RESUMEN

The validity of the extended Tanaka column characterization procedure against the retention behavior of 101 analytes of widely differing properties chromatographed on five differing stationary phase chemistries has been established using a chemometric technique called principal component analysis (PCA). It was concluded that the simple and conveniently determined column characterization parameters covered the same space in the PCA loading plot as the retention times for the 101 differing analytes. This confirms that the ten column characterization parameters of the extended Tanaka protocol encode the same information as the retention times of the 101 analytes. Significant selectivity differences were observed between stationary phases and the mobile-phase modifiers - MeOH and MeCN. PCA contribution plots served as a convenient way to highlight specific selectivity differences between stationary phases. logD values exhibited a poor correlation with retention indicating that retention in RP-LC is not solely dictated by the analyte's hydrophobicity. The use of MeOH was found to generate greater selectivity differences with the five stationary phases than when MeCN is used.

RESUMEN

A strategy for rapid optimization of liquid chromatography column temperature and gradient shape is presented. The optimization as such is based on the well established retention and peak width models implemented in software like e.g. DryLab and LC simulator. The novel part of the strategy is a highly automated processing algorithm for detection and tracking of chromatographic peaks in noisy liquid chromatography-mass spectrometry (LC-MS) data. The strategy is presented and visualized by the optimization of the separation of two degradants present in ultraviolet (UV) exposed fluocinolone acetonide. It should be stressed, however, that it can be utilized for LC-MS analysis of any sample and application where several runs are conducted on the same sample. In the application presented, 30 components that were difficult or impossible to detect in the UV data could be automatically detected and tracked in the MS data by using the proposed strategy. The number of correctly tracked components was above 95%. Using the parameters from the reconstructed data sets to the model gave good agreement between predicted and observed retention times at optimal conditions. The area of the smallest tracked component was estimated to 0.08% compared to the main component, a level relevant for the characterization of impurities in the pharmaceutical industry.

Asunto(s)

RESUMEN

A method for tracking of sample components during liquid chromatography-mass spectrometry (LC-MS) method development has been proposed. The method manages to, fully automatically and without user intervention, find the chromatographic peaks in the data sets, discriminate them to sample components and track them when the separation conditions have been changed. The algorithm utilises the resolution obtained from all considered data sets and has the ability to discriminate the non informative parts. The technique has a great sensitivity even in cases where a majority of the tracked components cannot easily be spotted by means of traditional total ion chromatogram (TIC) or base peak chromatogram (BPC) representations. The method was tested on an experimental sample using six different columns and an average of 79% of the suggested sample components could be successfully tracked at a minimum area of 0.05% of the main component in the sample. 66 components with 79-92% of the total suggested component area were able to be tracked between all data sets. The method could be used to rapidly investigate selectivity during different types of separation conditions.

Asunto(s)

RESUMEN

Electrospray ionization performs best with volatile buffers. However, generally the best separation performance for capillary electrophoresis (CE) is achieved with non-volatile buffers. Hyphenation of CE with mass spectrometry (MS) utilizing atmospheric pressure photoionization (APPI) enables use of a wider range of separation buffers without compromising detection sensitivity. As APPI is considered to be mass flow sensitive, the use of a larger inner diameter separation capillary (75 microm) allows larger volumes to be injected, without decreased separation performance, thus providing improved sensitivity (approx. a factor of 10), compared to the use of a 25 microm capillary. However, nebulizing gas flow and position of capillary tip in the sprayer have to be carefully optimized to prevent excessive band broadening. Further improvement in sensitivity (approx. a factor of 2) was obtained by decreasing the distance between the sprayer and ionization region, indicating that a specially designed CE/APPI-MS interface for low flow rates will be favourable.

Asunto(s)

RESUMEN

A highly automated procedure for localising and characterising peaks in the chromatographic time domain of LC-MS data has been developed. The work was initiated by an identified need to facilitate the detection and tracking of chromatographic peaks during method development for the analysis of impurities in pharmaceutical products. The algorithm is mainly based on a digital filter for which the settings are automatically adapted to the data set under study. The procedure was evaluated for synthetic data sets with various S/N levels, peak widths and baseline properties. It was found that even for the worst case tested with S/N=10 and a high variability in the baseline, 94% of the simulated analytical peaks could be detected without producing any false-positive identifications. Furthermore, the number of correctly estimated peak heights and peak widths falling within a 10% error of the true values were 94 and 91%, respectively. For experimental data sets, peak height, and width estimations were more difficult, but the processed reconstructions showed an excellent agreement with the analytical signals of the raw data, and also a clearly improved visualisation in total ion- and base-peak chromatograms.

Asunto(s)

RESUMEN

Recently, we introduced a new approach to chiral separation and analysis of amino acids by chiral complexation and electrospray high-field asymmetric waveform ion mobility spectrometry coupled to mass spectrometry (ESI-FAIMS-MS). In the present work, we extended this approach to the separation of the drug compound terbutaline. Terbutaline enantiomers were complexed with metal ions and an amino acid to form diastereomeric complexes of the type [M(II)(L-Ref)2((+)/(-)-A)-H](+), where M(II) is a divalent metal ion, L-Ref is an amino acid in its L-form, and A is the terbutaline analyte. When metal and reference compound were suitably chosen, these complexes were separable by FAIMS. We also detected and characterized larger clusters that were transmitted at distinct FAIMS compensation voltages (CV), disturbing data analysis by disintegrating after the FAIMS separation and forming complexes of the same composition [M(II)(L-Ref)2((+)/(-)-A)-H](+), thus giving rise to additional peaks in the FAIMS CV spectra. This undesired phenomenon could be largely avoided by adjusting the mass spectrometer skimmer voltages in such a way that said larger clusters remained intact. In the quantitative part of the present work, we achieved a limit of detection of 0.10% (-)-terbutaline in a sample of (+)-terbutaline. The limit of detection and analysis time per sample compared favorably to literature values for chiral terbutaline separation by HPLC and CE.

Asunto(s)

RESUMEN

Four data pre-processing methods have been applied with different settings to data sets obtained from the analysis of a pharmaceutical drug and its degradation products by liquid chromatography-mass spectrometry (LC-MS). The methods compared were the frequently used component detection algorithm (CODA) and three kinds of digital filters--matched filtration (MF), Gaussian second derivative (GSD) and Savitzky-Golay. The aim was to evaluate the performance and robustness of these methods for extracted ion chromatogram (XIC), total ion chromatogram (TIC) and base peak chromatogram (BPC) in the presence of different types of noise. In accordance with theory, the best improvements in signal-to-noise ratio (S/N) of the XICs were obtained with MF under the ideal case with random white noise. However, when highly coloured noise was present, it was found that no improvements in XIC S/N could be obtained with any of the pre-processing methods studied. GSD and CODA did, however, improve the S/N for both TIC and BPC. GSD and CODA also significantly reduced the background in the spectral domain, thereby facilitating the interpretation of the mass spectra. Another advantage associated with CODA and to some extent also with GSD is their data reduction ability.

Asunto(s)

RESUMEN

In this study, peak-broadening effects caused by nebulizing gas flow and lack of temperature control have been investigated for separation capillaries with three different inner diameters. The study was performed with serial UV/ESI-MS detection in an effort to distinguish between peak broadening arising in the separation and peak broadening arising in the ion source. The nebulizing gas was found to significantly affect both migration time and separation efficiency when using capillaries with 50 and 75 microm id. If the nebulizing gas is on during injection, the injection volume increases to such an extent that significant peak broadening is induced. Reducing the id to 25 microm minimizes the parabolic flow induced by the nebulizing gas. Results indicate that the nebulizing gas pressure can be optimized to minimize peak broadening in the ion source. A decrease in detection sensitivity, possibly related to the orthogonal design of the interface, was observed when the nebulizing gas pressure was increased. A tapered capillary tip was found to provide superior separation efficiency as well as sensitivity.

Asunto(s)

RESUMEN

The airway retention of inhaled glucocorticosteroids (GCs) depends largely on their lipophilicity. Inhaled budesonide (BUD) becomes highly lipophilic reversibly by the formation of esters acting as a reservoir of active BUD. Ciclesonide (CIC) was also reported to form esters after hydrolysis to active metabolite (CIC-AM). We have investigated lipophilicity and airway retention of BUD, CIC/CIC-AM, fluticasone propionate (FP), and mometasone furoate (MF), and compared esterification of BUD and CIC-AM and its contribution to GC airway retention. Rat tracheas were preincubated with the esterification inhibitor cyclandelate or vehicle. A (3)H-GC ( approximately 10(-7) M: BUD, CIC, CIC-AM, FP, MF) was added for 20 min. After incubation, one half of the trachea was used for analysis of GC uptake and the other to analyze GC release during 3 h in drug-free medium. GC species in trachea halves were analyzed by radiochromatography. At 20 min, the uptake of BUD was similar to that of CIC/CIC-AM; however, the BUD-ester pool was 9-fold greater (p < 0.01). BUD overall retention in trachea at 3 h was greater than that of other GCs (p < 0.01), and the BUD-ester pool was 3-fold greater than the CIC-AM-ester pool (p < 0.01). Cyclandelate decreased the initial BUD- and CIC-AM-ester pools (p < 0.01), and reduced the overall retention of BUD at 3 h (p < 0.01) but not of CIC-AM. Thus, BUD becomes esterified in the airways more promptly and to a greater extent than CIC-AM, and BUD esterification prolongs BUD airway retention. In contrast, airway retention of CIC-AM and CIC seems to be determined mainly by their lipophilicity, similar to FP and MF, which are not esterified.

Asunto(s)

RESUMEN

We present a new method for separation of enantiomers with high-field asymmetric waveform ion mobility spectrometry (FAIMS), coupled to mass spectrometric detection. Upon addition of an appropriate chiral reference compound to the analyte solution and subsequent ionization of the solution by electrospray ionization, analyte enantiomers formed diastereomeric complexes, which were potentially separable by FAIMS. The methodology being developed is intended to be general, but here amino acid analytes are specifically considered. In the examples presented herein, six pairs of amino acid enantiomers were successfully separated as metal-bound trimeric complexes of the form [MII(L-Ref)2(D/L-A)-H]+, where MII is a divalent metal ion, L-Ref is an amino acid in its L form acting as chiral reference compound, and A is the amino acid analyte. For example, D- and L-tryptophan were separated in FAIMS as [NiII(L-Asn)2(D-Trp)-H]+ and [NiII(L-Asn)2(L-Trp)-H]+. As FAIMS separation typically takes place over a time scale of only a few hundred milliseconds, the presented separation method opens new possibilities for rapid analysis of one analyte enantiomer in the presence of the other enantiomer. Preliminary quantification results are presented, which suggest that fast and sensitive quantitative chiral analyses can be performed with FAIMS. Method limitations are discussed in terms of diverse phenomena, which are not yet understood.

Asunto(s)