High-performance liquid chromatography of fat-soluble vitamins: separation and identification of vitamins D2 and D3 and their isomers in food samples in the presence of vitamin A, vitamin E and carotene

Vitamins D2 and D3 and their corresponding previtamins and provitamins were resolved by reversed-phase high-performance liquid chromatography using a ternary solvent system (acetonitrile-methanol-water) pumped according to a gradient elution programme. The D vitamins were also resolved in the presence of other lipid-soluble vitamins (A, E and K1) and carotene. The peaks were monitored with a UV-visible variable-wavelength detector and were detected at their maximum absorbance, resulting in maximum sensitivity. Lipid-soluble vitamins and carotene were resolved in extracts obtained from oils and butter, thus permitting their identification in a single chromatographic run.     

Simultaneous determination of fat-soluble vitamins and provitamins in dairy products by liquid chromatography with a narrow-bore column

A reversed-phase high-performance liquid chromatographic method is described for the simultaneous determination of vitamins A, D2, D3, E and K1, retinyl acetate, retinyl palmitate, tocopherol acetate, ergosterol and 7-dehydrocholesterol in milk and butter. Narrow-bore columns are recommended because this alternative provides a good separation and efficiency, plus greater economy and sensitivity. Detection limits for individual vitamins range from 0.14 to 6.9 ng. All vitamins are separated in less than 33 min. For the simultaneous determination of these vitamins and provitamins we use two sample pre-treatment methods, a liquid-liquid extraction with hexane or a solid-phase extraction with a C18 cartridge. Recovery studies show good results for all solutes (84-108% and 85-108% for milk and butter, respectively) and the intra-day coefficients of variations range from 1.6 to 4.5%. These methods permit the simple determination of fat-soluble vitamins using a small sample volume.     

Simultaneous quantification of vitamins A, D3 and E in fortified infant formulae by liquid chromatography-mass spectrometry

A novel method for the simultaneous quantification of Vitamins A, D3 and E in fortified infant formulae has been developed using isocratic normal-phase liquid chromatography with positive atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS). Food products were saponified and the vitamins were extracted by solid-phase extraction (SPE) on a Chromabond XTR cartridge. Quantification of Vitamins D3 and E were performed with Vitamin D2 and 5,7-dimethyltocol (DMT) as internal standards (IS), respectively while no IS was used for Vitamin A. Detection of the vitamins was made in the selected ion monitoring (SIM) mode. MS calibration curves were linear between 0.15 and 12 mg/l for Vitamin A, 5-400 microg/l for Vitamin D3 and 0.25-20 mg/l for Vitamin E with regression coefficient r2 > 0.996 and the limits of detection were below 1.4 ng. The repeatability (CV) obtained on a reference dietetic infant formula was 2.3% for Vitamin A, 2.6% for Vitamin E and 5.9% for Vitamin D3. The between-day variations (CV) over 6 days were in the ranges of 2.4-6.9% for the three vitamins. The mean recoveries from a reference infant formula spiked with all three vitamins ranged from 96 to 105% with a relative standard error less than 9%. The applicability of the method was demonstrated by analyzing a set of infant formula and infant cereals; similar results were obtained with the LC-MS method and reference HPLC methods.     

Determination of vitamins in food-matrix Standard Reference Materials

In recent years, the National Institute of Standards and Technology (NIST) has developed several food-matrix Standard Reference Materials (SRMs) characterized for vitamins and other organic nutrients. NIST uses several "modes" for assignment of analyte concentrations in SRMs, one of which includes the use of data provided by collaborating laboratories. Certification modes and liquid chromatographic methods that were used by NIST for value assignment of vitamin concentrations in recently introduced food-matrix SRMs are described in this paper. These materials and methods include vitamins D and E in coconut oil (SRM 1563) by gravimetry and multi-dimensional liquid chromatography (LC); vitamins A, E, and several B vitamins by reversed-phase LC and vitamin C by ion-exchange chromatography in infant formula (SRM 1846); and carotenoids and vitamins A and E by reversed-phase liquid chromatography in a baby food composite (SRM 2383).     

Determination of vitamins D2, D3, K1 and K3 and some hydroxy metabolites of vitamin D3 in plasma using a continuous clean-up-preconcentration procedure coupled on-line with liquid chromatography-UV detection

A semi-automatic procedure for the continuous clean-up and concentration of several fat-soluble vitamins prior to their separation by HPLC and UV detection is reported. The procedure is based on the use of a minicolumn packed with aminopropylsilica as sorbent located prior to the chromatographic detection system. The overall process was developed and applied to the main liposoluble vitamins (A, D2, D3, E, K1, K3) and several hydroxy metabolites of vitamin D3 [25-(OH)-D3,24,25-(OH)2-D3 and 1,25-(OH)2-D3]. All the analytes were monitored at a compromise wavelength of 270 nm. Calibration graphs were constructed between 0.01 and 100 ng ml-1 for vitamin D2 and D3 and their hydroxy metabolites, between 0.1 and 100 ng ml-1 for vitamin A, K1 and K3 and between 1 and 100 ng ml-1 for vitamin E, with excellent regression coefficients (> or = 0.9901) in all cases. The precision was established at two concentration levels with acceptable RSDs in all instances (between 3.6 and 8.7%). The method was appropriate for the determination of vitamin D2, D3, K1 and K3 and the 24,25-dihydroxy and 25-hydroxy metabolites of vitamin D3 in human plasma. The method was applied to plasma samples spiked with the target analytes and the recoveries ranged between 78 and 109%.     

Determination of vitamins D2 and D3 in pharmaceuticals by supercritical-fluid extraction and HPLC separation with UV detection

Summary The supercritical-fluid extraction of vitamins D₂ and D₃ with carbon dioxide is reported for the first time. The extraction recovery was enhanced by direct addition of diethyl ether to sample contained in the extraction cell. Separation and detection of the analytes was performed off-line by reversed-phase liquid chromatography with UV-detection. The quantification limit of the method is 4.1 μg for both analytes, with precision, expressed as relative standard deviation, of 3.8 and 6.3% for vitamins D₂ and D₃, respectively (η=7). The proposed method has been applied to the determination of vitamin D in different pharmaceutical products; recoveries were between 85 and 105%.     

Development of a green chromatographic method for determination of fat-soluble vitamins in food and pharmaceutical supplement

A green chromatographic analytical method for determination of fat-soluble vitamins (A, E, D3 and K1) in food and pharmaceutical supplement samples is proposed. The method is based on the modification of a C18 column with a 3.00% (w/v) sodium dodecyl sulphate (SDS) aqueous solution at pH 7 (0.02 mol L(-1) phosphate buffer solution) and in the usage of the same surfactant solution as mobile phase with the presence of 15.0% (v/v) butyl alcohol as an organic solvent modifier. After the separation process, the vitamins are detected at 230 nm (K1, D3 and E), 280 nm (A, E, D3 and K1) and 300 nm (K1, D3 and E). The chromatographic procedure yielded precise results (better than 5%) and is able to run one sample in 25 min, consuming 1.5 g of SDS, 90 mg of phosphate and 7.5 mL of butyl alcohol. When the flow rate of the mobile phase is 2 mL min(-1) the retention times are 4.0, 9.6, 13.0 and 22.7 min for D3, A, E and K1 vitamins, respectively; and all peak resolutions are higher than 2. The analytical curves present the following linear equations: area=6290+34852 (vitamin A), R2=0.9998; area=4092+36333 (vitamin E), R2=0.9997; area=-794+30382 (vitamin D3) R2=0.9998 and area=-7175+82621 (vitamin K1), R2=0.9996. The limits of detection and quantification for vitamins A, E, D(3) and K(1) were estimated for a test pharmaceutical vitamin supplement sample as 0.81, 1.12, 0.91 and 0.83 mg L(-1) and 2.43, 3.36, 2.73 and 2.49, respectively. When the proposed method was applied to food and pharmaceutical sample analysis, precise results were obtained (R.S.D.<5% and n=3) and in agreement with those obtained by using the classical chromatographic method that uses methanol and acetonitrile as mobile phase. Here, the traditional usage of toxic organic solvent as mobile phase is avoided, which permits to classify the present method as green.     

Analysis of water soluble vitamins by high pressure liquid chromatography.

Resolution of the water-soluble vitamins--pyridoxin, riboflavin, niacinamide, vitamin B12, thiamin, ascorbic acid, niacin and folic acid--by high pressure liquid chromatography was examined on two bonded-phase columns, muBondapak C18 and muBondapak NH2. The effect on the retention times of individual vitamins and the separation of a multivitamin sample was determined using varying proportions of water/methanol as the eluting solvent and by addition of various salts, buffer solutions and PIC reagents to the water/methanol. Each vitamin was able to be eluted satisfactorily from muBondapak C18. It was found that seven vitamins could be resolved from a multivitamin mixture in a single analysis in several solvent systems with the total time for the analyses being always less than 40 min. With muBondapak NH2, all the vitamins except folic acid were eluted and six vitamins could be resolved from a mixture in a single analysis. The speed of analysis was greater with muBondapak NH2 with all compounds eluted in 15 min and the peaks were sharper. The order of elution was essentially the reverse of that obtained with muBondapak C18.     

Determination of vitamins A and E in milk powder using supercritical fluid extraction for sample clean-up

A method for the analysis of the natural contents of vitamins A and E in milk powder has been developed. The method utilises supercritical fluid extraction, a miniaturised alkaline saponification procedure and reversed-phase HPLC with UV detection. Modifications of the sample matrix, combinations of static and dynamic modes of extraction and effects of changes in extraction parameters such as temperature, flow-rate, time, collection solvent and collection temperature were studied to optimise the extraction efficiency and selectivity. Supercritical CO2 at 80 degrees C and 37 MPa, modified with 5% methanol and pumped at a flow-rate of 1.0 ml/min, gave recoveries of 99 and 96% for vitamins A and E, respectively, using a 15 min static followed by a 15 min dynamic extraction. The measurements gave a within-day RSD of 4% for both vitamin A and E, and between-day RSDs of 4 and 8% for vitamins A and E, respectively.     

Provitamins and vitamins D2 and D3 in Cladina spp. over a latitudinal gradient: possible correlation with UV levels.

Provitamin D2, vitamin D2 and vitamin D3 were identified in the thallus of a lichen species, Cladina arbuscula (Wallr.) Hale and W.L. Culb. The identification of vitamin D3 was supported by: (1) co-chromatography in both reverse and straight phase HPLC (high performance liquid chromatography), (2) ultraviolet absorption spectrum, and (3) molecular ion peaks demonstrated by ESI (electrospray ionisation) mass spectrometry. The contents of vitamin D3 range from 0.67 to 2.04 microg g(-1) dry matter in the thalli of C. arbuscula specimens grown under different natural conditions, while provitamin D3 could not be detected. The ranges for provitamin D2 and vitamin D2 were 89-146 and 0.22-0.55 microg g(-1) dry matter, respectively, while the contents of provitamin D3 were below the detection limit (0.01 microg g(-1) dry matter). When C. arbuscula thalli collected at different latitudes from northern Finland to Greece were compared, a positive correlation of vitamin D2 and D3 contents with modelled UV-B radiation at the collection sites was found. A single sample of C. rangiferina from northern Finland gave much higher values for the vitamins. A possible reason could be the lower content of UV-B absorbing pigment in the latter species.     

Determination of fat-soluble vitamins in yogurt by HPLC with electrochemical detection

A method employing HPLC with electrochemical detection for the rapid and simultaneous determination of vitamins A, D(3) and E is described. The method uses a C-18 reverse phase column and 2.5 mM HAcO-NaAcO in methanol-water (99:1, v/v) solution as the mobile phase. The compounds are quantified using amperometric detection with a glassy carbon electrode at a potential of + 1300 mV (vs. Ag/AgCl) and the results are compared with those obtained using UV detection at a wavelength of 280 nm. The method was successfully applied to the analysis of vitamins A, D(3) and E in yogurt samples. After saponification, fat-soluble vitamins were extracted and the methanolic solution of the extracts was injected directly into the chromatographic system, avoiding the clean-up step which is necessary when no electrochemical detection is used. Good recovery percentages were obtained.     

High-performance liquid chromatography with electrochemical detection for the simultaneous determination of vitamin A, D3 and E in milk.

A high-performance liquid chromatographic electrochemical detection for the rapid and simultaneous determination of the vitamin A, D3 and E is described. The separation is carried out by using a C18 reversed-phase column and 0.1 M LiClO4 in methanol-water (99:1, v/v) as the mobile phase. The compounds are eluted with good resolution in the above order within about 15 min and are determined by amperometric detection with a glassy carbon electrode at +1050 mV (vs. Ag/AgCl). The method gave reproducible results and the detection limits were of the order of 0.07, 4 and 0.2 ng of vitamin A, D3 and E, respectively. The method was successfully applied to the determination of vitamin A, D3 and E in liquid cow milk and milk powder samples. After saponification, fat-soluble vitamins were extracted with hexane and a methanolic solution of the dried extract was injected directly into the chromatographic system, avoiding the clean-up step that is necessary for vitamin D3 when electrochemical detection is not used. Good recoveries were obtained.     

Identification of vitamins A, D and E by particle beam liquid chromatography-mass spectrometry

Particle beam (PB) high performance liquid chromatography (HPLC)-mass spectrometry (MS) has been studied to investigate its suitability for the analysis of fat-soluble vitamins, as an example of thermally labile substances. The PB LC-MS system has been used to detect and identify vitamins A, D and E in foods and multi-vitamin preparations. The qualitative performance of the PB interface has been evaluated by an examination of the working parameters. A rapid elution of the vitamins was obtained on a C8 reversed-phase column using an aqueous methanol mobile phase. Analyses have been carried out using both, electron (EI) and chemical (CI) ionization. Positive and negative-ion CI spectra of the compounds are discussed. When PB-MS conditions are optimized, the detection limits for the fat-soluble vitamins tested have been typically in the 0.6–25 ng range when using the selected-ion monitoring technique.     

微乳液相色谱法同时测定4种脂溶性维生素

建立了一种新的微乳体系,并成功地应用于微乳液相色谱法(MELC)快速分析脂溶性维生素VA、VD2、VD3和VE。通过对影响分离选择性的主要因素进行考察,得到最佳微乳体系组成为98%(v/v)(50 g/L十二烷基硫酸钠(SDS)-10%(质量分数)正丁醇-1.0%(质量分数)正辛烷-84%水(质量分数))-2%(v/v)乙腈。该微乳体系中,表面活性剂类型和浓度、油相正辛烷的含量、有机添加剂乙腈对脂溶性维生素的分离起到了重要的作用。以Venusil ASB C18色谱柱(150 mm×4.6 mm, 5 μm)为分离柱,流速为0.7 mL/min,检测波长为265 nm,柱温为40 ℃, VA、VD2、VD3和VE在20 min内达到基线分离。4种脂溶性维生素的保留时间和峰面积的相对标准偏差(RSD) (n=5)分别小于2.3%和3.0%; VA、VD2、VD3和VE的线性范围分别为22.0～88.0 mg/L、20.2～81.0 mg/L、24.3～97.2 mg/L和125.0～500.0 mg/L,相应的线性相关系数r2分别为0.9996、0.9994、0.9998、0.9998;检出限(S/N=3)分别为0.37、0.34、0.41和2.12 mg/L。本方法已成功应用于多维元素片(21)中VA与VE的测定,结果令人满意。     

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.     

在线二维液相色谱法同时测定婴幼儿和成人配方营养品中的维生素A、D3和E

建立了在线二维液相色谱同时快速测定婴幼儿配方乳品和成人强化乳品中维生素A、D₃和E含量的方法。首先,依据疏水减法模型,选择C8柱和极性嵌合的反相C18柱分别作为一维和二维分离柱,构成正交分离体系,并均以甲醇、乙腈和水作为流动相,检测波长设为263 nm(维生素D₃)、296 nm(维生素E)和325 nm(维生素A)。采用双三元液相色谱的左泵作为一维分析泵,完成维生素A、E的定量和维生素D₃的净化;根据维生素D₃在一维色谱柱上的保留时间,确定切割时间窗口,并以500 μL定量环收集含有维生素D₃的馏分,由双三元液相色谱的右泵将馏分带到二维色谱柱中,以维生素D₂作为内标物,采用内标法完成维生素D₃的定量分析,整个过程在密闭系统中自动化完成。在上述优化条件下测定了婴幼儿和成人奶粉、奶酪及酸奶等强化乳品中3种维生素的含量。经过1.25 kg/L KOH溶液的热皂化和石油醚的萃取,样品萃取液直接进样分析,得到维生素D₃的加标回收率为75.50%~85.00%,并通过配对t检验法与标准方法测定结果进行比较分析,结果差异无统计学意义,表明本方法可同时快速、准确测定婴幼儿及其他配方营养品中维生素A、D₃、E的含量,提高了样品分析效率。     

Determination of vitamins A, D and E in a small volume of human plasma by a high-throughput method based on liquid chromatography/tandem mass spectrometry

We have developed an automated high-throughput assay for the determination of vitamin A (retinol), ergocalciferol (25-OH D2), cholecalciferol (25-OH D3) and vitamin E (α-tocopherol) in a small volume of human plasma. Sample preparation involved mixing 50 μL of plasma with 100 μL of ethanol containing isotope-labelled internal standards, followed by mixing with isooctane/chloroform (3:1, 300 μL). The organic phase was evaporated, and the sample reconstituted in 50 μL methanol. The analysis was performed using reversed-phase liquid chromatography with a gradient mobile phase containing water, methanol and ammonium formate. Chromatographic run-time was 5 min, and positive mode electrospray tandem mass spectrometry (MS/MS) was used for detection. The limits of detection were 0.10 μM for all-trans retinol and 3.3 nM for 25-OH D2 and 25-OH D3. Recoveries were 91.9-105.0%, and within- and between-day coefficients of variance (CVs) 2.4-5.3 and 3.1-8.2, respectively. The assay is presently being used in large-scale studies.     

Measurements of the major isoforms of vitamins A and E and carotenoids in the blood of people with spinal-cord injuries

We used reversed-phase HPLC with diode array detection to simultaneously measure the major isoforms of vitamins A, E, and the carotenoids in serum from 55 healthy people with spinal cord injuries. Typically, the method measured retinol (vitamin A), alpha-tocopherol (vitamin E) and beta-carotene, alpha-carotene, lutein, lycopene, and cryptoxanthin (carotenoids). gamma-Tocopherol (vitamin E), 25-hydroxycalciferol (vitamin D), and the carotenoid zeaxanthin could also be measured when they were present in high concentrations. Healthy people with spinal cord injuries were more likely than similar people without injuries to have low concentrations of alpha-tocopherol, and to a lesser extent retinol and beta-carotene.     

Determination of eight water- and fat-soluble vitamins in multi-vitamin pharmaceutical formulations by high-performance liquid chromatography

In the present work, a reversed-phase high-performance liquid chromatographic procedure has been developed for the determination of water-soluble vitamins (thiamine hydrochloride, pyridoxine hydrochloride, nicotinamide, riboflavin phosphoric ester and cyanocobalamine) and fat-soluble vitamins (retinol palmitate, cholecalciferol, -tocopherol acetate) in multi-vitamin pharmaceutical formulations. The sample treatment proposed consists of a solid-phase extraction with C₁₈ AR cartridges that allow the separation of fat-soluble vitamins, which were retained on the sorbent, from water-soluble vitamins. Afterwards, the water-soluble vitamins were analysed by HPLC on a Nova-Pack C₁₈ (150×3.9 mm, 4 μm) analytical column, using CH₃OH–0.05 M CH₃COONH₄ as mobile phase The chromatographic analysis of the fat-soluble vitamins was carried out after their sequential elution with methanol and chloroform from C₁₈ sorbent, on the above column. The mobile phase employed was MeOH–CH₃CN (95:5, v/v) working at a flow-rate of 2 ml min⁻¹ in isocratic mode. The solid-phase extraction for these vitamins had been previously optimised. The experimental variables studied were: application volume, elution solvents and cleaning solutions. The UV–Vis detection of vitamins was made at 270 nm for all the water-soluble vitamins (362 nm for B₁₂) and 285 nm for the water-soluble and fat-soluble vitamins present in real samples at different concentration levels. The accuracy of the method was tested obtaining an average recovery ranging between 78 and 116%.     

Spectrofluorimetric determination of some water-soluble vitamins

Two simple and sensitive spectrofluorimetric methods were developed for determination of three water-soluble vitamins (B1, B2, and B6) in mixtures in the presence of cyanocobalamin. The first one was for thiamine determination, which depends on the oxidation of thiamine HCl to thiochrome by iodine in an alkaline medium. The method was applied accurately to determine thiamine in binary, ternary, and quaternary mixtures with pyridoxine HCl, riboflavin, and cyanocobalamin without interference. In the second method, riboflavin and pyridoxine HCl were determined fluorimetrically in acetate buffer, pH 6. The three water-soluble vitamins (B1, B2, and B6) were determined spectrofluorimetrically in binary, ternary, and quaternary mixtures in the presence of cyanocobalamin. All variables were studied in order to optimize the reaction conditions. Linear relationship was obeyed for all studied vitamins by the proposed methods at their corresponding lambda(exc) or lambda(em). The linear calibration curves were obtained from 10 to 500 ng/mL; the correlation ranged from 0.9991 to 0.9999. The suggested procedures were applied to the analysis of the investigated vitamins in their laboratory-prepared mixtures and pharmaceutical dosage forms from different manufacturers. The RSD range was 0.46-1.02%, which indicates good precision. No interference was observed from common pharmaceutical additives. Good recoveries (97.6 +/- 0.7-101.2 +/- 0.8%) were obtained. Statistical comparison of the results with reported methods shows excellent agreement and indicates no significant difference in accuracy and precision.