SYMPOSIUM 2010

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Sport is te mooi voor Doping

Olivier de Hon, Nederlandse Dopingautoriteit

Miljoenen mensen genieten iedere dag van sport – door het te doen, door er naar te kijken of door er over te praten of te lezen. Maar zoals alle mooie dingen in het leven, heeft ook sport een keerzijde. Sommige excessen uit de samenleving zijn uitvergroot terug te vinden in de sportwereld. Dopinggebruik is slechts één klein voorbeeld hiervan. In hun jacht naar “sneller, hoger, sterker” zijn sommige sporters, en hun begeleiders, bereid om grote risico’s te nemen. Risico’s omtrent de gezondheid van sporters en (gezien de strenge dopingregels) omtrent hun carrière.

In Nederland gebruiken naar schatting zo’n 160.000 mensen per jaar geneesmiddelen zonder medische indicatie, met als doel om hun sportprestatie te verbeteren. De meeste doen dit in niet-competitief verband: in de sportschool, voornamelijk gericht op het verfraaien van hun eigen uiterlijk. Maar zodra men besluit om deel te nemen aan wedstrijden, kan men in aanraking komen met dopingcontroles. In de introductie van deze dag, zal ingegaan worden op de soorten dopinggebruikers die er zijn, op de middelen en methoden die gebruikt worden, en op de wijze waarop dopingcontroles en dopinganalyses plaatsvinden.

Doping bestaat niet

Berend Nikkels, huisarts te Breda

Geen abstract ontvangen. 

Het doseren van recombinant erythropoëtine: een moeizaam verhaal

Prof. Dr. J. Delanghe, Afd. Klinische chemie, Universitair Ziekenhuis Gent

Aan het eind van de twintigste eeuw werd de toename van het aantal verdachte overlijdens van sporters in verband gebracht met erytropoëtine doping. Het opsporen van recombinant erytropoëtine (epo) is technisch niet eenvoudig. Zowel indirecte (op fysiologie gebaseerde combinatie van testen) bloedanalyse als directe (analyse van recombinant epo via iso electric focusing/immunoblot) werden aangewend.

De wereldantidopingsorganisatie WADA koos in 2001 voor de directe opsporingsmethode. Deze methode vergt een enorme concentratie van de urine. Evoluties in de dosageschema's, het vinden van caveats en het verschijnen van tweede en derde generatiepreparaten maakten aanpassingen van de test noodzakelijk. De interpretatieregels werden sinds het ontstaan (2000) herhaaldelijk aangepast. In ongeveer één vijfde van de geteste atleten vindt men geen epo in het urinemonster.

De analytische gevoeligheid is aan de lage kant. Het aantal positief geteste atleten is verrassend laag. De immunologische detectiestap werd het voorwerp van kritiek. Bepaalde atleten toonden openlijk hoe

men kan frauderen. Een aantal betwistingen brachten pijnlijke mankementen van de directe test aan het licht (vals positieve resultaten). Het indirect testen (bloedpaspoort) wint de laatste jaren terug terrein. Het recent dalen van de gemiddelde hematocrietwaarden bij bepaalde sporttakken (o.a. wielrennen) toont het belang van de test aan.

Building the Global Bioanalysis Consortium – Working towards a functional globally acceptable and harmonized guideline on bioanalytical method validation

Peter van Amsterdam, Philip Timmerman et al

Although harmonization of bioanalytical method validation (BMV) and application has been discussed at scientific forums on several occasions, it was not until the European Bioanalysis Forum (EBF) conference in December 2009 [1], where European Medicines Agency’s (EMA) intended draft bioanalytical method validation guideline [2] was discussed, that the bioanalytical scientists present expressed their strong wish for international harmonization. A first action by AAPS, APA, CVG & EBF in this direction was to publish an open letter requesting global harmonization of existing or emerging guidances for BMV and sample analysis and offering to support the processes needed [3].

To transfer words into actions, it was decided to set-up an all inclusive Global Bioanalysis Consortium (GBC) consisting of scientific associations with world wide recognition and influence in order to create one unified document that, after thorough discussion in (a) global conference(s), can be presented to the decision makers in each country or region. The intention and expectation is that the document must be influential enough that all agencies will want to adopt it and harmonize their auditing and reviewing approaches. The founding members will continue to build the GBC in the coming months and will present their plans at bioanalytical meetings. In those meetings, representatives from the already assigned GBC Steering Committee or one of the founding members will present and seek input from the broader audience.

Bibliography

1. Richard Abbott
European Bioanalysis Forum, 2nd Open Symposium: “The Broadening Scope of Validation: Towards Best Practices in the World of Bioanalysis”, Bioanalysis 2(4), 703 (2010)

2. EMA Draft Guideline on validation of bioanalytical methods
EMA/CHMP/EWP/192217/2009, http://www.ema.europa.eu/pdfs/human/ewp/19221709en.pdf

3. Philip Timmerman, Steve Lowes, Douglas M Fast, Fabio Garofolo
Request for Global Harmonization of the Guidance for Bioanalytical Method Validation and Sample Analysis, Bioanalysis 2(4), 683 (2010)

Trends in doping analyse

Saskia S. Sterk

Ook in de doping analyse, van oudsher een tradioneel veld waarin de methoden langzaam veranderen, is de afgelopen jaren een en ander gebeurd.

Nieuwe analieten, nieuwe matrices, nieuwe technieken en nieuwe benaderingen.

Op alle fronten zijn er ontwikkelingen. Een aantal van deze nieuwe trends zullen behandeld worden met enkele praktijk voorbeelden.

Gene doping: fact or fiction?

HJ Haisma, Pharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy
Groningen University

Genetic therapies are a promising new branch of regular and sports medicine. This raises the issue whether these techniques might be abused in the field of elite sports. Both the World Anti-Doping Agency (WADA) and the International Olympic Committee (IOC) have expressed concerns about this possibility. As a result, the method of gene doping has been included in the list of prohibited classes of substances and prohibited methods.

Many genes are readily available and evaluated in gene therapy strategies in animals and patients. Some of these may potentially be used to improve healing of sports injuries whereas others could have an effect on athletic performance. These include erythropoietin for increasing endurance or Insulin-like growth factor for improving muscle strength.

Previous experiences have learned that drugs that are still in the experimental phases of research may find their way into the athletic world. The sporting world will therefore eventually be faced with the phenomena of gene doping to improve athletic performance. Current detection methods are insufficient to detect gene doping because they are limited to the detection of foreign drugs or proteins and unable to measure small changes in native human protein levels.

New detection methods based on gene expression arrays or proteomics capable of measuring global pattern changes in combination with a clear education program on the associated risks seems to be the most promising preventive method to counteract the possible application of gene doping.

A New Approach for General Screening of Complex Mixtures on Ion Trap MSMS Library Search

Roman Mylonas [1], Yann Mauron [1], Alexandre Masselot [2], Olivier Philippe [2],
Pierre-Alain Binz [1,2], Veronique Viette [3,4], Marc Fathi [3], Denis F Hochstrasser [3,5],
Frederique Lisacek [1], Sebastian Goetz [6], Birgit Schneider [6], Jens Vagts [6],
Carsten Baessmann [6], Patrick van Houts [7], Rob van der Heijden [7]

[1] Swiss Institute of Bioinformatics, Geneva, Switzerland;
[2] Geneva Bioinformatics (GeneBio), Geneva ,Switzerland;
[3] Geneva University Hospital, Geneva, Switzerland;
[4] ADMed Fundation, La Chaux-de-Fonds, Switzerland;
[5] Swiss Center for Applied Human Toxicology, Geneva, Switzerland;
[6] BrukerDaltonik GmbH, Bremen, GERMANY;
[7] Bruker Nederland BV, Wormer, NL

Liquid Chromatography Tandem Mass Spectrometry (LC-MSMS) combined with library search appears in clinics, forensic, and food testing applications with the aim at replacing a LC-UV-DAD library search system. The approach does not require any chemical derivatization steps during the sample preparation. Compared to GC-MS, LC-MSMS covers a complementary in some respect, but clearly broader range of analytes. In routine laboratories, there is a need for an easy to use hardware and software solution coupled with simple SOPs, robust statistics and secured data storage and reporting methods. The system should also easily enable the creation, annotation and enhancement of spectral libraries.

Here we present an integrated hardware and software approach for acute clinical toxicology screening. The solution includes an ion trap MS instrument, a spectral library, a specially adapted search algorithm (SmileMS by Genebio) and predefined SOPs. Various SPE fractions of patient samples suspected for different drug intoxications as well as purified compounds were analyzed with an ion trap instrument coupled online with a modern ultra performance LC system. Both sample submission and evaluation of results is performed via a dedicated easy to use open access type software. The system was used to enrich a spectral library of dedicated analytes as well.

The implemented workflow allows a user to log in to open access software environment, register one or more samples and choose a processing method (including MS acquisition settings, data processing options and library search parameters). A technician launches the acquisition locally on the MS system. The recorded spectra are automatically sent to data processing. The user accesses the results in a summarized form via a web based browser and can visualize, validate and report the outcome of the analysis via hyperlinks. Finally, commented reports can be sent to the analysis requester via email. The summary results from the library search are made available in a simple table, while the details can be evaluated via hyperlinks. Due to its client-server architecture data can be accessed from any PC connected to the intranet. A library containing 300 common drugs and toxins is being created in collaboration with a clinical laboratory. Additionally an SOP was created that allows sample preparation, analysis and automated result evaluation in less than 30 min. In this work a selected set of clinical blood and urine samples has been spiked with known drug molecules. The identification quality was evaluated at different concentration levels.

Evaluation of Performance and Benefit of Ultrahigh-Resolution ESI-TOFMS Coupled to Fast Chromatography in the Application of Forensic Screening

Rob van der Heijden [1], Patrick van Houts [1], Anna Pelander [2], James Hillis [3] and
Petra Decker [4]

[1} Bruker Nederland BV, P.O. Box 88, 1530 AB Wormer, The Netherlands;
[2] University of Helsinki, Helsinki, Finland;
[3] Bruker Daltonics Ltd, Coventry, United Kingdom;
[4] Bruker Daltonik GmbH, Bremen, Germany

Comprehensive forensic screening of a large target database using full scan data from LC/TOFMS technology has already been established as a routine method. Compound identification relies on accurate mass, isotope pattern and (optionally) retention time information.

Nevertheless, although the method has proven to be very reliable in practice on a large number of samples, the target database contains a few compound pairs of similar retention time, which require mass resolution higher than the standard TOF MS specification (> 10,000), thus potentially preventing an unambiguous identification. These compound pairs are the ideal test cases for the enhanced capabilities of the latest UHR-TOF technology. Since mass accuracy, isotope pattern and resolution on TOF instruments are independent of acquisition rate coupling to an UHPLC additionally allows for faster methods.

The analytical system consisted of an Ultimate 3000 Rapid Separation LC (Dionex) and a maXis UHR-TOF MS (Bruker Daltonics). Flow injection analyses were used to study mass resolution; fast LC-methods were used to study aspects of speed of data acquisition (1-20 Hz) and quantitation.

It is concluded that UHR-TOF MS technology is successfully applied to the forensic screening examples: compounds, which cannot be resolved on standard TOF-MS systems can be unambiguously identified. In addition it is shown that mass accuracy, isotope pattern and mass resolution are independent of acquisition rates, so that also combinations with fast LC methods can be used for application in forensic screening.

New Strategy for Quantification of THC Carboxylic Acid Direct from Hair

Detlef Thieme [2], Hans Sachs [3], Axel Besa [1], Birgit Schlutt [1], Daniel Leigh [1],
Detlev Schleuder [1]

[1] AB SCIEX, Europe;
[2] Department of Sports Medicine and Doping Analysis, Kreischa, Germany;
[3] FTC Munich, Munich, Germany

Regarding 11-nor-9-carboxy-D9-tetrahydrocanabinol (THC carboxylic acid) in hair matrix as an example for the highly selective and sensitive analyses of matrix defective MRMs we would like to present a new strategy of operation.

The sensitive MRM traces of THC carboxylic acid are disturbed by many interfering signals as well as high background noise around the expected retention time. The ability to use fragments of second order, MRM3 (MS/MS/MS), will show a significant increase in selectivity in comparison to classical MRM mode.

The combination of a triple quad in conjunction with a linear ion trap in Q3 is devoid of compromise and offers highly increased sensitivity in MS/MS/MS, MRM3, applications compared to classical ion traps. In QTRAP technology first precursor is selected and fragmented as known in any triple quad MRM mode. Notwithstanding MRM mode, Q3 is accumulating desired fragment, second precursor, for additional fragmentation in linear ion trap mode. Hence, the outstanding sensitivity on principle of AB Sciex 5500 series in combination with increased capacity and scan speed of re-designed Q3 linear ion trap result in a highly selective and sensitive LC-MS/MS/MS, LC-MRM3, experiment.

Breakthrough productivity for ADME studies with the new AB SCIEX TripleTOF™ 5600 System

S. J. Lock, Technical Support, AB SCIEX, Pudsey, United Kingdom

Abstract: There are several challenges in Qualitative / Quantitative workflows in the pharmaceutical industry.  These include simultaneous metabolic stability and soft spot analysis where high throughput providing relative quantitation of the parent drug and identification of the major metabolites is required.  Another area is high throughput in-vivo pharmacokinetic studies where metabolite information is needed together with quantitative information on the same sample.

In both these areas the ideal instrument would have:
- Acquisition speeds fast enough to be UPLC compatibility;
- Be capable of running generic methods;
- Have triple quadrupole sensitivity;
- Give High resolution with High mass accuracy.

Current technology does not provide all these attributes at one time.  For example, orbital trapping gives high resolution but not at high speed and a high mass accuracy but has limited sensitivity and dynamic range for quantitation.  On the other hand triple quadrupole technology has excellent linearity and quantitation sensitivity but has lower resolution and low mass accuracy.

The New TripleTOF™ 5600 System from AB SCIEX provides both high sensitivity with dynamic Range and speed (tens of milliseconds) to give data where the resolution is ~30K or higher (across the mass range) with a high mass accuracy (1-2 ppm).  This talk will describe this new technology and give examples where it has already been applied to the qualitative / quantitative pharmaceutical workflows above.

Extending LC-MS/MS linear dynamic range for quantitative analysis of drugs in Dried Blood Samples

Suma Ramagiri [1], Feng Zhong [1], Mauro Aiello [1], Hesham Ghobarah [1],
Neil Spooner [2], Iain Gibb [3]

[1] ABSCIEX, Canada;
[2] GlaxoSmithKline Pharmaceutical, Welwyn, United Kingdom;
[3] ABSCIEX, Europe                                                                              

Demonstrating dilution integrity in dried blood spot (DBS) sample analysis is quite different from serum, or plasma samples. The latter sample types can be diluted using similar blank matrix.  However, this is not possible for DBS samples. Current approach use dilution with the extract of a blank DBS sample and is not appropriate for approaches involving the direct analysis of DBS samples. It is therefore necessary to maximize the linear dynamic range of the LC-MS/MS system. To address this, we have proposed two different methods: 1) by monitoring parent to most intense product ion to cover low concentrations in calibration curve and parent to least intense product ion transition high concentration in the calibration range 2) post acquisition data processing by a novel SignalFinder™  Algorithm to correct for detector saturation.

The determination of ketoprofen in human whole blood using dried blood spot sampling and LC-MS/MS

E. Mallat, M.J. Dröge, R. Wessel, F. Oostebring, H. Rutgers, K. de Jonge and E. Oosting
ABL, Assen, The Netherlands

A sensitive bioanalytical LC-MS/MS method for the determination of ketoprofen in whole blood and in whole blood collected on filter cards has been developed and compared to an in-house validated plasma method. The results will be presented.

Extensive utilisation of Dried Blood Spot sampling in early clinical development studies: pharmacokinetics, pharmacogenomics and safety assessments

Theo de Boer [1], Jaap Wieling [1], Erik Meulman [1], Gonda Renkema [1], Johan Wemer [1], Izaak den Daas [1], Thijs van Iersel [1], Mariska Reuvers [1] and LingSing Chen [2]

[1] QPS Netherlands B.V., Hanzeplein 1-53, 9713 GZ Groningen, The Netherlands;
[2] QPS, Delaware Technology Park, 3 Innovation Way, Suite 240, Newark, DE 19711, USA

For bioanalysis of drugs or genotyping, dried blood spot (DBS) sampling can be an alternative for venous sampling. Possible advantages include stabilization of the analyte by the DBS matrix and storage without the need of freezers in less storage room. More over, the technique allows for less invasive sampling in non-clinical, i.e. ambulant, conditions.

Comparison of accurate mass and nominal mass MSMS for the simultaneous acquisition of qualitative and quantitative data in DMPK studies.

Joanne Mather, Marian Twohig, Paul D. Rainville, Ronan O’Malley, and Robert S. Plumb

Introduction

A crucial phase of the drug discovery process is the quantification and qualitative analysis of candidate pharmaceuticals and their metabolites in biological fluids. Traditionally this would involve the use of a triple quadrupole mass spectrometer for the quantitative analysis of low exposure compounds, qualitative data is derived from either hybrid quadrupole time of flight (QToF) or ion trap instrumentation.

In this poster we present a comparison of the benefits and drawbacks for the simultaneous acquisistion of qualitative and quantitative LC/MS/MS data using either a QToF instrument or a triple quadrupole instrument. The throughput, sensitivity and spectral quality were compared using 20 structurally diverse compounds in blood products.
     

Methods

Calibration lines of model drug compounds and their associated metabolites were spiked into rat plasma and whole blood. Plasma samples were prepared by protein precipitation (2:1 acetonitrile: plasma), centrifugation and dilution (1:1 with water). Whole blood samples were prepared by spotting 15 µL of blood onto Whatman FTA DMPK cards and extracted with methanol. Samples were then injected onto 2.1x50mm 1.7 µm BEH C18 columns and eluted under gradient LC conditions with either 2 or 5 minute analysis times. Mass spectrometry was performed on a QToF and a triple quadruple mass spectrometer, both operating in positive electrospray mode.

Data

The triple quadrupole mass spectrometer was used to simultaneously acquire LC/MS data in two different modes;  i)  MRM to acquire quantitative data for the analytes and ii) full scan data to obtain qualitative data for metabolite detection.

The QToF was operated in alternating low high collision energy mode allowing the simultaneous collection of precursor and product ion data. The QToF is capable of acquiring data at fast acquisition speeds without reduction in mass accuracy or spectral quality. This stability allows the use of a narrow mass window for data processing, the effect of lowering the mass window around the target analytes accurate mass showed that it was possible to employ a 0.5mDa window. This produced excellent signal to noise values with 50pg/mL standards. The use of a hybrid TDC/ADC detector allowed for stable mass measurements and a wide dynamic range.

The average LOD for the triple quadrupole mass spectrometer in MRM mode was approximately 10pg/mL whilst the QToF achieved an average of approximately 50pg/mL for all 20 compounds. The fast data capture rate of the triple quadrupole mass spectrometer ensured that the LOD was not changed by the use of very fast gradient analysis.

In Contrast the QToF LOD was reduced by approximately 5 fold when using 2 minute gradients compared to a 5 minute gradient. This is due to the faster sampling rate required affecting the ion statistics. Whilst the triple quadrupole mass spectrometer was more sensitive for quantitation, the full scan data used for metabolite detection with this instrument was approximately 50 times less sensitive than the QToF data. The QToF also provides greater spectral quality data than the triple quadrupole mass spectrometer.

Both instruments are ideal for DMPK studies with the triple quadrupole mass spectrometer being more suited to high throughput quantitation.

Addressing the Issues of Matrix Resolution and Measurement in Bioanalytical Assays

Geneen Baynham [1], Paul D Rainville [1], Marian Twohig [1], Robert S Plumb [1] and
Ian Wilson [2]

[1] Waters Corporation, Milford, MA 01757
[2] AstraZeneca, Macclesfield, UK.

In quantitative bioanalysis the analytical technique of choice is LC/MS/MS due to the high sensitivity and selectivity that it affords.  Quantitative bioanalytical methods development is complicated by matrix interferences which can alter the response of the analyte. 

Matrix effects, resulting from co-eluting matrix components that compete for charge in the ionisation process, manifest themselves as suppression or occasionally enhancement of the analyte signal.  Matrix effects are caused by numerous factors all of which can cause significant errors in the accuracy and precision of a bioanalytical assay, Phospholipids, Subject differences, Impurities, Degradation Products, Co-eluting metabolites

Monitoring the presence and impact of matrix effects can complicate and prolong the development of a robust, sensitive assay. 

In this poster we evaluate the use sub 2um particle LC on the matrix effects observed.  We will also demonstrate how the background matrix interference can be monitored in a single run analytical using a mass spectrometer equipped with a novel fast switching quadrupole MS.  We will also demonstrate how the matrix effects can be determined and quantified by an integrated matrix calculator tool.  We will show how the use of these tools can significantly shorten the time taken to develop a sensitive, robust bioanalytical LC/MS/MS assay. 

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