Fast,simple, and sensitive high‐performance liquid chromatography method for measuring vitamins A and E in human blood plasma

Vitamins A and E are fat‐soluble vitamins that play important roles in several physiological processes. Monitoring their concentrations is needed to detect deficiency and guide therapy. In this study, we developed a high‐performance liquid chromatography method to measure the major forms of vitamin A (retinol) and vitamin E (α‐tocopherol and γ‐tocopherol) in human blood plasma. Vitamins A and E were extracted with hexane and separated on a reversed‐phase column using methanol as the mobile phase. Retinol was detected by ultraviolet absorption, whereas tocopherols were detected by fluorescence emission. The chromatographic cycle time was 4.0 min per sample. The analytical measurement range was 0.03–5.14, 0.32–36.02, and 0.10–9.99 mg/L for retinol, α‐tocopherol, and γ‐tocopherol, respectively. Intr‐aassay and total coefficient of variation were <6.0% for all compounds. This method was traceable to standard reference materials offered by the National Institute of Standards and Technology. Reference intervals were established using plasma samples collected from 51 healthy adult donors and were found to be 0.30–1.20, 6.0–23.0, and 0.3–3.2 mg/L for retinol, α‐tocopherol, and γ‐tocopherol, respectively. In conclusion, we developed and validated a fast, simple, and sensitive high‐performance liquid chromatography method for measuring the major forms of vitamins A and E in human plasma.     

二阶导数-同步荧光法同时测定微量维生素B_1,B_2和烟酰胺

建立了二阶导数-同步荧光法同时测定微量维生素B1,B2和烟酰胺含量的方法.在最佳实验条件下,维生素B1,B2和烟酰胺的检测限分别为2.1,2.6,和2.8 ng/mL,相对标准偏差为1.5%～4.9%,加标回收率为92%～105%.该方法可用于样品中微量维生素的测定.     

Profiling of rat plasma by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, a novel tool for biomarker discovery in nutrition research

The recent development of high-throughput proteomic technologies has given us new methods to analyze how an organism responds to changes in its nutritional environment. The analysis of plasma samples by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was investigated as a novel approach to the identification of new biomarkers of nutrient status. Pre-fractionation of rat plasma by anion-exchange chromatography in 96-well filter plates markedly increased the total number of unique peptides and proteins that could be observed in SELDI-TOF mass spectra. Replicate fractionations generated nearly identical pH fractions, not only in terms of peptide and protein composition but also in respect to the ion signal intensity of replicate SELDI-TOF mass spectra. The feasibility of this approach was tested with samples from retinol-sufficient and retinol-deficient rats. The comparative analysis revealed reduced levels of three proteins with molecular masses between 10,000 and 20,000 in plasma of retinol-deficient rats. These results demonstrate that plasma profiling by anion-exchange fractionation and SELDI-TOF-MS may be a promising surveillance tool to detect changes in nutritional status and whole body physiology.     

Simultaneous determination of water- and fat-soluble vitamins in pharmaceutical preparations by high-performance liquid chromatography coupled with diode array detection

Water- and fat-soluble vitamins were separated on a MetaChem Polaris C18-A (150 mm×4.6 mm, 3 μm particle size) in a single run using combined isocratic and linear gradient elution with a mobile phase consisting of 0.010% trifluoroacetic acid of pH 3.9 (solvent A) and methanol (solvent B) at the flow rate 0.7 ml min⁻¹. A linear gradient profile (A:B) started at 95:5 and was constant in the first 4 min, then linearly decreased up to 2:98 during the next 6 min, then it was constant in the next 20 min and finally linearly increased up to 95:5 ratio of water phase in the last 5 min of the separation. The most suitable detection wavelength for simultaneous vitamin determination was 280 nm. The method was applied for the solid sample of pharmaceutical preparation (B-Komplex), fortified powdered drinks (multi-vitamin) and food samples. The results were in good agreement with the declared values.     

Development of a validated HPLC method for the determination of B-complex vitamins in pharmaceuticals and biological fluids after solid phase extraction

An HPLC method was developed for the simultaneous determination of seven water-soluble vitamins, viz. thiamine, riboflavin, nicotinic acid, nicotinamide, pyridoxine, cyanocobalamin, and folic acid, in multivitamin pharmaceutical formulations and biological fluids (blood serum and urine). Separation was achieved at ambient temperature on a Phenomenex Luna C18 (150 x 4.6 mm) analytical column. Gradient elution was performed starting at a 99:1 A:B v/v composition, where A: 0.05 M CH3COONH4/CH3OH (99/1) and B: H2O/CH3OH (50/50), at a flow rate of 0.8 mL/min. After a 4-min isocratic elution the composition was changed to 100% of B in 18 min and elution continued isocratically for 8 min. Detection was performed with a photodiode array detector at 280 nm. Each vitamin was quantitatively determined at its maximum wavelength. Spectral comparison was used for peak identification in real samples. Detection limits were in the range of 1.6-3.4 ng, per 20-microL injection, while linearity held up to 25 ng/microL. Accuracy, intra-day repeatability (n = 6), and inter-day precision (n = 7) were found to be satisfactory. Theobromine (2 ng/microL) was used as internal standard. Sample preparation of biological fluids was performed by SPE on Supelclean LC-18 cartridges with methanol-water 85/15 v/v as eluent. Extraction recoveries from biological matrices ranged from 84.6% to 103.0%.     

Miniaturized membrane-based reversed-phase chromatography and enzyme reactor for protein digestion,peptide separation,and protein identification using electrospray ionization mass spectrometry

Separation and determination of water- and fat-soluble vitamins by micellar (MEKC) and microemulsion electrokinetic chromatography (MEEKC) are compared. MEKC is only useful in the quantitative analysis of water-soluble vitamins when sodium dodecylsulfate (SDS) is used as the surfactant. However, the separation of mixtures containing water- and fat-soluble vitamins is only achieved by MEEKC using a microemulsion prepared by mixing SDS as the surfactant, butanol as the co-surfactant, octane as the non-polar modifier and propanol as the second co-surfactant. The injection time and the solvent used for the dilution of samples have a significant effect on the analysis of lypophilic compounds. The most reproducible results in the analysis of fat-soluble vitamins are obtained by using the same microemulsion electrolyte as the solvent for samples and an injection time of 10 s.     

Electrochemical behaviour of pyridoxine hydrochloride (vitamin B6) at carbon paste electrode modified with crown ethers

The electrochemical behaviour of pyridoxine hydrochloride (pyridoxine HCl) at the plain carbon paste electrode and the electrode modified with oxa crown ether has been studied using voltammetric and impedance measurements. The macrocycles used as modifiers were 18-crown-6, dibenzo-18-crown-6 (DB18C6), dicyclohexano-18-crown-6 and dibenzo-24-crown-8, out of which DB18C6 gave better response for pyridoxine HCl. Tris buffer (pH 10.3) was chosen as an appropriate medium among the several supporting electrolytes of varying pH studied. The characterization of the DB18C6-modified electrode (CME-DB18C6) using kinetic parameters such as number of electrons (n) and electron transfer coefficient (α) is studied by cyclic voltammetry. Electrochemical impedance spectroscopic measurements obtained confirm the current enhancement over the modified electrode. Analytical applications of this electrode have been studied for the determination of pyridoxine HCl. A sensitive linear working range of 0.6 to 100 μg cm⁻³ with a detection limit of 0.4 μg cm⁻³ by differential pulse voltammetry was observed for pyridoxine HCl on CME-DB18C6. However, on decreasing the scan rate to 5 mV s⁻¹, the detection limit lowered to 0.2 μg cm⁻³. Interference from some vitamins like thiamine hydrochloride, riboflavin, nicotinamide, para-aminobenzoic acid, cyanocobalamin, folic acid and d-biotin and amino acid l-tryptophan was studied, and simultaneously, riboflavin, thiamine hydrochloride and pyridoxine HCl were determined over the modified electrode, CME-DB18C6. The modified electrode is successfully used for the determination of pyridoxine HCl in multivitamin pharmaceutical preparations.     

Chelation of CeIII and PrIII by N,N′‐Bis(pyridoxylideneiminato)ethylene,a Schiff Base Ligand Derived from Vitamin B6: Synthesis,Structural Features and TGA Evaluations of [M(Hpyr2en)(pyr2en)]·nH2O (M = CeIII,n = 2; PrIII,n = 8)

The neutral Schiff base N,N′‐bis(pyridoxylideneiminato)ethylene (H₂pyr₂en) reacts with CeCl₃ · 4H₂O and PrCl₃ · 4H₂O to give [Ce(Hpyr₂en)(pyr₂en)] · 2H₂O ( 1 ) and [Pr(Hpyr₂en)‐(pyr₂en)] · 8H₂O ( 2 ). In the two bimolecular chelate systems, the endo hydroxyl groups of the rings undergo deprotonation by transferring one proton to the pyridinic nitrogen atoms. This confirms the ability of the pyridoxal containing ligand H₂pyr₂en to form stable heavy metal chelates with unusual coordination polyhedra. Complexes 1 and 2 show coordination number 8, and represent a distorted quadratic antiprismatic arrangement. To date no other references have been made to the complexation of Ce and Pr by any vitamin B₆ derivative. The synthesis and the structure elucidation of 1 and 2 represent a contribution to the research on prevention and therapy of damages caused by heavy elements.     

Determination of cobalamins (hydroxo-, cyano-, adenosyl- and methyl-cobalamins) in seawater using reversed-phase liquid chromatography with diode-array detection

A high-performance liquid chromatography method was developed for the separation and determination of four cobalamins in seawater. Chromatographic separation was performed on a reversed-phase discovery RP-amide C₁₆ column with buffer potassium dihydrogenphosphate and acetonitrile as the mobile phases in linear gradients elution mode. Cobalamins were previously preconcentrated in C₁₈ resins cartridges. Detection was performed using UV-diode array detector in a range of λ of 200–400 nm. The method showed to be linear over a range of 1–300 ng mL⁻¹ with acceptable precision and accuracy. The detection limits ranged between 0.07 pg mL⁻¹ for 5′-deoxyadenosylcobalamin and 0.5 pg mL⁻¹ for hydroxocobalamin. The mean cobalamins recoveries for direct determination ranged between 76 and 93% for hydroxo-, cyano- and methylcobalamin, while the recovery for 5′-deoxyadenosylcobalamin was only 31% suggesting that the preconcentration method was not valid for this cobalamin. The method was successfully applied to coastal seawater where the concentrations ranged from 4.2 to 7.3 ng L⁻¹ for hydroxo-, 1.4–3.9 ng L⁻¹ for cyano-, 2.1–4.6 ng L⁻¹ for 5′-deoxyadenosyl- and 33–83.5 ng L⁻¹ for methylcobalamin.