High-performance Size-exclusion Chromatographic Method Research Articles

High-performance liquid chromatographic determination of 2-hydroxypropyl-γ-cyclodextrin in different biological fluids based on cyclodextrin enhanced fluorescence

A high-performance size exclusion chromatographic method with analyte enhanced fluorescence detection is described for the analysis of 2-hydroxypropyl-γ-cyclodextrin (HPGCD) in different biological fluids. The principle of detection was the in situ complexation of 8-anilinonaphthalene-1-sulfonic acid (ANS) by HPGCD. When HPGCD eluted from the column the increased fluorescence was measured at excitation and emission wavelengths of 270 and 512 nm, respectively. Solid-phase extraction cleanup and concentration of samples resulted in higher than 78% recovery of HPGCD for each of the studied biological fluids. Some important details of the method development as well as the validation of the method for rabbit plasma, rabbit aqueous humour, monkey plasma and monkey urine are given. The limits of quantification varied between 1 and 10 nmol/ml (correspond to 1.5–15 μg/ml) depending on the biological matrix used. The method was successfully adapted in another laboratory proving that HPGCD had not absorbed into aqueous humour and plasma after topical application of HPGCD containing eye drop in rabbits.

Determination of albumin and myoglobin in dialysate and ultrafiltrate samples by high-performance size-exclusion chromatography

A high-performance size-exclusion chromatographic method was developed, validated and implemented for simultaneous and quantitative determination of albumin and myoglobin along with inulin, vancomycin and creatinine in dialysate and ultrafiltrate samples from in vitro hemodialysis experiments. The experimental parameters including mobile phase pH, ionic strength, detection wavelength, flow-rate, injection volume were first optimized for the determination of albumin, myoglobin, inulin, vancomycin and creatinine. The peak height ratio and detection limits of the proteins were then comparatively studied at 210, 254 and 280 nm by UV and diode array detection. The method was further validated by evaluating the linearity, precision and accuracy of the proteins. The assay was finally implemented to the simultaneous and quantitative determination of the proteins in dialysate and ultrafiltrate samples.

Improved potency assay for recombinant bovine somatotropin by high-performance size-exclusion chromatography

A high-performance size-exclusion chromatographic method was developed for the determination of potency of recombinant bovine somatotropin (rbST) monomer and the estimation of dimer and soluble aggregates in bulk drug substances. These proteins can be completely extracted from bulk drug substances with sodium borate-ethylenediamine-tetraacetic acid disodium salt (EDTA) at pH 9.5 and separated on TSK G3000SW column with a mobile phase of pH 7.3 sodium borate-EDTA. The results demonstrated that this method was a non-denaturing assay for the determination of potency of rbST monomer and the data obtained in this study correlated well with data of the hypophysectomized rat body weight gain bioassay. The rbST monomer and dimer in the separation were verified by liquid chromatography—electrospray mass spectrometry. This method was optimized and validated.

Non-denaturing assay for the determination of the potency of recombinant bovine somatotropin by high-performance size-exclusion chromatography

A non-denaturing high-performance size-exclusion chromatographic method has been developed for the determination of the potency of recombinant bovine somatotropin (rbST) in bulk materials. A Spherogel TSK 3000 PW column containing a polymer base packing material with very hydrophilic bonded surface was used in this method. Ammonium hydrogencarbonate buffer pH 9.0, was used as the mobile phase. This method was shown to be non-denaturing by rat mass-gain assay and radioreceptor assay. The optimization for the separation and determination of rbST has been investigated. The method was validated for the determination of rbST in bulk materials. In addition, rbST soluble aggregates formed in the production process due to protein association, used to be found in bulk materials. The behavior of rbST soluble aggregates in ammonium hydrogencarbonate solutions have been studied. The bio-inactive aggregates can be separated by the method developed in this study. The high—low chromatographic technique has been used to estimate rbST soluble aggregates in bulk materials.

Evaluation of sodium dodecyl sulfate non-acrylamide, polymer gel-filled capillary electrophoresis for molecular size separation of recombinant bovine somatotropin

Sodium dodecyl sulfate (SDS) non-acrylamide, polymer gel-filled capillary electrophoresis was examined as an alternative to the high-performance size-exclusion chromatographic (HPSEC) method for the analysis of recombinant bovine somatotropin (bovine growth hormone, rbSt). A calibration curve for the molecular mass of protein standards ( M r 14 000 to 97 000) was linear ( r 2= 0.998) when the molecular mass of the proteins and their peak migration time were plotted on a logarithmic (log-log) scale. Relative standard deviation (R.S.D.) of the molecular man determination was approximately 2–3%. A pre-production as well as a production lot of the SDS gel-filled capillary columns were examined. Performance of both of these columns were equivalent. Peak migration time remained relatively constant beyond 140 sample injections. Gradual loss of theoretical plates was noted over the course of the assay; however, the peak resolution remained adequate for the analysis of rbSt. Peaks corresponding to monomer, dimer, trimer, and tetramer of rbSt were base-line resolved by the SDS gel-filled capillary electrophoresis. Theoretical plate of the monomer peak was approximately 28 000–30 000 per 40 cm column. Both the monomer and the dimer recovery studies indicated that the calibration curves are linear ( r 2 > 0.993) and the slopes are no different from one. Amounts of the components in rbSt determined by the SDS gel-filled capillary electrophoresis compared well with those of the HPSEC method. The results of this study indicated that the SDS non-acrylamide gel-filled capillary electrophoresis may be a viable alternative to the HPSEC method for the molecular size separation and analysis of recombinant proteins.

Rapid characterization of cellular retinoid-binding proteins by high-performance size-exclusion chromatography

A high-performance size-exclusion chromatographic method was developed for the analysis of cellular retinol and retinoic acid-binding proteins. The chromatographic analysis of the retinol-retinol-binding protein complex or the retinoic acid-retinoic acid-binding protein complex requires 15 min. The use of high-specific-radioactivity retinoid ligand (30–40 Ci/mmol) allows routine detection of 25 fmol of retinoid-binding protein/mg of cytosolic protein. Thus, this method provides a rapid alternative to sucrose density gradient sedimentation analysis of the cellular retinoid-binding proteins. High-performance size-exclusion chromatography is well suited to screening novel tissues, tumors, and cell lines for the presence of retinoid-binding proteins and to the further characterization of these cellular proteins. This method was applied to the characterization of cellular retinol- and retinoic acid-binding proteins in fetal rabbit tissues. Both retinol- and retinoic acid-binding proteins were detected in fetal rabbit brain, intestine, kidney, and lung at a gestational age of 28 days. Neither retinoidbinding protein was detected in 28-day-old fetal rabbit placenta. Specific retinoic acid binding in fetal intestinal cytosol decreased as a function of increasing gestational age.