Dorothy J. Phillips

Simple and rugged SPE method for the determination of tetracycline antibiotics in serum by HPLC using a volatile mobile phase

SummaryA simple and rugged SPE method for the determination of tetracycline (TC), minocycline (MC) and demeclocycline (DCC) in porcine serum by high performance liquid chromatography (HPLC) was developed. The spiked serum sample was pretreated with 2% phosphoric acid followed by a simple and rugged solid-phase extraction procedure using the OasisTM HLB extraction cartridges. High and reproducible recoveries were obtained even though the cartridges were run dry. The extracted sample analytes were injected onto a Waters SymmetryShieldTM RP8 column. The mobile phase was a simple volatile solution containing 0.1% TFA, 2% methanol and 7% acetonitrile in Water. The antibiotics were detected at 350 nm. The calibration curves were linear from 2.0 to 25.0 μg mL−1 of TC and MC with DCC as the internal standard at a concentration of 25.0 μg mL−1. For six replicate analyses, the average recoveries of TC and MC from porcine serum sample fortified at the level of 2.5 μg mL−1 were 96.1% with 1.3% RSD and 101% with 0.54% RSD; at level of 0.5 μg mL−1 the average recoveries were 88% with 1.6% RSD and 97.8% with 1.4% RSD.

A new small particle packing for faster analysis with high resolution

The need for fast and efficient separations of complex samples such as pharmaceuticals and biologicals led to the development of fast, efficient, and reproducible 3.5 microns columns. Separations using 3.5 microns column are 30-50% faster at equal plate-count compared to 5 microns columns. The results show that the 3.5 microns columns (100 mm length) give the same efficiency and resolution of drug impurities as the 5 microns columns (150 mm length). For many analytical methods, switching to 3.5 microns columns saves time and reduces costs. Separation methodologies using 5 microns columns are easily modified to accommodate 3.5 microns columns of the same chemistry because efficiency, resolution and sensitivity remain the same. It is shown that 3.5 microns columns have lifetimes comparable to 5 microns columns of the same brand.

Solid-Phase Extraction for the Determination of Tricyclic Antidepressants in Serum Using a Novel Polymeric Extraction Sorbent

A fast and easy solid-phase extraction method was developed for the determination of amitriptyline, doxepin and their metabolites (nortriptyline and nordoxepin) in porcine serum matrix by high performance liquid chromatography. The spiked serum sample was pretreated with 2% phosphoric acid followed by a simple and rugged SPE procedure using OasisTM HLB extraction cartridges. The SPE method requires only one mL of simple solvent throughout the entire SPE process. High and reproducible recoveries were obtained even though the cartridges ran dry. For five replicate analyses, the average recoveries of parent tricyclic antidepressants and their metabolites were all greater than 94%, and the RSDs were all less than 4.0%.

Role of mass spectrometry in the purification of peptides and proteins

Experiments were carried out to evaluate the fractionation of proteins and peptides according to mass. Model mixtures were separated by either reversed-phase or ion-exchange chromatography with mass spectrometry-compatible mobile phase additives. Fraction collection was triggered by the mass/charge ratio of each one of the components of the mixture. Chromatography was additionally monitored with a UV-Vis detector in order to compare the new technique with generally accepted in separations. The results indicated that adequate purification is achieved by this new technique. Fraction collection triggered by changes in the mass/charge ratio reduces sample handling and analysis time. This study demonstrates the utility of mass-directed fractionation of peptides and proteins when mass spectrometry-compatible mobile phase additives are used.