Synthesis of a fluorescent probe for rhodamine B and determination of Zn2+ content in pharmaceutical zinc gluconate

The intermediate LBXJ was prepared by Rhodamine B, hydrazine hydrate and HCl, and then the probe Y2ST was prepared by LBXJ, chromone 3-formaldehyde and absolute ethanol. NMR characterized the structures of the intermediate and probe, IR and MS. Compared the fluorescence selectivity of ions after the system was determined and tested the response time, pH value, cation interference, reversibility, linearity, detection limit, accuracy, coordination ratio, and recovery rate of the samples. The detection limit of Y2ST was 0.7×10-6 mol/L, and the detection rate of Zn2+ in zinc gluconate was 96.1% at 5×10-6 mol/L--5×10-5 mol/L. It can be used for the content determination of Zn2+.


Introduction
In recent years, fluorescent probes have developed rapidly, and the design of suitable fluorescent probes to detect targets or target environments has become an indispensable analytical method for scientific research [1][2] .Fluorescent probes are a tool for analytical sensing based on detecting target-induced or modulated changes in optical signals.The detector is fast, simple and widely used.It consists of three main components, a fluorophore that generates a fluorescent signal under a specific excitation, a linking group that connects the reactive group to the fluorophore, and a recognition group that can be identified with the substance to be measured by chemical reaction or complexation, etc.After the fluorescent probe is added to the target to be measured, the recognition group reacts or complexes with the target to be measured and other ways to change the fluorescent properties of the system.When the recognition group binds to the target to be measured, the fluorescent probe can undergo chemical reactions or structural changes, so that the target's type and content can be expressed in the form of fluorescent signals.Fluorescence analysis has been widely used in scientific research because of its high selectivity, sensitivity, low cost, in situ real-time monitoring and ease of operation [3][4][5][6][7][8][9] .Zn 2+ plays an extremely important role in living organisms and can cause disease in excess or deficiency [10] .Zinc is the second most abundant metal in living organisms (after iron).It is not only part of the catalytic activity center of many biological enzymes but an element that makes up the structure of several macromolecules [11] .A series of zinc ion fluorescent probe molecules have been successfully prepared, however, the currently reported zinc ion probes still do not adequately meet the detection needs.Therefore, the selectivity, sensitivity and adaptability of fluorescent probes to complex environments still need to further improve further.The increasing complexity of detection needs makes the design of suitable zinc ion probes for specific identification environments critical [12] .
Many scientists favor and select Rhodamine-based fluorescent dyes as fluorophores because of their good photostability, high fluorescence quantum yield, and long emission and absorption wavelengths [13] .
Yang's [14] group designed and synthesized a simple single-and two-photon probe HL to detect Zn 2+ .The probe was highly selective for Zn 2+ and at 500 nm, the fluorescence intensity was significantly enhanced, suggesting a possible reason for this [15] .This paper designed and synthesized a new structure of rhodamine fluorescent probe Y2ST with rhodamine as the parent.NMR, IR and mass spectrometry characterized and structurally analyzed its structure.It was determined that the probe was as expected.In this paper we will examine the performance of the Y2ST probe and the influencing factors, and validate the assay method by methodological examination.Zinc in zinc gluconate was extracted by the dry ashing method, and the content of the treated zinc ions was successfully determined.The Zn 2+ and Y2ST probes were combined and explored for response time, pH value, cation interference, reversibility, ion concentration, linearity, detection limit, precision, accuracy, ligand ratio, and spiked sample recovery.

Methods
Y2ST Probe preparation：Put 0.174g of chromone-3-formaldehyde in a round bottom flask and add 20mL of absolute ethanol for dissolution.At this time, the solution is turbid yellow.Add 0.48g of LBXJ to the flask and put it on a magnetic stirrer for stirring until the solution becomes clear pink.Raise the temperature to 75 ℃, vacuum and inject nitrogen.After an 8h of reflux, many white solids in the solution precipitate out to stop the reaction.Cool it to room temperature.Vacuum and filter and wash it with absolute methanol three times to obtain white powder after drying.

A subsection Ion fluorescence selectivity
Figure 1 shows the fluorescence spectra of Y2ST reacting with different metal ions.To investigate the selectivity of the Y2ST probe for different metal ions, We added 3.5×10 -5 mol/L Pb 2+ , Zn 2+ , Ag + , Ca 2+ , Mn 2+ , Cu 2+ , Al 3+ , Fe 3+ , K + , Na + , Mg 2+ , Cr 6+ and Hg 2+ metal solutions to the blank Y2ST probe solution for fluorescence detection.It can be observed from the test results that when Zn 2+ was added to the Y2ST probe solution, the solution color changed from transparent to pale yellow, and there was a strong fluorescence peak at 465 nm.A weak fluorescence peak appeared at 465 nm when Cr 6+ was added, but it did not affect the Zn 2+ selectivity of the probe.Compared with the fluorescence intensity of the Y2ST probe itself, almost no fluorescence was produced by other metal ions.Therefore, the experiment shows that Y2ST fluorescent probe is selective and specific to Zn 2+ .reacting with different metal ions

Reversibility
Figure 2 shows the fluorescence spectra when EDTA was added after the reaction of Y2ST with Zn 2+ .In order to test whether the reaction of the Y2ST probe with Zn 2+ is reversible, we designed an EDTA reversibility experiment .We performed a fluorescence test after 5 min of the reaction of Y2ST with Zn 2+ .The fluorescence value was measured after adding EDTA for 1 h and found that the fluorescence decreased nearly 6 times.