Feasibility of supercritical fluid chromatography/mass spectrometry of polypeptides with up to 40-mers.

Supercritical fluid chromatography (SFC) provides a number of advantages over traditional HPLC such as speed, practical use of longer columns, a normal-phase retention mechanism, and reduced use of organic solvents. Yet, it has been a technique traditionally limited to relatively nonpolar compounds. The nature of SFC mobile and stationary phases did not allow the elution of ionic compounds or of peptides, except, in the latter case, for the most hydrophobic peptides. The characterization of peptides is critically important for drug discovery and development in the pharmaceutical industry, as well as for a variety of other important applications. Here, for the first time to our knowledge, we show that relatively large peptides (at least 40 mers), containing a variety of acidic and basic residues, can be eluted in SFC. We used trifluoroacetic acid as additive in a CO2/methanol mobile phase to suppress deprotonation of peptide carboxylic acid groups and to protonate peptide amino groups. A 2-ethylpyridine bonded silica column, which was specifically developed for SFC, was used for the majority of this work. The relatively simple mobile phase was compatible with mass spectrometric detection.

Quantitation of PGE9509924, a novel, nonfluorinated quinolone, in rat plasma using liquid chromatography electrospray-tandem mass spectrometry following solid-phase extraction sample clean-up in a 96-well format.

PGE9509924, a novel nonfluorinated quinolone, is a potent antibacterial agent with a broad spectrum of activity. A semi-automated method using 96-well format, solid-phase extraction has been developed for quantitating PGE9509924 in rat plasma. The Waters Oasis HLB extraction plate containing a polymeric packing material was found to give the best overall recoveries. All liquid transfer steps other than aliquoting the plasma are accomplished using a 96-channel pipettor. Reverse-phase HPLC with electrospray/MS/MS detection using selective reaction monitoring is used to quantitate the samples. Stable isotopically labeled PGE9509924 is used as the internal standard. The assay is linear over the range from 0.01 to 10 ug/ml. Excellent precision is obtained within a single run and between multiple runs performed on different days. CVs of <6% were observed. The combination of the semi-automated, 96-well parallel sample processing and the short runtime on the LC/MS/MS results in a high throughput assay with reduced operator interaction.

Development of a chiral assay for a novel, nonfluorinated quinolone, PGE-9509924, in dog plasma using high performance liquid chromatography with electrospray tandem mass spectrometry or fluorescence detection.

PGE-9509924 is a nonfluorinated quinolone and is active against a variety of susceptible and drug resistant bacteria in vitro and in animal infection models. A method for determining both enantiomers of PGE-9509924 in dog plasma has been developed. The enantiomers are derivatized with a chiral derivatizing agent, (-)-1-(9-fluorenyl)ethyl chloroformate (FLEC) and the resulting diastereomers are separated by reverse phase chromatography. Plasma samples are prepared via solid phase extraction (SPE) in a 96-well format prior to being derivatized. Samples are then analyzed by electrospray-LC/MS/MS with multiple reaction monitoring or by HPLC with fluorescence detection. Results of a side-by-side validation of the method with LC/MS/MS and HPLC/Fl detection are presented. Over the range selected for validation (0.025-10 micro g/ml), both methods give similar results with identical limits of quantitation. Due to the selectivity of LC/MS/MS and the use of a stable-isotopically labeled internal standard, significantly shorter chromatographic runtimes are achieved with LC/MS/MS, making it the method of choice for sample analysis.

Preparation and antimicrobial assessment of 2-thioether-linked quinolonyl-carbapenems.

This reports the synthesis and in vitro antimicrobial properties of a series of 2-thioether-linked quinolonyl-carbapenems. Although the title compounds exhibited broad spectrum activity, the MICs were generally higher than those observed for selected benchmark carbapenems, quinolonyl-penems, and quinolones. Enzyme assays suggested that the title compounds are potent inhibitors of penicillin binding proteins and inefficient inhibitors of bacterial DNA-gyrase. Uptake studies indicated that the new compounds are not substrates for the norA encoded quinolone efflux pump.

Reversed-phase high-performance liquid chromatography of the cardiac glycoside LNF-209 with refractive index detection.

LNF-209 is a glycoside, similar to digoxin, which has potential for use in the treatment of congestive heart failure. However, unlike digoxin it exhibits virtually no useful UV absorption spectra, making detection difficult. One means of detection is the refractive index detector, but like most bulk property detectors it has certain limitations. Its sensitivity is limited and it is sensitive to small changes in a number of parameters, such as temperature, mobile phase composition, and flow-rate. These parameters must be closely controlled to obtain a stable baseline. This paper describes the steps taken to control the system and the development and validation of an assay for LNF-209 in dosing solutions. The method developed is capable of quantitating LNF-209 in solutions of sterile water and 5% dextrose at concentrations ranging from 8 to 6000 micrograms/ml. The method is linear over this range and quantitative recovery is obtained. The overall average relative standard deviation for replicate analysis of several samples at various concentrations assayed over two days was 2.3%.

The determination of minoxidil in human serum by high-performance liquid chromatography with amperometric detection.

An ion-pairing reversed-phase HPLC assay employing amperometric detection has been developed for the determination of free minoxidil (MNX) in human serum. The drug is isolated from the serum and concentrated by solid phase extraction with a disposable cartridge column containing ethylsilane (C2) bonded phase packing material. The average absolute recovery from serum was 85%. The HPLC separation is performed on a Sperisorb-C(8) column, using n-octanesulphonic acid as the pairing ion. The method exhibits linear behaviour from 0.3 to 100 ng/ml for spiked serum samples. The average daily relative standard deviations of replicate samples at the 0.75 and 2.0 ng/ml levels were 9.6 and 6.4%, respectively. Utilizing a 1 ml sample the limit of detection (S/N > or = 3) was 0.3 ng/ml.

Quantitative determination of acivicin in bulk drug and pharmaceutical formulations by ion-pair high-performance liquid chromatography.

An ion-pair reversed-phase high-performance liquid chromatographic method for the determination of the purity of acivicin and the amount of drug in a sterile powder and two sterile solution formulations is described. The method displays good recoveries (98.4-100.4%) for all formulations and a linear range of 0.002-20 micrograms of drug injected. Estimates of assay precision were 1.3% for the bulk drug, 0.6% for sterile solution formulations and 1.6% for the sterile powder formulations.

Quantitative determination of alprostadil (PGE) in bulk drug and pharmaceutical formulations by high-performance liquid chromatography.

An adsorption high-performance liquid chromatographic method suitable for the quantitative determination of the purity of alprostadil (prostaglandin E1) bulk drug and of the concentration of alprostadil in Prostin V.R. Pediatric Sterile Solution is described. A variety of isomers and related compounds including 8-iso-PGE1, 11-epi-PGE1, 15-epi-PGE1, PGE2, 5,6-trans-PGE2, and 8-iso-PGE2 are separated from alprostadil and are quantifiable. The 2-naphthacyl ester derivatives are employed and derivatization conditions providing maximum response are described.

Quantitative determination of prostaglandins A1 and B1 in alprostadil (PGE1) by high-performance liquid chromatography.

An absorption high-performance liquid chromatography assay for the quantitative determination of prostaglandins A1 and B1 in alprostadil (prostaglandin E1) and in Prostin VR Pediatric Sterile Solution was developed. Prostaglandins A1 and B1 have been shown to be the major degradation products of prostaglandin E1. The adsorption system provided baseline resolution of the 2-naphthacyl esters of prostaglandin A1 from prostaglandin B1. Derivatization conditions providing maximal response for prostaglandins A1 and B1 while minimizing the conversion of prostaglandin E1 to prostaglandins A1 and B1 were established.