Angelos Dovletoglou

Enantiomeric separation of the novel growth hormone secretagogue MK-0677 by capillary zone electrophoresis

Abstract MK-0677 {(R)-2-amino-N-[2-(1,2-dihydro-1-(methylsulfonyl) [3H-indole-3,4′-piperidin]-1′yl]-2-oxo-1-[(phenylmethoxy)methyl]ethyl-2-methylpropanamide-monomethane sulfonate} is a novel orally-active growth hormone secretagogue. The R- and S-enantiomers were separated by capillary zone electrophoresis (CZE) using β-cyclodextrin (β-CD) as the chiral selector in a phosphate buffer containing l -tartaric acid and ethanol. Resolution of the enantiomers requires optimizing the buffer constituent concentrations, apparent pH (pHapp) and temperature, T. The presence of an ion-pairing reagent such as tartaric acid is essential to achieve separation. The log of the separation factor, ln α, increases linearly with 1/T over the range from 10°C to 45°C. It is shown that the equilibrium 1:1 CD–enantiomer complex model of Wren and Rowe is a limiting case of the multiple equilibria model of Rawjee and Vigh. An analytical method for MK-0677 based on the CZE separation of it from its S-enantiomeric impurity was validated in terms of limit of detection, limit of quantitation, UV detector linearity, precision, accuracy and ruggedness. The best working conditions include a background electrolyte containing 40 mM l -tartaric acid, 24 mM NaH2PO4, 30 mM β-CD and 25% (v/v) ethanol at pHapp 4.2; a UV detector at 200 nm; and a 52 cm effective length×76 μm I.D. fused-silica capillary operated at 25°C.

Practical capillary electrophoresis method for the quantitation of the acetate counter-ion in a novel antifungal lipopeptide

This is the first report of the validation of a capillary ion electrophoresis method for the quantitative determination of acetate (CH3COO-) levels in the acetate salt of a basic lipopeptide. The acetate counter-ion was detected using indirect photometric detection, 4.0 mM 4-hydroxybenzoic acid (HOC6H4COOH) as the carrier electrolyte and the chromophore mobile phase, and OFM Anion-BT as the electroosmotic flow (EOF) modifier. The EOF modifier is primarily a 20 mM myristyltrimethylammonium bromide (TTAB) solution. The apparent pH (pHapp) of the electrophoretic buffer was 6.0. The response was linear from 0.9 microgram/ml to 46 micrograms/ml (r2 = 0.9997). The capillary electrophoresis (CE) system is very efficient and stable, giving R.S.D. values of about 1.0% for the injection-to-infection precision without using internal standards. The method is accurate as judged by comparison to an ion-exchange HPLC method and by the 99.7% recovery of spiked acetate ion in the aqueous solution of the lipopeptide. The method is rugged with regard to critical method parameters, such as different operational voltages and effective capillary lengths. The overall precision is acceptable with a R.S.D. of 1.5% between 0.2-12% (w/w) acetate ion present in the drug substance without using an internal standard. Comparison to the ion-exchange chromatography and advantages of the CE method are discussed. This CE method has been submitted to and accepted by the regulatory agencies and is now in routine use within our laboratories.

Moisture assay of an antifungal by near-infrared diffuse reflectance spectroscopy

Near-infrared (NIR) diffuse reflectance spectroscopy was employed in the method development and validation of a moisture assay for the novel antifungal caspofungin acetate. Spectra were obtained over the entire spectral region available (950-1650 nm) using an InGaAs photodiode array detector equipped with a diffuse reflectance probe. No sample pre-treatment was required and the analysis time was less than 1 min. Primary reference data were obtained using a Karl Fischer (KF) titration (coulometric, volumetric or both). The investigated range of water content was 2.6-9.9% (w/w) with a standard error of prediction (SEP) of 0.2%. The predictive capabilities of the partial least-squares (PLS) regression calibration model used in the moisture assay were verified using independent test sets. The NIR predicted values of the developed method were equivalent to the reference method sets and the prediction error was equivalent to the reference method error. These results reveal that the predictive model constructed by means of a PLS regression is valid, rugged and could be used to determine moisture levels on-line in caspofungin acetate drug substance.

Development of Practical HPLC Methods for Analysis and Quality Assessment of the Novel Carbonic Anhydrase Inhibitor MK-0507 and the Acetamidosulfonamide Intermediate

Abstract MK-0507 (Dorzolamide HCl, (4S,6S)-4-Ethylamino-5,6-dihydro-6-methyl-4H-thieno-[2,3-b] thiopyran-2-sulfonamide 7,7-dioxide hydrochloride) is the first topically active, water-soluble, carbonic anhydrase inhibitor to be developed for the treatment of glaucoma and ocular hypertension. Dorzolamide HCl is an effective and well tolerated agent as monotherapy for patients who cannot tolerate ophthalmic beta-blockers, and for those who need add-on therapy to beta-blockers.1 The steps taken in the development and validation of a fast and rugged reverse-phase HPLC method for the analysis and quality assessment of MK-0507 drug substance and acetamidosulfonamide intermediate are described. Four columns were used during the development: Du Pont Zorbax C-18, Rainin Microsorb C-8, Perkin Elmer CR-C8 and YMC4. The last two columns showed excellent resolution, peak shape, and precision. The selected HPLC method for acetamidosulfonamide, using the Perkin Elmer CR-C8 column, was validated with respect to linearity,...

Evaluating HPLC Assay Robustness with Experimental Design

Abstract A twelve run screening factorial experimental design was used to study the instrumental robustness of an HPLC weight percent assay for a fermentation derived pneumocandin B0. The factors varied were the instrumental settings of wavelength, injection volume, flow rate, mobile phase composition, column temperature, and column lot. The measured responses were the fundamental liquid chromatographic parameters of: retention time (RT); capacity factor (k′); theoretical plates (N); tailing factor (T); and resolution (Rs). The effect of each factor on the responses was calculated and significance determined by analysis of variance (ANOVA).

Mechanistic Studies of the Separation of an HIV Protease Inhibitor from Its Piperazine Diastereomer by Reversed Phase High Performance Liquid Chromatography

Abstract The HPLC separation and retention behavior of the piperazine diastereomers of an Human Immunodeficiency Virus (HIV) protease inhibitor on a commercially available C18 chromatographic phase is discussed. The pH and mobile phase composition have the greatest effect on the selectivity of this system. Evidence is presented that the selective interaction involves hydrogen bonding. As the mobile phase pH was varied, a reversal in the elution order corresponding to the change in the protonation state of the molecule is observed. It is proposed, that the selectivity is governed by coordination of the active piperazine amine as a proton donor or proton acceptor with the organic modifier, while interacting with the stationary phase. It is shown, that longer chain alcohols produce poorer selectivities than shorter chain alcohols. It is proposed, that this is because it is more difficult for the longer hydrophobic chains to form stable complexes with the solute through hydrogen bonding. The hydrogen bonding moiety of the solute may, thus, coordinate with water and not selectively interact with the stationary phase. The effects of stationary phase chemistry, ionic strength, and modifier strength are also described.

MODELING THE SELECTION OF FRACTIONS DURING PREPARATIVE HPLC OF A SEMISYNTHETIC PNEUMOCANDIN

Analytical high-performance liquid chromatography (HPLC) data collected during developmental scale-up studies were used to design an empirical model that was applied to the selection of HPLC fractions obtained during preparative scale reverse-phase HPLC of a semisynthetic lipopeptide antifungal. These data resulted in the ability to collect fractions containing the target compound at acceptable purity and with minimal yield loss, based on the ultraviolet data generated by the preparative column detector. The methodology used was successfully integrated from a developmental scale to a manufacturing scale.