Gemini NX Research Articles

Design of experiments as a tool for LC–MS/MS method development for the trace analysis of the potentially genotoxic 4-dimethylaminopyridine impurity in glucocorticoids

The present study reports on a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method development strategy supported by design of experiments (DoE) for the trace analysis of 4-dimethylaminopyridine (DMAP). The conventional approaches for development of LC-MS/MS methods are usually via trial and error, varying intentionally the experimental factors which is time consuming and interactions between experimental factors are not considered. The LC factors chosen for the DoE study include flow (F), gradient (G) and injection volume (V(inj)) while cone voltage (E(con)) and collision energy (E(col)) were chosen as MS parameters. All of the five factors were studied simultaneously. The method was optimized with respect to four responses: separation of peaks (Sep), peak area (A(peak)), length of the analysis (T) and the signal to noise ratio (S/N). A quadratic model, namely central composite face (CCF) featuring 29 runs was used instead of a less powerful linear model since the increase in the number of injections was insignificant. In order to determine the robustness of the method a new set of DoE experiments was carried out applying robustness around the optimal conditions was evaluated applying a fractional factorial of resolution III with 11 runs, wherein additional factors - such as column temperature and quadrupole resolution - were considered. The method utilizes a Phenomenex Gemini NX C-18 HPLC analytical column with electrospray ionization and a triple quadrupole mass detector in multiple reaction monitoring (MRM) mode, resulting in short analyses with a 10min runtime. Drawbacks of derivatization, namely incomplete reaction and time consuming sample preparation, have been avoided and the change from SIM to MRM mode resulted in increased sensitivity and lower LOQ. The DoE method development strategy led to a method allowing the trace analysis of DMAP at 0.5 ng/ml absolute concentration which corresponds to a 0.1 ppm limit of quantification in 5mg/ml mometasone furoate glucocorticoid. The obtained method was validated in a linear range of 0.1-10 ppm and presented a %RSD of 0.02% for system precision. Regarding DMAP recovery in mometasone furoate, spiked samples produced %recoveries between 83 and 113% in the range of 0.1-2 ppm.

Lipophilicity assessment of basic drugs (log Po/w determination) by a chromatographic method

A previously reported chromatographic method to determine the 1-octanol/water partition coefficient (log P o/w) of organic compounds is used to estimate the hydrophobicity of bases, mainly commercial drugs with diverse chemical nature and p K a values higher than 9. For that reason, mobile phases buffered at high pH to avoid the ionization of the solutes and three different columns (Phenomenex Gemini NX, Waters XTerra RP-18 and Waters XTerra MS C 18) with appropriate alkaline-resistant stationary phases have been used. Non-ionizable substances studied in previous works were also included in the set of compounds to evaluate the consistency of the method. The results showed that all the columns provide good estimations of the log P o/w for most of the compounds included in this study. The Gemini NX column has been selected to calculate log P o/w values of the set of studied drugs, and really good correlations between the determined log P o/w values and those considered as reference were obtained, proving the ability of the procedure for the lipophilicity assessment of bioactive compounds with very different structures and functionalities.

High-throughput toxicological analysis of propofol in human whole blood by LC-MS

A new high-throughput liquid chromatography / mass spectrometry (LC-MS) method for the quantification of propofol in human whole blood was developed and validated. The separation was performed on a Gemini NX column under isocratic conditions using a mixture of 0.05 % ammonium hydroxide in water and methanol (23:77, v/v) with a flow rate of 0.6 mL/min. The detection was performed in SIM mode using an ion trap MS equipped with an APCI ion source using negative mode ionization. The whole blood samples (0.2 mL) were deproteinated with methanol before the analysis. The method showed a good linearity (r > 0.997), precision and accuracy (< 10.2%) over the concentration ranges of 0.05 -1.2 μg/mL. The method is not expensive, requires a minimum time for sample preparation and has a run-time of 1.5 min for instrument analysis (retention times for propofol was 0.78 min). The developed and validated LC-MS method is very simple and more rapid than other similar methods. It was successfully applied for the rapid quantification of propofol in human whole blood in forensic toxicology analyses.

Kinetic performance of reversed-phase C18 high-performance liquid chromatography columns compared by means of the Kinetic Plot Method in pharmaceutically relevant applications

Four fully porous C18 columns (Hypersil Gold, ACE3, Xbridge and Gemini NX), widely employed in the pharmaceutical industry, were compared in terms of efficiency and analysis speed with the Kinetic Plot Method. Weakly basic, medium-sized, N-containing pharmaceutical compounds from GlaxoSmithKline Research and Development were used as test molecules. Isocratic elution was carried out at pH 4.5 and pH 8.0 with acetonitrile as organic modifier. The columns under evaluation included highly pure silica supports (Hypersil Gold, ACE3) and hybrid polymer-silica supports (XBridge, Gemini NX). Both types of columns claim for nearly absent secondary interactions with ionized silanol groups and are therefore applicable in a wide pH range. This is an important feature for method development purposes in pharmaceutical industries. The Kinetic Plot Method was used to compare the support characteristics and assess the kinetic performance of the columns in different experimental conditions. Although the evaluated columns have roughly identical particle diameters (from 3.0 to 3.5 μm) according to their manufacturers, large differences in kinetic performance were observed at pH 4.5 that can be accounted for by different flow resistances, porosities and average particle diameters, experimentally determined from scanning electron microscopy and laser light scattering experiments on loose stationary phase material. The ACE3 column was the best performing support among the evaluated columns, due to its excellent efficiency and average flow resistance. The better performance of the ACE3 column was due to its better packing quality, as could be derived from its impedance plot. Kinetic plots of resolution of a critical pair versus analysis time and column length were established at pH 8.0. These plots can be used as a method development tool to tailor the separation conditions to the required resolution of a given critical pair, combining efficiency and selectivity features of the column.