Based on Cu 2+ -OPD colorimetric and fluorescence dual-mode detection method for D-penicillamine content in penicillamine tablets

Abstract

The present invention discloses a Cu-based 2+ A method for colorimetric and fluorescent dual-mode detection of D-penicillamine content in penicillamine tablets using OPD is provided, comprising the steps of: taking an OPD solution, adding water and a CuCl2 solution, fixing the volume, and mixing to obtain a reference solution, wherein the absorbance is determined as A0 and the fluorescence intensity is determined as F0; taking multiple OPD solutions, adding D-penicillamine solutions of different concentrations to each OPD solution, and then adding a CuCl2 solution, fixing the volume, and mixing to obtain multiple standard solutions, wherein the absorbance of the multiple standard solutions is determined as A and the fluorescence intensity is determined as F; drawing a standard curve with the concentration of the D-penicillamine solution as the horizontal axis and the relative absorbance intensity / relative fluorescence intensity as the vertical axis; measuring the absorbance and fluorescence intensity of the solution to be tested, and substituting the absorbance and fluorescence intensity of the solution to be tested into the standard curve equation to obtain the D-penicillamine concentration in the solution to be tested. The present invention has the advantages of simple and easy-to-obtain detection reagents, simple operation, short experimental reaction time, and high reliability of the results.

Description

Technical Field

[0001] The present invention relates to the technical field of content determination in drug quality control. More specifically, the present invention relates to a method based on Cu 2+ -OPD dual-mode colorimetric and fluorescence detection method for D-penicillamine content in penicillamine tablets. Background Art

[0002] Penicillamine is an important sulfur-containing amino acid and the primary hydrolysis product of penicillin antibiotics. Due to its low toxicity, the D-enantiomer of penicillamine tablets is commonly used as an effective clinical treatment for a variety of diseases, including primary biliary cirrhosis, Wilson's disease, scleroderma, rheumatoid arthritis, cystinuria, and heavy metal poisoning. However, improper use of this drug can lead to adverse reactions such as abdominal pain, loss of taste, anorexia, agranulocytosis, kidney problems, and bone marrow suppression. Therefore, establishing a simple, rapid, low-cost, accurate, sensitive, and specific method for determining the content of D-penicillamine in pharmaceuticals is of great significance for the quality control of penicillamine tablets. Summary of the Invention

[0003] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0004] Another object of the present invention is to provide a Cu-based 2+ -OPD colorimetric and fluorescence dual-mode detection method for D-penicillamine content in penicillamine tablets, using an indirect method with the help of Cu 2+ -OPD color development system and Cu 2+ Complexation with D-penicillamine allows for convenient and effective colorimetric and fluorescence dual-mode detection of D-penicillamine concentration.

[0005] In order to achieve these objects and other advantages according to the present invention, there is provided a Cu-based 2+ The method for detecting the content of D-penicillamine in penicillamine tablets using colorimetric and fluorescence dual-mode OPD comprises the following steps:

[0006] Prepare OPD solution, CuCl2 solution, and multiple D-penicillamine solutions of different concentrations;

[0007] Take a portion of OPD solution, add a predetermined volume of water to it, then add CuCl2 solution, make up to volume and mix well to obtain a reference solution. The absorbance of the reference solution is measured as A0 and the fluorescence intensity is measured as F0.

[0008] Take multiple OPD solutions, add different concentrations of predetermined volume of D-penicillamine solution to each OPD solution, then add CuCl2 solution, mix after dilution, and obtain multiple standard solutions. Determine the absorbance of multiple standard solutions as A and the fluorescence intensity as F. The molar ratio of OPD to CuCl2 in the reference solution and the standard solution is 10:1. The CuCl2 content in multiple standard solutions is 10:1. 2+ The molar ratio with D-penicillamine ranges from 1:0.02 to 0.4;

[0009] A UV absorption intensity standard curve was drawn with the added D-penicillamine solution concentration as the abscissa and the relative absorbance intensity ΔA / A0 as the ordinate, and the UV absorption intensity standard curve equation was obtained, where ΔA=A-A0;

[0010] A fluorescence intensity standard curve was drawn with the concentration of the added D-penicillamine solution as the abscissa and the relative fluorescence intensity ΔF / F0 as the ordinate, and the equation of the fluorescence intensity standard curve was obtained, where ΔF=F-F0;

[0011] Determine the absorbance and fluorescence intensity of the test solution, and substitute them into the UV absorption intensity standard curve equation and the fluorescence intensity standard curve equation to obtain the D-penicillamine concentration in the test solution.

[0012] Preferably, the OPD solution is prepared by using ethanol as a solvent to prepare 5.0×10 -3 mol / L OPD solution.

[0013] Preferably, the CuCl2 solution is prepared by taking copper chloride dihydrate and using water as solvent to prepare 1.0×10 -3 mol / L CuCl2 solution.

[0014] Preferably, the preparation of multiple D-penicillamine solutions of different concentrations is specifically as follows: weighing a D-penicillamine standard, dissolving it in water, and diluting it to prepare 5 μmol / L, 8 μmol / L, 10 μmol / L, 20 μmol / L, 50 μmol / L, 80 μmol / L, and 100 μmol / L D-penicillamine solutions.

[0015] Preferably, the reference solution and the standard solution are both diluted to 5 mL, one portion of the OPD solution is 0.5 mL, and the predetermined volume is 1 mL.

[0016] Preferably, the reference solution and the standard solution are mixed and then allowed to stand at 15-30° C. for 5-10 minutes.

[0017] Preferably, the reference solution and the standard solution are mixed and then allowed to stand at 20°C.

[0018] Preferably, the reference solution and the standard solution are mixed and then allowed to stand at room temperature.

[0019] Preferably, the reference solution and the standard solution are mixed and then allowed to stand for 5 minutes.

[0020] Preferably, the reference solution and the standard solution are mixed and then allowed to stand for 10 minutes.

[0021] The present invention has at least the following beneficial effects:

[0022] By indirect method, Cu 2+ -OPD color development system and Cu 2+ By combining the complexation with D-penicillamine, a convenient and effective colorimetric and fluorescence dual-mode detection method for D-penicillamine has been developed. The detection reagents are simple and easy to obtain, the operation is simple, the experimental reaction time is short, and it can be performed under simple conditions, which greatly reduces the reagent cost, time cost and equipment cost, and effectively reduces the detection error results and improves the reliability of the results.

[0023] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the principle of the present invention;

[0025] Figure 2 This is the absorption spectrum of the verification experiment of the present invention;

[0026] Figure 3 This is the fluorescence spectrum of the verification experiment of the present invention;

[0027] Figure 4 The effects of different reaction times and temperatures on the OPD-Cu 2+ -D-penicillamine system relative absorbance and relative fluorescence intensity effect diagram;

[0028] Figure 5 Figure 5A is the absorption spectrum of the detection system in the presence of different concentrations of D-penicillamine; Figure 5B is the standard curve of the relative absorbance of the detection system in the presence of different concentrations of D-penicillamine and the concentration of D-penicillamine; Figure 5C is the fluorescence spectrum of the detection system in the presence of different concentrations of D-penicillamine; Figure 5D is the standard curve of the relative fluorescence intensity of the detection system in the presence of different concentrations of D-penicillamine and the concentration of D-penicillamine;

[0029] Figure 6 Graph showing the effect of coexisting substances on the absorbance and fluorescence intensity of the detection system. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0031] 1. Solution preparation

[0032] 1.1, weigh an appropriate amount of OPD (o-phenylenediamine) and ethanol as solvent to prepare 5.0×10 -3 mol / L OPD solution, stored in a refrigerator at 4°C away from light;

[0033] 1.2. Weigh copper chloride dihydrate and water as solvent to prepare 1.0×10 -3 mol / L CuCl2 solution, wherein the water used in this application is ultrapure water;

[0034] 1.3. Accurately weigh D-penicillamine standard, dissolve in water, and dilute to prepare 5μmol / L, 8μmol / L, 10μmol / L, 20μmol / L, 50μmol / L, 80μmol / L, and 100μmol / L D-penicillamine solutions;

[0035] 2. Verification experiment

[0036] 2.1. Prepare the solution as required, corresponding to Figure 2 The A, B, C, D, and E marks in the table are:

[0037] A is 1.0×10 configured in <1.2> -3 mol / L CuCl2 solution;

[0038] B is the 5.0×10 -3 mol / L OPD solution;

[0039] C is to add OPD solution (0.5 mL, 5.0 × 10 -3 mol / L) and 1 mL of water, and then add CuCl2 solution (0.25 mL, 1.0×10 -3 mol / L), and finally make up to 2.5 mL with water, shake well, and let it stand at room temperature for 10 min;

[0040] D is to add OPD solution (0.5 mL, 5.0 × 10 -3 mol / L) and D-penicillamine solution (1 mL, 5 μmol / L), and then add CuCl2 solution (0.25 mL, 1.0×10 -3 mol / L), and finally fill up to 2.5 mL with the corresponding volume of water, shake well, and let it stand at room temperature for 10 min;

[0041] E is to add OPD solution (0.5 mL, 5.0 × 10 -3 mol / L) and D-penicillamine solution (1 mL, 100 μmol / L), and then add CuCl2 solution (0.25 mL, 1.0×10 -3 mol / L), and finally fill up to 2.5 mL with the corresponding volume of water, shake well, and let it stand at room temperature for 10 min;

[0042] 2.2. Detect the absorption and fluorescence spectra of A, B, C, D, and E. The absorption spectra are shown in the figure below: Figure 2 The fluorescence spectrum is shown in Figure 3 shown

[0043] Depend on Figure 2 、 Figure 3 It can be seen that there is only Cu in the system 2+ (A) and only OPD (B), the solution is colorless and has no fluorescence, and the absorbance value is also very weak. However, when Cu is added to OPD 2+ (C), the system showed a strong UV absorption peak at 420nm, and the fluorescence intensity at 556nm was significantly enhanced. When 5μmol / L D-penicillamine (D) and 100μmol / L D-penicillamine (E) were added, the absorbance and fluorescence intensity decreased. When 100μmol / L D-penicillamine was added, the signal decreased more significantly. This shows that D-penicillamine and Cu 2+ Can effectively combine to oxidize the Cu of OPD 2+ The decrease leads to a decrease in the amount of OPDox generated. In this experiment, the UV absorption peak is 420nm, the excitation wavelength is 420nm, and the fluorescence emission peak is 556nm.

[0044] Further, if Figure 1 As shown, OPD can 2+ Under the catalytic action of Cu, OPDox is oxidized to form OPDox, which has a strong UV absorption peak at 420nm and a strong fluorescence peak at 556nm. The addition of D-penicillamine (D-PA) can react with Cu 2+ The complexation reaction produces Cu 2+ 2+ Reduce, reduce the ultraviolet absorption peak and fluorescence peak intensity.

[0045] 3. Temperature determination

[0046] Prepare 5 portions of solution according to the ratio of E in <2.1>, shake well, and react at 20°C, 25°C, 30°C, 35°C, and 40°C for 10 min, respectively, to obtain reaction solutions at different temperatures;

[0047] The UV absorption spectrum and fluorescence spectrum of the reaction system were tested using a UV-visible spectrophotometer and a fluorescence spectrophotometer, such as Figure 4 C, 4D, where:

[0048] Figure 4 In C and 4D, the horizontal axis is the reaction temperature in °C;

[0049] Figure 4 The vertical axis in C is the relative absorbance ΔA / A0, where A0 is the reference solution (prepared as described in Example 1, except that the reaction temperature is the same as A W The absorbance of the corresponding temperature, ΔA=A W -A0,A W is the absorbance of the solution measured at the corresponding reaction temperature, that is, A0 and A W The reaction temperature is the same;

[0050] Figure 4 The vertical axis of D is the relative fluorescence intensity ΔF / F0, where F0 is the reference solution (prepared as described in Example 1, except that the reaction temperature is the same as A W The fluorescence intensity of the corresponding temperature, ΔF = F W -F0,F W is the fluorescence intensity of the solution measured at the corresponding reaction temperature;

[0051] Depend on Figure 4 C and 4D show that the best result is obtained at 20°C, that is, the optimal reaction temperature is 20°C.

[0052] 4. Determine reaction time

[0053] Prepare 1 portion of solution according to the ratio of E in step <2.1>, shake well, react at 20℃ for 5 minutes, and then start measuring its absorbance and fluorescence intensity. Then measure it every 10 minutes for one hour to optimize the reaction time. The results are as follows: Figure 4 A, 4B, wherein:

[0054] Figure 4 In A and 4B, the horizontal axis is the reaction time in min;

[0055] Figure 4 The vertical axis of A is the relative absorbance intensity ΔA / A0, A0 is the reference solution (prepared as described in Example 1, except that the reaction time is the same as A T The absorbance at the corresponding time, ΔA=A T -A0,A T is the absorbance of the solution measured at the corresponding reaction time, that is, A0 and A T The reaction time is the same;

[0056] Figure 4 The vertical axis of B is the relative fluorescence intensity ΔF / F0, where F0 is the reference solution (prepared as described in Example 1, except that the reaction time is the same as that of A). T The fluorescence intensity of the corresponding time, ΔF = F T -F0,F T is the fluorescence intensity of the solution measured at the corresponding reaction time;

[0057] in accordance with Figure 4 As shown in A, for UV absorption, the reaction effect is best when the reaction time is 10 minutes. Figure 4 As shown in B, for fluorescence, the best reaction effect is achieved when the reaction time is 5 minutes. Taking into account the experimental cost and convenience, the ultraviolet absorption and fluorescence collection in this experiment are both reacted for 10 minutes and collected at room temperature.

[0058] <Example 1>

[0059] Based on Cu 2+ The method for detecting the content of D-penicillamine in penicillamine tablets using colorimetric and fluorescence dual-mode OPD comprises the following steps:

[0060] S1. Preparation of penicillamine stock solution

[0061] Randomly select 10 penicillamine tablets, grind them, weigh them, weigh 0.4091g of sample, dissolve them in water, dilute to VL, filter, dilute, and finally prepare 1.0×10 -3 mol / L (theoretical concentration) penicillamine stock solution, where the theoretical concentration is calculated based on the instructions for penicillamine tablets;

[0062] S2. Prepare a standard curve;

[0063] ⅰ. Preparation of reference solution and standard solution

[0064] Prepare eight 5 mL centrifuge tubes and label them G0, G1, G2, G3, G4, G5, G6, and G7.

[0065] OPD solution (0.5 mL, 5.0 × 10 -3 mol / L) and water (1 mL), and then CuCl2 solution (0.25 mL, 1.0×10 -3 mol / L), and finally make up to 2.5 mL with the corresponding volume of water, shake well, and let it stand at room temperature for 10 min to obtain the reference solution;

[0066] For G1, G2, G3, G4, G5, G6, and G7 centrifuge tubes, add OPD solution (0.5 mL, 5.0 × 10 -3mol / L) and D-penicillamine solution (1 mL), and then add CuCl2 solution (0.25 mL, 1.0×10 -3 mol / L), and finally made up to 2.5 mL with the corresponding volume of water, shaken and allowed to stand at room temperature for 10 min to obtain 7 standard solutions, wherein the concentration of the D-penicillamine solution added to the G1 centrifuge tube is 5 μmol / L, the concentration of the D-penicillamine solution added to the G2 centrifuge tube is 8 μmol / L, the concentration of the D-penicillamine solution added to the G3 centrifuge tube is 10 μmol / L, the concentration of the D-penicillamine solution added to the G4 centrifuge tube is 20 μmol / L, the concentration of the D-penicillamine solution added to the G5 centrifuge tube is 50 μmol / L, the concentration of the D-penicillamine solution added to the G6 centrifuge tube is 80 μmol / L, and the concentration of the D-penicillamine solution added to the G7 centrifuge tube is 100 μmol / L;

[0067] II. Absorbance and fluorescence intensity determination and construction of standard curve

[0068] Transfer the reference solution and standard solution into quartz cuvettes respectively, and record the absorbance at 420 nm and the fluorescence intensity at 556 nm under 420 nm excitation. Figure 5 A and 5C are shown to establish a standard curve. Specifically:

[0069] The UV absorption intensity standard curve is constructed as follows: the concentration of the added D-penicillamine solution is used as the horizontal axis and the relative absorbance intensity (ΔA / A0) is used as the vertical axis to draw a UV absorption intensity standard curve, wherein A0 is the absorbance of the reference solution, ΔA=A-A0, and A is the absorbance of the standard solution; Figure 5 As shown in B, the standard curve equation of UV absorption intensity is

[0070] ΔA / A0=0.0063X+0.0444,R 2 =0.9974, the unit of X is μmol / L, that is, the concentration of D-penicillamine, the detection limit is 0.76 μmol / L (3 times the relative standard deviation / sensitivity (slope)), and the linear range is 5-100 μmol / L;

[0071] The fluorescence intensity standard curve is constructed as follows: the concentration of the added D-penicillamine solution is used as the horizontal axis and the relative fluorescence intensity (ΔF / F0) is used as the vertical axis to draw a fluorescence intensity standard curve, wherein F0 is the fluorescence intensity of the reference solution, ΔF=F-F0, and F is the fluorescence intensity of the standard solution; Figure 5 As shown in D, the standard curve equation of fluorescence intensity is ΔF / F0=0.0081C+0.0944, R 2=0.9901, the unit of C is μmol / L, that is, the concentration of D-penicillamine, the detection limit is 0.54 μmol / L (3 times the relative standard deviation / sensitivity (slope)), and the linear range is 5-100 μmol / L;

[0072] S3. Determination of penicillamine stock solution content

[0073] OPD solution (0.5 mL, 5.0 × 10 -3 mol / L) and penicillamine stock solution (1 mL), and then add CuCl2 solution (0.25 mL, 1.0×10 -3 mol / L), and finally make up to 2.5mL with the corresponding volume of water, shake well, and let it stand at room temperature for 10min to obtain the test solution;

[0074] Transfer the test solution to a quartz cuvette, record the absorbance A1 at 420 nm and the fluorescence intensity F1 at 556 nm under 420 nm excitation, and substitute the standard curve equations of ultraviolet absorption intensity and fluorescence intensity into the standard curve equations to obtain:

[0075] |A1-A0| / A0=0.0063X1+0.044, where X1 is the concentration of D-penicillamine in the test solution, in μmol / L. Therefore, the concentration of D-penicillamine in the penicillamine stock solution is 5*X1. Furthermore, the content of D-penicillamine in the penicillamine tablet is 12.22*V*X1 μmol / g.

[0076] |F1-F0| / F0=0.0081C1+0.0944, where C1 is the concentration of D-penicillamine in the test solution, in μmol / L. Therefore, the concentration of D-penicillamine in the penicillamine stock solution is 5*C1. Furthermore, the content of D-penicillamine in the penicillamine tablet is 12.22*V*C1μmol / g.

[0077] The two test results of 12.22*V*X1μmol / g and 12.22*V*C1μmol / g verify each other and are jointly used as the results of the D-penicillamine content in penicillamine tablets.

[0078] test

[0079] The precision, recovery and sensitivity of the method of this application were investigated using methodology, and the specificity of the method was investigated using other excipients of penicillamine tablets to ensure the reliability of the method.

[0080] Experiment 1: Precision experiment

[0081] According to the ratio of E in step 2.1, 6 solutions were prepared and numbered 1, 2, 3, 4, 5, and 6 in sequence. The absorbance and fluorescence intensity of the 6 solutions were measured in parallel. Precision experiments were performed and the RSD of the absorbance was 0.002% and the RSD of the fluorescence intensity was 1.17%, indicating that the method has good precision, as shown in Table 1 below:

[0082] Table 1 Precision determination results

[0083]

[0084]

[0085] Note: RSD is relative standard deviation, which is usually used to indicate the precision of analytical test results. Relative standard deviation (RSD) = standard deviation (SD) / arithmetic mean of calculated results * 100%;

[0086] Test 2: Spiked recovery rate determination: Spiked recovery rate is to add a certain amount of standard substance to the sample subsample at the same time as the sample is measured, and the sample measurement value is deducted from the measurement result to obtain the recovery rate of the added standard substance. The calculation formula is spiked recovery rate P = (spike test measurement value - test measurement value) / spiked amount * 100%;

[0087] Ten penicillamine tablets were randomly selected, ground, and weighed. 0.4091 g was accurately weighed and dissolved in ultrapure water. The solution was filtered to remove insoluble matter and finally fixed to volume in a 100 mL volumetric flask to prepare a penicillamine sample solution.

[0088] OPD solution (0.5 mL, 5.0 × 10 -3 mol / L) and a certain volume of penicillamine tablet sample solution, and then add CuCl2 solution (0.25mL, 1.0×10 -3 mol / L), and finally made up to 2.5 mL with ultrapure water and shaken well. After standing at room temperature for 10 min, the concentration of the penicillamine sample solution was tested;

[0089] Take a certain volume of penicillamine tablet sample solution, add ultrapure water and a series of penicillamine standard solutions of different concentrations to it to obtain pre-spiked solution;

[0090] OPD solution (0.5 mL, 5.0 × 10 -3 mol / L) and pre-spiked solution, and then CuCl2 solution (0.25 mL, 1.0 × 10 -3mol / L), and finally make up to 2.5 mL with ultrapure water and shake well. After standing at room temperature for 10 min, the spiked solution was obtained (each pre-spiked solution corresponds to a spiked solution), and the concentration of the spiked solution was tested;

[0091] The results are shown in Tables 2 and 3 below. The recovery rates measured by the colorimetric method were between 96.2% and 105.0%, and the recovery rates measured by the fluorescence method were between 95.4% and 103.2%, indicating that the method is accurate in determining the content of D-penicillamine in penicillamine tablets.

[0092] Table 2 Results of colorimetric determination of D-penicillamine content in tablets

[0093]

[0094] Table 3 Results of fluorescence determination of D-penicillamine content in tablets

[0095]

[0096] Test 3: Excipient Interference Determination

[0097] In order to explore the specificity of this method, this experiment selected some common excipients in tablet drugs, such as starch, dextrin, lactose, talc, sodium carboxymethyl cellulose, polyethylene glycol, anhydrous ethanol, Tween 80, and penicillin potassium as coexisting substances for analysis to investigate whether their presence would interfere with the detection of D-penicillamine. Specifically:

[0098] ① The concentration obtained in step 1.3 is 5×10 -5 mol / L D-penicillamine solution;

[0099] ② Accurately weigh the above excipients, mix with D-penicillamine standard, dissolve in water, and dilute to prepare the concentration of excipients and D-penicillamine standard to be 5×10 -5 μmol / L solution, a single excipient coexistence solution is obtained. Specifically, the single excipient coexistence solution includes:

[0100] When the excipient is starch, it is defined as a starch coexistence solution;

[0101] When the excipient is dextrin, it is defined as a dextrin coexistence solution;

[0102] When the excipient is lactose, it is defined as a lactose coexistence solution;

[0103] When the excipient is talc, it is defined as a talc co-existing solution;

[0104] When the excipient is sodium carboxymethyl cellulose, it is defined as a sodium carboxymethyl cellulose coexistence solution;

[0105] When the excipient is polyethylene glycol, it is defined as a polyethylene glycol coexistence solution;

[0106] When the excipient is anhydrous ethanol, it is defined as an anhydrous ethanol coexistence solution;

[0107] When the excipient is Tween 80, it is defined as Tween 80 coexistence solution;

[0108] When the excipient is penicillin potassium, it is defined as penicillin potassium coexistence solution;

[0109] ③ All excipients were mixed at a concentration of 5×10 -5 mol / L to obtain a mixed solution;

[0110] ④, D-penicillamine standard and all excipients were added at a concentration of 5×10 -5 mol / L to obtain a D-penicillamine mixed solution;

[0111] ⑤. Prepare 12 5mL centrifuge tubes with the corresponding solutions of ①, ②, ③, and ④ as the initial solutions. Add OPD solution (0.5mL, 5.0×10 -3 mol / L) and the corresponding initial solution (1 mL), and then add CuCl2 solution (0.25 mL, 1.0×10 -5 mol / L), and finally make up to 2.5 mL with the corresponding volume of water, shake well, and let it stand at room temperature for 10 min to obtain the final solution;

[0112] ⑥ Transfer the final solution to a quartz cuvette, record the absorbance at 420 nm and the fluorescence intensity at 556 nm under 420 nm excitation, and calculate the relative absorbance of each final solution (ΔA / A D ) and relative fluorescence intensity (ΔF / F D ), where ΔA=A Z -A D , A Z is the absorbance of the final solution, A D The absorbance of the final solution prepared from the initial solution corresponding to solution ①, ΔF = F Z -F D , F Z is the fluorescence intensity of the final solution, F D is the fluorescence intensity of the final solution prepared by using the initial solution as the corresponding solution in step ①;

[0113] The results are as follows Figure 6 As shown, from Figure 6 Medium ΔA / A D and ΔF / F DThe calculation results show that, except for slight interference from penicillin potassium and talc, other substances have little interference on the detection of D-PA, proving that this method has good specificity for the detection of D-penicillamine, among which:

[0114] The test results of the final solution corresponding to the D-penicillamine solution in ① are Figure 6 It is shown as D-PA;

[0115] The test results of the final solution corresponding to the starch coexistence solution in ② are Figure 6 It is shown as Starch;

[0116] The test results of the final solution corresponding to the dextrin coexistence solution in ② are Figure 6 It is shown as Dextrin in

[0117] The test results of the final solution corresponding to the lactose coexistence solution in ② are Figure 6 Lactin is shown in

[0118] The test results of the final solution corresponding to the talc coexistence solution in ② are Figure 6 It is displayed as Tale;

[0119] The test results of the final solution corresponding to the coexistence solution of sodium carboxymethyl cellulose in ② are Figure 6 It is shown as CMC-Na;

[0120] The test results of the final solution corresponding to the polyethylene glycol coexistence solution in ② are Figure 6 It is shown as PEG-400;

[0121] The test results of the final solution corresponding to the anhydrous ethanol coexistence solution in ② are Figure 6 It is shown as Absolutealcohol;

[0122] The test results of the final solution corresponding to the Tween 80 coexistence solution in ② are Figure 6 It is shown as Tween 80;

[0123] The test results of the final solution corresponding to the coexisting solution of potassium penicillin in ② are Figure 6 It is shown as Benzylpenicillin potassium;

[0124] The test results of the final solution corresponding to the mixed solution in ③ are Figure 6 It is shown as mixed solution;

[0125] The test results of the final solution corresponding to the D-penicillamine mixed solution in ④ are Figure 6 It is displayed as D-PA+mixed solution;

[0126] Figure 6 In A, D-PA and D-PA+mixed solution are the absorbances of the corresponding final solutions. Figure 6 In B, D-PA and D-PA+mixed solution are the fluorescence intensities of the corresponding final solutions.

[0127] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. Based on Cu 2+ -OPD colorimetric and fluorescence dual-mode detection method for D-penicillamine content in penicillamine tablets, characterized in that, The following steps are involved: Prepare OPD solution, CuCl2 solution, and multiple D-penicillamine solutions of different concentrations; Take a portion of OPD solution, add a predetermined volume of water to it, then add CuCl2 solution, make up to volume and mix well to obtain a reference solution. The absorbance of the reference solution is measured as A0 and the fluorescence intensity is measured as F0. Take multiple OPD solutions, add different concentrations of predetermined volume of D-penicillamine solution to each OPD solution, then add CuCl2 solution, mix after dilution, and obtain multiple standard solutions. Determine the absorbance of multiple standard solutions as A and the fluorescence intensity as F. The molar ratio of OPD to CuCl2 in the reference solution and the standard solution is 10:

1. The CuCl2 content in multiple standard solutions is 10:

1. 2+ The molar ratio with D-penicillamine ranges from 1:0.02 to 0.4; A UV absorption intensity standard curve was drawn with the added D-penicillamine solution concentration as the abscissa and the relative absorbance intensity ΔA / A0 as the ordinate, and the UV absorption intensity standard curve equation was obtained, where ΔA=A-A0; A fluorescence intensity standard curve was drawn with the concentration of the added D-penicillamine solution as the abscissa and the relative fluorescence intensity ΔF / F0 as the ordinate, and the equation of the fluorescence intensity standard curve was obtained, where ΔF=F-F0; Determine the absorbance and fluorescence intensity of the test solution, and substitute them into the UV absorption intensity standard curve equation and the fluorescence intensity standard curve equation to obtain the D-penicillamine concentration in the test solution.

2. The Cu-based 2+ -OPD colorimetric and fluorescence dual-mode detection method for D-penicillamine content in penicillamine tablets, characterized in that, The OPD solution was prepared by using ethanol as solvent and preparing 5.0×10 -3 mol / LOPD solution.

3. Cu-based as claimed in claim 2 2+ -OPD colorimetric and fluorescence dual-mode detection method for D-penicillamine content in penicillamine tablets, characterized in that, The specific preparation of CuCl2 solution is as follows: take copper chloride dihydrate, use water as solvent, and prepare 1.0×10 -3 mol / L CuCl2 solution.

4. Cu-based as claimed in claim 3 2+ -OPD colorimetric and fluorescence dual-mode detection method for D-penicillamine content in penicillamine tablets, characterized in that, The preparation of multiple D-penicillamine solutions of different concentrations is specifically as follows: weigh a D-penicillamine standard, dissolve it in water, and dilute it to prepare 5 μmol / L, 8 μmol / L, 10 μmol / L, 20 μmol / L, 50 μmol / L, 80 μmol / L, and 100 μmol / L D-penicillamine solutions.

5. The Cu-based 2+ -OPD colorimetric and fluorescence dual-mode detection method for D-penicillamine content in penicillamine tablets, characterized in that, The reference solution and standard solution were both diluted to 5 mL, one portion of OPD solution was 0.5 mL, and the predetermined volume was 1 mL.

6. The Cu-based 2+ -OPD colorimetric and fluorescence dual-mode detection method for D-penicillamine content in penicillamine tablets, characterized in that, After mixing the reference solution and standard solution, let it stand at 15-30℃ for 5-10 minutes.

7. The Cu-based 2+ -OPD colorimetric and fluorescence dual-mode detection method for D-penicillamine content in penicillamine tablets, characterized in that, The reference solution and standard solution were mixed and allowed to stand at 20°C.

8. The Cu-based 2+ -OPD colorimetric and fluorescence dual-mode detection method for D-penicillamine content in penicillamine tablets, characterized in that, The reference solution and standard solution were mixed and allowed to stand at room temperature.

9. The Cu-based 2+ -OPD colorimetric and fluorescence dual-mode detection method for D-penicillamine content in penicillamine tablets, characterized in that, The reference solution and standard solution were mixed and allowed to stand for 5 min.

10. The Cu-based 2+ -OPD colorimetric and fluorescence dual-mode detection method for D-penicillamine content in penicillamine tablets, characterized in that, The reference solution and standard solution were mixed and allowed to stand for 10 min.

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