Amino acid analysis by capillary gas chromatography

Two developments have enabled major advancements in the use of capillary gas chromatography (GC), the result being its much more widespread use in investigations on a broad range of chemical and biological problems. The 2 technological developments were the introduction of fused silica capillary columns and the development of immobilized stationary phases for capillary GC columns. Because fused silica columns with immobilized stationary phases of varying polarities are offered by numerous vendors of chromatographic equipment, they have become widely used for many analytical tasks. We conducted a study to compare the effectiveness of commercially available fused silica capillary columns with the classical ion-exchange method in the separation and quantitation of amino acids. We selected the N-trifluoroacetyl (TFA) n-butyl and the N-heptafluorobutyryl (HFB) isobutyl ester derivatives for this study because of the extensive research and application of these derivatives during the past 20 years. The amino acid content of hydrolysates of 5 materials was measured: ribonuclease, beta-lactoglobulin, lysozyme, soybean meal, and a commercial poultry feed. Single 6N HCl hydrolysates of each material were prepared to minimize sample preparation differences, and 3 independent analyses of each hydrolysate were made by each of 3 techniques: the N-TFA n-butyl and N-HFB isobutyl ester methods using capillary gas chromatography and the ion-exchange chromatographic method using a Beckman 121 M amino acid analyzer. Our results clearly demonstrate that capillary GC analysis of amino acids using fused silica bonded-phase columns provides data with good precision and in general excellent agreement with ion-exchange analyses.     

Lipophilic extracts from banana fruit residues: a source of valuable phytosterols

The chemical composition of the lipophilic extracts of unripe pulp and peel of banana fruit 'Dwarf Cavendish' was studied by gas chromatography-mass spectrometry. Fatty acids, sterols, and steryl esters are the major families of lipophilic components present in banana tissues, followed by diacylglycerols, steryl glucosides, long chain fatty alcohols, and aromatic compounds. Fatty acids are more abundant in the banana pulp (29-90% of the total amount of lipophilic extract), with linoleic, linolenic, and oleic acids as the major compounds of this family. In banana peel, sterols represent about 49-71% of the lipophilic extract with two triterpenic ketones (31-norcyclolaudenone and cycloeucalenone) as the major components. The detection of high amounts of steryl esters (469-24405 mg/kg) and diacylglycerols (119-878 mg/kg), mainly present in the banana peel extract, explains the increase in the abundance of fatty acids and sterols after alkaline hydrolysis. Several steryl glucosides were also found in significative amounts (273-888 mg/kg), particularly in banana pulp (888 mg/kg). The high content of sterols (and their derivatives) in the 'Dwarf Cavendish' fruit can open new strategies for the valorization of the banana residues as a potential source of high-value phytochemicals with nutraceutical and functional food additive applications.     

Capillary electrophoresis for evaluating orange juice authenticity: a study on Spanish oranges

Fruit juices have very distinct organic acid profiles that can be used as fingerprints for establishing possible adulteration. Recently, our group developed and validated a capillary electrophoresis method using UV detection for determining citric, isocitric, tartaric, and malic acids in natural and commercial orange juices. Sample treatment consisted of only dilution and centrifugation or filtration. This method has been applied to evaluate these acids and their ratios in 63 samples of Navelina, the most common variety of Spanish oranges, over a three month period. This evaluation has been conducted to establish ranges of acid concentrations and to compare them with those found in commercial juices. The more reliable parameter, because of the lower variability in fresh samples, was found to be the citrate/isocitrate ratio with a value of 113 (RSD = 10%). Only one of nine ramdonly selected commercial juices presented values within the range of those of the population of just-pressed Navelina orange juice. Moreover, three of them had measurable tartrate values, which is not a natural component of orange juice, showing mixtures with cheaper fruits.     

Extraction parameters and capillary electrophoresis analysis of limonin glucoside and phlorin in citrus byproducts.

Limonin glucoside (LG) and phlorin were extracted from citrus fruit tissues and assayed by capillary electrophoresis (CE). LG was determined in dried [1.20 +/- 0.10 mg of dry weight (dw)] and wet peel residues (1.16 +/- 0.04 mg of dw), orange juice finisher pulp (0.58 +/- 0.03 mg of dw), dried grapefruit seeds (2.70 +/- 0.15 mg of dw), and 50 degrees Brix molasses (2225 +/- 68 mg/L). Phlorin was purified from orange peel residue and grapefruit albedo, and concentrations were determined in some citrus products. Phlorin and LG were extracted from residues with water/pectinase or with water solutions of methanol and ethanol. Efficient LG extraction from grapefruit seeds (2.40 +/- 0.15 mg/g) was achieved with 50-65% methanol, solvent polarity P' approximately equal to 7-8. Extracts were purified and concentrated by adsorptive resins and HPLC to obtain 95% pure compounds of LG and phlorin. CE analysis did not require extract purification beyond filtration. LG and phlorin migrated as anions in electropherograms containing peaks representing other citrus flavonoids and limonoid glucosides.     

Simplified cleanup and gas chromatographic determination of organophosphorus pesticides in crops

A simple and efficient cleanup method for gas chromatographic determination of 23 organophosphorus pesticides in crops including onion is described. The sample was extracted with acetone. The extract was purified with coagulating solution, which contained ammonium chloride and phosphoric acid, and then filtered by suction. The filtrate was diluted with NaCl solution and reextracted with benzene. The organic layer was evaporated and injected into a gas chromatograph equipped with a flame photometric detector (FPD) and fused silica capillary columns (0.53 mm id) coated with silicone equivalent to OV-1701, OV-1, and SE-52. Onion extract, which contained FPD interferences, was cleaned up on a disposable silica cartridge. Recoveries of most organophosphorus pesticides from spiked crops: mandarin orange, tomato, spinach, sweet pepper, broccoli, lettuce, and onion at levels of 0.02-0.28 ppm, exceeded 80%, but the water-soluble pesticides dichlorvos and dimethoate gave poor recoveries in all crops; the nonpolar pesticides disulfoton, chlorpyrifos, fenthion, prothiophos, and leptophos were not recovered quantitatively in spinach, sweet pepper, broccoli, and lettuce. IBP, edifenphos, phosmet, and pyridaphenthion were not recovered from onion because of adsorption to the silica cartridge. The detection limits ranged from 1.25 to 17.5 ppb on a crop basis.     

Quantitative multiresidue analyses for volatile organics in water and milk, using a fused silica open-tubular wide-bore capillary column and automated headspace gas chromatography

A modified multiresidue capillary gas chromatographic (GC) procedure has been developed using automated headspace sampling and a wide-bore fused silica open-tubular (FSOT) capillary column for the determination of volatiles in water and milk. Compounds are quantitated by the method of standard additions. An IBM System 9000 computer with the CAPMC3 chromatographic applications package and a BASIC linear regression program are used for data reduction. Data are presented for solutions prepared by fortifying water and milk with volatile solvents such as acetone, methyl ethyl ketone, benzene, methylene chloride, and chloroform, which are commonly used in the manufacture of packaging materials and adhesives. The wide-bore FSOT capillary columns showed dramatically improved detection for certain compounds, compared with normal-bore capillary GC columns. Data presented for various chemicals demonstrate the improved limits of detection from the use of automated headspace gas chromatography with wide-bore capillary columns and flame ionization detection.     

Simultaneous analysis of free phytosterols/phytostanols and intact phytosteryl/phytostanyl fatty acid and phenolic acid esters in cereals

An approach based on solid-phase extraction for the effective separation of free phytosterols/phytostanols and phytosteryl/phytostanyl fatty acid and phenolic acid esters from cereal lipids was developed. The ester conjugates were analyzed in their intact form by means of capillary gas chromatography. Besides free sterols and stanols, up to 33 different fatty acid and phenolic acid esters were identified in four different cereal grains via gas chromatography-mass spectrometry. The majority (52-57%) of the sterols and stanols were present as fatty acid esters. The highest levels of all three sterol and stanol classes based on dry matter of ground kernels were determined in corn, whereas the oil extract of rye was 1.7 and 1.6 times richer in fatty acid esters and free sterols/stanols than the corn oil. The results showed that there are considerable differences in the sterols/stanols and their ester profiles and contents obtained from corn compared to rye, wheat, and spelt. The proposed method is useful for the quantification of a wide range of free phytosterols/phytostanols and intact phytosteryl/phytostanyl esters to characterize different types of grain.     

Determination of cis, cis-methylene interrupted polyunsaturated fatty acids in fats and oils by capillary gas chromatography

A capillary gas chromatographic (CGC) method is described for the determination of cis,cis-methylene interrupted polyunsaturated fatty acids (cis-PUFA) in fats and oils. The sample is saponified and the liberated fatty acids are esterified to the corresponding methyl esters. The latter are analyzed by CGC using a 60 M. SP2340 capillary column. Area percent values for 9,12-cis,cis-C18:2 and 9,12,15-cis,cis,cis-C18:3 fatty acid methyl esters are summed to give the total cis-PUFA content. Gas chromatographic results agreed well with those obtained by an enzymatic lipoxygenase method at the 31-48% cis-PUFA levels with a correlation coefficient of 0.98. The method has a precision (relative standard deviation) of 0.33% at a 44.4% cis-PUFA level in margarine oil.     

Liquid chromatographic methodology for the characterization of orange juice

Liquid chromatographic (LC) methodology potentially useful for the characterization of orange juice, with particular regard to detecting adulteration of orange juice by computer pattern recognition analysis, has been developed. After dilution with methanol the juice is extracted with hexane to remove the carotenoids, which are chromatographed on a C18 column with an acetonitrile-methanol-methylene chloride mobile phase and detection at 450 nm. Further extraction of the juice with methylene chloride isolates the methoxylated flavones, which are chromatographed by reverse phase LC with an acetonitrile-methanol-water mobile phase and detection at 280 nm. The flavanone glycosides remaining in solution are chromatographed on a C18 column with an acetonitrile-water mobile phase and detection at 280 nm. The precisions of the heights of the 32 LC peaks selected for pattern recognition analysis were determined from 5 replicate analyses of a single juice. Coefficients of variation of the replicates ranged from 0.3 to 4.5%, with an average of 2.1%. Adulteration of products with sodium benzoate-fortified pulpwash or grapefruit juice can be detected by this method. Pattern recognition analysis of the data obtained for 80 authentic and 19 adulterated orange juices showed that the method is potentially useful for distinguishing between authentic and adulterated products.     

Lipid [corrected] classes, fatty acids, and sterols in seafood from Gilbert Bay, southern labrador

Seafood from Gilbert Bay, southern Labrador, was sampled for lipid classes, fatty acid, and sterol composition. Gilbert Bay is a proposed Marine Protected Area, and the composition of seafood from this region is interesting from both human health and ecological perspectives. Analyses included four species of bivalves and flesh and liver samples from four fish species. Lipids from a locally isolated population of northern cod (Gadus morhua) were also compared to lipids from other cod populations. Lipid classes were analyzed by Chromarod/Iatroscan TLC-FID, fatty acids by GC, and sterols by GC-MS. Three cod populations had similar levels of total lipid per wet weight (0.6%) with triacylglycerols (TAG), sterols, and phospholipids comprising on average 13, 11, and 51%, respectively, of their total lipids. Fatty fish such as capelin and herring contained on average 8.4% lipid with 86% present as TAG. Fish livers from cod and herring showed opposite trends, with cod having elevated lipid (27%) and TAG (63%) and herring containing only 3.8% lipid and 20% TAG. Shellfish averaged 0.6% lipid; however, significant lipid class differences existed among species. Fatty acid analysis showed few significant differences in cod populations with on average 57% polyunsaturated fatty acids (PUFA), 18% monounsaturated fatty acids (MUFA), and 24% saturated fatty acids (SFA). Cod livers had lower PUFA (34%) and elevated MUFA (44%) relative to flesh. Bivalves averaged 25% SFA, 18% MUFA, and 57% PUFA, whereas scallop adductor muscle had the highest PUFA levels (63%). Bivalves contained 20 different sterols with cholesterol present as the major sterol (19-39%). trans-22-Dehydrocholesterol, brassicasterol, 24-methylenecholesterol, and campesterol individually accounted for >10% in at least one species. High levels of PUFA and non-cholesterol sterols observed in Gilbert Bay seafood demonstrate their positive attributes for human nutrition.     

Food matrix effects on in vitro digestion of microencapsulated tuna oil powder

Tuna oil, containing 53 mg of eicosapentaenoic acid (EPA) and 241 mg of docosahexaenoic acid (DHA) per gram of oil, delivered as a neat microencapsulated tuna oil powder (25% oil loading) or in food matrices (orange juice, yogurt, or cereal bar) fortified with microencapsulated tuna oil powder was digested in simulated gastric fluid or sequentially in simulated gastric fluid and simulated intestinal fluid. The level of fortification was equivalent to 1 g of tuna oil per recommended serving size (i.e., per 200 g of orange juice or yogurt or 60 g of cereal bar). The changes in particle size of oil droplets during digestion were influenced by the method of delivery of the microencapsulated tuna oil powder. Lipolysis in simulated gastric fluid was low, with only 4.4-6.1% EPA and ≤1.5% DHA released after digestion (as a % of total fatty acids present). After sequential exposure to simulated gastric and intestinal fluids, much higher extents of lipolysis of both glycerol-bound EPA and DHA were obtained (73.2-78.6% for the neat powder, fortified orange juice, and yogurt; 60.3-64.0% for the fortified cereal bar). This research demonstrates that the choice of food matrix may influence the lipolysis of microencapsulated tuna oil.     

Rapid quantitation and confirmation of aflatoxins in corn and peanut butter, using a disposable silica gel column, thin layer chromatography, and gas chromatography/mass spectrometry

A simple, rapid, and solvent-efficient method for determining aflatoxins in corn and peanut butter is described. Aflatoxins B1, B2, G1, and G2 were extracted from 50 g sample with 200 mL methanol-water (85 + 15). A portion of the extract was diluted with 10% NaCl solution to a final concentration of 50% methanol, and then defatted with hexane. The aflatoxins were partitioned into chloroform. The chloroform solution was evaporated, and the residue was placed on a 0.5 g disposable silica gel column. The column was washed with 3 mL each of hexane, ethyl ether, and methylene chloride. Aflatoxins were eluted with 6 mL chloroform-acetone (9 + 1). The solvent was removed by evaporation on a steam bath, and the aflatoxins were determined using thin layer chromatography (TLC) with silica gel plates and a chloroform-acetone (9 + 1) developing solvent. Overall average recovery of aflatoxin B1 from corn was 82%, and the limit of determination was 2 ng/g. For mass spectrometric (MS) confirmation, aflatoxin B1 in the extract from 3 g sample (20 ng/g) was purified by TLC and applied by direct on-column injection at 40 degrees C into a 6 m fused silica capillary gas chromatographic column. The column was connected directly to the ion source. After injection, the temperature was rapidly raised to 250 degrees C, and the purified extract was analyzed by negative ion chemical ionization MS.     

Capillary electrophoresis analysis of orange juice pectinesterases

Pectinesterase (PE) was extracted from orange juice and pulp with 1 M NaCl, desalted, and separated using capillary electrophoresis (CE) gel procedures (CE-SDS-CGE) and isoelectric focusing (CE-IEF). PE resolved as a single peak using noncoated fused silica columns with CE-SDS-CGE. CE-IEF separation of PE required acryloylaminoethoxyethanol-coated columns, which had limited stability. Thermal stability of PE extracts before and after heating at 75 degrees C for 30 min and at 95 degrees C for 5 min established heat labile and heat stabile fractions with identical PE migration times by CE-SDS-CGE or CE-IEF. Peak magnitude decreased to a constant value as heating time increased at 75 degrees C. Regression analysis of CE-SDS-CGE peak migration times of molecular weight (MW) standards estimated both heat labile and heat stable PE at MW approximately 36 900. Traditional SDS-PAGE gel separation of MW standards and active PE isolated by IEF allowed estimation of MW approximately 36 000. CE-SDS-CGE allowed presumptive, but not quantitative, detection of active PE in fresh juice.     

Sensitive micellar electrokinetic chromatography-laser-induced fluorescence method to analyze chiral amino acids in orange juices

In this work a new method to detect the existence of chiral amino acids in orange juice is presented. The method employs beta-cyclodextrins and micellar electrokinetic chromatography with laser-induced fluorescence (MEKC-LIF) to separate and detect L- and D-amino acids (L-aa and D-aa) previously derivatized with fluorescein isothiocianate (FITC). A systematic optimization of the chiral-MEKC conditions is done bringing about in less than 20 min a good separation of the main amino acids found in orange juice (i.e., Pro, Asp, Ser, Asn, Glu, Ala, Arg, and the nonchiral GABA, i.e., gamma-aminobutyric acid). Using this procedure, the analysis time reproducibility for the 15 standard compounds (L-aa, D-aa, and GABA) has been determined to be better than 0.2% (n = 5) for the same day and better than 0.7% (n = 15) for three different days. Corrected peak area reproducibility is somewhat lower, providing values better than 3.3% (n = 5) for the same day and 6.9% (n = 15) for three different days. The limit of detection using this procedure was determined to be 0.86 attomoles for L-Arg. The optimized FITC derivatization method allows the easy and straightforward detection of amino acids in orange concentrates and juices (i.e., only centrifugation of diluted samples for 5 min is needed prior to their derivatization). D-Ala, D-Asp, D-Arg, and D-Glu were determined in orange juices and orange concentrates from different geographical origins using this new method. Moreover, the effect of different temperature treatments (50, 92, and 150 degrees C) on the content of D-aa in orange juice was evaluated.     

Gas chromatography/electron-capture negative ion mass spectrometry for the quantitative determination of 2- and 3-hydroxy fatty acids in bovine milk fat

2- and 3-hydroxy fatty acids (2- and 3-OH-FAs) are bioactive substances reported in sphingolipids and bacteria. Little is known of their occurrence in food. For this reason, a method suitable for the determination of OH-FAs at trace levels in bovine milk fat was developed. OH-FAs (and conventional fatty acids in samples) were converted into methyl esters and the hydroxyl group was derivatized with pentafluorobenzoyl (PFBO) chloride to give PFBO- O-FA methyl esters. These derivatives with strong electron affinity were determined by gas chromatography interfaced to mass spectrometry using electron-capture negative ion in the selected ion monitoring mode (GC/ECNI-MS-SIM). This method proved to be highly sensitive and selective for PFBO-O-FA methyl esters. For the analysis of samples, two internal standards were used. For this purpose, 9,10-dideutero-2-OH-18:0 methyl ester (ISTD-1) from 2-OH-18:1(9 c) methyl ester as well as the ethyl ester of 3-PFBO-O-12:0 (ISTD-2) was synthesized. ISTD-1 served as a recovery standard whereas ISTD-2 was used for GC/MS measurements. The whole-sample cleanup consisted of accelerated solvent extraction of dry bovine milk, addition of ISTD 1, saponification, conversion of fatty acids into methyl esters by use of boron trifluoride, separation of the methyl esters of OH-FAs from nonsubstituted FAs on activated silica, conversion of OH-FAs methyl esters into PFBO-O-FA methyl esters, addition of ISTD-2, and measurement by GC/ECNI-MS-SIM. By this method, ten OH-FAs were quantified in bovine milk fat with high precision in the range from 0.02 +/- 0.00 to 4.49 +/- 0.29 mg/100 g of milk fat.     

A gas chromatography/electron ionization-mass spectrometry-selected ion monitoring method for determining the fatty acid pattern in food after formation of fatty acid methyl esters

A method using gas chromatography/electron ionization-mass spectrometry (GC/EI-MS) in the selected ion monitoring (SIM) mode was developed for the analysis of fatty acids as methyl esters (FAMEs) in order to determine their percentage contribution to the fatty acid profile in food. In the GC/EI-MS-SIM mode, saturated fatty acids were determined with m/z 87, monoenoic fatty acids were determined with m/z 74, and polyenoic fatty acids were determined via the sum of m/z 79 and m/z 81. The ratios of these fragment ions and the GC retention data provided additional information for tentative structural assignments. The 28 FAME standards tested provided similar results for the novel GC/EI-MS-SIM method and GC/EI-MS in the full scan mode, both of which were slightly worse than GC/flame ionization detection (FID). Analysis of sunflower oil, suet, and cod liver oil verified that both major and minor fatty acids (20-60% and down to 0.001% contribution to the fatty acid pattern) were determined with sufficient quality that justifies application of the GC/EI-MS-SIM method for the analysis of food samples. Furthermore, the method was approximately 20- or approximately 10-fold more sensitive than GC/EI-MS in the full scan mode or GC/FID, respectively. The method is suited for both quantitative purposes and fatty acid identification in samples where only low amounts of lipids are available.     

Linalool in orange juice: origin and thermal stability

Linalool concentrations were determined in juice from three groups of 60 Valencia oranges using pentane:ether extraction and high-resolution capillary GC. The outer peel (flavedo) was removed from one group. The other two groups retained their peel intact. Juice was extricated from the halved fruits of the flavedo-less group and from one of the peel-intact groups using a hand reamer. A peel-cutting/macerating juice extractor was used for the other peel-intact group. Linalool concentrations were 0.004 mg/L in peeled fruit juice and 0.020 and 0.106 mg/L for hand-reamed and mechanically extracted peel-intact juice, respectively. Juice from peeled fruit contained significantly (P < 0.05) less linalool than peel-intact juice. Approximately 80% of the total juice linalool content was associated with peel using reamer design, and 96% was associated with peel-cutting/macerating design. Linalool increased with increasing peel oil levels; however, the increases were not proportionate. Since all commercial juices are mechanically extracted, the vast majority of linalool in commercial orange juice originates from the peel and not from the juice vesicle cytoplasm. Juice from peel-macerated, mechanically extracted fruit increased from 0.106 to 0.134 mg/kg after thermal processing, whereas juice from reamer extraction was essentially unchanged.     

超滤对血橙果汁中抗氧化成分及总抗氧化能力的影响

采用管式聚偏氟乙烯(PVDF)超滤膜，探讨了超滤对血橙果汁中抗氧化成分及总抗氧化能力的影响。结果表明，其对维生素C和花青素类成分的保存率约为85%左右，对几种酚酸和类黄酮组分（除槲皮素外）的保存率均在90%以上。同未杀菌的血橙原汁相比，巴氏杀菌汁的总抗氧化能力(TAA)损失率为23%，而超滤澄清汁仅为10%左右，主要与维生素C和花青素含量损失有关。     

锦橙汁辐照和巴氏灭菌处理后相关品质的分析

为探讨辐照和巴氏杀菌对橙汁品质的影响,对锦橙汁分别进行1.4、2.8、5.6kGy3种不同剂量辐照及巴氏灭菌处理,使用固相微萃取-气质联用技术对挥发性成分进行分析,测定橙汁色度、pH值和Vc含量,并对橙汁香气进行感官评定。在鲜橙汁、巴氏灭菌汁和3种辐照样品中分别检测到了54、47、57、55和53种成分,2.8kGy剂量辐照处理后橙汁挥发性成分总峰面积最高,β-月桂烯、柠檬烯和γ-松油烯等橙汁特征香气物质经辐照后保持率高于巴氏灭菌。各种处理后Vc含量均有所下降,橙汁橙香均减弱。1.4kGy辐照后的橙汁感官评价结果最好,因此可以对橙汁进行低剂量辐照灭菌。     

Determination of conjugated linoleic acid (CLA) concentrations in milk chocolate

The fatty acids from a series of milk-chocolate-based confectionery samples were analyzed as methyl esters by GC to determine the presence and amount of conjugated linoleic acid (CLA). A single peak corresponding to the 9-cis,11-trans isomer and ranging from less than 0.1% to nearly 0.2% of the total fatty acids, corresponding to up to 0.3 mg per g of chocolate, was observed. One of the chocolate extracts and a milk extract were subjected to silver ion HPLC and GC-MS in order to confirm the identity of the major isomer and tentatively identity minor isomers.