An improved method validation for rapid determination of acrylamide in foods by ultra-performance liquid chromatography combined with tandem mass spectrometry

An improved method was validated for the determination of acrylamide in foods using ultra-performance liquid chromatography (UPLC) coupled to eletrospray ionization tandem mass spectrometry (MS/MS) in the present study. This improved method supplies a rapid quantitative procedure of acrylamide with a run time of only 3 min. Results showed a good repeatability (RSD< or =4.5%) with 50, 100, 250, 500 and 1000 microg/kg spiked concentrations in potato crisps in within-day (n=5) and day-to-day (n=10) precision tests. Meanwhile, good recoveries (81.6-99.0%) were obtained with the same spiked concentrations in acrylamide-free cereal samples (n=3). The excellent method validation data and proficiency test results (Z-score: -0.1) of the official Food Analysis Performance Assessment Scheme (FAPAS) suggested that the present quantitative method could be applied for rapid determination of acrylamide in many investigations.     

Determination of hexanal as indicator of the lipidic oxidation state in potato crisps using gas chromatography and high-performance liquid chromatography

Hexanal (an oxidative state indicator) formed in the headspace of potato crisps during storage was evaluated using two different procedures. First, solid-phase microextraction, an innovative sampling preparation methodology was used. It consisted on the absorption of analytes directly from samples and subsequent thermal desorption on the gas chromatograph (GC) injector. Then, a reversed-phase high-performance liquid chromatographic technique (HPLC) was employed to quantify hexanal in the form of 2,4-dinitrophenylhydrazone derivative. Methods were evaluated in what concerns to validation parameters such as linearity, repeatibility and detection limit. GC (LOD = 1 ng/ml) method resulted in more sensitive method than HPLC (LOD = 9 ng/ml). The most suitable technique for hexanal measurement was selected.     

Extraction of maleic hydrazide residues from potato crisps and their determination using high-performance liquid chromatography with UV and atmospheric pressure chemical ionisation mass spectrometric detection

A method was required for the determination of maleic hydrazide residues in potato crisps. A published method for the extraction of the analyte from onions and potatoes was evaluated and found to be inappropriate due to the inability of the extracting solvent to penetrate the oily matrix. A method was developed to overcome this problem; the resulting recovery data (mean=92.9%. R.S.D.=8.3%, N=16) confirmed its efficiency, and was used to analyse 48 retail potato crisp samples. To confirm possible residues identified by screening with HPLC-UV, and HPLC-atmospheric pressure chemical ionization MS method was developed. There was good agreement between the data obtained from the detection techniques (R²=0.978, slope=1.11).     

Gas Chromatographic Determination of Glycerides in Potato Crisps Fried in Different Oils

Summary Glycerides in eight commercial potato crisps were identified after conversion to fatty acids. The profiles were analysed and compared, with regard to 14 fatty acids, by use of a gas chromatographic method. Results showed that the fatty acid profile is quite variable among brands and depends on the type of oil used in the frying process. To provide a means of identifying the frying oil correctly, brands were classified into three groups depending on the main fatty acid present. After the first analysis samples were stored at room temperature in the dark and analysed again 1 and 3 months later.Presented at: International Symposium on Seperation and Characterization of Natural and Synthetic Macromolecules, Amsterdam, The Netherlands, February 5–7, 2003     

Gas chromatography - optical fiber detector for assessment of fatty acids in urban soils

Fatty acids have been used as biomarkers of the microbial community composition of soils and they are usually separated and quantified by gas-chromatography coupled to a flame ionization detector (GC-FID). The aim of this study was to develop, validate and apply a methodology based on gas chromatography coupled to optical fiber detection (GC-OF) for screening five fatty acids used as indicators of fungal and bacterial communities in urban soils. The performance of the GC-OF methodology (optical fiber detector at 1550 nm) was evaluated by comparison with the GC-FID methodology and it was found that they were comparable in terms of linear range, detection limit and analytical errors. Besides these similar analytical characteristics, the GC-OF is much cheaper than the GC-FID methodology. Different concentrations were determined for each fatty acid indicator which in turn varied significantly between the soil samples analyzed from Lisbon ornamental gardens. Additionally, the GC-OF showed a great potential as alternative for determination of eleven or more fatty acids in urban soils.     

Rapid determination of acrylamide contaminant in conventional fried foods by gas chromatography with electron capture detector

Gas chromatography coupled with electron capture detector (GC-ECD) was successfully developed and applied for the rapid determination of acrylamide in conventional fried foods, such as potato crisps, potato chips, and fried chicken wings. The method included defatting with n-hexane, extraction with aqueous solution of sodium chloride (NaCl), derivatization with potassium bromate (KBrO3) and potassium bromide (KBr), and liquid-liquid extraction with ethyl acetate. The final acrylamide extract was analyzed by GC-ECD for quantification and by GC-MS for confirmation. The chromatographic analysis was performed on the HP-INNOWax capillary column, and good retention and peak response of acrylamide were achieved under the optimal conditions (numbers of theoretical plates N = 83,815). The limit of detection (LOD) was estimated to be 0.1 microg kg(-1) on the basis of ECD technique. Recoveries of acrylamide from conventional samples spiked at levels of 150, 500 and 1000 microg kg(-1) (n = 4 for each level) ranged between 87 and 97% with relative standard deviations (RSD) of less than 4%. Furthermore, the GC-ECD method showed that no clean-up steps of acrylamide derivative would be performed prior to injection and was slightly more sensitive than the MS/MS-based methods. Validation and quantification results demonstrated that this method should be regarded as a new, low-cost, and robust alternative for conventional investigation of acrylamide.     

Quantitation of the main constituents of vanilla by reverse phase HPLC and ultra‐high‐pressure‐liquid‐chromatography with UV detection: Method validation and performance comparison

Vanilla's main constituents, i. e., vanillin, para‐hydroxybenzaldehyde, and their corresponding acids, can be easily quantified by RP LC with UV detection and external calibration. This paper describes two methods that were developed using HPLC and ultra‐high‐pressure LC (UHPLC), respectively, and validated according to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) [1]. Both methods were highly specific, exhibited good linearities with high precision, and achieved good accuracies of quantitative results. The UHPLC method was more sensitive, five times shorter, and gave better peak resolutions than the HPLC alternative.     

Quantitation of phenacyl esters of retinal fatty acids by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for the separation and quantitation of retinal fatty acids containing long-chain polyunsaturated fatty acids is described. Fatty acids from frog retinal lipids were converted to the corresponding phenacyl derivatives which were separated on a C18 reversed-phase column and detected at 242 nm. Molar absorptivities (peak area units/nmol) of up to seventeen fatty acid phenacyl derivatives were determined and used for quantitation of fatty acids separated by HPLC. Compared with gas chromatography, the HPLC method gave a similar molar percent distribution of the fatty acids and was twenty to fifty times more sensitive. This HPLC method provides a useful means for the study of chemistry and metabolism of long-chain polyunsaturated fatty acids in retina and other tissues where amounts of material may be limited or recovery of individual components desirable.     

Chemical characterisation of old cabbage (Brassica oleracea L. var. acephala) seed oil by liquid chromatography and different spectroscopic detection systems

We report an extensive chemical characterisation of fatty acids, triacylglycerols, tocopherols, carotenoids and polyphenols contained in the oil extracted from old cabbage (Brassica oleracea L. var. acephala) by cold-pressing of the seeds. Analyses were performed by GC-FID combined with mass spectrometry, HPLC with photodiode array, fluorescence and mass spectrometry detection. The 94% of the total fatty acids were unsaturated, rappresented by erucic acid (more than 50%) followed by linoleic, linolenic and oleic acids accounting for approximately 10% each. The most abundant triacylglycerols (>13%) were represented by erucic–gadolenic–linoleic, erucic–eruci–linoleic and erucic–erucic–oleic. Among tocopherols, γ-tocopherol accounted for over 70% of the total content. Thirteen carotenoids and 11 polyphenols were identified and measured. In particular, the total content in carotenoids was 10.9 ppm and all-E-lutein was the main component (7.7 ppm); among polyphenols, six hydroxycinnamic acids and five flavonoids, were identified by combining information from retention times, PDA and MS data.     

Determination of butylated hydroxytoluene in food samples by high-performance liquid chromatography with ultraviolet detection and gas chromatography/mass spectrometry

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and compared with a gas chromatography/mass spectrometry (GC/MS) method for determining butylated hydroxytoluene (BHT) in foodstuffs as a result of migration from plastic packaging. Similar extraction procedures were used in both methods. BHT was quantitated using an external standard in the HPLC method and an internal standard in the GC/MS method. Both methods presented good linearity (r(2) > or = 0.9917) and low detection limits. Recoveries obtained with the HPLC method (chicken meat, 95.8%, and Gouda cheese, 83.9%) were better than with the GC/MS method (chicken meat, 85.6%, and Gouda cheese, 71.3%).     

Determination of caffeoylquinic acids and flavonoids inCynara scolymus L. by high performance liquid chromatography

Summary The main phenolic compounds in dried extracts fromCynara scolymus (artichoke)—monocaffeoylquinic acids, dicaffeoylquinic acid, and flavonoids–have been separated by high-performance liquid chromatography. By use of a narrow bore C₁₈ column and an acidic mobile phase this HPLC method enabled improved separation within 31 min with significantly reduced solvent consumption compared with other methods. The method was validated to demonstrate its linearity, precision, accuracy, and robustness. Twelve commercial samples were analyzed. Monocaffeoylquinic acids were the most abundant phenolic compounds; the amounts present ranged from 0.48 to 4.24%. The amounts of dicaffeoylquinic acids and flavonoids were smaller—from 0.03 to 0.52%. The method is a good combination of efficiency and economy and should be especially useful for commercial applications.     

Isolation of neutral and acidic lipid biomarker classes for compound-specific-carbon isotope analysis by means of solvent extraction and normal-phase high-performance liquid chromatography

An analytical method for the quantitative determination of neutral and acidic lipid biomarkers in particulate and sediment samples has been developed. The method involves a first step with solvent extraction to isolate the neutral from the acidic compounds and a second step using normal-phase HPLC on a Nucleosil silica column to separate four different classes of neutral compounds: (1) aliphatic hydrocarbons, (2) aromatic hydrocarbons, (3) ketone compounds and (4) sterol and alcohol compounds. Recoveries of the individual spiked lipid biomarkers for the whole analytical procedure ranged from 88 to 106% for fatty acids, from 50 to 60% for aliphatic hydrocarbons (> or = n-C17), from 50 to 60% for polycyclic aromatic hydrocarbons (PAHs) (> or = 3 rings), 83% for friedelin and 60-80% for the sterol and alcohol compounds. The isolated compound classes were analysed by gas chromatography-combustion-isotope ratio mass spectrometry to measure the stable carbon isotope ratios in the individual compounds. The method enables the isolation of compound classes without fractionation for compound-specific carbon isotope analysis (delta13C). This analytical protocol has been applied, and proved suitable, for the determination of lipid biomarkers (sterols, fatty alcohols and fatty acids) in marine particulate material and for the determination of PAHs in sediment samples.     

Calibration and determination of volatile fatty acids in waste leachates by gas chromatography

Low-level radioactive waste leachates were analyzed for volatile fatty acids by gas chromatography as part of the continuing waste management program at the Chalk River Laboratories. An existing method was optimized whereby carboxylic acids were detected at the mg/1 level with a precision of 5% or better for C₂---C₇ acids and an accuracy of 3% or better for acetic acid. Parameters such as sample handling, calibration and accuracy are discussed.     

Comparative analysis of lipophilic compounds in eggs of organically raised ISA Brown and Araucana hens

Hens?? eggs represent a rich source of important nutrients, including lipids and carotenoids. The lipid composition of hens?? eggs is influenced by genetic factors, age, and diet. The aim of this study was to compare the fatty acids, cholesterol, and carotenoids content of the egg yolk of ISA Brown and Araucana hens grown in free-range housing systems. Fatty acids and cholesterol were analysed by GC-FID and GC-MS and carotenoids were quantified by RP-HPLC-PDA. The Araucana egg yolk has a higher lipid content and higher egg-to-albumen ratio than the ISA Brown yolk, while the total cholesterol, carotenoids content and profile are not significantly different. The lipids of the Araucana egg yolk have a higher content of mono-unsaturated fatty acids (MUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) and a better n-6/n-3 ratio than the ISA Brown egg yolk lipids. The major carotenoids were lutein and zeaxanthin, which account for more than 83 % in egg yolk. Eggs of both breeds, when raised organically, represent very good sources of highly bio-available lutein and zeaxanthin, pigments which are related to lower risk of age-related macular degeneration. We report for the first time on the fatty acids composition in lipid fractions and the profile and content of carotenoids of the Araucana egg yolk.     

Microanalysis of free fatty acids in plasma of experimental animals and humans by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed for microanalysis of thirteen free fatty acids using 200 microliter of plasma. Fatty acids were derivatized with 9-anthryldiazomethane for HPLC analysis. Use of an ODS minicolumn for pretreatment of plasma gave a more accurate determination of free fatty acids in plasma than by chloroform extraction. Using this method, thirteen free fatty acids in the plasma of normal human, dog, rabbit, guinea pig and rat were determined.     

Pyrolysis-mass spectrometry and gas chromatography-flame ionization detection as complementary tools for soil lipid characterization

Lipid biomarker profiles are a powerful tool for assessing soil microbial community structure, but intensive laboratory work and data analysis are needed to construct profiles from phospholipid fatty acids and other common biomarkers. Pyrolysis mass spectrometry (Py-MS) is a alternative method that provides a rapid and sensitive ‘fingerprint’ of soil lipids and may be sufficient to characterize lipids from various sites. The objective of this work was to evaluate the capacity of pyrolysis metastable atom bombardment time-of-flight mass spectrometry (Py-MAB-TOF-MS) to provide replicable analysis of soil lipids, compared to a routine gas chromatography-flame ionization detection (GC-FID) method. Soils were collected from six agricultural fields under soybean, corn and asparagus production. Soil lipids extracted with 1:2 chloroform:methanol solvent were analyzed with Py-MAB-TOF-MS or transesterified into fatty acids and then analyzed by GC-FID. The two methods were complementary, but distinct: lipid fingerprints, generated from Py-MAB-TOF-MS spectra, included extractable soil lipids from microbial, animal and plant origins plus non-living organic matter in the samples, whereas fatty acid profiles generally represented lipids from soil bacteria and fungi. We conclude that the soil lipid fingerprints generated from Py-MAB-TOF-MS present more variability than lipid biomarker profiles from the GC-FID method because they include a broader group of extractable soil lipids. Further work is needed to identify the molecular fragment masses in Py-MAB-TOF-MS spectra that could precisely identify soil lipids of microbial origin.     

Determination of plasma lactic acid concentration and specific activity using high-performance liquid chromatography.

Assessment of lactate metabolism is of particular interest during exercise and in disease states such as diabetes, shock, and absorptive abnormalities of short-chain fatty acids by the colon. We describe an analytical method that introduces radio-active tracers and high-performance liquid chromatography (HPLC) to simultaneously analyze concentrations and specific activities (SAs) of plasma lactate. The HPLC conditions included separation on a reversed-phase column (octadecylsilane) and an isocratic buffer (30% acetonitrile in water). [3H]Acetate served as an internal standard. Lactate and acetate were extracted from plasma samples with diethyl ether following a pH adjustment to less than 1.0 and back-extracted into a hydrophilic phase with sodium carbonate (2 mM, pH greater than 10.0). Lactate is detected in the ultraviolet range (242 and 320 nm) by derivatization with alpha-bromoacetophenone. Control plasma samples were studied after an overnight fast for precision and analytical recovery. Calibration curves were linear in the range 0.18-6.0 mM (r = 0.92). The precision was 3% and the analytical recovery was 87%. The detection limit of the method was 36 pmol. Determination of lactate metabolism was performed in a patient with chronic congestive heart failure who was administered primed-continuous L-[U-14C]lactate (10 microCi bolus and 0.3 microCi/min continuously) during a 60-min rest period. Mean arterial lactate concentration and SA were 1.69 +/- 0.2 mM and 253.8 +/- 22 dpm/mumol, respectively. Systemic lactate turnover was 25.65 mumol/kg per min. Lactic acid systemic turnover, organ uptake and release rates can be accurately determined by isocratic HPLC.     

Simple high-performance liquid chromatography method for alpha-tocopherol measurement in Rosmarinus officinalis leaves. New data on alpha-tocopherol content.

A simple HPLC method for vitamin E (alpha-tocopherol) measurement in the leaves of Rosmarinus officinalis has been developed and validated. It has enabled new data for alpha-tocopherol content to be established. The leaves, recently harvested, were dried in a microwave oven and crushed; then, alpha-tocopherol was directly extracted from portions of ground material with acetone, by probe sonication. After centrifugation the acetonic extract was analysed by HPLC with ergocalciferol (vitamin D2) added as internal standard and a gradient elution with a Nucleosil C18 column at 35 degrees C. Validation parameters of the method can be considered adequate. For standards: linearity is r=0.999, recovery is 100+/-2%, intra-assay precision has RSD=+/-3% and inter-assay precision has RSD=+/-6%. For samples: linearity is r=0.99, recovery: 93+/-7%, intra-assay precision has RSD=+/-4% and inter-assay precision has RSD=+/-7%.     

Comparison of four extraction/methylation analytical methods to measure fatty acid composition by gas chromatography in meat

Four different extraction-derivatization methods commonly used for fatty acid analysis in meat (in situ or one-step method, saponification method, classic method and a combination of classic extraction and saponification derivatization) were tested. The in situ method had low recovery and variation. The saponification method showed the best balance between recovery, precision, repeatability and reproducibility. The classic method had high recovery and acceptable variation values, except for the polyunsaturated fatty acids, showing higher variation than the former methods. The combination of extraction and methylation steps had great recovery values, but the precision, repeatability and reproducibility were not acceptable. Therefore the saponification method would be more convenient for polyunsaturated fatty acid analysis, whereas the in situ method would be an alternative for fast analysis. However the classic method would be the method of choice for the determination of the different lipid classes.     

Determination of acrylamide in potato crisps by capillary electrophoresis with quantum dot-mediated LIF detection

Acrylamide or 2-propenamide (AAM), a water-soluble toxic contaminant, has recently caused health concern after it was found in food products made by high-temperature cooking. Due to its weak UV absorption and electrochemically inactive state, common analytical methods do not have sufficient sensitivity to meet the World Health Organization requirement. A LIF detection method mediated by water-soluble CdTe quantum dots capped with mercaptopropyl acid (MPA) is thus developed for AAM quantitation. The optimized conditions are as follows: 30 mmol/L SDS, 0.1 mmol/L quantum dot, and 40 mmol/L phosphate buffer solution at pH 8.0 under 18 kV run voltage with LIF detection at 473 nm excitation and 568 nm fluorescence. The linear quantitation range for AAM was found to vary from 1.0 to 100 mg/kg and a detection limit (S/N=2) at 0.1 mg/kg, showing sufficient sensitivity to meet the maximum AAM specified by the Joint Food and Agriculture Organization/World Health Organization Expert Committee for potato crisps. Recoveries for potato crisps sample spiked with 10, 20, and 100 mg/kg AAM were found to vary between 90 and 95% with RSD <5.7% (n=3).