Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography. Anal Biochem 211:279–287 You J, Lao W, Sun X, Ou QJ (1999) Carbazole-9-N-acetyl-N-hydroxysuccinimide (Cahs) as Pre-column derivatization agent for fluorimetric detection of amino aompounds with liquid chromatography. Liq Chromatogr Relat Technol 22:2907–2923 You J, Lao W, You J, Wang G (1999) Characterization and application of acridine-9-N-acetyl-N-hydroxysuccinimide as a pre-column derivatization agent for fluorimetric detection of amino acids in liquid chromatography. Analyst 124:1755–1760 You J, You J, Lao W, Wang G, Jia X (1999) Fluorescence properties of carbazole-9-ylpropionic acid and its application to the determination of amines via HPLC with fluorescence detection. Analyst 124:281–288 Aina M, Biel G, Anaïs I et al (2022) Data fusion approaches for the characterization of musts and wines based on biogenic amine and elemental composition. Sensors 22:2132–2132 Gao Y, Tan J, Lu J, Sun Z, Li Z, Ji Z, Zhang S, You (2020) A novel fluorescent labeling reagent, 2-(9-acridone)-ethyl chloroformate, and its application to the analysis of free amino acids in honey samples by HPLC with fluorescence detection and identification with online ESI-MS. Anal Bioanal Chem 412:8339–8350 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 13 Jan, 2025 Read the published version in Journal of Fluorescence → Version 1 posted Editorial decision: Revision requested 22 Nov, 2024 Reviews received at journal 22 Nov, 2024 Reviews received at journal 21 Nov, 2024 Reviewers agreed at journal 21 Nov, 2024 Reviewers agreed at journal 20 Nov, 2024 Reviewers agreed at journal 19 Nov, 2024 Reviewers invited by journal 19 Nov, 2024 Editor assigned by journal 08 Nov, 2024 Submission checks completed at journal 08 Nov, 2024 First submitted to journal 31 Oct, 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. 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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-5365168","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381395529,"identity":"44ee4d84-8cf3-4bb6-b4a9-8e68b7e0e7c0","order_by":0,"name":"Yingying Lou","email":"","orcid":"","institution":"Shaoxing University","correspondingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Lou","suffix":""},{"id":381395530,"identity":"722cac51-1928-4ba8-a7b2-b48d46dce906","order_by":1,"name":"Huiquan Xiao","email":"","orcid":"","institution":"Shaoxing University","correspondingAuthor":false,"prefix":"","firstName":"Huiquan","middleName":"","lastName":"Xiao","suffix":""},{"id":381395533,"identity":"07a1bf3f-58a8-4378-841c-7d67c142e389","order_by":2,"name":"Xiaocong Zou","email":"","orcid":"","institution":"Shandong Technician Institute","correspondingAuthor":false,"prefix":"","firstName":"Xiaocong","middleName":"","lastName":"Zou","suffix":""},{"id":381395534,"identity":"dde8f7eb-31db-41a5-b356-85ebe1151caa","order_by":3,"name":"Zenghui Xu","email":"","orcid":"","institution":"Qufu Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zenghui","middleName":"","lastName":"Xu","suffix":""},{"id":381395535,"identity":"bae41eb4-4945-47ab-9e06-bb52cc66256a","order_by":4,"name":"Zhiwei Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYJACZgYDBjk29uYDpGkx5uM5lkCKFgaGxHkSOQrEKZefkXv4c0HB4fQ2hhwGhh8V2whrMbiRl2A8w+BwbhvD2QOMPWduE6FFIscgmcfgdm4bY18CM2MbEVrkZ+QYHAZqSWdj5jEgTgvDjRzDZqCWBDY2YrUYnHljDDT/v2EbD1vCQaL8It+eY/yZ50+avPz8xwcf/KggxmHI4ACJ6kfBKBgFo2AU4AIAsNo2ud1QN9cAAAAASUVORK5CYII=","orcid":"","institution":"Qufu Normal University","correspondingAuthor":true,"prefix":"","firstName":"Zhiwei","middleName":"","lastName":"Sun","suffix":""},{"id":381395536,"identity":"4a0859f1-d65e-4a12-8beb-7cc66741a549","order_by":5,"name":"Zan Li","email":"","orcid":"","institution":"Qufu Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zan","middleName":"","lastName":"Li","suffix":""},{"id":381395537,"identity":"06890b34-b453-4da2-a028-3cda6e501e81","order_by":6,"name":"Zhongyin Ji","email":"","orcid":"","institution":"Qufu Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zhongyin","middleName":"","lastName":"Ji","suffix":""},{"id":381395538,"identity":"fae398ac-8acb-4290-9f85-b5f82ae0d430","order_by":7,"name":"Jinmao You","email":"","orcid":"","institution":"Shaoxing University","correspondingAuthor":false,"prefix":"","firstName":"Jinmao","middleName":"","lastName":"You","suffix":""}],"badges":[],"createdAt":"2024-10-31 06:23:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5365168/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5365168/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10895-024-04098-6","type":"published","date":"2025-01-13T15:57:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70031210,"identity":"0b9ea709-f67e-4cea-95c3-67c93147c2a1","added_by":"auto","created_at":"2024-11-27 16:24:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":136223,"visible":true,"origin":"","legend":"\u003cp\u003eScheme of the synthesis and derivatization reaction of APE-Cl with amine compounds\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5365168/v1/567b4624e457cdea3cc588db.png"},{"id":70030320,"identity":"3be92774-800c-412f-97d9-ec2e11ff0411","added_by":"auto","created_at":"2024-11-27 16:16:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":425396,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of acetonitrile concentration on emission spectra of APE-butylamine derivative\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5365168/v1/c18d0021a1b7a908de389d00.png"},{"id":70030321,"identity":"ebf36a9d-46c4-4ddb-a6d3-f729ed41bd05","added_by":"auto","created_at":"2024-11-27 16:16:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":169768,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence responses of APE-amine-derivatives in different solvents=containing pure ACN, ACN/DMSO (10:1,v/v), and ACN/DMF(10:1,v/v）\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5365168/v1/2f715fa888b8a639a734ab4d.png"},{"id":70030322,"identity":"f4003fa8-9ff8-494e-b8a8-0b862424be09","added_by":"auto","created_at":"2024-11-27 16:16:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":140967,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A, B)\u003c/strong\u003e Chromatogram for standard aliphatic amines derivatized with APE-Cl (injected amount 1.0 pmol). Column temperature is set at 30℃; excitation wavelength λex 254 nm, emission wavelength: λem 418 nm; reversed-phase Eclipse XDB-C8 column (5 µm); flow rate = 1.0 ml min\u003csup\u003e−1\u003c/sup\u003e. C1: methylamine; C2: ethylamine; C3: propylamine; C4: butylamine; C5: pentylamine; C6: hexylamine; C7: heptylamine; C8: octylamine; C9: nonylamine; C10: decylamine; C11: undecylamine (no detected); C12 dodecylamine ; APE-OH （4-(9-acridone)phenylethyl ) hydrogen carbonate); APE-Cl：unreacted 4-(9-acridone)-phenylethyl chloroformate;\u003c/p\u003e\n\u003cp\u003etop (A): reagent blank; bottom (B): the separation of derivatized standard amines.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5365168/v1/3d7adacc648c9dcc679951c3.png"},{"id":70030323,"identity":"e5586beb-402f-4335-a274-a58f0af1b684","added_by":"auto","created_at":"2024-11-27 16:16:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":530933,"visible":true,"origin":"","legend":"\u003cp\u003eChromatograms for the determination of aliphatic amines from real sample derivatized with APE-Cl.\u003c/p\u003e\n\u003cp\u003eColumn temperature is set at 30℃; column eclipse XDB-C8 (5µm); flow rate = 1.0 ml min\u003csup\u003e−1\u003c/sup\u003e; peaks as Fig. 5B.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5365168/v1/ba5877e771c6ec68ba2d6fe2.png"},{"id":74284896,"identity":"596d6201-1de5-4414-a655-871d52a5e526","added_by":"auto","created_at":"2025-01-20 16:13:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1811082,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5365168/v1/c34c5158-7f97-4676-9f1c-a95518f6d58d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel high-conjugated probe of 4-(acridone-10-yl)-phenylethyl chloroformate (ABE-Cl) for rapid detection of amino compounds Using HPLC with Fluorescence detection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAliphatic compounds are naturally occurring and widely distributed in nature, including aquatic environments, biological fluids, and industrial waste materials. They typically arise as metabolic products in microorganisms, plants, and animals, with the main pathways for amine formation being the decarboxylation of amino acids, amination of carbonyl compounds, and degradation of nitrogen-containing compounds [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Aliphatic amines also serve as important raw materials and intermediates in manufacturing, as well as in the chemical and pharmaceutical industries [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Analyzing aliphatic amines in various liquids is crucial due to their toxic or carcinogenic properties [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe analysis of amines has traditionally been challenging due to their unique physicochemical properties, such as high polarity, volatility, alkalinity, and water solubility. Gas chromatography is commonly employed to analyze amines using various derivatization reagents [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Other techniques, including enzymatic [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and ion-exchange chromatographic detection [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], have been reported for the determination of amines in different matrices. However, these methods often face limitations due to low sensitivity. Most aliphatic amines exhibit neither natural UV absorption nor fluorescence. Consequently, chemical derivatization is essential to enhance detection sensitivity and improve selectivity through pre-column or post-column HPLC derivatization methods. Additional techniques used to enhance detection limits include microcolumn LC [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and capillary electrophoresis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Currently, the most popular methods for determining aliphatic amines involve pre-column and post-column derivatization with fluorescence detection.\u003c/p\u003e \u003cp\u003eSeveral common fluorescent derivatizing reagents [\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] have been fully developed. Despite the popularity of these pre-column methods, numerous reports have highlighted various shortcomings in their application, and to date, no method has been demonstrated to address all these issues. For instance, the o-phthalaldehyde (OPA) method provides greater sensitivity overall, but it is restricted to primary amines. The 2-phthalimidylbenzoyl chloride (PIB-Cl) method offers high sensitivity; however, its derivatives experience slight decomposition during storage [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBDF) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) are more effective derivatization reagents for amino compounds; however, when these reagents are exposed to daylight for 25 minutes in an aqueous methanol solution, approximately 30\u0026ndash;50% decomposition occurs [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Recently, 9-fluorenylmethyl chloroformate (FMOC) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], 1-(9-fluorenyl)ethyl chloroformate (FLEC) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and 2-(9-anthryl)ethyl chloroformate (AEOC) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] reagents have been utilized for the derivatization of amines, amino acids, and peptides for chiral or nonchiral separation in LC or CE.\u003c/p\u003e \u003cp\u003eThese reagents provide excellent UV absorption and very high sensitivity with laser-induced fluorescence detection; however, current derivatization procedures using FMOC are cumbersome. For effective derivatization, an excess of reagent is required, and the derivatized solution must be extracted with pentane to eliminate the surplus reagent [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], as it interferes with the separation of derivatives and negatively impacts column performance. The 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) method is quick and convenient, yielding stable derivatives. Nevertheless, only 10% of the fluorescent intensity in aqueous solution compared to that in pure acetonitrile solution is observed for its derivatives. Consequently, the detection limits for early eluted amine derivatives are typically higher than those for later ones [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, developing convenient assay methods for highly sensitive monitoring of amino compounds is extremely valuable, not only for water quality assessment but also for soil organic matter research.\u003c/p\u003e \u003cp\u003eIn our previous studies [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], we described the preparation of several fluorescent probes and their applications for analyzing common amino compounds. The primary objective of this research is to further develop more sensitive fluorescent probes with relatively long emission wavelengths. The results indicated that the synthesized APE-Cl probe molecule can react swiftly with amines to form highly stable derivatives. These derivatives not only exhibit high fluorescence sensitivity but also demonstrate long emission wavelengths. To the best of our knowledge, this is the first report of the APE-Cl fluorescent probe for the determination of amino compounds. In the present work, we investigate the optimal labeling conditions, including derivatization time, reaction temperature, buffer pH, and solvent system. We also determine the linearity, detection limits, and precision of the entire procedure. The detection of trace amounts of amines in various real samples using APE-Cl as the labeling reagent yielded satisfactory results.\u003c/p\u003e"},{"header":"Experimental Section","content":"\u003cp\u003eApparatus\u003c/p\u003e \u003cp\u003eExperiments were conducted using an LC/MSD-Trap-SL electrospray ion trap liquid chromatography/mass spectrometry (1100 Series LC/MSD Trap, a complete LC/MS/MS). All components of the HPLC system were from the HP 1100 series. Derivatives were separated on a BDS-C18 column (200\u0026times;4.6 mm, 5 \u0026micro;m, Yilite Dalian, China). The HPLC system was operated using HP Chemstation software. The mass spectrometer from Bruker Daltonik (Bremen, Germany) was equipped with atmospheric pressure chemical ionization (APCI). The mass spectrometer system was managed by Esquire-LC NT software, version 4.1. Fluorescence excitation and emission spectra were recorded using an F-7000 fluorescence spectrophotometer (Hitachi). Both excitation and emission bandpass were set to 5 nm.\u003c/p\u003e \u003cp\u003eReagents\u003c/p\u003e \u003cp\u003eAcetonitrile and methanol were of chromatographic grade from Luguang Chemical Reagent Co. (Shandong, China). All solvents used for RP-LC were of analytical reagent grade and filtered through a 0.45 micron filtration disk. Acetone, DMF, DMSO and other reagents were also of analytical reagent grade. Methylamine (99%), ethylamine (99%), and diethylamine (99%) as hydrochlorides were sourced from Shanghai Chemical Reagent Co. (Shanghai, China). All other amines used in experiments were of chromatographic grade and purchased from Luguang Chemical Reagent Co. Hyaluronic acid fermentation broth was obtained from Focus Biology Co., Ltd (Qu, Shandong, China). Stinky tofu, yogurt, and shrimp paste were acquired from the local supermarket (Qufu, Shandong, China).\u003c/p\u003e \u003cp\u003eSynthesis of 4-(9-acridone)phenylethyl chloroformate (APE-Cl)\u003c/p\u003e \u003cp\u003eThe synthesis of N-(4-(2-hydroxyethyl)phenyl)acridone (HPA)\u003c/p\u003e \u003cp\u003eThe synthesis of APE-Cl is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Acridone (2.0 g, 10 mmol), 2-(4-iodophenyl)ethanol (3.0 g, 12 mmol), dipivaloylmethane (DPVM, 2 mL), K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (2.0 g), and CuI (0.2 g) were combined in 50 mL of DMSO in a 250 mL round-bottom flask. The mixture was rapidly heated to 135℃and stirred at that temperature for 24 h. Upon completion of the reaction, the mixture was cooled to room temperature and filtered to remove insoluble solids (such as CuI and K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e). Subsequently, 400 mL of water was added with vigorous stirring, and the pH was adjusted to 6.5 using 3M HCl. The precipitated solids were filtered and washed twice with water (2\u0026times;20 mL) to yield yellow products. The crude product was recrystallized from a mixed solvent of acetonitrile and DMF (1:3, v/v) to obtain 3.8 g of light yellow crystals, with a yield of 84.8%. Mp. 248.3 ℃. Found: C:75.34%, H:5.50%, N:4.75%. Calculated: C: 75.30%, H:5.48%, N:5.85%. 1H NMR (500 MHz, Chloroform ) δ 7.55 (d, J\u0026thinsp;=\u0026thinsp;70.0 Hz, 14H), 7.45 (s, 1H), 7.23 (d, J\u0026thinsp;=\u0026thinsp;50.0 Hz, 16H), 7.08 (s, 11H), 7.03 (s, 5H), 3.63 (s, 4H), 2.75 (s, 4H), 1.37 (s, 4H). LC-APCI-MS: \u003cem\u003em/z\u003c/em\u003e 315.4 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e in positive-ion mode.\u003c/p\u003e \u003cp\u003ePreparation of 4-(9-acridone)benzylethyl chloroformate (APE-Cl)\u003c/p\u003e \u003cp\u003eTo a solution containing 8.5 g of solid phosgene and 100 mL of dichloromethane (0 ℃) in a 500 mL round-bottom flask, a mixture of N-(4-(2-hydroxyethyl)phenyl)acridone (3.0 g) and triethylamine (1.0 g catalyst) in 100 mL of dichloromethane solution was added dropwise over 2 h under electromagnetic stirring. After stirring at 0 ℃ for 4 h, the mixture was allowed to react at ambient temperature for an additional 6 h with electromagnetic stirring. The solution was then concentrated using a rotary evaporator. The residue was extracted three times with acetone; the combined acetone layers were concentrated under vacuum to yield a faint yellow solid. The crude products were recrystallized twice from acetone/acetonitrile (3:1, v/v) to produce yellow crystals.(2.87 g, yield, 80.0%), m.p. 173.5 ℃. Found: C 63.69%, H 4.01%, N 4.64%, Cl 11.75; Calculated: C 63.72%, H 4.11%, N 4.58%, Cl 11.05%; \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, DMSO) δ 8.38 (dd, J\u0026thinsp;=\u0026thinsp;8.0, 1.6 Hz, 2H), 7.72\u0026ndash;7.64 (m, 2H), 7.62 (d, J\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 7.45 (dd, J\u0026thinsp;=\u0026thinsp;31.9, 8.2 Hz, 2H), 7.33 (dd, J\u0026thinsp;=\u0026thinsp;11.0, 3.9 Hz, 2H), 6.75 (dd, J\u0026thinsp;=\u0026thinsp;15.2, 8.6 Hz, 2H), 3.90 (dt, J\u0026thinsp;=\u0026thinsp;123.7, 6.9 Hz, 2H), 3.09\u0026ndash;2.91 (m, 2H).;LC-APCI-MS: \u003cem\u003em/z\u003c/em\u003e 377.8 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e in positive-ion mode.\u003c/p\u003e \u003cp\u003ePreparation of APE-butylamine derivative\u003c/p\u003e \u003cp\u003eAPE-butylamine derivative was synthesized by reacting APE-Cl with an excess of butylamine as follows: To a solution of butylamine (0.146 g, 2.0 mmol) and DMAP (0.122 g, 0.1 mmol) in acetonitrile (50 mL) in a 100 mL round-bottom flask, a solution of APE-Cl (0.307 g, 1.0 mmol) in acetonitrile (50 mL) was added dropwise under electromagnetic stirring. The mixture was rapidly heated to 60 ℃ for 5 minutes and then poured into 300 mL of ice-water, stirring vigorously for 10 minutes; the precipitated solid was collected by filtration, washed with water, and dried at room temperature for 48 hours. The crude product was recrystallized twice from acetonitrile (30 mL) to yield yellow crystals (0.179 g, 52.0%). LC-APCI-MS: m/z 339.0 [M\u0026thinsp;+\u0026thinsp;H]\u0026thinsp;+\u0026thinsp;in positive-ion mode.\u003c/p\u003e \u003cp\u003ePreparation of Standard Solutions\u003c/p\u003e \u003cp\u003eThe APE-Cl reagent solution (approximately 4.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e mol/L) was prepared by dissolving 1.5 mg of APE-Cl in 10 mL of acetonitrile. The concentration of all amine standard solutions (50 ng/mL) was achieved by diluting the stock solutions of each amine (1 mg/mL) with acetonitrile. For long-chain amines (i.e., \u0026gt; C10), the stock solution was prepared by dissolving the long-chain amine in a small amount of DMF and then diluting it with acetonitrile to reach a final concentration of 50 ng/mL\u003c/p\u003e \u003cp\u003eAPE-butylamine solution (4.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e mol/L) was prepared by dissolving 1.6 mg of APE-butylamine in 10 mL of acetonitrile. The working solution of APE-butylamine for fluorescence spectrometry evaluation was prepared by diluting the stock solution with acetonitrile.\u003c/p\u003e \u003cp\u003eChromatographic Method and MS condition\u003c/p\u003e \u003cp\u003eThe separation of APE-amine derivatives by HPLC was performed on a reversed-phase BDS-C18 column using a gradient elution. Eluent A consisted of 35% acetonitrile with 20 mM ammonium/formic acid buffer (pH\u0026thinsp;=\u0026thinsp;3.5), while eluent B was 100% acetonitrile. Gradient elution commenced with 70% B and 30% A, increasing to 100% B over 35 minutes. Before the injection of the next sample, the column needed to be re-equilibrated with mobile phase A for 10 minutes. The flow rate was maintained at 1.0 mL min⁻\u0026sup1;, and the column temperature was set to 30℃. The fluorescence excitation and emission wavelengths were established at Ex\u0026thinsp;=\u0026thinsp;256 nm and Em\u0026thinsp;=\u0026thinsp;418 nm, respectively. MS ion source: Dry gas temperature, 350℃; nebulizer pressure, 60.0 psi; dry gas flow, 5.0 L/min; APCI Vap Temp 450℃; corona current (nA) 4000 (pos); capillary voltage 3500 V.\u003c/p\u003e \u003cp\u003eSample preparation procedure\u003c/p\u003e \u003cp\u003eExtraction of amines from fermented shrimp sauce\u003c/p\u003e \u003cp\u003eTo minimize the loss of volatile amines, all collected samples were promptly stored in brown bottles and placed in a freezer at -18 ℃ until used in subsequent experiments. The sample preparation procedure was as follows: 5.0 g of fermented shrimp sauce was homogenized in 25 mL of cooled trichloroacetic acid (TCA) solution (5%, v/v) in a 50 mL centrifuge tube. The centrifuge tube was thoroughly vortexed, then ultrasonicated for 30 min and centrifuged at 6000 r/min for 15 min at 4 ℃. The supernatant was filtered through 0.22 \u0026micro;m syringe filters (Millipore) and stored in a brown glass bottle at -18 ℃ until HPLC analysis.\u003c/p\u003e \u003cp\u003eExtraction of amines from stinky tofu\u003c/p\u003e \u003cp\u003eTo a 50 mL centrifuge tube, 5.0 g of stinky tofu and 25 mL of the cooled trichloroacetic acid solution (5%, v/v) or cooled HClO\u003csub\u003e4\u003c/sub\u003e solution (1.0 M, with 100 mg/L EDTA) were added. After the mixture was vibrated for 3 min, it was ultrasonicated for 30 min and then centrifuged at 6000 r/min for 15 min. The supernatant was filtered through 2 \u0026micro;m Millipore filters. The resulting solution was stored in a brown glass bottle at -18 ℃ until HPLC analysis.\u003c/p\u003e \u003cp\u003eExtraction of amines from acidophilus milk\u003c/p\u003e \u003cp\u003eTo a 10 ml centrifugal tube, yogurt (2 ml), deionized water (2.0 ml), and sodium hydroxide solution (0.5 ml, 4M) were added continuously and vortexed thoroughly for 3 minutes. Subsequently, 20 ml of chloroform was added and sonicated for 10 minutes. The chloroform layer was then transferred to a 50-ml round-bottom flask and adjusted to pH 3.0 with 4 M HCl solution. The solvent was evaporated to dryness using a rotary evaporator. The residue was re-dissolved in 2.0 ml of aqueous acetonitrile (ACN:H\u003csub\u003e2\u003c/sub\u003eO, 4:1, v:v) and stored in a brown glass bottle at -18 ℃ until HPLC analysis.\u003c/p\u003e \u003cp\u003eExtraction of amines from hyaluronic acid fermentation fluid.\u003c/p\u003e \u003cp\u003eTo a solution containing 5 mL of acetonitrile in a 10 mL round-bottom flask, 5 mL of the filtered hyaluronic acid fermentation fluid was added. The contents of the flask were ultrasonicated for 5 minutes. The extraction was performed twice, and the resulting solutions were combined and adjusted to pH\u0026thinsp;=\u0026thinsp;3.0 using a 4.0 M HCl solution. The combined extracts were then evaporated to dryness at 60 ℃ under reduced pressure. The residue was re-dissolved in 50% aqueous acetonitrile and stored at -18 ℃ until HPLC analysis.\u003c/p\u003e \u003cp\u003eDerivatization Procedure\u003c/p\u003e \u003cp\u003eTo a 2 ml screw-capped test tube, a standard amine mixture (50 \u0026micro;L), borate buffer (200 \u0026micro;L, pH\u0026thinsp;=\u0026thinsp;9.0), acetonitrile (50 \u0026micro;L), and 100 \u0026micro;L of APE-Cl solution (1.0\u0026times;10⁻\u0026sup3; mol/L) were successively added. The mixture was allowed to react at room temperature for 10 minutes. After the reaction was complete, a 10 \u0026micro;L volume of the mixture was diluted to 100 \u0026micro;L with acetonitrile. The diluted solution (10 \u0026micro;L) was then injected directly onto the chromatograph (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eStabilities of APE-butylamine Derivative\u003c/p\u003e\n\u003cp\u003eThe stabilities of APE-amine derivatives were assessed by analyzing the stored APE-amine solution over a week. As anticipated, when the derivative was stored in pure acetonitrile or methanol, daylight had no impact on its stability. However, the aqueous solution of the derivatives showed slight degradation (approximately 2.6\u0026ndash;3.8%) after being exposed to daylight for one week. This indicates that the derivatives were sufficiently stable to be effectively analyzed during the HPLC analysis period.\u003c/p\u003e\n\u003cp\u003eUltraviolet Absorption of APE-butylamine\u003c/p\u003e\n\u003cp\u003eAcridone and its derivatives represent one of the most significant classes of organic photochromic molecules, characterized by a large molar absorption coefficient. They display intriguing photochromic properties. In previous studies, the 10-phenyl-acridone-2-sulfonyl chloride (PASC) synthesized in our laboratory demonstrated strong UV properties \u003csup\u003e[4]\u003c/sup\u003e. In this study, APE derivatives exhibited significantly stronger UV and fluorescence properties compared to PASC derivatives. This enhancement can be attributed to the introduction of a benzene group at the N position of the acridone ring via an N-linked mode, which increases the \u0026pi;-conjugated system of the entire APE molecule. Consequently, some acridone derivatives can also function as high-sensitivity UV or fluorescence materials. The maximum ultraviolet absorption responses of APE derivatives are observed at wavelengths of 254 nm, with a molar absorption coefficient (\u0026epsilon;) exceeding 7.7\u0026times;10\u003csup\u003e4\u003c/sup\u003e (L mol\u003csup\u003e-1\u003c/sup\u003e cm\u003csup\u003e-1\u003c/sup\u003e). Previous studies indicated that PASC has an absorption maximum at 252 nm in acetonitrile, with a comparatively lower molar absorption coefficient of 3.85\u0026times;10\u003csup\u003e4\u003c/sup\u003e (L mol\u003csup\u003e-1\u003c/sup\u003e cm\u003csup\u003e-1\u003c/sup\u003e). Clearly, due to the enhancement of the molecular conjugation system, UV absorption was significantly improved, with the maximum molar absorption coefficient for the APE molecule being approximately twice that of the PASC molecule.\u003c/p\u003e\n\u003cp\u003eFluorescence for Representative ABE-butylamine Derivative.\u003c/p\u003e\n\u003cp\u003eFluorescence spectra of APE derivatives in aqueous acetonitrile were obtained with an excitation maximum at \u0026lambda;ex 254 nm and an emission maximum at \u0026lambda;em 418 nm. The excitation spectra of APE derivatives showed no significant change when varying the polarity of the solvent. An increase in solvent polarity resulted in a slight red shift in the fluorescence emission spectra. The emission intensities in 100% acetonitrile were 2.6 times stronger than those in pure water (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The fluorescence intensities in pure acetonitrile showed a significant increase compared to pure water. These results were in excellent agreement with some nitrogen-containing fluorescence derivatives previously reported in our experiments [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. This can be attributed to the fact that most fluorophores were only partially dissolved in low concentrations of aqueous methanol or acetonitrile. The fluorescence emission exhibited a slight blue shift with the addition of progressively increasing amounts of acetonitrile. A peak blue shift of about 17 nm (from 428 nm to 410 nm) for APE derivatives was observed. Similar results were also noted in methanol.\u003c/p\u003e\n\u003cp\u003eTo evaluate the effects of inorganic anions (such as sulfate, nitrate, and phosphate), organic anions (such as citrate), and divalent cations (Ca\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Ni\u003csup\u003e2+\u003c/sup\u003e, Co\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, and Hg\u003csup\u003e2+\u003c/sup\u003e) on fluorescence emission intensities, aqueous acetonitrile solutions of APE-butylamine (10 mol/L, 80:20, v/v) were utilized. Experiments were conducted by gradually adding increasing amounts of the corresponding anions or divalent cations to the APE-butylamine solution. The addition of progressively increasing amounts of Ca\u003csup\u003e2+\u003c/sup\u003e and Mg\u003csup\u003e2+\u003c/sup\u003e from 1.0 to 100 mol/L to the APE-butylamine solution did not result in a significant change in fluorescence emission intensities. However, the addition of progressively increasing amounts of Ni\u003csup\u003e2+\u003c/sup\u003e and Co\u003csup\u003e2+\u003c/sup\u003e beyond 40 mol/L to the APE-butylamine solution caused a decrease in emission intensities of approximately 4.2\u0026ndash;5.3% (the intensity was compared to those obtained in the absence of divalent cations). The addition of progressively increasing amounts of Cu\u003csup\u003e2+\u003c/sup\u003e and Hg\u003csup\u003e2+\u003c/sup\u003e (0\u0026ndash;20 mol/L) to the solution did not lead to a significant change in emission intensities. A notable decrease in emission intensities was observed when progressively increasing amounts of Cu\u003csup\u003e2+\u003c/sup\u003e and Hg\u003csup\u003e2+\u003c/sup\u003e beyond 100 mol/L were added, resulting in about a 10% decrease in emission intensities. Typically, the inorganic ions present in environmental samples did not interfere with the measurement results.\u003c/p\u003e\n\u003cp\u003eEffects of Co-solvents on Derivatization Efficiency.\u003c/p\u003e\n\u003cp\u003eIn general, acetonitrile or aqueous acetonitrile was selected as the derivatization solvent for amines using chloroformate reagents. Thus, the choice of co-solvents is vital to enhance derivatization yield and improve the solubility of non-water-soluble derivatives. In this study, DMF and methyl sulfoxide (DMSO) were assessed for their ability to boost derivatization yields and enhance the solubility of amine derivatives in aqueous acetonitrile, particularly for some non-water-soluble long-chain amine derivatives. Optimal efficiency was achieved by adding an appropriate amount of DMF or DMSO to the derivatization solution. The yields in acetonitrile solutions containing approximately 10% DMF or DMSO were 20% higher than those in pure acetonitrile. As shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, DMF as a co-solvent exhibited the strongest fluorescence and outperformed other solvents due to its low viscosity, effectively mitigating the issue of precipitation of hydrophobic long-chain amine derivatives (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eEffects of Temperature, Time and pH on Derivatization\u003c/p\u003e\n\u003cp\u003eAPE-Cl exhibits the same chloroformate reaction with primary and secondary amino compounds as BCEOC-Cl, CEOC-Cl, and AEC-Cl, as previously reported [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. The entire conjugation system of the APE-Cl molecule has been fully enhanced by an N-linked phenyl ring, with an excitation wavelength of \u0026lambda;ex 252 nm and an emission wavelength of \u0026lambda;em 414 nm. The corresponding derivatives displayed higher fluorescence responses. Complete derivatization of APE-Cl with amines could be performed, and the peak heights for all amine derivatives remained constant at 25 ℃ for 5 min. However, the derivatization yields slightly decreased when the temperature exceeded 30 ℃, likely due to the self-hydrolysis of the reagent at relatively high temperatures. When the reaction temperature was below 20 ℃, it took at least 10 minutes to ensure complete derivatization of the long-chain amines. Conversely, prolonged derivatization time also resulted in a slight decrease in signal for all derivatives, probably because the APE-amine derivatives could be hydrolyzed in an alkaline buffer solution environment. Therefore, subsequent derivatization was conducted at 25 ℃ for 5 min, and the solution was immediately neutralized to pH 6.0\u0026ndash;6.5 with 36% acetic acid.\u003c/p\u003e\n\u003cp\u003eThe fluorescence intensity of APE derivatives increased with the amount of derivatization reagent. A constant fluorescence intensity was achieved with the addition of a 3 to 4-fold molar excess of reagent relative to the total molar amines; further increasing the excess reagent beyond this level had no significant effect on yields. With as little as a 2.0-fold molar excess of derivatization reagent, the derivatization of amines was incomplete, resulting in low detection responses. Additionally, the hydrolysis of the excess APE-Cl produced the corresponding by-product APE-OH [MS, \u003cem\u003em/z\u003c/em\u003e 360.12 [M\u0026thinsp;+\u0026thinsp;H]]; MS/MS: \u003cem\u003em/z\u003c/em\u003e 342.12, \u003cem\u003em/z\u003c/em\u003e 315.12, \u003cem\u003em/z\u003c/em\u003e 285.0, \u003cem\u003em/z\u003c/em\u003e 196.1], and subsequently, the excess ABE-Cl reacted with APE-OH to form the di-substituted derivative APE-O-APE [MS: \u003cem\u003em/z\u003c/em\u003e 656.74 [M\u0026thinsp;+\u0026thinsp;H]]. The presence of APE-OH and (APE)2 did not interfere with the separation of amine derivatives. APE-Cl concentrations ranging from 5.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e to 5.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e mol/L did not significantly alter the derivatization time. When the derivatization solution was neutralized to pH 6.0\u0026ndash;6.5 with 36% acetic acid (w/w), the corresponding amine derivatives were found to be stable for more than 48 hours at room temperature. Therefore, the subsequent derivatization was carried out at 25 ℃ for 5 minutes, and the solution was immediately neutralized to pH 6.0\u0026ndash;6.5 with 36% acetic acid.\u003c/p\u003e\n\u003cp\u003eUsually, the derivatization reaction was conducted in the presence of basic catalysts; here, borate buffers, carbonate buffers, and phosphate buffers were evaluated. The results indicated that borate buffer was the optimal choice. The effects of pH on the derivatization reaction were then tested with borate buffers (0.2 mol/l) in the pH range of 8.5\u0026ndash;10.5. The maximum derivatization yields were achieved in the pH range of 9.0\u0026ndash;9.5. Consequently, all subsequent derivatization was performed in this pH range; however, outside this range, particularly in more acidic solutions, decreased responses were observed. At even higher pH values (\u0026gt;\u0026thinsp;10.0), PAE-Cl molecules were partially hydrolyzed to produce APE-OH forms; therefore, a 0.2 mol/l borate buffer solution at pH 9.0 was selected for all amine derivatization.\u003c/p\u003e\n\u003cp\u003eLinearity and detection limits and for derivatized amines\u003c/p\u003e\n\u003cp\u003eA standard solution containing 50 pmol of the APE-amine derivative was prepared to assess method repeatability. The relative standard deviations (R.S.D; n\u0026thinsp;=\u0026thinsp;10) of the peak areas and retention times ranged from 0.44\u0026ndash;1.19% and 0.023\u0026ndash;0.067%, respectively. For precision and accuracy, interday precision for 12 amines was evaluated using three identical shrimp paste samples, with amines spiked at low, medium, and high concentration levels, and analyses were conducted in triplicate. The experimental recoveries ranged from 96.2\u0026ndash;105.9%.\u003c/p\u003e\n\u003cp\u003eThe detection limit is a crucial parameter for assessing the sensitivity of an analytical method, particularly for analyzing trace amounts of components. Establishing a method with a low detection limit is a prerequisite for ensuring accurate determination. Based on an injection of 1.0 pmol amines, the detection limits (at a signal-to-noise ratio of 3:1) for each amine ranged from 1.68 to 11.2 fmol. Linearities were established over a 1000-fold concentration range for amines, with serial standard solutions analyzed from 1.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e to 1.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol/l (corresponding injected amounts of amines from 500 to 5.0 pmol). All amines exhibited linear responses over this range, with correlation coefficients exceeding 0.9994. The linear regression analysis for higher concentrations of amines was not conducted due to significant peak overrun. The linear regression equations are presented in Table 3.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eLinear regression equations, correlation coefficients and detection limits\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAmine\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;AX\u0026thinsp;+\u0026thinsp;B, X: injected amount (pmol): Y: peak area\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDetection limits\u003c/p\u003e\n \u003cp\u003e(fmol)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR.S.D.s\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;46.83X \u0026minus;\u0026thinsp;2.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;71.06X \u0026minus;\u0026thinsp;0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;93.43X -2.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;128.71X \u0026minus;\u0026thinsp;4.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;122.79X \u0026minus;\u0026thinsp;1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9994\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;146.82X \u0026minus;\u0026thinsp;8.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;151.09X \u0026minus;\u0026thinsp;10.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;165.75X \u0026minus;\u0026thinsp;11.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;169.04X \u0026minus;\u0026thinsp;11.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;170.29X \u0026minus;\u0026thinsp;11.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;171.21X \u0026minus;\u0026thinsp;12.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;171.29X \u0026minus;\u0026thinsp;12.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eHPLC separation of APE-amine derivatives\u003c/p\u003e\n\u003cp\u003eThe separation of the derivatized amine standards could be achieved under slightly acidic conditions (pH from 3.5 to 5.5). When the separation was conducted at pH\u0026thinsp;\u0026gt;\u0026thinsp;7.0, a noticeable increase in retention time for PAE-amine derivatives was observed, accompanied by slight peak tailing. Subsequently, the separation was performed on a BDS-C18 chromatographic column (200 x 4.6 mm, 5 \u0026micro;m) with gradient elution using aqueous acetonitrile containing 20 mM ammonium/formic acid as the mobile phase. The separation of the blank and sample is illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e (A and B).\u003c/p\u003e\n\u003cp\u003eSample analysis.\u003c/p\u003e\n\u003cp\u003eThe analysis of real samples of hyaluronic acid fermentation fluid (A), shrimp paste (B), stinky tofu (C), and yogurt (D) using fluorescence detection is presented in Fig.\u0026nbsp;6 (A, B, C and D). Amine compositional data are provided in Table\u0026nbsp;3. In the sample of hyaluronic acid fermentation fluid (A), fatty amines C\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e5\u003c/sub\u003e, and C\u003csub\u003e6\u003c/sub\u003e were found, with amounts of 5.23 ng/L, 9.71 ng/L, and 0.94 ng/L, respectively. Other fatty amines, including C\u003csub\u003e1\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003e-C\u003csub\u003e4\u003c/sub\u003e, and C\u003csub\u003e7\u003c/sub\u003e-C\u003csub\u003e12\u003c/sub\u003e, were not detected or were below detection limits. For shrimp paste samples (B), fatty amines C\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e5\u003c/sub\u003e, and C\u003csub\u003e9\u003c/sub\u003e were identified, with amounts of 2.54 ng/g, 5.32 ng/g, 4.60 ng/g, and 0.53 ng/g, respectively. Other fatty amines, including C\u003csub\u003e1\u003c/sub\u003e, C\u003csub\u003e4\u003c/sub\u003e, C\u003csub\u003e6\u003c/sub\u003e, C\u003csub\u003e7\u003c/sub\u003e, C\u003csub\u003e8\u003c/sub\u003e, and C\u003csub\u003e10\u003c/sub\u003e-C\u003csub\u003e12\u003c/sub\u003e, were either not detected or below detection limits. For stinky tofu samples (C), high concentrations of fatty amines were detected, except for C\u003csub\u003e3\u003c/sub\u003e and C\u003csub\u003e7\u003c/sub\u003e. The amine amounts were as follows: C\u003csub\u003e1\u003c/sub\u003e (17.98 ng/g), C\u003csub\u003e2\u003c/sub\u003e (57.91 ng/g), C\u003csub\u003e4\u003c/sub\u003e (75.22 ng/g), C\u003csub\u003e5\u003c/sub\u003e (89.33 ng/g), C\u003csub\u003e6\u003c/sub\u003e (1.20 ng/g), C\u003csub\u003e8\u003c/sub\u003e (23.18 ng/g), C\u003csub\u003e9\u003c/sub\u003e (18.58 ng/g), and C\u003csub\u003e10\u003c/sub\u003e (2.26 ng/g). Amines C\u003csub\u003e3\u003c/sub\u003e and C\u003csub\u003e7\u003c/sub\u003e were not detected. Among these fatty amines, C\u003csub\u003e4\u003c/sub\u003e and C\u003csub\u003e5\u003c/sub\u003e exhibited the highest levels. For yogurt samples (D), high concentrations of fatty amines C\u003csub\u003e1\u003c/sub\u003e and C\u003csub\u003e4\u003c/sub\u003e-C\u003csub\u003e8\u003c/sub\u003e were detected, with amounts of 2.07 ng/g, 28.03 ng/g, 3.54 ng/g, 2.89 ng/g, 11.92 ng/g, and 16.71 ng/g, respectively. In all these samples, C\u003csub\u003e5\u003c/sub\u003e amine was detected in relatively high amounts, while C\u003csub\u003e11\u003c/sub\u003e-C\u003csub\u003e12\u003c/sub\u003e were absent. It was clear that the high concentration of amine primarily originated from fermented stinky tofu and yogurt. Generally, the amine levels varied depending on the sample type, with C\u003csub\u003e4\u003c/sub\u003e and C\u003csub\u003e5\u003c/sub\u003e consistently showing relatively high concentrations across all fermented samples. C\u003csub\u003e3\u003c/sub\u003e amine was not detected in any samples except for the shrimp sauce.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAmine amount in different samples\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003eAmines\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHyaluronic acid fermentation fluid ( A)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eShrimp\u003c/p\u003e\n \u003cp\u003esauce (B)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStinky tofu (C)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eYogurt\u003c/p\u003e\n \u003cp\u003e(D)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eng/L\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eng/g\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eng/g\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eng/L\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eC3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eC4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eC5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eC6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eC7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eC8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.71\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eC9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eC10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eC11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eC12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eNo: detection or below detection limits.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAlthough the analysis of amine compounds is a well-established technique, current liquid chromatography methods still need improvements in analysis speed and sensitivity for a diverse range of samples. In this study, APE-Cl was employed to analyze amines, offering not only high sensitivity but also a simple and convenient derivatization process. The method offers several advantages: (1) the derivatization reaction proceeds quickly and smoothly with a basic catalyst; (2) the reagent and its derivatives remain stable for at least one week during HPLC analysis; (3) the reagent exhibits excellent fluorescence and a high ionization potential in the MS ion chamber.The derivatization reaction of APE-Cl with amines is simple, sensitive, and reproducible. Complete derivatization for higher chain amines (\u0026gt;\u0026thinsp;C12) can be easily achieved under the proposed derivatization conditions. This method has been successfully applied to determine free amines in various real samples with satisfactory results. Further work will focus on improving experimental conditions and investigating trace-level analysis of specific biogenic and aromatic amines in diverse environmental samples.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthical Approval Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYingying Lou: data collection, purification, methodology, and analysis, writing-original draft. Jinmao You: conceptualization, writing-review, and editing. Huiquan Xiao: methodology. Xiaocong Zou: HPLC-FLD analysis. Zenghui Xu: data curation. Zhiwei Sun, Zan Li, and Zhongyin Ji: conceptualization. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the Major Innovation Fund of Shandong Province (No. 2021ZDSYS23) and supported by the National Natural Science Foundation of China (No. 21976105).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated or analyzed for this study are available from reasonable requests to the corresponding author. The results presented in this manuscript have neither been published before nor submitted for publication elsewhere.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYingying Lou declares that she has no conflict of interest. Jinmao You declares that he has no conflict of interest. Zhiwei Sun declares that he has no conflict of interest. Zan Li declares that he has no conflict of interest. Zhongyin Ji declares that he has no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLu Xxin, Wang et al (2021) Metagenomes of polyaminetransforming bacterioplankton along a nearshore\u0026ndash;open ocean transect. Mar Life Sci Technol 4:163\u0026ndash;178\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakano-Fujii S, Kamata T, Kakehashi H et al (2022) Automated simultaneous identification of methamphetamine, precursor compounds to methamphetamine and their metabolites in urine for proof of methamphetamine use. Japanese J Forensic Sci Technol 27:151\u0026ndash;160\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBing li L, Ling yun W, Xue guang R et al (2022) Recent advances in fluorescent methods for polyamine detection and the polyamine suppressing strategy in tumor treatment. 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Anal Bioanal Chem 412:8339\u0026ndash;8350\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":"HPLC, Amino compounds, Fluorescence detection, Mass spectrometry, 4-(9-acridone)benzylmethyl carbonochloride (APE-Cl)","lastPublishedDoi":"10.21203/rs.3.rs-5365168/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5365168/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe fluorescence detection of amino compounds and the evaluation of their content in environmental samples are vital, not only for assessing food quality but also for studying soil organic matter. Here, we present the synthesis and application of a novel fluorescent probe, 4-(9-acridone)benzylmethyl carbonochloride (APE-Cl), for detecting amino compounds via a chloromate reaction with fluorescence detection. The complete derivatization reaction of APE-Cl with amino compounds can be accomplished in aqueous acetonitrile within 5 minutes at room temperature, using 0.2 M borate buffer (pH\u0026thinsp;=\u0026thinsp;9.0). APE-amine derivatives exhibited intense fluorescence with an excitation maximum at λex 254 nm and an emission maximum at λem 418 nm. All derivatives demonstrated high stability, strong fluorescence, and elevated ionization potential under atmospheric pressure chemical ionization (APCI-MS) in positive ion detection mode. The method, combined with gradient elution, provides baseline resolution of common amine derivatives on a reversed-phase C18 column. The LC separation for the derivatized amines shows good reproducibility with aqueous acetonitrile as the mobile phase. The relative standard deviations (n\u0026thinsp;=\u0026thinsp;6) for each amine derivative are \u0026lt;\u0026thinsp;3.99%. The detection limits (at a signal-to-noise ratio of 3) per injection ranged from 1.68 to 11.2 femtomole. The established pre-column derivatization method for determining amino compounds in practical samples proved to be satisfactory.\u003c/p\u003e","manuscriptTitle":"A novel high-conjugated probe of 4-(acridone-10-yl)-phenylethyl chloroformate (ABE-Cl) for rapid detection of amino compounds Using HPLC with Fluorescence detection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-27 16:16:50","doi":"10.21203/rs.3.rs-5365168/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-22T12:13:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-22T12:08:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-21T07:52:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79250498578072919978309480384205516856","date":"2024-11-21T06:11:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321305224915975405469229742179566813516","date":"2024-11-20T08:12:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299488639012590638877473137886016148748","date":"2024-11-20T03:02:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-19T14:38:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-08T18:07:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-08T18:05:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2024-10-31T06:08:15+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":"11fd359b-1b8a-4505-8daa-78e5e25800a1","owner":[],"postedDate":"November 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-20T16:07:15+00:00","versionOfRecord":{"articleIdentity":"rs-5365168","link":"https://doi.org/10.1007/s10895-024-04098-6","journal":{"identity":"journal-of-fluorescence","isVorOnly":false,"title":"Journal of Fluorescence"},"publishedOn":"2025-01-13 15:57:59","publishedOnDateReadable":"January 13th, 2025"},"versionCreatedAt":"2024-11-27 16:16:50","video":"","vorDoi":"10.1007/s10895-024-04098-6","vorDoiUrl":"https://doi.org/10.1007/s10895-024-04098-6","workflowStages":[]},"version":"v1","identity":"rs-5365168","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5365168","identity":"rs-5365168","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}