Trace level impurity method development with high-field asymmetric waveform ion mobility spectrometry: systematic study of factors affecting the performance.

For the determination of trace level impurities, analytical chemists are confronted with complex mixtures and difficult separations. New technologies such as high-field asymmetric waveform ion mobility spectrometry (FAIMS) have been developed to make their work easier; however, efficient method development and troubleshooting can be quite challenging if little prior knowledge of the factors or their settings is available. We present the results of an investigation performed in order to obtain a better understanding of the FAIMS technology. The influence of eight factors (polarity of dispersion voltage, outer bias voltage, total gas flow rate, composition of the carrier gas (e.g. %He), outer electrode temperature, ratio between the temperatures of the inner and outer electrodes, flow rate and composition of the make-up mobile phase) was assessed. Five types of responses were monitored: value of the compensation voltage (CV), intensity, width and asymmetry of the compensation voltage peak, and resolution between two peaks. Three types of studies were performed using different test mixtures and various ionisation modes to assess whether the same conclusions could be drawn across these conditions for a number of different types of compounds. To extract the maximum information from as few experiments as possible, a Design of Experiment (DoE) approach was used. The results presented in this work provide detailed information on the factors affecting FAIMS separations and therefore should enable the user to troubleshoot more effectively and to develop efficient methods.

The use of shift reagents in ion mobility-mass spectrometry: studies on the complexation of an active pharmaceutical ingredient with polyethylene glycol excipients.

Gas-phase ion mobility studies of mixtures containing polyethylene glycols (PEG) and an active pharmaceutical ingredient (API), lamivudine, have been carried out using electrospray ionization-ion mobility spectrometry-quadrupole-time-of-flight mass spectrometry (ESI-IMS-Q-TOF). In addition to protonated and cationized PEG oligomers, a series of high molecular weight ions were observed and identified as noncovalent complexes formed between lamivudine and PEG oligomers. The noncovalent complex ions were dissociated using collision induced dissociation (CID) after separation in the ion mobility drift tube to recover the protonated lamivudine free from interfering matrix ions and with a drift time associated with the precursor complex. The potential of PEG excipients to act as "shift reagents," which enhance selectivity by moving the mass/mobility locus to an area of the spectrum away from interferences, is demonstrated for the analysis of lamivudine in a Combivir formulation containing PEG and lamivudine.

Development, validation and transfer into a factory environment of a liquid chromatography tandem mass spectrometry assay for the highly neurotoxic impurity FMTP (4-(4-fluorophenyl)-1-methyl-1,2,3,6-tetrahydropyridine) in paroxetine active pharmaceutical ingredient (API).

This work describes the development of a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for a highly toxic impurity, FMTP (4-(4-fluorophenyl)-1-methyl-1,2,3,6-tetrahydropyridine), in paroxetine active pharmaceutical ingredient (API), followed by the subsequent validation of the methodology and transfer into a global production/quality control environment. The method was developed to achieve a detection limit of 10ppb mass fraction of FMTP in paroxetine API. An LC-MS/MS method was chosen because it provided the required sensitivity and selectivity with minimal sample preparation. This paper discusses the issues with transferring such complex methodology to a production environment. Linearity, repeatability and reproducibility of the method were demonstrated. This work shows that it is possible using the same approach that would be used for the transfer of any analytical method from R&D to a manufacturing environment.

Investigation of the advanced functionalities of a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer.

Time-of-flight mass spectrometry (ToF-MS) has gained wide acceptance in many fields of chemistry, proteomics, metabolomics and small molecule analysis. ToF-MS, however, has some inherent advantages and drawbacks. Numerous developments have been made to hybrid ToF instruments to improve their capabilities. We have used a quadrupole orthogonal acceleration ToF (Q-oa-ToF) instrument to assess developments made to improve resolution, dynamic range and signal-to-noise (S/N) ratios (i.e. sensitivity). Higher mass resolution can improve the analysis of mixtures containing compounds with similar m/z values and improved mass accuracy gives greater confidence for structural elucidation applications. Wide dynamic ranges are necessary for the analysis of unknown samples or samples that vary widely in analyte concentrations. The performance of the advanced functionalities for routine structural elucidation in terms of resolution, dynamic range and S/N ratios was investigated using test compounds. The results presented in this work demonstrate and validate the use of these new enhancements for Q-ToF instruments and also show their limitations.

Evaluating the utility of ion mobility separation in combination with high-pressure liquid chromatography/mass spectrometry to facilitate detection of trace impurities in formulated drug products.

Many formulated products contain complex polymeric excipients such as polyethylene glycols (PEGs). Such excipients can be readily ionized by electrospray and may be present at very high concentrations, thus making it very difficult to identify trace level impurities such as degradants in samples, even if hyphenated techniques such as liquid chromatography/mass spectrometry (LC/MS) are used. Ion mobility (IM) spectrometry is a very rapid gas-phase separation technique and offers additional separation capability within the LC timeframe. This work investigates the use of an IM separator in combination with high-pressure liquid chromatography (HPLC) and MS, to improve the separation of drug-related materials from excipients, thus aiding the identification of trace-level impurities in an anti-HIV medication, Combivir.

Investigation into the factors affecting accuracy of mass measurements on a time-of-flight mass spectrometer using Design of Experiment.

The results of an investigation of the parameters which have the most significant effect on the accuracy of mass measurements on a quadrupole orthogonal acceleration time-of-flight mass spectrometer (q-oaToF) are reported. The influence of eight factors is investigated: ion abundances of reference and analyte compounds, mass difference between analyte and reference compounds, quality of calibration, number of reference acquisitions averaged and TDC (time-to-digital converter) settings (resolution, Np multiplier (number of pushes correction factor), minimum number of points, i.e. minimum acquisition width which defines a peak). To extract the maximum information from as few experiments as possible, a Design of Experiment approach was used. The data will be used as a basis for developing guidance on accurate mass measurement on q-oaToF instruments.

'On-the-fly' hydrogen/deuterium exchange liquid chromatography/mass spectrometry using a dual-sprayer atmospheric pressure ionisation source.

In-source 'on-the-fly' hydrogen/deuterium (H/D) exchange liquid chromatography mass spectrometry (LC/MS) has been investigated. The work was performed using a dual-sprayer source. The analyte was introduced through an electrospray ionisation sprayer and D2O was introduced through an atmospheric pressure chemical ionisation sprayer. To achieve H/D exchange sufficient to determine the number of exchangeable H atoms of a compound, a saturated 'steady-state' D2O atmosphere had to be created in the ion source by having a 2:1 or higher D2O-to-analyte flow rate ratio. Under these conditions H/D exchange levels of 32-90% were achieved. In most cases the H/D exchange was sufficient to measure the number of exchangeable H atoms in some antiulcerative and anthelmintic pharmaceuticals. The concept of in-source 'on-the-fly' H/D exchange by introducing the deuterating agent via a second sprayer has been shown. It allows the integrity of the chromatographic separation to be kept, since the H/D exchange takes place post-separation.