Determination of equilibrium constant of amino carbamate adduct formation in sisomicin by a high pH based high performance liquid chromatography.

Amino carbamate adduct formation from the amino group of an aminoglycoside and carbon dioxide has been postulated as a mechanism for reducing nephrotoxicity in the aminoglycoside class compounds. In this study, sisomicin was used as a model compound for amino carbamate analysis. A high pH based reversed-phase high performance liquid chromatography (RP-HPLC) method is used to separate the amino carbamate from sisomicin. The carbamate is stable as the breakdown is inhibited at high pH and any reactive carbon dioxide is removed as the carbonate. The amino carbamate was quantified and the molar fraction of amine as the carbamate of sisomicin was obtained from the HPLC peak areas. The equilibrium constant of carbamate formation, Kc, was determined to be 3.3 × 10(-6) and it was used to predict the fraction of carbamate over the pH range in a typical biological systems. Based on these results, the fraction of amino carbamate at physiological pH values is less than 13%, and the postulated mechanism for nephrotoxicity protection is not valid. The same methodology is applicable for other aminoglycosides.

The evaluation of 25 chiral stationary phases and the utilization of sub-2.0µm coated polysaccharide chiral stationary phases via supercritical fluid chromatography.

A rapid screening method to identify the best conditions for chiral separations is described. We analyzed a representative set of 80 racemic compounds against 25 different chiral stationary phases with three different mobile phases to identify the combination of columns and mobile phases that will separate the most compounds on the initial screen. While the OD separated the largest number of compounds, we found the best combination of six columns to be the AD, AS, AY, CC4, ID and Whelk-O1. The second team included the CCC, Cellulose-1, Cellulose-3 or OJ, IA, IE and IF. All 80 compounds were separated with a resolution range of 0.65-15.36. Screening the covalently bonded phases provided separation for 79 of the 80 compounds. We also found ethanol (0.1% NH4OH) separated more compounds than methanol (0.1% NH4OH) or isopropanol (0.1% NH4OH). As part of this study, we also compared the effectiveness of stationary phases that have the same chiral selector. Finally, we demonstrated the effectiveness of using a fast, 1.5-min screening method that utilizes a 1.7µm coated polysaccharide chiral stationary phase.

A high pH based reversed-phase high performance liquid chromatographic method for the analysis of aminoglycoside plazomicin and its impurities.

A reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed for the aminoglycoside (AG) plazomicin (ACHN-490). This method employed a high pH mobile phase (pH>11) with a gradient of 0.25 M ammonium hydroxide in water and acetonitrile, an XBridge C(18) column and UV detection at 210 nm. Although the molar UV absorption of plazomicin is weak, the high pH conditions of this method allow for higher loadings, which compensates for the inherent low UV sensitivity. Under these high pH conditions, impurities and degradants were base line separated from plazomicin. The mobile phases used for this method allowed for on-line mass detection for the impurities and degradants. The RP-HPLC method has been validated in terms of specificity, linearity and range, accuracy, and precision. The analytical method met specificity requirements of a homogenous peak with no interferences from the blank or from the known impurities in plazomicin. The linearity of the method for the plazomicin impurity determination was excellent, with a coefficient of determination (r(2)) of 0.9993, over the freebase (FB) concentration range of 0.0025-3.0 mg/mL. The method is capable of detecting impurities down to 0.1% of the peak area of plazomicin. A single point standard at a concentration of 1.0 mg/mL FB was validated over the range of 50-150% for quantitation of the freebase content (the assay) in bulk drug substance. The mean recoveries of FB are in the range 98.6-102.0% with a mean RSD (relative standard deviation) <1.0%. The study also examined the method precision for purity, impurities and the assay with two instruments on two different days. The method showed adequate accuracy and precision for the intended use. This high pH method was successfully used to determine the impurity and measure the drug content in the final plazomicin drug substance. In addition, the method with an on-line mass spectrometry detector has been used to characterize the structures of the impurities in plazomicin.

The use of ammonium hydroxide as an additive in supercritical fluid chromatography for achiral and chiral separations and purifications of small, basic medicinal molecules.

This study describes using 0.1% of a 28-30% ammonium hydroxide solution as an additive to alcohol modifiers in SFC to improve chromatographic peak shapes for basic molecules. Ammonium hydroxide's high volatility leaves no residual additive in the purified sample unlike classical additives in preparative chromatography such as diethylamine and triethylamine. We demonstrate that the silica support is stable despite having ammonium hydroxide in the modifier by running a durability study for over 350 h (105 L of solvent, 105,000 column volumes) on an analytical Chiralcel OJ column and a second study for 30 h (7.2 L, 14,400 column volumes) on an analytical Lux Cellulose-1 column. The peak shape of small, basic molecules is greatly improved with the use of ammonium hydroxide and this improvement is very similar to those having 0.1% diethylamine as a mobile phase additive. Electrospray ionization is also enhanced with the presence of ammonium hydroxide compared with that of diethylamine. We have found that the age of the 28-30% bottle of ammonium hydroxide solution can have significant effects on the chromatography and we describe how this can be overcome. Finally, we analyzed 23 racemic and basic compounds on six different chiral stationary phases and found there to be very little chiral selectivity difference between ammonium hydroxide and diethylamine, triethylamine, ethanolamine and isopropylamine.

Telavancin, a multifunctional lipoglycopeptide, disrupts both cell wall synthesis and cell membrane integrity in methicillin-resistant Staphylococcus aureus.

The emergence and spread of multidrug-resistant gram-positive bacteria represent a serious clinical problem. Telavancin is a novel lipoglycopeptide antibiotic that possesses rapid in vitro bactericidal activity against a broad spectrum of clinically relevant gram-positive pathogens. Here we demonstrate that telavancin's antibacterial activity derives from at least two mechanisms. As observed with vancomycin, telavancin inhibited late-stage peptidoglycan biosynthesis in a substrate-dependent fashion and bound the cell wall, as it did the lipid II surrogate tripeptide N,N'-diacetyl-L-lysinyl-D-alanyl-D-alanine, with high affinity. Telavancin also perturbed bacterial cell membrane potential and permeability. In methicillin-resistant Staphylococcus aureus, telavancin caused rapid, concentration-dependent depolarization of the plasma membrane, increases in permeability, and leakage of cellular ATP and K(+). The timing of these changes correlated with rapid , concentration-dependent loss of bacterial viability, suggesting that the early bactericidal activity of telavancin results from dissipation of cell membrane potential and an increase in membrane permeability. Binding and cell fractionation studies provided direct evidence for an interaction of telavancin with the bacterial cell membrane; stronger binding interactions were observed with the bacterial cell wall and cell membrane relative to vancomycin. We suggest that this multifunctional mechanism of action confers advantageous antibacterial properties.

Increasing the efficiency of pharmacokinetic sample procurement, preparation and analysis by liquid chromatography/tandem mass spectrometry.

The movement towards a 96-well format has greatly increased productivity and throughput in bioanalytical laboratories. Improvements in automated sample preparation and analytical methods have further contributed to increased productivity. We have focused on sample collection and transfer to the bioanalyst and have found improvements to the current available methods. The problem of manual transfers and plasma clotting issues can be overcome with the use of microtainers. Specifically, for illustrative purposes, three proprietary Theravance compounds were tested for stability, non-specific binding, and electrospray ion suppression in microtainers. There were no issues with stability, non-specific binding or ion suppression for the above compounds even after leaving plasma samples in the microtainers over long periods of time. The microtainers are robot-compatible and the resulting plasma can be transferred without clotting issues. To date, all in-house compounds successfully analyzed and tested using the microtainers have mass ranges between 200 and 1800 Da, pK(a) ranges between 3.8 and 10.3, and logD ranges between -1.7 and 4.2. Once samples are transferred into 96-well plates, flexibility in preparation and analysis is available. Together with automated sample preparation and the use of liquid chromatography/tandem mass spectrometry (LC/MS/MS) as an analytical tool, the use of microtainers as sample collection tubes and for sample storage saved considerable time, cost and effort in both of our pharmacokinetic (PK) and bioanalytical groups. This in turn has led to an increased efficiency and overall throughput in support of our drug discovery effort.