HPLC enantiomeric resolution of novel aromatase inhibitors on cellulose- and amylose-based chiral stationary phases under reversed phase mode

The chiral resolution of seven aromatase inhibitors (four triazole derivatives (Ia, Ib, Ic, and Id) and three tetrazole derivatives (IIa, IIb, and IIc)) was achieved on Chiralcel OJ-R [cellulose tris (4-methyl benzoate)], Chiralcel OD-RH [cellulose tris (3,5-dimethylphenyl carbamate)], and Chiralpak AD-RH [amylose tris (3,5-dimethylphenyl carbamate)] chiral stationary phases. The mobile phases used were A: 2-PrOH-MeCN (90:10, v/v); B: 2-PrOH-MeCN (50:50, v/v); C: MeCN-H(2)O (50:50, v/v); D: MeCN-H(2)O (80:20, v/v); and E: MeCN-H(2)O (95:05, v/v). The flow rate was 0.5 mL/min for all the mobile phases. The resolution capability of these chiral stationary phases were in the order Chiralpak AD-RH > Chiralcel OD-RH > Chiralcel OJ-R. The values of alpha and Rs of the resolved enantiomers of the aromatase inhibitors varied from 1.02-5.63 and 1. 12-6.72, respectively.     

Liquid chromatographic direct resolution of tocainide and its analogs on a (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6-based chiral stationary phase containing residual silanol protecting n-octyl groups

Optically active (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6-based chiral stationary phase (CSP) containing residual silanol protecting n-octyl groups on silica surface was applied to the liquid chromatographic direct resolution of tocainide and its analogs. The chiral recognition ability of the CSP was excellent, the separation (alpha) and the resolution factors (R(S)) for 15 analytes including tocainide being in the range of 3.02-22.92 and 3.94-20.41, respectively. In addition, the chiral recognition ability of the CSP was much greater than that of (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6-based CSP containing residual silanol groups on the silica surface. The chromatographic behaviors for the resolution of tocainide and its analogs were found to be dependent on the content and the type of organic and acidic modifiers and the ammonium acetate concentration in aqueous mobile phase.     

Liquid chromatographic direct resolution of beta-amino acids on a doubly tethered chiral stationary phase containing N--H amide linkage based on (+)-(18-crown-6)- 2,3,11,12-tetracarboxylic acid

A doubly tethered chiral stationary phase (CSP) prepared by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to doubly tethered primary aminoalkyl silica gel was used for the resolution of various beta-amino acids. All the beta-amino acids tested were resolved quite well, the separation (alpha) and the resolution factors (RS) being in the ranges 1.34-2.09 and 2.52-7.45, respectively, with a mobile phase of methanol-water (50:50, v/v) containing 10 mM acetic acid. The chiral recognition efficiency of the doubly-tethered CSP was found to be generally superior to that of the corresponding singly-tethered CSP in the resolution of beta-amino acids. The chiral recognition behaviors for the resolution of beta-amino acids on the doubly tethered CSP were examined by varying the type and content of organic and acidic modifiers in the aqueous mobile phase and the column temperature.     

Normal phase chiral HPLC of methylphenidate: comparison of different polysaccharide-based chiral stationary phases.

A comparison of the enantiomeric resolution of (+/-)-threo-methylphenidate (MPH) (Ritalin) was achieved on different polysaccharide based chiral stationary phases. The mobile phase used was hexane-ethanol-methanol-trifluoroacetic acid (480:9.75:9.75:0.5, v/v/v/v). Benzoic acid and phenol were used as the mobile phase additives for the enantiomeric resolution of MPH on Chiralcel OB column only. The alpha values for the resolved enantiomers were 1.34, 1.29, 1.30, and 1.24 on Chiralpak AD, Chiralcel OD, Chiralcel OB (containing 0.2 mM benzoic acid in mobile phase), and Chiralcel OB (containing 0.2 mM phenol in mobile phase) columns, respectively. The R(s) values were 1.82, 1.53, 1.19, and 1.10 on Chiralpak AD, Chiralcel OD, Chiralcel OB (containing 0.2 mM benzoic acid in mobile phase), and Chiralcel OB (containing 0.2 mM phenol in mobile phase), respectively. The role of benzoic acid and phenol as mobile phase additives is discussed.     

Enantiomeric determination of D- and L-lactate in rat serum using high-performance liquid chromatography with a cellulose-type chiral stationary phase and fluorescence detection

A method is described for the quantitative determination of d- and L-lactate in 10 microl of rat serum, which includes fluorescence derivatization of D- and L-lactate with 4-(N, N-dimethylaminosulfonyl)-7-piperazino-2,1,3- benzoxadiazole (DBD-PZ) followed by O-acetylation. The derivatives are separated by HPLC on an octadecylsilica, and, via column switching, on a cellulose-type chiral column. Levulinic acid was used as the internal standard. The enantiomers of lactate were separated with the separation factor (alpha) of 1.27 and the resolution (Rs) of 2.72, while the linearity for the detection was over the range of 10 nmol/ml to 20 micromol/ml (r = 0.999). Interday precision values for D-lactate in rat serum were 5.8, 5.3, and 4.1% for 10, 100, and 1000 nmol/ml, and accuracy values were 109.6, 98.2, and 103.1%, respectively (n = 5). The reduction of d-lactate concentration in rat serum by fasting was observed with the method.     

Single-step organic extraction of leukotrienes and related compounds and their simultaneous analysis by high-performance liquid chromatography

A method for the simultaneous single-step organic extraction from biological matrices of peptido- and dihydroxyleukotrienes as well as 5-hydroperoxy- and 5-hydroxyeicosatetraenoic acid followed by separation and quantitation in a single run on reversed-phase high-performance liquid chromatography was evaluated. Using an extraction system comprising 400/1200/4800 (v/v/v) aqueous phase/isopropanol/dichloromethane, pH 3.0, absolute recoveries of 82.3 +/- 2.0, 89.7 +/- 1.0, 93.7 +/- 1.4, 92.8 +/- 1.4, 90 +/- 4, and 90 +/- 4% for prostaglandin B1 (PGB1), leukotriene C4 (LTC4), leukotriene B4 (LTB4), leukotriene D4 (LTD4), 5-hydroperoxyeicosatetraenoic acid (5-HETE), respectively, were achieved. Separation and quantitation of products were performed on a Nucleosil 100 C18 column (5 microns, 4.6 X 250 mm) using, at pH 6.0, a gradient system comprising 72/28/0.02 (v/v/v) methanol/water/glacial acetic acid from 0 to 15 min, followed by a convex gradient to 76/24/0.02 (v/v/v) methanol/water/glacial acetic acid, followed by a 10-min hold at this methanol concentration. The method was used to investigate the profile of leukotrienes synthesized by rat hepatocyte homogenates from 5-HPETE or leukotriene A4 in absence or presence of glutathione (GSH). During a 5-min incubation with 100 microM 5-HPETE, 9.6 ng LTB4/mg protein and 2.2 micrograms 5-HETE/mg protein were formed in the absence of GSH. In the presence of 0.4 mM GSH, 3.7 ng LTB4/mg protein and 11.0 micrograms 5-HETE/mg protein were formed. Using 20 microM LTA4 as a substrate, 17.3 and 324.0 ng LTC4/mg protein X min and 14.3 and 19.3 ng LTB4/mg protein X min were formed in the presence of 0.4 and 10 mM GSH, respectively.     

Enantiomer separation of imidazo-quinazoline-dione derivatives on quinine carbamate-based chiral stationary phase in normal phase mode

The normal phase mode liquid chromatographic enantiomer separation capability of a quinine tert-butyl-carbamate-type chiral stationary phase (CSP) has been investigated for a set of polar [1,5-b]-quinazoline-1,5-dione derivatives. This class of chiral heterocycles is currently under development as potential alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and/or N-methyl-D-aspartic acid (NMDA) receptor antagonists. The effect of the nature and concentration of polar modifier, i.e., ethanol and isopropanol, in n-hexane-based mobile phases, as well as the substituent pattern of the phenyl ring attached to the quinazolone framework on retention factor, enantioselectivity, and resolution was investigated. The Soczewiński competitive adsorption model was used to describe the relationship between the retention and the binary mobile phase compositions. According to this model, linear plots of the logarithms of retention factor versus molar fractions of the polar modifiers were obtained over a wide concentration range (X(B) between 0.15 and 0.35). Addition of equimolar ethanol yields higher resolution than isopropanol, R(S) values ranging between 1.54 and 2.75, whereas the latter allows to achieve moderately increased enatioselectivity. The resolution was further improved by using a ternary mixture of n-hexane:methanol:isopropanol/85:5:10 (v/v). The most pronounced selectivity factor alpha and resolution R(S) values were obtained for the para-hydroxy substituted compound, indicating that chiral recognition is sensitive to steric and stereoelectronic factors. In the course of optimization, the temperature-dependence on the chiral separation was also investigated. It turned out that the enantiomer separation is predominantly enthalpically driven in normal phase mode.     

Permeabilization of metabolites from biologically viable soybeans (Glycine max)

Chemical permeabilization has been widely studied for the release useful metabolites from many types of plant cells and tissues. In this study, the effect of 0-30% (v/v) of aqueous methanol solutions were used to permeabilize soybeans for the release of two isoflavonoids: daidzein and genistein. The release of these metabolites increases with increasing methanol concentrations. The amounts of daidzein and genistein released can increase up to 40- and 86-fold, respectively, when incubated in a 30% (v/v) methanol solution for 24 h compared with those incubated with water only. The effect of methanol on the release rates is primarily due to an increase in solubility of the stored daidzein and genistein (14- to 18-fold) inside the seeds, thus maximizing the concentration gradients for metabolite release. However, the viability of the seeds dropped with increase in methanol concentrations and the incubation time. The viability of soybeans (indicated by their ability to germinate) after permeabilization treatment with 0-20% (v/v) methanol solutions was maintained above 80% throughout the 24 h, whereas no seeds were found to be viable when 30% (v/v) methanol solution was used. The permeability coefficients (P) of daidzein and genistein were found to increase as the methanol concentration used was increased. These P values were estimated to range from 1.1 x 10(-)(9) to 1.9 x 10(-)(8) m/s and 1.0 x 10(-)(9) to 1.7 x 10(-)(8) m/s, respectively. The increase in P can be attributed primarily to an increase in the partition coefficient of the metabolites in the soybean seedcoats. An empirical correlation is proposed in which the log P values are described as a function of the metabolite molecular weights and the partition coefficients of the metabolites between octanol and water, K(oct/water), which was modified to include the effect of methanol present. Knowledge obtained from this study will help provide useful selection criteria for chemical permeabilization of plant tissues, such as seeds, with minimal loss in their viability.     

Liquid chromatographic direct resolution of aryl alpha-amino ketones on a residual silanol group-protecting chiral stationary phase based on optically active (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6

A residual silanol group-protecting chiral stationary phase (CSP) based on optically active (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6 was successfully applied to the resolution of racemic cathinone and it analogue aryl alpha-amino ketones. The separation factors (alpha) and the resolutions (Rs) for 12 analytes were in the ranges of 2.85-16.12 and 6.49-19.64, respectively. The chromatographic resolution behaviors were investigated as a function of the content and type of organic and acidic modifiers and the ammonium acetate concentration in aqueous mobile phase. The practical usefulness of the CSP in the determination of the enantiomeric purity of optically active cathinone and in the preparative resolution of racemic cathinone was demonstrated.     

Enantioselective resolution of thyroxine hormone by high-performance liquid chromatography utilizing a highly fluorescent chiral tagging reagent

A highly fluorescent chiral tagging reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole, [R(-)-DBD-PyNCS], was employed to develop an indirect resolution method for efficient separation of thyroxine enantiomers,D-T(4) and L-T(4). The reaction of R(-)-DBD-PyNCS with the thyroxine enantiomers proceeds effectively at 40 degrees C for 20 min in the presence of basic medium to produce the corresponding pair of diastereomers. No racemization occurs during the tagging reaction under the optimized conditions. Various experimental parameters for derivatization reaction including the species of catalyst, the concentration of tagging reagent and reaction temperatures, have been examined to get a highest yield for T(4) derivatives. The structure of T(4) derivatives was identified based on ESI-MS/MS measurements in negative mode. The efficient separation of D-, L-T(4) derivatives was achieved by isocratic elution with water-acetonitrile mobile phase containing 1% AcOH on a reversed phase column utilizing a conventional fluorescence detector. The resolution (Rs) value of the diastereomers derived from thyroxine was 5.1. The calibration curves of both the D-T(4) and L-T(4) were linear over the concentration range of 0.1-20 microg/ml. The limits of detection (S/N = 3) for both D-T(4) and L-T(4) were 0.2 ng per injection. The proposed method was applied to the determination of D-T(4) and L-T(4) in pharmaceutical formulations and human serum samples.     

Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of the enantiomers of a novel anticonvulsant, N-(4-chlorophenyl)-1-(4-pyridyl)ethylamine, in rats

A selective chiral high performance liquid chromatographic (HPLC) method was developed and validated to separate and quantify the enantiomers of a novel anticonvulsant agent, N-(4-chlorophenyl)-1-(4-pyridyl)ethylamine (AAP-Cl), in rat plasma. After extraction of the plasma samples with ethyl acetate, the separation was accomplished by an HPLC system consisting of a Chirex chiral column (250 mm x 4.6 mm i.d.) and a mobile phase of hexane:ethanol:tetrahydrofuran (280:20:40 (v/v)) containing trifluroacetic acid (0.3% (v/v)) and triethylamine (0.018% (v/v)) at a flow rate of 0.8 ml/min with UV detection. Male Sprague-Dawley rats were given (+)-AAP-Cl (10 and 20 mg/kg), (-)-AAP-Cl (10 mg/kg) or the racemic mixture (20 mg/kg) by i.v. bolus injection and serial blood samples were collected at different times after drug administration. (+)-AAP-Cl and (-)-AAP-Cl were separated with a resolution factor, Rs, of at least 1.4, and a separation factor, alpha, greater than 1.09. Linear calibration curves were obtained over the concentration range of 0.5-30 microg/ml in plasma for both (+)-AAP-Cl and (-)-AAP-Cl (R2 > or = 0.996) with a limit of quantitation of 100 ng/ml and the recovery was greater than 80% for both enantiomers. The accuracy and precision for both enantiomers ranged from 96 to 102% (+/-0.2-7%) at upper and lower concentrations. The plasma concentration-time profiles of the enantiomers of AAP-Cl were best described by a two-compartment open model with a mean terminal half-life of about 5h, volume of distribution at steady state of 3 l/kg and clearance of about 0.6l/(hkg) in rats. There was no significant difference between the pharmacokinetic parameters of (+)-AAP-Cl and (-)-AAP-Cl, suggesting that the disposition of AAP-Cl in rats is not enantioselective. In addition, no chiral inversion of (+)-AAP-Cl to (-)-AAP-Cl or vice versa was observed. The results of this investigation have shed some light on the mechanism of action and disposition of AAP-Cl in rats.     

A reverse phase HPLC method for the separation of two stereo isomers of 2-[4-(methylsulfonyl)phenyl]-3-(3(R)-oxocyclopentyl)propanoic acid

This study describes successful method development and separation of two stereo isomers of 2-[4-(methylsulfonyl)phenyl]-3-(3(R)-oxocyclopentyl)propanoic acid by reverse phase high-performance liquid chromatography. Baseline resolution was achieved on a J'sphere-ODS-H80 (150 mm × 4.6 mm, 4 μm) column using mobile phase consisting of 0.05% triflouroacetic acid in water-acetonitrile (85:15, v/v) at a flow rate of 1.0 ml/min. The detection was carried out at 228 nm. The title compound, in turn, can be obtained by C-alkylation of methyl 2-[4-(methylthio)phenyl]acetate with 2(S)-iodomethyl-8,8-dimethyl-6,10-dioxaspiro[4.5]decane followed by consecutive hydrolysis and oxidation. The partially validated analytical method (system suitability, peak homogeneity, linearity, precision, robustness, and solution stability) has limit of detection and limit of quantification, 0.15 and 0.50 μg/ml respectively. Alternatively, the new method is being routinely utilized to monitor epimerization of α-carbon of the propanoic acid in the title compound by crystallization-induced dynamic resolution.     

Preparation of enantiopure Wieland-Miescher ketone and derivatives by the MalphaNP acid method: substituent effect on the HPLC separation

Enantiopure Wieland-Miescher ketone (4, W-M ketone) and derivatives were prepared by the enantioresolution with 2-methoxy-2-(1-naphthyl)propionic acid (MalphaNP acid 1). Various racemic derivatives of 4 were esterified with acid (S)-(+)-1 yielding diastereomeric MalphaNP esters, which were separated by HPLC on silica gel. It was clarified that the HPLC separation of diastereomers depended on the substituent of the derivatives, leading to the working hypothesis that MalphaNP acid esters of alcohols with less polar and more bulky aliphatic substituents are more effectively separated. The best separation was obtained in the case of tert-butyldimethylsilyl (TBDMS) ether derivative (12a/12b): separation factor alpha=1.80, and resolution factor, Rs=1.30. The (1)H NMR spectra of separated MalphaNP esters showed anomalously large magnetic anisotropy effects, from which their absolute configurations were determined. Solvolysis or reduction of the separated MalphaNP esters yielded alcohols, which were converted to enantiopure W-M ketones 4. The results thus provided another route for preparation of enantiopure ketones (8aR)-(-)-4 and (8aS)-(+)-4.     

Characterization of the metabolite produced by Mycosphaerella pinodes, the causal agent of mycosphaerella blight on field peas (Pisum sativum L.)

The metabolite produced by Mycosphaerella pinodes, the causal agent of mycosphaerella blight on field peas, was detected by thin layer chromatography (TLC) and was analyzed for its chemical and pathogenic characteristics. One blue dot was detected using 254nm UV light on TLC plate, and a spray of rho-anisaldehyde (110 degrees C, 30 min) also produced a blue dot. The solvent systems used for TLC analysis were ethyl acetate/water/acetone (5/2/5), chloroform/methanol/glacial acetic acid (19/10/2), toluene/ethyl acetate/90% formic acid (6/3/1), diethylether/methanol/water/90% formic acid (95/4/1/1), and bezene/methanol/acetic acid (24/2/1), with R(f) values (min-max) of 0.09-0.18, 0.88-0.95, 0.06-0.15, 0.39-0.47 and 0.05-0.12, respectively. The recovered metabolite from the TLC plate displayed UV absorption peaks at 212, 244, 250, 256 and 261 nm. The proposed formula of the main component of the metabolite was C16H12N3O6. The TLC-purified metabolite induced symptom of discoloration on detached pea leaves.     

Influences of salts on high-performance liquid chromatography of leukotrienes

The effects of concentrations and kinds of salts on the resolution of leukotrienes (LT) C4, D4, E4, and B4 were investigated by two kinds of reversed-phase high-performance liquid chromatography columns (muBondapak C18 and Novapak C18). When a mobile phase (acetonitrile/methanol/water) with a lower concentration of acetic acid (0.02-0.1%) at pH 5.6 was used, LTC4 and LTD4 were not eluted from the muBondapak column. On the Novapak column, LTC4 and LTD4 were eluted, but they were poorly resolved. When the concentration of acetic acid in the mobile phase was raised to 1.0% and adjusted to pH 5.6 with ammonium hydroxide or triethylamine, excellent resolution of LTs was obtained. Sodium hydroxide was, to some extent, useful for the pH adjustment of the mobile phase. Sodium chloride could not be substituted for acetic acid-ammonium hydroxide or -triethylamine salt. The resolution of LTC4, LTD4, and LTE4 was affected more strongly than that of LTB4 by changes of concentrations and kinds of salts. When the acetonitrile/methanol/water/acetic acid solvent system adjusted to pH 5.6 with triethylamine was applied to the analysis of the leukotrienes produced from rat peritoneal cells with stimulation of calcium ionophore A23187, de novo-synthesized LTC4, LTD4, LTB4, and isomers were clearly separated. This solvent system may be useful for the investigation of variations in the synthesis of subclasses of LTs with different stimuli and under different circumstances.     

Quantification of Gluten Exorphin A5 in cerebrospinal fluid by liquid chromatography-mass spectrometry

In the present work, for the first time, a method for the quantification of the alimentary opioid peptide Gluten Exorphin A5 (GE-A5; Gly-Tyr-Tyr-Pro-Thr) in cerebrospinal fluid (CSF) was developed. Aliquots (5 microL) of CSF were injected into a liquid chromatography-mass spectrometry (LC-MS) instrument equipped with a reversed-phase C18 column at a flow-rate of 0.4 mL/min. The mobile phase consisted of Eluent A water with 0.6% acetic acid as an ion-pairing reagent and Eluent B acetonitrile/methanol (75:25, v/v). The LC-MS system was programmed to divert column flow to waste for 4 min after injection, after which time flow was directed into the mass spectrometer that operated in positive ion mode. No significant interfering peaks were detected at the retention times of GE-A5 in CSF blanks. The lower limit of detection and the lower limit of quantitation values for GE-A5 in CSF were established at 0.60 and 1.50 ng/mL, respectively. The intra- and inter-day precision values were <5% relative standard deviation. The intra- and inter-day accuracy were 99.6-102.8% and 100.0-101.9%, respectively. The reported assay employs extremely small volumes of CSF, thus allowing the analysis of GE-A5 from both small and large animal models.     

Comparison of performance of C18 monolithic rod columns and conventional C18 particle-packed columns in liquid chromatographic determination of Estrogel and Ketoprofen gel

The performance of monolithic HPLC columns Chromolith (made by Merck, Germany) and conventional C18 columns Discovery (Supelco, Sigma-Aldrich, Prague, Czech Republic) was tested and the comparison for two topical preparations Ketoprofen gel and Estrogel gel was made. The composition of mobile phases - for Ketoprofen analysis a mixture of acetonitrile, water and phosphate buffer adjusted to pH 3.5 (40:58:2) and for Estrogel analysis a mixture of acetonitrile, methanol, water (23:24:53) - was usually not optimal for analyses at all types of columns. Thus an adjustment of components ratio was necessary for sufficient resolution of the compounds analysed. Various flow rates (1.0-5.0 ml/min) and mobile phases (usually increasing ratio of water content) were applied. Determination of active substances, preservatives and impurities and comparison of retention times and system suitability test parameters was accomplished. For Estrogel gel, following chromatographic conditions were found: using Chromolith Flash RP-18e monolith column, mobile phase was acetonitrile, methanol, water (13:24:63, v/v/v) and flow-rate 3.0 ml/min. Using monolith column ChromolithSpeedROD RP-18e, the mobile phase was acetonitrile, methanol, water (18:24:58, v/v/v) and flow-rate 4.0 ml/min. For the monolith column Chromolith Performance RP-18e, the mobile phase was acetonitrile, methanol, water (23:24:53, v/v/v), flow-rate 3.0ml/min. Analysis of Ketoprofen gel gave the best results using following analytical conditions: for monolith column Chromolith Flash RP-18e, mobile phase as a mixture of acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) was used, at flow-rate 2.0 ml/min. For ChromolithSpeedROD RP-18e monolith column, acetonitrile, water, phosphate buffer pH 3.5 (35:63:2, v/v/v) was used as a mobile phase at flow-rate 3.0 ml/min. Chromolith Performance RP-18e gave the best results using mobile phase acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) at the flow-rate 5.0 ml/min. It was proved that monolith columns, due to their porosity and low back-pressure, can save analysis time by about a factor of three with sufficient separation efficiency. Thus, for example 11 min long analysis can be performed in 4 min with comparable results.     

Development of a high-performance liquid chromatography method for the simultaneous quantification of four organoarsenic compounds in the feeds of swine and chicken

A high-performance liquid chromatography (HPLC) method with UV detection was developed for the simultaneous determination of arsanilic acid, roxarsone, nitarsone, and carbarsone in the feeds of swine and chicken. Feed samples were extracted with methanol/1% acetic acid (90:10, v/v) in an ultrasonic bath and the protein was precipitated with 2% Cu(2)SO(4). The samples were further purified by solid phase extraction (SPE) on SAX cartridges. Separation was performed on a Zorbax SB-Aq C18 HPLC column using an isocratic procedure with methanol and 1% acetic acid (3:97, v/v) at a flow-rate of 0.7 mL min(-1), and the UV detector was set at a wavelength of 260 nm. The recoveries of organoarsenic compounds spiked at levels of 2, 20 and 200 μg g(-1) ranged from 81.2% to 91.3%; the inter-day relative standard deviation values were less than 7.0%. The limits of quantification for four organoarsenic compounds were 1.0-2.0 μg g(-1). This simple and fast method could be applied to the determination of multi-residues of organic arsenic compounds in animal feeds.     

Chemical synthesis of all-trans-beta-retinoyl phosphat.

all-trans-beta-Retinoic acid is phosphorylated to retinoyl phosphate by bis(triethylamine) phosphate with yields of 10-15%. The product is soluble in methanol and is eluted from DEAE-cellulose acetate at a concentration of 0.1M-ammonium acetate in 99% (v/v) methanol. Its phosphate/retinoic acid molar ratio is 1. Retinoyl phosphate has an absorption maximum at 360nm in methanol, whereas retinoic acid has a maximum at 350 nm. The compound is hydrolysed at pH2 and pH13 for 20 min at 37 degrees C, but is relatively stable under the same conditions at pH4, 6, 8 and 10. Retinoyl phosphate (RF 0.1) can be separated from retinyl phosphate (RF 0.2) by chromatography on thin layers of silica gel in chloroform/methanol/water (60:25:4, by vol.).