A new analytical method based on anti-EPO monolith column and LC-FAIMS-MS/MS for the detection of rHuEPOs in horse plasma and urine samples.

Recombinant human erythropoietin (rHuEPO) is a 30-34 kDa glycoprotein banned by the racing authorities. For some years this molecule has been detected in race horses in USA and in Europe, and even in racing camels. Although direct methods to differentiate horse endogenous EPO and rHuEPO have been developed either by LC-MS/MS or by isoelectric focusing (IEF) with double-blotting, the short confirmation time of such prohibited hormone in plasma remains a problem for horseracing doping control laboratories. In order to improve the rHuEPOs confirmation process in horse plasma or urine in terms of reliability and delay, a small anti-EPO monolith membrane contained in a disposable column (anti-EPO monolith column) has been successfully used and validated (n = 10). This new sample preparation, combined with LC-FAIMS-MS/MS, has been performed on plasma and urine samples collected from one horse which received an Eprex® treatment during six consecutive days and a second one with a single injection of Aranesp®. This inventive technology allowed the possibility to confirm the presence of rHuEPO within one day with a limit of detection validated for both urine and plasma at 250 pg mL(-1) by means of a disposable, ready to use immunoaffinity column. The lower limit of detection (LLOD) obtained for each matrix was 100 pg mL(-1). These results provide an important improvement for rHuEPO doping control in horseracing especially the possibility to confirm these banned molecules in both matrices, urine and plasma, with a confidence of two specific target peptides.

Evaluation of laser diode thermal desorption (LDTD) coupled with tandem mass spectrometry (MS/MS) for support of in vitro drug discovery assays: increasing scope, robustness and throughput of the LDTD technique for use with chemically diverse compound libraries.

Within the drug discovery environment, the key process in optimising the chemistry of a structural series toward a potential drug candidate is the design, make and test cycle, in which the primary screens consist of a number of in vitro assays, including metabolic stability, cytochrome P450 inhibition, and time-dependent inhibition assays. These assays are often carried out using multiple drug compounds with chemically diverse structural features, often in a 96 well-plate format for maximum time-efficiency, and are supported using rapid liquid chromatographic (LC) sample introduction with a tandem mass spectrometry (MS/MS) selected reaction monitoring (SRM) endpoint, taking around 6.5 h per plate. To provide a faster time-to-decision at this critical point, there exists a requirement for higher sample throughput and a robust, well-characterized analytical alternative. This paper presents a detailed evaluation of laser diode thermal desorption (LDTD), a relatively new ambient sample ionization technique, for compound screening assays. By systematic modification of typical LDTD instrumentation and workflow, and providing deeper understanding around overcoming a number of key issues, this work establishes LDTD as a practical, rapid alternative to conventional LC-MS/MS in drug discovery, without need for extensive sample preparation or expensive, scope-limiting internal standards. Analysis of both the five and three cytochrome P450 competitive inhibition assay samples by LDTD gave improved sample throughput (0.75 h per plate) and provided comparable data quality as the IC50 values obtained were within 3 fold of those calculated from the LC-MS/MS data. Additionally when applied generically to a chemically diverse library of over 250 proprietary compounds from the AstraZeneca design, make and test cycle, LDTD demonstrated a success rate of 98%.

Validated quantitation method for a peptide in rat serum using liquid chromatography/high-field asymmetric waveform ion mobility spectrometry.

The analysis of peptides presents serious challenges for bioanalytical scientists including low total ion current and non-selective fragmentation during tandem mass spectrometry (MS/MS). During method validation of a peptide in rat serum matrix some interferences could not be easily removed and thus prevented accurate and precise measurement. These problems associated with peptide quantitation were resolved by using FAIMS (high-Field Asymmetric waveform Ion Mobility Spectrometry). This selectivity-enhancing technique filters out matrix interferences, and the resulting pseudo-selected reaction monitoring (pseudo-SRM) chromatograms were nearly free from interferences. Control blank matrix samples contained an acceptable level of interference (only 7% signal as compared to the lower level of quantitation). Chromatographic peaks were easily, accurately and precisely integrated resulting in a validated liquid chromatography (LC)/FAIMS-MS/MS method for the analysis of a peptide drug in rat serum according to United States Food and Drug Administration (US FDA) bioanalytical guidelines. These results confirm that new selectivity-enhancing technologies aid the pharmaceutical industry in reliably producing acceptable pharmacokinetic data.