Vincent Antonucci

Development of LC chiral methods for neutral pharmaceutical related compounds using reversed phase and normal phase liquid chromatography with different types of polysaccharide stationary phases

The enantioselectivity of a collection of neutral pharmaceutical compounds on six different types of polysaccharide chiral stationary phases (CSPs), Chiralpak AD (and AD-RH), Chiralcel OD (and OD-RH), Chiralpak OJ (and OJ-R), Chiralcel AS (and AS-RH), Sepapak-2 and Sepapak-4 are compared using reversed phase (RPLC) and normal phase liquid chromatography (NPLC). Screening strategies for maximizing the probability of achieving an initial chiral separation hit for neutral compounds using both RPLC and NPLC are described. Further method optimizations are demonstrated by modifying parameters such as organic modifier composition, eluent pH or CSP particle size. Several practical examples of the application of chiral methods for the study of synthetic reaction mixtures are presented. The most critical validation aspects, including limit of detection, specificity, and ruggedness, are also briefly presented.

Use of a Quality-by-Design approach to justify removal of the HPLC weight % assay from routine API stability testing protocols

Due to the high method variability (typically > or = 0.5%, based on a literature survey and internal Merck experience) encountered in the HPLC weight percent (%) assays of various active pharmaceutical ingredients (APIs), it is proposed that the routine use of the test in stability studies should be discouraged on the basis that it is frequently not sufficiently precise to yield results that are stability-indicating. The high method variability of HPLC weight % methods is not consistent with the current ICH practice of reporting impurities/degradation products down to the 0.05% level, and it can lead to erroneous out-of-specification (OOS) results that are due to experimental error and are not attributable to API degradation. For the vast majority of cases, the HPLC impurity profile provides much better (earlier and more sensitive) detection of low-level degradation products. Based on these observations, a Quality-by-Design (QbD) approach is proposed to phase out the HPLC weight % assay from routine API stability testing protocols.

A simple quantitative FT-IR approach for the study of a polymorphic transformation under crystallization slurry conditions

The pharmaceutical compound (2R,3S)-2-([(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl]oxy)-3-(4-fluorophenyl)morpholine hydrochloride (denoted here as Compound X), has been found to crystallize in at least two polymorphic forms. Using only two frequencies (1009 and 1058 cm(-1)) in the infrared, a linear (R=0.998) calibration plot, consisting of the ratio of the peak absorbances plotted against polymorph concentration, was constructed. This plot allowed the quantification of binary mixtures of polymorphs ranging from <3 to approximately 100 wt% Form II in Form I. Spectra were acquired in transmission mode using mineral oil (Nujol) mull sample preparation, for reasons of compatibility with wet cake and slurry samples. The transformation of the less thermodynamically stable polymorph (Form II) to the more stable form (Form I), in stirred methyl isobutyl ketone (MIBK) slurries, was monitored spectroscopically as a function of time. Performing the experiment at various temperatures allowed the energy of activation for the process to be estimated (42 kJ/mol).

High throughput screening of active pharmaceutical ingredients by UPLC

Ultra performance LC (UPLC) was evaluated as an efficient screening approach to facilitate method development for drug candidates. Three stationary phases were screened: C-18, phenyl, and Shield RP 18 with column dimensions of 150 mm x 2.1 mm, 1.7 microm, which should theoretically generate 35,000 plates or 175% of the typical column plate count of a conventional 250 mm x 4.6 mm, 5 microm particle column. Thirteen different active pharmaceutical ingredients (APIs) were screened using this column set with a standardized mobile-phase gradient. The UPLC method selectivity results were compared to those obtained for these compounds via methods developed through laborious trial and error screening experiments using numerous conventional HPLC mobile and stationary phases. Peak capacity was compared for columns packed with 5 microm particles and columns packed with 1.7 microm particles. The impurities screened by UPLC were confirmed by LC/MS. The results demonstrate that simple, high efficiency UPLC gradients are a feasible and productive alternative to more conventional multiparametric chromatographic screening approaches for many compounds in the early stages of drug development.

Practical Applications of Monolithic Columns to Pharmaceutical Process Development

Abstract High performance liquid chromatography (HPLC) is among the most widely used analytical techniques in the process development of drug substances. With the goal of reducing analysis time, there has been considerable focus on high‐speed HPLC separations. Recently, commercially available monolithic columns have proven to be one of the most promising developments in the area of fast chromatographic separations. In this work, pressure drop, column efficiency, and retention behavior of monolithic columns (Chromolith type) were evaluated and compared with those of conventional columns packed with porous stationary‐phase particles. It was demonstrated that high‐speed HPLC separations could be achieved with monolithic columns at acceptable pressure drops without significantly compromising column efficiency. The applications of monolithic columns in pharmaceutical process development, such as reaction monitoring, column fraction screening, and analysis of mother liquors and unstable analytes, are described with respect to the quality of separations, as well as, enhanced throughput and speed of analysis. It is concluded that the use of monolithic columns offers a significant advancement over currently available techniques in the high‐speed and high‐throughput analysis of pharmaceutical compounds. Some practical aspects in utilizing monolithic columns for fast separations are also discussed.

Employment of on-line FT-IR spectroscopy to monitor the deprotection of a 9-fluorenylmethyl protected carboxylic acid peptide conjugate of doxorubicin

A method for accurately determining the end-point, >98% conversion, of the deprotection reaction of a highly toxic 9-fluorenylmethyl (Fm) ester 1b to its corresponding carboxylate 1d in real time by FT-IR spectroscopy is reported. Advantages of this method over analysis by conventional chromatographic means include real time determination of the end-point of a reaction that is time sensitive to by-product formation, and elimination of sampling a highly toxic reaction mixture. The FT-IR method is based on monitoring, in real time, the disappearance of the Fm ester carbonyl band for 1b at 1737 cm(-1), during deprotection by piperidine, and calibration models were established by Partial Least Squares (PLS) regression analysis with high performance liquid chromatography (HPLC) as reference. The best calibration model was built with 5 PLS factors in the spectral range of 1780-1730 and 1551-1441 cm(-1) and resulted in a standard error of cross validation (SECV) of 0.63 mM 1b and a standard error of prediction (SEP) of 0.51 mM 1b in the range of 0-25 mM. This error of prediction is approximately 0.8% of the initial concentration of 1b and is well within our specifications of <2% initial concentration.

Efficient HPLC method development using structure-based database search, physico-chemical prediction and chromatographic simulation.

Development of a robust HPLC method for pharmaceutical analysis can be very challenging and time-consuming. In our laboratory, we have developed a new workflow leveraging ACD/Labs software tools to improve the performance of HPLC method development. First, we established ACD-based analytical method databases that can be searched by chemical structure similarity. By taking advantage of the existing knowledge of HPLC methods archived in the databases, one can find a good starting point for HPLC method development, or even reuse an existing method as is for a new project. Second, we used the software to predict compound physicochemical properties before running actual experiments to help select appropriate method conditions for targeted screening experiments. Finally, after selecting stationary and mobile phases, we used modeling software to simulate chromatographic separations for optimized temperature and gradient program. The optimized new method was then uploaded to internal databases as knowledge available to assist future method development efforts. Routine implementation of such standardized workflows has the potential to reduce the number of experiments required for method development and facilitate systematic and efficient development of faster, greener and more robust methods leading to greater productivity. In this article, we used Loratadine method development as an example to demonstrate efficient method development using this new workflow.

Gas chromatographic analysis of the thermally unstable dimethyl methylphosphonate carbanion via trimethylsilyl derivatization.

A sensitive gas chromatographic method was developed to monitor the reaction of lithium diisopropylamide (LDA) with dimethyl methylphosphonate (DMMP) to generate the phosphonate carbanion (DMMPA). Analysis of the DMMPA was complicated due to its thermal instability and lack of a chromophore. To overcome these problems, samples were derivatized with trimethylsilylchloride (TMSCI) to form DMMPA-TMS which was sufficiently volatile and thermally stable for GC analysis. Work-up of the derivatized solution with 10 vol.% 2-propanol in hexanes was necessary to quench residual TMSCI prior to GC analysis. The presence of DMMPA-TMS and other sample components was confirmed by GC-MS analysis. This method was utilized to profile the synthesis of DMMPA as DMMP was added to LDA and then aged at -78 degrees C. Method precision for DMMPA-TMS of less than 0.2% RSD was achieved for repeat injections after normalization of the response with n-dodecane contained in the sample. Due to the thermal instability of the DMMPA, subambient derivatization temperatures were essential to the stability, and consequently, accurate quantification. Under optimized conditions, this derivatization was successfully utilized as a process monitoring tool.

DEVELOPMENT OF PRACTICAL CHROMATOGRAPHIC METHODS FOR THE ANALYSIS OF ACTIVE ESTERS

Two differing approaches to the liquid chromatographic analysis of active esters used in pharmaceutical processing were developed. Selection of methodology was dictated by the intrinsic chemistries involved and the processing requirements. The first approach (chromatographic systems A and B) described was employed to monitor a mesylation reaction with a relatively simple HPLC impurity profile to a minimum of 99.0% conversion. Chromatographic conditions, designed as a compromise to achieve adequate analyte solubility, specificity, and minimized on-column decomposition within a short analysis time cycle, included an aprotic mobile phase used with a Zorbax SB-CN column at ambient temperature. The second approach (chromatographic system C) described was employed to monitor a triflation reaction to a minimum 97.0% conversion. The impurity profile of this reaction was far more complex than the former reaction, and as a result a more sophisticated analysis method was necessary. A chemical derivatization using tetrabutylammonium bromide in sieve-dried acetonitrile was developed to stabilize the reaction product as its bromo analog, permitting longer column residence times to achieve the necessary method specificity.

Development of chromatographic methods to monitor the synthesis of (tributylstannyl)methanol through unstable lithiated intermediates

Abstract Simple and robust derivatization methods for monitoring the formation of (tributylstannyl)methanol from tributyltin hydride via unstable lithiated intermediates have been developed and validated. These analytes present both chromatographic and detection difficulties in their native states due to low volatility, poor aqueous solubility/stability, and lack of a chromophore. Derivatization of these analytes to trimethylsilyl analogues for gas chromatographic analysis or to benzenesulfonyl urethane analogues for reversed-phase liquid chromatographic analysis was evaluated. The derivatization/gas chromatographic methods developed were demonstrated to be more specific and sensitive for impurity monitoring during (tributylstannyl)methanol preparation.