New chiral high-performance liquid chromatographic methodology used for the pharmacokinetic evaluation of dexfenfluramine.

A new chiral high-performance liquid chromatographic (HPLC) method utilizing ultraviolet (UV) detection has been developed for determining plasma and urinary concentrations of d-fenfluramine and its major metabolite d-norfenfluramine, while being able to determine the possible presence of l-fenfluramine after oral administration of enantiopure d-fenfluramine hydrochloride. Sensitivity, stability, and specificity were enhanced by derivatizing the extracted analytes with 3,5-dinitrophenylisocyanate while utilizing a Pirkle-type chiral recognition approach. In vitro and in vivo statistical data are analogous. Overall plasma inter-assay precision was less than 7% with a minimum quantitation limit of 10 ng/ml. Overall urine inter-assay precision was also less than 7% with a minimum quantitation limit of 25 ng/ml.

Determination of pituitary and recombinant human growth hormone molecular weights by modern high-performance liquid chromatography with low angle laser light scattering detection.

The determination of molecular weight for pituitary and recombinant human growth hormone (p-hGH/Crescormon and r-hGH/Protropin) has been performed. This has involved on-line coupling of size-exclusion chromatography (SEC) and gradient elution, reversed-phase high-performance liquid chromatography (RP-HPLC) with low-angle laser light scattering (LALLS) detection. A 5-microns, 300 A, Delta-bond octyl column was used. Traditional specific refractive index increment (dn/dc) and refractive index (n) measurements have been performed in order to derive absolute weight-average molecular weight (Mw) information for p-hGH and r-hGH. Known concentrations of each protein have been separated using reversed-phase gradients utilizing acetonitrile with on-line LALLS determination of excess Rayleigh scattering factors. Accurate Mw data has been obtained for both proteins under conventional RP-HPLC gradient elution conditions. SEC data of both hGHs were found to be concentration, mobile phase, and column dependent for the particular analyses. Both medium- and high-resolution SEC-LALLS studies were performed, and all of these determinations further confirmed our RP-HPLC results. On-line LALLs provides certain advantages in identifying aggregates that may be present, even in medium-resolution SEC, where incomplete resolution occurs. The on-line coupling of modern RP-HPLC for biopolymers with LALLS detection represents a major step forward in the ability of bioanalytical chemists to determine the nature (monomer versus higher-order aggregate) of such materials. Other classes of biopolymers should prove suitable for studies with the same RP-HPLC-LALLS-UV approaches.

Complete on-line determination of biopolymer molecular weight via high-performance liquid chromatography coupled to low-angle laser light scattering, ultraviolet, and differential refractive index detection.

An optically modified high-performance liquid chromatography refractive index detector was developed to allow complete on-line determinations for biopolymer molecular weights. On-line concentration, refractive index, specific refractive index increment (dn/dc2)mu, and Rayleigh factor were determined under flow injection analysis (FIA) and size exclusion chromatography (SEC) conditions using low-angle laser light scattering, ultraviolet, and modified refractive index detection. This instrumental system is capable of determining absolute on-line molecular weights. The error and time requirements involved in conventional methodologies for proteins have been reduced. Sample quantities have been reduced from 150 to 200 mg, in conventional off-line methods, to less than 2 mg for on-line FIA and 0.5 mg for on-line SEC, if mass absorptivities (a) are known. Otherwise, the determination of a will be the most sample-demanding step, requiring about 3 mg of the pure protein. On-line measurements of (dn/dc2)mu are in good agreement with traditional off-line values established at Donnan equilibrium (usually within 5%). In addition, this technique provides true injected mass as determined by the UV detector, after chromatographic exposure where losses may occur, which is then used in the calculation of biopolymer molecular weight.

Determination of biopolymer (protein) molecular weights by gradient elution, reversed-phase high-performance liquid chromatography with low-angle laser light scattering detection.

The determination of molecular weights for certain proteins has been performed. This has involved the on-line coupling of gradient elution, reversed-phase high-performance liquid chromatography (RP-HPLC) with low-angle laser light scattering (LALLS) detection. A new 1.5-micron, non-porous, Monosphere RP-C8 column has been used in order to perform fast and conventional RP-HPLC gradients (5-45 min). Traditional specific refractive index increment (dn/dc) and refractive index (n) measurements have been performed in order to derive absolute weight-average molecular weight (Mw) information for ribonuclease A, lysozyme, and bovine serum albumin. Standard mixtures of known concentrations of each protein have been separated using reversed-phase gradients utilizing acetonitrile with on-line LALLS determination of excess Rayleigh scattering factors. Accurate Mw data have been obtained for all three proteins, but only under certain, conventional reversed-phase gradient elution conditions. Between 5-10 min of fast gradient elution, each protein appears to exhibit unusual Mw values, suggestive of aggregate formations. Methods have been developed to define the nature of such aggregates. The on-line coupling of modern RP-HPLC for biopolymers with LALLS represents a major step forward in the ability of bioanalytical chemists to determine the nature (monomer versus aggregate) of such materials. Other classes of biopolymers should prove suitable for studies with the same RP-HPLC-LALLS-UV approaches.

Conformational studies of bovine alkaline phosphatase in hydrophobic interaction and size-exclusion chromatography with linear diode array and low-angle laser light scattering detection.

Alkaline phosphatase has been studied in hydrophobic interaction chromatography (HIC), using a bonded C1-ether phase on a silica gel support, together with an aqueous salt gradient. Its behavior under various gradient elution conditions has demonstrated good chromatographic performance and retention of enzymatic activity under aqueous conditions. It has now been studied using linear photodiode array (LDA) spectroscopy in combination with low-angle laser light scattering (LALLS) in gradient elution HIC. HIC-LALLS permitted the use of routine salt gradients for collection of molecular weight information, despite small changes in the baseline, via computerized baseline subtraction. Size-exclusion chromatography (SEC)-LALLS measurements, under various isocratic conditions, meant to mimic HIC elution, have indicated the presence of monomer/dimer, dimer/trimer, or mainly trimer. aggregates of alkaline phosphatase can also be detected under salt gradient HIC conditions, but at lower levels relative to the monomer. This paper also describes the behavior of alkaline phosphatase when detected using LDA under various chromatographic, temperature, and concentration (injected) conditions. The results indicate a facile equilibrium of at least two monomeric forms of alkaline phosphatase of the same molecular weight, which change relative populations as a function of operational conditions. Most interesting is the suggestion that alkaline phosphatase undergoes rapid conformational interconversions on the chromatographic detection time scales, and that these interconverting conformations, concentration dependent, produce a novel dual wavelength ratioing, viz., a pseudo-Gaussian peak mimicking the chromatographic elution profile at either wavelength. The reasons for these observations and their possible use in future high-performance liquid chromatographic biopolymer studies are discussed and described.