Robert Hartman

Analytical Method Volume Intensity (AMVI): A green chemistry metric for HPLC methodology in the pharmaceutical industry

Analytical Method Volume Intensity (AMVI), a simple metric developed to measure the total solvent consumption of an HPLC method, can help chemists make informed decisions on how to lower an analytical methods environmental impact.

Examination of rofecoxib solution decomposition under alkaline and photolytic stress conditions

Rofecoxib is a highly active and selective cyclo-oxygenase II inhibitor. A stability-indicating method for the assay of rofecoxib has been developed using reverse-phase high-performance liquid chromatography (HPLC). Stress testing of rofecoxib was conducted during the method development and validation. HPLC analysis of rofecoxib solutions stressed under alkaline and photolytic conditions revealed the presence of several degradates. Two main degradates were determined to be the cyclization product formed by photo-cyclization and the dicarboxylate formed by ring opening in the presence of base and oxygen. The identities of these degradates were confirmed by comparison of UV spectra and HPLC retention time with the independently synthesized products. The mechanistic pathways for the formation of these degradates are discussed. Further improvement of the HPLC methods ruggedness has been made based on these studies.

Effect of certain antihistamine and antiserotonin agents upon experimental trichinosis in mice.

Abstract Diets containing an antihistamine drug, 2-[ P -chloro-α-(2-dimethylamino-ethyl)benzyl] pyridine maleate (chlorpheniramine maleate), or an antiserotonin drug, 1-benzyl-2-methyl-5-methoxytryptamine (B.A.S.), or a drug possessing both properties, 1-methyl-4-(5-dibenzo-[α,e]-cycloheptatrienylidine)-piperidine hydrochloride monohydrate (cyproheptadine), were fed to mice during the acute intestinal phase of experimental trichinosis. In each instance the number of worms remaining in the small intestine on days 17 and 21 of infection was greater in the treated than in the untreated mice. It is suggested that the cellular reaction responsible for the expulsion of Trichinella from the gut may be mediated, to some extent, by histamine.

Induction of Immunity to Trichinosis in Mice by means of Chemically Abbreviated Infections.

Abstract In two experiments mice were exposed to four heavy infections of Trichinella spiralis within a period of 11 days. Each infection was terminated by 2-[4′-thiazolyl]-benzimid-azole (thiabendazole) therapy within 1 day after exposure. In both experiments the mice were subjected to a challenge infection approximately 1 month after the last immunizing exposure. On day 7 after challenge the number of adult worms in the immunized mice was 63–72% less than that in the control mice. In a third experiment thiabendazole therapy was applied from days 14 to 21 after exposure of mice to a severe Trichinella infection. The mice were exposed to a severe challenge infection 1 month after the initial exposure. On day 7 after challenge, the number of adult worms in the previously infected mice was 63% less than that in the control mice. In one experiment, in which multiple infections were used for immunization, the number of larvae in the muscles of the immunized mice was 50% less than that in the control mice. In the other two experiments, however, no reduction in the number of larvae in the muscles of the immunized mice was demonstrated. These experiments have demonstrated that (1) protective immunity against reinfection with Trichinella can be stimulated by repeated intestinal infections of only 1 day duration, and (2) a similar degree of protection can be elicited by a single infection of 14 days duration; (the effect of a single 1-day-old infection was not studied).

Development and Validation of an HPLC Method for the Impurity and Quantitative Analysis of Etoricoxib

Abstract Etoricoxib (5‐chloro‐6′‐methyl‐3[4‐(methanesulfonyl)phenyl]‐2,3′‐bipyridine) is a highly active and selective cyclo‐oxygenase II inhibitor. A single, stability‐indicating HPLC method has been developed and validated for both the impurity and quantitative analysis of etoricoxib. Method development incorporated the optimization of stationary phase, pH, temperature, and mobile phase composition for the resolution of thirteen process impurities and three major degradation products. Further optimization of pH and mobile phase composition was aided by the use of DryLab®, a computer‐based simulation program. The stability‐indicating capability of the method was proven through the identification of photolytic and oxidative decomposition products. Method validation produced excellent results for linearity, precision, limit of quantitation and limit of detection, specificity, accuracy, recovery, and robustness. The identities of etoricoxib decomposition products were confirmed by UV, LC/MS, and NMR spectra.

Changes in the efficacy of three anthelmintics during the maturation of a nematode (Trichinella spiralis).

A study was made of the changes observed in the sensitivity of Trichinella spiralis to anthelmintic treatment during the parasites period of maturation in the gut. Young adult worms (24 hr old) were removed by two of the medications (tetramisole at 25 mg/kg subcutaneously, methyridine at 250 mg/kg subcutaneously) but the early (2 hr) enteral stages were not removed. In contrast, a third medication (thiabendazole at 50 mg/kg orally) was not active against the young adults, but was highly active against the early stages. The regimens used had been selected specifically to reveal changes in drug sensitivity; given sufficiently large dosages, all three drugs were at least partially active against both the earlier and the later developmental stages. The results suggest either that the mode of action of thiabendazole against trichinella is fundamentally different from that of the other two compounds, or that the relationship between efficacy and the route of administration of a given drug changes during the maturation phase of trichinella. Of the compounds which possess activity against the enteral phase of trichinella, four are capable of eradicating the worms when given as a single dose, namely, thiabendazole (Campbell and Cuckler, 1964), methyridine (Denham, 1965), tetramisole, and pyrantel tartrate. The efficacy of tetramisole against trichinella has been reported (Thienpont et al., 1966) but the dosage and time of treatment were not given. The anthelmintic activity of pyrantel tartrate has been reported, without reference to trichinosis (Austin et al., 1966). In the present study pyrantel was not used except in supplementary experiments designed to clarify the results obtained with the other drugs. The efficacy of the above compounds, and the rapid maturation of trichinella, presented a convenient opportunity to compare the changes in efficacy of anthelmintics during the development of a nematode. MATERIALS AND METHODS The strain of albino mouse, the strain of parasite, and the parasitological procedures used, were all as described previously (Campbell, 1965). Mice were housed in experimental groups of 10, except for a group of 16 mice (Experiment 1), which were housed as subgroups of 10 and 6. Each drug was administered by the route known to be most effective, viz., orally in the case of thiabendazole, and subcutaneously in the case of tetramisole and methyridine. For each drug a single dosage was used, and was selected (on the basis of preliminary experiments) as a suitable dosage for demonstrating changes in efficacy during maturation. Thiabendazole was administered as a 0.5-ml volume of a suspension of the base Received for publication 10 November 1967. in 2% methylcellulose. Tetramisole was administered as 0.25 ml of an aqueous solution, and methyridine as 0.25 ml of an aqueous dilution of a commercial preparation containing 90% active ingredient (Promintic, ICI Ltd.). In each experiment and at each treatment period, a control group of mice received a dose of water. The administration of water was designed (on the basis of the preliminary experiments) as a control for drug efficacy rather than the lack of efficacy. Thus 0.5 ml water, containing 2% methylcellulose, was administered orally when the inoculation-treatment interval was 8 hr or less, and 0.25 ml distilled water was given subcutaneously when the interval was greater. The mice were inoculated at the rate of one group (10 mice) per 90 sec (approximately). In Experiments 1 and 3 the time of inoculation was recorded separately for each group. In Experiment 2 the time was recorded for every fourth group. It was thus possible to adhere to the scheduled intervals between inoculation and treatment with a relatively high degree of accuracy. At necropsy, worm burdens were routinely estimated on the basis of 4 or 6 aliquots of 5 ml each; they were drawn from 200 ml of stirred suspension by means of a widemouthed pipette. Where the worm counts were very low (less than 10 worms/ aliquot) the 5-ml aliquots were discarded in favor of 15-ml aliquots.

Macrocyclic glycopeptide chiral selectors bonded to core-shell particles enables enantiopurity analysis of the entire verubecestat synthetic route

Verubecestat is an inhibitor of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) being evaluated in clinical trials for the treatment of Alzheimers disease. Synthetic route development involves diastereoselective transformations with a need for enantiomeric excess (ee) determination of each intermediate and final active pharmaceutical ingredient (API). The analytical technical package of validated methods relies on enantioselective SFC and RPLC separations using multiple 3 and 5 μm coated polysaccharide-based chiral stationary phases (CSPs) and mobile phases combinations. Evaluation of recently developed chiral columns revealed a single chiral selector (Teicoplanin) bonded to 2.7 μm core-shell particles using H3PO4 in H2O/ACN and triethylammonium acetate: methanol based eluents at different isocratic compositions allowed good enatioseparation of all verubecestat intermediates. EE determination of verubecestat is easily performed on NicoShell, another macrocyclic glycopeptide chiral selector bonded to 2.7 μm superficially porous particles. This approach enables fast and reliable enantiopurity analysis of the entire verubecestat synthetic route using only two chiral columns and mobile phases on a conventional HPLC system, simplifying technical package preparation, method validation and transfer to manufacturing facilities.

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.

DEVELOPMENT OF A DERIVATIZATION METHOD, COUPLED WITH REVERSE PHASE HPLC, FOR MONITORING THE FORMATION OF AN ENOLATE INTERMEDIATE

A sensitive liquid chromatographic method has been developed to monitor the formation of an enolate intermediate in a synthetic route to Etoricoxib, a drug candidate for the treatment of arthritis. The method requires the derivatization of the enolate with methyl iodide to form a stable methylketosulfone derivative followed by reverse phase HPLC analysis. Parameters affecting the derivatization, including the nature of derivatizing agent, reaction solvent, amount of derivatizing agent, reaction time, reaction temperature, and amount of excess base in the reaction were investigated. The derivatization reaction was shown to give selective C-alkylation. The linear range of the chromatographic method for the determination of the starting material, ketosulfone, and the derivative, methylketosulfone, was determined. Finally, the accuracy of the method was established based on recovery experiments.

Generic gas chromatography-flame ionization detection method for quantitation of volatile amines in pharmaceutical drugs and synthetic intermediates

Volatile amines are among the most frequently used chemicals in organic and pharmaceutical chemistry. Synthetic route optimization often involves the evaluation of several different amines requiring the development and validation of analytical methods for quantitation of residual amine levels. Herein, a simple and fast generic GC-FID method on an Agilent J&W CP-Volamine capillary column (using either He or H2 as the carrier gas) capable of separating over 25 volatile amines and other basic polar species commonly used in pharmaceutical chemistry workflows is described. This 16min method is successfully applied to the analysis and quantitation of volatile amines in a variety of pharmaceutically-related drugs and synthetic intermediates. Method validation experiments showed excellent analytical performance in linearity, recovery, repeatability, and limit of quantitation and detection. In addition, diverse examples for the application of this method to the simultaneous determination of other amine-related chemicals in reaction mixtures are illustrated, thereby indicating that these GC-FID method conditions can be effectively used as starting point during method development for the analysis of other basic polar species beyond the validated list of amines described in this study.