Assessment of atmospheric solids analysis probe as a tool for the rapid determination of drug purity.

The ability to determine the purity (% controlled compound) of drug-of-abuse samples is necessary for public health and law enforcement. Here, we describe the assessment of atmospheric solids analysis probe (ASAP) for the rapid determination of drug purity for a set of formulated pharmaceuticals, chosen due to their availability, uncontrolled status and consistency. Paracetamol and loratadine were used as models of high and low purity compounds being ~90% and ~10% active ingredient, respectively. Individual tablets were ground up and diluted in an internal standard solution. The resulting samples were analysed by ASAP coupled to a Waters QDa mass spectrometer followed by confirmatory testing by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The inclusion of a non-matched internal standard (quinine) improved linearity and repeatability of drug analysis by ASAP-MS. Levels of drug purity using formulated pharmaceutical tablets were found to be highly comparable with results produced by the 'gold standard' LC-MS/MS technique. Rapid determination of drug purity is therefore possible with ASAP-MS for highly concentrated samples with minimal sample preparation. It may be possible to use this deployable system to determine drug purity outside of a laboratory setting.

Investigating microwave hydrolysis for the traceable quantification of peptide standards using gas chromatography-mass spectrometry.

Over the past decade, a number of endogenous peptides and endogenous peptide analogs have been employed in therapeutics and as diagnostic markers. The use of peptides as standards for the absolute quantification of proteins has become commonly accepted. Consequently, the requirement for standard peptides traceable to the International System of Units with low associated measurement uncertainty, and for accurate methods of peptide quantification, has increased. Here we describe a method of peptide quantification involving microwave-assisted acid hydrolysis followed by gas chromatography-mass spectrometry that enables traceable quantification of a peptide by exact matching isotope dilution mass spectrometry where the total hydrolysis time required is only 3h. A solution of angiotensin I was quantified using this method, and the results were in agreement with those obtained previously using an oven hydrolysis liquid chromatography-tandem mass spectrometry method.

On-line reaction monitoring by extractive electrospray ionisation.

The design and development of a novel extractive electrospray ionisation (EESI) device for on-line reaction monitoring is described. The EESI apparatus uses a secondary, grounded nebuliser to produce an analyte aerosol and a Venturi pump is then used to transfer a sample of the aerosol to an electrospray source where it is ionised. The EESI apparatus was then tested with a variety of small, organic molecules to assess sensitivity, linearity and dynamic range. The performance of the technique will depend on the mass spectrometer used for the experiments; in the configurations used here it has a usable dynamic range of around 3.5 orders of magnitude with a linear range of around 2.5 orders of magnitude and is capable of analysing species present down to low µg/mL with signal-to-noise ratio greater than 2.5. The use of EESI for reaction monitoring was validated using a series of mock reaction mixtures and then used to monitor the base hydrolysis of ethyl salicylate to salicylic acid.

Atmospheric Solids Analysis Probe Coupled to a Portable Mass Spectrometer for Rapid Identification of Bulk Drug Seizures.

The emergence of ambient ionization techniques and their combination with smaller, cheaper mass spectrometers is beginning to make real the possibility of mass spectrometry measurements being made routinely outside of traditional laboratory settings. Here, we describe the development of an atmospheric solids analysis probe (ASAP) source for a commercially available miniaturized, single-quadrupole mass spectrometer and subsequent modification of the instrument to allow it to run as a deployable system; we further go on to describe the application of this instrument to the identification of the contents of drug seizures. For the drug seizure analysis, a small quantity of the material (powder, tablet, resin, etc.) was dissolved in ethanol and shaken to extract the analytes, the resulting solutions were then sampled by dipping a sealed glass capillary into the solution prior to analysis by ASAP-MS. Identification of the contents of the seizures was carried out using a NIST searching approach utilizing a bespoke spectral library containing 46 compounds representative of those most commonly encountered in UK forensic laboratories. In order to increase confidence in identification the library sample and subsequent analyses were carried out using a four-channel acquisition method; each channel in this method used a different cone voltage (15, 30, 50, and 70 V) inducing differing levels of in-source fragmentation in each channel; the match score across each channel was then used for identification. Using this developed method, a set of 50 real-life drug samples was analyzed with each of these being identified correctly using the library searching method.

High accuracy determination of malachite green and leucomalachite green in salmon tissue by exact matching isotope dilution mass spectrometry.

A high accuracy method for the quantification of malachite green (MG) and leucomalachite green (LMG) in salmon is described. Analytical challenges including the effects of analyte instability and matrix suppression were minimised by the use of exact matching isotope dilution mass spectrometry. The developed method included overnight extraction in acidified acetonitrile/ammonium acetate buffer and analysis by LC-MS/MS utilising isotopic internal standards. This method was used to determine the level of MG and LMG in a sample of salmon used in an international inter-comparison organised by the Comité Consultatif pour la Quantité de Matière (CCQM). The sum of MG and LMG was found to be 9.32+/-0.98ngg(-1) at the 95% confidence interval (relative expanded uncertainty 10.5% (k=2)). This encompassed the mean and median of the CCQM inter-comparison.

Development of a LC-MS method for the discrimination between trace level Prunus contaminants of spices.

The need for an analytical procedure for the identification of allergens present at trace levels in foods was highlighted by conflicting results in a case of contamination of the spice cumin. The application of a bottom-up proteomics experiment was investigated to identify marker peptides for potential contaminant nuts which could then be monitored with high specificity and sensitivity by selective reaction monitoring experiments. The method developed allowed for the distinction between two closely related Prunus species, almond and mahaleb, in two different spices, cumin and paprika. The paprika sample was found to be contaminated with almond and the cumin sample, contaminated at a much lower level, was found to be contaminated with mahaleb. The method could be applied to any protein-dense food matrix allergen so long as suitable control and reference samples can be acquired.

Towards a universal product ion mass spectral library - reproducibility of product ion spectra across eleven different mass spectrometers.

Product ion spectra produced by collision-induced dissociation (CID) in tandem mass spectrometry experiments can differ markedly between instruments. There have been a number of attempts to standardise the production of product ion spectra; however, a consensus on the most appropriate approach to the reproducible production of spectra has yet to be reached. We have previously reported the comparison of product ion spectra on a number of different types of instruments - a triple quadrupole, two ion traps and a Fourier transform ion cyclotron resonance mass spectrometer (Bristow AWT, Webb KS, Lubben AT, Halket JM. Rapid Commun. Mass Spectrom. 2004; 18: 1). The study showed that a high degree of reproducibility was achievable. The goal of this study was to improve the comparability and reproducibility of CID product ion mass spectra produced in different laboratories and using different instruments. This was carried out experimentally by defining a spectral calibration point on each mass spectrometer for product ion formation. The long-term goal is the development of a universal (instrument independent) product ion mass spectral library for the identification of unknowns. The spectra of 48 compounds have been recorded on eleven mass spectrometers: six ion traps, two triple quadrupoles, a hybrid triple quadrupole, and two quadrupole time-of-flight instruments. Initially, 4371 spectral comparisons were carried out using the data from eleven instruments and the degree of reproducibility was evaluated. A blind trial has also been carried out to assess the reproducibility of spectra obtained during LC/MS/MS. The results suggest a degree of reproducibility across all instrument types using the tuning point technique. The reproducibility of the product ion spectra is increased when comparing the tandem in time type instruments and the tandem in space instruments as two separate groups. This may allow the production of a more limited, yet useful, screening library for LC/MS/MS identification using instruments of the same type from different manufacturers.

The analysis of thyroxine in human serum by an 'exact matching' isotope dilution method with liquid chromatography/tandem mass spectrometry.

A method for the analysis of thyroxine in human serum, utilising 'exact matching' isotope dilution mass spectrometry (IDMS) in combination with liquid chromatography/tandem mass spectrometry (LC/MS/MS), has been developed with a limit of quantification of 0.5 ng g(-1) of thyroxine in human serum. The extraction and clean-up of thyroxine from serum involves an efficient protein-precipitation stage followed by a solid-phase extraction procedure to produce an extract essentially free from interfering compounds. The method is reproducible, with expanded uncertainties of less than 2%, and is relatively fast to perform.