Synthetic studies of vitamin D analogues. IX. Synthesis and differentiation-inducing activity of 1 alpha,25-dihydroxy-23-oxa-, thia-, and azavitamin D3.

Three vitamin D3 analogues, 1 alpha,25-dihydroxy-23-oxavitamin D3 (3), 1 alpha,25-dihydroxy-23-thiavitamin D3 (4) and 1 alpha,25-dihydroxy-23-azavitamin D3 (5) were synthesized. In the differentiation-inducing activity of human myeloid leukemia cells into macrophages in vitro, the 23-aza analogue (5) showed the least activity, while no remarkable differences were observed between the 23-oxa analogue (3) and the 23-thia analogue (4), which were less active than 1 alpha,25-dihydroxyvitamin D3 (1).     

Synthesis of nucleosides of 5-substituted-1,2,4-triazole-3-carboxamides

Nucleosides of 5-substituted-1,2,4-triazole-3-carboxamides were prepared by the acid-catalyzed fusion procedure and by glycosylation of the appropriate trimethylsilyl derivative. The following nucleosides were obtained in two steps starting from methyl 4-substituted-1,2,4-triazole-3-carboxylates: 5-chloro-1-β- D -ribofuranosyl-1,2,4-triazole-3-carboxamide ( 6 ), 3-chloro-1-β- D -ribofuranosyl-1,2,4-triazole-5-carboxamide ( 5 ), 3-nitro-1-β- D -ribofuranosyl-1,2,4-triazole-5-carboxamide ( 12 ), 3-amino-1-β- D -ribofuranosyl-1,2,4-triazole-5-carboxamide ( 13 ), 5-methyl-1-β- D -ribofuranosyl-1,2,4-triazole-3-carboxamide ( 15 ), and 3-methyl-1-β- D -ribofuranosyl-1,2,4-triazole-5-carboxamide ( 16 ). In addition, 5-amino-1-β- D -ribofuranosyl-1,2,4-triazole-3-carboxamide ( 7 ), and 1-β- D -ribofuranosyl-1,2,4-triazole-3-carboxamide-5-thiol ( 8 ) were prepared from 6 .     

Effect on carbon lengthening at the side chain terminal of 1 alpha,25-dihydroxyvitamin D3 for calcium regulating activity

A series of analogs of 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3 (1)] with alkyl substitutions in 26- and 27-positions have been tested for their activity 1) in competing with 1,25-(OH)2D3 for binding to chick intestinal cytosol receptor, 2) in ability for formation of multinucleated cells (MNC) with various osteoclastic cell characteristics from blast cells, and 3) in stimulating bone calcium mobilization in vitamin D-deficient rats. The relative potencies of 1,25-(OH)2D3, 1 alpha,25-dihydroxy-26,27-dimethylvitamin D3 (2), 1 alpha,25-dihydroxy-26,27-diethylvitamin D3 (3), and 1 alpha,25-dihydroxy-26,27-dipropylvitamin D3 (4) in competing for intestinal cytosolic binding were 1:1.1:0.25:0.05. The similar order of the abilities on formation of the multinucleated cells in the same series was observed. In a bone calcium mobilization test with vitamin D-deficient rats, 1 alpha,25-dihydroxy-26,27-dimethylvitamin D3 showed slightly less activity than 1,25-(OH)2D3 at 12 h after administration, but long lasting activity was observed during time course experiments. 1 alpha,25-Dihydroxy-26,27-diethylvitamin D3, and 1 alpha,25-dihydroxy-26,27-dipropylvitamin D3 were found to be much less active than 1,25-(OH)2D3 in a bone calcium mobilization test.     

Synthesis and mesomorphism of novel multi-arm liquid crystals with cholic acid as chiral centre linking Schiff base moieties as mesogens

A new series of multi-arm chiral liquid crystals (LCs) D1–D3 were synthesised and characterised. Cholic acid was used as the core and ω-[4-(p-alkoxybenzoloxy)phenoxycarbonyl]valeric acid (B1–B3) was used as the mesogenic arms, containing different terminal substituent X (B1: X= -OCH₃, B2:X= -CH₃, B3: X= -Cl). Their structures and mesomorphic properties were investigated by Fourier-transform infrared spectroscopy, nuclear magnetic resonance hydrogen spectrometer, differential scanning calorimetry, polarised optical microscopy and X-ray diffraction, respectively. The mesogenic B1–B3 displayed smectic B phase. The multi-arm LC D1 displayed cholesteric, while D2 and D3 exhibited nematic phase. The formation of cholesteric phase of D1 was affected by both the chiral core – the bulky cholic acid and the polarity of the terminal substituent of the mesogenic arm. That D1 displayed cholesteric phase but D2–D3 did not indicated that the stronger polarity of the terminal group OCH₃ of D1 played an important part in stabilising the cholesteric phase. The multi-arm LCs D1–D3 all showed ultraviolet activity. The wavelength of maximum absorption of D1–D3 was affected by the terminal substituent of the mesogenic arm.     

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.     

Coordination networks with fluorinated backbones

Fluorine-containing ligands 2,3,5,6-tetrafluoro-1,4-bis(imidazol-1-yl-methyl)benzene (1) and 2,3,5,6-tetrafluoro-1,4-bis(2-methylimidazol-1-yl-methyl)benzene (2) were prepared and coordinated with AgNO3, Co(ClO4)2 x 6 H2O, and Cd(NO3)2 x 4 H2O, respectively, to form the following structures: 3D channel polymer [Ag2(1)2(NO3)2 x H2O x MeOH]n (3), 2D sheet polymer [Co(1)3(ClO4)2]n (4), 1D chain polymer [Cd(1)3(NO3)2 x 4 H2O]n (5), and a 2D herringbone sheet polymer [Ag(2)NO3 x 1.5 MeOH]n (6). The solid-state crystal structures of 3-6 were studied by single-crystal X-ray crystallography.     

Catalytic asymmetric syntheses and biological activities of singly dehydroxylated 19-nor-1alpha,25-dihydroxyvitamin D(3) A-ring analogs in cancer cell differentiation and apoptosis

BACKGROUND: 1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) has been shown to modulate not only proliferation and differentiation, but also apoptosis in malignant cells, indicating that it could be useful for treating cancer. Little information is available concerning the structural motifs of the 1alpha, 25(OH)(2)D(3) molecule responsible for modulation of differentiation and apoptosis, however. We set out to synthesize singly dehydroxylated A-ring analogs of 19-nor-1alpha,25(OH)(2)D(3) in a catalytic asymmetric fashion, and to investigate their biological activities in leukemia HL-60 cells. RESULTS: A series of singly dehydroxylated 19-nor-1alpha,25-dihydroxyvitamin D(3) A-ring analogs were synthesized using a combinatiorial sequence of regioselective propiolate-ene reaction and catalytic asymmetric carbonyl-ene cyclization. Surprisingly, the analogs could be clearly divided into two categories; one group, bearing 1alpha-hydroxy or 3beta-hydroxy groups in the A-ring, were potent differentiators and the second group, bearing 1beta-hydroxy or 3alpha-hydroxy groups, were potent stimulators of apoptosis. CONCLUSIONS: We have clearly identified the structural motifs of 19-nor-1alpha,25(OH)(2)D(3) analogs responsible for differentiation and apoptosis in HL-60 cells. These findings will provide useful information not only for development of therapeutic agents for treatment of leukemia and other cancers, but also for structure-function studies of 1alpha,25(OH)(2)D(3).     

An LC-MS/MS Method for Analysis of Vitamin D Metabolites and C3 Epimers in Mice Serum: Oral Supplementation Compared to UV Irradiation

Introduction: The most common forms of vitamin D in human and mouse serum are vitamin D3 and vitamin D2 and their metabolites. The aim of this study is to determine whether diet and sunlight directly affect the circulating concentrations of vitamin D metabolites in a mouse model. We investigated the serum concentrations of eight vitamin D metabolites—vitamin D (vitamin D3 + vitamin D2), 25OHD (25OHD3 + 25OHD2), 1α25(OH)₂D (1α25(OH)₂D2, and 1α25(OH)₂D3)—including their epimer, 3-epi-25OHD (3-epi-25OHD3 and 3-epi-25OHD2), and a bile acid precursor 7alpha-hydroxy-4-cholesten-3-one (7αC4), which is known to cause interference in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Method: The LC-MS/MS method was validated according to FDA-US guidelines. The validated method was used for the analysis of mouse serum samples. Forty blood samples from mice were collected and divided into three groups. The first group, the DDD mice, were fed a vitamin D-deficient diet (25 IU VD3/kg of diet) and kept in the dark; the second group, the SDD mice, were maintained on a standard-vitamin D diet (1000 IU VD3) and kept in the dark; and the third group, SDL, were fed a standard-vitamin D diet (1000 IU VD3) but kept on a normal light/dark cycle. LC-MS/MS was used for the efficient separation and quantitation of all the analytes. Results: The validated method showed good linearity and specificity. The intraday and interday precision were both <16%, and the accuracy across the assay range was within 100 ± 15%. The recoveries ranged between 75 and 95%. The stability results showed that vitamin D metabolites are not very stable when exposed to continuous freeze–thaw cycles; the variations in concentrations of vitamin D metabolites ranged between 15 and 60%. The overlapping peaks of vitamin D, its epimers, and its isobar (7αC4) were resolved using chromatographic separation. There were significant differences in the concentrations of all metabolites of vitamin D between the DDD and SDL mice. Between the groups SDD (control) and SDL, a significant difference in the concentrations of 3-epi-25OHD was noted, where C3 epimer was about 30% higher in SDL group while no significant differences were noted in the concentrations of vitamin D, 25OHD, 1α25(OH)₂D, and 7αC4 between SDD and SDL group. Conclusions: A validated method, combined with a simple extraction technique, for the sensitive LC-MS/MS determination of vitamin D metabolites is described here. The method can eliminate the interferences in LC-MS/MS analysis caused by the overlapping epimer and isobar due to them having the same molecular weights as 25OHD. The validated method was applied to mouse serum samples. It was concluded that a standard-vitamin D diet causes an increase in the proportion of all the vitamin D metabolites and C3 epimers and isobar, while UV light has no pronounced effect on the concentrations of the majority of the vitamin D metabolites except 3-epi-25OHD. Further studies are required to confirm this observation in humans and to investigate the biochemical pathways related to vitamin D’s metabolites and their epimers.     

Syntheses,Characterization, and Fluorescence Properties of Four Coordination Polymers based on Double Betaine Ligands

Four novel In³⁺ and Cd²⁺ based 1D coordination compounds constructed by double betaine ligands were synthesized and characterized structurally and optically. They assemble into 3D supra‐molecular architectures via different stacking or entanglement of 1D zigzag shaped chains, in which C–H ··· Cl and C–H ··· O hydrogen bonding interactions play a dramatic impact. Compound 1 displays a 1D + 1D → 3D four‐connect lvt net with 4² · 8⁴ topology. Compound 2 assembles into a 3D architecture by inclined stacking of the adjacent zigzag chains. Compound 3 displays a 2D + 2D → 3D inclined polycatenation based on the resulting 2D (6, 3) layers that constructed by 1D chains. Compound 4 displays a 3D supra‐molecular architecture based on 1D chains, which were connected via the hydrogen bonding. Meanwhile, four compounds emit in the range of visible region owing to the intra‐ligand π*→π and/or π*→n electron transition induced florescence.     

Computer simulation of sedimentation of ionic systems using the Wolf method

We present computer simulation results for 1:1 and 2:1 electrolyte solutions in the presence of a gravitational field, using the Monte Carlo method in the NVT ensemble for the restrictive primitive model. Coulombic interactions were taken into account comparing the Ewald and Wolf methods. Three variations of Ewald summations were considered: the exact method for slab geometries (EW2D), and the three-dimensional (3D) versions with and without a dipolar correction (EW3DC and EW3D, respectively). The equivalent 3D Wolf protocols were applied under the same conditions (WF3DC and WF3D, respectively). The Wolf and Ewald methods agree accurately in the prediction of several thermodynamic and structural properties for these inhomogeneous systems: excess internal energies, isochoric heath capacities, and density and electrostatic potential profiles. The main advantage using the Wolf method is the significant saving in computing time, which is approximately six times faster than EW3D and EW3DC, and sixty times faster than EW2D.     

Two new compounds from Coriaria nepalensis

Two new compounds,corialins A(1) and B(2) were isolated from Coriaria nepalensis Wall.These new compounds were established as 7-hydroxy-3-[2,3-acetonide-(3-methylbutane)]coumarin(1) and 3-O-β-D-glucopyranosyl-3,4,5-trihydroxy-1-(3- methyl-2-butenyl)-benzene(2),on the basis of 1D and 2D NMR techniques.     

Four new ursane‐type saponins from Morina nepalensis var. alba

Four new ursane‐type saponins, monepalosides C–F, together with a known saponin, mazusaponin II, were isolated from Morina nepalensis var. alba Hand.‐Mazz. Their structures were determined to be 3‐O‐α‐L ‐arabinopyranosyl‐(1 → 3)‐&[alpha;‐L ‐rhamnopyranosyl‐(1 → 2)]‐α‐L ‐arabinopyranosylpomolic acid 28‐O‐β‐D ‐glucopyranosyl‐(1 → 6)‐β‐D ‐glucopyranoside (monepaloside C, 1 ), 3‐O‐α‐L ‐arabinopyranosyl‐(1 → 3)‐&[alpha;‐L ‐rhamnopyranosyl‐(1 → 2)]‐β‐D ‐xylopyranosylpomolic acid 28‐O‐β‐D ‐glucopyranosyl‐(1 → 6)‐β‐D ‐glucopyranoside (monepaloside D, 2 ), 3‐O‐α‐L ‐arabinopyranosyl‐(1 → 3)‐&[beta;‐D ‐glucopyranosy‐(1 → 2)]‐α‐L ‐arabinopyranosylpomolic acid 28‐O‐β‐D ‐glucopyranosyl‐(1 → 6)‐β‐D ‐glucopyranoside (monepaloside E, 3 ) and 3‐O‐β‐D ‐xylopyranosylpomolic acid 28‐O‐β‐D ‐glucopyranoside (monepaloside F, 4 ) on the basis of chemical and spectroscopic evidence. 2D NMR techniques, including ¹H–¹H COSY, HMQC, 2D HMQC‐TOCSY, HMBC and ROESY, and selective excitation experiments, including SELTOCSY and SELNOESY, were utilized in the structure elucidation and complete assignments of ¹H and ¹³C NMR spectra. Copyright © 2002 John Wiley & Sons, Ltd.     

Determination of Solubility Parameters of Ionic Liquids and Ionic Liquid/Solvent Mixtures from Intrinsic Viscosity

The total and partial solubility parameters (dispersion, polar and hydrogen‐bonding solubility parameters) of ten ionic liquids were determined. Intrinsic viscosity approaches were used that encompassed a one‐dimensional method (1D‐Method), and two different three‐dimensional methods (3D‐Method1 and 3D‐Method2). The effect of solvent type, the dimethylacetamide (DMA) fraction in the ionic liquid, and dissolution temperature on solubility parameters were also investigated. For all types of effect, both the 1D‐Method and 3D‐Method2 present the same trend in the total solubility parameter. The partial solubility parameters are influenced by the cation and anion of the ionic liquid. Considering the effect on partial solubility parameters of the solvent type in the ionic liquid, it was observed that in both 3D methods, the dispersion and polar parameters of a 1‐ethyl‐3‐methylimidazolium acetate/solvent (60:40 vol %) mixture tend to increase as the total solubility parameter of the solvent increases.     

Crossed beam reactions of methylidyne [CH(X2Π)] with D2-acetylene [C2D2(X1Σg(+))] and of D1-methylidyne [CD(X2Π)] with acetylene [C2H2(X1Σg(+))

The crossed molecular beam reactions of ground state methylidyne, CH(X(2)Π), with D2-acetylene, C(2)D(2)(X(1)Σ(g)(+)), and of D1-methylidyne, CD(X(2)Π), with acetylene, C(2)H(2)(X(1)Σ(g)(+)), were conducted under single collision conditions at a collision energy of 17 kJ mol(-1). Four competing reaction channels were identified in each system following atomic 'hydrogen' (H/D) and molecular 'hydrogen' (H(2)/D(2)/HD) losses. The reaction dynamics were found to be indirect via complex formation and were initiated by two barrierless-addition pathways of methylidyne/D1-methylidyne to one and to both carbon atoms of the D2-acetylene/acetylene reactant yielding HCCDCD/DCCHCH and c-C(3)D(2)H/c-C(3)H(2)D collision complexes, respectively. The latter decomposed via atomic hydrogen/deuterium ejection to form the thermodynamically most stable cyclopropenylidene species (c-C(3)H(2), c-C(3)D(2), c-C(3)DH). On the other hand, the HCCDCD/DCCHCH adducts underwent hydrogen/deuterium shifts to form the propargyl radicals (HDCCCD, D(2)CCCH; HDCCCH, H(2)CCCD) followed by molecular 'hydrogen' losses within the rotational plane of the decomposing complex yielding l-C(3)H/l-C(3)D. Quantitatively, our crossed beam studies suggest a dominating atomic compared to molecular 'hydrogen' loss with fractions of 81 ± 23% vs. 19 ± 10% for the CD/C(2)H(2) and 87 ± 30% vs. 13 ± 4% for the CH/C(2)D(2) systems. The role of these reactions in the formation of interstellar isomers of C(3)H(2) and C(3)H is also discussed.     

3s Rydberg and cationic States of pyrazine studied by photoelectron spectroscopy

We have studied 3s(n-1 and pi-1) Rydberg states and D0(n-1) and D1(pi-1) cationic states of pyrazine [1,4-diazabenzene] by picosecond (2 + 1) resonance-enhanced multiphoton ionization (REMPI), (2 + 1) REMPI photoelectron imaging, He(I) ultraviolet photoelectron spectroscopy (UPS), and vacuum ultraviolet pulsed field ionization photoelectron spectroscopy (VUV-PFI-PE). The new He(I) photoelectron spectrum of pyrazine in a supersonic jet revealed a considerably finer vibrational structure than a previous photoelectron spectrum of pyrazine vapor. We performed Franck-Condon analysis on the observed photoelectron and REMPI spectra in combination with ab initio density functional theory and molecular orbital calculations to determine the equilibrium geometries in the D0 and 3s(n-1) states. The equilibrium geometries were found to differ slightly between the D0 and 3s states, indicating the influence of a Rydberg electron on the molecular structure. The locations of the D1-D0 and 3s(pi-1)-3s(n-1) conical intersections were estimated. From the line width in the D1 <-- S0 spectrum, we estimated the lifetime of D1 to be 12 fs for pyrazine and 15 fs for fully deuterated pyrazine. A similar lifetime was estimated for the 3s(pi-1) state of pyrazine by REMPI spectroscopy. The vibrational feature of D1 observed in the VUV-PFI-PE measurement differed dramatically from that in the UPS spectrum, which suggests that the high-n Rydberg (ZEKE) states converging to the D1 vibronic state are short-lived due to electronic autoionization to the D0 continuum.     

Observation and analysis of the 2g(1D) ion-pair state of I2: the g/u mixing between the 1u(1D) and 2g(1D) states

We report the analysis of the 2g(1D) ion-pair state of I2 by perturbation-facilitated optical-optical double resonance. The present study began with the observation of the 2g(1D)-A' 3Pi(2u) emission at around 230 nm during the analysis of the ultraviolet emissions originating form the 1u(1D) ion-pair state. The identification of this new transition helped us to specify the wavelengths for detecting the 2g(1D) state by emission, and also to estimate its absolute position. The intermediate states used to observe the 2g(1D) state were the B 3Pi(0u(+))-b' 2u mixed states by the hyperfine interaction, which allowed us to combine the X 1Sigmag(+) ground state with the 2g(1D) state in the (1+1) photon excitation following the optical selection rules for one-photon transitions: 2g(1D)<--b' 2u-B 3Pi(0u(+))<--X 1Sigmag(+). Our analysis covered the 2g(1D) state in the 0< or =v< or =12 and 9< or =J< or =40 ranges. The molecular constants and Rydberg-Klein-Rees (RKR) potential of the 2g(1D) state were reported. We discussed the occurrence of the 2g(1D)-A' 3Pi(2u) emission, when exciting to the 1u(1D) v=0 state, and attributed it to the g/u mixing between the 2g(1D) and 1u(1D) states by the hyperfine interaction. The effect of the perturbation on measured line intensities and lifetimes was evident.     

Syntheses,Structures and Properties of Three Transition Metal Coordination Polymers Based on Semi-rigid 3-Pyridylnicotinamide and S/N-Containing Dicarboxylate Mixed Ligands

Three new coordination complexes, [Co（L）（ADTZ）]·H2O（1）, [Cd（L）（ADTZ）]·H2O（2） and [Zn（L）（ADTZ）]·H2O（3）[L=3-pyridylnicotinamide, H2ADTZ=2,5-（s-acetic acid）dimercapto-1,3,4-thiadiazole], were synthesized under hydrothermal conditions. These complexes were structurally characterized by single-crystal X-ray diffraction analysis and further characterized by infrared spectroscopy（IR）, powder X-ray diffraction （PXRD） and thermogravimetric analysis（TGA）. Complexes 1-3 exhibit the similar 2D double-layer networks based on 1D [M-L], zigzag chains and 1D [M-ADTZ]2n double-chains with different distances between metal ions and with various conformations of ADTZ anions. In complexes 1 and 3, the 2D sheets are extended into a 3D supramolecular frameworks by hydrogen bonding interactions. The subtle effects of the central metal atoms on the structures of the title coordination polymers were discussed. The electrochemical properties of complex 1 and luminescent properties of complexes 2 and 3 were investigated. In addition, complexes 1-3 exhibit photocatalytic activity for dye methylene blue degradation under UV light and show good stability toward photoca- talysis.     

Chlorinated briarane-type diterpenoids from the gorgonian coral Ellisella robusta (Ellisellidae)

Four chlorinated metabolites featuring briarane carbon skeletons have been isolated from the gorgonian coral Ellisella robusta, which was collected off the coast of southern Taiwan: two new natural products, robustolides D (1) and E (2), and two known metabolites, robustolides F (3) and G (4). The structures of metabolites 1–4 were determined by spectroscopic methods, using 1D and 2D NMR in particular. The structures and absolute stereochemistry of robustolides D (1), F (3), and G (4) were directly established by X-ray diffraction analysis. Robustolide D (1) is the first metabolite of briarane-related natural products found to possess two halogen atoms.     

Two New Triterpene Saponins from Acanthophyllum laxiusculum

Two new triterpene glycosides, 1 and 2 , together with three known ones, were isolated from roots of Acanthophyllum laxiusculum Schiman ‐Czeika . The structures of the new compounds were established by extensive 1D‐ and 2D‐NMR spectroscopic experiments and MS analyses as 23‐O‐β‐D ‐galactopyranosylgypsogenic acid 28‐O‐{β‐D ‐glucopyranosyl‐(1→2)‐6‐O‐[4‐carboxy‐3‐hydroxy‐3‐methyl‐1‐oxobutyl]‐β‐D ‐glucopyranosyl‐(1→6)}‐[β‐D ‐glucopyranosyl‐(1→3)]‐β‐D ‐galactopyranosyl ester ( 1 ) and gypsogenic acid 28‐O‐{β‐D ‐glucopyranosyl‐(1→2)‐6‐O‐[4‐carboxy‐3‐hydroxy‐3‐methyl‐1‐oxobutyl]‐β‐D ‐glucopyranosyl‐(1→6)}‐[β‐D ‐glucopyranosyl‐(1→3)]‐β‐D ‐galactopyranosyl ester ( 2 ).     

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%.