Liquid Chromatographic Method for Quantitative Determination of Free Fatty Acids in Butter

Abstract

A liquid chromatographic method has been developed for the determination of free fatty acids in butter. The fatty acids are converted to the p-bromophenacyl esters, via a crown ether-catalyzed reaction, without separation from the other butter components. The esters are separated on a Cis-bonded silica column by using an acetonitrile-water solvent gradient and quantitated using the ester of heptadecanoic acid as internal standard. Cu, and C ,S:i co-elute in the acetonitrile-water system but are separated using an isocratic methanol-acetonitrile-water system. Limits of detection range from 7 ng for butyric acid to 45 ng for linoleic acid. The average coefficient of variation (n = 10) for 10 free fatty acids from,a butter was 5.83%.

Re‐evaluation of fatty acids (E 570) as a food additive

“…• high-performance liquid chromatography (for the estimation of total free and bound in glycerides fatty acids) equipped with a fluorescence detector after (1) formation of their p-bromophenacyl esters (Reed et al, 1984 as referred to by Mukherjee and Weber, 1993;Elliot et al, 1989; as referred to by Mukherjee and Weber, 1993), (2) a post-column reaction with a pH indicator (Bigley and Grob, 1986 as referred to by Mukherjee and Weber, 1993), (3) derivatisation with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone and limit of detection (LOD) of 10-15 fmol/10 lL (Yamaguchi et al, 1985 as referred to by Sun et al, 2011) or 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide and LOD of 3-6 fmol/10 lL (Yamaguchi et al, 1990 as referred to by Sun et al, 2011), (4) derivatisation with 4-aminomethyl-6,7-dimethoxycoumarin and LOD of 20-50 fmol/10 lL (Sasamoto et al, 1996 as referred to by Sun et al, 2011), ( 5) derivatisation with 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene and LOD of 26-77 fmol/10 lL (You et al, 2007; as referred to by Sun et al, 2011), (6) derivatisation with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole and LOD of 110-660 fmol/10 lL (Tsukamoto et al, 2005 as referred to by Sun et al, 2011), ( 7) derivatisation with 6-oxy-(acetyl piperazine) Du et al, 2008), with a LOD between 1 and 2 nmol of fatty acid/L edible oil equal to 0.14 lg/L for caprylic acid and 0.57 lg/L for stearic acid; and (9) derivatisation with 2-(2-(anthracen-10-yl)-1H-phenanthro[9,10-d]imidazol-1-yl)ethyl 4-methylbenzenesulfonate and identification with online post-column atmospheric pressure chemical ionisation/mass spectrometry (APCI/MS) with a LOD of 23 fmol/10 lL equal to 0.65 lg stearic acid/L extract from raisins and hawthorn (Sun et al, 2011);…”

“…(). These methods include: thin‐layer chromatography (TLC) for lauric‐ and oleic acid for detection purposes only (Purghazi et al., ); high‐performance liquid chromatography (for the estimation of total free and bound in glycerides fatty acids) equipped with a fluorescence detector after (1) formation of their p ‐bromophenacyl esters (Reed et al., as referred to by Mukherjee and Weber, ; Elliot et al., ; as referred to by Mukherjee and Weber, ), (2) a post‐column reaction with a pH indicator (Bigley and Grob, as referred to by Mukherjee and Weber, ), (3) derivatisation with 3‐bromomethyl‐6,7‐dimethoxy‐1‐methyl‐2(1 H )‐quinoxalinone and limit of detection (LOD) of 10–15 fmol/10 μL (Yamaguchi et al., as referred to by Sun et al., ) or 6,7‐dimethoxy‐1‐methyl‐2(1 H )‐quinoxalinone‐3‐propionylcarboxylic acid hydrazide and LOD of 3–6 fmol/10 μL (Yamaguchi et al., as referred to by Sun et al., ), (4) derivatisation with 4‐aminomethyl‐6,7‐dimethoxycoumarin and LOD of 20–50 fmol/10 μL (Sasamoto et al., as referred to by Sun et al., ), (5) derivatisation with 1‐[2‐( p ‐toluenesulfonate)‐ethyl]‐2‐phenylimidazole‐[4,5‐ f ]‐9,10‐phenanthrene and LOD of 26–77 fmol/10 μL (You et al., ; as referred to by Sun et al., ), (6) derivatisation with 4‐[2‐( N,N ‐dimethylamino)ethylaminosulfonyl]‐7‐(2‐aminoethylamino)‐2,1,3‐benzoxadiazole and LOD of 110–660 fmol/10 μL (Tsukamoto et al., as referred to by Sun et al., ), (7) derivatisation with 6‐oxy‐(acetyl piperazine) fluorescein and LOD of 1–64 fmol/10 μL (Du et al., as referred to by Sun et al., ), (8) derivatisation with 6‐oxy‐(ethylpiperazine)‐9‐(2‐methoxycarbonyl) fluorescein (Du et al., ), with a LOD between 1 and 2 nmol of fatty acid/L edible oil equal to 0.14 μg/L for caprylic acid and 0.57 μg/L for stearic acid; and (9) derivatisation with 2‐(2‐(anthracen‐10‐yl)‐1 H ‐phenanthro[9,10‐d]imidazol‐1‐yl)ethyl 4‐methylbenzenesulfonate and identification with online post‐column atmospheric pressure chemical ionisation/mass spectrometry (APCI/MS) with a LOD of 23 fmol/10 μL equal to 0.65 μg stearic acid/L extract from raisins and hawthorn (Sun et al., ); gas chromatography (GC) with flame ionisation detector (FID) following an initial isolation step, i.e. via ion exchange resins, alumina (Mukherjee and Weber, ) or via magnetic solid‐phase extraction with LODs 7.22−26.26 ng/mL oil (Wei et al., ).…”

Analysis of Derivatized N-Acyl Amino Acid Surfactants Using HPLC and HPLC/MS

“…Analytica 2020, 1, FOR PEER REVIEW 4 lack of chromophores necessary for UV/Vis spectrometric detection, we used 2,4′-dibromoacetophenone as a derivatization agent for the preparation of the corresponding 4′-bromophenacyl esters (Figure 1b). Reaction of carboxylic acid group with 2,4′-dibromoacetophenone yielding corresponding 4′-bromophenacyl esters is well known for the quantitative determination of fatty acids in several matrices [16,17], however, it is not commonly applied to amino acids samples. In UV spectra, the phenacyl esters exhibit strong absorption bands between 230 and 290 nm, due to the presence of a benzene ring in conjugation with the carbonyl group, and an electronegative bromine atom in para-position on the benzene ring.…”

Total and Free Fatty Acids Analysis in Milk and Dairy Fat

“…In fact, one of the first HPLC methods to resolve major FFA in milk fat used RP-HPLC. In this method, Reed et al [92] separated the p-bromophenacyl esters of FFA. Two chromatographic separations were required due to problems of co-elution of some medium-and long-chain FFA.…”