A novel approach to the rapid analysis of pharmaceutical drug formulations using hyphenated ion mobility spectrometry (IMS) and time-of-flight mass spectrometry (ToF-MS) that requires no sample pretreatment or chromatographic separation is described. A modified quadrupole time-of-flight mass spectrometer containing an ion mobility drift cell was used for gas-phase electrophoretic separation of ions prior to ToF-MS detection. The generation of sample ions directly from tablets and cream formulations was effected by desorption electrospray ionization (DESI) using a modified electrospray ion source. The analysis of a range of over-the-counter and prescription tablet formulations is described, including histamine H2 receptor antagonist (ranitidine), analgesic (paracetamol), opiate (codeine), and aromatase inhibitor anticancer (anastrozole) drugs. The successful determination of active drugs from soft formulations, such as an antiseptic cream (chlorhexidine) and a nicotine-containing skin patch, is also presented. Limits of detection for the active drugs using the DESI/IMS/ToF-MS method fell within the high-picomole to nanomole range. In all cases, the use of ion mobility drift tube separation showed increased selectivity for active drug responses (present as low as 0.14% w/w) over excipient responses such as poly(ethylene glycol). Tandem mass spectrometric analysis of precursor ions separated by IMS allowed positive confirmation of active drugs with little loss of ion mobility efficiency. The ability to analyze hard or soft pharmaceutical formulations directly by DESI combined with ion mobility spectrometry/mass spectrometry in approximately 2 min demonstrates the potential applicability of this novel method to pharmaceutical screening of low-molecular-weight drug formulations with high selectivity over the formulation vehicle.
Ion mobility spectrometry (IMS) is an electrophoretic technique that allows ionised analyte molecules to be separated on the basis of their mobilities in the gas phase. The technique has found widespread application as a detector, most noticeably for chemical warfare agents on the battlefield and for explosives and narcotics at ports and airports. The application of IMS to structural studies of small molecules has also been recognised since the advent of the technique in the 1970s. The coupling of IMS with electrospray (ESI) and MALDI ion sources has opened up exciting possibilities for the study of the conformations and structures of a wide range of biomolecules in the gas-phase, including proteins, peptides and oligonucletoides. This tutorial review discusses the principles, instrumentation and applications of IMS for the elucidation of molecular structural characteristics.If the time taken to traverse a drift cell of length d (cm) is t d
A rapid acetonitrile (ACN)-based extraction method has been developed that reproducibly depletes high abundance and high molecular weight proteins from serum prior to mass spectrometric analysis. A nanoflow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) multiple reaction monitoring (MRM) method for 57 high to medium abundance serum proteins was used to characterise the ACN-depleted fraction after tryptic digestion. Of the 57 targeted proteins 29 were detected and albumin, the most abundant protein in serum and plasma, was identified as the 20th most abundant protein in the extract. The combination of ACN depletion and one-dimensional nano-LC/MS/MS enabled the detection of the low abundance serum protein, insulin-like growth factor-I (IGF-I), which has a serum concentration in the region of 100 ng/mL. One-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the depleted serum showed no bands corresponding to proteins of molecular mass over 75 kDa after extraction, demonstrating the efficiency of the method for the depletion of high molecular weight proteins. Total protein analysis of the ACN extracts showed that approximately 99.6% of all protein is removed from the serum. The ACN-depletion strategy offers a viable alternative to the immunochemistry-based protein-depletion techniques commonly used for removing high abundance proteins from serum prior to MS-based proteomic analyses.
Using a multi-layer perceptron Artificial Neural Network (ANN) (Neuroshell 2) with a back propagation algorithm we have developed a prototype approach that uses a model system (comprising five low and seven high-grade human astrocytomas) to identify mass spectral peaks whose relative intensity values correlate strongly to tumour grade. Analyzing data derived from MALDI mass spectrometry in conjunction with Ciphergen protein chip technology we have used relative importance values, determined from the weights of trained ANNs (Balls et al., Water, Air Soil Pollut., 85, 1467-1472, 1996), to identify masses that accurately predict tumour grade. Implementing a three-stage procedure, we have screened a population of approximately 100000-120000 variables and identified two ions (m/z values of 13454 and 13457) whose relative intensity pattern was significantly reduced in high-grade astrocytoma. The data from this initial study suggests that application of ANN-based approaches can identify molecular ion patterns which strongly associate with disease grade and that its application to larger cohorts of patient material could potentially facilitate the rapid identification of validated biomarkers having significant clinical (i.e. diagnostic/prognostic) potential for the field of cancer biology. AVAILIBILITY: Neuroshell 2 is commercially available from ward systems.
Ion mobility spectrometry (IMS), using standalone instrumentation and hyphenated with mass spectrometry (IM-MS), has recently undergone significant expansion in the numbers of users and applications, particularly in sectors outside its established user base; predominantly military and security applications. Although several IMS reference standards have been proposed, there are no currently universally recognised reference standards for the calibration and evaluation of mobility spectrometers. This review describes current practices and the literature on chemical standards for validating IMS systems in positive and negative ion modes. The key qualities and requirements an 'ideal' reference standard must possess are defined, together with the instrumental and environmental factors such as temperature, electric field, humidity and drift gas composition that may need to be considered. Important challenges that have yet to be resolved are also identified and proposals for future development presented.
Validation of these findings may enable proteomic profiling to become a valuable tool for identifying high-risk melanoma patients eligible for adjuvant therapeutic interventions.
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