Method for detecting polymer impurities in desmopressin injection

Abstract

The invention discloses a method for detecting polymer impurities in a desmopressin injection. Aiming at the problems that polymer impurities in the desmopressin injection cannot be detected in the prior art and method, and the quality control of a desmopressin injection product is difficult, the invention establishes a method for detecting a polymer in the desmopressin injection. Verification experiments of specificity, system applicability, destruction test, quantitation limit and detection limit, linearity and range, accuracy, precision (repeatability and intermediate precision), solution stability and durability show that the method can separate the desmopressin polymer impurities in a high performance liquid chromatogram, and can achieve the purpose of accurate quantitative analysis; and strict quality control on the desmopressin injection is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical testing technology and relates to an analytical method for active polypeptide injections, particularly a method for detecting polymer impurities in desmopressin injections. Background Technology

[0002] Desmopressin injection is a synthetic antidiuretic hormone analogue primarily used to treat central diabetes insipidus and control nocturnal enuresis. It can also be used as adjunctive therapy for certain hemorrhagic diseases. Its mechanism of action involves selectively binding to V2 receptors in the kidneys, reducing urine output, and regulating coagulation function.

[0003] During the production and preparation of desmopressin injection, dimer, trimer, and even polymeric impurities may be generated. Common impurities are listed in the table below:

[0004] Polymer impurities in drugs can affect the quality of desmopressin injection. To strictly control the quality of desmopressin injection and ensure its safe and effective clinical use, an effective method for detecting polymers in desmopressin injection needs to be established. Currently, due to the small specifications of desmopressin injection [specifications: 1ml 13.4μg and 1ml 3.56μg], existing polymer detection methods for desmopressin injection cannot effectively separate and detect polymers, thus hindering strict quality control. Therefore, a suitable detection method for polymers in desmopressin injection is urgently needed. Summary of the Invention

[0005] The purpose of this invention is to provide a high-performance liquid chromatography (HPLC) method for detecting polymeric impurities in the aforementioned desmopressin injection. Addressing the problem that existing technologies and methods cannot detect polymers in desmopressin injection, thus hindering quality control, the inventors, through extensive experimental research, have established a method for detecting polymeric impurities in desmopressin injection. This method can be used for quality research and quality control of desmopressin injection products.

[0006] This invention employs size exclusion chromatography to detect polymeric impurities in desmopressin injection. Under this method, the elution time of polymeric impurities is related to their molecular weight; larger molecular weight impurities elute first, followed by smaller molecular weight impurities. Specific chromatographic conditions are as follows: Spherical hydrophilic modified silica gel (molecular weight range of polymer 500~15000) was used as the packing material, and the chromatographic column specifications were TSK gel G2000SWXL, 7.8mm×300mm, 5μm; The mobile phase was trifluoroacetic acid-acetonitrile-water (0.05:30:70), and the pH was adjusted to 3.0 with 1 mol / L sodium hydroxide solution. The column temperature for the above polymer detection method is 30℃-40℃, preferably 35℃.

[0007] The flow rate of the above polymer detection method is 0.4-0.6 ml / min, preferably 0.5 ml / min.

[0008] The mobile phase pH value of the above polymer detection method is 2.8-3.2, preferably 3.0.

[0009] The trifluoroacetic acid ratio in the above polymer detection method is 0.04%-0.06%, preferably 0.05%.

[0010] The injection volume for the above polymer detection method is 200 µl.

[0011] The present invention uses a fluorescence detector to measure the excitation wavelength of 276 nm and the emission wavelength of 305 nm.

[0012] This invention determines the polymer impurity content in desmopressin injection using the area normalization method. The method employed achieves baseline separation between polymer impurities and the main peak of desmopressin, enabling accurate determination of the polymer impurity content in desmopressin injection. This method is simple, rapid, accurate, efficient, and highly reproducible, providing a solid foundation for the development of quality standards for desmopressin injection. Attached Figure Description

[0013] Figure 1 The peaks for the [1.1', 6.6'] deaminoplastin dimer are shown in the diagram.

[0014] Figure 2 The peaks for the [1.6', 6.1'] deaminoplastin dimer are shown in the diagram.

[0015] Figure 3 The diagram shows the separation of desmopressin and [1.1',6.6']desmopressin dimer. Detailed Implementation

[0016] The present invention is further illustrated by the following examples, but these are not intended to limit the invention. Example 1

[0017] Preparation of system suitability solution: Take appropriate amounts of desmopressin acetate reference standard and [1.1',6.6']desmopressin dimer reference standard, place them in the same volumetric flask, add mobile phase to dissolve and dilute to prepare a mixed solution containing approximately 7.5 μg of desmopressin acetate (6.7 μg based on desmopressin) and 0.075 μg of [1.1',6.6']desmopressin dimer per 1 ml.

[0018] Impurity localization solution: Accurately weigh appropriate amounts of [1.1',6.6']deammonia-vasopressin dimer and [1.6',6.1']deammonia-vasopressin dimer reference standards, dissolve and dilute them in the mobile phase to prepare a solution containing approximately 0.6 μg of each per ml.

[0019] Determination of polymeric impurities in desmopressin was performed under the following high-performance liquid chromatography (HPLC) conditions. Accurately measure 200 μL of each of the above solutions, inject them into the HPLC system, and record the chromatograms. (See figure below.) Figures 1-3 .

[0020] Chromatographic conditions: Spherical hydrophilic modified silica gel (molecular weight range of polymer 500~15000) was used as the packing material (TSKgel G2000SWXL, 7.8mm×300mm, 5μm or equivalent column); the mobile phase was trifluoroacetic acid-acetonitrile-water (0.05:30:70), with the pH adjusted to 3.0 by 1mol / L sodium hydroxide solution; the flow rate was 0.5ml per minute; fluorescence detection was used, with an excitation wavelength of 276nm and an emission wavelength of 305nm; the column temperature was 35℃; and the injection volume was 200μl.

[0021] Test results

[0022] The method of this invention can achieve a separation degree of greater than 1.0 between the dimer impurities and the main peak of desaminoglycan, thus enabling effective separation. Example 2

[0023] In the following examples, the substance measured was a polymer.

[0024] 1. System Applicability Preparation of injection precision solution: Take appropriate amounts of desmopressin acetate reference standard and [1.1',6.6']desmopressin dimer reference standard, place them in the same volumetric flask, add mobile phase to dissolve and dilute to prepare a mixed solution containing approximately 7.5 μg of desmopressin acetate (6.7 μg based on desmopressin) and 0.075 μg of [1.1',6.6']desmopressin dimer per 1 ml.

[0025] Accurately measure 200 µl of the injection precision solution (6 consecutive determinations), inject it into the liquid chromatograph, and record the chromatogram.

[0026] System suitability test results

[0027] Conclusion: The injection precision was good when the solution was injected in parallel for 6 times. The RSD of the retention time of [1.1', 6.6'] desaminoglycan dimer was 0.11% and the RSD of the peak area was 1.7%. The RSD of the retention time of desaminoglycan was 0.035% and the RSD of the peak area was 0.21%.

[0028] 2. Destructive test (1 ml: 3.56 μg) Undamaged (1ml: 3.56μg): Accurately measure 0.5ml of this product and add 0.5ml of acetonitrile, mix well, and the product is ready.

[0029] Acid destruction (1ml: 3.56μg): Accurately measure 0.5ml of this product, add 0.5ml of acetonitrile and mix well. Add 100μl of 2mol / L hydrochloric acid solution, let stand at room temperature for 2 minutes, then add 100μl of 2mol / L sodium hydroxide solution and shake well to obtain the product.

[0030] Alkali destruction (1 ml: 3.56 μg): Accurately measure 0.5 ml of this product, add 0.5 ml of acetonitrile and mix well. Add 125 μl of 0.1 mol / L sodium hydroxide solution, let stand at room temperature for 13 minutes, then add 125 μl of 0.1 mol / L hydrochloric acid solution and shake well to obtain the product.

[0031] Light damage (1ml: 3.56μg): Take one vial of this product and place it in a high-intensity light stability test chamber (4500LX±500 LX, 84μW / cm²). 2 Irradiate for 10 days; accurately measure 0.5 ml of this product and add 0.5 ml of acetonitrile, mix well, and the product is ready.

[0032] Oxidative degradation (1 ml: 3.56 μg): Accurately measure 0.5 ml of this product, add 0.5 ml of acetonitrile and mix well. Add 100 μl of 15% hydrogen peroxide solution, let stand at room temperature for 40 minutes, and shake well to obtain the product.

[0033] High-temperature destruction (1ml: 3.56μg): Place this product in a 90℃ oven for high-temperature destruction for 10 hours, then allow it to cool to room temperature; accurately measure 0.5ml and add 0.5ml of acetonitrile, mix well, and the product is ready.

[0034] Undamaged (1ml: 13.4μg): Accurately measure 0.5ml of this product and add 0.5ml of acetonitrile, mix well, and the product is ready.

[0035] Acid destruction (1 ml: 13.4 μg): Accurately measure 0.5 ml of this product, add 0.5 ml of acetonitrile, add 100 μl of 2 mol / L hydrochloric acid solution, let stand at room temperature for 20 minutes, then add 100 μl of 2 mol / L sodium hydroxide solution, shake well, and the product is ready.

[0036] Alkali destruction (1 ml: 13.4 μg): Accurately measure 0.5 ml of this product, add 0.5 ml of acetonitrile, add 10 μl of 1 mol / L sodium hydroxide solution, let stand at room temperature for 2 minutes, then add 10 μl of 1 mol / L hydrochloric acid solution, shake well, and the product is ready.

[0037] Light damage (1ml: 13.4μg): Take one vial of this product and place it in a high-intensity light stability test chamber (4500LX±500 LX, 84μW / cm²). 2 Irradiate for 10 days; accurately measure 0.5 ml of this product and add 0.5 ml of acetonitrile, mix well, and the product is ready.

[0038] Oxidative degradation (1 ml: 13.4 μg): Accurately measure 0.5 ml of this product, add 0.5 ml of acetonitrile, add 110 μl of 15% hydrogen peroxide solution, let stand at room temperature for 60 minutes, shake well, and the product is ready.

[0039] High-temperature destruction (1ml: 13.4μg): Place this product in a 90℃ oven for high-temperature destruction for 10 hours, then allow it to cool to room temperature; accurately measure 0.5ml and add 0.5ml of acetonitrile, mix well, and the product is ready.

[0040] Accurately measure 200 µl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram.

[0041] Forced degradation test results (1 ml: 3.56 μg)

[0042] Forced degradation test results (1 ml: 13.4 μg)

[0043] Conclusions: (1) The blank solvent and blank excipient solutions under various degradation conditions do not interfere with the determination of desmopressin and polymers. (2) 1 ml: 3.56 μg: the minimum resolution between desmopressin and adjacent peaks is 1.41; 1 ml: 13.4 μg: the minimum resolution between desmopressin and adjacent peaks is 1.43. (3) 1 ml: 3.56 μg: the mass conservation under various degradation conditions is in the range of 86.2% to 100.5%; 1 ml: 13.4 μg: the mass conservation under various degradation conditions is in the range of 88.0% to 97.8%.

[0044] 3. Limit of Quantitation and Limit of Detection Limit of quantitation: Accurately weigh appropriate amounts of [1.1',6.6']demopressin dimer, [1.6',6.1']demopressin dimer, and demopressin acetate reference standard, and gradually dilute them. The limit of quantitation is the concentration at a signal-to-noise ratio of 10:1 or the amount injected into the instrument.

[0045] Accurately measure 200 µl of the limit of quantitation solution (6 consecutive determinations), inject it into the liquid chromatograph, and record the chromatogram.

[0046] Quantification limit results -1

[0047]

[0048] Quantification limit results -2

[0049] Quantification limit results -3

[0050] Limit of detection: Take the limit of quantitation solution from the limit of quantitation section, dilute it step by step until the concentration corresponding to the signal-to-noise ratio (S / N) is about 3. This concentration is the limit of detection concentration.

[0051] Accurately measure 200 µl of the detection limit solution, inject it into the liquid chromatograph, and record the chromatogram.

[0052] Detection limit results

[0053] Conclusion: This method has high sensitivity.

[0054] 4. Linearity and Range [1.1',6.6']Desmopressin Dimer Reference Stock Solution: Accurately weigh approximately 3.6 mg of [1.1',6.6']Desmopressin Dimer Reference Standard, place it in a 200 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0055] [1.6',6.1']Desmopressin Dimer Reference Stock Solution: Weigh approximately 3.4 mg of [1.6',6.1']Desmopressin Dimer Reference Standard accurately, place it in a 200 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0056] Desmopressin reference standard stock solution: Weigh approximately 3.0 mg of desmopressin acetate reference standard accurately, place it in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0057] [1.1',6.6'] Preparation of linear solution of desammopressin dimer: Prepare according to the table below.

[0058] [1.6',6.1'] Preparation of linear solution of desammopressin dimer: Prepare according to the table below.

[0059] Preparation of linear desmopressin solution: Follow the table below for preparation.

[0060] Accurately measure 200 µl of each of the above solutions and inject them into the liquid chromatograph, recording the chromatograms. For polymeric impurities within the LOQ~200% limit concentration range and desmopressin within the LOQ~200% test solution concentration range, perform linear regression analysis with concentration as the abscissa (C) and peak area as the ordinate (A).

[0061] Linear test results

[0062] Conclusion: The method showed good linearity between desmopressin and [1.1',6.6']desmopressin dimer and [1.6',6.1']desmopressin dimer.

[0063] 5. Accuracy [1.6',6.1']Desmopressin Dimer Reference Stock Solution (1 ml: 3.56 μg): Weigh approximately 3.4 mg of [1.6',6.1']Desmopressin Dimer Reference Standard accurately, place it in a 200 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0064] Preparation of 50% accuracy stock solution (1 ml: 3.56 μg): Accurately measure 1 ml of [1.6', 6.1'] desaminoglycan dimer reference stock solution (1 ml: 3.56 μg), place it in a 150 ml volumetric flask, dilute to the mark with mobile phase, and shake well.

[0065] Preparation of 100% accuracy stock solution (1 ml: 3.56 μg): Accurately measure 2 ml of [1.6', 6.1'] desaminoglycan dimer reference stock solution (1 ml: 3.56 μg), place it in a 150 ml volumetric flask, dilute to the mark with mobile phase, and shake well.

[0066] Preparation of 150% accuracy stock solution (1 ml: 3.56 μg): Accurately measure 3 ml of [1.6', 6.1'] desaminoglycan dimer reference stock solution (1 ml: 3.56 μg), place it in a 150 ml volumetric flask, dilute to the mark with mobile phase, and shake well.

[0067] Preparation of 50% accuracy solution (1 ml: 3.56 μg): Accurately measure 5 ml of this product and place it in a 10 ml volumetric flask. Add 1 ml of 50% accuracy stock solution (1 ml: 3.56 μg), dilute to the mark with acetonitrile, and mix well. (Prepare 3 parallel portions) Preparation of 100% accuracy solution (1 ml: 3.56 μg): Accurately measure 5 ml of this product and place it in a 10 ml volumetric flask. Add 1 ml of 100% accuracy stock solution (1 ml: 3.56 μg), dilute to the mark with acetonitrile, and mix well. (Prepare 3 parallel portions) Preparation of 150% accuracy solution (1 ml: 3.56 μg): Accurately measure 5 ml of this product and place it in a 10 ml volumetric flask. Add 1 ml of 150% accuracy stock solution (1 ml: 3.56 μg), dilute to the mark with acetonitrile, and mix well. (Prepare 3 parallel portions) Preparation of background solution (1 ml: 3.56 μg): Accurately measure 5 ml of this product, place it in a 10 ml volumetric flask, dilute with acetonitrile to the mark, and shake well.

[0068] Reference solution (1 ml: 3.56 μg): Accurately measure 5 ml of 100% accuracy stock solution (1 ml: 3.56 μg), place it in a 50 ml volumetric flask, dilute to the mark with the mobile phase, and shake well.

[0069] [1.6',6.1']Desmopressin Dimer Reference Stock Solution (1 ml: 13.4 μg): Weigh approximately 3.4 mg of [1.6',6.1']Desmopressin Dimer Reference Standard accurately, place it in a 200 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0070] Preparation of 50% accuracy stock solution (1 ml: 13.4 μg): Accurately measure 2.5 ml of [1.6', 6.1'] desaminoglycan dimer reference stock solution (1 ml: 13.4 μg), place it in a 100 ml volumetric flask, dilute to the mark with mobile phase, and shake well.

[0071] Preparation of 100% accuracy stock solution (1 ml: 13.4 μg): Accurately measure 2.5 ml of [1.6', 6.1'] desaminoglycan dimer reference stock solution (1 ml: 13.4 μg), place it in a 50 ml volumetric flask, dilute to the mark with mobile phase, and shake well.

[0072] Preparation of 150% accuracy stock solution (1 ml: 13.4 μg): Accurately measure 1.5 ml of [1.6', 6.1'] desaminoglycan dimer reference stock solution (1 ml: 13.4 μg), place it in a 20 ml volumetric flask, dilute to the mark with mobile phase, and shake well.

[0073] Preparation of 50% accuracy solution (1 ml: 13.4 μg): Accurately measure 5 ml of this product and place it in a 10 ml volumetric flask. Add 1 ml of 50% accuracy stock solution (1 ml: 13.4 μg), dilute to the mark with acetonitrile, and mix well. (Prepare 3 parallel portions) Preparation of 100% accuracy solution (1 ml: 13.4 μg): Accurately measure 5 ml of this product and place it in a 10 ml volumetric flask. Add 1 ml of 100% accuracy stock solution (1 ml: 13.4 μg), dilute to the mark with acetonitrile, and mix well. (Prepare 3 parallel portions) Preparation of 150% accuracy solution (1 ml: 13.4 μg): Accurately measure 5 ml of this product and place it in a 10 ml volumetric flask. Add 1 ml of 150% accuracy stock solution (1 ml: 13.4 μg), dilute to the mark with acetonitrile, and mix well. (Prepare 3 parallel portions) Preparation of background solution (1 ml: 13.4 μg): Accurately measure 5 ml of this product, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0074] Reference solution (1 ml: 13.4 μg): Accurately measure 5 ml of 100% accuracy stock solution (1 ml: 13.4 μg), place it in a 50 ml volumetric flask, dilute to the mark with the mobile phase, and shake well.

[0075] Accurately measure 200 µl each of the accuracy solution, background solution, and reference solution, inject them into the liquid chromatograph, and record the chromatogram.

[0076] Accuracy (1ml: 3.56μg)

[0077] Accuracy (1ml: 13.4μg)

[0078] Conclusion: The method has good accuracy.

[0079] 6. Precision (repeatability and intermediate precision) [1.6',6.1']Desmopressin Dimer Reference Stock Solution (1 ml: 3.56 μg): Weigh approximately 3.4 mg of [1.6',6.1']Desmopressin Dimer Reference Standard accurately, place it in a 200 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0080] Preparation of 100% accuracy stock solution (1 ml: 3.56 μg): Accurately measure 2 ml of [1.6', 6.1'] desaminoglycan dimer reference stock solution (1 ml: 3.56 μg), place it in a 150 ml volumetric flask, dilute to the mark with mobile phase, and shake well.

[0081] Preparation of 100% accuracy solution (1 ml: 3.56 μg): Accurately measure 5 ml of this product and place it in a 10 ml volumetric flask. Add 1 ml of 100% accuracy stock solution (1 ml: 3.56 μg), dilute to the mark with acetonitrile, and mix well. (Prepare 6 parallel portions) Reference solution (1 ml: 3.56 μg): Accurately measure 5 ml of 100% accuracy stock solution (1 ml: 3.56 μg), place it in a 50 ml volumetric flask, dilute to the mark with the mobile phase, and shake well.

[0082] [1.6',6.1']Desmopressin Dimer Reference Stock Solution (1 ml: 13.4 μg): Weigh approximately 3.4 mg of [1.6',6.1']Desmopressin Dimer Reference Standard accurately, place it in a 200 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0083] Preparation of 100% accuracy stock solution (1 ml: 13.4 μg): Accurately measure 2.5 ml of [1.6', 6.1'] desaminoglycan dimer reference stock solution (1 ml: 13.4 μg), place it in a 50 ml volumetric flask, dilute to the mark with mobile phase, and shake well.

[0084] Preparation of 100% accuracy solution (1 ml: 13.4 μg): Accurately measure 5 ml of this product and place it in a 10 ml volumetric flask. Add 1 ml of 100% accuracy stock solution (1 ml: 13.4 μg), dilute to the mark with acetonitrile, and mix well. (Prepare 6 parallel portions) Reference solution (1 ml: 13.4 μg): Accurately measure 5 ml of 100% accuracy stock solution (1 ml: 13.4 μg), place it in a 50 ml volumetric flask, dilute to the mark with the mobile phase, and shake well.

[0085] Repeatability and intermediate precision were measured by different personnel at different times using different instruments.

[0086] Precision (repeatability and intermediate precision) test results (1 ml: 3.56 μg)

[0087] Precision (repeatability and intermediate precision) test results (1 ml: 13.4 μg)

[0088] Conclusion: The average recovery rates of the six parallel test solutions were all between 90.0% and 108.0%, and the RSD of the polymer recovery rate in the 12 test solutions were all below 10.0%, indicating good method precision.

[0089] 7. Solution stability At room temperature, from the time of solution preparation until a certain period of time, accurately measure 200µl each of the 100% accuracy solution preparation (1ml: 3.56μg) and the 100% accuracy solution preparation (1ml: 13.4μg) under the accuracy section, inject them sequentially into the liquid chromatograph, and record the chromatogram.

[0090] Stability results of the test solution (1 ml: 3.56 μg)

[0091] Stability results of the test solution (1 ml: 13.4 μg)

[0092] Conclusion: When the spiked test solution (1 ml: 3.56 μg) was incubated at room temperature for 46 hours, the absolute value of the change in the detected amount at each time point compared to 0 h was 0.03%. When the spiked test solution (1 ml: 13.4 μg) was incubated at room temperature for 37 hours, the absolute value of the change in the detected amount at each time point compared to 0 h was 0.01%.

[0093] 8. Durability Durability and fine-tuning of chromatographic conditions

[0094] Durability Results - Separation

[0095] Robustness results - test solution (1 ml: 3.56 μg)

[0096] Robustness results - test solution (1 ml: 13.4 μg)

[0097] Conclusions: Different flow rates, column temperatures, pH values, trifluoroacetic acid dosages, chromatographic columns, blank solvents, and blank excipients did not interfere with the detection of polymers in the test solution. In the system suitability solution chromatogram, [1.1', 6.6'] desaminoglycin dimer and desaminoglycin eluted sequentially, with a minimum resolution of 1.82. The absolute value of the difference between the detected amount of polymer in the spiked test solution (1 ml: 3.56 μg) and the standard conditions was 0.04%. The absolute value of the difference between the detected amount of polymer in the spiked test solution (1 ml: 13.4 μg) and the standard conditions was 0.07%, indicating good method robustness.

[0098] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for detecting polymeric impurities in desmopressin injection, characterized in that, High-performance liquid chromatography (HPLC) was used for detection. The specific chromatographic conditions were as follows: the chromatographic column was filled with spherical hydrophilic modified silica gel with a molecular weight of 500-15000; a fluorescence detector was used for determination; the mobile phase was 0.05:30:70 trifluoroacetic acid-acetonitrile-water; and isocratic elution was performed by adjusting the pH value to 3.0 with 1 mol / L sodium hydroxide solution.

2. The detection method according to claim 1, characterized in that: The specifications of the chromatographic column are: TSK gelG2000SWXL, 7.8mm×300mm, 5μm.

3. The detection method according to claim 1, characterized in that: The column temperature is 35℃.

4. The detection method according to claim 1, characterized in that: The flow rate was 0.5 ml / min.

5. The detection method according to claim 1, characterized in that, The injection volume was 200 µl.

6. The detection method according to claim 1, characterized in that, The excitation wavelength is 276 nm and the emission wavelength is 305 nm.

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