Determination of vitamin K1 in medical foods by liquid chromatography with postcolumn reduction and fluorometric detection

A liquid chromatographic (LC) method is described for the determination of vitamin K1 in medical foods. The sample is enzymatically digested with lipase and alpha-amylase and extracted with 1% sodium bicarbonate solution-isopropanol (1 + 1). After C18 solid-phase extraction, vitamin K1 is separated by nonaqueous reversed-phase LC, converted to the hydroquinone by postcolumn zinc reduction, and quantitated by fluorescence detection. The limit of detection is 8 pg (3 sigma), and the limit of quantitation is 27 pg (10 sigma) on column. Linear response ranged from 0.1 to 1.0 ng vitamin K1 (r= 0.9999). The mean recovery (n = 38) for all spiking levels was 101.6 +/- 2.85%. Analysis of Standard Reference Material 1846, Infant Formula, gave a mean value of 0.95 +/- 0.088 mg vitamin K/kg (K or K1?) (n = 31) with a coefficient of variation of 9.26.     

Accelerated solvent extraction of vitamin K1 in medical foods in conjunction with matrix solid-phase dispersion

An extraction technique is described for vitamin K1 in medical foods, using accelerated solvent extraction (ASE) in conjunction with matrix solid-phase dispersion (MSPD). The medical food sample is treated as it would be with MSPD extraction, followed by ASE for a hands-free automated extraction. The vitamin K1 in the ASE extract is then quantitated by reversed-phase liquid chromatography with fluorescence detection. The chromatography specifications are identical to those in previous work that used MSPD only, with a limit of detection of 6.6 pg and a limit of quantitation of 22 pg on column. Recoveries, which were determined for an analyte-fortified zero control reference material for medical foods, averaged 97.6% (n = 25) for vitamin K1. The method provides a rapid, automatic, specific, and easily controlled assay for vitamin K1 in fortified medical foods with minimal solvent usage.     

Liquid chromatographic analysis of vitamin B6 in soy-based infant formula

A liquid chromatographic (LC) method is described for determination of total vitamin B6 in soy-based infant formula. Total vitamin B6 is quantitated by using ion-pair LC after precolumn transformation of phosphorylated and free vitamers into pyridoxol. The limit of detection is 0.3 ng and the limit of quantitation is 1.0 ng on-column (injection volume = 100 microL). Linear response ranged from 39 to 616 ng/mL (r2 = 0.99986). Analysis of a soy-based infant formula control fortified at 6 different concentration levels gave recoveries that averaged 104%. Assay of SRM 1846 gave results within the certified range (8.6 +/- 0.086 mg/kg versus the certified value of 8.4 +/- 1.0 mg/kg). The method provides a rapid and specific assay for the analysis of total vitamin B6 in fortified soy-based infant formula.     

Determination of folate in infant formula and adult/pediatric nutritional formula by optical biosensor assay: First Action 2011.05

After a review of data from a single-laboratory validation (SLV) study published in the International Dairy Journal 21, 783-789 (2011), a method for folate in infant formula and adult/pediatric nutritional formula was submitted for consideration of adoption by AOAC as an automated assay that is rapid and simple. The method uses an optical biosensor assay to quantitate total folate content in milk and milk-based pediatric and adult nutritional products. The assay uses folate binding protein and a functionalized sensor surface. The SLV showed an instrumental LOD of 0.1 ng/mL (equivalent to 2.5 microg/100 g for a typical infant formula). The method detection limit was 6.5 microg/100 g with a repeatability of 3.48% and an intermediate reproducibility of 4.63% RSD.     

Determination of vitamin K1 in emulsified nutritional supplements by solid-phase extraction and high-performance liquid chromatography with postcolumn reduction on a platinum catalyst and fluorescence detection

Determination of small amounts of vitamin K1 (0.8 microg/g) in nutritional supplements with high fat content (20 mg/g) was performed by solid-phase extraction and high-performance liquid chromatography (HPLC) with fluorescence detection after reduction on a platinum oxide catalyst. The concentration ratio of plant oils to vitamin K1 (0.8 microg/g) was about 25,000:1. A sample solution was applied to a solid-phase extraction cartridge and vitamin K1 was eluted with ethanol, followed by HPLC. The proposed method was simple, rapid (analysis time: ca. 12 min), sensitive [detection limit: ca. 0.1 pg per injection (100 microl) at a signal-to-noise ratio of 3:1], highly selective and reproducible [relative standard deviation: ca. 1.3%. (n=5)]. The calibration graph of vitamin K1 was linear in the range of 0-2 pg per injection (100 microl). Recovery of vitamin K1 was over 90% by the standard addition method.     

Determination of vitamin B12 in infant formula and adult nutritionals using HPLC after purification on an immunoaffinity column: first action 2011.09

During the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting," on June 29, 2011, the method "Determination of vitamin B12 in infant formula and adult nutritionals using HPLC after purification on an immunoaffinity column" was recommended by an Expert Review Panel and adopted as AOAC Official First Action status. The method is applicable for the determination of vitamin B12 in milk-based infant formula. Vitamin B12 is extracted from the sample in sodium acetate buffer in the presence of potassium cyanide. After purification and concentration with an immunoaffinity column (IAC), vitamin B12 is determined by LC with UV detection (361 nm). Data supplied by CLF demonstrated linear response over a wide range of concentrations (1.4-39 microg/100 mL). The analytical range is 0.2-10 microg/100 g, depending on the capacity of the IACs (0.01-0.5 microg), the input weight, and dilutions. Recovery rates were assessed using National Institute of Standards and Technology SRM 1849, and determined to be 95.1%, with SD of 0.34 and CV of 9.0. Measurement uncertainty (UE) was 0.8 microg/100 g, which was calculated from the validation data. It is an expanded measurement uncertainty and was obtained through multiplication with a coverage factor k. LOQ values were reported as 0.10 microg/100 g. The performance characteristics of the method met the standard method performance requirements set forth by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals; thus, the method was determined to be appropriate for First Action status.     

Determination of vitamin A (retinol) in infant and medical nutritional formulas with AOAC method 992.06 using a modified extraction procedure: single-laboratory validation

The applicability of AOAC Official Method 992.06, vitamin A (retinol) in milk-based infant formula can be extended to specialty infant formulas, and medical and adult nutritional products with a few minor modifications to the sample preparation procedure. Currently, AOAC Official Method 992.06 is only applicable to milk-based infant formulas containing >500 IU vitamin A per reconstituted quart. When this method is used as written to test specialty infant formulas, vitamin A recoveries are low compared to results generated with alternate validated vitamin A methods. AOAC Method 992.06 vitamin A recoveries can be improved significantly in specialty infant formulas if the amount of potassium hydroxide used during the saponification step is doubled. With this one minor modification to the sample preparation procedure, AOAC Method 992.06 demonstrates acceptable precision and accuracy for the quantitation of vitamin A (retinol) in specialty infant formulas, milk- and soy-based infant formulas, and adult and medical nutritionals. Because increasing the amount of potassium hydroxide can cause emulsions to form, 2-4 mL aliquots of reagent alcohol may need to be added to some samples to separate the organic and aqueous layers during the extraction step. A single-laboratory validation of these modifications was completed. During validation, 15 different product matrixes were analyzed. The intermediate precision averaged 2.70% RSD, and spike recovery data averaged 96.3%.     

Fully automated determination of vitamins A and E in milk-based products using a robotic station-HPLC arrangement

An automated method, which includes an extraction procedure based on the AOAC official method of analysis for vitamins A and E in milk-based infant formula, was developed. The whole procedure is carried out automatically in a robotic station coupled to a liquid chromatograph with a UV detector for individual separation and quantification of the vitamins. Different commercial products (milk, infant formula and nutritional supplements) were analysed, and the data were compared with those obtained using the manual AOAC extraction method. Relative standard deviations using the automated method were 5.92 and 2.57% for vitamin A and E, respectively.     

Simultaneous determination of vitamin K1, vitamin K1 2,3-epoxide and menaquinone-4 in human plasma by high-performance liquid chromatography with fluorimetric detection

A highly sensitive method for measuring endogenous vitamin K1, menaquinone-4 (which is one of the K2 vitamins) and vitamin K1 2,3-epoxide in human plasma was developed, based on high-performance liquid chromatography with coulometric reduction and fluorimetric detection, following extraction from plasma and purification on a Sep-Pak silica cartridge. The detection limits of vitamin K1, menaquinone-4 and vitamin K1 2,3-epoxide were 5, 5 and 8 pg per injection for the standard substances and 30, 30 and 50 pg/ml in human plasma, respectively.     

LC-UV Column-Switching for Quantitation of Vitamin K1 in Infant Formula

This study was undertaken in order to develop an analytical method for vitamin K₁ in infant formula. The content of vitamin K₁ was investigated by using a column-switching LC-UV method. A Certified Reference Material sample of infant formula containing 0.94 ± 0.04 mg kg^?1 of vitamin K₁ was extracted with hexane followed by enzymatic digestion of fat and precipitation of the fatty acids. The linearity of this method was calculated using five consecutive standard curves, and the coefficient of determination (r ²) was found to be 0.9995. The limit of detection (LOD) and the limit of quantitation (LOQ) was 3.31 and 11.12 μg L^?1, respectively. The accuracy of intra- and inter-day measurements was in the range from 96.67 to 108.67%, and the precision of intra- and inter-day measurements was less than 5.13%. The recoveries were 109.27 ± 5.92%, and the recoveries of inter-laboratory results were in the range from 97.59 ± 1.29 to 109.27 ± 5.92%. The newly developed method uses the optimum conditions required to determine the content of vitamin K₁ in infant formula.     

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.     

Total arsenic determination and speciation in infant food products by ion chromatography-inductively coupled plasma-mass spectrometry

Health risk associated with dietary arsenic intake may be different for infants and adults. Seafood is the main contributor to arsenic intake for adults while terrestrial-based food is the primary source for infants. Processed infant food products such as rice-based cereals, mixed rice/formula cereals, milk-based infant formula, applesauce and puree of peaches, pears, carrots, sweet potatoes, green beans, and squash were evaluated for total and speciated arsenic content. Arsenic concentrations found in rice-based cereals (63-320 ng/g dry weight) were similar to those reported for raw rice. Results for the analysis of powdered infant formula by inductively coupled plasma-mass spectrometry (ICP-MS) indicated a narrow and low arsenic concentration range (12 to 17 ng/g). Arsenic content in puree infant food products, including rice cereals, fruits, and vegetables, varies from <1 to 24 ng/g wet weight. Sample treatment with trifluoroacetic acid at 100 degrees C were an efficient and mild method for extraction of arsenic species present in different food matrixes as compared to alternative methods that included sonication and accelerated solvent extraction. Extraction recoveries from 94 to 128% were obtained when the summation of species was compared to total arsenic. The ion chromatography (IC)-ICP-MS method selected for arsenic speciation allowed for the quantitative determination of inorganic arsenic [As(III) + As(V)], dimethylarsinic acid (DMA), and methylarsonic acid (MMA). Inorganic arsenic and DMA are the main species found in rice-based and mixed rice/formula cereals, although traces of MMA were also detected. Inorganic arsenic was present in freeze-dried sweet potatoes, carrots, green beans, and peaches. MMA and DMA were not detected in these samples. Arsenic species in squash, pears, and applesauce were not detected above the method detection limit [5 ng/g dry weight for As(III), MMA, and DMA and 10 ng/g dry weight for As(V)].     

Determination of vitamin B12 in food products by liquid chromatography/UV detection with immunoaffinity extraction: single-laboratory validation

A fast and simple method to determine vitamin B12 in foods is presented. The method allows, in addition to the determination of added cyanocobalamin, the determination of natural vitamin B12 forms, making it also applicable to nonfortified products, especially those that are milk-based. Vitamin B12 is extracted in sodium acetate buffer in the presence of sodium cyanide (100 degrees C, 30 min). After purification and concentration with an immunoaffinity column, vitamin B12 is determined by liquid chromatography with UV detection (361 nm). The method has been validated in analyses of a large range of products: milk- and soy-based infant formulas, cereals, cocoa beverages, health care products, and polyvitamin premixes. The method showed appropriate performance characteristics: linear response over a large range of concentrations, recovery rates of 100.8 +/- 7.5% (average +/- standard deviation), relative standard deviation of repeatability, RSDr, of 2.1%, and intermediate reproducibility, RSDiR, of 4.3%. Limits of detection and quantitation were 0.10 and 0.30 microg/100 g, respectively, and correlation with the reference microbiological assay was good (R2 = 0.9442). The proposed method is suitable for the routine determination of vitamin B12 in fortified foods, as well as in nonfortified dairy products. It can be used as a faster, more selective, and more precise alternative to the classical microbiological determination.     

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.     

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.     

Rapid determination of vitamin B12 concentration with a chemiluminescence lab on a chip

This paper reports a novel method for the rapid determination of vitamin B(12) concentration in a continuous-flow lab-on-a-chip system. This new method is based on luminol-peroxide chemiluminescence (CL) assays for the detection of cobalt(II) ions in vitamin B(12) molecules. The lab-on-a-chip device consisted of two passive micromixers acting as microreactors and a double spiral microchannel network serving as an optical detection region. This system could operate in two modes. In the first mode, samples are acidified and evaluated directly in the microchip. In the second mode, samples are treated externally by acidification prior to detection in the microchip. In the first mode, the linear range obtained was between 1.00 ng ml(-1) to 10 μg ml(-1), R(2) = 0.996, with a relative standard deviation (RSD) of 1.23 to 2.31% (n = 5) and a limit of detection (lod) of 0.368 pg ml(-1). The minimum sample volume required and the analytical time were 30 μl and 3.6 s, respectively. In the second mode, the linear range obtained was between 0.10 ng ml(-1) to 10 μg ml(-1), R(2) = 0.994, with the RSD of 0.90 to 2.32% (n = 6) and a lod of 0.576 pg ml(-1). The minimum sample and the analytical time required were 50 μl and 6 s, respectively. The lab on a chip working in mode II was successfully used for the determination of vitamin B(12) concentrations in nutritional supplemental tablets and hen egg yolks.     

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。     

Analysis of nucleosides and nucleotides in infant formula by liquid chromatography–tandem mass spectrometry

A method for the simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography–tandem mass spectrometry is described. This approach is advantageous for compliance testing of infant formula over other LC-MS methods in which only nucleotides or nucleosides are measured. Following sample dissolution, protein was removed by centrifugal ultrafiltration. Chromatographic analyses were performed using a C₁₈ stationary phase and gradient elution of an ammonium acetate/bicarbonate buffer, mass spectrometric detection and quantitation by a stable isotope-labelled internal standard technique. A single laboratory validation was performed, with spike recoveries of 80.1–112.9 % and repeatability relative standard deviations of 1.9–7.2 %. Accuracy as bias was demonstrated against reference values for NIST1849a certified reference material. The method has been validated for the analysis of bovine milk-based, soy-based, caprine milk-based and hydrolysed milk protein-based infant formulae.

Sample preparation for routine high-performance liquid chromatographic determination of retinol palmitate in emulsified nutritional supplements by solid-phase extraction using monosodium L-glutamate as dissolving agent

Retinol palmitate (vitamin A, 73.3 microg/g) in an emulsified nutritional supplement was determined by solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with fluorescence detection (excitation 350 nm, emission 480 nm) using monosodium L-glutamate as a dissolving agent to obtain higher recovery of vitamin A from the emulsified sample solution. A Bond Elut C2 cartridge (500 mg) was chosen for SPE after comparison with 16 other types. A sample solution was applied to a conditioned Bond Elut C2 cartridge and then vitamin A was eluted with ethanol followed by HPLC. The proposed method was simple, rapid (sample preparation time by SPE: ca. 8 min, retention time: ca. 8 min), sensitive [detection limit: ca. 0.1 pg/injection (100 microl) at a signal-to-noise ratio of 3:1], highly selective and reproducible (relative standard deviation (RSD): ca. 2.9% (n = 5), between-day RSD ca. 3.7 (5 days). The recovery of vitamin A was over 90% by the standard addition method.     

Determination of vitamin B12 in multivitamin tablets and fermentation medium by high-performance liquid chromatography with fluorescence detection

A novel method for the determination of vitamin B12 by high-performance liquid chromatography with fluorescence detection is reported. The method was simple and highly sensitive with good precision. Vitamin B12 was analyzed by HPLC on a muBondapak C18 column (300x3.9 mm, 10 microm) with methanol-water (30:70) as mobile phase and fluorescence detection at 305 nm (with excitation at 275 nm). The calibration graph was linear from 1.000 to 100.0 ng ml(-1) for vitamin B12 with a correlation coefficient of 0.998 (n=6). The detection limit was 0.1 ng ml(-1). The method was successfully applied to the determination of vitamin B12 in vitamin B12 tablets, multivitamin tablets and fermentation medium. The recovery was from 94 to 102% and the relative standard deviation was in the range of 1.8 to 4.1%.