Naphthacyl

Naphtolc Acid (4). 1 and pyndine 2 were heated on a water bath for a short time The product was dissolved in ElOH and precipitated with EtjO Recrystalhzation from 10 volumes ol water affoided pure a-naphthacyl pyndinium bromide 3 A solution of 3 (0 5 g IS mmol) m waler (40 mL), EtOH (12 mL) and 10 N NaOH (2 ml) after healing for 12 min on a waler bath, gave on acidification and extraction with EtzO 4 mp 160-162°C... 

Naphthacyl ester of pelargonic acid, prepared as described previously [22]. 

Br-AMN (0.05 ml), 0.2 ml BA solution (or serum) and 0.3 ml TDeABr solution (10 mM in phosphate buffer, pH 7.0) are incubated for 10 min at 40°C under ul-trasonication. The reaction mixture is spiked with 0.3 ml of the respective internal standard solution and ultrasonicated at room temperature for 1 min. Then, 50-pi aliquots of the BA naphthacyl ester mixture are injected into the HPLC. 

The chromatogram of free BA standard mixture is reported in Fig. 5.4.7. The Br-AMN degradation products are eluted at lower retention times than derivatised BA, close to the solvent front, so they do not impair BA separation. Free BA fraction also encloses taurine conjugates, previously enzymatically hydrolysed. The separation of glycine conjugated BA is illustrated in Fig. 5.4.8. In both chromatograms, the peaks of BA naphthacyl esters are fully resolved and separated from the reagent peaks. 

Fig. 5.4.7 HPLC resolution at 35°C of a standard mixture of free BA naphthacyl esters (3.18 x 10 6 M). Chromatogram (a) 1 UDCA, 2 CA, 3 CDCA, 4 DCA, 5 lauric acid (the internal standard), 6 LCA. Chromatogram (b) blank, consisting of reagent kept at reaction conditions. R is the reagent peak in both chromatograms (reprinted from [20])...

The direct analysis of small amounts of Cig-Cjo long-chain fatty acids by HPLC is not feasible because of the weak UV absorption of these compounds. The formation of 2-naphthacyl esters provides greatly increased UV sensitivity and makes possible HPLC analysis of trace amounts of fatty acids [40]. 

Method. The fatty acid (10 jumoles) is dissolved in 1 ml of dimethylformamide. To this solution are added 20 jumoles of 2-naphthacyl bromide and 40 mmoles of ethyldiisopropyl-... 

Fig.4.16. Separation of some 2-naphthacyl esters of fatty adds. A, C, adds B, C adds. Peaks 1 = oleic add 2 = linoleic add 3 a-Unolenic add 4 = 7-linolenic add 5 = dihomo-7-linolenic add 6 = arachidonic add 7 = 2-naphthacyl bromide. (From ref. 40 with permission of the American Chemical Society, Washington.)...

The separation of the 2-naphthacyl esters of several fatty acids is shown in Fig.4.16. Corasil-Cig (3 ft. X 0.07 in. I.D. column) was used as the stationary phase and the fatty acid esters were eluted with methanol—water (17 3) at a flow-rate of 0.2 ml/min. Nanogram quantities of the fatty acids may be detected by this procedure. 

Examples of such visualising scents for carboxylic acids (also short-chain acids) include 2-naphthacyl esters (Cooper and Anders, 1974), phenacyl esters (Borch, 1975 Durst et al., 1975), p-nitrobenzyl esters (Knapp and Krueger, 1975), p-methoxyanilides (Hoffman and Liao, 1976), p-bromophenacyl... 

In the quantitative analysis of fatty acids by HPLC a plethora of reagents have been used to increase the sensitivity of detection. The most popular derivatives formed include benzyl derivatives (Polizer et al., 1973) 2-naphthacyl derivatives (Cooper and Anders, 1974) o-and p-nitrobenzyl derivatives (Knapp and Krueger, 1975) phenacyl derivatives (Borch, 1975) p-bromophenacyl derivatives (Durst et al, 1975) methoxyphenacyl derivatives (Miller et al., 1978), and 1-naph-thylamide derivatives (Ikeda et al., 1983). The benzyl, nitrobenzyl, phenacyl, p-bromophenacyl, methoxyphenacyl, 1-naphthylamide and... 

A wide variety of other esters has been made, or may potentially be accessible, by the type of procedure outlined in this section. To give a couple of examples, the naphthacyl esters were made for improved detectability in HPLC analysis in the same way as the phenacyl esters mentioned earlier [102], the fluorescent esters of 4-methyl-7-methoxycoumarin were made from the 4-bromomethyl-7-methoxycoumarin by the potassium salt procedure [103] and anthrylmethyl esters were obtained by an extractive alkylation procedure [104]. 

Phenacyl bromide, naphthacyl bromide and their analogues... 

The molar absorption coefficients [72] of some phenacyl and naphthacyl bromides at 2 or the detection wavelength are given in Table 3. To improve the sensitivity and selectivity of the alkylation reaction of carboxylic... 

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. 

Alkylation can also be carried out on other acidic compounds, such as barbiturates. 2-Naphthacyl derivatives of barbiturates [103] were formed in acetone at 30 °C in 30 min, using caesium carbonate as catalyst. The derivatives of six barbiturates were separated in 10 min by RP-HPLC. Down to 1 ng of N,N-dinaphthacyl phenobarbitore could be detected at 249 nm, providing the potential for the determination of barbiturates in small plasma or serum samples at concentrations well below the therapeutic range. 

The following fluorescent alkylating agents have been introduced as pre-chromatographic reagents 9-bromo-methylacridine [480], 3-bromomethyl-6,7-dimethoxy-l-methyl-2(lH)-quinoxalinone [481], naphthacyl bromide (2-bromoacetonaphthone) [482, 483], p-(anthroyloxy)-phenacyl bromide (panacyl bromide) [484], 1-bromo-acetylpyrene [485], 9-chloromethy[anthracene [486], and 9-anthryldiazomethane [487, 488]. 

Derivatives of HFAs can be hydroxamic acids, phenacyl derivatives, nitrobenzoyl derivatives, and naphthacyl derivatives. Derivatization of the hydroxy group can be achieved using 1-anthroylnitrile. The naphthacyl derivatives can be separated by reversed-phase chromatography and detected at 246 nm, with a detection limit of 0.1 ng. 

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