Applicability of supercritical fluid chromatography for oligonucleotide analysis: A proof-of-concept study.

We evaluated the suitability of supercritical fluid chromatography (SFC) for oligonucleotide analysis using 4-mer oligonucleotides with various phosphorothioate (PS) contents as model compounds. Column screening showed that the diol-modified column was able to separate sequences with different PS contents. Optimization of the column body and additives allowed us to analyze polar oligonucleotides using SFC. Various sequences were also analyzed using the optimized method. A good peak shape was obtained when the guanine plus cytosine content of the analyte was two or less in the 4-mer oligonucleotides. Furthermore, we found that the retention times of the selected sequences were positively correlated with polar surface areas, indicating that oligonucleotides interact with polar stationary phases. In contrast, more hydrophobic full PS sequences were retained more strongly in the diol column than the full phosphodiester (PO) sequences. This suggests that the diol column has unique selectivity for PO and PS linkages. These results indicate that SFC is potentially applicable to oligonucleotide analysis with a separation mechanism that is different from that of ion-pair reversed-phase liquid chromatography.

Facilitated on-line supercritical fluid extraction - supercritical fluid chromatography for nonpolar and polar compounds from milk thistle seeds.

Plant seeds, as those from milk thistle (Silybum marianum), are a valuable source of nonpolar and polar compounds with potentially interesting biological activity. The main nonpolar compounds are triglycerides, which are also the main components of all vegetable oils. In addition, specific polar compounds - flavonolignans, called silymarin, have been found in large amounts in milk thistle seeds extract. These flavonoids derivatives have different biological activity, for instance hepatoprotective effects. In order to extract and analyze both nonpolar (triglycerides) and polar compounds (flavonolignans) from milk thistle seeds through a sequential methodology, an on-line supercritical fluid extraction - supercritical fluid chromatography (SFE-SFC) method was developed. Different ways of transferring the extracts from SFE to SFC (i.e. direct on-column transfer and loop transfer) were compared, and particularly for their effect on chromatographic quality. In this respect, nonpolar and polar compounds caused different issues, especially as polar compounds required a significant portion of co-solvent in the extraction step, favoring early elution in the chromatographic column. First, on-line SFE-SFC was used for triglycerides analysis and allowed the comparison of transfer modes. Then, on-line kinetics were performed to measure defatting time before polar molecules extraction. Finally, the eventual benefit of loop transfer was also investigated for the analysis of flavonolignans, polar molecules whose analysis can be difficult by on-line SFE-SFC. The aim of this paper is to discuss the versatility of on-line SFE-SFC and how challenging the coupling can be, especially when both non-polar and polar molecules must be analyzed independently in a single sample.

Online SFE-SFC-MS/MS colony screening: A high-throughput approach for optimizing (-)-limonene production.

Conventional analysis of microbial bioproducers requires the extraction of metabolites from liquid cultures, where the culturing steps are time consuming and greatly limit throughput. To break through this barrier, the current study aims to directly evaluate microbial bioproduction colonies by way of supercritical fluid extraction-supercritical fluid chromatography-triple quadrupole mass spectrometry (SFE-SFC-MS/MS). The online SFE-SFC-MS/MS system offers great potential for high-throughput analysis due to automated metabolite extraction without any need for pretreatment. This is the first report of SFE-SFC-MS/MS as a method for direct colony screening, as demonstrated in the high-throughput screening of (-)-limonene bioproducers. Compared with conventional analysis, the SFE-SFC-MS/MS system enables faster and more convenient screening of highly productive strains.

Defining a generic column set for achiral supercritical fluid chromatography applied to pharmaceuticals or natural products.

When starting a method development in supercritical fluid chromatography (SFC), the first step is usually to screen several stationary phases based on previous experience or simply based on what is available in the laboratory. However, as there are now a large number of stationary phases available for SFC, the choice of an adequate set of columns to rapidly achieve a satisfying result can be difficult. In this project, 16 columns comprising a wide diversity of stationary phases and polarities ranging from the most polar (like bare silica gel) to the least polar (like octadecylbonded-silica) were compared, based on the gradient analysis of 129 probe compounds. The set mostly comprised active pharmaceutical ingredients, natural products and a few metabolites. The columns were ranked with the help of Derringer desirability functions taking account of (i) the number of compounds eluted from the column, (ii) the elution time in a suitable time frame, (iii) the average peak width, (iv) the average peak symmetry and (v) the spreading of retention along the gradient time. The five criteria selected showed no correlation. Overall, it appeared that those columns that had a high overall score were good for several reasons, like bare silica gel, propanediol-bonded silica or pentabromobenzyloxy-bonded silica. Initially, the columns had been screened with a gradient elution starting from 5% co-solvent and ending with 50% co-solvent in CO2. However, for some most retentive columns like amide-bonded silica, too many compounds remained non-eluted from the column. To examine this column more fairly, a second elution gradient was applied that ended with 100% co-solvent. This proved effective in restoring good overall performance through the elution of the most polar compounds.

A simple and rapid method to simultaneously analyze ciclesonide and its impurities in a ciclesonide metered-dose inhaler using on-line supercritical fluid extraction/supercritical fluid chromatography/quadrupole time-of-flight mass spectrometry.

A simple and rapid on-line SFE/SFC/quadrupole TOF-MS method to simultaneously analyze active pharmaceutical ingredients and impurities from metered-dose inhalers (MDIs) was developed using ciclesonide MDI (CIC-MDI) as an example. CIC-MDI, as drug Alvesco®, has been approved for the treatment of bronchial asthma, and its major impurities are listed in the European Pharmacopoeia and in the supplementary package inserts of Alvesco® (called as "Pharmaceutical interview form" in Japan). In the developed method, CIC-MDI was manually sprayed only once on a glass disc prior to the SFE/SFC/quadrupole TOF-MS. In the SFE, CIC and its impurities and other impurities having various polarities and hydrophobicity, were extracted in 3.5 min and subsequently separated on a CHIRALPAK IE-3 column to be detected by quadrupole TOF-MS in 6.5 min. This method would be applicable to the analysis of other inhalable pharmaceutical products whose sample preparation requires complicated procedures, as well as to the analysis of general pharmaceutical products for profiling impurities.

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.