A qualitative and quantitative detection method for lithium isobutyrate-L-proline salt based on fluorescence properties

Abstract

The present invention discloses a method for qualitative and quantitative detection of lithium isobutyrate-L-proline salt based on fluorescence properties. The method is used for qualitative detection of synthetic products during the production and synthesis process of lithium isobutyrate-L-proline salt. During the preparation process of lithium isobutyrate and L-proline using a solvent method, the obtained white solid is added to a non-fluorescent solvent to prepare a solution. The solution is detected by one or more of the following methods: 1. irradiation under a 365nm dark box ultraviolet analyzer to observe whether fluorescence occurs; 2. ultraviolet-visible absorption spectroscopy to observe its absorption peak; 3. fluorescence spectroscopy to observe its fluorescence peak; 4. fluorescence spectrometer detection to observe characteristic peaks in a two-dimensional spectrum; and quantification using a standard curve method. Lithium isobutyrate-L-proline salt has unique fluorescence properties when formed into a solution in a non-fluorescent solvent. Based on these unique properties, lithium isobutyrate-L-proline salt is qualitatively and quantitatively detected.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescence detection, and in particular relates to a qualitative and quantitative detection method of lithium isobutyrate-L-proline salt based on fluorescence characteristics. Background Art

[0002] Lithium salts can be used in the field of mental illness, and have a good therapeutic and preventive effect on the recurring episodes of mania and depression in bipolar disorder, and have a unique effect in preventing suicide risk. Recent studies have also found that lithium salts can play a neuroprotective role by acting on GSK-3, WNT, AKT and neurotransmitters. Lithium salts have selective activity on GSK-3β. There are two main ways in which lithium salts inhibit GSK-3β: (1) as Mg 2+ Competitive inhibitors directly inhibit the activity of GSK-3β; (2) they indirectly inhibit the activity of GSK-3β by increasing the expression of phosphorylated AKT, phosphorylated GSK-3β (ser9), and MCL-1. This protective effect has potential applications in the prevention and treatment of neurodegenerative diseases including Alzheimer's disease. Currently, lithium salts commonly used in clinical practice are lithium carbonate, lithium citrate, lithium acetate, and long-acting lithium salts, among which lithium carbonate is the most commonly used.

[0003] Long-term use of inorganic lithium salts in the treatment of psychiatric disorders can potentially cause kidney damage and thyroid dysfunction, leading to blood pH disturbances, metabolic acidosis, and increased burden on the kidneys. Research and development has revealed that a variety of small-molecule organic acids, including butyric acid, isobutyric acid, valproic acid, and folic acid, have significant effects on the central nervous system, effectively alleviating mood disorders such as anxiety and depression, and delaying degenerative changes in the central nervous system. Therefore, the development of organic acid salts is expected to address the in vivo distribution defects of existing inorganic lithium salts.

[0004] Our company has developed a novel organic acid lithium amino acid salt, patented in Chinese patent CN114081881, "An organic lithium amino acid salt, crystal form, composition, and application." This salt is prepared by adding an organic acid lithium (lithium isobutyrate) and an amino acid (L-proline) to an appropriate amount of solvent, using either a single solvent method or a mixed solvent method. The resulting white solid enhances the efficacy of treatments for central nervous system diseases while also offering improved safety. During research and development, we discovered that lithium isobutyrate-L-proline salt exhibits unique fluorescence properties. To this end, we have developed a method for qualitatively and quantitatively detecting the fluorescence properties of this organic acid lithium amino acid salt (lithium isobutyrate-L-proline). Summary of the Invention

[0005] The object of the present invention is to provide a qualitative and quantitative detection method for lithium isobutyrate-L-proline salt based on fluorescence characteristics.

[0006] In order to achieve the above object, the present invention adopts the following technical means:

[0007] A first aspect of the present invention provides a method for qualitatively detecting lithium isobutyrate-L-proline salt based on fluorescence characteristics. The method is used for qualitatively detecting the synthetic product during the production and synthesis process of lithium isobutyrate-L-proline salt. A white solid is obtained during the preparation of lithium isobutyrate-L-proline salt using lithium isobutyrate and L-proline. The white solid is added to a non-fluorescent solvent to prepare a solution, and the solution is detected using one or more of the following methods:

[0008] ① Place the solution in a box-type UV analyzer, irradiate with 365nm UV light, and observe its fluorescence phenomenon. If fluorescence is observed, the prepared white solid contains lithium isobutyrate-L-proline salt;

[0009] ② The solution is subjected to UV-visible absorption spectrum detection using a UV-visible absorption spectrometer to observe its absorption peak; if there are absorption peaks at 260 nm and 320 nm, and the 320 nm absorption peak is higher than the 260 nm absorption peak, then the prepared white solid contains lithium isobutyrate-L-proline salt;

[0010] ③ The solution is subjected to fluorescence spectrum detection using a fluorescence spectrometer to observe its fluorescence peak; if there is a fluorescence peak at 370 nm, the prepared white solid contains lithium isobutyrate-L-proline salt;

[0011] ④ The solution is subjected to fluorescence spectrum detection using a fluorescence spectrometer to observe its two-dimensional spectrum; if two characteristic peaks of excitation / emission at 280 / 380 nm and 380 nm / 427 nm are present in the two-dimensional spectrum, and the 380 nm / 427 nm excitation / emission characteristic peak is stronger than the 280 / 380 nm excitation / emission characteristic peak, then the prepared white solid contains lithium isobutyrate-L-proline salt.

[0012] Furthermore, there are two characteristic peak bands in the two-dimensional spectrum, characteristic peak band 1: excitation wavelength 260~300nm, corresponding emission wavelength 350~400nm; characteristic peak band 2: excitation wavelength 360~400nm, corresponding color emission wavelength 410~450nm; the characteristic peaks at the excitation / emission wavelengths of 280 / 380nm and 380nm / 427nm in the two characteristic peak bands have significant characteristics.

[0013] The second aspect of the present invention provides a method for quantitatively detecting lithium isobutyrate-L-proline salt based on fluorescence characteristics, comprising the following steps:

[0014] ① Weigh lithium isobutyrate-L-proline salt powder, set the milligram concentration, and prepare standard lithium isobutyrate-L-proline salt solution samples of different dilutions by the serial dilution method. Perform fluorescence spectroscopy on the standard lithium isobutyrate-L-proline salt solution samples using 380 / 427 nm excitation / emission wavelengths to obtain the corresponding fluorescence values. The standard curve of the lithium isobutyrate-L-proline salt solution is obtained by fitting the fluorescence values ​​with the concentrations;

[0015] ② Perform fluorescence spectrum analysis on the lithium isobutyrate-L-proline salt solution to be tested using 380 / 427 nm excitation / emission wavelengths to obtain the fluorescence value;

[0016] ③ The fluorescence value of the lithium isobutyrate-L-proline salt solution to be tested obtained in step ② is used to obtain the concentration of the lithium isobutyrate-L-proline salt solution to be tested using a standard curve.

[0017] Furthermore, if the concentration of the lithium isobutyrate-L-proline salt solution to be tested is at the microgram level, further testing is performed using the following method:

[0018] ① Weigh lithium isobutyrate-L-proline salt powder, set the concentration at the microgram level, and prepare standard lithium isobutyrate-L-proline salt solution samples of different dilutions by the serial dilution method. Perform fluorescence spectrum detection on the standard lithium isobutyrate-L-proline salt solution samples using excitation / emission wavelengths of 280 / 380 nm to obtain the corresponding fluorescence values. The standard curve of the lithium isobutyrate-L-proline salt solution is obtained by fitting the fluorescence values ​​with the concentrations;

[0019] ② Perform fluorescence spectrum analysis on the lithium isobutyrate-L-proline salt solution to be tested using an excitation / emission wavelength of 280 / 380 nm to obtain the fluorescence value;

[0020] ③ The fluorescence value of the lithium isobutyrate-L-proline salt solution to be tested obtained in step ② is used to obtain the concentration of the lithium isobutyrate-L-proline salt solution to be tested using a standard curve.

[0021] The third aspect of the present invention provides a method for quantitatively detecting lithium isobutyrate-L-proline salt based on fluorescence characteristics, which can also be performed using the following steps:

[0022] ① Weigh lithium isobutyrate-L-proline salt powder, set a concentration gradient and prepare standard lithium isobutyrate-L-proline salt solution samples of different dilutions, perform ultraviolet-visible absorption spectroscopy on the standard lithium isobutyrate-L-proline salt solution samples at a wavelength of 260 nm or 320 nm to obtain the light absorption intensity, and obtain a standard curve of the lithium isobutyrate-L-proline salt solution by fitting the light absorption intensity and concentration;

[0023] ② Perform UV-visible absorption spectrum detection on the lithium isobutyrate-L-proline salt solution to be tested at a wavelength of 260nm or 320nm to obtain the light absorption intensity;

[0024] ③ According to the light absorption intensity of the lithium isobutyrate-L-proline salt solution to be tested obtained in step ②, the concentration of the lithium isobutyrate-L-proline salt solution to be tested can be obtained using a standard curve.

[0025] Furthermore, the standard lithium isobutyrate-L-proline salt solution sample is prepared by dissolving lithium isobutyrate-L-proline salt powder in a non-fluorescent solvent.

[0026] Beneficial effects of the present invention

[0027] The invention has the following beneficial effects: when an aqueous solution of lithium isobutyrate-L-proline salt is irradiated under a 365nm dark box ultraviolet analyzer, a fluorescence phenomenon is emitted, and obvious absorption peaks are present at 260nm and 320nm of the ultraviolet-visible absorption spectrum, and the intensity of the absorption peak shows a positive correlation with the concentration; the lithium isobutyrate-L-proline salt is detected by fluorescence spectroscopy, and an obvious fluorescence peak is present at 370nm, and the fluorescence value shows a positive correlation with the concentration of the solution; in a two-dimensional graph of the fluorescence spectrometer, two significant characteristic peaks are present at excitation / emission wavelengths of 280 / 380nm and 380nm / 427nm, and at the excitation / emission wavelengths of 380 / 427nm, the fluorescence value shows a positive correlation with the concentration of the solution; based on these unique characteristics, during the production and synthesis process, qualitative detection is performed on the synthetic product of lithium isobutyrate-L-proline salt; and quantitative detection is performed on the concentration of the synthetic product based on the positive correlation between the absorption peak and the concentration and the positive correlation between the fluorescence value and the concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The results of the dark box UV analyzer test of lithium isobutyrate-L-proline salt solution and comparison compound solution are shown;

[0029] Figure 2 The results of ultraviolet-visible absorption spectroscopy are shown for a gradient concentration of lithium isobutyrate-L-proline salt aqueous solution;

[0030] Figure 3 Shows the detection results of aqueous solutions of different compounds under UV-visible absorption spectroscopy;

[0031] Figure 4 The fluorescence spectrum detection results of lithium isobutyrate-L-proline salt with gradient concentration in different solvents are shown;

[0032] Figure 5 Shows the fluorescence spectrum detection results of lithium isobutyrate-L-proline salt in different solvents;

[0033] Figure 6 Shows a two-dimensional contour map of a lithium isobutyrate-L-proline salt aqueous solution under fluorescence spectroscopy;

[0034] Figure 7 Shows the three-dimensional spectrum of lithium isobutyrate-L-proline salt aqueous solution under fluorescence spectrum;

[0035] Figure 8 The figure shows the linear relationship between the absorption peak at 320 nm and the concentration of lithium isobutyrate-L-proline salt solution in UV-visible absorption spectrum detection;

[0036] Figure 9 The figure shows the linear relationship between the concentration y and the fluorescence value x of a high concentration lithium isobutyrate-L-proline salt solution at an excitation / emission wavelength of 380 / 427 nm.

[0037] Figure 10 The figure shows the linear relationship between the concentration y and the fluorescence value x of a low concentration lithium isobutyrate-L-proline salt solution at an excitation / emission wavelength of 280 / 380 nm;

[0038] in, Figure 1 In the figure, from left to right, there are purified water, lithium diisobutyrate, lithium diproline, an equivalent mixture of lithium isobutyrate + proline, and lithium isobutyrate-L-proline salt. DETAILED DESCRIPTION

[0039] Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages in this application are based on weight, and the test and characterization methods used are current as of the filing date of this application. Where applicable, the contents of any patents, patent applications, or publications referred to in this application are incorporated herein by reference in their entirety, and their equivalent patent families are also incorporated by reference, especially with respect to definitions of synthetic techniques, product and processing designs, polymers, comonomers, initiators, or catalysts disclosed in these documents in the art. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0040] Numerical ranges in this application are approximate values, so unless otherwise stated, they may include numerical values ​​outside the scope. Numerical ranges include all numerical values ​​from the lower limit to the upper limit increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For example, if the description component, physical or other properties (such as molecular weight, melt index, etc.) is 100 to 1000, it means that all individual numerical values ​​are clearly enumerated, such as 100, 101, 102, etc., and all subranges, such as 100 to 166, 155 to 170, 198 to 200, etc. For the scope comprising a numerical value less than 1 or comprising a fraction greater than 1 (such as 1.1, 1.5, etc.), 1 unit is appropriately considered as 0.0001, 0.001, 0.01 or 0.1. For the scope comprising a single digit less than 10 (such as 1 to 5), 1 unit is usually considered as 0.1. These are only specific examples of what is intended, and all possible combinations of numerical values ​​between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

[0041] When used with respect to chemical compounds, unless expressly stated otherwise, the singular includes all isomeric forms and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively). In addition, nouns using "a," "an," or "the" also include their plural forms unless expressly stated otherwise.

[0042] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, excipients or compounds unless expressly stated otherwise. In contrast, the term "essentially consisting of" excludes any other components, steps or processes from the scope of any subsequent description of the term, except those necessary for operational performance. The term "consisting of" does not include any components, steps or processes that are not specifically described or listed. Unless expressly stated otherwise, the term "or" refers to the listed members alone or in any combination.

[0043] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments.

[0044] Example

[0045] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials they cite are hereby incorporated by reference.

[0047] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0048] Preparation and synthesis method of lithium isobutyrate-L-proline salt:

[0049] Single solvent method: Add an appropriate amount of good solvent n-butanol to equal chemical equivalents of lithium isobutyrate and L-proline, heat under reflux to dissolve it just enough, stir for about 3 hours, filter while hot, cool naturally to crystallize, filter the solid, and vacuum dry to constant weight to obtain a white solid.

[0050] Mixed solvent crystallization method: Add an appropriate amount of good solvent (ethanol) to equal chemical equivalents of lithium isobutyrate and L-proline, heat and reflux to dissolve them, then add an appropriate amount of poor solvent (tetrahydrofuran) until solid is precipitated, add more good solvent to redissolve it, reflux for 3 hours, cool naturally to crystallize, filter the solid, and vacuum dry to constant weight to obtain a white solid.

[0051] 1. Qualitative detection of fluorescence characteristics of lithium isobutyrate-L-proline salt

[0052] (1) Dark box UV analyzer detection

[0053] Example 1

[0054] A lithium isobutyrate-L-proline salt sample was dissolved in purified water to prepare a sample solution with a concentration of 100 g / ml and placed in a sample tube.

[0055] The sample tube was placed in a dark box UV analyzer and detected using 365 nm UV light to observe its fluorescence phenomenon.

[0056] Comparative Example 1

[0057] Take purified water and put it into a sample tube, place the sample tube in a dark box UV analyzer, use 365nm UV light to detect, and observe its fluorescence phenomenon.

[0058] Comparative Example 2

[0059] Take a lithium diisobutyrate sample and dissolve it in purified water to prepare a sample solution with a concentration of 100 g / ml, and then put it into a sample tube.

[0060] The sample tube was placed in a dark box UV analyzer and detected using 365 nm UV light to observe its fluorescence phenomenon.

[0061] Comparative Example 3

[0062] A lithium diproline sample was dissolved in purified water to prepare a sample solution with a concentration of 100 g / ml and placed in a sample tube.

[0063] The sample tube was placed in a dark box UV analyzer and detected using 365 nm UV light to observe its fluorescence phenomenon.

[0064] Comparative Example 4

[0065] An equivalent amount of lithium isobutyrate and proline was mixed and dissolved in purified water to prepare a sample solution with a concentration of 100 mg / ml and placed in a sample tube.

[0066] The sample tube was placed in a dark box UV analyzer and detected using 365 nm UV light to observe its fluorescence phenomenon.

[0067] The results show that: Figure 1 As shown, purified water, lithium diisobutyrate, lithium diproline, and equivalent mixtures of lithium isobutyrate and proline do not exhibit fluorescence. Only the sample tube of lithium isobutyrate-L-proline salt solution has obvious fluorescence, showing unique fluorescence characteristics.

[0068] (2) UV-visible absorption spectroscopy detection

[0069] Example 2

[0070] Prepare the stock solution: weigh 0.3 g of lithium isobutyrate-L-proline salt powder sample and dissolve it in 9 ml of purified water solvent to a concentration of 33.33 g / L.

[0071] The original solution was diluted 2 times, 3 times, 4 times, 5 times, 6 times, 10 times, 20 times, 40 times, 60 times, 80 times, 100 times, 150 times, and 200 times to set different concentration gradients.

[0072] The lithium isobutyrate-L-proline salt solution was detected by UV-visible absorption spectroscopy.

[0073] The results show that: Figure 2 As shown, lithium isobutyrate-L-proline salt solution has obvious absorption peaks at 260nm and 320nm, and the intensity of the absorption peak is positively correlated with the concentration;

[0074] Example 3

[0075] Preparation of stock solutions: Prepare aqueous solutions of proline, lithium proline, lithium isobutyrate, isobutyric acid, an equivalent mixture of lithium isobutyrate and proline, and lithium isobutyrate-L-proline salt at specific concentrations using purified water. The proline concentration was 0.28986 mol / L, the lithium proline concentration was 0.27548 mol / L, the lithium isobutyrate concentration was 0.3546 mol / L, the isobutyric acid concentration was 2.69886 mol / L, and the lithium isobutyrate-L-proline salt concentration was 0.03987 mol / L. The aqueous solutions of the different compounds were analyzed by UV-visible absorption spectroscopy.

[0076] The results show that: Figure 3 As shown in the UV-visible absorption spectrum scanning diagrams of different compounds in aqueous solution, the absorption peaks are obviously different. The lithium isobutyrate-L-proline salt solution has obvious absorption peaks at 260nm and 320nm, which is specific.

[0077] Comparative Example 5

[0078] To prepare a solution, weigh 0.3 g of lithium diisobutyrate powder and dissolve it in 9 ml of purified water to a concentration of 33.33 g / L. Detection was performed using UV-visible absorption spectroscopy.

[0079] The results showed that there were no obvious characteristic absorption peaks of lithium diisobutyrate solution at 260nm and 320nm.

[0080] Comparative Example 6

[0081] To prepare a solution, weigh 0.3 g of lithium bisproline powder and dissolve it in 9 ml of purified water to a concentration of 33.33 g / L. Detection was performed using UV-visible absorption spectroscopy.

[0082] The results showed that there were no obvious characteristic absorption peaks of lithium diisobutyrate solution at 260nm and 320nm.

[0083] (3) Fluorescence spectrum detection

[0084] Example 4

[0085] Prepare the stock solution: weigh 0.3 g of lithium isobutyrate-L-proline salt powder sample and dissolve it in 9 ml of purified water solvent to a concentration of 33.33 g / L.

[0086] The stock solution was diluted 2 times, 3 times, 4 times, 5 times, 6 times, 10 times, and 20 times to set different concentration gradients.

[0087] The lithium isobutyrate-L-proline salt solution was subjected to fluorescence spectrum detection using a Hitachi fluorescence spectrometer.

[0088] The fluorescence spectrum detection results showed that Figure 4 As shown, lithium isobutyrate-L-proline salt solution has an obvious fluorescence peak at 370 nm, and the fluorescence value is positively correlated with the concentration.

[0089] Comparative Example 7

[0090] Prepare the stock solution: weigh 0.3 g of lithium isobutyrate-L-proline salt powder sample and dissolve it in 9 ml of methanol solvent to a concentration of 33.33 g / L.

[0091] The lithium isobutyrate-L-proline salt solution was subjected to fluorescence spectrum detection using a Hitachi fluorescence spectrometer.

[0092] Comparative Example 8

[0093] Preparation of stock solution: Weigh 0.3 g of lithium isobutyrate-L-proline salt powder sample and dissolve it in 9 ml of a solvent formed by a 1:1 mixture of methanol and water, with a concentration of 33.33 g / L.

[0094] The lithium isobutyrate-L-proline salt solution was subjected to fluorescence spectrum detection using a Hitachi fluorescence spectrometer.

[0095] The results show that Figure 5 As shown, in different solvents, lithium isobutyrate-L-proline salt solutions all have fluorescent characteristics. In different solvents, the fluorescence wavelength and the corresponding fluorescence value are different, which has a certain impact on the fluorescence spectrum detection of the lithium isobutyrate-L-proline salt solution, causing a certain shift in its fluorescence emission wavelength. At the same time, the effect of the solvent on the fluorescence wavelength shift of the lithium isobutyrate-L-proline salt solution can be used to optimize the fluorescence spectrum detection method, so that it has a better effect in specific detection.

[0096] Comparative Example 9

[0097] 0.3 g of isobutyric acid sample was weighed and dissolved in 9 ml of purified water solvent to a concentration of 33.33 g / L; the isobutyric acid solution was subjected to fluorescence spectrum detection using a Hitachi fluorescence spectrometer.

[0098] The fluorescence spectrum detection results showed that the isobutyric acid solution had no obvious fluorescence characteristics at the emission wavelength of 370nm.

[0099] Comparative Example 10

[0100] 0.3 g of lithium isobutyrate sample was weighed and dissolved in 9 ml of purified water solvent to a concentration of 33.33 g / L; the lithium isobutyrate solution was subjected to fluorescence spectrum detection using a Hitachi fluorescence spectrometer.

[0101] Fluorescence spectrum detection results show that lithium isobutyrate solution has no fluorescence characteristics at the emission wavelength of 370nm.

[0102] Comparative Example 11

[0103] 0.3 g of proline sample was weighed and dissolved in 9 ml of purified water solvent at a concentration of 33.33 g / L; the proline solution was subjected to fluorescence spectrum detection using a Hitachi fluorescence spectrometer.

[0104] The results of fluorescence spectrum detection showed that the proline solution had no fluorescence characteristics at the emission wavelength of 370nm.

[0105] Comparative Example 12

[0106] 0.3 g of lithium proline sample was weighed and dissolved in 9 ml of purified water solvent at a concentration of 33.33 g / L; the lithium proline solution was subjected to fluorescence spectrum detection using a Hitachi fluorescence spectrometer.

[0107] The results of fluorescence spectrum detection showed that the lithium proline solution had no fluorescence characteristics at the emission wavelength of 370nm.

[0108] Comparative Example 13

[0109] An isobutyric acid solution, a lithium isobutyrate solution, a proline solution, and a lithium proline solution are formed into a mixed solution, and the mixed solution is subjected to fluorescence spectrum detection using a Hitachi fluorescence spectrometer.

[0110] The results of fluorescence spectrum detection showed that the mixed solution had no fluorescence characteristics at the emission wavelength of 370nm.

[0111] The results showed that only lithium isobutyrate-L-proline salt solution had a fluorescence peak at 370nm, which was its unique fluorescence characteristic.

[0112] (4) Fluorescence spectrometer two-dimensional contour map and three-dimensional map detection

[0113] Example 5

[0114] The lithium isobutyrate-L-proline salt solution in Example 4 was detected using a F97PRO fluorescence spectrometer, with the vertical axis representing the excitation wavelength in the range of 200 nm to 900 nm and the horizontal axis representing the emission wavelength in the range of 200 nm to 900 nm, to obtain a two-dimensional graph;

[0115] The lithium isobutyrate-L-proline salt solution in Example 4 was detected using an F98 fluorescence spectrometer, with the vertical axis representing the excitation wavelength in the range of 250 nm to 500 nm and the horizontal axis representing the emission wavelength in the range of 250 nm to 600 nm, to obtain a three-dimensional spectrum, as shown in FIG. Figure 7 shown.

[0116] The result shows 1: Figure 6As shown, lithium isobutyrate-L-proline salt has two characteristic peaks of excitation / emission at 280 / 380nm and 380nm / 427nm; among them, the yellow high and low fields are fluorescence values, and the red line represents the scattering peak of the excitation wavelength; it can represent Raman scattering. Figure 7 As shown, lithium isobutyrate-L-proline salt has excitation / emission wavelength peaks near 280 / 380nm and 380nm / 427nm. The closer to the excitation / emission wavelength position in this band, the higher the corresponding fluorescence response value.

[0117] The result shows 2: Figure 6 and Figure 7 The two-dimensional contour map and the three-dimensional map mutually confirm the existence of two characteristic fluorescence peaks for lithium isobutyrate-L-proline salt. The 280 / 380nm characteristic peak is far from the red Raman scattering peak, making it less susceptible to interference from Raman scattering and more suitable for qualitative and quantitative analysis of ultra-low concentration samples. The 380nm / 427nm characteristic peak is closer to the Raman scattering peak and is therefore more susceptible to interference from scattering peaks, making it suitable for qualitative and quantitative analysis of high-concentration sample solutions. The three-dimensional map shows that under the same concentration conditions, the fluorescence response of the 380nm / 427nm characteristic peak is higher than that of the 280 / 380nm characteristic peak, and the characteristic peak shape is more distinct.

[0118] The result shows 3: Figure 6 and Figure 7 It can be seen that the characteristic peak range 1 of lithium isobutyrate-L-proline salt is: excitation wavelength 260~300nm, corresponding emission wavelength 350~400nm; characteristic peak range 2 is: excitation wavelength 360~400nm, corresponding color emission wavelength 410~450nm; there are fluorescence response values ​​in these two corresponding wavelength ranges, and the excitation / emission wavelengths of 280 / 380nm and 380nm / 427nm are characteristic peaks at the top of the peak.

[0119] When a lithium isobutyrate-L-proline salt solution is exposed to 365nm ultraviolet light in a box-type ultraviolet analyzer, fluorescence is observed; when ultraviolet-visible absorption spectrometer is used for ultraviolet-visible absorption spectrum detection, it has absorption peaks at 260nm and 320nm, and the 320nm absorption peak is higher than the 260nm absorption peak; when fluorescence spectrum is detected by a fluorescence spectrometer, it has a fluorescence peak at 370nm; and in the two-dimensional fluorescence spectrum, there are two characteristic peaks of excitation / emission at 280 / 380nm and 380nm / 427nm, and the 380nm / 427nm excitation / emission characteristic peak is stronger than the 280 / 380nm excitation / emission characteristic peak. These fluorescence characteristics are unique to lithium isobutyrate-L-proline salt and can therefore be used for qualitative detection during the preparation and synthesis of lithium isobutyrate-L-proline salt.

[0120] 2. Quantitative Detection of Fluorescence Characteristics of Lithium Isobutyrate-L-Proline Salt

[0121] Example 6

[0122] The original solution of the lithium isobutyrate-L-proline salt solution in Example 2 and solutions thereof diluted 2 times, 3 times, 4 times, 5 times, 6 times and 10 times in different concentration gradients were detected by ultraviolet-visible absorption spectroscopy using purified water.

[0123] The results showed that the absorption peak of lithium isobutyrate-L-proline salt solution at 260nm was interfered by miscellaneous peaks, and the characteristic peak at 260nm was not as clear as the characteristic peak at 320nm. The characteristic peak at 320nm was preferred for quantitative detection.

[0124] like Figure 8 As shown, the absorption peak at 320 nm is linearly related to the concentration, the linear equation is: y = 0.0052 + 0.01382x, and the correlation coefficient is 0.9997.

[0125] Example 7

[0126] Lithium isobutyrate-L-proline salt solution was prepared into milligram-level high concentration using purified water. The concentrations were set to 0, 1 mg / ml, 2 mg / ml, 4 mg / ml, and 8 mg / ml using the serial dilution method. The fluorescence values ​​were detected using an F97PRO fluorescence spectrometer at excitation / emission wavelengths of 380 / 427 nm, and were 0.01, 25.6, 53.64, 99.76, and 195.2, respectively.

[0127] The results show that: Figure 9 As shown, at fixed excitation / emission wavelengths of 380 / 427 nm, the concentration y and fluorescence value x exhibit a good linear relationship. The linear equation is: y = -0.081426 + 0.041172x, with a correlation coefficient of 0.9997, indicating a good linear correlation. This indicates that at high concentrations, the characteristic peak exhibits a good linear relationship.

[0128] Example 8

[0129] Lithium isobutyrate-L-proline salt solution was prepared to a low concentration at the microgram level with purified water. The serial dilution method was used to set the concentrations to 0, 0.31ug / ml, 0.61ug / ml, 1.22ug / ml, 2.44ug / ml, 4.88ug / ml, 9.77ug / ml, 19.53ug / ml, 39.06ug / ml, 78.13ug / ml, and 156.25ug / ml. The fluorescence values ​​were detected by F98 fluorescence spectrometer at excitation / emission wavelengths of 280 / 380nm. The fluorescence values ​​were -0.19, 17.6, 22.21, 27.98, 40.84, 67.57, 120, 227.9, 433.5, 833.7, and 1579, respectively.

[0130] The results show that: Figure 10 As shown, at fixed excitation / emission wavelengths of 280 / 380 nm, the concentration y and fluorescence value x exhibit a linear relationship. Within the range of 0.31 μg / ml to 156.25 μg / ml, the linear relationship is excellent, with the linear equation being: y = -2.2366 + 0.0992x, and a correlation coefficient of 0.9997. This indicates that at low sample concentrations, this characteristic peak exhibits a good linear relationship over a wide linear range, with minimal interference at 280 / 380 nm, resulting in high accuracy and precision.

[0131] In summary, the results of Examples 1 to 8 show that lithium isobutyrate-L-proline salt is an organic metal coordination salt with a specific fluorescence phenomenon, indicating that there is a metal coordination bond within the organic metal compound molecule and it is an organic bimolecular metal coordination salt, thereby generating the fluorescence phenomenon.

[0132] Lithium isobutyrate-L-proline coordination salt can be qualitatively and quantitatively detected by spectral instruments such as UV analyzer, UV-visible absorption spectroscopy and fluorescence spectrometer; and qualitative and quantitative detection can be performed at both high and low concentrations.

[0133] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for qualitative detection of lithium isobutyrate-L-proline salt based on fluorescence characteristics, which is used for qualitative detection of synthetic products during the production and synthesis process of lithium isobutyrate-L-proline salt, characterized in that: A white solid is obtained during the preparation of lithium isobutyrate-L-proline salt using lithium isobutyrate and L-proline. The white solid is added to a non-fluorescent solvent to prepare a solution, and the solution is detected using one or more of the following methods: ① Place the solution in a box-type UV analyzer, irradiate with 365nm UV light, and observe its fluorescence phenomenon. If fluorescence is observed, the prepared white solid contains lithium isobutyrate-L-proline salt; ② The solution was subjected to UV-visible absorption spectrum detection using a UV-visible absorption spectrometer to observe its absorption peak; If there are absorption peaks at 260 nm and 320 nm, and the 320 nm absorption peak is higher than the 260 nm absorption peak, the prepared white solid contains lithium isobutyrate-L-proline salt; ③ Using a fluorescence spectrometer to detect the fluorescence spectrum of the solution and observe its fluorescence peak; If there is a fluorescence peak at 370 nm, the prepared white solid contains lithium isobutyrate-L-proline salt; ④ The solution is subjected to fluorescence spectrum detection using a fluorescence spectrometer to observe its two-dimensional spectrum; if two significant characteristic peaks of 280 / 380 nm and 380 nm / 427 nm excitation / emission are present in the two-dimensional spectrum, and the 380 nm / 427 nm excitation / emission characteristic peak is stronger than the 280 / 380 nm excitation / emission characteristic peak, then the prepared white solid contains lithium isobutyrate-L-proline salt.

2. The method for qualitative detection of lithium isobutyrate-L-proline salt based on fluorescence characteristics according to claim 1, wherein: There are two characteristic peak bands in the two-dimensional spectrum, characteristic peak band 1: excitation wavelength 260~300nm, corresponding emission wavelength 350~400nm; characteristic peak band 2: excitation wavelength 360~400nm, corresponding color emission wavelength 410~450nm; the characteristic peaks at the excitation / emission wavelengths of 280 / 380nm and 380nm / 427nm in the two characteristic peak bands have significant characteristics.

3. A fluorescence-based quantitative detection method for lithium isobutyrate-L-proline salt, characterized in that: The steps include: ① Weigh lithium isobutyrate-L-proline salt powder, set the milligram concentration, and prepare standard lithium isobutyrate-L-proline salt solution samples of different dilutions by the serial dilution method. Perform fluorescence spectroscopy on the standard lithium isobutyrate-L-proline salt solution samples using 380 / 427 nm excitation / emission wavelengths to obtain the corresponding fluorescence values. The standard curve of the lithium isobutyrate-L-proline salt solution is obtained by fitting the fluorescence values ​​with the concentrations; ② Perform fluorescence spectrum analysis on the lithium isobutyrate-L-proline salt solution to be tested using 380 / 427 nm excitation / emission wavelengths to obtain the fluorescence value; ③ The fluorescence value of the lithium isobutyrate-L-proline salt solution to be tested obtained in step ② is used to obtain the concentration of the lithium isobutyrate-L-proline salt solution to be tested using a standard curve.

4. The method for quantitative detection of lithium isobutyrate-L-proline salt based on fluorescence characteristics according to claim 3, characterized in that: If the concentration of the lithium isobutyrate-L-proline salt solution to be tested is at the microgram level, further testing is performed using the following method: ① Weigh lithium isobutyrate-L-proline salt powder, set the concentration at the microgram level, and prepare standard lithium isobutyrate-L-proline salt solution samples of different dilutions by the serial dilution method. Perform fluorescence spectrum detection on the standard lithium isobutyrate-L-proline salt solution samples using excitation / emission wavelengths of 280 / 380 nm to obtain the corresponding fluorescence values. The standard curve of the lithium isobutyrate-L-proline salt solution is obtained by fitting the fluorescence values ​​with the concentrations; ② Perform fluorescence spectrum analysis on the lithium isobutyrate-L-proline salt solution to be tested using an excitation / emission wavelength of 280 / 380 nm to obtain the fluorescence value; ③ The fluorescence value of the lithium isobutyrate-L-proline salt solution to be tested obtained in step ② is used to obtain the concentration of the lithium isobutyrate-L-proline salt solution to be tested using a standard curve.

5. The method for quantitative detection of lithium isobutyrate-L-proline salt based on fluorescence characteristics according to claim 3, characterized in that: You can also take the following steps: ① Weigh lithium isobutyrate-L-proline salt powder, set a concentration gradient and prepare standard lithium isobutyrate-L-proline salt solution samples of different dilutions, perform ultraviolet-visible absorption spectroscopy on the standard lithium isobutyrate-L-proline salt solution samples at a wavelength of 260 nm or 320 nm to obtain the light absorption intensity, and obtain a standard curve of the lithium isobutyrate-L-proline salt solution by fitting the light absorption intensity and concentration; ② Perform UV-visible absorption spectrum detection on the lithium isobutyrate-L-proline salt solution to be tested at a wavelength of 260nm or 320nm to obtain the light absorption intensity; ③ According to the light absorption intensity of the lithium isobutyrate-L-proline salt solution to be tested obtained in step ②, the concentration of the lithium isobutyrate-L-proline salt solution to be tested can be obtained using a standard curve.

6. The method for quantitative detection of lithium isobutyrate-L-proline salt based on fluorescence characteristics according to any one of claims 3 to 5, characterized in that: The standard lithium isobutyrate-L-proline salt solution sample is prepared by dissolving lithium isobutyrate-L-proline salt powder in a non-fluorescent solvent.

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