Spezielle Probleme bei der Vitamin D-Analytik in Futtermitteln mit HPLC

Special Problems of the Vitamin D-Analysis in Feedstuff by HPLC The protection of vitamin D by new coatings requires the saponification of the sample. The advantages of digestion at room temperature fail, for example the separation of unchanged vitamin A-esters. Saponification involves additional difficulties: the displacement of equilibrium vitamin D/previtamin D to more previtamin, the separation vitamin D from vitamin A-alcohols and the separation from citranaxanthin and canthaxanthin in poultry feedstuff. These substances interfere in HPLC by absorption at 265 nm, the absorption maximum of vitamin D. A special method for poultry feedstuff is described: 1. saponification and extraction, 2. Al₂O₃ column chromatography, 3. thin-layer chromatography, 4. reversed phase-HPLC, 5. straight phase-HPLC.     

Determination of the conjugated linoleic acid-containing triacylglycerols in New Zealand bovine milk fat

Reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection at 233 nm was used to separate, quantify, and identify the triacylglycerols (TAG) of milk fat that contain conjugated linoleic acid (CLA). The absorbance at 233 nm was substantially due to CLA-TAG (chromatography of some representative TAG devoid of CLA, such as tripalmitin and triolein, showed poor responses at 233 nm, 1/800th that of CLA-TAG). A CLA molar extinction coefficient at 233 nm of 23 360 L mol⁻¹ cm⁻¹ and an HPLC UV response factor were obtained from a commercially available cis-9, trans-11-CLA standard. This molar extinction coefficient was only 86% reported literature values. Summation of all chromatographic peaks absorbing at 233 nm using the corrected response factor gave good agreement with independent determinations of total CLA by gas chromatography and UV spectrophotometry. This agreement allowed quantification of individual CLA-TAG peaks in the HPLC separation of a typical New Zealand bovine milk fat. Three CLA-containing TAG, CLA-dipalmitin, CLA-oleoyl-palmitin and CLA-diolein, were prepared by interesterification of tripalmitin with the respective fatty acid methyl esters and used to assign individual peaks in the reversed-phase chromatography of total milk fat, of which CLA-oleoyl-palmitin was coincident with the largest UV peak. Band fractions from argentation thin-layer chromatography of total milk fat were similarly employed to identify five predominant CLA-TAG groups in total milk fat: CLA-disaturates, CLA-oleoyl-saturates, CLA-vaccenyl-saturates, CLA-vaccenyl-olein, and CLA-diolein.     

Analysis of Vitamin K1 in Soybean Seed: Assessing Levels in a Lineage Representing Over 35 Years of Breeding

Recommendations by the Organization of Economic Cooperation and Development (OECD) for compositional assessments of new genetically modified (GM) soybean now include measurement of vitamin K₁. Here we employ an HPLC method to provide information on variation in levels of vitamin K₁ in seed from soybean representing over 35 years of breeding. Vitamin K₁ analysis was conducted on seed from six conventional lines and three GM glyphosate‐tolerant lines grown concurrently at two sites in the US. Vitamin K₁ levels in seed were influenced by both location and germplasm. Levels were higher in the newer higher‐yielding lines when compared with the older varieties. The effect of GM was negligible. Overall, our study showed, using a method suitable for compositional assessments under OECD guidelines, that (i) variation in levels of vitamin K₁ in soybean is associated with a safe history of consumption and (ii) that modern breeding strategies can be employed for soybean varietal development without adversely impacting vitamin K₁ levels.     

Dihydro-vitamin K1: Primary food sources and estimated dietary intakes in the American diet

Dihydro-vitamin K₁ was recently identified as a dietary form of vitamin K produced during the hydrogenation of vitamin K₁-rich vegetable oils. Dihydro-vitamin K₁ is absorbed, with measurable levels in human plasma following dietary intake. To determine the primary food sources of dihydro-vitamin K₁ in the American diet, 261 foods from the U.S. Food and Drug Administration's (FDA) Total Diet Study (TDS) were analyzed by high-performance liquid chromatography. Of these foods, 36 contained dihydro-vitamin K₁. Fast-food items that were otherwise poor sources of vitamin K₁, such as french fries and fried chicken, contained appreciable amounts of dihydro-vitamin K₁ (36 and 18 μg/100 g, respectively). These nutrient values were then applied to the FDA TDS consumption model to determine average dietary intake of dihydro-vitamin K₁ in 14 age-gender groups. With the exception of infants, all age-gender groups had estimated mean daily dihydro-vitamin K₁ intakes of 12–24 μg, compared to mean daily vitamin K₁ intakes of 24–86 μg. The vitamin K₁ and dihydro-vitamin K₁ intakes were summed, and the dietary contribution of dihydro-vitamin K₁ was expressed as a percentage of total vitamin K intake. Children reported the highest intakes of dihydro-vitamin K₁ (30% of total vitamin K intake), followed by a progressive decrease in percentage contribution with age. There are currently no data on the relative bioavailability of dihydro-vitamin K₁ but, given its abundance in the American diet, this hydrogenated form of vitamin K warrants further investigation.     

Separation of vitamin E (tocopherol,tocotrienol, and tocomonoenol) in palm oil

Previous reports showed that vitamin E in palm oil consists of various isomers of tocopherols and tocotrienols [α-tocopherol (α−T), α-tocotrienol, γ-tocopherol, γ-tocotrienol, and δ-tocotrienol), and this is normally analyzed using silica column HPLC with fluorescence detection. In this study, an HPLC-fluorescence method using a C₃₀ silica stationary phase was developed to separate and analyze the vitamin E isomers present in palm oil. In addition, an α-tocomonoenol (α−T₁) isomer was quantified and characterized by MS and NMR. α−T₁ constitutes about 3–4% (40±5 ppm) of vitamin E in crude palm oil (CPO) and is found in the phytonutrient concentrate (350±10 ppm) from palm oil, whereas its concentration in palm fiber oil (PFO) is about 11% (430±6 ppm). The relative content of each individual vitamin E isomer before and after interesterification/transesterification of CPO to CPO methyl esters, followed by vacuum distillation of CPO methyl esters to yield the residue, remained the same except for α−T and γ−T₃. Whereas α−T constitutes about 36% of the total vitamin E in CPO, it is present at a level of 10% in the phytonutrient concentrate. On the other hand, the composition of γ−T₃ increases from 31% in CPO to 60% in the phytonutrient concentrate. Vitamin is present at 1160±43 ppm, and its concentrations in PFO and the phytonutrient concentrate are 4,040±41 and 13,780±65 ppm, respectively. The separation and quantification of α−T₁ in palm oil will lead to more in-depth knowledge of the occurrence of vitamin E in palm oil.     

Removal of fat from Cow's milk decreases the vitamin E contents of the resulting dairy products

The present study was undertaken to determine whether decreases in fat contents result in lower vitamin E contents. Milk samples of varying fat contents (half and half, whole milk, reduced-fat milk low-fat milk, and nonfat milk) were obtained from a local dairy on six different occasions, α-locopherol was the major form of vitamin E (>85%); γ-tocopherol and α-tocotrienol were present to a lesser extent. As the fat contents of milk products decreased from 11 to 0.3%, the vitamin E contents decreased. For example, raw milk as compared to nonfat milk had both higher α-tocopherol contents (45.5+-4.6 vs. 4.5±0.5 μg/100 g; P<-0.0001) and higher total lipids ( 3.46±0.49 vs. 0.30±0.07 g/100 g; P≤0.0001). Vitamin E, cholesterol, and total lipids increased as cream was added back to nonfat milk during production. For every 1 mg cholesterol increase, there was an increase of approximately 4 μg of α-tocopherol; for every 1 g total lipids increase, the α-tocopherol content increased by 17 μg. These data demonstrate that removal of milk fat markedly decreases the vitamin E content of various milk products     

Riboflavin-photosensitized singlet oxygen oxidation product of vitamin D<Subscript>2</Subscript>

The riboflavin-photosensitized singlet oxygen oxidation of vitamin D₂ in a model system of 12% water and 88% acetone was studied to understand the possible oxidation of fortified vitamin D in milk. Only the samples containing vitamin D₂ and riboflavin under light storage showed two new peaks in the HPLC chromatogram, indicating that singlet oxygen oxidation of vitamin D₂ had occurred. UV analysis indicated that a new compound was formed from the reaction of the triene of vitamin D₂ with oxygen. The mass spectrum showed that one of the two compounds had a molecular ion at m/z=412, which was an increase of the mass of vitamin D₂ by the mass of exactly one oxygen. The IR spectrum suggested the presence of a hydroxyl group and no carbonyl group in the product. The combned information from UV, MS, and FTIR spectra and the chemical mechanisms of singlet oxygen oxidation of vitamin D₂ indicated that a 5,6-epoxide of vitamin D₂ was formed from vitamin D₂ in the presence of riboflavin under light.     

Cloud Point-Dispersive Liquid–Liquid Microextraction for Extraction of Organic Acids from Biological Samples

A new method combining cloud point extraction (CPE) with dispersive liquid–liquid microextraction (DLLME) named “cloud point‐dispersive liquid–liquid microextraction (CP‐DLLME)” was presented in this work for the first time for the determination of organic acids as model compounds in biological samples using high performance liquid chromatography and UV detection (HPLC–UV). Water (disperser solvent) containing tert‐octylphenol ethoxylate (7.5EO) as extraction solvent, was rapidly injected into a warmed sample solution and cloudy state formed immediately. After that, phase separation was performed by centrifugation. The surfactant‐rich phase was diluted with acetonitrile and injected into a HPLC–UV apparatus. The influence of several important parameters on the extraction efficiencies was evaluated. Under optimized experimental conditions, the calibration graphs were linear in the range of 0.06–1,500 µg L^?1 for target analytes. Coefficient of determination (r²) ranged from 0.9985 to 0.9994. The limits of detection were in the range of 0.05–17.1 µg L^?1. The new method was successfully applied with satisfactory results for analysis of target analytes in spiked samples. The relative mean recoveries of the spiked samples ranged from 91.8 to 107.1 % with relative standard deviations in the range of 2.1 to 4.3 %.     

Plasma concentrations of dihydro-vitamin K1 following dietary intake of a hydrogenated vitamin K1-rich vegetable oil

Dihydro-vitamin K₁ is a dietary form of vitamin K₁ (phylloquinone) produced during the hydrogenation of vegetable oils. To determine if dihydro-vitamin K₁ is present in plasma following dietary intake of a hydrogenated fat, eight healthy adults consumed each of two diets containing 30% of calories from fat, of which 20% was either soybean oil or a partially hydrogenated soybean oil-based stick margarine. Of the fats and oils analyzed, dihydro-vitamin K₁ was only found in the hydrogenated products. The soybean oil diet contained 180 ±12 μg (mean±SD) of vitamin K₁/day and nondetectable levels of dihydro-vitamin K₁, whereas the stick margarine diet contained 199±7 μg of vitamin K₁/day and 23±2 μg of dihydrovitamin K₁/day. After consuming each diet for five weeks, plasma dihydro-vitamin K₁ concentrations were higher (P=0.002) in all eight subjects when consuming the stick margarine diet (0.56 ±0.33 nmol/L) compared to the soybean oil diet (0.12±0.11 nmol/L). There was no significant change in plasma vitamin K₁ concentrations when the two diets were compared. In conclusion, dihydro-vitamin K₁ is detectable in plasma following dietary intake of a hydrogenated vitamin K₁-rich vegetable oil.     

Quantitative Bestimmung der Vitamine D2 und D3 in Margarine mittels HPLC

Quantitative Determination of the Vitamins D₂ and D₃ in Margarine by HPLC After mild saponification of the sample, extraction of the unsaponifiable fraction and its clean-up by application of 2 TLC-systems the isolated vitamin D-zone is eluated and once more separated by reversed-phase HPLC on a Zorbax ODS column. Good basis-line separation of D₂ and D₃ results hereby (18.64 resp. 20.04 min). The D-content of 10 samples of margarine was determined. The variation coefficient varied from 1.49 to 4.40 (average ± 3.25) corresponding to the sort of margarine; recovery was 88% on an average.     

高效液相色谱在检测维生素B2中的应用

本文综述了应用高效液相色谱法测定维生素B2过程中样品处理及分离、检测条件，并展望了今后的工作方向。     

Gas chromatography of the fat-soluble vitamins: A review

The application of gas liquid chromatography (GLC) as an analytical tool for the determination of the fat-soluble vitamins A, D, E and K has yet to be utilized to its full potential. A review of the published work of many researchers in this field is presented. GLC methods to measure the vitamin A isomers have not been developed to any appreciable practical extent. Liquid liquid chromatography might well be the technique of choice. In the field of vitamin D there are indications that a practical GLC analysis is feasible for pharmaceutical preparations. The GLC applications for vitamin E are diverse, well defined and generally widely accepted in research laboratory situations and for regulatory and quality control usage. Vitamins K₁ and K₂ have been measured with limited success in a few research laboratories, but the GLC methods have not developed on a practical basis. However GLC is used for measuring vitamin K₃ (menadione and menadione sodium bisulfite) on a fairly routine basis in quality control laboratories.     

中药材中维生素C含量测定方法的对比分析

通过对比分析的方法,确定在不同的中药材中维生素C含量的测定方法。将五味子、枸杞、山楂、大枣等药材进行研究,采用紫外分光光度法、二氯靛酚滴定法和高效液相色谱法3种方法进行维生素C的测定,对测定的过程和结果进行分析,选择最合适的维生素C测定的方法。结果表明,采用高效液相色谱法对维生素C进行测定的准确性最高,滴定法最简单,但是测试的结果不是特别准确,特别在测试颜色较深的样品中误差偏大。紫外分光光度法测定的结果与其他的两种方法比偏高,容易受到杂质的影响。高效液相色谱法在对中药中的维生素C测定中最合适,滴定法的局限性比较多,只能对颜色较浅的样品进行测定,紫外分光光度法在维生素C含量测定中不合适。     

On-line coupling of solid-phase extraction of phenols on porous graphitic carbon and LC separation on C18 silica gel column via subcritical water desorption

An approach to the on-line coupling of solid-phase extraction on porous graphitic carbon (PGC) column and high performance liquid chromatography (HPLC) separation on octadecyl silica column is proposed. The approach includes extraction of phenols from 10 ml of water sample on PGC column; desorption with subcritical water at 175°C, injection of the eluate to the HPLC column and separation at ambient temperature with acetonitrile–water eluent. Limits of detection for UV detection of phenols were 1–5 μg l^?1, and analyte peaks were 20–50% narrower than for direct injection of 20 μl of sample.     

A Simple and Direct Procedure for the Evaluation of Triglyceride Composition of Cocoa Butters by High Performance Liquid Chromatography - A Comparison with the Existing TLC-GC Method

A high performance liquid chromatography (HPLC) method for the separation of triglycerides in cocoa butters at nanogram sensitivity is described. This procedure is based on UV detection at 220 nm. This study demonstrates the feasibility of using UV detection system for the quantitation of triglycerides employing a chromatographic separation system coupled with stop flow technique. HPLC analysis of triglycerides from various cocoa butters compared well with values obtained by existing TLC-GC method. This appears to represent the first report to describe the quantitative analysis of triglycerides employing short wavelength UV detection. The rapid quantitative analysis of triglycerides by HPL C holds promise in the qualitative evaluation of cocoa butters.     

HPLC von Vitamin A-Estern in Margarine

HPLC of Vitamin A-Esters in Margarine In many countries, margarines are fortified by the addition of fatsoluble vitamins. Vitamin A is usually added in the form of its palmitate or acetate esters or as β-9carotene. The addition of vitamin A-esters to margarines can be controlled by HPLC using silica columns. Various approaches for this analysis are discussed in this paper. The analysis of added vitamin A in the ester form (without saponification) provides a number of advantages which are also discussed. In gradient-elution HPLC the separation of several lipid-soluble vitamins (Vitamin A esters, β-carotene and tocopherols) is possible.     

Separation and Detection of Plasmalogen in Marine Invertebrates by High‐Performance Liquid Chromatography with Evaporative Light‐Scattering Detection

We have developed a new method for determining ethanolamine plasmalogen contents in marine invertebrates. This quantification method involves derivatization of ethanolamine glycerophospholipid (EtnGpl) subclasses, alkenylacyl (plasmalogen), diacyl, and alkylacyl subclasses, by enzyme treatment and acetylation, followed by separation and detection by high‐performance liquid chromatography (HPLC) with evaporative light‐scattering detection (ELSD). This method enabled complete separation of the subclasses, and the limit of detection for plasmalogen was 200 ng (260 pmol). The peak area of plasmalogen by ELSD was unaffected by the degree of unsaturated fatty acids in EtnGpl, in contrast to ultraviolet (UV) detection. Thus, this method enables accurate determination of plasmalogen contents in various species containing marine products possessing abundant polyunsaturated fatty acids (PUFA). The method developed here was applied to marine invertebrates available in Japan. The examined marine invertebrates showed a wide range of plasmalogen contents ranging from 19 to 504 μmol/100 g wet wt. The plasmalogen levels in samples except those of class Cephalopoda and Crustacea were more than 60 mol% of EtnGpl.     

高效液相色谱法测定维生素C中糠酸含量

建立了液相色谱法测定维生素C中糠酸方法。采用液相色谱检测,紫外检测器,C18色谱柱,柱温30℃,流动相采用乙腈/磷酸盐缓冲溶液,对检测方法的检出限、定量限、线性、精密度、稳定性进行考察。结果表明该方法线性方程为:Y=51403X+2142.9,在0.5~10μg·mL-1内,线性关系良好(r=0.9999)。该方法适合于维生素C中糠酸的测定。     

SPE萃取-离子对高效液相色谱法测定可乐中4-甲基咪唑

建立了可乐中4-甲基咪唑(4-MI)的SPE萃取-离子对试剂高效液相色谱(HPLC)测定法:采用SPE固相萃取小柱分离并富集样品中4-MI,用高效液相色谱-紫外检测器分析,以甲醇/磷酸二氢钾缓冲液为流动相,在反相C18色谱柱上进行等度淋洗,进样量为25.00μL,检测波长215 nm。该方法的检出限为0.4 mg/kg(5 S),相对标准偏(RSD)均小于2%,具有良好的准确性和精密度,可满足可乐中4-MI含量测定分析。应用该方法测定多种品牌可乐中4-MI含量,范围为0.18~2.01 mg/L。     

Triglyceride composition of ewe,cow, and goat milk fat

The separation and identification of the components in milk fat, which are mainly triglycerides, is a challenge due to its complex composition. A reverse-phase high-performance liquid chromatography (HPLC) method with gradient elution and light-scattering detection is described in this paper for the triglyceride analysis in ewes’ milk fat. Triglyceride identification was carried out by combining HPLC, gas-liquid chromatography (GLC), and the calculated equivalent carbon numbers of several triglyceride standards. Quantitation of partially resolved peaks in the HPLC chromatogram was accomplished by applying a peak deconvolution program. Forty-four fatty acids were identified by GLC analysis, but only 19 were used for the following prediction of triglyceride molecular species; 181 triglycerides were identified, some of which were grouped at the same peak and needed application of the deconvolution program. Consequently, coefficients of variation were close to or lower than 5%. Moreover, the triglyceride composition of ewe, cow, and goat milk fat were compared by using these methods. These results show that ewe milk fat is richer in short- and medium-chain triglycerides, and cow milk fat is richer in long-chain and unsaturated triglycerides.