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.

Interlaboratory study of a supercritical fluid chromatography method for the determination of pharmaceutical impurities: Evaluation of multi-systems reproducibility.

Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an 'instrument type' effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis.

Characterization of stationary phases in supercritical fluid chromatography including exploration of shape selectivity.

Achiral packed column supercritical fluid chromatography (SFC) has shown an important regain of interest in academic and industrial laboratories in the recent years. In relation to this increased concern, major instrument manufacturers have designed some stationary phases specifically for SFC use. SFC stationary phases have been widely examined over the last two decades, based on the use of linear solvation energy relationships (LSER), which relate analyte retention to its properties and to the interaction capabilities of the chromatographic system. The method provides some understanding on retention mechanisms (normal phase, reversed phase or mixed-mode) and the possibility to compare stationary phases on a rational basis, especially through a spider diagram providing a visual classification. The latter can be used as a primary tool to select complementary stationary phases to be screened for any separation at early stages of method development, before optimization steps. In this context, the characterization of the 14 columns from the Shim-pack UC series (Shimadzu Corporation, Kyoto, Japan), which are dedicated to SFC and more broadly to unified chromatography (UC), was performed, using the LSER methodology. As in previous works, seven descriptors, including five Abraham descriptors (E, S, A, B, V) and two descriptors describing positive and negative charges (D- and D+) were first employed to describe interactions with neutral and charged analytes. Secondly, two more descriptors were introduced, which were previously employed solely for the characterization of enantioselective systems and expressing shape features of the analytes (flexibility F and globularity G). They brought additional insight into the retention mechanisms, showing how spatial insertion of the analytes in some stationary phases is contributing to shape separation capabilities and how folding possibilities in flexible molecules is unfavorable to retention in other stationary phases.

Chromatographic analysis of biomolecules with pressurized carbon dioxide mobile phases - A review.

Biomolecules like proteins, peptides and nucleic acids widely emerge in pharmaceutical applications, either as synthetic active pharmaceutical ingredients, or from natural products as in traditional Chinese medicine. Liquid-phase chromatographic methods (LC) are widely employed for the analysis and/or purification of such molecules. On another hand, to answer the ever-increasing requests from scientists involved in biomolecules projects, other chromatographic methods emerge as useful complements to LC. In particular, there is a growing interest for chromatography with a mobile phase comprising pressurized carbon dioxide, which can be named either (i) supercritical (or subcritical) fluid chromatography (SFC) when CO2 is the major constituent of the mobile phase, or (ii) enhanced fluidity liquid chromatography (EFLC) when hydro-organic or purely organic solvents are the major constituents of the mobile phase. Despite the low polarity of CO2, supposedly inadequate to solubilize such biomolecules, SFC and EFLC were both employed in many occasions for this purpose. This paper specifically reviews the literature related to the SFC/EFLC analysis of free amino acids, peptides, proteins, nucleobases, nucleosides and nucleotides. The analytical conditions employed for specific molecular families are presented, with a focus on the nature of the stationary phase and the mobile phase composition. We also discuss the potential benefits of combining SFC/EFLC to LC in a single gradient elution, a method sometimes designated as unified chromatography (UC). Finally, detection issues are presented, and more particularly hyphenation to mass spectrometry.

Analysis of flavonoids with unified chromatography-electrospray ionization mass spectrometry-method development and application to compounds of pharmaceutical and cosmetic interest.

In this project, we aimed at analysing flavonoid-type compounds with unified chromatography (joining supercritical fluid chromatography and enhanced fluidity liquid chromatography with carbon dioxide-methanol mobile phases covering a wide range of compositions) and diode-array and electrospray ionization mass spectrometric detection (UC-DAD-ESI-MS). First, the chromatographic method was developed for 9 standard flavonoid molecules from three different families (flavanols, flavanones and flavonols, glycosylated or not), with a strong focus on mobile phase composition to achieve the elution of a wide range of flavonoids with good chromatographic quality (efficiency and resolution). For this purpose, two stationary phases were selected (ACQUITY UPC2 DEA and Diol), and five different additives (formic acid, citric acid, phosphoric acid, methanesulfonic acid and ammonium hydroxide) were successively introduced in the methanol co-solvent. The composition containing 0.1% methanesulfonic acid in methanol was retained as it provided the best chromatographic quality together with the possibility of hyphenating the chromatography to mass spectrometry. The DEA column appeared to provide the best efficiency and was retained for further method development. The gradient method was then optimized to achieve a fast analysis, which involved elution with a wide range of mobile phase composition (from 20 to 100% co-solvent in methanol) together with reversed flow rate and reversed pressure gradients at fixed temperature. The final gradient lasted 10 min, followed by 2.5 min of re-equilibration. Then, ESI-MS detection was optimized. Because the single-quadrupole mass spectrometer employed (ACQUITY UPC2 QDa) allowed the variation of only a few parameters, a design of experiments was used to define the best compromise for three parameters (probe temperature, cone voltage and capillary voltage). The make-up fluid introduced before entering the MS was also varied: different compositions of methanol-water containing either formic acid, ammonium hydroxide or sodium chloride were tested. The best results in terms of signal-to-noise ratio were obtained with methanol containing 20 mM ammonium hydroxide and 2% water. The optimal UC-DAD-ESI-MS method was then applied to two different flavonoid formulation ingredients. The first one, hidrosmin (5-O-(ß-hydroxyethyl)diosmin), is known for its vasoprotective properties and therefore employed in pharmaceutical formulations. The second one, α-glucosyl-hesperidin (sometimes referred to as vitamin P), is employed in cosmetic formulations. Identification of the major compounds in each sample was achieved with the help of MS detection. Graphical abstract.