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.

Online 2D-LC for Complex N-Glycan Analysis from Biopharmaceuticals.

EPO has a complex glycosylation pattern with differently branched and charged glycans. A combination of hydrophilic interaction chromatography (HILIC) with weak anion exchange chromatography (WAX) enables highly orthogonal separation. Comprehensive 2D-LC analysis with HILIC in the first and WAX in the second dimension provides high resolution 2D chromatography together with simultaneous charge profiling. Meanwhile, multiple heart-cutting 2D-LC analysis combining WAX and HILIC separation provides a flexible alternative whereby the user can select multiple peaks to be analyzed in the second dimension and, moreover, run longer gradients in the second dimension.

Ion exchange in supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS): Application for polar and ionic drugs and metabolites in forensic and anti-doping analysis.

Supercritical fluid chromatography (SFC) is an orthogonal technique to UHPLC. In recent years, SFC has demonstrated potential for use in the analysis of a broad variety of analytes of different polarities, if modifiers and additives are utilized as additional mobile phase constituents. However, to date, little research has been carried out on ion-exchange separation of highly polar and ionic analytes using SFC. The objective of this work was to investigate the elution characteristics of polar compounds using SFC combined with tandem mass spectrometry. Highly polar and even ionic drugs and metabolites, with a diversity of functional groups such as gamma-hydroxybutyrate (GHB), gamma-butyrolactone, GHB-glucuronide, ethyl sulfate, ethyl glucuronide as well as meldonium and gamma-butyrobetaine, were selected for the study. To investigate the chromatographic behavior of the solutes using SFC, a systematic chromatographic method development workflow including a basic validation in human urine was implemented. To ensure the best selectivity, columns with different stationary phase chemistries (silica, NH2, CN, SCX, Diol, EP and amide) were screened. Furthermore, different modifier compositions were evaluated, including pure methanol and methanol with various additives (ammonium acetate or ammonium formate, water, and/or ammonia or formic acid in varying amounts). Trends in retention time shifts were investigated by the systematic variation of gradients, backpressure and column compartment temperature. The quality and reliability of the method has been tested in the course of a basic validation by evaluation of selectivity, linearity, limit of identification, limit of quantification, carry-over, precision, and matrix effect. The highest chromatographic selectivity (especially with respect to the separation of the critical pair of zwitterionic meldonium and γ-butyrobetaine) and suitable peak shapes for all target analytes were obtained using the strong cation exchange column. We observed that increasing buffer concentrations improved the peak shape. Ion-exchange stationary phase and polar additives (buffers, bases, acids and water) mixed with organic modifiers enabled separation of small ionic molecules using SFC. Hence, SFC further demonstrated its wide applicability in small molecule analysis, and may be considered an alternative separation technique to improve the separation of polar analytes.

SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control.

HPLC is considered the method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact insufficiently on conventional reversed-phase (RP) columns. Especially in combination with mass spectrometric detection, limitations apply for alterations of stationary phases. Some highly polar sympathomimetic drugs and their metabolites showed almost no retention on different RP columns. Their retention remains poor even on phenylhexyl phases that show different selectivity due to π-π interactions. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. All compounds showed sharp peaks and good retention even for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations as a result of the orthogonality. Short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, precise regulation of temperature and backpressure is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced. Graphical Abstract SFC-MS/MS vs. LC-MS/MS.