Determination of tertatolol enantiomers in biological fluids by high-performance liquid chromatography.

A stereospecific high-performance liquid chromatographic method for the quantification of (-)- and (+)-tertatolol in plasma and urine is described. The method involves solid-phase extraction followed by derivatization with S(+)-naphthylethylisocyanate to form the urea derivative, which is more sensitive to fluorescence detection. The separation of the diastereomeric derivatives was performed by reversed-phase high-performance liquid chromatography. Fluorimetric detection (lambda excitation = 220 nm, lambda emission = 320 nm) allows the quantification of tertatolol enantiomers down to 6 ng/ml. The assay was used to study the pharmacokinetic profile of tertatolol enantiomers following oral administration of racemic tertatolol; preliminary results suggest enantioselective absorption and/or disposition of tertatolol.     

Chiral separation and determination of propranolol enantiomers in rat or mouse blood and tissue by column switching high performance liquid chromatography with ovomucoid bonded stationary phase

Resolution of propranolol (PL) enantiomers in biological samples was accomplished by column switching high performance liquid chromatography using a short precolumn and an analytical column of ovomucoid chiral phase. Plasma, whole blood or tissue homogenate sample was directly injected into the precolumn, and PL was adsorbed on Butyl Toyopearl 650-M. After column switching, the PL was backflushed and transferred to the analytical column (Ultron ES-OVM) by the eluant. Fluorometric detection was carried out at lambda ex = 297 nm and lambda em = 340 nm with a detection limit of 0.5 pmol (signal to noise ratio = 2). The recovery (98.8-103%), reproducibility (coefficient of variance less than 3%) and enantiomer resolution (separation factor 1.15) were satisfactory using as eluant 50 mM sodium dihydrogenphosphate (pH 4.6) containing 12% ethanol. The time course of elimination of PL enantiomers in rat or mouse blood and tissues was also studied.     

Chiral analysis of baclofen by alpha-cyclodextrin-modified capillary electrophoresis and laser-induced fluorescence detection

An enantioselective method for baclofen (4-amino-3-p-chlorophenylbutyric acid) based on capillary electrophoresis (CE) separation and laser-induced fluorescence (LIF) detection has been developed. Naphthalene-2,3-dicarboxaldehyde (NDA) was used for precolumn derivatization of the nonfluorescent drug. alpha-Cyclodextrin (alpha-CD) was included in the buffer as a chiral selector for the separation of NDA-labeled S-(+)- and R-(-)-baclofen. Optimal resolution and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) containing 7 mM alpha-CD and a He-Cd laser (lambda ex = 442 nm, lambda em = 500 nm). Combined with a simple cleanup procedure, this method can be applied to the analysis of baclofen enantiomers in human plasma. The relative standard deviation (RSD) values on peak areas of a plasma sample containing 1.0 microM racemic baclofen were 6.4 and 4.9% (n = 8) for the S-(+)- and R-(-)-enantiomer, respectively. The RSD value on migration times of both enantiomers was 0.5% (n = 8). Calibration graphs for S-(+)- and R-(-)-baclofen in plasma showed a good linearity (r > or = 0.999) in the concentration range of 0.1-2.0 microM. The limit of detection of baclofen in plasma was about 10 ng/mL.     

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in plasma after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate.

A sensitive high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat plasma has been developed. Racemic atenolol and practolol (internal standard) were extracted from alkalinized plasma (pH 12) into dichloromethane containing 3% (v/v) heptafluoro-1-butanol, and the organic layer was evaporated. The samples were derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate at pH 8.5 for 30 min. After removal of excess reagent, the diastereomers were extracted into dichloromethane. The diastereomers were separated on a Microspher C18 column (3 microns) with a mobile phase of acetonitrile-sodium acetate buffer (0.01 M, pH 7) (50:50, v/v) at a flow-rate of 0.8 ml/min. Fluorescence detection (lambda ex = 227 nm, lambda em = 310 nm) was used. When 100 microliters of plasma were used, the quantitation limit was 10 ng/ml for the atenolol enantiomers. The assay was applied to measure concentrations of atenolol enantiomers in plasma after intravenous administration of racemic atenolol to rats.     

Diastereoselectivity and site dependency in the photochemistry of ketoprofen in the bovine serum albumin matrix

The photodegradation of the S(+)- and R(-)-ketoprofen (KP) enantiomers in the bovine serum albumin matrix was studied by steady-state photolysis with the use of lambda(irr) > 320 nm and transient absorption spectroscopy with lambda(exc) = 355 nm, at 1/1 and 2/1 KP/BSA molar ratios. R(-)-KP was found to be more labile than S(+). Triplet ketoprofen species were evidenced with lifetimes of 400 ns for S(+) and 600 ns for R(-)-KP. Further longer-lived transients with lifetimes of 2.6 and 6.0 mus for S(+) and R(-), respectively, were detected. On the basis of the binding constants of the drug enantiomers to the two main binding sites of the protein, obtained from circular dichroism experiments, the individual disappearance quantum yields of the 1:1 and 2:1 diastereomeric KP:BSA complexes could be estimated. The photoreactivity in the BSA matrix was rationalized on the basis of diastereoselective photodecarboxylation in the two main protein sites.     

柱前手性衍生化反相高效液相色谱法分离拉贝洛尔对映体

以乙酰葡萄糖异硫氰酸酯（GITC）作柱前手性衍生化试剂,用反相高效液相色谱法成功地分离了拉贝洛尔的两对对映异构体,并以荧光检测和紫外检测作对照,确认了4个衍生物的色谱峰。     

Simultaneous stereoselective analysis of tramadol and its primary phase I metabolites in plasma by liquid chromatography. Application to a pharmacokinetic study in humans

This paper describes a bioanalytical method involving a simple liquid-liquid extraction for the simultaneous HPLC determination of the enantiomers of tramadol, the active metabolite O-desmethyltramadol (M1), and the other main metabolite N-desmethyltramadol (M2) in biological samples. Chromatography was performed at 5 degrees C on a Chiracel OD-R column containing cellulose tris(3,5-dimethylphenylcarbamate) as chiral selector, preceded by a achiral end-capped C8 column (LiChrospher 60-RP-selected B 5 microm, 250 mm x 4 mm). The mobile phase was a mixture of phosphate buffer containing sodium perchlorate (1 M) adjusted to pH 2.5-acetonitrile-N,N-dimethyloctylamine (74.8:25:0.2). The flow rate was 0.5 ml/min. Fluorescence detection (lambda(ex) 200 nm/lambda(em) 301 nm) was used. Fluconazol was selected as internal standard. The limit of quantitation of each enantiomer of tramadol and their metabolites was 0.5 ng/ml (sample size = 0.5 ml). The chiral conditions and the LC optimisation were investigated in order to select the most appropriate operating conditions. The method developed has also been validated. Mean recoveries above of 95% for each enantiomer were obtained. Calibration curves for tramadol enantiomers (range 1-500 ng/ml), M1 enantiomers (range 0.5-100 ng/ml), and M2 enantiomers (range 0.5-250 ng/ml) were linear with coefficients of correlation better than 0.996. Within-day variation determined on four different concentrations showed acceptable values. The relative standard deviation (R.S.D.) was determined to be less than 10%. This method was successfully used to investigate plasma concentration of enantiomers of tramadol, O-desmethyltramadol and N-desmethyltramadol in a pharmacokinetic study.     

Fluorescent high-performance liquid chromatographic analysis of vigabatrin enantiomers after derivatizing with naproxen acyl chloride

Vigabatrin is widely used as an anticonvulsant in the treatment of seizures. Vigabatrin is usually supplied as racemate in formulation, but only the (S)-(+)-enantiomer of vigabatrin is pharmacologically active. A simple and sensitive liquid chromatographic method is described for the separation and quantification of vigabatrin enantiomers. The method is based on derivatizing racemic vigabatrin with a fluorescent chiral reagent (naproxen acyl chloride). The resulting diastereomeric derivatives are highly responsive to a fluorimetric detector (lambda(ex)=230 nm, lambda(em)=350 nm). The lower quantitation limit of the method is attainable at 25 nM for (S)-(+)-vigabatrin or (R)-(-)-vigabatrin with a detection limit of about 2.5 nM (S/N=3 with 10 microl injected). Application of the method to the analysis of vigabatrin in serum of dosed patients proved feasible.     

Bioluminescence color determinants of Phrixothrix railroad-worm luciferases: chimeric luciferases, site-directed mutagenesis of Arg 215 and guanidine effect

Chimeric proteins were produced using the green light-emitting luciferase of Phrixothrix vivianii (PxGr: lambda max = 548 nm) and the red light-emitting luciferase of Phrixothrix hirtus (PxRe: lambda max = 623 nm). Constructs containing residues 1-344 of the red light-emitting luciferase with residues 345-545 of the green light emitting one emitted red light (PxReGr; lambda max = 613 nm), while the reverse emitted green light (PxGrRe; lambda max = 552 nm). From these results we conclude that the region 1-344 determines the color of bioluminescence (BL) in railroad-worm luciferases, and that residues above 344 are not involved. The substitution R215S in the green light-emitting luciferase (PxGr) resulted in a approximately 40 nm redshift on the BL spectrum (lambda max = 585 nm) and an associated decrease of activity, whereas the same mutation in PxRe luciferase had little effect. Guanidine was shown to cause blueshifts in the BL spectra and stimulate the activity of the red-emitting luciferases (from lambda max = 623 to lambda max = 600 nm) and in PxGr R215S (from lambda max = 585 to lambda max = 560 nm) mutant luciferase, but not in the green-emitting luciferases, suggesting that guanidine can simulate positively charged residues involved in BL color determination.     

Photometric and amperometric flow-injection determination of triazolam and clotiazepam

Triazolam and clotiazepam can be determined by flow-injection analysis with photometric and amperometric detection. With photometric detection, the signals are a linear function of the drug concentration from 3 to 55muM (lambda = 228 nm) for triazolam and from 31 to 502muM (lambda = 390 nm) and from 6 to 125muM (lambda = 260 nm) for clotiazepam. FIA systems with amperometric detection allow the determination of these drugs in the range 6-116muM (triazolam) and 16-162muM (clotiazepam).     

Coloured complexes of all-trans-retinal with benzocaine and other local anaesthetics

Coloured Schiff base complexes with lambda max values of 500 nm or above were formed between the visual pigment all-trans-retinal and the local anesthetics procaine (lambda max = 533 nm), benzocaine (4-amino-benzoic acid ethyl ester, lambda max = 535 nm), 3-amino-benzoic acid ethyl ester (lambda max = 500 nm) and 2-amino-benzoic acid ethyl ester (lambda max = 509 nm) in methanol acidified with HCl. The anaesthetics lidocaine and urethane failed to form coloured compounds with lambda max values greater than 500 nm under the same conditions. The relevance of these observations to the effect of anaesthetics on the visual pigments is discussed.     

Enantioselective high-performance liquid chromatography with fluorescence detection of methamphetamine and its metabolites in human urine.

A simple, rapid and highly sensitive high-performance liquid chromatographic method with fluorescence detection for determining the enantiomers of methamphetamine and its major metabolites, amphetamine and p-hydroxymethamphetamine, in urine samples was developed. Using a newly developed reagent for amines, namely, 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride, six enantiomers were derivatized under mild conditions (i.e., 10 min at room temperature, pH 9.0) and separated isocratically on a cellulose tris(3,5-dimethylphenylcarbamate) coated silica gel column following a pre-separation on an ODS column within 42 min, and the effluent was monitored at 440 nm (lambda ex 330 nm). Calibration curves for these derivatives using spiked human urine were linear in the range 0.05-100 mumol dm-3 with correlation coefficients > or = 0.999. The detection limits at a signal-to-noise ratio of 3 were 2.8-8.8 fmol per 5 microliters injection. The relative standard deviations of within- (n = 6) and between-day (n = 5) variations were < or = 7.4%. The method was successfully applied to discriminate between (S)-(+)-methamphetamine and its corresponding metabolites found in abusers' urine and their antipodes in a sample taken from a Parkinsonian patient on selegiline (Deprenyl) therapy.     

Determination of pimozide in oral preparations by spectrofluorometry

The intense native fluorescence of pimozide is reported. It is due to the benzo-1,3-diazolin-2-one fraction of the molecule, also present in the structurally analogous benperidol molecule. The fluorescence intensity is nearly independent of solvent acidity. This substance can be determined in oral preparations by its native fluorescence in diethyl ether (lambda(EXC) = 290 nm. lambda = 319 nm) with a detection limit of 0.01 mug/ml.     

Rapid, sensitive and fully automated high-performance liquid chromatographic assay with fluorescence detection for sulmazole and metabolites

Sulmazole (2-[(2-methoxy-4-methylsulfinyl)phenyl]-3H-imidazo [4,5-b] pyridine; AR-L 115 BS) and two metabolites (sulfide, sulfone) were quantified from directly injected body fluids (plasma, urine, bile) after high-performance liquid chromatographic separation. No internal standard is needed, which is particularly advantageous when fluorescence detection is established. After automated pre-column enrichment on Corasil C18 (37-50 microns), the parent compound and biotransformation products could be backflushed and chromatographed on ODS-Hypersil (5 microns) with a mixture of 0.075 mol/l phosphate buffer-acetonitrile (2:1), an elution rate of 2.0 ml/min and fluorimetric detection (lambda ex = 330 nm; lambda em = 370 nm). A hydroxylated metabolite of sulmazole which occurs preferentially in urine (and bile) can be quantified in the above-mentioned solvent system diluted 1:1 with water, but with different fluorescence characteristics (lambda ex = 345 nm; lambda em = 515 nm). The assay was linear in the range 8-1000 ng/ml. The lower limit of detection was about 8 ng/ml or 80 pg with coefficients of variation between 0.4 and 5.8% for sulmazole.     

HYDRATED ELECTRON FORMATION IN NANOSECOND and PICOSECOND LASER FLASH PHOTOLYSIS OF HEMATOPORPHYRIN IN AQUEOUS SOLUTION

Nanosecond (lambda exc = 266, 355 and 532 nm) and picosecond (lambda exc = 355 nm) laser flash photolysis of hematoporphyrin (Hp) was performed in neutral (pH 7.4) and alkaline (pH 12) aqueous solution, as well as in the presence of 0.1% Triton X-100. The dependence of the yield of photoproduced hydrated electrons (e-aq) on laser pulse energy was studied over a wide range of energies (0.2 to greater than 1000 mJ cm-2). The results show that e-aq are predominantly formed in a two-photon process at lambda exc = 266 and 355 nm. One-photon quantum yields are higher at lambda exc = 266 nm than at lambda exc = 355 nm. Both one-photon and two-photon pathways are less efficient at higher Hp concentration, reflecting the influence of Hp self-aggregation. Two-photon e-aq formation is more efficient when 30 ps pulses are used for excitation, as compared to 10 ns pulses. No e-aq could be detected at lambda exc = 532 nm. Nanosecond pulse-induced transient spectra obtained at pH 7.4 are also discussed.     

Formation and characterization of the gallium and indium subhydride molecules Ga2H2 and In2H2: a matrix isolation study

Ga2 reacts spontaneously with H2 in solid Ar matrixes at 12 K to form the cyclic molecule Ga(mu-H)2Ga. In2 does not react with H2 under similar conditions, but irradiation at wavelengths near 365 nm induces the formation of the corresponding indium hydride, In(mu-H)2In. The molecules have been identified and characterized by the IR spectra displayed by matrixes containing the metal and H2, D2, HD, or H2 + D2; they each have planar, dihydrido-bridged structures with D2h symmetry, as endorsed by comparison of the measured spectra (i) with the properties forecast by quantum chemical calculations and (ii) with the spectra of known gallium and indium hydrides. Both are photolabile under visible light (lambda > 450 nm): green light (lambda = ca. 546 nm) causes Ga(mu-H)2Ga to isomerize to a mixture of HGaGaH and H2GaGa, whereas broad-band visible irradiation (lambda > 450 nm) of In(mu-H)2In gives rise to the isomer HInInH, together with InH. The isomerization can be reversed by UV photolysis (lambda = ca. 365 nm) of HGaGaH, H2GaGa, and HInInH or by near-IR photolysis (lambda > 700 nm) of HGaGaH and H2GaGa.     

Enantioselective drug monitoring of (R)- and (S)- propranolol in human plasma via derivatization with optically active (R,R)-O,O-diacetyl tartaric acid anhydride

A sensitive high-performance liquid chromatographic method was developed for the stereoselective assay of (R)- and (S)-propranolol in human plasma. The method involves diethyl ether extraction of the drugs and a racemic internal standard, N-tert.-butylpropranolol, followed by derivatization of the compounds with the chiral reagent (R,R)-O,O-diacetyl tartaric acid anhydride. The resulting diastereomeric derivatives were separated isocratically on a reversed-phase column. Quantitation was achieved by the peak-height ratio method with reference to the internal standard. The assay was accurate and reproducible in the concentration range 1-100 ng of (R)- and (S)-propranolol per ml plasma, using fluorescence detection at lambda ex 290 nm and lambda em 335 nm. The applicability of this method was demonstrated for the determination of concentration-time profiles of propranolol enantiomers in the course of comparative pharmacokinetic studies.     

HPLC-fluorescence determination of equilin and equilenin in postmenopausal women's urine

A high-performance liquid chromatographic (HPLC) method with fluorescence detection (lambda(ex) = 280 nm; lambda(em) = 410 and 312 nm) in combination with a post-column on-line photochemical derivatization is described for the determination of equilin and equilenin in urine from normal postmenopausal women after therapy with conjugated oestrogens. The column effluents were subjected on-line to UV irradiation (254 nm) and the photo-induced modifications were useful for the identification of the analytes. The conjugated (sulphate and glucuronide) forms were analysed after enzymatic or chemical hydrolysis and extracted with chloroform. Solid-phase extraction using strong anion-exchange sorbent was applied to the analysis of unconjugated oestrogen fraction to obtain a practical and reliable sample clean-up. The HPLC separations were achieved using ODS columns with a mobile phase consisting of 0.05 M triethylamine phosphate buffer (pH 4.0)-acetonitrile (64:36, v/v) at a flow rate of 1.0 mL/min. The method was accurate and reproducible; for the equilin and equilenin separation isocratic conditions were satisfactory, allowing a sensitive detection in urine samples with a detection limit of about 50 fmol for equilin (lambda(ex) = 280 nm; lambda(em) = 312 nm, after photoderivatization) and 10 fmol for equilenin (lambda(ex) = 280 nm; lambda(em) = 410 nm).     

Isomerizations between nitrosyl halides X-N=O and isonitrosyl halides X-O-N: a matrix-spectroscopic study

Irradiation of nitrosyl bromide BrNO (4) with light of the wavelength lambda = 248 nm and nitrosyl chloride CINO (6) with lambda = 193 nm in an argon matrix at 10 K leads to the corresponding isomers isonitrosyl bromide BrON (5) and isonitrosyl chloride CION (7). Both new compounds 5 and 7 have been identified by comparison of the experimental and calculated (BLYP/6-311 + G*) IR spectra. Nitrosyl fluoride FNO (8) could not be transferred into isonitrosyl fluoride FON (9). The back reactions 5-->4 and 7-->6 can be initiated by UV (lambda > 310 nm), visible or IR light. The retransformation also occurs spontaneously in the matrix at 10 K under exclusion of any UV/Vis or IR radiation. Surprisingly, the reaction rates of these spontaneous back reactions are temperature independent between 8.5 and 25 K. The mechanism of these processes is discussed.     

Silica-coated Ln3+-Doped LaF3 nanoparticles as robust down- and upconverting biolabels

The preparation of nearly monodisperse (40 nm), silica-coated LaF(3):Ln(3+) nanoparticles and their bioconjugation to FITC-avidin (FITC=fluorescein isothiocyanate) is described in this report. Doping of the LaF(3) core with selected luminescent Ln(3+) ions allows the particles to display a range of emission lines from the visible to the near-infrared region (lambda=450-1650 nm). First, the use of Tb(3+) and Eu(3+) ions resulted in green (lambda=541 nm) and red (lambda=591 and 612 nm) emissions, respectively, by energy downconversion processes. Second, the use of Nd(3+) gave emission lines at lambda=870, 1070 and 1350 nm and Er(3+) gave an emission line at lambda=1540 nm by energy downconversion processes. Additionally, the Er(3+) ions gave green and red emissions and Tm(3+) ions gave an emission at lambda=800 nm by upconversion processes when codoped with Yb(3+) (lambda(ex)=980 nm). Bioconjugation of avidin, which has a bound fluorophore (FITC) as the reporter, was carried out by means of surface modification of the silica particles with 3-aminopropyltrimethoxysilane, followed by reaction with the biotin-N-hydroxysuccinimide activated ester to form an amide bond, imparting biological activity to the particles. A 25-fold or better increase in the FITC signal relative to the non-biotinylated silica particles indicated that there is minimal nonspecific binding of FITC-avidin to the silica particles.