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Analytical Methods and Quality Standards for Dihydroartemisinin
CHAPTE R 19
Analytical Methods and Quality Standards for Dihydroartemisinin Chapter Outline 1 Physical and Chemical Parameters of Dihydroartemisinin 303 1.1 Description 303 1.2 Identification 303 1.3 Inspection 304
2 Thin-Layer Chromatography (TLC) 304 3 High-Performance Liquid Chromatography (HPLC) 304 4 UV Spectrophotometry 306
1 Physical and Chemical Parameters of Dihydroartemisinin 1.1 Description White needle-like crystal, odorless, with a bitter taste. Easily soluble in chloroform, soluble in dimethyl sulfoxide, ether, and acetone, slightly soluble in methanol and ethanol, and practically insoluble in water. Melting point: Melting and decomposition of crystals occur simultaneously at 145–150°C. Specific rotation: [α] 20D = +140° to +146° (c = 1.0026, CHCl3)
1.2 Identification 1. Dissolve 5 mg in 0.5 mL of dehydrated ethanol, add 2.5 mL of acidic potassium iodide solution (dissolve 0.4 g of potassium iodide in 2.5 mL of sulfuric acid at a concentration of 2.5 mol/L), add 1–2 drops of 2% starch solution. The blank control solution is prepared with anhydrous ethanol following the same procedures. The blue and violet color in test solution is immediately produced, while only light violet color is produced in blank control solution. 2. Add a few drops of chloroform solution and evaporate to dryness, add a drop of 2% vanillin sulfuric acid solution, the red color is produced immediately and become brown after placement. 3. The infrared absorption spectrum is concordant with the reference spectrum. From Artemisia annua L. to Artemisinins http://dx.doi.org/10.1016/B978-0-12-811655-5.00019-2
303
Copyright © 2017 Chemical Industry Press, published by Elsevier Inc. All rights reserved.
304 Chapter 19
1.3 Inspection Loss on drying: Dry over phosphorus pentoxide R under reduced pressure (not exceeding 2.67 kPa); it loses not more than 0.5%. Residue on ignition: <0.1%.
2 Thin-Layer Chromatography (TLC) Dissolve the product in chloroform to produce 10 mg/mL solution (test solution), and dissolve dihydroartemisinin in chloroform to produce 10 mg/mL solution (control solution). Apply 10 µL of aforementioned two solutions in the same silica gel G plate, respectively. Develop with benzene–ethyl acetate (8:2), dry the plate, and spray with 2% vanillin sulfuric acid solution. The spot of dihydroartemisinin in sample is not darker than the reference spots, and no other individual impurity spot is observed. Dihydroartemisinin contains not less than 98.0% and not more than the equivalent of 102.0% of C15H24O5.
3 High-Performance Liquid Chromatography (HPLC) Preparation of internal standard solution: Accurately weigh about 10 mg biphenyl (spectroscopic ally pure), transfer into a 10 mL volumetric flask, dissolve in methanol and dilute to the mark, and shake thoroughly. Preparation of reference solution: Accurately weigh about 10 mg dihydroartemisinin reference, transfer into a 10 mL volumetric flask, dissolve in methanol, add 20 mL of above internal standard solution, dilute to the mark, and shake thoroughly. Preparation of test solution: The same as the preparation of reference solution. Assay: Waters-201 high performance liquid chromatography, variable wavelength UV monitor Mode 1481, wavelength: 210 nm, sensitivity: 0.05AUFS, column: µ-Bondapa KC13 (4.0 mm × 300 mm), mobile phase: methanol–water (8:2), flow rate: 1.0 mL/ min, and injection volume: 10 µL. Transfer about 10 mL of methanol into a 10 mL volumetric flask, add 20 µL of internal standard, and dilute with methanol to the mark (as the blank control). Conduct the assay. Peak time is within 15 min, and the content is calculated based on peak area and response factor (or calibration factor) with the following equation (Fig. 19.1): 1. Correction factor method C% = Fi × AiAis × WisWi × 100%
Analytical Methods and Quality Standards for Dihydroartemisinin 305
Figure 19.1: A Chromatogram.
where Fi (correction factor) = W′iW′is × A′isA′i; A′is and Ais are the peak area of internal standard in reference solution and test solution, respectively; A′i and Ai are the peak area of certain substance in reference solution and test solution, respectively; W′is and Wis are the weight of internal standard in reference solution and test solution, respectively (µg/µL); W′i and Wi are the weight of certain substance in reference solution and test solution, respectively (µg/µL). 2. Response coefficient method (the spike amount in reference solution is the same as that in internal standard) C% = A ′isAis × A ′iWi × rF1000 × 100%
where rF (response factor) = W′iA′i × 1000. Other symbols labels are the same as (1).
Laboratory samples and pilot trial samples are measured with the aforementioned methods, and the results are shown in Table 19.1.
306 Chapter 19 Table 19.1: Laboratory and pilot trial results of dihydroartemisinin content Measured Value (%) Batch No.
I
II
Mean
Laboratory 1 Laboratory 2 Laboratory 3 Laboratory 4 Laboratory 5 Pilot trial 1 Pilot trial 2 Pilot trial 3
99.38 99.51 98.22 98.78 99.32 98.82 99.85 98.29
101.18 99.74 96.87 98.58 100.10 98.11 99.85 99.82
100.53 99.63 98.79 98.68 99.71 98.97 99.85 99.06
4 UV Spectrophotometry Preparation of control solution: Accurately weigh about 10 mg of dihydroartemisinin reference substance, place into a 50 mL volumetric flask, add ethanol solution to dissolve and dilute to the mark, shake, and then stand for 2 h. Preparation of test solution: Accurately weigh 10 tablets, porphyrize, transfer appropriate amount (about equivalent to 10 mg of dihydroartemisinin) into a 50 mL volumetric flask, add ethanol, and shake to dissolve, dilute to the mark, stand for 2 h, and filtrate. Discard the primary filtrate, and use the subsequent filtrate. Measuring procedures: Accurately transfer each of 1 mL of the reference solution and the test solution into a 10-mL volumetric flask, respectively, add 1 mL of ethanol, shake thoroughly, add 2% sodium hydroxide solution to the mark, shake, allow it to react in a water bath at 60°C for 30 min, and then cool to room temperature. With 2% sodium hydroxide– ethanol (4:1) as the blank, the absorbance was measured at a wavelength of 238 nm using UV–visible spectrophotometer.
Analytical Methods and Quality Standards for Dihydroartemisinin Chapter Outline 1 Physical and Chemical Parameters of Dihydroartemisinin 303 1.1 Description 303 1.2 Identification 303 1.3 Inspection 304
2 Thin-Layer Chromatography (TLC) 304 3 High-Performance Liquid Chromatography (HPLC) 304 4 UV Spectrophotometry 306
1 Physical and Chemical Parameters of Dihydroartemisinin 1.1 Description White needle-like crystal, odorless, with a bitter taste. Easily soluble in chloroform, soluble in dimethyl sulfoxide, ether, and acetone, slightly soluble in methanol and ethanol, and practically insoluble in water. Melting point: Melting and decomposition of crystals occur simultaneously at 145–150°C. Specific rotation: [α] 20D = +140° to +146° (c = 1.0026, CHCl3)
1.2 Identification 1. Dissolve 5 mg in 0.5 mL of dehydrated ethanol, add 2.5 mL of acidic potassium iodide solution (dissolve 0.4 g of potassium iodide in 2.5 mL of sulfuric acid at a concentration of 2.5 mol/L), add 1–2 drops of 2% starch solution. The blank control solution is prepared with anhydrous ethanol following the same procedures. The blue and violet color in test solution is immediately produced, while only light violet color is produced in blank control solution. 2. Add a few drops of chloroform solution and evaporate to dryness, add a drop of 2% vanillin sulfuric acid solution, the red color is produced immediately and become brown after placement. 3. The infrared absorption spectrum is concordant with the reference spectrum. From Artemisia annua L. to Artemisinins http://dx.doi.org/10.1016/B978-0-12-811655-5.00019-2
303
Copyright © 2017 Chemical Industry Press, published by Elsevier Inc. All rights reserved.
304 Chapter 19
1.3 Inspection Loss on drying: Dry over phosphorus pentoxide R under reduced pressure (not exceeding 2.67 kPa); it loses not more than 0.5%. Residue on ignition: <0.1%.
2 Thin-Layer Chromatography (TLC) Dissolve the product in chloroform to produce 10 mg/mL solution (test solution), and dissolve dihydroartemisinin in chloroform to produce 10 mg/mL solution (control solution). Apply 10 µL of aforementioned two solutions in the same silica gel G plate, respectively. Develop with benzene–ethyl acetate (8:2), dry the plate, and spray with 2% vanillin sulfuric acid solution. The spot of dihydroartemisinin in sample is not darker than the reference spots, and no other individual impurity spot is observed. Dihydroartemisinin contains not less than 98.0% and not more than the equivalent of 102.0% of C15H24O5.
3 High-Performance Liquid Chromatography (HPLC) Preparation of internal standard solution: Accurately weigh about 10 mg biphenyl (spectroscopic ally pure), transfer into a 10 mL volumetric flask, dissolve in methanol and dilute to the mark, and shake thoroughly. Preparation of reference solution: Accurately weigh about 10 mg dihydroartemisinin reference, transfer into a 10 mL volumetric flask, dissolve in methanol, add 20 mL of above internal standard solution, dilute to the mark, and shake thoroughly. Preparation of test solution: The same as the preparation of reference solution. Assay: Waters-201 high performance liquid chromatography, variable wavelength UV monitor Mode 1481, wavelength: 210 nm, sensitivity: 0.05AUFS, column: µ-Bondapa KC13 (4.0 mm × 300 mm), mobile phase: methanol–water (8:2), flow rate: 1.0 mL/ min, and injection volume: 10 µL. Transfer about 10 mL of methanol into a 10 mL volumetric flask, add 20 µL of internal standard, and dilute with methanol to the mark (as the blank control). Conduct the assay. Peak time is within 15 min, and the content is calculated based on peak area and response factor (or calibration factor) with the following equation (Fig. 19.1): 1. Correction factor method C% = Fi × AiAis × WisWi × 100%
Analytical Methods and Quality Standards for Dihydroartemisinin 305
Figure 19.1: A Chromatogram.
where Fi (correction factor) = W′iW′is × A′isA′i; A′is and Ais are the peak area of internal standard in reference solution and test solution, respectively; A′i and Ai are the peak area of certain substance in reference solution and test solution, respectively; W′is and Wis are the weight of internal standard in reference solution and test solution, respectively (µg/µL); W′i and Wi are the weight of certain substance in reference solution and test solution, respectively (µg/µL). 2. Response coefficient method (the spike amount in reference solution is the same as that in internal standard) C% = A ′isAis × A ′iWi × rF1000 × 100%
where rF (response factor) = W′iA′i × 1000. Other symbols labels are the same as (1).
Laboratory samples and pilot trial samples are measured with the aforementioned methods, and the results are shown in Table 19.1.
306 Chapter 19 Table 19.1: Laboratory and pilot trial results of dihydroartemisinin content Measured Value (%) Batch No.
I
II
Mean
Laboratory 1 Laboratory 2 Laboratory 3 Laboratory 4 Laboratory 5 Pilot trial 1 Pilot trial 2 Pilot trial 3
99.38 99.51 98.22 98.78 99.32 98.82 99.85 98.29
101.18 99.74 96.87 98.58 100.10 98.11 99.85 99.82
100.53 99.63 98.79 98.68 99.71 98.97 99.85 99.06
4 UV Spectrophotometry Preparation of control solution: Accurately weigh about 10 mg of dihydroartemisinin reference substance, place into a 50 mL volumetric flask, add ethanol solution to dissolve and dilute to the mark, shake, and then stand for 2 h. Preparation of test solution: Accurately weigh 10 tablets, porphyrize, transfer appropriate amount (about equivalent to 10 mg of dihydroartemisinin) into a 50 mL volumetric flask, add ethanol, and shake to dissolve, dilute to the mark, stand for 2 h, and filtrate. Discard the primary filtrate, and use the subsequent filtrate. Measuring procedures: Accurately transfer each of 1 mL of the reference solution and the test solution into a 10-mL volumetric flask, respectively, add 1 mL of ethanol, shake thoroughly, add 2% sodium hydroxide solution to the mark, shake, allow it to react in a water bath at 60°C for 30 min, and then cool to room temperature. With 2% sodium hydroxide– ethanol (4:1) as the blank, the absorbance was measured at a wavelength of 238 nm using UV–visible spectrophotometer.