Effect of dilution solvent and injection volume on the analysis of basic hydrophilic therapeutic polypeptide salts with pressurized carbon dioxide mobile phases.

The chromatographic analysis of long-chain hydrophilic therapeutic peptides, with molecular weight mostly in the 3500-4500 Da range (31-34 amino acids), is explored with pressurized CO2 in the mobile phase. The optimal method was obtained on a Torus 2-PIC column, with a gradient elution of 50-90% co-solvent in CO2, which is relevant of enhanced-fluidity liquid chromatography (EFLC). Both UV (210 nm) and mass spectrometric detection modes were employed to assess the purity of the major peak and its resolution from impurities. Ten out of the eleven peptides in this set were basic, thus they were analyzed as acetate or trifluoroacetate salts. As significant peak distortion was observed in some cases, thorough examination of dilution solvent and injection volume was conducted to improve peak shape and resolution from impurities. Finally, the best injection volume was 1 µL, as any other volume (smaller or larger) yielded distorted peaks, and the best dilution solvent composition was the same as the mobile phase co-solvent (methanol comprising 5% water and 0.1 % methanesulfonic acid). However, not all peptide salts were fully soluble in this solvent so other alternatives (including more water in the dilution solvent), offering adequate dissolution but slightly inferior chromatographic performance should be chosen in such cases.

Analysis of short-chain bioactive peptides by unified chromatography-electrospray ionization mass spectrometry. Part II. Comparison to reversed-phase ultra-high performance liquid chromatography.

In the first part of this study, a unified chromatography (UC) analysis method, which is similar to supercritical fluid chromatography (SFC) but with wide mobile phase gradients of pressurized CO2 and solvent, was developed to analyse short-chain peptides, with UV and mass spectrometry (MS) detection. In this second part, the method is compared to a reference reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) method, based on the analysis of 43 peptides, including 10 linear peptides and 33 cyclic ones. First, the orthogonality between the two methods was examined, based on the retention patterns. As the UC method was developed on a polar stationary phase (Ascentis Express OH5), the elution orders and selectivities were expected to be significantly different from RPLC on a non-polar stationary phase (ACQUITY CSH C18). Secondly, the success rate of the methods was examined, based on successful retention / elution of the peptides and the absence of observed co-elutions between the main peak and impurities. A successful analysis was obtained for 81% of the peptides in UC and 67% in RPLC. Thirdly, the performance of the methods for the intended application of impurity profiling of peptide drug candidates was assessed, based on the comparison of peak purities, the number of impurities detected and the thorough examination of impurity profiles. Excellent complementarity of the two methods for the specific task of impurity profiling, and for the separation of isomeric species was observed, with only one isomeric pair in this set remaining unresolved. The method sensitivity was however better with RPLC than UC. Finally, the operational costs in terms of solvent cost per analysis were the same between the two methods.

Analysis of short-chain bioactive peptides by unified chromatography-electrospray ionization mass spectrometry. Part I. Method development.

A method to analyse short-chain bioactive peptides (MW < 800 Da) and their impurities was developed with a unified chromatography (UC) analysis, including a wide mobile phase gradient ranging from supercritical fluid to near-liquid conditions, with UV and electrospray ionization mass spectrometry detection (ESI-MS). Four stationary phases and three mobile phase compositions were examined. Ten model peptides were first selected to identify the best operating conditions, including five linear tripeptides and five cyclic pentapeptides, with log P values ranging from -5.9 to 3.6, and including isomeric species. Derringer desirability functions were designed to identify optimal operating conditions based on 7 criteria, namely the number of peaks detected (including all impurities resolved), the proportion of the chromatogram occupied by target peaks, the least favourable resolution observed between the main peptide and impurities, peak shape features (asymmetry and peak width at half height), and finally the signal-to-noise ratio observed both with UV (210 nm) and ESI-MS in positive ionization mode. The optimum conditions were obtained on Ascentis Express OH5 stationary phase, with a mobile phase composed of carbon dioxide and methanol, comprising 2% water and 20 mM ammonium hydroxide. The final gradient program ranged from 5 to 80% co-solvent in CO2, with a reversed flow rate gradient ranging from 3.0 to 1.5 mL/min. Back-pressure was set at 120 bar and the column oven temperature at 60°C. Optimal conditions were applied to a large set of 76 peptides (34 linear tripeptides and 42 cyclic pentapeptides) and provided adequate scattering of the peaks in the retention space, together with some separation of isomeric species, particularly for the cyclic peptides.

Purification of drug degradation products supported by analytical and preparative supercritical fluid chromatography.

A stressed degradation (oxidation) was employed to produce metabolites from an active pharmaceutical ingredient (API) with large molecular weight (about 900 g/mol). An analytical chromatographic method was desired to compare the products generated by different degradation methods while a multi-gram-scale preparative chromatographic method was necessary to purify the produced metabolites. Supercritical fluid chromatography (SFC) was selected for both tasks as no other chromatographic method had achieved the resolution of the API and metabolites (two isomeric mono-oxide species and one di-oxide). First, an analytical-scale method was developed with ultra-high performance supercritical fluid chromatography (UHPSFC). Achiral stationary phases containing sub-2 µm fully porous particles or sub-3 µm superficially porous particles, and chiral phases containing 3 and 5 µm fully porous particles were selected for a first screening with gradient elution (carbon dioxide - methanol containing additives). The stationary phase providing the most promising results was ACQUITY Torus 2-PIC (100 x 3 mm, 1.7 µm, Waters). A central composite design (CCD) was conducted to optimize the gradient program and oven temperature. Final gradient conditions were as follows: 50-70% methanol in 3.8 min with oven temperature set at 36 °C, back-pressure set at 11 MPa and flow-rate at 0.8 mL/min. The optimized method was employed to analyze samples obtained with different degradation conditions. Then the method was adapted and transferred to preparative-scale SFC on a 5 µm-particles Torus 2-PIC stationary phase (150 x 30 mm). The method was modified to comprise an isocratic step followed by a gradient, favoring peak shape of the last eluting compound and minimal volume of collected fractions. Batch injections in gradient mode were carried out to purify six grams of crude product.

First inter-laboratory study of a Supercritical Fluid Chromatography method for the determination of pharmaceutical impurities.

Supercritical Fluid Chromatography (SFC) has known a strong regain of interest for the last 10 years, especially in the field of pharmaceutical analysis. Besides the development and validation of the SFC method in one individual laboratory, it is also important to demonstrate its applicability and transferability to various laboratories around the world. Therefore, an inter-laboratory study was conducted and published for the first time in SFC, to assess method reproducibility, and evaluate whether this chromatographic technique could become a reference method for quality control (QC) laboratories. This study involved 19 participating laboratories from 4 continents and 9 different countries. It included 5 academic groups, 3 demonstration laboratories at analytical instrument companies, 10 pharmaceutical companies and 1 food company. In the initial analysis of the study results, consistencies within- and between-laboratories were deeply examined. In the subsequent analysis, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. The results obtained were compared with the literature values for liquid chromatography (LC) in the context of impurities determination. Repeatability and reproducibility variances were found to be similar or better than those described for LC methods, and highlighted the adequacy of the SFC method for QC analyses. The results demonstrated the excellent and robust quantitative performance of SFC. Consequently, this complementary technique is recognized on equal merit to other chromatographic techniques.

Impurity profiling of drug candidates: Analytical strategies using reversed-phase and mixed-mode high-performance liquid chromatography methods.

The development of new active pharmaceutical ingredients (API) requires accurate impurity profiling. Nowadays, reversed-phase HPLC (RPLC) on C18 stationary phase is the method of first choice for this task and usually employed in generic screening methods. However, this method sometimes fails, especially when the target analyte is not sufficiently retained, making impurity analysis difficult or even impossible. In such cases, a second method must be available. In the present paper, we compare the merits of RPLC on C18 phase to those of previously optimized alternative methods, based on the analysis of a large and diverse set of small-molecule drug candidates. Various strategies are considered: RPLC on C18 phase but with different mobile phase composition (acidic or basic), RPLC with a pentafluorophenyl stationary phase, or mixed-mode HPLC with both bimodal and trimodal stationary phases. First, method performances were compared in terms of response rate (proportion of compounds eluted) and peak shapes for a large set of synthetic drugs (140) with structural diversity and their orthogonality was evaluated. Then a subset of compounds (25) containing varied impurity profiles was used to compare the methods based on the capability to detect impurities and evaluate the relative purity of the API.

Interest of achiral-achiral tandem columns for impurity profiling of synthetic drugs with supercritical fluid chromatography.

To achieve the most complete impurity profiling of synthetic drugs with a single chromatographic technique, high resolution is required, which may be gained with a combination of high efficiency and versatile selectivity, allowing to separate most similar analytes. Compared to a single-column chromatographic method, coupling complementary stationary phases promises both an increase in efficiency and an increase in selectivity possibilities. With supercritical fluid chromatography (SFC), the use of long columns is facilitated by the low viscosity of the mobile phase. In this paper, we investigate the interest of coupling two achiral stationary phases (Acquity UPC2 HSS C18 SB and Nucleoshell HILIC) that were previously observed to have excellent complementarity in SFC to carry out impurity profiling on 25 individual drug substances containing varied numbers and amounts of impurities. The single-column gradient methods are compared to tandem-column gradient methods with the two possible ordering of columns (C18 phase in first or second position) based on selectivity, peak capacity, sensitivity, UV-estimated purity of the active pharmaceutical ingredient and number of impurities detected with UV-estimated concentration >0.04%. It appears that it could be more beneficial to have two columns coupled in a single analysis than two consecutive methods with the single columns. The overall analysis time are nearly the same, but with more informative chromatograms in about 35% cases.

Comparison of ultra-high performance methods in liquid and supercritical fluid chromatography coupled to electrospray ionization - mass spectrometry for impurity profiling of drug candidates.

Impurity profiling of organic products synthesized as possible drug candidates represents a major analytical challenge. Complementary analytical methods are required to ensure that all impurities are detected. Both high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) can be used for this purpose. In this study, we compared ultra-high performance HPLC (UHPLC) and ultra-high performance SFC (UHPSFC) using a large dataset of 140 pharmaceutical compounds. Four previously optimized methods (two on each technique) were selected to ensure fast high-resolution separations. The four methods were evaluated based on response rate, peak capacity, peak shape and capability to detect impurities (UV). The orthogonality between all methods was also assessed. The best UHPLC method and UHPSFC methods provided comparable quality for the 140 compounds included in this study. Moreover, they were found to be highly orthogonal. At last, the potential of the combined use of UHPLC and UHPSFC for impurity profiling is illustrated with practical examples.

An improved classification of stationary phases for ultra-high performance supercritical fluid chromatography.

Supercritical fluid chromatography (SFC) has recently benefited of new instrumentation, together with the availability of many ultra-high performance columns (sub -2µm fully porous particles or sub -3µm superficially porous particles), rendering it more attractive than ever. Most of these columns commonly used in SFC were initially developed for HPLC use, with an increasing number of stationary phases specifically designed for SFC. While the availability of different stationary phase chemistries is an advantage to achieve successful SFC separations, selecting a column for method development remains difficult. For this reason, we have previously developed a classification of stationary phases dedicated to SFC use. It is based on linear solvation energy relationships (LSER) with Abraham descriptors (for neutral species). While current interest in SFC is strong in the pharmaceutical industry, the need to take account of interactions occurring with ionisable species is pressing. We have previously shown how a modified version of the solvation parameter model, adapted to take account of ionic and ionizable species, could be applied to the characterization of SFC systems. In the present paper, based on this modified LSER model, and on the analysis of 109 neutral and ionisable species, we propose an improved classification of 31 ultra-high performance stationary phases to facilitate method development with SFC.

Development of an achiral supercritical fluid chromatography method with ultraviolet absorbance and mass spectrometric detection for impurity profiling of drug candidates. Part I: Optimization of mobile phase composition.

Supercritical fluid chromatography (SFC) is a very useful tool in the purpose of impurity profiling of drug candidates, as an adequate selection of stationary phases can provide orthogonal separations so as to maximize the chances to see all impurities. The purpose of the present work is to develop a method for chemical purity assessment. The first part, presented here, focuses on mobile phase selection to ensure adequate elution and detection of drug-like molecules, while the second part focuses on stationary phase selection for optimal separation and orthogonality. The use of additives in the carbon dioxide - solvent mobile phase in SFC is now commonplace, and enables in particular to increase the number of eluted compounds and to improve peak shapes. The objective of this first part was to test different additives (acids, bases, salts and water) for their chromatographic performance assessed in gradient elution with a diode-array detector, but also for the mass responses obtained with a single-quadrupole mass detector, equipped with an electrospray ionization source (Waters ACQUITY QDa). In this project, we used a selection of one hundred and sixty compounds issued from Servier Research Laboratories to screen a set of columns and additives in SFC with a Waters ACQUITY UPC(2) system. The selected columns were all high-performance columns (1.7-1.8µm with totally porous particles or 2.6-2.7µm with superficially porous particles) with a variety of stationary phase chemistries. Initially, eight additives dissolved in the methanol co-solvent were tested on a UPC(2) ACQUITY UPC(2) HSS C18 SB column. A Derringer desirability function was used to classify the additives according to selected criteria: elution capability, peak shapes, UV baseline drift, and UV and mass responses (signal-to-noise ratios). Following these tests, the two best additives (ammonium acetate and ammonium hydroxide) were tested on a larger number of columns (10) where the two additives appeared to provide very comparable overall scores. However, ammonium acetate was selected for slightly better chromatographic quality. In a second step, we investigated the effects of ammonium acetate concentration (between 0 and 25mM in the methanol co-solvent) on retention and peak efficiency. Two types of silica supports were tested by working with ACQUITY UPC(2) HSS C18 SB and BEH columns. 20mM ammonium acetate in methanol with 2% water was finally selected as the best co-solvent composition.

Development of an achiral supercritical fluid chromatography method with ultraviolet absorbance and mass spectrometric detection for impurity profiling of drug candidates. Part II. Selection of an orthogonal set of stationary phases.

Impurity profiling of organic products that are synthesized as possible drug candidates requires complementary analytical methods to ensure that all impurities are identified. Supercritical fluid chromatography (SFC) is a very useful tool to achieve this objective, as an adequate selection of stationary phases can provide orthogonal separations so as to maximize the chances to see all impurities. In this series of papers, we have developed a method for achiral SFC-MS profiling of drug candidates, based on a selection of 160 analytes issued from Servier Research Laboratories. In the first part of this study, focusing on mobile phase selection, a gradient elution with carbon dioxide and methanol comprising 2% water and 20mM ammonium acetate proved to be the best in terms of chromatographic performance, while also providing good MS response [1]. The objective of this second part was the selection of an orthogonal set of ultra-high performance stationary phases, that was carried out in two steps. Firstly, a reduced set of analytes (20) was used to screen 23 columns. The columns selected were all 1.7-2.5µm fully porous or 2.6-2.7µm superficially porous particles, with a variety of stationary phase chemistries. Derringer desirability functions were used to rank the columns according to retention window, column efficiency evaluated with peak width of selected analytes, and the proportion of analytes successfully eluted with good peak shapes. The columns providing the worst performances were thus eliminated and a shorter selection of columns (11) was obtained. Secondly, based on 160 tested analytes, the 11 columns were ranked again. The retention data obtained on these columns were then compared to define a reduced set of the best columns providing the greatest orthogonality, to maximize the chances to see all impurities within a limited number of runs. Two high-performance columns were thus selected: ACQUITY UPC(2) HSS C18 SB and Nucleoshell HILIC.

MT3/QR2 melatonin binding site does not use melatonin as a substrate or a co-substrate.

Quinone reductase 2 (QR2, E.C. 1.10.99.2) is implicated in cell reactive oxygen species production. The catalytic activity of this enzyme is inhibited by 1 microM of melatonin. QR2 was identified as the third melatonin binding site (MT3). It is of major importance to understand the exact roles of melatonin and QR2 in oxidative stress. A fascinating possibility that melatonin could serve as a co-substrate or substrate of QR2 was hypothesized recently. In the current investigation, nuclear magnetic resonance studies of the QR2 catalytic reaction were performed, the results led us to conclude that, whatever the conditions, melatonin is not cleaved off to form N1-acetyl-N2-formyl-5-methoxykynurenine by a catalytically active QR2, very strongly indicating that melatonin is neither a substrate nor a co-substrate of this enzyme. Further studies are needed in order to better understand the relationship between MT3/QR2, melatonin and redox status of the cells, in order to better explain the anti-oxidant activities of melatonin at pharmacological concentrations (>1 microM).

New substrate analogues of human serotonin N-acetyltransferase produce in situ specific and potent inhibitors.

Melatonin is synthesized by an enzymatic pathway, in which arylalkylamine (serotonin) N-acetyltransferase catalyzes the rate-limiting step. A previous study reported the discovery of bromoacetyltryptamine (BAT), a new type of inhibitor of this enzyme. This compound is the precursor of a potent bifunctional inhibitor (analogue of the transition state), capable of interfering with both the substrate and the cosubstrate binding sites. This inhibitor is biosynthesized by the enzyme itself in the presence of free coenzyme A. In the present report, we describe the potency of new N-halogenoacetyl derivatives leading to a strong in situ inhibition of serotonin N-acetyltransferase. The new concept behind the mechanism of action of these precursors was studied by following the biosynthesis of the inhibitor from tritiated-BAT in a living cell. The fate of tritiated-phenylethylamine (PEA), a natural substrate of the enzyme, in the presence or absence of [(3)H]BAT was also followed, leading to their incorporation into the reaction product or the inhibitor (N-acetyl[(3)H]PEA and coenzyme A-S[(3)H]acetyltryptamine, respectively). The biosynthesis of this bifunctional inhibitor derived from BAT was also followed by nuclear magnetic resonance during its catalytic production by the pure enzyme. In a similar manner we studied the production of another inhibitor generated from N-[2-(7-hydroxynaphth-1-yl)ethyl]bromoacetamide. New derivatives were also screened for their capacity to inhibit a purified enzyme, in addition to enzyme overexpressed in a cellular model. Some of these compounds proved to be extremely potent, with IC(50)s of approximately 30 nM. As these compounds, by definition, closely resemble the natural substrates of arylalkylamine N-acetyltransferase, we also show that they are potent ligands at the melatonin receptors. Nevertheless, these inhibitors form a series of pharmacological tools that could be used to understand more closely the inhibition of pineal melatonin production in vivo.

Stereospecific synthesis of 5-substituted 2-bisarylthiocyclopentane carboxylic acids as specific matrix metalloproteinase inhibitors.

The synthesis and structure-activity relationship (SAR) studies of a series of cyclopentane carboxylic acid matrix metalloproteinase (MMP) inhibitors are described. Potent and specific MMP-2, -3, -9, -13 inhibitors were obtained by regio- and stereoselective substitutions at positions 2 and 5 on the cyclopentane ring. Compounds 2a and 2e are active in the mouse B16-F10 metastasis model and display very good pharmacokinetic parameters.