Trace determination of bisphenol A and phytoestrogens in infant formula powders by gas chromatography-mass spectrometry

This investigation describes a reliable and sensitive method for simultaneously determining bisphenol A (BPA) and two major phytoestrogens, daidzein and genistein, in powdered milks and infant formulas by gas chromatography-mass spectrometric analysis after trimethylsilylation. To reduce the matrix interference associated with the constituents of the formulas, the dissolved formula solutions were firstly ultra-centrifuged and the analytes in the supernatant were then extracted using a C18 solid-phase extraction column. The accuracy and precision of the method were determined and the technique was successfully employed to measure trace concentrations of BPA, daidzein and genistein in powdered formulas. The results show that BPA, daidzein and genistein were detected in all the testing samples (n = 6) at concentrations from 45 to 113 ng/g (except one infant formula), 20 to 2050 ng/g and 21 to 6510 ng/g, respectively. The highest concentrations of daidzein and genistein (i.e., 2050 and 6510 ng/g) were detected in a soy-based powdered infant formula. The quantitation limits were 1.0 ng/g for BPA, and 10 ng/g for daidzein and genistein using 0.5 g powdered milk samples.     

Determination of lipids in infant formula powder by direct extraction methylation of lipids and fatty acid methyl esters (FAME) analysis by gas chromatography.

Fatty acid methyl esters (FAMEs) of pure triglyceride standards, oils, and fat from dry matrixes were formed by transesterification using sodium methoxide in methanol-hexane. FAMEs were produced by direct addition of sodium methoxide-hexane to samples and heating to simultaneously extract and transesterify acyl lipids. FAMEs were quantitated by capillary gas chromatography (GC) over a fatty acid concentration range of 0 to 1.7 mg/mL (r > or = 0.9997). Total fat was calculated as the sum of individual fatty acids expressed as triglyceride equivalents, in accordance with nutrition labeling guidelines. Saturated, polyunsaturated, and monounsaturated fats were calculated as sums of individual free fatty acids. Absolute recoveries determined from individual fatty acids in test samples ranged from 69.7 to 106%. Recoveries (relative to the C13:0 internal standard) for individual fatty acids in test samples ranged from 95 to 106%. Reproducibility was constant at each fatty acid level in the reaction mixture (n = 5, coefficient of variation [CV] < 2%). Absolute recovery determined from the sum of total fatty acids in standard reference material (SRM) 1846 (powdered infant formula) was 96.4%. Analysis of SRM 1846 gave results that agreed closely with the certified fat and fatty acid values. Analysis of commercial infant formula gave results that were comparable to those obtained with AOAC Method 996.01. The direct extraction methylation procedure is rapid, and the transesterification of acyl lipids to form FAMEs is complete within 15 min. Classical saponification and refluxing are not required. This method provides FAMEs free of interferences and easily quantitated by GC or confirmed by GC/mass spectrometry (MS). Unambiguous MS identification of individual FAMEs derived from pure standards, SRM 1846, and powdered infant formula product was obtained.     

Simultaneous determination of free carnitine and total choline by liquid chromatography/mass spectrometry in infant formula and health-care products: single-laboratory validation

A single-laboratory validation study was conducted for a liquid chromatographic/mass spectrometric (LCIMS) method for the simultaneous determination of the free carnitine and total choline in milk-based infant formula and health-care products. The sample preparation used for both carnitine and choline was adapted from AOAC Official Method 999.14, with an acidic and enzymatic hydrolysis of esterified forms of choline. Carnitine and choline were quantified by ion-pair chromatography with single-quadrupole MS detection, using their respective deuterated internal standards. The repeatability relative standard deviation was < or =2.5 and 2.1%, respectively, for carnitine and choline. The intermediate reproducibility relative standard deviation was <4.7 and 2.4%, respectively, for carnitine and choline. The ranges of the average product-specific recoveries were 92-98 and 94-103%, respectively, for carnitine and choline. Choline concentration determined in infant formula reference material SRM 1846 was in agreement with the reference value. The proposed method was compared with the enzymatic methods for a range of products; good correlation (r = 0.99) was obtained, although a significant bias was observed for both analytes. The method, with a short chromatographic run time (7 min), is convenient for routine analysis to enhance analytical throughput and is a good alternative to enzymatic assays.     

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.     

Extension of AOAC Official Method 996.01 to the analysis of Standard Reference Material (SRM) 1846 and infant formulas.

There is currently no official method for the analysis of fatty acids (including trans fatty acids) in infant formulas. AOAC Official Method 996.01 for Fat Analysis in Cereal Products was extended to the analysis of milk-based infant formula Standard Reference Material (SRM)1846 to determine its applicability for use with infant formulas. Following the analysis of SRM 1846, 2 infant formulas, one milk-based liquid and one soy-based powdered infant formula, were analyzed for total fatty acid composition. Fatty acid methyl esters were prepared and analyzed by gas chromatography. The results of the analysis of SRM 1846 show that the mean analyzed values were highly reproducible as indicated by low coefficients of variation (CV). The CVs were <5% for the major fatty acids. Mean analyzed values for individual fatty acids in SRM 1846 were within +/- 1 standard deviation of the certificate values. The analyzed value for total fat as triglycerides (26.27 +/- 0.25%) agreed well with the certificate value (27.1 +/- 0.59%). Analyses of infant formulas showed that the concentrations of linoleic acid and fat meet the requirements for such formulas.     

Determination of lead and cadmium in milk by electrothermal atomic absorption spectrophotometry

The concentration of lead and cadmium in different kinds of milk samples (powdered, infant formula, market, buffalo, condensed and human) were determined using electrothermal atomic absorption spectrophotometric technique. Among all the varieties of milk analysed, condensed milk was found to contain much higher amount of lead. Human milk as expected was found to have lowest concentration of these elements. The results were compared with the reported values of other countries. Daily intake of these toxic elements by adults and babies up to the age of six months through the consumption of various types of milk was estimated and compared with the tolerance levels.     

Ion chromatographic determination of inositol in infant formulae and clinical products for enteral feeding

An ion chromatographic method is described for the determination of inositol in infant formula and products for enteral feeding. A two-step procedure for hydrolysis and extraction of total inositol has been developed, involving alkaline hydrolysis with 3 M potassium hydroxide and enzymatic dephosphorylation. Substances having a long chromatographic retention time were removed with an ion-exchange resin. Inositol was separated on a high-resolution ion-exchange column and detected by pulsed amperometric detection. Phytic acid interferes only slightly in the analysis. This method can be used for determination of total inositol in infant formulae, and enteral feeding products. The analytical method gave an average recovery of 94% from infant formula samples spiked with inositol and a recovery of 86+/-3% from products spiked with lecithin.     

An Ion Exchange Separation-Atomic Absorption Spectrophotometric Method for the Determination of Microgram Quantities of Copper,Iron and Zinc in Infant Milk Formula: Powder Form.

Abstract

An atomic absorption spectrophotometric method is described for the determination of microgram quantities of copper, iron and zinc in infant formula powdered milks. After sample digestion in concentrated nitric acid in a pressure vessel, the solution is evaporated till dryness, and then a solution of 6M HCl is added, in order to form the chlorocomplexes of the metals. This acid solution is passed through an ion-exchange column (anion exchange resin, chloride form). The metals are eluted from the column with diluted acid mixture of 0.005M HCl + 0.5% (v/v) HNO₃, and then the eluate is evaporated till dryness. The residue is dissolved in 1% (v/v) HNO₃, and then atomized into an air-acetylene flame. A standard addition procedure was employed as a back-up technique. The results obtained by this proposed method and those obtained by the standard addition technique were statistically treated, compared and discussed in this paper.     

Evaluation of nutritional parameters in infant formulas and powdered milk by Raman spectroscopy

It has been made a critical evaluation of the application of near infrared Fourier transform-Raman spectroscopy for the simultaneous determination of the most important nutritional parameters such as energetic value, carbohydrate, protein and fat contents of infant formula and powdered milk samples based on the use of partial least squares (PLS) regression analysis. A highly heterogeneous population of 23 samples, covering a wide range of infant food formula and powdered milk, were obtained from the Spanish market. Raman spectra, obtained by excitation with a Nd:YAG laser at 1064 nm, show no disturbing fluorescence effects; therefore sample spectra can be recorded without any previous preparation step. After correcting the spectra, hierarchical cluster analysis was performed in order to select a representative calibration set and the corresponding validation sample set. Different PLS models and several spectral windows were tested in order to evaluate their prediction capabilities for the validation set. Considering a calibration set comprised of three replicate spectra of 15 samples and a validation data set of eight samples, the procedure developed provided figures of merit which complied with the statutory values declared by the United States Food and Drug Administration (US FDA).     

Determination of Lead and Cadmium in Milk by Electrothermal Atomic Absorption Spectophotometry

Abstract

The concentrations of lead and cadmium in different kinds of milk samples (powdered, infant formula, market, buffalo, condensed and human) were determined using electrothermal atomic absorption spectrophotometric technique. Among all the varieties of milk analysed, condensed milk was found to contain much higher amount of lead. Human milk as expected was found to have lowest concentration of these elements. The results were compared with the reported values of other countries. Daily intake of these toxic elements by adults and babies up to the age of six months through the consumption of various types of milk was estimated and compared with the tolerance levels.     

固相萃取-离子色谱法测定甘蔗糖蜜及糖蜜酒精废液中的非氮有机酸和无机阴离子

建立了固相萃取-离子色谱测定甘蔗糖蜜及糖蜜酒精废液中乙酸、乳酸、琥珀酸、苹果酸、酒石酸、草酸、富马酸、柠檬酸、乌头酸等非氮有机酸和盐酸根、硫酸根、磷酸根等3种无机阴离子的方法。样品稀释液经强阴离子(SAX)固相萃取小柱净化除去糖类和色素等干扰基质,再用稀KOH溶液洗脱,经0.45 μm水膜过滤后,用IonPac AS15阴离子分离柱、KOH溶液梯度淋洗-抑制电导检测分离分析。考察了固相萃取小柱对待测离子的保留和洗脱条件。实验结果表明,除乙酸和乳酸的分离不完全、苹果酸与琥珀酸的组分重叠外,其余组分可达到完全分离,被测组分的浓度与其峰高在一定的范围呈良好的线性关系,检出限均低于0.20 mg/L,相对标准偏差(RSD)小于6.7%。测定了2种甘蔗糖蜜和1种糖蜜酒精废液中有机酸及无机阴离子,结果满足检测的要求,样品中各组分的加标回收率为94%～109%。     

Fast Determination of Ciprofloxacin by Batch Injection Analysis with Amperometric Detection and Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection

We report fast, precise, selective, and sensitive electroanalytical methods for the determination of ciprofloxacin in milk and pharmaceutical samples by batch‐injection analysis with amperometric detection (BIA‐AMP) and by capillary electrophoresis with capacitively‐coupled contactless conductivity detection (CE‐C⁴D). Both methods required simple sample preparation protocols before analysis (milk samples were just diluted and tablets powdered and dissolved in electrolyte/water). The analytical features of BIA‐AMP and CE‐C⁴D methods include, respectively, low relative standard deviation values for repetitive measurements (2.8 % and 1.7 %, n=10), low detection limits (0.3 and 5.0 µmol L^?1), elevated analytical frequency (80 and 120 h^?1) and satisfactory accuracy (based on comparative determinations by HPLC and recovery values for spiked samples).     

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 total fat in milk- and soy-based infant formula powder by supercritical fluid extraction

Commercially available simple benchtop systems using CO2 supercritical fluid extraction (SFE) eliminate expensive organic solvent disposal problems and offer potential to meet a demand for rapid, accurate high-volume gravimetric determinations of total fat content of infant formula powders. A Data Quality Objectives (DQOs) approach was used to evaluate the performance characteristics of instrumental SFE extraction for determination of total gravimetric fat in infant formula. The established DQOs included the following: ACCURACY: Correct values were obtained for a suitable reference material, SRM 1846 Infant Formula [National Institute of Standards and Technology (NIST), Gaithersburg, MD]. RUGGEDNESS: Variables were defined as (1) extraction time (35 min optimum); (2) ratio of sample size to diatomaceous earth support material (1 g sample/2 g support); (3) ratio of distilled water to alcohol (50% isopropanol optimum for both milk- and soy-based infant formula samples); (4) extraction flow rate was 3-3.5 mL/min optimum. PRECISION: Relative standard deviations of multiple determinations fell within the Horwitz limits of acceptability of < or = 2.8% at the level of analyte determined (0.34-2.5% obtained). SCOPE OF APPLICABILITY: Includes milk- and soy-based infant formula powders. Research data were obtained by use of a commercially available fat analyzer. Samples of the SRM, 2 commercial milk-based and 3 commercial soy-based infant formula products were distributed to 2 additional collaborating laboratories. Very good agreement was obtained among the submitting and collaborating laboratories for these samples. The use of clearly defined DQOs to establish method performance characteristics, along with the commercially available reference material, provided the mechanism for verification and validation of analytical methodology.     

Application of multiwalled carbon nanotubes as sorbents for the extraction of mycotoxins in water samples and infant milk formula prior to high performance liquid chromatography mass spectrometry analysis

In this work, a simple and environmental friendly methodology has been developed for the analysis of a group of six mycotoxins with estrogenic activity produced by Fusarium species (i.e. zearalanone, zearalenone, α‐zearalanol, β‐zearalanol, α‐zearalenol, and β‐zearalenol), using microdispersive SPE (μ‐dSPE) with multiwalled carbon nanotubes as sorbent. Separation, determination, and quantification were achieved by HPLC coupled to ion trap MS with an ESI interface. Parameters affecting the extraction efficiency of µ‐dSPE such as pH of the sample, amount of multiwalled carbon nanotubes, and type and volume of elution solvent, were studied and optimized. The methodology was validated for mineral, pond, and wastewater as well as for powdered infant milk using 17β‐estradiol‐2,4,16,16,17‐d₅ (17β‐E₂‐D₅) as internal standard, obtaining recoveries ranging from 85 to 120% for the three types of water samples and from 77 to 115% for powdered infant milk. RSD values were lower than 10%. The LOQs achieved were in the range 0.05–2.90 μg/L for water samples and 2.02–31.9 μg/L for powdered infant milk samples.     

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.     

Determination of quinolone residues in infant and young children powdered milk combining solid-phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry

The present work describes a method based on solid-phase extraction (SPE) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the simultaneous determination of three quinolones (pipemidic acid, oxolinic acid and flumequine) and twelve fluoroquinolones (marbofloxacin, fleroxacin, pefloxacin, levofloxacin, norfloxacin, ciprofloxacin, enrofloxacin, danofloxacin, lomefloxacin, difloxacin, sarafloxacin, and moxifloxacin) in different infant and young children powdered milks. After suitable deproteination of the reconstituted powdered samples, a SPE procedure was developed providing recovery values higher than 84% (RSDs lower than 13%) for all the analytes, with limits of detection between 0.04 and 0.52 μg/kg. UPLC-MS/MS analyses were carried out in less than 10 min. Sixteen infant and young children powdered milk samples of different origin, type and composition bought at Spanish markets were analyzed. Residues of the selected antibiotics were not detected in any of the analyzed samples.     

阀切换-离子色谱法测定1,2-环氧丁烷产品中有机酸

建立了阀切换-离子色谱法测定1,2-环氧丁烷（1,2-BO）产品中有机酸（甲酸、乙酸和丙酸）的方法。样品经乙醇稀释后,通过阀切换技术,有机酸被IonPac TAC-ULP1捕集柱捕集,然后进入IonPac AS11分析柱分离,并由电导检测器检测。结果表明：甲酸、乙酸和丙酸均能实现较好地分离,在各自的范围内其色谱峰面积呈现良好的线性关系。实际样品的加标回收率为92.5%~111.8%,相对标准偏差小于5.6%（n=3）,检出限为0.60~4.80 μg/L。该方法具有前处理简单、快速、准确等特点,适用于不溶于水的有机体系中有机酸的分析。     

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.     

Direct determination of silicon in powdered aluminium oxide by use of slurry sampling with in situ fusion graphite-furnace atomic-absorption spectrometry

A direct method for determination of silicon in powdered high-purity aluminium oxide samples, by slurry sampling with in situ fusion graphite-furnace atomic-absorption spectrometry (GF-AAS), has been established. A slurry sample was prepared by 10-min ultrasonication of a powdered sample in an aqueous solution containing both sodium carbonate and boric acid as a mixed flux. An appropriate portion of the slurry was introduced into a pyrolytic graphite furnace equipped with a platform. Silicon compounds to be determined and aluminium oxide were fused by the in situ fusion process with the flux in the furnace under optimized heating conditions, and the silicon absorbance was then measured directly. The calibration curve was prepared by use of a silicon standard solution containing the same concentration of the flux as the slurry sample. The accuracy of the proposed method was confirmed by analysis of certified reference materials. The proposed method gave statistically accurate values at the 95% confidence level. The detection limit was 3.3 microg g(-1) in solid samples, when 300 mg/20 mL slurry was prepared and a 10 microL portion of the slurry was measured. The precision of the determination (RSD for more than four separate determinations) was 14% and 2%, respectively, for levels of 10 and 100 microg g(-1) silicon in aluminium oxide.