Richard Victor Vivilecchia

Considerations of small particles in different modes of liquid chromatography

Abstract The use of column packing with particle sizes of 10 μ in different modes of column chromatography, liquid—solid (LSC), bonded phase (BPC) and gel permeation (GPC), was investigated. LSC columns of very high efficiency, 10 effective plates/sec. and high reproducibility were produced by packing μPorasil, a silica gel with a surface area of 400 m 2 /g and an average particle size of μ, into drilled stainless-steel tubes. Bonded phases were prepared by the reactionof monomeric organosilanes with μPorasil to form monomolecular bonded layers. Octadecyl, aminopropyl and cyanopropyl groups were bound to the surface. Packed columns of the monomolecular, bonded phases gave very high efficiencies. More than 300 theoretical plates and 10 effective plates/sec can easily be obtained on a single 30-cm-long column at a carrier velocity of 0.7 cm/sec. High-speed GPC was performed by using packed columns of μStyragel 2 , a porous polymer of styrene and divinylbenzene, with average particle sizes of 20 μ. Columns packed with μStyragel ( dp ??? 10μ) gave 4000–10,000 theoretical plates/30 cm of column, depending upon the carrier velocity. Molecular-weight distributions of many polymers can be obatined in 10–15 min. In addition, the columns were also run at lower carrier velocities to separate small molecules which differ by as little as one methylene group.

Content uniformity determination of pharmaceutical tablets using five near-infrared reflectance spectrometers : A process analytical technology (PAT) approach using robust multivariate calibration transfer algorithms

Near-infrared calibration models were developed for the determination of content uniformity of pharmaceutical tablets containing 29.4% drug load for two dosage strengths (X and Y). Both dosage strengths have a circular geometry and the only difference is the size and weight. Strength X samples weigh approximately 425 mg with a diameter of 12 mm while strength Y samples, weigh approximately 1700 mg with a diameter of 20mm. Data used in this study were acquired from five NIR instruments manufactured by two different vendors. One of these spectrometers is a dispersive-based NIR system while the other four were Fourier transform (FT) based. The transferability of the optimized partial least-squares (PLS) calibration models developed on the primary instrument (A) located in a research facility was evaluated using spectral data acquired from secondary instruments B, C, D and E. Instruments B and E were located in the same research facility as spectrometer A while instruments C and D were located in a production facility 35 miles away. The same set of tablet samples were used to acquire spectral data from all instruments. This scenario mimics the conventional pharmaceutical technology transfer from research and development to production. Direct cross-instrument prediction without standardization was performed between the primary and each secondary instrument to evaluate the robustness of the primary instrument calibration model. For the strength Y samples, this approach was successful for data acquired on instruments B, C, and D producing root mean square error of prediction (RMSEP) of 1.05, 1.05, and 1.22%, respectively. However for instrument E data, this approach was not successful producing an RMSEP value of 3.40%. A similar deterioration was observed for the strength X samples, with RMSEP values of 2.78, 5.54, 3.40, and 5.78% corresponding to spectral data acquired on instruments B, C, D, and E, respectively. To minimize the effect of instrument variability, calibration transfer techniques such as piecewise direct standardization (PDS) and wavelet hybrid direct standardization (WHDS) were used. The PDS approach, the RMSEP values for strength X samples were lowered to 1.22, 1.12, 1.19, and 1.08% for instruments B, C, D, and E, respectively. Similar improvements were obtained using the WHDS approach with RMSEP values of 1.36, 1.42, 1.36, and 0.98% corresponding to instruments B, C, D, and E, respectively.

Chiral separation of primary amino compounds using a non-chiral crown ether with β-cyclodextrin by capillary electrophoresis

A non-chiral crown ether (18-crown-6) along with beta-cyclodextrin (beta-CD) was used to achieve enantioselective separations of primary amino compounds in capillary electrophoresis. In this new method, the amino group of these compounds is protonated in a low pH separation buffer and forms a selective host-guest complex with the crown ether (amino compound+18-crown-6). The hydrophobic portion of the host-guest complex is then incorporated into the cavity of the beta-cyclodextrin. The amino compound is sandwiched between the crown ether and the cyclodextrin (18-crown-6+amino compound+beta-CD) and thus determines or enhances the enantioselective recognition. It is postulated that the formation of this sandwich results in a more selective chiral interaction between the molecule and beta-cyclodextrin. The chiral recognition is dependent upon the formation of this sandwich complex. This method has been used to achieve enantioselectivity of primary amino compounds with a wide variety of substitutions.

Achievement of enantioselectivity of non-polar primary amines by a non-chiral crown ether

A non-chiral crown ether (18-crown-6), as a molecular modifier, along with cyclodextrin (CD) was used to achieve or enhance enantioselective separations of non-polar primary amines in capillary electrophoresis. Chiral separations of the primary amines are rarely achieved using cyclodextrins alone in capillary electrophoresis, because the three-point intermolecular interaction between the chiral amine and the cyclodextrin is not selective enough to yield resolution of the enantiomers of interest. To resolve these types of compounds, the non-chiral crown ether (18-crown-6) has been used with cyclodextrins (CDs) in the buffer. In the method, the amino group of the compounds is protonated using a low pH buffer solution and locked into 18-crown-6 ring forming a host-guest complex (amine+18-crown-6). The hydrophobic portion of the complex is then incorporated into the cavity of the cyclodextrin forming a secondary sandwich complex (18-crown-6+ amine+CD), in which the chiral center of the amine molecule is recognized. It is postulated that the sandwich complex results in a more rigid structure around the chiral center and more selective interaction between the chiral amine and cyclodextrin. This method can be used to separate a variety of non-polar primary amines, even when the chiral center is three-carbon atoms or more from the hydrophobic group.

Enhancement of chiral recognition by formation of a sandwiched complex in capillary electrophoresis.

For chiral primary amino compounds not separable by cyclodextrins alone, chiral recognition was successfully achieved by the formation of a sandwiched complex of the non-chiral 18-crown-6, the chiral amine and cyclodextrin (CD) [18-crown-6+amino compound+CD]. The separation of 1-methyl-3-phenylpropylamine and 1,2,3,4-tetrahydro-1-naphthylamine racemates showed the special function of the non-chiral 18-crown-6 on chiral recognition. By formation of the sandwiched complex, the chiral center of 1-methyl-3-phenylpropylamine was successfully recognized, and resolution of 1,2,3,4-tetrahydro-1-naphthylamine dramatically increased. In these studies, the mobility differences of the enantiomers were evaluated as a function of the concentration of cyclodextrins with and without the 18-crown-6, and as a function of the concentration of the 18-crown-6. In addition, the separations by this method were compared to those by the chiral 18-crown-6 reagent.

Analysis of a recombinant granulocyte macrophage colony stimulating factor dosage form by capillary electrophoresis, capillary isoelectric focusing and high-performance liquid chromatography

Abstract The analysis of a recombinant granulocyte macrophage colony stimulating factor (GM-CSF) dosage form by free solution capillary electrophoresis (FSCE), capillary isoelectric focusing (cIEF), and high-performance liquid chromatography (HPLC) is described. The quantitative use of capillary electrophoresis, whether FSCE or cIEF, will always be prone to special problems, such as sensitivity to differences in salt concentrations between the standard and sample, and can not match the ruggedness of HPLC. The usable quantitative linear range for both HPLC and FSCE surpass that achieved for cIEF methods by a factor of 10 or greater. The FSCE system, utilizing an octyl bonded/Brij-35 coated capillary, did not work for all proteins examined. This is probably due to an interaction of the protein with the bonded phase or the absorbed Brij-35. In contrast, the cIEF method worked well for all proteins tested thus far, yielding high efficiency and resolution comparable to slab gel isoelectric focusing. This paper addresses the potential for using free solution capillary electrophoresis and capillary isoelectric focusing as a quantitative analytical tool. Also, the effect of salt in the dosage form on quantitation, reproducibility, and efficiency of capillary electrophoresis methods is also discussed.

Strategy for identification of leachables in packaged pharmaceutical liquid formulations

Drug stability is one of the key properties to be monitored in pharmaceutical drug development. Drug degradation products, impurities and/or leachables from the drug product and packages may have significant impacts on drug efficacy, safety profile and storage conditions. In the registration stability samples of an ophthalmic pharmaceutical drug product, an unknown compound was found at a level of 0.19% by HPLC analysis. Subsequent liquid chromatography/mass spectrometry (LC/MS) analysis with electrospray ionization (ESI) indicated that the unknown was not related to the drug substance and was most likely a leachable. Identification of this unknown leachable was needed to evaluate the impact on drug safety. Through systematic extraction of various components or component combination of the packaging materials, and subsequently LC/MS analysis, the unknown was found to be a leachable coming from the varnish applied to the label. In general, using LC/MS alone is not sufficient to elucidate the structure of a complete unknown. Gas chromatography/mass spectrometry (GC/MS) was then conducted with a chemical ionization (CI) source to determine the retention time and mass of the compound of interest. Both CI and ESI sources generated the same protonated molecular ion [M+H] and similar fragmentation ions, which provides a good correlation of the unknown eluted in the liquid chromatogram and in the gas chromatogram. GC/MS with electron impact (EI) was then conducted to obtain the EI mass spectrum of this unknown. It was identified as monomethyl derivative of mephenesin through the NIST library search. The identification strategy utilized electrospray LC/MS and GC/MS with chemical and electron ionization sources which provided complimentary information for structure elucidation of this unknown compound. This combination approach in conjunction with systematic extraction was necessary for the determination of the source of this unknown in the pharmaceutical drug stability studies.

Comprehensive Validation Scheme for In Situ Fiber Optics Dissolution Method for Pharmaceutical Drug Product Testing

There has been a growing interest during the past decade in the use of fiber optics dissolution testing. Use of this novel technology is mainly confined to research and development laboratories. It has not yet emerged as a tool for end product release testing despite its ability to generate in situ results and efficiency improvement. One potential reason may be the lack of clear validation guidelines that can be applied for the assessment of suitability of fiber optics. This article describes a comprehensive validation scheme and development of a reliable, robust, reproducible and cost-effective dissolution test using fiber optics technology. The test was successfully applied for characterizing the dissolution behavior of a 40-mg immediate-release tablet dosage form that is under development at Novartis Pharmaceuticals, East Hanover, New Jersey. The method was validated for the following parameters: linearity, precision, accuracy, specificity, and robustness. In particular, robustness was evaluated in terms of probe sampling depth and probe orientation. The in situ fiber optic method was found to be comparable to the existing manual sampling dissolution method. Finally, the fiber optic dissolution test was successfully performed by different operators on different days, to further enhance the validity of the method. The results demonstrate that the fiber optics technology can be successfully validated for end product dissolution/release testing.

Enantiomeric separation of N-tert.-butoxycarbonyl amino acids by capillary electrophoresis using hydroxypropyl-substituted cyclodextrins

Abstract The separation of the enantiomers of N-tert.-butoxycarbonyl (N-t-Boc) amino acids by capillary electrophoresis employing hydroxypropyl-substituted cyclodextrins is described. The enantiomeric separation of the N-t-Boc derivatized amino acids studied was successfully accomplished by employing hydroxypropyl-β-cyclodextrin. The N -t- Boc - d,l -3-(2- naphthyl)alanine enantiomers were separated using hydroxypropyl-γ-cyclodextrin. The different forms and concentrations of cyclodextrins, and the effects of organic solvent addition to the separation buffer are investigated. In addition, the pH of the separation buffer and the effect of pH on the enantioselectivity of the N-t-Boc amino acids are discussed.

Enhancing Productivity in the Analytical Laboratory Through the Use of Ultra Fast-HPLC in Preformulation/Formulation Development

Abstract The pharmaceutical industry today is driven to create new, more efficient ways to discover, develop, deliver and monitor drugs. Pharmaceutical companies are being faced with major challenges in reducing drug discovery and development timelines. Automation and the introduction of new analytical technologies that increase speed of analysis are integral in the analytical laboratory. The development of rapid chromatographic methods in preformulation and formulation development is playing an increasing role to support this drive in efficiency and productivity. The introduction of ultra fast-HPLC systems that can operate at pressures of up to 15,000 psi with columns packed with sub-2-µm particles have allowed for high speed and efficient separations. The consequent reduction of time, solvent, and waste disposal, and the analysis of more samples per unit time makes ultra-fast HPLC a very attractive technology. Faster method development and decision making can be achieved during late-phase preformulation/formulation development for the analysis of both singly charged and multiply charged basic compound analysis. The use of ultra fast-HPLC (UHPLC) for pH scouting experiments and the determination of the analytes ionogenic nature was shown to be an effective tool for rapid and systematic method development. The implementation of this technology was also evaluated for the analysis of drug product formulations and excipient compatibility studies. Increased speed of analysis and significant gains in resolution per unit time were obtained compared to separations performed using conventional HPLC systems (operating pressures of <5,500 psi). Also, the use of liophilic ions as mobile phase additives with operation under high pressures led to enhanced separation selectivity, retention, and peak symmetry of multiply charged basic compounds.