Convenient analysis of vitamin D in cheese and other food matrixes by liquid chromatography/mass spectrometry

A convenient method is presented for determination of vitamin D in natural cheese, processed cheese, milk, cereals, noncarbonated soft drinks, and juice by liquid chromatography/ mass spectrometry (LC/MS). Samples were saponified, extracted, evaporated, redissolved in acetonitrile, and injected into an LC-atmospheric pressure chemical ionization-MS system with no preparative chromatographic steps. Vitamin D was determined by selected ion monitoring. MS response was linear for vitamin D3 and its internal standard vitamin D2, and overall average recoveries ranged from 98 to 105%. A blending experiment in which shredded vitamin D3-fortified cheddar was mixed with control nonfortified cheddar showed linearity. The limit of detection for vitamin D was 1.3 ng and the limit of quantitation was 3 ng. The method gave good accuracy and precision, with a standard deviation of 9.5 and relative standard deviation of 6.7%. Results for vitamin D3 obtained with this method for different food matrixes, at different levels, were in agreement with those obtained with the reference LC/UV method currently used by many laboratories and derived from AOAC Official Method 982.29.     

液相色谱-大气压化学电离串联质谱法测定婴幼儿配方奶粉中的维生素D

建立了婴幼儿配方奶粉中维生素D的液相色谱-大气压化学电离串联质谱(LC-APCI-MS/MS)分析方法。样品经正己烷和甲基叔丁基醚混合溶液提取,ProElut VDC固相萃取柱净化,Kinetex C_(18)色谱柱分离,采用大气压化学电离(APCI)源、正离子扫描和多反应监测(MRM)模式对维生素D_2和维生素D_3进行检测,内标法定量。结果表明维生素D_2和维生素D_3在5~5 000μg/L范围内均具有良好的线性关系,检出限为2μg/kg,定量限为5μg/kg。在5、10和100μg/kg添加水平下,维生素D_2和维生素D_3的回收率为85.2%~105.3%,相对标准偏差为4.7%~8.1%。该方法简便准确,灵敏度高,适用于婴幼儿奶粉中维生素D的测定。     

Liquid chromatographic analysis of vitamin B6 in reconstituted infant formula: collaborative study

A liquid chromatographic (LC) method was validated for the determination of total vitamin B6 in infant formula. Total vitamin B6 was quantified by converting the phosphorylated and free vitamers into pyridoxine. Pyridoxine was determined by ion pair reversed-phase LC with fluorescence detection. The method was subjected to an AOAC collaborative study involving a factory-manufactured, milk- and soy-based infant formula. Each was spiked at 3 concentrations in the range of 0-1 microg/g and sent as blind duplicate to participant laboratories. Nine laboratories returned valid data which were statistically analyzed for outliers and precision parameters. The repeatability relative standard deviation (RSD(r)) ranges were 2.0-4.0 and 3.5-5.9% for fortified milk- and soy-based formulas, respectively. The reproducibility relative standard deviation (RSD(R)) ranges were 8.2-8.4 and 6.7-11.2% for fortified milk- and soy-based formulas, respectively. HORRAT values ranged from 0.42 to 0.53, indicating that the precision of the method is acceptable. The mean RSD(r):RSD(R) values were 0.60 and 0.55 for milk- and soy-based formulas, respectively. As expected, RSDs for the unfortified samples were higher, but their HORRAT values (0.81 and 2.06) helped define a realistic limit of quantitation as 0.05 microg/g. Recovery data were quantitative and varied between 81.4 and 98.0% (mean = 89.8%) for each of 6 spiked materials.     

Application of ultra-performance liquid chromatography/tandem mass spectrometry for the measurement of vitamin D in foods and nutritional supplements

Ultra-performance liquid chromatography (UPLC)/MS/MS was applied to measure vitamin D in various foods and nutritional supplements. The run-time of the chromatographic separation was cut from 20 min in HPLC/MS/MS to 10 min in UPLC/MS/MS, while equal or better separation efficiency was achieved to deal with complex food matrixes. Under the optimized conditions, all the previtamins of vitamin D3, D2, and isotope-labeled vitamin D3 were baseline-separated from their corresponding vitamins. It was also demonstrated that many sterol isomers in complex food matrixes that interfere in the analysis could be well-separated from the analytes. Accuracy of this method was evaluated by analysis of NIST SRM 1849 infant formula reference material. With eight replicates, the average vitamin D3 concentration was 0.251 +/- 0.012 mg/kg, an excellent agreement with the certified value of 0.251 +/- 0.027 mg/kg. In addition, spike recovery from a commercial infant formula matrix was in the range of 100 to 108% for both vitamins D3 and D2 at three spike concentration levels. The spike recovery for an even more complex matrix, pet food, was 101-105%. LOQ values were 0.026 and 0.033 IU/g, or 0.086 and 0.11 IU/mL in solution, for vitamins D3 and D2, respectively. The dynamic range had three orders of magnitude, which made the method flexible and useful to deal with the wide concentration range of vitamin D in various samples. The method was robust based on the results of changing the parameters of LC separation and MS measurement. This accurate and reliable vitamin D method increased instrument efficiency and analysis productivity significantly.     

Application of ultra-high-performance liquid chromatography/tandem mass spectrometry for the measurement of vitamin D in infant formula and adult/pediatric nutritional formula: First Action 2011.11

In an effort to measure vitamin D, ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) was applied to samples. The use of UHPLC-MS/MS decreased the run time by 50%. The UHPLC-MS/MS achieved equal or better separation efficiency with complex food matrixes compared to HPLC-MS/MS. It was also observed that under the optimized conditions of UHPLC, all previtamins of vitamin D3, D2, and isotope-labeled vitamin D3 were baseline-separated from their corresponding vitamins. The sterol isomers found in complex food matrixes that interfere in the analysis were well separated from the analytes. The accuracy of the method was evaluated by analyzing National Institute of Standards and Technology Standard Reference Material 1849 infant reference material. The average vitamin D3 concentration was 0.251 +/- 0.012 microg/g. This showed excellent agreement with the certified value of 0.251 +/- 0.027 microg/g. The spike recovery study of a commercial infant formula matrix showed a range of recovery from 100 to 108%. The LOQ values determined were 0.0022 and 0.0028 microg/g for vitamins D3 and D2, respectively; LOD values were 0.00065 and 0.00083 microg/g for vitamins D3 and D2, respectively.     

Determination of vitamin A (retinol) in infant formula and adult nutritionals by liquid chromatography: First Action 2011.15

During the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting," held on June 29, 2011, an Expert Review Panel (ERP) reviewed the method for the "Determination of Vitamins A (Retinol) and E (alpha-Tocopherol) in Foods by Liquid Chromatography: Collaborative Study," published by Jonathan W. DeVries and Karlene R. Silvera in J. AOAC Int. in 2002. After evaluation of the original validation data, an ERP agreed in June 2011 that the method meets standard method performance requirements (SMPRs) for vitamin A, as articulated by the Stakeholder Panel on Infant Formula and Adult Nutritionals. The ERP granted the method First Action status, applicable to determining vitamin A in ready-to-eat infant and adult nutritional formula. In an effort to achieve Final Action status, it was recommended that additional information be generated for different types of infant and adult nutritional formula matrixes at varied concentration levels as indicated in the vitamin A (retinol) SMPR. Existing AOAC LC methods are suited for specific vitamin A analytical applications. The original method differs from existing methods in that it can be used to assay samples in all nine sectors of the food matrix. One sector of the food matrix was powdered infant formula and gave support for the First Action approval for vitamin A in infant and adult nutritional formula. In this method, standards and test samples are saponified in basic ethanol-water solution, neutralized, and diluted, converting fats to fatty acids and retinol esters to retinol. Retinol is quantitated by an LC method, using UV detection at 313 or 328 nm for retinol. Vitamin concentration is calculated by comparison of the peak heights or peak areas of retinol in test samples with those of standards.     

A rapid analytical method for cholecalciferol (vitamin D3) in fortified infant formula, milk and milk powder using Diels-Alder derivatisation and liquid chromatography-tandem mass spectrometric detection

A method for analysing vitamin D₃ (VD3, cholecalciferol) has been established and validated. This method is rapid and cost effective and is intended for use in quality control in the manufacture of fortified infant formulae and milk powders. Milk or reconstituted milk powder was solubilised in methanol and extracted in one step into isooctane, which was separated by centrifugation. A portion of the isooctane layer was then transferred, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione was added to derivatise VD3. The analyte was then re-extracted into a small volume of acetonitrile and analysed by reverse-phase chromatography. Detection was by triple quadrupole mass spectrometer using a selective transition, m/z 560 → 298. An internal standard, deuterium-labelled VD3, was used to correct for losses in extraction and any variation in derivatisation and ionisation efficiencies. The method has been subjected to a single-laboratory validation and has been found to be linear, highly selective and accurate with respect to National Institute of Standards and Technology Standard Reference Material 1849, analyte spiking experiments and comparison with an LC–UV-based method. The repeatability standard deviation was 4.23 %. Significantly for routine laboratories, the method returns results within 2 h, generates minimal waste and minimises health and safety concerns to the analyst.     

Determination of pantothenic acid in foods by optical biosensor immunoassay

An optical biosensor inhibition immunoassay was developed using a specific pantothenic acid-binding protein for the quantitation of free pantothenic acid (vitamin B5) in foodstuffs. Samples were prepared by a simple extraction procedure in buffer, and vitamin content was estimated against authentic calibrants in the same buffer. Performance parameters included a working range of 10-5000 ng/mL, a limit of detection of 4.4 ng/mL, precision relative standard deviation of 5.4-7.1% over a range of concentrations, and recoveries > 95% in the matrixes tested. A wide range of foodstuffs, including National Institute of Standards and Technology reference samples, were tested in 3 independent laboratories and the results were compared with microbiological assay and liquid chromatography/mass spectrometry (LC/MS) methods. The results indicate that the biosensor technique is appropriate for the estimation of pantothenic acid in a wide range of foodstuffs.     

Determination of zearalenone in barley, maize and wheat flour, polenta, and maize-based baby food by immunoaffinity column cleanup with liquid chromatography: interlaboratory study

An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the effectiveness of an affinity column cleanup liquid chromatography (LC) method for the determination of zearalenone (ZON) in a variety of cereals and cereal products at proposed European regulatory limits. The test portion is extracted with acetonitrile:water. The sample extract is filtered, diluted, and applied to an affinity column. The column is washed, and ZON is eluted with acetonitrile. ZON is quantified by reversed-phase LC with fluorescence detection. Barley, wheat and maize flours, polenta, and a maize-based baby food naturally contaminated, spiked, and blank (very low level) were sent to 28 collaborators in 9 European countries and 1 collaborator in New Zealand. Participants were asked to spike test portions of all samples at a ZON concentration equivalent to 100 microg/kg. Average recoveries ranged from 91-111%. Based on results for 4 artificially contaminated samples (blind duplicates) and 1 naturally contaminated sample (blind duplicate), the relative standard deviation for repeatability (RSDr) ranged from 6.9-35.8%, and the relative standard deviation for reproducibility (RSDR) ranged from 16.4-38.2%. The method showed acceptable within- and between-laboratory precision for all 5 matrixes, as evidenced by HorRat values <1.7.     

Interlaboratory study of a multiresidue gas chromatographic method for determination of organochlorine and pyrethroid pesticides and polychlorobiphenyls in milk,fish, eggs,and beef fat

An interlaboratory study was conducted to validate a gas chromatographic (GC) method for determination of 21 organochlorine pesticides, 6 pyrethroid pesticides, and 7 polychlorobiphenyl (PCB) congeners in milk, beef fat, fish, and eggs. The method was performed at low contamination levels, which represent relevant contents in food, and is an extension of the European standard (method NF-EN-1528, Parts 1-4). It enlarges the applicable scope of the reference EN method to pyrethroid pesticides and proposes the use of solid-phase extraction (SPE) as a cleanup procedure. Cryogenic extraction was made, and SPE cleanup was performed with 2 successive SPE cartridges: C18 and Florisil. After injection of the purified extract onto a GC column, residues were measured by electron capture detection. Food samples (liquid milk, beef fat, mixed fish, and mixed eggs) were prepared, tested for homogeneity, and sent to 17 laboratories in France. Test portions were spiked with 27 pesticides and 7 PCBs at levels from 26 to 45, 4 to 27, 31 to 67, and 19 to 127 ng/g into milk, eggs, fish, and fat, respectively. Based on results for spiked samples, the relative standard deviation for repeatability ranged from 1.5 to 6.8% in milk, 3 to 39% in eggs, 4.5 to 12.2% in fish, and 7 to 13% in fat. The relative standard deviation for reproducibility ranged from 33 to 50% in milk, 29 to 59% in eggs, 31 to 57% in fish, and 30 to 62% in fat. This method showed acceptable intra- and interlaboratory precision data, as corroborated by HORRAT values at low levels of pesticide and PCB contamination. The statistical evaluation of the results was performed according to the International Organization for Standardization (ISO; ISO 3534 standard) and 5725-2 Guideline.     

Development and validation of a method for the quantification of milk proteins in food products based on liquid chromatography with mass spectrometric detection

The protection of allergic consumers is crucial to the food industry. Therefore, accurate methods for the detection of food allergens are required. Targeted detection of selected molecules by MS combines high selectivity with accurate quantification. A confirmatory method based on LC/selected reaction monitoring (SRM)-MS/MS was established and validated for the quantification of milk traces in food. Tryptic peptides of the major milk proteins beta-lactoglobulin, beta-casein, alphaS2-casein, and K-casein were selected as quantitative markers. Precise quantification was achieved using internal standard peptides containing isotopically labeled amino acids. For each peptide, qualifier and quantifier fragments were selected according to Commission Decision 2002/657/EC. A simple sample preparation method was established without immunoaffinity or SPE enrichment steps for food matrixes containing different amounts of protein, such as baby food, breakfast cereals, infant formula, and cereals. Intermediate reproducibility, repeatability, accuracy, and measurement uncertainty were determined for each matrix. LOD values of 0.2-0.5 mg/kg, e.g., for beta-lactoglobulin, were comparable to those obtained with ELISA kits. An LOQ of approximately 5 mg/kg, expressed as mass fraction skim milk powder, was validated in protein-rich infant cereals. The obtained validation data show that the described LC/SRM-MS/MS approach can serve as a confirmatory method for the determination of milk traces in selected food matrixes.     

Determination of deoxynivalenol in cereals and cereal products by immunoaffinity column cleanup with liquid chromatography: interlaboratory study

An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatographic (LC) method for the determination of deoxynivalenol in a variety of cereals and cereal products at proposed European regulatory limits. The test portion was extracted with water. The sample extract was filtered a applied to an immunoaffinity column. After being washed with water, the deoxynivalenol was eluted with acetonitrile or methanol. Deoxynivalenol was quantitated by reversed-phase LC with UV determination. Samples of artificially contaminated wheat-flour, rice flour, oat flour, polenta, and wheat based breakfast cereal, naturally contaminated wheat flour, and blank (very low level) samples of each matrix were sent to 13 collaborators in 7 European countries. Participants were asked to spike test portions of all samples at a range of deoxynivalenol concentrations equivalent to 200-2000 ng/g deoxynivalenol. Average recoveries ranged from 78 to 87%. Based on results for 6 artificially contaminated samples (blind duplicates), the relative standard deviation for repeatability (RSDr) ranged from 3.1 to 14.1%, and the relative standard deviation for reproducibility (RSDR) ranged from 11.5 to 26.3%. The method showed acceptable within-laboratory and between-laboratory precision for all 5 matrixes, as evidenced by HorRat values < 1.3.     

An interlaboratory-verified method for the determination of vitamins A and E in milk- and soy-based infant formula by liquid chromatography with matrix solid-phase dispersion extraction

An interlaboratory-verified, liquid chromatographic (LC) method is presented for determination of all-racemic alpha-tocopheryl acetate and retinyl palmitate in infant formula. The extraction procedure uses matrix solid-phase dispersion. A sample is mixed with C18, and the mixture is packed into a reservoir and eluted with selective solvents to extract the analytes. After evaporation and filtration, the sample extract is injected directly into a normal-phase LC system with fluorescence detection. All-racemic alpha-tocopheryl acetate and retinyl palmitate are quantitated isocratically with a mobile phase of hexane containing isopropanol at 0.2% (v/v) and 0.125% (v/v), respectively. A nonfortified zero control reference material (ZRM) was spiked at 5 levels, with 5 replicate analyses of 1/2x, x, 2x, 4x, and 16x where "x" represents the minimum levels of 250 IU/100 kcal (vitamin A) and 0.7 IU/100 kcal (vitamin E) as specified in Title 21 of the Code of Federal Regulations, part 107.100. Recoveries of retinyl palmitate ranged from 83.8 to 107%, and those of all-racemic alpha-tocopheryl acetate ranged from 87.7 to 108%. Two additional laboratories analyzed the ZRM samples at 4 spiking levels with 6 replicates. Recoveries of retinyl palmitate and all-racemic alpha-tocopheryl acetate ranged from 92.2 to 104% and from 91.7 to 101%, respectively, in the second laboratory. Recoveries of retinyl palmitate and all-racemic alpha-tocopheryl acetate ranged from 85.3 to 97.0% and from 86.6 to 110%, respectively, in the third laboratory. Relative standard deviations for all 3 laboratories ranged from 0.2 to 7.5% with an average of 2.9%. In addition, each laboratory analyzed a commercial milk- and commercial soy-based infant formula. Excellent agreement in results was obtained between the 3 laboratories for vitamins A and E in all matrixes.     

Determination of aflatoxin B1 in baby food (infant formula) by immunoaffinity column cleanup liquid chromatography with postcolumn bromination: collaborative study

A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for determination of aflatoxin B, in a milk powder based infant formula at a possible future European regulatory limit (0.1 ng/g). The test portion was extracted with methanol-water (8 + 2 [v + v]), filtered, diluted with water, and applied to an immunoaffinity column. The column was washed with water to remove interfering compounds, and the purified aflatoxin B1 was eluted with methanol. The separation and determination of the aflatoxin B1 was performed by reversed-phase LC and detected by fluorescence after postcolumn derivatization (PCD) involving bromination. PCD was achieved with either pyridinum hydrobromide perbromide (PBPB) or an electrochemical (Kobra) cell by addition of bromide to the mobile phase. The baby food (infant formula) test samples, both spiked and naturally contaminated with aflatoxin B1, were sent to 14 laboratories in 13 different European countries. Test portions were spiked at levels of 0.1 and 0.2 ng/g for aflatoxin B1. Recoveries ranged from 101 to 92%. Based on results for spiked test samples (blind pairs at 2 levels) and naturally contaminated test samples (blind pairs at 3 levels), the relative standard deviation for repeatability (RSDr) ranged from 3.5 to 14%. The relative standard deviation for reproducibility (RSDR) ranged from 9 to 23%. Nine participants used PBPB derivatization, and     

Determination of retinyl palmitate (vitamin A) in fortified fluid milk by liquid chromatography: collaborative study

A liquid chromatographic (LC) method was developed for fast and simple measurement of retinyl palmitate (vitamin A) in fortified milk. Retinyl acetate internal standard was added to a test portion of milk followed by extraction into hexane. The hexane extract was analyzed by LC using a normal-phase silica gel column equilibrated with mobile phase (conditioned hexane-isopropanol, 99.85 + 0.15, v/v) about 1 h before injections. The retinyl palmitate concentration was calculated by using a relative response factor determined with calibration standards. In the collaborative study, 11 laboratories analyzed 13 pairs of fluid milk materials in blind duplicate. Twelve of the materials were composed of skim milk (< 0.5% fat), 1% fat milk, 2% fat milk, and 1% fat chocolate milk. Each material was fortified at 3 concentrations of retinyl palmitate of approximately 581 microg/L (1000 IU/qt), 1163 microg/L (2000 IU/qt), and 2236 microg/L (4000 IU/qt). The 13th material, unfortified skim milk, served as a matrix blank. Repeatability standard deviations (RSDr) without outliers ranged from 1.5 to 5.7% and reproducibility standard deviations (RSDR) without outliers ranged from 5.0 to 22.7%. cis-Isomers co-eluted with the predominant trans-retinyl palmitate isomer and were included in the results reported by all the collaborative laboratories. Endogenous long-chain esters from milk fat were also measured with the retinyl palmitate additive. The Study Director recommends that this method for determination of retinyl palmitate in fluid milk by LC be adopted First Action.     

Determination of methylcellulose and hydroxypropyl methylcellulose food gums in food and food products: collaborative study

A collaborative study was performed to determine the reproducibility of a method for the determination of methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC) in food. These widely used food gums possess unusual solubility characteristics and cannot accurately be determined by existing dietary fiber methods. The new method uses the enzyme-digestion procedure of AOAC Official Method 991.43. Digestate solutions must be refrigerated to fully hydrate MC or HPMC. The chilled solutions are filtered and analyzed by size-exclusion liquid chromatography. Collaborating laboratories received 28 samples containing MC or HPMC in the range of 0-100%. The sample set included blind duplicates of 5 food matrixes (bread, milk, fish, potato, and powdered juice drink). Cochran and Grubbs tests were used to eliminate outliers. For food samples containing MC, values for within-laboratory precision, repeatability relative standard deviation (RSDr), ranged from 4.2 to 16%, and values for among-laboratories precision, reproducibility relative standard deviation (RSDR), ranged from 11 to 20%. For HPMC samples, RSDr values ranged from 6.4 to 27%, and RSDR values ranged from 17 to 39%. Recoveries of MC and HPMC from the food matrixes ranged from 78 to 101%. These results show acceptable precision and reproducibility for the determination of MC and HPMC, for which no Official AOAC Methods exist. It is recommended that this method be adopted as AOAC Official First Action.     

Determination of isopropylthioxanthone (ITX) in milk, yoghurt and fat by HPTLC-FLD, HPTLC-ESI/MS and HPTLC-DART/MS

Two new HPTLC methods for quantification of isopropyl-9H-thioxanthen-9-one (ITX) in milk, yoghurt and fat samples have been developed. Extraction of ITX from milk and yoghurt was performed with a mixture of cyclohexane and ethyl acetate by employment of accelerated solvent extraction (ASE). For soy bean oil and margarine, a simple partitioning of ITX into acetonitrile was used. ITX and 2,4-diethyl-9H-thioxanthen-9-one (DTX) used as internal standard have been separated on silica gel 60 HPTLC plates with a mixture of toluene and n-hexane (4:1, v/v) and on RP18 HPTLC plates with a mixture of acetonitrile and water (9:1, v/v). Development was performed anti-parallel from both plate sides leading to a throughput of 36 separations in 7 min. Fluorescence measurement at 254/>400 nm was used for quantification. Limits of detection (S/N of 3) have been established to be 64 pg for ITX and DTX on both types of HPTLC plates. In fatty matrix (spiked butter) LOD of ITX was determined to be 1 μg kg⁻¹. In the working range monitored (20–200 μg kg⁻¹) polynomial regression of ITX showed a relative standard deviation (sdv) of ±1.51 % (r=0.99981). Starting with the limit of quantification the response was linear (sdv=±2.18 %, r=0.99893). Regarding repeatability (n=9) a coefficient of variation (CV) of 1.1 % was obtained for ITX at 32 ng on silica gel plates and of 2.9 % on reversed-phase plates. Repeatabilities (n=4) of ITX determination at 20, 50 and 100 μg kg⁻¹ in milk, yoghurt, soybean oil and margarine showed CVs between ±1.0 and 6.4 %. The results prove that modern planar chromatography is a rapid and cost-efficient alternative method to quantify ITX in milk-based or fatty matrices. Only positive results are confirmed by online ESI/MS in the SIM mode (LOQ 128 pg) and by DART/MS involving a minimal employment of the MS device, which is a further advantage of HPTLC. Overall mean recovery rates of ITX at 20 or 50 and 100 μg kg⁻¹ (n=8) were 41 % for milk, 70 % for yoghurt, 6 % for margarine and 12 % for soy bean oil. However, with the internal standard correction recoveries were about 130 % for milk and yoghurt and 70 and 97 % for margarine and soy bean oil, respectively.      

Determination of alpha-tocopherol in infant foods by liquid chromatography combined with atmospheric pressure chemical ionisation mass spectrometry

A novel, sensitive and specific method for the quantification of alpha-tocopherol in two infant foods (milk and cereals) using liquid chromatography on-line with positive atmospheric pressure chemical ionisation mass spectrometry detection (LC/APCI-MS) has been developed. The samples were first saponified in order to eliminate fats and to transform tocopherol esters into free tocopherol, followed up by a liquid-liquid extraction of the analyte in petroleum benzine/diisopropyl ether (75:25, v/v) prior to injection onto the LC system. For the quantification, deuterium-labelled tocopherol was used as internal standard and the samples were monitored in selected ion monitoring (SIM) mode. Calibration curves between 1-40 microg/mL of alpha-tocopherol showed a good linear correlation (r(2) = 0.99994), and the detection limit was determined to be 2.5 ng/mL. The within-day and between-day precision were determined for several dietetic infant formulae and certified reference samples, and found to be below 3.5%. The accuracy determined on a Nestlé reference sample (milk powder) was calculated to be 115.2 +/- 1.2%, which confirms the robustness of the proposed method. This study shows that single quadrupole LC/MS can be applied for the quantification of vitamins in food and the method offers better sensitivity and selectivity than traditional method such as LC-UV. This would simplify the preparation of the food samples and consequently enhance the vitamin analysis throughput in the food area.     

Determination of ochratoxin A in currants, raisins, sultanas, mixed dried fruit, and dried figs by immunoaffinity column cleanup with liquid chromatography: interlaboratory study

An interlaboratory study was performed on behalf of the Food Standards Agency to evaluate the effectiveness of an affinity column cleanup liquid chromatographic (LC) method for the determination of ochratoxin A in a variety of dried fruit at European regulatory limits. To ensure homogeneity before analysis, laboratory samples are normally slurried with water in the ratio of 5 parts fruit to 4 parts water, and test materials in this form were used in the study. The test portion was extracted with acidified methanol. The extract was filtered, diluted with phosphate-buffered saline, and applied to an affinity column. The column was washed and ochratoxin A was eluted with methanol. Ochratoxin A was quantified by reversed-phase LC. The use of post-column pH shift to enhance the fluorescence of ochratoxin A by the addition of 1.1 M ammonia solution to the column eluant is optional. Determination was by fluorescence. Currants, sultanas, raisins, figs, and mixed fruit (comprising dried pineapple, papaya, sultanas, prunes, dates, and banana chips), both naturally contaminated and blank (very low level), were sent to 24 collaborators in 7 European countries. Participants were asked to spike test portions of all test samples at a level equivalent to 5 ng/g ochratoxin A. Average recoveries ranged from 69 to 74%. Based on results for 5 naturally contaminated test samples (blind duplicates) the relative standard deviation for repeatability (RSDr) ranged from 4.9 to 8.7%, and the relative standard deviation for reproducibility (RSDR) ranged from 14 to 28%. The method showed acceptable within- and between-laboratory precision for all 5 matrixes, as evidenced by HORRAT values <1.3.     

Determination of vitamin A in infant formula and adult nutritionals by UPLC-UV: First Action 2011.07

Vitamin A, a fat-soluble vitamin, is essential for health and plays an important part in vision, bone growth, reproduction, regulating the immune system, cell function, and skin health. Due to the advances in technology and the expansion of its uses, LC technologies are being studied for effectiveness in detecting and quantifying vitamin A in an effort to help determine the amount of vitamin A in various types of samples. For this reason, an Expert Review Panel agreed on June 29, 2011, at the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting," to approve "Determination of Vitamin A in Infant Formula and Adult Nutritionals by UPLC-UV" as AOAC Official Method 2011.07. To move from First to Final Action status, it was recommended that additional information be generated for all types of infant formulas and adult nutritional formula matrixes at varied concentration levels, as indicated in the standard method performance requirements. International units or retinol equivalents typically represent the concentration of vitamin A in food and supplements. However, for the purpose of this method, the concentration represented is presented in microg/100 g.