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Abstracts van Lezingen

Quality Assurance in Drug Development: Criteria for safety assessments
G. De Schrijver
Labcore, Belgium

The presentation explains how to make a rational approach when selecting and conducting safety pharmacology studies. It emphasizes on how the actual clinical laboratory will implement and use scientifically valid methods, preferable internationally recognized.
Achieve a reliable and accurate result within the actual economical constraints is the major challenge. The requirements of the applicable guidelines are discussed within this context and some practical application examples are given. Special attention is paid to the pre-analytical, analytical and post-analytical processes and how to control them. More details are given on the following basic processes:

- the analytical process from receipt of the sample to the provision of the result;
- the analytical procedure: from method validation to the regular (daily) performance of the analysis

The link with the requirements of international guidelines such as ISO 17025 and ISO 15189 is also documented together with the indication that the implementation of an integrated quality management system is the absolute condition to assure the best possible clinical valid result with a level of quality within the limitations of the actual technology.
A short overview of the different validation elements is explained together with the practical approach in the laboratories of the 21st century. To finish, the most common compliance issues are also discussed.

Validation of assays to measure macromolecules
Uschi Rose
Organon, Oss, The Netherlands

< no abstract received yet >

Pharmacokinetics - Instrumental Bioanalysis
Philip Timmerman
Johnson & Johnson, Beerse, Belgium

< No abstract available yet >

< TITLE >
Nico van Gorkum
Analytico, Breda, Netherlands

< No abstract available yet >

Calibration and Specificity: Fit-for-Purpose Method Development for Successful Biomarker Measurement
Foka Venema
ABL, Netherlands

< No abstract available yet >

< TITLE >
Ronald de Vries
Johnson & Johnson, Beerse, Belgium

< No abstract available yet >

Abstracts van Posters

Impact on Ion Suppression by Eliminating Phospholipid Interferences Using a Generic TurboFlow® Method
François A. Espourteille; Catherine LaFontaine
Thermo Fisher Scientific, Franklin, MA, USA

• Phospholipid interference is a major component of matrix effect in bioanalysis.
• During method development it is necessary to account for phospholipid impact on analytes.
• Two dimensional chromatography, on-line extraction plus analytical separation, was identified as a promising technology to control phospholipids during routine bioanalysis.
• Developing an on-line method which segregates phospholipids away from the gradient elution window was achieved.
• Good recoveries from plasma standards over a wide range of common drugs were achieved. Matrix effects were significantly reduced.

A Systematic Approach To Reducing Matrix Effects In Bioanalytical Studies
Uwe D. Neue and Patrick D. McDonald
Waters Corporation

Ion suppression - a common phenomenon in the LC/MS analysis of complex samples - can be effectively eliminated by good solid-phase extraction methods. Today, with tools such as SPE, coupled with the availability of methods for the quantitative assessment of ion suppression, significant errors in LC/MS analysis due to ion suppression are avoidable and inexcusable.

How to determine matrix effects and extraction recovery in online solid-phase extraction – liquid chromatography – mass spectrometry
Alex Berhitu, Emile Koster
Spark System Solutions B.V., P.O. Box 388, 7800 AJ Emmen

Introduction:
Regulation requires that matrix effects for the analysis of biological samples are investigated. When MS is used no false positives or negatives may occur due to either ionization suppression or enhancement.
When off-line SPE is used for sample clean-up before utilizing LC-MS, matrix effects are determined by comparing the response of a neat solution with that of a spiked extract obtained form a blank biological sample. However, for online SPE such is not possible because the sample is automatically introduced into
the LC-MS without collecting it first. In this poster a new approach is proposed for the determination of matrix effects and recovery utilizing online SPE-LC-MS/MS.

Overview of the poster:
• For online SPE-LC-MS the total workflow from sample to data file is automated.
• Consequently the methodology used for the determination of recovery and matrix effects in off-line SPE before LC-MS analysis can no longer be used.
• An example is given to demonstrate that the determination of total recovery by means of comparing LC-MS with SPE-LC-MS data is not always valid.
• A new methodology is proposed that enables automated online SPE-LC-MS determination of extraction recovery and matrix effects.
• Online SPE is performed over two cartridges in series allowing visualization of the extraction process.
• The extraction recovery and matrix effects can be calculated by means of two new equation.

A systematic online SPE-LC-MS/MS method development strategy for the analysis of biological samples
Rob Castien, Martin Sibum, Emile Koster
Spark System Solutions B.V., P.O. Box 388, 7800 AJ Emmen

Introduction:
High throughput analysis of biological samples by means of LC-MS/MS has become the norm in today's laboratory. To permit high-speed analysis an elevated degree of automation is preferred. Classical sample preparation techniques such as liquid-liquid extraction and protein precipitation are no longer of first choice as they are less suitable for automation. Therefore, automated solid-phase extraction has gained lots of interest over the last years. The difficulty faced by many chemists is, however, that solid-phase extraction method development is too difficult or takes too much time. In this poster a systematic online SPE method development strategy is proposed that allows for generating new methods easy and relatively fast.

Overview:
• Automated SPE-LC-MS allows for high throughput analysis.
• SPE method development is experienced to be the time limiting process.
• A strategy is proposed that enables quick and simple method development.
• The approach includes sorbent screening, wash optimization (% organic modifier and wash volume) and finding the best elution conditions.
• A new full optimized online SPE-LC-MS/MS method was developed for the determination of propranolol in fish plasma in about 2 hours.

Accurate mass and isotopic pattern based routine drug screening in horse urine using liquid chromatography/time-of-flight mass spectrometry.
Matthias Pelzing¹; Geoff Beresford²; Chris Hendrix³; Rob Howitt¹; Ian Sanders⁴
1. Bruker Biosiences Pty Ltd, Brunswick, Australia
2. New Zealand Racing Laboratory Services, Auckland, New Zealand
3. Bruker Daltonics BVBA, Brussels, Belgium
4. Bruker Daltonik GmbH, Bremen, Germany

The LC/MSTOF methods described allow equine urine drug screening for a wide range of analytes with sufficient sensitivity to meet required performance specifications in almost all cases. In addition, the data acquired can be processed retrospectively as new compounds are added to the database. A mass accuracy averaging less than ±3mDa has been achieved allowing a maximum tolerance of 10mDa to be applied to screening results. Isotopic pattern evaluation (SigmaFitTM) has been shown to reduce the incidence of false positives. Where pseudomolecular ions are misidentified due to the presence of near-mass interferences, in-source CID can be used to create qualifier ions to discriminate between true and false positive results. The basic method was used to analyse 118 basic analytes. Of these 110 were successfully detected. Of the 110 analytes detected using the basic method, 106 were found at or below 10ng/mL and 93 analytes were found at or below 5ng/mL. The general method was used to analyse 118 basic analytes and 49 additional analytes including diuretic drugs, caffeine and its metabolites, acid drugs and barbiturates. Many of these compounds are usually analysed using negative mode ionisation. However, an adequate response was achieved for the majority using ESI in positive mode. The general method in particular demonstrates that ESI/TOF techniques can identify low-level analytes in the presence of very complex matrices. This has the potential to allow fewer and more simplified extraction procedures and facilitate broad-spectrum screening using a single instrumental run. An enzyme hydrolysed urine sample can be extracted and analysed within 1hr using this method. It is expected that these methods will also be suitable for canine and human urine and plasma samples. A full validation of these methods using these matrices will follow any method refinements.

Metabolic Profiling of Amino Acids in Urine using CE-ESI-TOF-MS
Oleg Mayboroda²; Rico Derks²; Gabriela Zurek¹; Matthias Pelzing³; Christian Neusuess⁴; Chris Hendrix⁵; Andree Deelder²
1. Bruker Daltonik GmbH, Bremen, Germany
2. LUMC, Leiden, Netherlands
3. Bruker Daltonics Australia, Melbourne, Australia
4. HTW Aalen, Aalen, Germany
5. Bruker Daltonics BVBA, Brussels, Belgium

A method for the direct analysis of amino acids in urine is presented. The direct analysis of several amino acids in biofluids simultaneously is advantageous as conventional methods are either limited to a single compound or require derivatisation. The sensitivity of the CE-MS method is improved by increasing of injection volume with help of pH mediated stacking. Detection limits below 50nM can be achieved for most amino acids representing an improvement of 1-2 orders of magnitude compared to previous studies. Linearity of the method is demonstrated for concentrations between 50nM and 250µM. Mass accuracy and stability of the ESI-TOF instrument provide the basis for compound identification via the molecular formula and migration time. Real urine samples from osteoarthritis patients (ten patients each of 2 different disease stages and 10 controls) have been analyzed. Introduction of principle component analysis is evaluated on spiked urine samples and real clinical material. The presented work demonstrates that CE-TOF MS is a useful tool for the characterization of body fluids in clinical context based on profiling of amino acids, amines, small peptides, and related metabolites.

Detailed Investigation of the Fragmentation of aminopyridinyl-sulfanyl-imidazole-based MAPKinase Inhibitors using accurate MS(n) and isotopic pattern data
Holger Scheible¹; Ilmari Krebs²; Sebastian Goetz²; Gabriela Zurek²; Wolfgang Albrecht³; Chris Hendrix⁴; Christoph Gleiter¹; Stefan Laufer¹; Bernd Kammerer¹
1. University of Tuebingen, Tuebingen, Germany
2. Bruker Daltonik GmbH, Bremen, Germany
3. Merckle GmbH, Ulm, Germany
4. Bruker Daltonics BVBA, Brussels, Belgium

In structure elucidation, the confident determination of the molecular formula of the parent ion is the key to all interpretation. MS/MS and also MS3 shed light on fragmentation pathways. When doing IS-CID combined with subsequent isolation and fragmentation of a first generation product ion, MS3 experiments can be performed easily in a Qq-TOF MS. In this entire evaluation process, the generation of formulae is the initial step towards interpretation. Formula generation is supported by chemical knowledge (nitrogen rule, rings and double bonds, H/C ratio) and by fitting the measured isotopic pattern against the theoretical one. In a top down/bottom up approach, the number of candidates for the precursor ion is narrowed down as formulae of low mass ions are often assigned unequivocally. Furthermore, neutral losses are used to crosscheck the relationship of precursor and product ions. User interaction is allowed to delete wrong formulae due to knowledge of the chemical context of the sample. For the complete series of formulae from precursor and product ions, a combined quality value is calculated. This tool was then applied to investigate the influence of structural variations in an aminopyridinyl-sulfanyl-imidazole core structure on the fragmentation behaviour. Structural modifications comprised: 1. sulfanyl- to sulfenyl- group, 2. N-substitutions at the imidazole; 3. different substituents at amino function of aminopyridinyl-group. When varying the oxidation state of sulphur (case 1), a methyl radical can be observed in the initial fragmentation of the molecule. This changes the entire fragmentation pathway. A second interesting fact is how the direct binding of a phenyl-group at the amino function (case 3) changes the fragmentation compared to other substituents (phenylethyl- and cyclohexyl group). Other variations (cases 2 and 3) are also described.

Determination of loratadine in dog plasma by high-performance liquid chromatography with fluorescence detection
J. De Smet¹, W. Goeteyn¹, J.-P. Remon², and J.F.P. Van Bocxlaer¹
1. Laboratory of Medical Biochemistry and Clinical Analysis, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium
2. Laboratory of Pharmaceutical Technology, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium

A high-performance liquid chromatographic (HPLC) method was developed for the determination of loratadine in dog plasma. Diazepam was used as internal standard. These compounds were extracted with n-butyl alcohol/n-hexane 2/98 v/v in alkaline conditions and the upper organic phase was evaporated under stream of nitrogen. The dry residue is then re-dissolved in eluent A (KH2PO4 0.05M pH 2.5 in water/acetonitrile 90/10 v/v). Chromatography was carried out using a Waters XTerra® MS C8 column (2.1 x 150 mm, 3.5µm) and a gradient mobile phase consisting of methanol-acetonitrile-phosphate buffer. The fluorescence detection was performed at excitation and emission wavelengths of respectively 280 and 460 nm. During validation, calibration curves were prepared in both human and dog plasma, in an effort to cross-validate the analysis for calibration of dog samples with human plasma calibrators. Statistical tests comparing regression lines, however, indicated a statistically significant slope difference. Linearity was nevertheless assessed in the range of 0.50-40.00 ng/ml in dog plasma with correlation coefficients (r²) > 0.99, using a 1/X weighing. Accuracy for loratadine ranged from 103.0% to 115.1% at low, mid and high levels, while the intra-day precision ranged from 3.86% to 15.7%. The method proved specific and sensitive with a quantification limit of 0.50 ng/ml and a detection limit of 0.41 ng/ml. The purpose of this analysis was the evaluation of the bio-availability of loratadine after buccal application in dogs in a limited feasibility study. Plasma concentration-time data evaluation showed that inadequate absorption of loratadine was obtained.

Quantitative Analysis on a Novel MALDI Triple Quadrupole Platform
Jean-François Alary, George Scott, Feng Zhong, Jay Corr
Applied Biosystems/MDS Sciex, 71 Four Valley Drive, Concord, Ontario, Canada, L4K 4V8

The FlashQuant™ Workstation consists of the FlashLaser Source compatible with either an API 4000™ or 4000 Q TRAP® MS/MS System and newly designed application specific software to complement the increasing demand for higher bioanalytical throughput and productivity in early Discovery ADME screening applications.

Key Features
- MALDI Source swappable back to Turbo V™ Source
- Operated in vacuum for maximum sensitivity
- High repetition solid state UV laser (349 nm @ 1000 Hz)
- 96 & 384 plate formats
- Fast moving stages (40 mm/sec) for high-speed plate reading
- Reduced plate loading and pump down time (<2 minutes)
- Robust integration with MS (>150,000 samples between cleanup)
- Integrated camera
- Sample analysis time ~ 3 seconds per sample (variable rastering speeds possible)
- Level of detection = Better than 50 nM at Mwt 500 with a signal-to-noise of 3:1 (25 pg/μL)
- Linear dynamic range up to 4 orders in magnitude
- Precision (spot to spot) typically less than 10 %CV

FlashQuant™ Workstation – Bringing Speed to Standard ADME Assays
Applied Biosystems/MDS Sciex, 71 Four Valley Drive, Concord, Ontario, Canada, L4K 4V8

Metabolic Stability Screening
Metabolic stability profiles from more than 40 compounds can be obtained with the FlashQuantTM Workstation. The conventional metabolic stability assay uses LC/MS/MS based methods where sample throughput is limited by the LC separation time. The FlashQuant Workstation demonstrates its high throughput potential for this class of application, reaching routine analysis speed of 1 sec/sample and producing sensitivity, accuracy and precision adequate for in vitro ADME quantification. A workflow suitable for high throughput metabolic stability screening purposes based on the FlashQuant Workstation can be enabled using commercially available liquid handlers as illustrated below.
High Throughput CYP 3A4 Inhibition Screening
The FlashQuant Workstation can efficiently process Drug-Drug Interaction (DDI) experiments. This example illustrates a DDI experiment using midazolam as substrate and Ketoconazole as a test inhibitor. Reaction was stopped by acetonitrile precipitation and reaction solutions were cleaned up with solid phase extraction cartridge followed by mixing with MALDI matrix. Four MRMs were monitored to probe substrate, substrate metabolite, test inhibitor and internal standard.
Since the typical DDI experiment requires monitoring the same probe metabolites for all the test inhibitors, no further method development is required to run additional inhibitors. This application class can therefore take full advantage of the speed of the FlashQuant Workstation.

Evaluation of MALDI Triple Quadrupole Mass Spectrometry for High Throughput Drug-Drug Interaction Screening
Feng Zhong, Hesham Ghobarah, Daniel Lebre, George Scott, Gary Impey and Jay Corr
Applied Biosystems/MDS Sciex, 71 Four Valley Drive, Concord, Ontario, Canada, L4K 4V8

MALDI-QqQ mass spectrometry was evaluated for potential high throughput CYP450 inhibition screening. Nonfluorescent probe substrates of CYP1A4, 2C19, 2D6 and 3A4 enzymes in human liver microsomes and their respective metabolites were quantified with the FlashQuantTM workstation. The results meet the quantitative requirements for CYP450 inhibition assay. Potential interference from substrates through in-source fragmentation were also studied along with ion suppression from substrates and inhibitors. CYP3A4 inhibition samples using midazolam as a substrate and ketoconazole as a test inhibitor were analyzed. The results show a good correlation with literature.

High Throughput Automated LC/MS/MS Strategies for ADME Workflows: Software Based Automation for Method Development and Data Collection.
Loren Y Olson¹, Anthony J Romanelli¹, Christopher Borton¹, Wen-Chen Hsu², Pengdeth Lim², Huafen Liu¹, May Young², Jane Huang², Peadar Cremin³, Mark Warren³, Ying Bao³, Manping Ji³, Kerry Koller³, Quincey Wu³ and Elliott B Jones¹
1. Applied Biosystems, 850 Lincoln Center Drive, Foster City, CA 94404
2. Roche, Palo Alto, CA, USA
3. Xenoport Inc., Santa Clara, CA, USA

Novel experimental software algorithms were designed and evaluated for advantages in throughput, ease of use, and data quality in typical ADME workflows. Future revisions of DiscoveryQuantTM Software are to include these novel algorithms. The automated software package was capable of tuning compounds, storing tune data and method info in a database, making LC/MRM method files and submitting batches. The automated flow injection tune algorithm that was evaluated is capable of choosing ion path voltages within 2 eV of the optimal values resulting in MRM sensitivity equal to or better than traditional infusion methodologies. Two traditional ADME assays were analyzed using the novel software, an MDCK permeability assay and a metabolic stability screen using human hepatocytes. The results from these assays match previously observed results.

Quantitative determination of glycopyrrolate in human plasma by liquid chromatography – electrospray ionization mass spectrometry
R. t’Kindt, M. Storme, W. Goeteyn, J. Van Bocxlaer
Laboratory of Medical Biochemistry and Clinical Analysis, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium

Glycopyrrolate (GLY), as a synthetic quaternary ammonium (QA) compound, has been used for decades as an antisialogogue, vagolytic and gastric volume reducing drug [a]. Irrespective of the fact that the use of glycopyrrolate has been cut back during the last twenty years, many anesthesiologists still routinely make use of GLY, particularly by painful and anxiety-provoking intramuscular injection [b].
The analysis of QA drugs through liquid chromatography is not straightforward due to their cationic character. Ion-pair chromatography, coupled to MS, might be the only appropriate approach. Nevertheless, a high concentration of non-volatile conventional ion-pair reagents would preclude this approach. Here we report a reversed-phase liquid chromatography electrospray ionization mass spectrometry method for quantitative determination of the QA compound GLY by using volatile ion-pair reagents. Mepenzolate was used as an internal standard. The method running on a QTOF instrument allows quantitation of GLY between 0.1 and 100.9 ng/ml plasma. The chromatography, using the volatile ion-pair reagent heptafluorobutyric acid (HFBA), takes only 10 minutes, allowing a fast sample turnover time. Extraction from plasma was based on a simple and relatively fast liquid liquid ion-pair extraction, also using HFBA as ion-pairing agent.
The assessment of matrix effect is a crucial step during the bioanalytical method validation. According to Matuszewski et al. [c], the degree of ion suppression for an analyte and an internal standard may be different in different lots of the same plasma. In that respect, absolute and relative matrix effect, absolute recovery and process efficiency were determined for our method using four different lots of plasma. As a result, mean absolute matrix effect was 125.0 ± 19.18% (mean ± sd; n=3), while mean relative matrix effect was 15.94 ± 7.225% (mean ± sd; n=4). Also, absolute recovery and process efficiency were acceptable with values ranging from 78.27% to 90.76% and 83.14% to 132.5%, respectively.

[a] C. A. Bernstein, J.H. Waters, M.C. Torjman, D. Ritter, J. Clin. Anesth. 1996, 8: 515-518
[b] E. Kentala, M. Salonen, J. Kanto, Acta Anaesthesiol. Scand. 1990, 34: 17-20
[c] B. K. Matuszewski, M. L. Constanzer, C. M. Chavez-Eng, Anal. Chem. 2003, 75: 3019-3030

BIOMARKERS: FROM DIAGNOSTIC TOOL TO GLP VALIDATED EFFICACY DETERMINATIONS
Jan Dankers, Ole Lagatie, Marcel van der Linden, Jean-Paul Boon
Eurofins Medinet, Bergschot 71, 4817PA, Breda, The Netherlands

As for many biomarkers, like immunoassays, many commercially available (diagnostic) assay kits are designed for research only and are not always suitable for efficacy or pharmacodynamic examination.
We will present our approach how to evaluate, modify and validate commercially available diagnostic tests to turn into assays suitable for PD determinations. An assay life cycle can be categorized into 3 general phases: method development (evaluation and optimization phase), pre-study validation and in-study validation. During method development, an assay concept is evaluated, which will be confirmed during the pre-study validation phase, and applied during the in-study validation.
All three phases are conducted under and supervised by an assigned Study Director.
The evaluation phase includes an assessment of assay range, minimum required sample dilution, potential matrix interferences, and approaches for minimizing matrix interference when necessary.
Depending on the outcome of this evaluation phase, work will progress to modify the method in order to become suitable for a formal pre-study GLP validation according to the FDA Guidance for industry: Bioanalytical Methods Validation Center for Drug Evaluation and Research (May 2001).
Pre-study validation characteristics are applied as quality control parameters for in-study validation.

Evaluation of a novel phenotyping cocktail for phenotyping three human cytochromes P450 iso-enzymes CYP2D6, CYP2C19 and CYP3A4 in healthy volunteers.
M.P. van Iersel, T. de Boer, J. B. Hak, J. Wieling, R. S. Schwietert, I. den Daas and J. Wemer.
Xendo Drug Development BV, Hanzeplein 1, 9713 GZ Groningen, The Netherlands

Variability in drug metabolism attributes to the variability in drug response and incidence and severity of adverse events. Cytochrome P450 iso-enzymes play an important role in drug metabolism and genetic polymorphism has been identified for some of these enzymes. Poor metabolisers have low drug metabolizing enzyme activity compared to the majority of individuals defined as extensive metabolisers. This variability may result in a different pharmacokinetic, pharmacodynamic and adverse event profile. For phenotyping procedures drugs are either given alone or in combinations of CYP specific substrates. Phenotype cocktails to evaluate cytochrome P450 activity have been used widely by measuring the parent compound and its metabolite(s) in plasma or in urine.
Genotyped and phenotyped healthy volunteers are important in clinical pharmacological studies to help elucidate the aethiology of side effects or deviations in pharmacokinetics and/or pharmacodynamics. Phenotyping cocktails for phenotyping human cytochromes which can be given routinely with a simple administration and sampling scheme are essential.
In this study a phenotyping cocktail of dextromethorphan, omeprazole and alprazolam for phenotyping healthy volunteers for three human cytochrome P450 enzymes (viz. CYP2D6, CYP2C19 and CYP3A4/5) was studied to see if the combination could be given without changing the activity of one of the enzymes by the concurrent drugs given together.
In addition, evaluation was done to see if the phenotype indices of metabolic ratios could be obtained in urine 0-8 h and/or in a single (or as few) plasma samples (as possible) for all three substrates.