HPLC determination of ketoprofen enantiomers in human serum using a nonporous octadecylsilane 1.5 microns column with hydroxypropyl beta-cyclodextrin as mobile phase additive.

A sensitive and stereospecific high-performance liquid chromatography (HPLC) method for the quantitation of ketoprofen enantiomers in human serum was developed. The assay involves the use of an octadecylsilane solid-phase extraction for serum sample clean-up prior to HPLC analysis. Chromatographic resolution of the ketoprofen enantiomers was performed on a nonporous octyldecylsilane column with hydroxypropyl beta-cyclodextrin as the mobile phase additive. The composition of the mobile phase was 98:2 v/v aqueous 0.1% trifluoroacetic acid (TFA), pH 4.00 (adjusted with triethylamine (TEA))/acetonitrile containing 10 mM hydroxypropyl beta-cyclodextrin (beta-CD) at a flow rate of 0.8 ml min-1. Recoveries of R(-)-ketoprofen was 95.4 +/- 2.16% and for S(+)-ketoprofen 96.2 +/- 1.31%. Linear calibration curves were obtained in the range 0.025-15 micrograms ml-1 range for each enantiomer in serum. The detection limit based on a S/N = 3 ratio was 10 ng ml-1 for each enantiomer in serum with ultraviolet detection at 220 nm. The limit of quantitation for each enantiomer was 25 ng ml-1. Precision calculated as % relative standard deviation (%R.S.D.) and accuracy calculated as % error were in the range 0.2-5.2% and 0.3-2.2%, respectively, for the R enantiomer and 0.3-6.2 and 0.2-3.2%, respectively, for the S enantiomer.

Resolution and quantitation of pentazocine enantiomers in human serum by reversed-phase high-performance liquid chromatography using sulfated beta-cyclodextrin as chiral mobile phase additive and solid-phase extraction.

A sensitive and stereospecific HPLC method was developed for the analysis of (-)- and (+)-pentazocine in human serum. The assay involves the use of a phenyl solid-phase extraction column for serum sample clean-up prior to HPLC analysis. Chromatographic resolution of the pentazocine enantiomers was performed on a octadecylsilane column with sulfated-beta-cyclodextrin (S-beta-CD) as the chiral mobile phase additive. The composition of the mobile phase was aqueous 10 mM potassium dihydrogenphosphate buffer pH 5.8 (adjusted with phosphoric acid)-absolute ethanol (80:20, v/v) containing 10 mM S-beta-CD at a flow-rate of 0.7 ml/min. Recoveries of (-)- and (+)-pentazocine were in the range of 91-93%. Linear calibration curves were obtained in the 20-400 ng/ml range for each enantiomer in serum. The detection limit based on S/N=3 was 15 ng/ml for each pentazocine enantiomer in serum with UV detection at 220 nm. The limit of quantitation for each enantiomer was 20 ng/ml. Precision calculated as R.S.D. and accuracy calculated as error were in the range 0.9-7.0% and 1.2-6.2%, respectively, for the (-)-enantiomer and 0.8- 7.6% and 1.2-4.6%, respectively, for the (+)-enantiomer (n=3).

Stereoselective determination of apomorphine enantiomers in serum with a cellulose-based high-performance liquid chromatographic chiral column using solid-phase extraction and ultraviolet detection.

A novel and rapid method for the separation and determination of R-(-)- and S-(+)-enantiomers of apomorphine in serum by high-performance liquid chromatography with UV detection is reported. The method involved a solid-phase extraction of the R-(-)- and S-(+)-enantiomers of apomorphine and the internal standard R-(-)-propylnorapomorphine from serum using a C8 Bond-Elut column. The HPLC system consisted of a reversed-phase cellulose-based chiral column (Chiralcel OD-R, 250 x 4.6 mm I.D.) with a mobile phase of 35:65 (v/v) acetonitrile-0.05 M sodium perchlorate (pH 2.0, adjusted with 60-62% perchloric acid) at a flow-rate of 0.5 ml/min with UV detection at 273 nm. The detection and quantitation limits were 10 ng/ml for each enantiomer using 1 ml of serum. Linear calibration curves from 10 to 1000 ng/ml for both R-(-)- and S-(+)-enantiomers show coefficient of determination of more than 0.9995. Precision calculated as %R.S.D. and accuracy calculated as % error were 0.2-4.7 and 3.1-6.9%, respectively, for the R-(-)-enantiomer and 1.3-4.2 and 0.3-6.8%, respectively, for the S-(+)-enantiomer.

The function and properties of the iron-sulfur center in spinach ferredoxin: thioredoxin reductase: a new biological role for iron-sulfur clusters.

Thioredoxin reduction in chloroplasts is catalyzed by a unique class of disulfide reductases which use a [2Fe-2S]2+/+ ferredoxin as the electron donor and contain an Fe-S cluster as the sole prosthetic group in addition to the active-site disulfide. The nature, properties, and function of the Fe-S cluster in spinach ferredoxin:thioredoxin reductase (FTR) have been investigated by the combination of UV/visible absorption, variable-temperature magnetic circular dichroism (MCD), EPR, and resonance Raman (RR) spectroscopies. The results indicate the presence of an S = 0 [4Fe-4S]2+ cluster with complete cysteinyl-S coordination that cannot be reduced at potentials down to -650 mV, but can be oxidized by ferricyanide to an S = 1/2 [4Fe-4S]3+ state (g = 2.09, 2.04, 2.02). The midpoint potential for the [4Fe-4S]3+/2+ couple is estimated to be +420 mV (versus NHE). These results argue against a role for the cluster in mediating electron transport from ferredoxin (Em = -420 mV) to the active-site disulfide (Em = -230 mV, n = 2). An alternative role for the cluster in stabilizing the one-electron-reduced intermediate is suggested by parallel spectroscopic studies of a modified form of the enzyme in which one of the cysteines of the active-site dithiol has been alkylated with N-ethylmaleimide (NEM). NEM-modified FTR is paramagnetic as prepared and exhibits a slow relaxing, S = 1/2 EPR signal, g = 2.11, 2.00, 1.98, that is observable without significant broadening up to 150 K. While the relaxation properties are characteristic of a radical species, MCD, RR, and absorption studies indicate at least partial cluster oxidation to the [4Fe-4S]3+ state. Dye-mediated EPR redox titrations indicate a midpoint potential of -210 mV for the one-electron reduction to a diamagnetic state. By analogy with the properties of the ferricyanide-oxidized [4Fe-4S] cluster in Azotobacter vinelandii 7Fe ferredoxin [Hu, Z., Jollie, D., Burgess, B. K., Stephens, P. J., & Münck, E. (1994) Biochemistry 33, 14475-14485], the spectroscopic and redox properties of NEM-modified FTR are interpreted in terms of a [4Fe-4S]2+ cluster covalently attached through a cluster sulfide to a cysteine-based thiyl radical formed on one of the active-site thiols. A mechanistic scheme for FTR is proposed with similarities to that established for the well-characterized NAD(P)H-dependent flavin-containing disulfide oxidoreductases, but involving sequential one-electron redox processes with the role of the [4Fe-4S]2+ cluster being to stabilize the thiyl radical formed by the initial one-electron reduction of the active-site disulfide. The results indicate a new biological role for Fe-S clusters involving both the stabilization of a thiyl radical intermediate and cluster site-specific chemistry involving a bridging sulfide.

Oxidation-reduction properties of the ferredoxin-linked glutamate synthase from spinach leaf.

Oxidation-reduction titrations have been conducted to determine the midpoint potential (Em) values of the three electron-carrying prosthetic groups of the ferredoxin-linked glutamate synthase isolated from spinach leaves. Titrations using electron paramagnetic resonance (EPR) signals to monitor the oxidation state of the [3Fe-4S]+,0 cluster found in the enzyme, indicated the presence of a single n = 1 component with Em = -170 mV at pH 7.7. Titrations using absorbance changes in the visible region to monitor the oxidation states of the FAD and FMN groups present in the enzyme could be fit to a single n = 2 Nernst curve with Em = -180 mV at pH 7.7. The magnitude of the absorbance change observed during this titration accounts for all of the FMN and FAD found in the enzyme, indicating that the two flavins are either isopotential or differ in Em by less than about 30 mV. Neither optical nor EPR titrations gave any evidence for the presence of stable flavin free radicals. These results represent the first characterization of the redox properties of the prosthetic groups of a ferredoxin-dependent glutamate synthase.

Spectroscopic evidence for a [3Fe-4S] cluster in spinach glutamate synthase.

The combination of low temperature EPR, magnetic circular dichroism, and resonance Raman spectroscopies reveals the presence of a single [3Fe-4S]+,0 center as the sole iron-sulfur prosthetic group in glutamate synthase from spinach leaves. The electronic, magnetic, and structural properties of the oxidized and reduced cluster are analogous with those of similar clusters in bacterial ferredoxins. It was not possible to convert the [3Fe-4S] cluster to a [4Fe-4S] cluster by incubating with iron under reducing conditions. Taken together with the published amino acid sequence data for plant and bacterial glutamate synthases, this suggests that the [3Fe-4S] cluster is not an isolation artifact resulting from oxidative degradation of a [4Fe-4S] cluster. The likelihood that a [3Fe-4S] cluster is an intrinsic component of all plant and bacterial glutamate synthases is discussed.