Derivatization with bromophenacyl bromide

The most common fluorotags (fluorescent reagents) are dansyl chloride and o-phthalaldehyde (OPA). Chromotags include p-bromophenacyl bromide (PBPB) for derivatization of carboxylic acids (K-salts) with a crown ether catalyst, ninhydiin for primary amines forming complexes that have their adsorption maxima at about 570 nm, and dansyl chloride for primary and secondary... 

Fatty acids can be derivatized with p-bromophenacyl (73, 85, 86], phenacyl [87-89], naphthacyl [90, 91] or p-nitrophenacyl [74] bromide. To prepare p-bromophenacyl esters [73], fatty acids (0.001—0.5 mM) were dissolved in methanol or water and neutralized to a phenolphthalein end-point by methanolic KOH. The solvent was removed by either a rotary evaporator or lyophilization. A 3—10-fold molar excess of alkylating agent, p-bromophenacyl bromide/18-crown-6 (20 1) in acetonitrile, was then added. The mixture was stirred continuously in a sealed Reacti-Vial at 80 °C for 15 min. After cooling, the solution containing the derivatives was directly subjected to RP-HPLC with UV detection at 260 nm. The mobile phase was usually acetonitrile/ water [87, 92, 93], methanol/water [73, 88, 94, 95] or acetonitrile/methanol/water (86, 89], The separation of phenacyl derivatives of palmitoleic (Ci, ) and arachi-donic ( 20 4) acids was not achieved with the acetonitrile/ water system [87, 89], and elution with methanol/water could not resolve linolenic (Cig.3) and myristic (C]4.q) acids [88, 89]. A ternary mobile phase [89] containing a mixture of acetonitrile, methanol and water seemed to be best for the separation of phenacyl derivatives of fatty acids. 

Biotin [97], its biogenetic precursor dethiobiotin and its sulphoxide and sulphone metabolites have been derivatized with p-bromophenacyl bromide or 4-bromo-methylmethoxycoumarin with dibenzo-18-crown-6 as the catalyst. The reaction products were separated on a C18 column. Both UV (254 nm) and fluorescence detection were used, with detection limits of 10 and 1 ng ml respectively. The derivatization has been applied to the analysis of valproic acid in serum and plasma [76, 77,98, 99]. The acid was extracted from biological matrices with organic solvents. Alric et al. [76] developed a procedure involving solvent demixing in which the extraction of valproic acid was achieved by saturation with potassium chloride following addition of acetonitrile and HCl to the plasma. The derivatization was performed in the acetonitrile phase after separation from the aqueous phase. The recovery was over 80%. The analysis was carried out on a Cig column with acetonitrile/water as the mobile phase. 

Comparable phenacyl esters are synthesized with p-bromophenacyl bromide or p-nitrophenacyl bromide with a similar spectroscopic behavior (both p-Bondapak C18). Useful derivatization by tagging the COOH group can also be done with a-bromo-2 -acetonaphthone resulting in well-soluble and strongly UV-absorbing (254 nm) naphthanyl esters after a reaction time of only 10 min at ambient temperature (LiChrosorb Si-100). Furthermore, coumarin derivatives (substituted at position 7 for higher fluorescence quantum yields) are predestinated to... 

Bile acids can also be converted into phenacyl [81] and p-bromophenacyl [102] esters. A bile acid sample with 50% excess of 0.15 M triethylamine in acetonitrile was warmed briefly and 0.1 M phenacyl bromide (50% molar excess) in acetonitrile was added. The reaction mixhire was maintained at 80—90°C for 45—60 min. The separation of phenacyl esters of lithocholic, deoxy-cholic, chenodeoxycholic, ursodeoxycholic hyodeoxy-cholic and cholic acids was carried out on an ODS column. The excess derivatizing agents and interfering biological compounds from bile samples were first eluted with n-heptane/dioxane (90 10 v/v), and the separation of derivatives of bile adds was accomplished with n-heptane/dioxane/isopropanol (70 25 5, by vol). The detection limits at 254 nm for the derivatives were 5-lOng [81]. 

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