{"id":29016,"date":"2025-11-10T11:13:07","date_gmt":"2025-11-10T04:13:07","guid":{"rendered":"https:\/\/nhathuocngocanh.com\/bp\/?p=29016"},"modified":"2025-11-10T11:13:07","modified_gmt":"2025-11-10T04:13:07","slug":"riboflavin","status":"publish","type":"post","link":"https:\/\/nhathuocngocanh.com\/bp\/riboflavin\/","title":{"rendered":"Riboflavin"},"content":{"rendered":"

(Ph. Eur. monograph 0292)<\/em><\/p>\n

C17<\/sub>H20<\/sub>N4<\/sub>O6<\/sub> 376.4 83-88-5<\/p>\n

Action and use<\/strong><\/p>\n

Vitamin B2<\/sub>.<\/p>\n

DEFINITION<\/h2>\n

7,8-Dimethyl-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]benzo[g]pteridine-2,4(3H,10H)-dione.<\/p>\n

This monograph applies to riboflavin produced by fermentation.<\/p>\n

Content<\/h3>\n

97.0 per cent to 103.0 per cent (dried substance).<\/p>\n

CHARACTERS<\/h2>\n

Appearance<\/h3>\n

Yellow or orange-yellow, crystalline powder.<\/p>\n

Solubility<\/h3>\n

Very slightly soluble in water, practically insoluble in ethanol (96 per cent).<\/p>\n

Solutions deteriorate on exposure to light, especially in the presence of alkali.<\/p>\n

It shows polymorphism (5.9).<\/p>\n

IDENTIFICATION<\/h2>\n

A. Specific optical rotation (see Tests).<\/p>\n

B. Thin-layer chromatography (2.2.27).<\/p>\n

Test solution: Suspend 25 mg of the substance to be examined in 10 mL of water R, shake for 5 min and filter the suspension to remove the undissolved material.<\/p>\n

Reference solution: Suspend 25 mg of riboflavin CRS in 10 mL of water R, shake for 5 min and filter the suspension to remove the undissolved material.<\/p>\n

Plate: TLC silica gel plate R (2-10 \u03bcm).<\/p>\n

Mobile phase: water R.<\/p>\n

Application: As follows, drying in a current of cold air after each individual application:<\/p>\n

\u2014 1 application: 2 \u03bcL of methylene chloride R then 2 \u03bcL of the test solution;<\/p>\n

\u2014 2 application: 2 \u03bcL of methylene chloride R then 2 \u03bcL of the reference solution.<\/p>\n

Development: Over a path of 6 cm.<\/p>\n

Drying: In a current of cold air.<\/p>\n

Detection: Examine in ultraviolet light at 365 nm.<\/p>\n

Results: The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.<\/p>\n

C. Dissolve about 1 mg in 100 mL of water R. The solution has, by transmitted light, a pale greenish-yellow colour, and, by reflected light, an intense yellowish-green fluorescence which disappears on the addition of mineral acids or alkalis.<\/p>\n

TESTS<\/h2>\n

Specific optical rotation (2.2.7)<\/h3>\n

-115 to -135 (dried substance).<\/p>\n

Dissolve 50.0 mg in 0.05 M sodium hydroxide free from carbonate and dilute to 10.0 mL with the same alkaline solution.<\/p>\n

Measure the optical rotation within 30 min of dissolution.<\/p>\n

Absorbance (2.2.25)<\/h3>\n

Test solution: Dilute the final solution prepared for the assay with an equal volume of water R.<\/p>\n

Absorption maxima: At 223 nm, 267 nm, 373 nm and 444 nm.<\/p>\n

Absorbance ratios:<\/p>\n

\u2014 A373\/A267 = 0.31 to 0.33;<\/p>\n

\u2014 A444\/A267 = 0.36 to 0.39.<\/p>\n

Related substances<\/h3>\n

Liquid chromatography (2.2.29). Prepare the solutions immediately before use and protect from light.<\/p>\n

Solution A: 13.6 g\/L solution of sodium acetate R.<\/p>\n

Test solution: With the aid of ultrasound, dissolve 0.120 g of the substance to be examined in 10 mL of 0.1 M sodium hydroxide and dilute to 100 mL with solution A.<\/p>\n

Reference solution (a): Dilute 1.0 mL of the test solution to 10.0 mL with solution A. Dilute 1.0 mL of this solution to 100.0 mL with solution A.<\/p>\n

Reference solution (b): With the aid of ultrasound, dissolve the contents of a vial of riboflavin for peak identification CRS (containing impurities C and D) in 1.0 mL of a mixture of 1 volume of mobile phase B and 9 volumes of mobile phase A.<\/p>\n

Reference solution (c): In order to prepare in situ impurities A and B, dissolve 10 mg of the substance to be examined in 1 mL of 0.5 M sodium hydroxide. Expose to daylight for 1.5 h. Add 0.5 mL of acetic acid R and dilute to 100 mL with water R.<\/p>\n

Column:<\/p>\n

\u2014 size: l = 0.25 m, \u00d8 = 4.6 mm;<\/p>\n

\u2014 stationary phase: end-capped octadecylsilyl silica gel for chromatography R (5 \u03bcm).<\/p>\n

Mobile phase:<\/p>\n

\u2014 mobile phase A: phosphoric acid R, water R (1:1000 V\/V);<\/p>\n

\u2014 mobile phase B: acetonitrile R;<\/p>\n\n\n\n\n\n\n\n\n
Time<\/strong>
\n(min)<\/strong><\/td>\n		Mobile phase A<\/strong>
\n(per cent V\/V)<\/strong><\/td>\n		Mobile phase B<\/strong>
\n(per cent V\/V)<\/strong><\/td>\n<\/tr>\n
0 – 5<\/td>\n90<\/td>\n10<\/td>\n<\/tr>\n
5 – 20<\/td>\n90 \u2192 80<\/td>\n10 \u2192 20<\/td>\n<\/tr>\n
20 – 25<\/td>\n80<\/td>\n20<\/td>\n<\/tr>\n
25 – 35<\/td>\n80 \u2192 50<\/td>\n20 \u2192 50<\/td>\n<\/tr>\n
35 – 45<\/td>\n50<\/td>\n50<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Flow rate: 1.0 mL\/min.<\/p>\n

Detection: Spectrophotometer at 267 nm.<\/p>\n

Injection: 10 \u03bcL.<\/p>\n

Identification of impurities: Use the chromatogram supplied with riboflavin for peak identification CRS and the chromatogram obtained with reference solution (b) to identify the peaks due to impurities C and D.<\/p>\n

Relative retention: With reference to riboflavin (retention time = about 16 min): impurity C = about 0.2; impurity D = about 0.5; impurity A = about 1.4; impurity B = about 1.9.<\/p>\n

System suitability:<\/p>\n

\u2014 resolution: minimum 5 between the peaks due to impurities A and B in the chromatogram obtained with reference solution (c);<\/p>\n

\u2014 the chromatogram obtained with reference solution (b) is similar to the chromatogram supplied with riboflavin for peak identification CRS.<\/p>\n

Limits:<\/p>\n

\u2014 correction factors: for the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity A = 0.7; impurity B = 1.4; impurity C = 2.3; impurity D = 1.4;<\/p>\n

\u2014 impurity A: not more than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.025 per cent);<\/p>\n

\u2014 impurities B, C, D: for each impurity, not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent);<\/p>\n

\u2014 unspecified impurities: for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.10 per cent);<\/p>\n

\u2014 total: not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent);<\/p>\n

\u2014 disregard limit for peaks other than those due to impurity A: 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent).<\/p>\n

Loss on drying (2.2.32)<\/h3>\n

Maximum 1.5 per cent, determined on 1.000 g by drying in an oven at 105 \u00b0C.<\/p>\n

Sulfated ash (2.4.14)<\/h3>\n

Maximum 0.1 per cent, determined on the residue obtained in the test for loss on drying.<\/p>\n

ASSAY<\/h2>\n

Carry out the assay protected from light.<\/p>\n

In a brown-glass 500 mL volumetric flask, suspend 65.0 mg in 5 mL of water R ensuring that it is completely wetted and dissolve in 5 mL of dilute sodium hydroxide solution R. As soon as dissolution is complete, add 100 mL of water R and 2.5 mL of glacial acetic acid R and dilute to 500.0 mL with water R. Place 20.0 mL of this solution in a 200 mL brown-glass volumetric flask, add 3.5 mL of a 14 g\/L solution of sodium acetate R and dilute to 200.0 mL with water R. Measure the absorbance (2.2.25) at the absorption maximum at 444 nm.<\/p>\n

Calculate the content of C17<\/sub>H20<\/sub>N4<\/sub>O6<\/sub> taking the specific absorbance to be 328.<\/p>\n

STORAGE<\/h2>\n

In an airtight container, protected from light.<\/p>\n

IMPURITIES<\/h2>\n

Specified impurities A, B, C, D.<\/p>\n

\"Rifampicin\"<\/p>\n

A. 7,8,10-trimethylbenzo[g]pteridine-2,4(3H,10H)-dione (lumiflavine),<\/p>\n

\"Rifampicin\"<\/p>\n

B. 7,8-dimethylbenzo[g]pteridine-2,4(1H,3H)-dione,<\/p>\n

\"Rifampicin\"<\/p>\n

C. 6,7-dimethyl-8-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]pteridine-2,4(3H,8H)-dione,<\/p>\n

\"Rifampicin\"<\/p>\n

D. 8-(hydroxymethyl)-7-methyl-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]benzo[g]pteridine-2,4(3H,10H)-dione.<\/p>\n","protected":false},"excerpt":{"rendered":"

(Ph. Eur. monograph 0292) C17H20N4O6 376.4 83-88-5 Action and use Vitamin B2. DEFINITION 7,8-Dimethyl-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]benzo[g]pteridine-2,4(3H,10H)-dione. This monograph applies to riboflavin produced by fermentation. Content 97.0 per cent to 103.0 per cent (dried substance). CHARACTERS Appearance Yellow or orange-yellow, crystalline powder. Solubility Very slightly soluble in water, practically insoluble in ethanol (96 per cent). Solutions deteriorate on…<\/p>\n","protected":false},"author":4,"featured_media":29017,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[174],"tags":[],"class_list":["post-29016","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-medicinal-substances"],"acf":[],"_links":{"self":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/29016","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/users\/4"}],"replies":[{"embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/comments?post=29016"}],"version-history":[{"count":2,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/29016\/revisions"}],"predecessor-version":[{"id":29025,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/29016\/revisions\/29025"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/media\/29017"}],"wp:attachment":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/media?parent=29016"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/categories?post=29016"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/tags?post=29016"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}