{"id":14971,"date":"2025-10-16T11:17:27","date_gmt":"2025-10-16T04:17:27","guid":{"rendered":"https:\/\/nhathuocngocanh.com\/bp\/?p=14971"},"modified":"2025-11-15T15:39:51","modified_gmt":"2025-11-15T08:39:51","slug":"fluorescein","status":"publish","type":"post","link":"https:\/\/nhathuocngocanh.com\/bp\/fluorescein\/","title":{"rendered":"Fluorescein"},"content":{"rendered":"<p><em>(Ph. Eur. monograph 2348)<\/em><\/p>\n<p>C<sub>20<\/sub>H<sub>12<\/sub>O<sub>5<\/sub> 332.3 2321-07-5<\/p>\n<p><strong>Action and use<\/strong><\/p>\n<p>Detection of corneal lesions, retinal angiography and pancreatic function testing.<\/p>\n<h2>DEFINITION<\/h2>\n<p>3\u2032,6\u2032-Dihydroxy-3H-spiro[isobenzofuran-1,9\u2032-xanthen]-3-one.<\/p>\n<h3>Content<\/h3>\n<p>97.0 per cent to 102.0 per cent (dried substance).<\/p>\n<h2>CHARACTERS<\/h2>\n<h3>Appearance<\/h3>\n<p>Orange-red, fine powder.<\/p>\n<h3>Solubility<\/h3>\n<p>Practically insoluble in water, soluble in hot ethanol (96 per cent). It dissolves in dilute solutions of alkali hydroxides.<\/p>\n<h2>IDENTIFICATION<\/h2>\n<p>First identification: A, D.<\/p>\n<p>Second identification: B, C, D.<\/p>\n<p>A. Infrared absorption spectrophotometry (2.2.24).<\/p>\n<p>Comparison: fluorescein CRS.<\/p>\n<p>Dissolve the substance to be examined and the reference substance separately in the minimum volume of ethanol (96 per cent) R, evaporate to dryness and record the spectra using the residues.<\/p>\n<p>B. Dilute 0.1 mL of solution S (see Tests) to 10 mL with water R. The solution shows a yellowish-green fluorescence. The fluorescence disappears on addition of 0.1 mL of dilute hydrochloric acid R and reappears on addition of 0.2 mL of dilute sodium hydroxide solution R.<\/p>\n<p>C. The absorption by a piece of filter paper of 0.05 mL of the solution prepared for identification B (before the addition of dilute hydrochloric acid R) colours the paper yellow. On exposing the moist paper to bromine vapour for 1 min and then to ammonia vapour, the colour becomes deep pink.<\/p>\n<p>D. Suspend 0.5 g in 50 mL of water R and shake for 10 min. The substance does not completely dissolve.<\/p>\n<h2>TESTS<\/h2>\n<h3>Solution S<\/h3>\n<p>Suspend 1.0 g in 35.0 mL of water R and add dropwise with shaking 4.5 mL of 1 M sodium hydroxide. Adjust to pH 8.5-9.0 with 1 M sodium hydroxide and dilute to 50.0 mL with water R to obtain a clear solution.<\/p>\n<h3>Appearance of solution<\/h3>\n<p>Solution S is clear (2.2.1) and orange-red with yellowish-green fluorescence.<\/p>\n<h3>Related substances<\/h3>\n<p>Liquid chromatography (2.2.29).<\/p>\n<p>Solvent mixture acetonitrile for chromatography R, mobile phase A (30:70 V\/V).<\/p>\n<p>Test solution (a): Disperse 50.0 mg of the substance to be examined in 15.0 mL of ethanol (96 per cent) R.<\/p>\n<p>Sonicate and dilute to 50.0 mL with the solvent mixture.<\/p>\n<p>Test solution (b): Dilute 5.0 mL of test solution (a) to 250.0 mL with the solvent mixture.<\/p>\n<p>Reference solution (a): Disperse 50.0 mg of fluorescein CRS in 15.0 mL of ethanol (96 per cent) R. Sonicate and dilute to 50.0 mL with the solvent mixture. Dilute 5.0 mL of this solution to 250.0 mL with the solvent mixture.<\/p>\n<p>Reference solution (b): Dissolve 10.0 mg of phthalic acid CRS (impurity B) and 10.0 mg of resorcinol CRS (impurity A) in the solvent mixture and dilute to 100.0 mL with the solvent mixture. Dilute 1.0 mL of this solution to 100.0 mL with the solvent mixture.<\/p>\n<p>Reference solution (c): Dilute 5.0 mL of test solution (b) to 20.0 mL with the solvent mixture.<\/p>\n<p>Reference solution (d): Dilute 10.0 mL of reference solution (c) to 100.0 mL with the solvent mixture.<\/p>\n<p>Reference solution (e): Dissolve the contents of a vial of fluorescein impurity C CRS in 1 mL of the solvent mixture.<\/p>\n<p>Column:<\/p>\n<p>\u2014 size: l = 0.25 m, \u00d8 = 4.6 mm;<\/p>\n<p>\u2014 stationary phase: octylsilyl silica gel for chromatography R3 (5 \u03bcm);<\/p>\n<p>\u2014 temperature: 35 \u00b0C.<\/p>\n<p>Mobile phase:<\/p>\n<p>\u2014 mobile phase A: dissolve 0.610 g of potassium dihydrogen phosphate R in water for chromatography R, adjust to pH 2.0 with phosphoric acid R and dilute to 1000.0 mL with water for chromatography R;<\/p>\n<p>\u2014 mobile phase B: acetonitrile for chromatography R;<\/p>\n<table style=\"border-collapse: collapse; width: 100%; height: 85px;\">\n<tbody>\n<tr style=\"height: 43px;\">\n<td style=\"width: 33.3333%; height: 43px; text-align: center;\"><strong>Time<\/strong><\/p>\n<p><strong>(min)<\/strong><\/td>\n<td style=\"width: 33.3333%; height: 43px; text-align: center;\"><strong>Mobile phase A<\/strong><\/p>\n<p><strong>(per cent V\/V)<\/strong><\/td>\n<td style=\"width: 33.3333%; height: 43px; text-align: center;\"><strong>Mobile phase B<\/strong><\/p>\n<p><strong>(per cent V\/V)<\/strong><\/td>\n<\/tr>\n<tr style=\"height: 21px;\">\n<td style=\"width: 33.3333%; height: 21px; text-align: center;\">0 &#8211; 20<\/td>\n<td style=\"width: 33.3333%; height: 21px; text-align: center;\">85 \u2192 20<\/td>\n<td style=\"width: 33.3333%; height: 21px; text-align: center;\">15 \u2192 80<\/td>\n<\/tr>\n<tr style=\"height: 21px;\">\n<td style=\"width: 33.3333%; height: 21px; text-align: center;\">20 &#8211; 29<\/td>\n<td style=\"width: 33.3333%; height: 21px; text-align: center;\">20<\/td>\n<td style=\"width: 33.3333%; height: 21px; text-align: center;\">80<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>Flow rate: 1.0 mL\/min.<\/p>\n<p>Detection: Spectrophotometer at 220 nm.<\/p>\n<p>Injection: 20 \u03bcL of test solution (a) and reference solutions (b), (c), (d) and (e).<\/p>\n<p>Identification of impurity C: Use the chromatogram obtained with reference solution (e) to identify the peak due to<br \/>\nimpurity C.<\/p>\n<p>Relative retention: With reference to fluorescein (retention time = about 15 min): impurity A = about 0.42; impurity B = about 0.48; impurity C = about 0.86.<\/p>\n<p>System suitability: Reference solution (b):<\/p>\n<p>\u2014 resolution: minimum 2.0 between the peaks due to impurities A and B.<\/p>\n<p>Limits:<\/p>\n<p>\u2014 correction factor: for the calculation of content, multiply the peak area of impurity C by 1.9;<\/p>\n<p>\u2014 impurity C: not more than 1.2 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.6 per cent);<\/p>\n<p>\u2014 impurities A, B: for each impurity, not more than the area of the corresponding peak in the chromatogram obtained with reference solution (b) (0.1 per cent);<\/p>\n<p>\u2014 unspecified impurities: for each impurity, not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.10 per cent);<\/p>\n<p>\u2014 sum of impurities other than A, B and C: not more than 0.4 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.2 per cent);<\/p>\n<p>\u2014 disregard limit: the area of the principal peak in the chromatogram obtained with reference solution (d) (0.05 per cent).<\/p>\n<h3>Chlorides (2.4.4)<\/h3>\n<p>Maximum 0.25 per cent.<\/p>\n<p>To 10.0 mL of solution S add 90.0 mL of water R and 3.0 mL of dilute nitric acid R, wait for at least 30 min and filter. Dilute 10.0 mL of the filtrate to 15.0 mL with water R.<\/p>\n<h3>Loss on drying (2.2.32)<\/h3>\n<p>Maximum 1.0 per cent, determined on 1.000 g by drying in an oven at 105 \u00b0C.<\/p>\n<h2>ASSAY<\/h2>\n<p>Liquid chromatography (2.2.29) as described in the test for related substances with the following modification.<\/p>\n<p>Injection: Test solution (b) and reference solution (a).<\/p>\n<p>Calculate the percentage content of C<sub>20<\/sub>H<sub>12<\/sub>O<sub>5<\/sub> taking into account the assigned content of fluorescein CRS.<\/p>\n<h2>STORAGE<\/h2>\n<p>Protected from light.<\/p>\n<h2>IMPURITIES<\/h2>\n<p>Specified impurities A, B, C.<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"alignnone size-medium wp-image-14987\" src=\"https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/2-207-300x163.jpg\" alt=\"Fluorescein\" width=\"300\" height=\"163\" srcset=\"https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/2-207-300x163.jpg 300w, https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/2-207-1024x555.jpg 1024w, https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/2-207-768x416.jpg 768w, https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/2-207.jpg 1200w\" sizes=\"auto, (max-width: 300px) 100vw, 300px\" \/><\/p>\n<p>A. benzene-1,3-diol (resorcinol),<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"alignnone size-medium wp-image-14988\" src=\"https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/3-194-300x163.jpg\" alt=\"Fluorescein\" width=\"300\" height=\"163\" srcset=\"https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/3-194-300x163.jpg 300w, https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/3-194-1024x555.jpg 1024w, https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/3-194-768x416.jpg 768w, https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/3-194.jpg 1200w\" sizes=\"auto, (max-width: 300px) 100vw, 300px\" \/><\/p>\n<p>B. benzene-1,2-dicarboxylic acid (phthalic acid),<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"alignnone size-medium wp-image-14989\" src=\"https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/4-176-300x163.jpg\" alt=\"Fluorescein \" width=\"300\" height=\"163\" srcset=\"https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/4-176-300x163.jpg 300w, https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/4-176-1024x555.jpg 1024w, https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/4-176-768x416.jpg 768w, https:\/\/nhathuocngocanh.com\/bp\/wp-content\/uploads\/2025\/10\/4-176.jpg 1200w\" sizes=\"auto, (max-width: 300px) 100vw, 300px\" \/><\/p>\n<p>C. 2-(2,4-dihydroxybenzoyl)benzoic acid.<\/p>\n","protected":false},"excerpt":{"rendered":"<p>(Ph. Eur. monograph 2348) C20H12O5 332.3 2321-07-5 Action and use Detection of corneal lesions, retinal angiography and pancreatic function testing. DEFINITION 3\u2032,6\u2032-Dihydroxy-3H-spiro[isobenzofuran-1,9\u2032-xanthen]-3-one. Content 97.0 per cent to 102.0 per cent (dried substance). CHARACTERS Appearance Orange-red, fine powder. Solubility Practically insoluble in water, soluble in hot ethanol (96 per cent). It dissolves in dilute solutions of&#8230;<\/p>\n","protected":false},"author":4,"featured_media":14986,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[174],"tags":[],"class_list":["post-14971","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\/14971","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=14971"}],"version-history":[{"count":2,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/14971\/revisions"}],"predecessor-version":[{"id":14992,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/14971\/revisions\/14992"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/media\/14986"}],"wp:attachment":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/media?parent=14971"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/categories?post=14971"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/tags?post=14971"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}