A sodium derivative of phenylaminopropanoic acid, its preparation method and application

Abstract

Disclosed are a sodium phenylamino propionic acid derivative, a preparation method and application thereof. Specifically, disclosed is sodium 3-(4-(2-(9H-carbazol-9-yl)ethoxy)phenyl)-2-((2-(4-(4-(2-sodium carboxylate-2-((2-(4-fluorobenzoyl)phenyl)amino)ethyl)phenoxy)benzoyl)phenyl)amino)propanoate as shown in formula (I), a preparation method thereof and a use thereof for quality control of a raw material or preparation of siglitinide or a derivative thereof. In particular, the compound of formula (I) can be used as a control or standard for impurity / related substance inspection in siglitinide or a sodium salt drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical pharmacy, and particularly relates to a phenylaminopropionic acid sodium derivative, and further relates to a preparation method of the phenylaminopropionic acid sodium derivative and a use of the phenylaminopropionic acid sodium derivative as a raw material drug or quality control of a preparation in a drug of siglitinide or a derivative thereof; in particular, the phenylaminopropionic acid sodium derivative can be used as a control or standard for impurity or related substance inspection in a raw material drug or preparation of siglitinide or a salt (such as a sodium salt) thereof. BACKGROUND

[0002] 2-(2-(4-fluorobenzoyl)phenylamino)-3-(4-(2-(9H-carbazol-9-yl)ethoxy)phenyl)propanoic acid, commonly known as siglitinide, is a phenylalanine compound having therapeutic and prophylactic activities on metabolic diseases, and has the chemical structural formula as follows:

[0003]

[0004] The pharmacological activities of the compound are described in Chinese Patent Application No. CN03126974.5 and US Patent Application No. US7,268,157. Siglitinide has the ability to selectively activate PPAR-alpha, PPAR-gamma and PPAR-delta, and can be used for treating diseases related to metabolic syndrome, such as diabetes, hypertension, obesity, insulin resistance, hypertriglyceridemia, hyperglycemia, high cholesterol, atherosclerosis, coronary heart disease, etc.

[0005] In the prior art, Chinese Patent Application No. 201610855107.3 and Chinese Patent Application No. 201410856282.5 disclose a synthesis method of siglitinide and its sodium salt.

[0006] A method for industrialized preparation of siglitinide is disclosed in Chinese Patent Application No. 201610855107.3, and the synthesis route is as follows:

[0007]

[0008]

[0009] The method is suitable for industrial production, and the obtained target compound has high purity. However, siglitinide has poor stability, and is prone to decomposition during drug manufacturing, storage and transportation, which seriously affects the safety and effectiveness of the drug, and therefore, it is necessary to prepare a sodium salt thereof, i.e., siglitinide sodium, which has better stability. A preparation method of siglitinide sodium is disclosed in Chinese Patent Application No. 201410856282.5, and the method is as follows:

[0010]

[0011] According to the preparation method in the prior art, the purity of sitagliptan sodium prepared is greater than 99%, and the process is stable and controllable, and is suitable for industrial production. However, the present inventors have unexpectedly found, through a large number of studies, that

[0012] HPLC analysis (chromatographic column: C 18 Column, Shim-pack VP-ODS 5 μm 250Lx4.6; mobile phase: methanol-water-tetrahydrofuran-acetic acid 40:30:30:0.5; detection wavelength: 236 nm; flow rate: 1.5 mL / min) shows that there is always an unknown structure impurity at a relative retention value of about 2.4, and the content of the impurity fluctuates within a certain range with the relative proportion of the use amount of raw material 1 and raw material 2. When the ratio of raw material 1 to raw material 2 is 1:1, the content of the impurity is about 0.18% (area normalization method); when the ratio of raw material 1 to raw material 2 is 1:1.5, the content of the impurity is about 0.06% (area normalization method). Since the existence and structure of the impurity have not been disclosed and reported in the prior art, and its pharmacological and toxicological properties are unknown, it poses a risk to safe medication. At the same time, since the structure of the impurity is unknown, and the prior art has not disclosed and reported any impurity information and separation method of sitagliptan sodium, it brings great difficulty to the separation and identification of the impurity.

[0013] On the one hand, in order to ensure the safety of patients, it is necessary to confirm the structure of the unknown impurity; on the other hand, it is necessary to study the preparation method of the impurity compound, obtain its reference substance or reference standard, and use it for quality control of sitagliptan or sitagliptan sodium and the like, especially as a reference substance or reference standard for related substance / impurity inspection. SUMMARY

[0014] Based on the above needs in the prior art, one of the purposes of the present application is to disclose the above unknown structure compound existing in sitagliptan or sitagliptan sodium salt and the like prepared by the process in the prior art.

[0015] The present inventors have found that the impurity is sodium 3-(4-(2-(9H-carbazol-9-yl)ethoxy)phenyl)-2-((2-(4-(4-(2-carboxylate sodium-2-((2-(4-fluorobenzoyl)phenyl)amino)ethyl)phenoxy)benzoyl)phenyl)amino)propanoate, and its structure is shown in formula (I):

[0016]

[0017] Without being limited by any theory, after a large number of detections and studies, the present inventors speculate that the above compound of formula (I) may be generated due to the following side reaction:

[0018]

[0019] In fact, the production of the above-mentioned compound of formula (I) is not limited to the above-mentioned reaction route for preparing sitagliptin sodium, but can also exist in the synthetic product of sitagliptin.

[0020] In another aspect, the object of the present application is to provide a method for preparing the above-mentioned compound of formula (I), an exemplary synthetic route of which is shown as follows:

[0021]

[0022] It should be noted that the reaction solvent and base used in the above-mentioned reaction route are exemplary rather than limiting, and the person skilled in the art can make appropriate changes and adjustments.

[0023] In an exemplary embodiment, compound (a) is subjected to condensation reaction with compound (b) to obtain compound (c). The reaction can be catalyzed by cesium carbonate, preferably in N,N-dimethylformamide as solvent, the reaction temperature can be 80-120°C, and the reaction time can be 20-30 hours. The obtained crude product can be directly used in the next step reaction without further purification.

[0024] Compound (c) is acidified to obtain compound (d). The acidification is preferably performed by hydrochloric acid. The reaction is preferably performed in ethyl acetate and water as solvent, the reaction temperature can be room temperature, and the reaction time can be 4-5 hours. The obtained crude product can be directly used in the next step reaction without further purification.

[0025] Compound (d) is subjected to hydrolysis in the presence of lithium hydroxide to obtain compound (e). The reaction is preferably performed in tetrahydrofuran and water as solvent, the reaction temperature can be room temperature, and the reaction time can be 12-16 hours. The obtained crude product can be directly used in the next step reaction without further purification.

[0026] Compound (e) is acidified to obtain compound (f). The acidification is preferably performed by hydrochloric acid. The reaction is preferably performed in ethyl acetate and water as solvent, the reaction temperature can be room temperature, and the reaction time can be 4-5 hours. In an exemplary embodiment, the obtained crude product is separated by semi-preparative liquid chromatography column (column: YMC-Pack ODS-AQ 5μm 250Lx20; mobile phase: methanol-water-tetrahydrofuran-glacial acetic acid 48:22:30:0.5; detection wavelength: 236nm; flow rate: 8ml / min) to obtain compound (f) with a purity greater than 97%.

[0027] Compound (f) is neutralized with sodium hydroxide to obtain the compound of formula (I). The reaction can be performed in methanol as solvent, the reaction temperature can be room temperature, and the reaction time can be 20-40min.

[0028] In another aspect, the present application also provides the use of the compound of formula (I) for quality control of the raw material or preparation of Sitagliptin or its derivatives. In particular, the present application provides the use of the compound of formula (I) as a control or standard for the examination of impurities or related substances in Sitagliptin or Sitagliptin sodium drugs.

[0029] In addition, the present application also provides a method for detecting the content of impurities or related substances in Sitagliptin or its derivatives drugs, which comprises using the compound of formula (I)

[0030]

[0031] as a control or standard.

[0032] Preferably, the above detection method is preferably an HPLC method.

[0033] In an exemplary embodiment of the present application, the conditions of the HPLC method are as follows:

[0034] Chromatographic column: C 18 column, Shim-pack VP-ODS 5 μm 250Lx4.6;

[0035] Mobile phase: methanol-water-tetrahydrofuran-acetic acid 40:30:30:0.5;

[0036] Detection wavelength: 236 nm; flow rate: 1.5 mL / min).

[0037] Using the compound of formula (I) as a control, an appropriate amount of the compound of formula (I) is added to the Sitagliptin sodium sample solution, and the chromatogram is recorded to confirm that the compound of formula (I) is an impurity with a relative retention value of about 2.4. The chromatograms of the Sitagliptin sodium sample solution and the standard solution of the compound of formula (I) are recorded respectively, and the content of the compound of formula (I) in the Sitagliptin sodium drug is calculated by using the external standard method.

[0038] The above HPLC detection method of the present application has the advantages of accurate and reliable determination results, strong specificity, strong practicability, and can effectively detect the above-mentioned impurities, and the impurities and Sitagliptin or its salt are well separated.

[0039] Impurity research is an important part of drug research and development, and is throughout the entire drug research and development process, directly affecting the quality and safety of drugs. In order to provide related substance control for the quality research of Sitagliptin or its derivatives, improve the quality standards of Sitagliptin or its derivatives, and compositions containing Sitagliptin or its derivatives (including pharmaceutical preparations), and provide important guidance for safe drug use, the present application studies, synthesizes and identifies the process impurities. DETAILED DESCRIPTION

[0040] The present application is further illustrated by the following examples, but the scope of the present application is not limited to these examples. The percentages described in the present application are percentages by weight unless otherwise specified. The numerical ranges described in the present specification in terms of measurement units or percentages are intended to serve as a deliberate limitation. The skilled person will be able to obtain the intended results using temperatures, concentrations, amounts, etc. outside the ranges described herein or different from the individual numerical values, based on the teachings and principles of the present application.

[0041] Terms and definitions:

[0042] "Derivative": In the present application, the derivatives of sitagliptin include not only sitagliptin free acid, but also salts thereof, such as inorganic salts, for example, sodium salt, and hydrates thereof.

[0043] "Impurity" and "relative retention value": Any substance that affects the purity of a drug is called an impurity. Generally, impurities refer to other chemical substances than the active pharmaceutical substance introduced or generated during production and storage. It is well known to the skilled person that minor products, by-products and additional reagents (collectively referred to as "impurities") can be identified using spectroscopic methods and by other physical methods, whereby the impurities are associated with a peak position in chromatography (or a spot on a thin layer chromatography plate) (Strobel, H. A.; Heineman, W. R., Chemical Instrumentation: A systematic Approach, 3rd. (Wiley & Sons: New York 1989)). Thereafter, the impurities can be identified by their position in the chromatography, which is usually expressed in minutes between the injection of the sample into the column and the elution of the specific component through the detector, and is called "retention time". This time period varies from day to day based on the conditions of the instrument used and many other factors. To mitigate the effect of such variations on the accurate identification of impurities, the skilled person uses "relative retention time" (or relative retention value) to identify impurities. The relative retention value of an impurity is the ratio of its retention time to the retention time of a reference marker, such as a control or a reference standard.

[0044] "Control" and "standard": As known to those skilled in the art of pharmaceutical development, a compound of reasonably high purity can be used as a "standard" or "control". A control is generally a standard substance used for the identification, testing, assay, and calibration of the performance of an assay instrument, while a standard is generally a standard substance used for the assay or potency determination of an antibiotic or biologic drug product. In the present invention, the two terms are not strictly distinguished. A standard can be used for qualitative analysis as well as for quantitative determination of the amount of a compound of interest in an unknown mixture. When a solution of a standard of known concentration is analyzed using the same technique as the unknown mixture, the standard is an "external standard". The amount of the compound in the mixture can be determined by comparing the size of the detector response. See also U.S. Patent 6,333,198, the contents of which are incorporated herein by reference. If the "response factor" that compensates for the difference in sensitivity of the detector for the two compounds has been previously determined, the standard can also be used to measure the amount of another compound in a mixture. For this purpose, the standard is added directly to the mixture, and is referred to as an "internal standard". A standard can also be used as an internal standard when the unknown mixture is caused to contain a detectable amount of a reference standard by a technique known as "standard addition", without the deliberate addition of the standard.

[0045] Starting materials and experimental instruments:

[0046] SGLT-1 : prepared according to the method of Chinese Patent Applications 201410856282.5 and 201610855107.3, purity >99%.

[0047] 2-[(2-(4-fluorobenzoyl)phenyl)amino]-3-(4-hydroxyphenyl)propionic acid methyl ester (Compound (b)): produced by Beijing Lwtech Pharmaceutical Technology Co., Ltd., purity >96%.

[0048] High performance liquid chromatography: instrument: UltiMate 3000; column: C 18 column, Shim-pack VP-ODS 5 μm 250 L x 4.6; detector: VWD-3100.

[0049] Semi-preparative liquid chromatography: instrument: UltiMate 3000; column: YMC-Pack ODS-AQ 5 μm 250 L x 20; detector: VWD-3100.

[0050] Proton nuclear magnetic resonance: instrument: Varian INOVA 500; solvent: DMSO-d6.

[0051] High resolution mass spectrometry: instrument: VG ZAB-HS mass spectrometer; detection method: fast atom bombardment ionization (FAB).

[0052] Example 1: Isolation, preparation and identification of the compound of formula (I)

[0053]

[0054] 1. Isolation

[0055] The 0.5 g of sitagliptin sodium (prepared according to the method of Chinese patent applications 201410856282.5 and 201610855107.3) was separated by semi-preparative liquid chromatography column (column: YMC-Pack ODS-AQ 5 μm 250L x 20; mobile phase: methanol-water-tetrahydrofuran-glacial acetic acid 48:22:30:0.5; detection wavelength: 236 nm; flow rate: 8 ml / min), and the effluent liquid collected from 30 to 42 min was neutralized to pH 7 with 1 mol / L aqueous sodium bicarbonate solution. The above separation operation was repeated 40 times, and the neutralized liquid from each time was combined, concentrated under vacuum to remove the organic solvent, neutralized to pH 5-6 with 1 mol / L dilute hydrochloric acid, filtered, washed with water, and the solid was collected and dried under vacuum at room temperature for 24 h to obtain 5 mg of compound (f) with a purity (HPLC) of 97.8%, LC-MS (m / z) 933 (M+1).

[0056] In a reaction bottle, 5 mg (0.0054 mmol) of compound (f) and 1 mL of methanol were added in sequence and stirred to dissolve. 0.43 mg (0.011 mmol) of sodium hydroxide was dissolved in 0.5 mL of methanol and added dropwise to the above solution, which was stirred at room temperature for 30 min. The reaction liquid was added dropwise to 15 mL of anhydrous ether, filtered, and dried under vacuum at 60°C for 8 h to obtain 5 mg of the compound of formula (I) with a purity (HPLC) of 98.4%.

[0057] Structure identification:

[0058] HRMS (M + +1) (C 58 H 45 N3O8FN2) calculated (%) : 976.2986; found (%) : 976.2992.

[0059] 1H NMR (DMSO-d6) δ 2.87 (dd, IH, one of CH2), 3.02 (dd, IH, one of CH2), 3.05 (dd, IH, one of CH2), 3.22 (dd, IH, one of CH2), 3.91 (m, IH, CH), 4.06 (m, IH, CH), 4.24 (t, 2H, CH2), 4.71 (t, 2H, CH2), 6.40 (m, 2H, Ar-H), 6.58 (d, 2H, Ar-H), 6.65 (d, IH, Ar-H), 6.69 (d, IH, Ar-H), 6.90 (d, 4H, Ar-H), 7.00 (d, 2H, Ar-H), 7.17 (t, 2H, Ar-H), 7.27 (m, 8H, Ar-H), 7.42 (m, 2H, Ar-H), 7.47 (m, 2H, Ar-H), 7.57 (m, 2H, Ar-H), 7.62 (d, 2H, Ar-H), 8.11 (d, 2H, Ar-H), 8.60 (d, IH, NH), 8.81 (dd, IH, NH).

[0060] In a reaction flask, 400 mL of N,N-dimethylformamide, 23.76 g (40 mmol) of sodium 2-(2-(4-fluorobenzoyl)phenylamino)-3-(4-(2-(9H-carbazol-9-yl)ethoxy)phenyl)propanoate (i.e., compound (a)), 19.65 g (50 mmol) of methyl 2-[(2-(4-fluorobenzoyl)phenyl)amino]-3-(4-hydroxyphenyl)propanoate (i.e., compound (b)), and 16.25 g (50 mmol) of cesium carbonate were added sequentially and reacted at 120 °C for 25 h. The filtrate was added to 4000 mL of saturated sodium chloride solution, filtered, washed with water, and the solid was collected and dried under vacuum to obtain crude compound (c) with a purity (HPLC) of 12.2% and LC-MS (m / z) 969 (M+1). The product was used directly in the next step without further purification.

[0061] In a reaction flask, 400 mL of N,N-dimethylformamide, 23.76 g (40 mmol) of sodium 2-(2-(4-fluorobenzoyl)phenylamino)-3-(4-(2-(9H-carbazol-9-yl)ethoxy)phenyl)propanoate (i.e., compound (a)), 19.65 g (50 mmol) of methyl 2-[(2-(4-fluorobenzoyl)phenyl)amino]-3-(4-hydroxyphenyl)propanoate (i.e., compound (b)), and 16.25 g (50 mmol) of cesium carbonate were added sequentially and reacted at 120 °C for 25 h. The filtrate was added to 4000 mL of saturated sodium chloride solution, filtered, washed with water, and the solid was collected and dried under vacuum to obtain crude compound (c) with a purity (HPLC) of 12.2% and LC-MS (m / z) 969 (M+1). The product was used directly in the next step without further purification.

[0062] The above compound (d) was dissolved in 350 mL of tetrahydrofuran, and 48 mL of 12 mol / L aqueous lithium hydroxide solution was added. The reaction was stirred at room temperature for 14 h, the organic phase was separated, and the compound (e) was obtained as a crude product by vacuum concentration. The product was used directly in the next step without further purification.

[0063] In a reaction flask, 480 mL of ethyl acetate and the above compound (e) were added successively, stirred for 30 min, 230 mL of water was added, 150 mL of 3 mol / L dilute hydrochloric acid was added dropwise, stirred for 4 h, the organic phase was separated, concentrated under vacuum to obtain the crude product of compound (f) with a purity (HPLC) of 18.9%, LC-MS (m / z) 933 (M+1).

[0064] The crude product of 0.5 g of compound (d) was separated by semi-preparative liquid chromatography column (column: YMC-Pack ODS-AQ 5 μm 250Lx20; mobile phase: methanol-water-tetrahydrofuran-glacial acetic acid 48:22:30:0.5; detection wavelength: 236 nm; flow rate: 8 ml / min), and the effluent liquid collected from 30 to 42 min was neutralized to pH 7 with 1 mol / L aqueous sodium bicarbonate solution. The above separation operation was repeated 5 times, and the neutralized liquid after each time was combined, concentrated under vacuum to remove the organic solvent, neutralized to pH 5-6 with 1 mol / L dilute hydrochloric acid, filtered, washed with water, and the solid was collected and dried under vacuum at room temperature for 24 h to obtain 230 mg of compound (f) with a purity (HPLC) of 98.0%, LC-MS (m / z) 933 (M+1).

[0065] In a reaction flask, 230 mg (0.247 mmol) of compound (f) and 5 mL of methanol were added successively and stirred to dissolve. 19.76 mg (0.494 mmol) of sodium hydroxide was dissolved in 1 mL of methanol and added dropwise to the above solution, which was stirred at room temperature for 30 min. The reaction liquid was added dropwise to 45 mL of anhydrous ether, filtered, and dried under vacuum at 60°C for 8 h to obtain 236 mg of compound of formula (I) with a purity (HPLC) of 98.6%.

[0066] Structural identification showed that the prepared compound was consistent with the HRMS and 1 HNMR of the compound separated above.

[0067] Example 2: Compound of formula (I) used as a control for determination of impurity content in sitagliptin sodium drug

[0068] I. Test conditions

[0069] Instrument: UltiMate 3000; column: Shim-pack VP-ODS 5 μm 250Lx4.6; detector: VWD-3100; mobile phase: methanol-water-tetrahydrofuran-acetic acid 40:30:30:0.5; detection wavelength: 236 nm; flow rate: 1.5 mL / min. 18 Instrument: UltiMate 3000; column: Shim-pack VP-ODS 5 μm 250Lx4.6; detector: VWD-3100; mobile phase: methanol-water-tetrahydrofuran-acetic acid 40:30:30:0.5; detection wavelength: 236 nm; flow rate: 1.5 mL / min.

[0070] II. Test method

[0071] (1) Take about 10 mg of seglitide sodium sample, accurately weigh, put into a 100 ml volumetric flask, dissolve and dilute to the mark with solvent methanol-water-tetrahydrofuran (40:30:30), shake well, as test solution A, accurately take 20 μl, inject into liquid chromatograph, record chromatogram.

[0072] (2) Take about 10 mg of compound of formula (I), accurately weigh, put into a 100 ml volumetric flask, dissolve and dilute to the mark with solvent methanol-water-tetrahydrofuran (40:30:30), shake well, accurately take 1 mL, put into a 100 ml volumetric flask, dissolve and dilute to the mark with solvent methanol-water-tetrahydrofuran (40:30:30), shake well, as test solution B, accurately take 20 μl, inject into liquid chromatograph, record chromatogram.

[0073] (3) Respectively take 0.5 mL of test solution A and 0.5 mL of test solution B, shake well, as test solution C, accurately take 20 μl, inject into liquid chromatograph, record chromatogram.

[0074] III. Test results

[0075] In the chromatogram of test solution A, the peak time of seglitide sodium is 15.1 min, at the same time, there is an impurity peak at 35.8 min, the relative area is 0.05%.

[0076] In the chromatogram of test solution B, the peak time of compound of formula (I) is 35.8 min.

[0077] In the chromatogram of test solution C, the peak time of seglitide sodium is 15.1 min, at the same time, there is an impurity peak at 35.8 min, the relative area is 0.7%.

[0078] Conclusion: Compound of formula (I) is confirmed to be an impurity with relative retention value of about 2.4 in seglitide sodium sample.

[0079] Example 3: Compound of formula (I) is used as standard for determination of impurity content in seglitide sodium drug

[0080] I. Test conditions

[0081] Instrument: UltiMate3000; chromatographic column: C 18 column, Shim-pack VP-ODS 5 μm 250Lx4.6; detector: VWD-3100, mobile phase: methanol-water-tetrahydrofuran-acetic acid 40:30:30:0.5; detection wavelength: 236 nm; flow rate: 1.5 mL / min.

[0082] II. Test method

[0083] About 10 mg of sitagliptin sodium sample was precisely weighed, dissolved and diluted to the mark in a 100 ml volumetric flask with solvent methanol-water-tetrahydrofuran (40:30:30), shaken well to serve as the test solution. About 10 mg of standard sample of compound of formula (I) was precisely weighed, dissolved and diluted to the mark in a 100 ml volumetric flask with solvent methanol-water-tetrahydrofuran (40:30:30), shaken well, 1 mL of which was precisely transferred into a 1000 ml volumetric flask, dissolved and diluted to the mark with solvent methanol-water-tetrahydrofuran (40:30:30), shaken well to serve as the reference solution. 20 μl of each of the above two solutions was precisely pipetted into the liquid chromatograph to record the chromatogram. The content of compound of formula (I) in the sitagliptin sodium sample was calculated by the peak area according to the external standard method.

[0084] III. Test Results

[0085] Three batches of sitagliptin sodium sample were determined, and the determination results are shown in Table 1.

[0086] Table 1 Content of compound of formula (I) in sitagliptin sodium sample

[0087] Situ samples Compound of formula (I) content (%) Batch 20160817 0.07 Batch 20160909 0.08 Batch 20160923 0.08

Claims

1. A compound of formula (I): ###0001### 2. Use of a compound of formula (I) according to claim 1 for the quality control of the bulk drug or the formulation of Sitagliptin or Sitagliptin Sodium.

3. Use according to claim 2, wherein the compound of formula (I) is used as a reference or standard for the impurity or related substances test in a Sitagliptin or Sitagliptin Sodium drug.

4. A method for detecting the content of impurities or related substances in a Sitagliptin or Sitagliptin Sodium drug, said method comprising the use of a compound of formula (I) ###0002### as a reference or standard.

6. A method for controlling the quality in the synthesis process of Sitagliptin or Sitagliptin Sodium, comprising the use of a compound of formula (I) ###0003### for the detection or control of the content of impurities or related substances.

5. The assay method of claim 4, which is HPLC method, wherein the compound of formula (I) is used as an external standard, and the assay conditions are: column: C 18 18 column; mobile phase: methanol-water-tetrahydrofuran-acetic acid 40:30:30:0.5; detection wavelength: 236 nm. ​ ​

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