Value assignment of nutrient concentrations in standard reference material 2384 baking chocolate

Standard Reference Material (SRM) Baking Chocolate was recently issued, and the process used for value assignment of nutrient concentrations is reported herein. SRM 2384 is intended for use as a primary control material for assigning values to in-house control materials and for validation of analytical methods for the measurement of fatty acids, proximates, vitamins, and elements in chocolate and similar high-fat matrices. The Certificate of Analysis for SRM 2384 provides assigned values for concentrations of fatty acids, proximates, vitamins, elements, and total dietary fiber, for which product labeling is required by the Nutrition Labeling and Education Act of 1990, as well as for catechins, caffeine, theobromine, and theophylline. These assigned values were based on measurements by NIST and/or collaborating laboratories.     

Quantification of the predominant monomeric catechins in baking chocolate standard reference material by LC/APCI-MS

Catechins are polyphenolic plant compounds (flavonoids) that may offer significant health benefits to humans. These benefits stem largely from their anticarcinogenic, antioxidant, and antimutagenic properties. Recent epidemiological studies suggest that the consumption of flavonoid-containing foods is associated with reduced risk of cardiovascular disease. Chocolate is a natural cocoa bean-based product that reportedly contains high levels of monomeric, oligomeric, and polymeric catechins. We have applied solid-liquid extraction and liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometry to the identification and determination of the predominant monomeric catechins, (+)-catechin and (-)-epicatechin, in a baking chocolate Standard Reference Material (NIST Standard Reference Material 2384). (+)-Catechin and (-)-epicatechin are detected and quantified in chocolate extracts on the basis of selected-ion monitoring of their protonated [M + H](+) molecular ions. Tryptophan methyl ester is used as an internal standard. The developed method has the capacity to accurately quantify as little as 0.1 microg/mL (0.01 mg of catechin/g of chocolate) of either catechin in chocolate extracts, and the method has additionally been used to certify (+)-catechin and (-)-epicatechin levels in the baking chocolate Standard Reference Material. This is the first reported use of liquid chromatography/mass spectrometry for the quantitative determination of monomeric catechins in chocolate and the only report certifying monomeric catechin levels in a food-based Standard Reference Material.     

Improved high-performance liquid chromatography method to determine theobromine and caffeine in cocoa and cocoa products

At present, the commonly used HPLC method for the analysis of caffeine and theobromine contents in aqueous cocoa extracts employs direct application of the extracts on the column. This practice gradually reduces the efficiency of the column and shortens its life. Also, this method gives inflated values due to interfering substances and difficulty in achieving baseline resolution. In the improved method, the interfering cocoa pigments are effectively removed by passing the aqueous extract through a Sep-pak C(18) cartridge. Subsequent injection on a C(18) reverse-phase column employing acetonitrile and water (20:80) as the mobile phase reduces the analysis time without affecting either resolution of the peak or the accuracy of caffeine and theobromine determination or achieving baseline resolution. Therefore, this method is ideally suited for rapid routine analysis of cocoa and its products.     

Determination of saccharin, sodium benzoate, and caffeine in beverages by reverse phase high-pressure liquid chromatography.

A reverse phase high-pressure liquid chromatographic method is presented for the simultaneous separation and determination of sacchrain, sodium benzoate, and caffeine in soft drinks, fruit juices, fruit cocktails, fruit punches, coffee, and artificial sweetener concentrates. Decarbonated soft drinks, fruit punches, and artificial sweetener concentrates are injected directly into the chromatograph. Fruit juices and coffee solutions require filtration through a 0.45 mum pore membrane filter prior to injection. Samples are eluted from a mu-Bondapak/C18 column with 5% glacial acetic acid and are quantitated with an ultraviolet detector. The results of saccharin, sodium benzoate, and caffeine determinations in 34 soft drinks (representing 11 manufacturers and 20 flavors); 8 fruit juices, cocktails, and punches; 7 coffees; and 6 artificial sweetener concentrates are presented. Average recoveries of saccharin, sodium benzoate, and caffeine from soft drinks are 99.0, 99.3, and 100.2%, respectively.     

Determination of anthraquinone in technical material,formulations, and lettuce by high performance liquid chromatography

Foraging on lettuce seeds and seedlings by horned larks (Eremophila alpestris) causes millions of dollars in losses to the California lettuce crop annually. Anthraquinone (AQ; 9,10-anthracenedione) has been shown to deter pest birds from consuming the seeds and seedlings of several plant species and was evaluated as a repellent to horned larks when applied to lettuce seedlings. A set of analytical methods using simple liquid extraction followed by high-performance liquid chromatography analysis were developed for the quantitation of AQ as technical material, as an active ingredient in a commercial formulation, and as a residue in lettuce plants. The methods were easy, reliable, and repeatable. AQ recoveries from control formulation fortified to concentrations of either 24 or 600 mg g(-)(1) were 99 (+/-1.2%) and 98% (+/-1.2%), respectively, with a control formulation method limit of detection (MLOD) of 0.50 mg g(-)(1). Control lettuce tissues from three growth stages were AQ-fortified to concentrations of 0.50 and 500 microg g(-)(1). The resulting AQ recoveries for the two fortification levels were 99 (+/-8.5) and 89% (+/-1.5%) for 11 day old seedlings, 95 (+/-2.6%) and 86% (2.1%) for 16 day old plants, and 92 (+/-1.4%) and 93% (+/-1.1%) for adult head lettuce cover leaves, respectively. The MLODs for the same three lettuce tissues were 0.055, 0.058, and 0.077 microg g(-)(1), respectively. These methods were used to quantify AQ residues from field-grown, treated lettuce and associated fortified quality control samples.     

Improved liquid chromatography methods for the separation and quantification of biotin in NIST standard reference material 3280: multivitamin/multielement tablets

Two independently developed liquid chromatography (LC) methods for the quantitative determination of biotin in multivitamin/multielement tablets (NIST Standard Reference Material 3280 (SRM 3280)) are described. The methods use distinctly different tablet extraction solvents (methanol vs 1.5% aqueous formic acid) and analyte detection principles (mass spectrometry (MS) versus evaporative light-scattering detection (ELSD)) to ensure quantitative reliability. The use of different extraction and detection procedures allows cross-validation of the methods and enhances confidence in the final quantitative results. Both methods yield highly comparable results for the mean level of biotin (LC/MS = 26.5 mg/kg +/- 0.3 mg/kg (n = 12); LC/ELSD = 24.7 mg/kg +/- 1.7 mg/kg (n = 12)) in SRM 3280, yet the methods differ considerably in their analytical characteristics. The isotope-dilution LC/MS method exhibits excellent linearity from 0.02 ng to 77 ng biotin on-column with a method limit of detection (LOD) and limit of quantification (LOQ) of 0.02 ng (S/N > 3) and 0.06 ng (S/N > 10) biotin on-column, respectively. The LC/ELSD method exhibits good linearity from 155 ng to 9900 ng biotin on-column with a method LOD and LOQ of 155 ng (S/N > 3) and 310 ng (S/N > 10) biotin on-column, respectively. Method performance data indicates that the LC/MS method is analytically superior to the LC/ELSD method; however, either method in combination with SRM 3280 should provide quality assurance, accuracy, and traceability for biotin levels in multivitamin/multielement dietary supplements.     

Determination of bisphenol a and bisphenol B residues in canned peeled tomatoes by reversed-phase liquid chromatography

Bisphenol A (BPA) and bisphenol B (BPB) concentrations were determined in peeled canned tomatoes of different brands bought in Italian supermarkets. Tomato samples analyzed were packaged in cans coated with either epoxyphenolic lacquer or low BADGE enamel. A solid phase extraction (SPE) was performed on C-18 Strata E cartridge followed by a step on Florisil cartridge. Detection and quantitation were performed by a reversed phase high-performance liquid chromatography (RP-HPLC) method with both UV and fluorescence detection (FD). On the total of 42 tested tomato samples, BPA was detected in 22 samples (52.4%), while BPB was detected in 9 samples (21.4%). BPA and BPB were simultaneously present in 8 of the analyzed samples. The levels of BPA found in this study are much lower than the European Union migration limits of 3 mg/kg food and reasonably unable to produce a daily intake exceeding the limit of 0.05 mg/kg body weight, established by European Food Safety Authority.     

Identification of procyanidins in cocoa (Theobroma cacao) and chocolate using high-performance liquid chromatography/mass spectrometry

Monomeric and oligomeric procyanidins present in cocoa and chocolate were separated and identified using a modified normal-phase high-performance liquid chromatography (HPLC) method coupled with on-line mass spectrometry (MS) analysis using an atmospheric pressure ionization electrospray chamber. The chromatographic separation was achieved using a silica stationary phase in combination with a gradient ascending in polarity. This qualitative report confirms the presence of a complex series of procyanidins in raw cocoa and certain chocolates using HPLC/MS techniques. Although both cocoa and chocolate contained monomeric and oligomeric procyanidin units 2-10, only use of negative mode provided MS data for the higher oligomers (i.e., >pentamer). Application of this method for qualitative analysis of proanthocyanidins in other food products and confirmation of this method as a reliable quantitative tool for determining levels of procyanidins in cocoa, chocolate, and other food products are currently being investigated.     

Determination of neomycin in animal tissues, using ion-pair liquid chromatography with fluorometric detection

A liquid chromatographic (LC) method is described for the determination of neomycin in animal tissues. Tissues are homogenized in 0.2M potassium phosphate buffer (pH 8.0); the homogenate is centrifuged, and the supernate is heated to precipitate the protein. The heat-deproteinated extract is acidified to pH 3.5-4 and directly analyzed by LC. The LC method consists of an ion-pairing mobile phase, a reverse phase ODS column, post-column derivatization with o-phthalaldehyde reagent, and fluorometric detection. The LC method uses paromomycin as an internal standard, and separates neomycin from streptomycin or dihydrostreptomycin because they have different retention times. The LC column separates neomycin in 25 min; the detection limit is about 3.5 ng neomycin. The overall recovery of neomycin from kidney tissues spiked at 1-30 ppm was 96% with a 9.0% coefficient of variation. The method was also applied to muscle tissue.     

Determination of vitamin D3 in liquid multivitamin preparation, using reverse phase, solid phase extraction liquid chromatography

A method is described for the determination of vitamin D3 in a liquid multivitamin preparation by liquid chromatography. Samples are purified on a disposable reverse phase extraction (SPE) column with a mobile phase of methanol-2-propanol (97 + 3) and are analyzed on a Zorbax ODS (5 micron) column with an acetonitrile-2-propanol-water (90 + 8 + 2) solvent system. Vitamin D3 is completely resolved from other interfering compounds within approximately 21 min and is detected with a UV detector at 254 nm. A mean of 98.5% of theory with a coefficient of variation of 3.8% was found for determination of vitamin D3 in a commercial preparation.     

Determination of tetracycline antibiotics in beef and pork tissues using ion-paired liquid chromatography

A simplified procedure was developed for determination of tetracycline antibiotics in tissues which improved stability of these compounds in sample extracts and eliminated the need for troublesome cleanup procedures. Tissues were homogenized in water. Acetonitrile (16 mL) and then 1 mL of 0.1 M H(3)PO(4) were added to 4 mL of homogenate and the clear supernatant was filtered. The filtrate was mixed with hexane and dichloromethane and the resulting water layer was collected, evaporated to 1-2 mL, and filtered into autosampler vials. Ion-pairing liquid chromatography was used to separate tetracyclines from interferences in sample extracts, eliminating the need for further cleanup. Analysis was isocratic using a Phenomonex Prodigy ODS(3) column with a mobile phase of 4 mM oxalic acid, 4 mM sodium oxalate, 10 mM sodium decanesulfonate-acetonitrile (70 + 30 for oxytetracycline and tetracycline; 66 + 34 for chlortetracycline). Recoveries were generally in the 90-100% range with limits of quantitation of 0. 05-0.1 ppm. The procedure was evaluated with beef and pork muscle, liver, and kidney.     

Determination of methylglyoxal in ruminal fluid by high-performance liquid chromatography using fluorometric detection

There is no reported method for the quantification of methylglyoxal in ruminal fluid. The method reported here is based on the conversion of methylglyoxal to 6-methylpterin, followed by quantification of the resulting pteridinic compound by fluormetric detection using liquid chromatography. Ruminal fluid was collected and preserved with 1 M HCl at -20 degrees C. Cation exchange prior to derivatization was used to eliminate possible interfering peaks. The detection limit of 0.125 microg/mL was calculated. The recoveries were >80%, and the coefficients of variation were <15%. This method has proven to be rugged and accurate for the detection of methylglyoxal concentration in ruminal fluid collected from cows fed diets deficient in degradable intake protein as a marker. Methylglyoxal is produced by ruminal bacteria in response to low nitrogen levels in the rumen. The ruminal methylglyoxal concentration has the potential to be a useful marker to assess ruminal nitrogen status to aid in more accurate diet formulation.     

Determination of levamisole in animal tissues using liquid chromatography with ultraviolet detection.

An efficient and sensitive liquid chromatographic method is described for the determination of the anthelminthic drug levamisole, in muscle, liver, kidney and fat of sheep, pigs and poultry, using thiabendazole as internal standard. Samples were extracted by homogenizing with chloroform, and were applied to Supelco Si solid-phase extraction columns and eluted with methanol. Chromatographic analysis was performed on a LiChrospher 60 RP-Select B column using methanol/ammonium acetate buffer 0.05 M (55/65, v/v) as mobile phase and reading at 220 nm. The quantification limit for the assay was 4 ng/g. Mean recoveries were about 84% for liver, 85% for kidney, 89% for muscle and 84% for fat. The assay has been used for statutory testing purposes.     

Analysis of pesticides in nuts by online reversed-phase liquid chromatography-gas chromatography using the through-oven transfer adsorption/desorption interface

A new method to determine pesticide residues in nuts is presented, in which the pesticides are extracted from samples with a small amount of ethyl acetate and anhydrous sodium sulfate. No additional cleanup or concentration steps are necessary. The extract is directly injected into the high-pressure liquid chromatograph, where preseparation of the pesticide residues from other components coextracted from the nuts is carried out using methanol/water as the eluent. The selected liquid chromatography fraction containing the pesticides is automatically transferred to the gas chromatograph using the through-oven transfer adsorption/desorption interface. The calculated limits of detection for each pesticide varied from 0.1 to 61.3 microg/kg. The repeatabilities of the analysis and the overall procedure (extraction and analysis) were satisfactory. No variations in the retention time were observed. The method was applied to the analysis of pistachio nut, peanut, walnut, hazelnut, and sunflower seed.     

Quantification of milk fat in chocolate fats by triacylglycerol analysis using gas-liquid chromatography

The development and in-house testing of a method for the quantification of milk fat in chocolate fats is described. A database consisting of the triacylglycerol profiles of 310 genuine milk fat samples from 21 European countries and 947 mixtures thereof with chocolate fats was created under a strict quality control scheme using 26 triacylglycerol reference standards for calibration purposes. Out of the individual triacylglycerol fractions obtained, 1-palmitoyl-2-stearoyl-3-butyroyl-glycerol (PSB) was selected as suitable marker compound for the determination of the proportion of milk fat in chocolate fats. By using PSB values from the standardized database, a calibration function using simple linear regression analysis was calculated to be used for future estimations of the milk fat content. A comparison with the widely used butyric acid method, which is currently used to determine the milk fat content in nonmilk fat mixtures, showed that both methods were equivalent in terms of accuracy. The advantage of the presented approach is that for further applications, i.e., determination of foreign fats in chocolate fats, just a single analysis is necessary, whereas for the same purpose, the C4 method requires two different analytical methods.     

Determination of naptalam and its metabolite in foods, as 1-naphthylamine, using liquid chromatography with oxidative electrochemical detection

A new method is described for the determination of the herbicide naptalam and its metabolite 1-naphthylamine in several foods. The method is sensitive, selective, and extremely rapid compared with previously reported methods. Liquid chromatography with electrochemical detection (LC/ECD) is used to determine 1-naphthylamine produced from the metabolism or base hydrolysis of naptalam in asparagus, peaches, and cranberries. These foods were spiked with naptalam at 0.05 and 0.11 ppm and hydrolyzed with 30% NaOH with concomitant distillation of 1-naphthylamine. Aliquots of the distillate were injected onto a reverse-phase PRP-1 LC column for separation of 1-naphthylamine from coextractives near the solvent front and detection at an applied potential of +0.83 V using an amperometric electrochemical detector in the oxidation mode. Recoveries ranged from 89% +/- 2% to 97% +/- 8% for all foods at both spiking levels. Accuracy of these recoveries was confirmed by use of 14C-radiolabeled naptalam and radioassay by liquid scintillation spectrometry of the 14C-1-naphthylamine released.     

Determination of novobiocin residues in milk, blood, and tissues by liquid chromatography

Residues of novobiocin in milk, blood, and tissues can be detected by microbiological tests but cannot be distinguished from other antibiotics. A simple liquid chromatographic (LC) method was developed for identification of residues. Tissues were blended and milk and blood serum were mixed with 0.2M NH4H2PO4. The mixture was deproteinized by adding aqueous methanol and filtering. The LC apparatus consisted of a variable wavelength detector, set at 340 nm, an automatic loop injector, and a C18 column with guard cartridge. The flow rate was 1 mL/min and the solvent mixture of 0.01M H3PO4-acetonitrile-methanol was programmed from 50 + 0 + 50 (0-1 min) to 20 + 80 + 0 (20 min). Novobiocin was concentrated directly by solid-phase extraction on the analytical column. Five or more 200 microL aliquots of the filtrate in water-methanol (1 + 1) (adjusted if necessary) were injected with the column solvent at 50 + 0 + 50. After the final injection, the program was run to completion. Recoveries were 90-100% with sensitivities of 0.05 ppm or less. The procedure should be adaptable for use with formulations and feeds.     

Optimization and validation of the reversed-phase high-performance liquid chromatography with fluorescence detection method for the separation of tocopherol and tocotrienol isomers in cereals, employing a novel sorbent material

The separation and determination of tocopherols (Ts) and tocotrienols (T3s) by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed and validated after optimization of various chromatographic conditions and other experimental parameters. Analytes were separated on a PerfectSil Target ODS-3 (250 × 4.6 mm, 3 μm) column filled with a novel sorbent material of ultrapure silica gel. The separation of Ts and T3s was optimized in terms of mobile-phase composition and column temperature on the basis of the best compromise among efficiency, resolution, and analysis time. Using a gradient elution of mobile phase composed of isopropanol/water and 7 °C column temperature, a satisfactory resolution was achieved within 62 min. For the quantitative determination, α-T acetate (50 μg/mL) was used as the internal standard. Detection limits ranged from 0.27 μg/mL (γ-T) to 0.76 μg/mL (γ-T3). The validation of the method was examined performing intraday (n = 5) and interday (n = 3) assays and was found to be satisfactory, with high accuracy and precision results. Solid-phase extraction provided high relative extraction recoveries from cereal samples: 87.0% for γ-T3 and 115.5% for δ-T. The method was successfully applied to cereals, such as durum wheat, bread wheat, rice, barley, oat, rye, and corn.