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A paradigm shift in toxicological risk assessment: more science, less animal testing
Bas Blaauboer (Universiteit Utrecht, NL)

The paradigm of toxicological risk assessment contains the elements of hazard identification, hazard characterization, exposure assessment and risk characterization. For different areas and applications of the process of risk assessment, these elements have a different weight. The basis for the process of determining risks is the ability to quantify toxicological endpoints, in most cases in animal models. The next steps are the interpretation of the results, assessment of the uncertainty in the data and extrapolation of the outcome of the experimental findings to the risk situation one needs to assess, e.g. for human health.
The heavy reliance on animal models in toxicity testing has prompted many ethical issues. Furthermore, the costs of animal experimentation becomes a serious burden. Also from a scientific point of view the usefulness of a risk assessment relying on the apical endpoints in animal studies is doubted in many cases.
Thus, the classical way of toxicological risk assessment needs redefinition in the light of the modern paradigm of toxicology, viz. that toxicity is depending on the proper dose metric (i.e. concentration or concentration in time) of the proper compound (might be a metabolite) at the most critical site (i.e. the target at which the primary mechanism occurs).
This implies that risk assessments will need to be more chemical-specific. This will be possible if we can make use of data on the mechanism of toxicity in combination with an estimation of the target concentration. The application of new technologies such as the “omics”, a better use of in vitro methodologies and of (physiologically-based) kinetic modelling will allow such an integrated approach.
What is necessary is the integral use of data from the above-mentioned areas in a transparent and logical way. Ideally, the new paradigm in toxicology would then allow a safety testing for compounds without the use of animal experimentation.

ALTERNATIVE TESTING STRATEGIES TO REDUCE ANIMAL USE IN PRECLINICAL DEVELOPMENT AT J&JPRD BELGIUM
Bas-Jan Van der Leede (Johnson & Johnson Pharmaceutical Research & Development, a division of Janssen Pharmaceutica N.V., Genetic and Exploratory Toxicology, Turnhoutseweg 30, Beerse, BE)

Animal studies are still an essential part of the preclinical safety assessment in the development of new pharmaceutical drugs. However the number of animals to be used can be reduced significantly by applying different study designs and introducing alternatives for animal testing. At J&JPRD several initiatives have been implemented that contribute to animal welfare by replacement, reduction and refinement of animal use in preclinical development of pharmaceutical drugs. An additional advantage of the initiatives replacing animal testing is that pharmaceutical drugs can be screened at higher throughput, thereby delivering results faster. In order to identify the toxicity potential of new drugs correctly, bio-analytical research can play an important factor in these screening assays. The following initiatives will be presented in more detail.

Integration of in vivo micronucleus test (MNT) in rat toxicity studies
The MNT and the 2-4 week rat toxicity study are both part of the preclinical safely package submitted to regulatory agencies for authorization of first-in-human trails. Integration of these studies saves approximately 120 animals per package.
In order to predict the potential human risk to develop cancer, the MNT needs to be conducted at the highest possible systemic exposure of the drug. Since the 2-4 week rat toxicity study is typically conducted at lower doses, acceptance criteria for dose selection are used at J&JPRD to decide whether integration of the MNT is justified.

HET-CAM
Due to the fact that injection site reactions can result in serious problems during preclinical i.v. infusion studies, there was a rising demand to develop and implement an in vitro screening model to identify vascular toxicity prior to animal testing. The HET-CAMVT(Vascular toxicity Hen’s Egg Test-Chorioallantoic Membrane), developed by J&JPRD, is an in vitro model in which three irritation effects (hemorrhage, coagulation and lysis) are evaluated after topical application of a formulation on the CAM of fertilized chicken eggs. Experiments in which formulations with known in vivo injection site reactions were applied, showed that the HET-CAMVT model is a very sensitive assay, able to generate reproducible and quantative results.

Zebrafish
Zebrafish have the potential to accelerate and de-risk the development process by reducing attrition rates and lowering the development cost of new pharmaceutical drugs. With a significant genetic similarity to humans and the presence of many vital organs including heart, brain and liver, the larval zebrafish is highly suitable for screening potential drug candidates for efficacy and safety effects. At J&JPRD, zebrafish are used to predict hepatotoxic properties of pharmaceutical drugs in development.

In vitro liver toxicity predicts in vivo hepatoxicity using the microarray technique
Peter Olinga (Solvay Pharmaceuticals, Clinical Candidate Selection, Weesp / University Groningen, NL)

We have developed an in vitro system to study drug metabolism and toxicity in the human and rat liver. Model compounds of liver toxicity were studied with rat liver slices. The effect of these different hepatotoxins was analyzed at the gene expression level in the rat liver in vitro. Microarray analysis in rat liver slices correctly predicts in vivo hepatotoxicity. This in vitro technique, the precision-cut liver slices, mimics the human and rat liver in vivo.

Optimal kinetic screening (in-vivo and in-vitro) and evaluation
Jessica Dijksman (Solvay Pharmaceuticals, Weesp, NL)

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Recent Developments Towards Green Chromatography 
Pat Sandra (Research Institute for Chromatography (RIC), Kennedypark 26, B-8500 Kortrijk, B)

Chemists are (should be) more and more concerned about the environmental impact of their activities and at the end of the 20th century the term "green chemistry" was introduced. The "green chemistry" principles should also be applied to analytical chemistry and more especially to chromatography and its sample preparation procedures.
Several analytical methods are, from an ecological point of view, very unfriendly and, without any doubt, liquid chromatography (LC) is the first technique to be considered. The present consumption of acetonitrile (an EPA pollutant) in LC is enormous. Moreover, the waste disposal is incinerated resulting in the formation of NO2 that is linked to acid rain.
The current trend of LC analyses is biased toward high throughput, high productivity and high resolution. In response to these increasingly demanding requirements, over recent years, innovative technologies and improvements in instrumentation have emerged which are having a significant impact on our daily work. Ultra High Pressure LC (UHPLC) and Elevated Temperature (ETLC) both have extended speed, productivity and peak capacity for pharmaceutical, environmental, food, and bio analyses. The application of both developments in combination with a more or less forgotten technique i.e. sub- and supercritical fluid chromatography, often allows to translate "unfriendly" methods into green methods using as mobile phase constituents only water, ethanol and carbon dioxide with formic acid and ammonia as additives. Several examples will be shown to illustrate that conventionally used LC mobile phase compositions can easily be replaced by these green alternatives.

The Use of Dried Blood Spot Samples and Direct Analysis for the Quantitative Bioanalysis of Drugs
Paul Abu-Rabie (GlaxoSmithKline, Bioanalytical Science and Development, UK)

The surge in interest in dried blood spot (DBS) techniques for supporting pharmaceutical exposure studies is due to the many advantages it offers over conventional plasma sampling. These include the reduction in blood volumes required, with associated cost and ethical advantages, the simplification of clinical sampling procedures and the reductions in sample processing, storage and transportation costs. DBS analysis is now GlaxoSmithKline’s default technique for all new oral drug candidates, where methods can be validated. This strategic movement from plasma based analysis to DBS analysis has achieved a significant step forward in the toxicological 3Rs (refined bleeding techniques, reduced animal usage).

However, while organisations as a whole will realise these benefits, the switch to DBS analysis would seem to provide little advantage for the bioanalyst. Non-optimised automation, low sensitivity and increased ion suppression compared to plasma assays are some of the disadvantages that can be encountered with DBS analysis. The Bioanalytical Science and Development group at GSK is investigating direct analysis techniques for DBS samples that could readily counter these disadvantages, resulting in a process that is simpler than that currently used for plasma analysis. It has been demonstrated that such techniques not only have the potential to make significant time and cost savings in sample preparation, but also to significantly increase assay sensitivity, potentially allowing DBS analysis to include a wider range of compounds. Quantitative analysis using direct analysis has also been demonstrated without the use of chromatographic separation.

Evaluation of experimental drugs by bioanalysis: reduction of needed animals
Ben Westerink and Thomas IFH Cremers (Groningen Research Institute of Pharmacy, RUG and Brains-on-line BV, NL)

Development of experimental drugs aimed at the central nervous sytems requires detailed information about their levels in brain and blood. In addition information about the effects of the compounds on the biochemistry of the brain is needed. For a full pharmacokinetic and pharmacodynamic evaluation of an experimental drug, levels in blood as well as in the extracellular space are required. In addition changes of the brain biochemistry are to be recorded. To that end we use - in freely moving animals - chronic implantations of blood cathethers and microdialysis probes or sensors in laboratory rats or mice.
To reduce the number of experimental animals for these experiments we have followed 2 strategies:
a. The established method to sample extracellular space is microdialysis. However the method lacks quantitative information as the recovery of the compounds through the dialysis membrane is low and unpredictable. Methods to circumvent this disadvantage require high amounts of experimental animals. We have recently developed a probe that samples exogeneous and endogeneous compounds with a recovery close to 100%.
b. A sensitive derivatization method was developed that enables simultaneous determination by LC-MS of a large series of amino acid and amine-derived neurotransmitters and related metabolites.
Combination of both methods reduces to amount of animals needed to about 10% of the traditional approach.

Whole blood analysis with LC-MS/MS using direct injection
Jaap Verweij (Schering-Plough, Toxicology and Drug Disposition, Oss, NL)

Mass spectrometric developments in the last decade enable (sub)nanomolar detection of drug compounds in biological matrices in a few microliters of blood. However, the sampling and especially the handling of these small blood volumes is not straightforward. We studied the feasibility of a recently developed 'sorbent sampling technique' to handle these small blood volumes and the application to support pharmacokinetic (PK) screening programs. This technique applies 5-10mL of blood on a fibrous material packed into a cartridge. Blood samples absorbed on these cartridges are eluted directly, on-line onto a solid-phase extraction liquid chromatography/tandem mass spectrometry (SPE-LC/MS/MS) system. It is shown that the sorbent sampling technique can be applied for a range of drug compounds. In spite of issues with recovery and sample clean-up that need further improvement, the sorbent sampling technique provided similar data as compared to conventional analytics. The technique was successfully applied to derive kinetic data from individual mice, thereby decreasing the number of required mice for a PK study from 21 to 3.

Addressing the Challenges of Limited Sample Volumes in Bioanalysis with Microscale LC-MS/MS
Paul Rainville (Waters, UK)

Small sample volumes from tail-bled rodents or pediatric studies place an extra challenge on the bioanalytical scientist; how to achieve the desired levels of sensitivity from limited sample volumes. The use of microscale separations have shown potential for the high-sensitivity analysis of limited-volume samples in the fields of proteomics and metabolite profiling. However, they have traditionally required a very experienced analyst and specialized instrument configurations. In this presentation, we will discuss the use of capillary-scale (300 µm) UPLC coupled to tandem quadrupole MS to achieve pg/mL levels of sensitivity from just a few microliters of sample. The analysis was performed on a standard tandem quadrupole high-sensitivity mass spectrometer with an easy-to-configure and exchange dedicated nano-spray source using a ceramic tile-based separations with integrated emitter. The design of the source and nano-tile correctly positions the separations device and emitter such that no user intervention is required. In these experiments with sample derived from rat plasma, the separations device showed excellent robustness towards the samples with greater than 1,000 injections obtained and maintained the chromatographic efficiency, such that the peak widths were similar or superior to standard microscale LC/MS.

Radioactive measurement: Combustion or Dissolving - Radioactive measurement of feces, blood, liver, brain or fat tissue, a Comparison of Dissolving with Combustion
E. Beek-van Velzen (Solvay Pharmaceutical Research Laboratories, C.J. van Houtenlaan 36, 1381 CP Weesp, The Netherlands)

Combustion of tissue is the most used technique to determine the amount of radioactivity. This combustion technique is very labour intensive and therefore a search for alternatives was made. Feces and blood were directly dissolved with SolueneÒ 350. Liver, brain and fat were dissolved with SolueneÒ 350 and the Adaptive Focused Acoustics wave technique of Covaris.
The solubility technique gave a recovery of 95-110% of the values found with combustion.

Bioanalytical Method Development for Therapeutic Peptides
Erin Chambers (Waters Corporation)

Developing bioanalytical methods for therapeutic peptides represents a significant challenge for many laboratories, particularly those that have historically worked with small molecules. This poster discusses the streamlining and the transition from small molecule bioanalysis to peptide bioanalysis by introducing logical step-wise MS, LC, and sample preparation method development approaches. Important considerations for chromatography of peptides for bioanalysis and an LC screening protocol are described. A highly selective SPE screening method which provides significant sample concentration while eliminating evaporation was developed based on 2 sorbents and 1 simple starting protocol. Results from 12 diverse peptide therapeutics extracted from human plasma are then provided as demonstration of the effectiveness of the methodologies outlined in the application. Emphasis is placed on maximum resource utilization and developing methods which are selective and robust enough to meet regulatory criteria typically associated with small molecule LC/MS/MS assays.
A bioanalytical method for the peptide desmopressin is developed using the screening methods and run through partial validation as an example of successful implementation of these concepts. The method uses 300 µL of human plasma and easily achieves sensitivity in the pg/mL range.

A Leap Forward in Sensitivity and Selectivity for PAH Analysis using GC-APLI-TOF/MS
Rob van der Heijden¹, Patrick van Houts¹, George McLeod², Thomas Arthen-Engeland³, Carsten Baessmann³, Armin Holle³ (1. Bruker Daltonics BV, PO Box 88, 1530 AB, Wormer, The Netherlands, 2. Bruker Daltonics, Coventry, United Kingdom and 3. Bruker Daltonik GmbH, Bremen, Germany)

Most polycyclic aromatic hydrocarbons (PAHs) containing more than three aromatic rings are considered to be genotoxic and carcinogenic to humans. Therefore, PAHs are classified as priority pollutants to be controlled in food and in the environment. PAH analysis has not been performed effectively with atmospheric pressure ionisation (API) mass spectrometry due to poor ionisation performance with standard ESI and APCI.
We present a major step forward in analytical capability for PAH analysis though Atmospheric Pressure Laser Ionisation (APLI). A novel APLI source is described for coupling of chromatography to Bruker TOF or Qq-TOF mass spectrometers. Efficient and highly selective ionisation is combined with a mass spectrometer offering stable and precise measurements of mass and isotopic patterns, plus the necessary dynamic range. The result is highly specific detection with order-of-magnitude improvements in sensitivity over existing techniques.

In this paper we report on an analytical method for the determination of the 16 PAHs by GC/APLI-MS with the aim to transfer this method into routine analysis. We report characteristic analytical method parameters and show applications in a (spiked) river sediment sample and in a real-life fish liver sample.

It is concluded that APLI-TOF-MS is a highly selective and efficient technique for PAH analysis. The high selectivity of ionization for PAHs enables ready detection in dirty samples. In addition, the formula-based identification based on accurate mass and isotope pattern gives greatly enhanced identification over GC-q-MS.

Improved Peptide Identification with an Ultra High Resolution Quadrupole Time of Flight MS (maXis)
Patrick van Houts¹, Rob van der Heijden¹, Markus Lubeck², Marina Behrens², Andrea Schneider², Carsten Baessmann² and Oliver Raether² (1. Bruker Daltonics, Wormer, The Netherlands and 2. Bruker Daltonik, Bremen, Germany)

Q-TOFs are widely used working horses for proteomics due to their superior mass accuracy in MS as well as in MSMS, making them ideal instruments for de novo sequencing and PTM characterization. For peptide identification at very low concentrations or for highly complex samples, MSMS sensitivity and duty cycle become especially critical. A new designed ultra high resolution Q-TOF MS (maXis) shows significant improvements for these types of samples.
In contrast to small molecule applications where MSMS sensitivity is usually defined via signal to noise of a single fragment, peptide identification has additional requirements: Completeness of a fragmentation pattern is more crucial than optimized intensities for single masses. Thus generation of fragment ions covering a broad mass range and their efficient transfer from the collision cell into the TOF part of the instrument were optimized. Using a higher order multipole as collision cell and an additional ion cooler for guiding ions into the orthogonal acceleration showed a dramatic improvement for proteomics applications.
As a model system for samples of high complexity, tryptic digests of E. coli, separated using a 90 min nanoLC gradient were used, leading to 7000-10000 fragment spectra and more than 600 significant protein identifications from 100ng sample. For single protein digests, analyzed with nanoLC, high sequence coverages could be obtained from sub fmol amounts.

Detection of Metabolites in Urine at Microdosing Levels in Rats Using LC-MS/MS
Carmai Seto¹, Tanya Gamble¹, Takeo Sakuma¹, Jinsong Ni², Fred Ouyang², Devin Welty² and Andrew Acheampong² (1. Applied Biosystems/MDS Analytical Technologies, 71 Four Valley Drive, Concord, Ontario, Canada and 2. Dept of Drug Safety Evaluation, Allergan, Irvine, CA, USA)

Many new chemical entities fail in Phase I clinical trials due to poor pharmacokinetics. Until Phase I, human pharmacokinetics can only be predicted from animal models. Microdosing is an approach which may make human clinical trails more effective, predictable and quicker because this approach allows for an earlier assessment of the drugs pharmacokinetics and metabolism in human sooner. Screening compounds for clinical trials using the microdosing approach can become more effective with the combination of the QTRAP® 5500 LC/MS/MS system and LightSight® software for metabolite identification.

Structure determination as part of metabolism and unknown ID necessitate sensitive and selective product ion spectra. These easily can be obtained by TripleQuadrupole instruments as well as IonTraps. Due to technical setup the former suffers from bad duty cycle while IonTraps typically show weak low mass fragments (70:30 low mass cut off). The latter can be bypassed by MSn fragmentation at the expense of loss in sensitivity and increase in cycle time. Nevertheless, these secondary or higher fragmentation pattern result in useful further information for structural elucidation.

Beside the use of MSn experiments for gathering additional structure information MS3 provides the capability to increase selectivity. Commonly Multiple-Reaction-Monitoring (MRM) is dedicated sensitive and selective mode for quantification on TripleQuad instruments. In complex matrices MRM selectivity sometimes suffers from high background and hence the sensitivity of method decrease. To prevent laborious development of new LC conditions the increase of MRM selectivity would be beneficial and time-saving to meet desired levels of sensitivity.

High Throughput Screening of GSH Adducts Using Hybrid Linear Ion Trap Systems Coupled with Fast Chromatography at Clinically Relevant Dose Concentrations
Hua-fen Liu¹, Hesham Ghobarah² , Renee Huang¹, Jens Dahlmann³ and Elliott Jones¹ (Applied Biosystems, part of Life Technologies, (1) Foster City, USA, (2) Concord, Canada, (3) Darmstadt, Germany)

Trazodone is a triazolopyridine derivative which is effective in the treatment of depressive disturbances, including depression associated with anxiety and sleep disturbances. Therapy is usually started at a total of 150 mg per day divided into two or three doses. Cmax was reached at 0.7 μg/ml (~1.82 μM), and 1.2 μg/ml (3.2 μM) after a single oral dose administration of 75 mg and 150 mg prolonged release trazodone, respectively. Overdosage of trazodone may cause an increased incidence or severity of any reported adverse reactions. Trazodone is highly protein-bound and rare but severe hepatic toxicity has been reported, which often described as idiosyncratic toxicity.

Trazodone is a 3A4 substrate and extensively metabolized by hydroxylation in humans.
Several substrate glutathione adducts (GSH) were reported in human liver microsomes at high substrate concentrations. Higher (10-50 μM) than clinical relevant substrate concentration and complex sample preparation were often needed in order to achieve the sensitivity desired. Investigation of GSH formation at lower substrate concentration (1-3 μM) which is at clinical Cmax relevant concentrations has not been explored due to the sensitivity limitation of current technologies.

A number of QTRAP® specific methods are possible for GSH detection. A combination constant neutral loss scan of 129 Daltons and a negative precursor ion of 272 has also been suggested as a superior method for GSH detection compared to single MS.
Historically, in order to detect all potential GSH adducts, two or more injections were often used due to instrument scan speed limitations. The QTRAP® 5500 system is the first LC/MS with the capability to do both negative precursor and positive neutral loss in one fast IDA (Information Dependent Acquisition) scan cycle and Linear Ion Trap (LIT) scan speed up to 20,000 Da/sec with superior sensitivity. Given these ground-breaking speeds, it is possible to perform a combined PI/NL with polarity switching in IDA, with 4 dependent ion trap MS/MS scans in approximately 2 seconds. Furthermore, the enhanced resolution and enhanced that all GSH formations and potential structure product ion scan ensure major information can be assigned from a single LCMS injection.

A second novel and highly effective method of GSH detection possible on the QTRAP® is pMRM (predictive MRM). This method uses the next generation MRM builder in LightSight software version 2.1 coupled with Excel™ to create a complete set of potential MRM transitions for any GSH biotransformation. In this case, a comprehensive set of greater than 80 phase I transformations can be created in a GSH specific way to produce a positive MRM list based on the neutral loss of 129 and a negative list based on the precursor of 272. This pMRM GSH method is fundamentally a pos/neg switching IDA experiment with 3 to 5 dependent LIT MS/MS scans. Since the MRM has a superior signal to noise ratio compared to all other survey scan modes, this often returns the greatest number of GSH confirmations in a single injection.

With these methods, a novel new approach with one injection as a generic high-throughput screening method can be achieved with sufficient sensitivity to ensure an accurate result at clinical relevant concentrations.

Simultaneous quantification of testosterone and androstenedione in human serum using LC-MS/MS
E. Joos^1,2, J. Van Bocxlaer², M. Vandermarliere¹ and K. Croes¹ (1. Clinical Laboratory, AZ Groeninge, Reepkaai, B-8500 Kortrijk. Belgium & 2. Laboratory for Medical Biochemistry and Clinical Analysis, Fac. Pharm. Sci., Ghent University, Harelbekestraat 72, B-9000 Gent. Belgium)

Testosterone (T) and androstenedione (AS) functionally belong to the androgen class of hormones. Androgens have a variety of effects on reproductive as well as non-reproductive tissues. T measurements consequently have many clinical applications, in men (e.g. hypogonadism), as well as in children (e.g. pubertas praecox) and women (e.g. hirsutism). T is extensively protein-bound in blood. AS, at the other hand, is a weak androgen but a chief precursor for many other androgens. AS measurements are equally diagnostic in many reproductive system disorders but also in certain tumours. T, AS and other androgens are generally quantified by immunoassay. Several literature reports, however, indicate that immunochemical measurements of T, more particularly very low T concentrations in woman, children and hypogonadal men, tend to overestimate the true concentration. Also for AS, poor correlations were found between RIA and e.g. LC-MS/MS. Cross-reactivity of closely related molecules and reduced overall selectivity of immunoassays have initiated the search for, generally chromatographic, alternatives.
Here, we describe the development of a fast and simple, fully validated LC-MS/MS method for the simultaneous quantification of T and AS. 400 µL of serum is extracted with diethylether and the extract is chromatographed using a Symmetry® C8 (2.1x100mm, 3.5µm) column with a turnover time of 7 min. MRM detection of T (m/z 289.2 > 96.9 and m/z 289.2 > 108.8), AS (m/z 287.2 > 96.9 and m/z 287.2 > 108.8) and their d3-labeled counterparts, used as internal standards, is achieved on a Waters Quattro Premier triple quadrupole system. Calibration curves (1/x weighting, in 0.1% BSA) were linear over the concentration range 0.05 to 15 ng/mL. Full validation according to the FDA guidelines revealed an LOQ of 0.05 ng/mL for both androgens, an intra-run precision of ≤7.2% (T) and ≤9.3% (AS) as well as a between-day precision of ≤8.7% (T) and ≤9.9% (AS) at 4 different QC levels (in human serum, n=6), and (between-day, n=6) accuracy data ranging from 97.0 to 105.8% (T) and from 100.5 to 105.3% (AS). External quality control samples were also evaluated using our method (n=12 for T; n=10 for AS). All but two results were situated within the (MS/MS method) mean ± 1 SD interval, all results within mean ± 2 SD. No significant matrix effect was demonstrated and recovery figures between 96.2 – 119.2% (T) and 99.7 – 122.8% (AS) were found. Freeze-thaw, short-term and long-term stability were all adequate. The impact of 4 different types of vacutainer tube was tested (n=5) and found to be not significant (multiple test corrected one-way Anova). Finally, the validated method was used in a method comparison with RIA (61 and 35 patient samples for T and AS, respectively). Passing-Bablok regression revealed statistically different regression slopes, precluding the cross platform use of reference intervals.

CHIRAL LC-MS-MS DETERMINATION OF (R)- AND (S)-CITALOPRAM IN HUMAN PLASMA SAMPLES
E. Mallat, V. Jas, J. Flik, M. Boterman, K. de Jonge (Analytisch Biochemisch Laboratorium (ABL) BV, Scholtenstraat 7, Assen, The Netherlands)

Introduction
Drug monitoring of antidepressants is a required issue in order to achieve optimal dosage of these compounds in patients and, therefore, avoid possible intoxications or any other medical complications. Citalopram is a selective serotonin reuptake inhibitor used as antidepressant. Its desired activity resides mainly in the (S)- enantiomer and, in less degree, in its metabolite (+)-(S)-desmethylcitalopram. In this work, the validation and application of a sensitive and enantioselective LC-MS-MS method for the chiral separation and quantitation of the enantiomers (R)- and (S)- citalopram in human plasma samples is presented.

Material and Methods
After homogenization, centrifugation and addition of internal standard (d6-(RS)-citalopram) to human plasma samples, liquid-liquid extraction at basic pH using t-butylmethylether (TBME) was performed to isolate citalopram from the matrix. A chiral chromatographic column CHIROBIOTIC V (Astec) modified covalently with macrocyclic glycoproteins has been used to separate both enantiomers [1]. The mobile phase consisted of a methanolic solution containing 0.05% formic acid and 0.01% ammonia. An API 4000 LC-MS-MS system (SCIEX) equipped with a Turbo Ion Spray interface working in the positive mode was used as detector.

Results
The assay was validated over the concentration range from 1 to 500 ng/mL for each enantiomer. Method validation was carried out to evaluate the accuracy and precision of the calibration range, and the quality control samples, the recovery of the enantiomers in plasma, the accuracy of the 10-fold dilution, and the stability of the racemic mixture and of each of the enantiomers (stability at 2-8oC, at room temperature, freeze/thaw stability and post-preparative stability). Accuracy of the method (expressed as bias) of the quality control samples was under 12%, whilst precision (expressed as C.V.) showed values from 2 to 5%. Stability studies revealed an acceptable low deviation (bias ≤15%) from the target value for the racemic mixture as well as for each of the enantiomers.

Discussion and conclusion
A sensitive and enantioselective bioanalytical LC-MS-MS method for the separation and quantitation of each enantiomer of citalopram has been developed and validated successfully. This method has been applied satisfactorily to determine (R)- and (S)- citalopram in clinical samples.

References
M. Kosel, C.B. Eap, M. Amey, P. Baumann (1998) J. Chromatogr. B 719 (1998) 234-238.

A sensitive LC-MS/MS method for the separate quantification of gentamicin C1, C1a, C2 and C2a in bovine tissue.
M. Boterman, M.J. Dröge, F. Oostebring, F. Venema & E. Oosting (Analytisch Biochemisch Laboratorium (ABL) BV, Assen, The Netherlands)

Introduction
Gentamicin is a broad-spectrum aminoglycoside antibiotic, consisting of a mixture of at least four components (C1, C1a, C2 and C2a) with three different molecular weights. In veterinary medicine, gentamicin has been used since many years for the treatment of bacterial infections in cattle. Therefore, tissues and milk intended for human consumption have to be analysed for their maximal residue limits (MRLs). The following MRLs have been established by the Committee for Veterinary Medicinal Products (EMEA) for total gentamicin (sum of C1, C1a, C2 and C2a) in bovine tissue: 200 µg/kg (liver), 750 µg/kg (kidney) and 50 µg/kg (muscle and fat) [1]. Consequently, sensitive analytical methods are required to determine these MRLs, and up till now, such a method was not available for the individual determination of gentamicin C1, C1a, C2 and C2a in bovine tissue. The ultimate goal of our work was to develop and validate a reliable and very sensitive LC-MS/MS method suitable for the determination of gentamicin C1, C1a, C2 and C2a in bovine tissue (kidney, liver, muscle and fat).

Materials and methods
After homogenisation of the tissue, the internal standard (tobramycin sulphate) was added and a protein precipitation was performed using trichloroacetic acid. After centrifugation, the samples were subjected to solid phase extraction (SPE) using Isolute® CBA SPE columns (3 mL, 100 mg). The evaporated residues were dissolved in injection solvent and injected into the LC-MS/MS system (Sciex API4000). Gentamicin C1, C1a, C2 and C2a were fully separated using a Hypersil® BDS C18 LC column (3 µm, 4.60 x 150 mm). The mass-spectrometer was equipped with a Turbo Ion Spray interface and operated in the positive ion mode. A full validation of the method was performed according the EC requirements [2]. The calibration range covered 0.10 MRL – 10.0 MRL.

Results
The assay was validated in the concentration range of 75.0–7500 µg/kg (kidney), 20.0–2000 µg/kg (liver) and 5.00–5000 µg/kg (muscle and fat) total gentamicin. The method showed acceptable accuracies (expressed as bias) and precisions (expressed as CV) for all four components in all tissues and, typically, a lower limit of quantification (LLOQ) of 0.10 MRL was achieved (Table 1). Moreover, the method was found valid with respect to recovery, specificity and stability (48h storage on the autosampler, one additional freeze/thaw cycle, 221 days long-term storage at -70°C) for all matrices.

Discussion and conclusion
At ABL, a sensitive bioanalytical LC-MS/MS method for the separate quantification of gentamicin C1, C1a, C2 and C2a in bovine kidney, liver, muscle and fat has been developed and validated successfully. The method produced accurate and precise results and very low LLOQs were achieved (down to 0.10 MRL) in comparison to other available methods (usually ≥0.50 MRL). Due to the low LLOQ and full separation of gentamicin C1, C1a, C2 and C2a, the assay has proved to be extremely suitable for quantitative bioanalysis and has, therefore, offered a competitive alternative to methods currently available for the investigation of the depletion kinetics of gentamicin in bovine tissue. Moreover, the method can be used as lead for the validation of the determination of gentamicin in other species.

References
1. EMEA 2001. Committee for veterinary Products. Gentamicin. Summary Report.
2. Volume 8 of The Rules governing Medicinal Products in the European Union. Notice to Applicants and Guideline. Establishment of maximum residue limits (MRLs) for residues of veterinary medicinal products in foodstuffs of animal origin, October 2005.

A sensitive automated online SPE-LC-MS/MS method for the quantitative determination of ivermectin B1a in bovine plasma.
M. Boterman¹, M.J. Dröge¹, F. Oostebring¹, M.A.H. Boezeman² and E. Oosting¹ (1. Analytisch Biochemisch Laboratorium (ABL) BV, Assen, The Netherlands and 2. Farma Research Animal Health (FRAH) BV, Nijmegen, The Netherlands)

Introduction
Ivermectin, a mixture of two homologous compounds ivermectin B1a and ivermectin B1b, is a widespread used antiparasitic drug in veterinary medicine and is routinely administered to millions of cattle per year. Over the years, the topical “pour-on” formulation has largely displaced the conventional injectable formulation in farming practices. Consequently, more sensitive analytical methods are required for the quantitative determination of ivermectin for pharmacokinetic purposes in bovine plasma samples. The objective of the present study was to develop and validate a sensitive and reliable bioanalytical LC-MS/MS method suitable for the determination of ivermectin B1a in bovine plasma.

Materials and Methods
Bovine plasma samples were supplemented with an internal standard (abamectin) and subjected to a protein precipitation using acetonitrile followed by online solid phase extraction (SPE) on HySphereTM C8 EC-SE cartridges using a SymbiosisTM Pharma system. Subsequently, the extracted samples were introduced into the LC-MS/MS system for quantification. The samples were chromatographed on a Zorbax Eclipse XDB-C8 column (3.5 µm, 75 x 4.6 mm). The mass-spectrometer consisted of a Sciex API 4000 equipped with an atmospheric pressure chemical ionization interface and was operating in the positive ion mode. A full validation of the method was performed according to the current guidelines for bioanalytical method validation [1,2]. The calibration range for ivermectin B1a covered 0.200 – 200 ng/mL in bovine plasma. The validation included the determination of the parameters: calibration, accuracy and precision, recovery, specificity, dilution, and stability.

Results
The assay for ivermectin B1a was validated in the concentration range of 0.200 – 200 ng/mL and a Lower Limit Of Quantification (LLOQ) of 0.200 ng/mL was achieved in bovine plasma. The method showed acceptable accuracies (expressed as bias) and precisions (expressed as Intra- and Total CV) for all QC levels (Table 1), which were determined in three validation runs. In addition, the method was found valid with respect to recovery (protein precipitation step), specificity, 20-fold dilution of samples with blank matrix and stability (bench-top and refrigerator stability (24 h.), on machine stability (72 h.) and stability after three additional freeze/thaw cycles).

Discussion and Conclusion
At ABL, a sensitive bioanalytical LC-MS/MS method for the determination of ivermectin B1a in bovine plasma has been developed and validated successfully. In contrast to other available methods for the determination of ivermectin B1a (usually with an LLOQ of 1.00 ng/mL), for the first time an automated online SPE technique was used, resulting in highly accurate and precise results and a very low LLOQ (0.200 ng/mL). Furthermore, the assay appeared to be extremely suitable for the quantification of ivermectin B1a in bovine plasma samples and thereby offered a competitive alternative to the methods available for the investigation of the pharmacokinetics of ivermectin B1a in bovine plasma. In addition, it should be noted that a similar method can be successfully applied for the determination of ivermectin B1a in other species, such as horse, dog and cat.

References
1. Shah et al. Workshop/Conference report; Bioanalytical Method Validation. A revisit with a decade of progress. Pharmaceutical Research 2000; 17: 551-1557.
2. Guidance for Industry, Bioanalytical Methods Validation. U.S. Department of Health and Human Services, Food and Drug Administration. June 2001.

New UHPLC systems from Eksigent Technologies for improved performance in LC/MS
Rein Reitsma (Separations Analytical Instruments)

Based on its Microfluidic Flow Control technology, Eksigent has recently introduced a new Nanochip- and Micro LC system that can operate at pressures up to 10,000 psi.

Our nanoLC-Ultra and cHiPLC systems have been developed to address the growing demand for higher resolution separations, smaller samples and complex analyses using nanoLC-MS. The increased maximum pressure allows for the use of longer columns or columns packed with smaller diameter particles.
The ExpressHT-Ultra system is designed for fast LC/MS of small molecules using microLC columns with an ID of 0.5 – 1 mm. Microfluidic Flow Control allows for excellent control for gradients as short as 30 sec. A newly designed active wash station ensures minimal carryover with cycle times as short as 1 min. Performance equals or exceeds conventional UHPLC systems, while saving up to 90% solvent. Applications of the new UHPLC system which demonstrate increased throughput and performance with mass spectrometric detection are presented.

DRIED BLOOD SPOTS FOR TK/PK AND BIOMARKER ANALYSIS AND 3R
Elwin Verheij (TNO Quality of Life, PO Box 360, 3700 AJ Zeist)

Dried blood spot sampling (DBS) as alternative to plasma collection contributes to 3R in a relatively simple straightforward manner as result of improved animal welfare (smaller sample volume) and reduction of the number of animals (less satellite animals). Acceptance and widespread application of DBS depends on the following issues (among others):
- quantitative data for TK/PK application
- very high sensitivity methods for PK of high potency drugs
- acceptance of whole blood values (plasma is the golden standard)
- compatibility with biomarker/omics analysis (disease/efficacy/safety)
- practical aspects, e.g. easy of use, robustness,..

We recently started investigating DBS for TK/PK, biomarker and omics applications in life sciences research and in this paper we will present some of our initial results.

Generic solid phase extraction procedure for the quantitative analysis of peptides in plasma
H. Korthals, H. Gerritsen, R. Brust-van Schaik and B. Buscher* (TNO Quality of Life, Zeist, The Netherlands)

The quantitative analysis of peptides in plasma can be very challenging compared to the bioanalysis of the more predictable ‘small molecules’. Method development is often a matter of trial and error. Problems encountered include, amongst others, adsorption of peptides to materials (glass, polypropylene, polyethylene), instability of peptides in standard solution and/or in plasma and strong binding of peptides to plasma proteins. A generic solid phase extraction (SPE) procedure has been developed for the quantitative LC-MS/MS analysis of peptides in plasma. The procedure is based on four different SPE materials and procedures. All peptides investigated with the developed SPE procedure had a recovery from plasma of > 80 % on at least one of the four SPE materials. Method development including a short pre-validation takes approximately one week.

EX VIVO HUMAN SKIN MODEL FOR PHOTOGENOTOXICITY TESTING
AA Reus, FAA van Acker, CAM Krul (TNO Quality of Life, Zeist, The Netherlands)

Photogenotoxicity testing aims at the early detection of the potential of compounds to induce tumors upon activation with UV (photocarcinogenicity). In contrast to standard genotoxicity testing, there are no equivalent in vivo photogenotoxicity assays for additional evaluation of a positive (or equivocal) result in vitro, or in vivo tests can not be used, as for personal care products, due to EU legislation. Moreover, oversensitivity and the occurrence of pseudo-effects with in vitro assays have become a major problem. Consequently, the number of false positives and unnecessary in vivo photocarcinogenicity studies will increase.
For these reasons, a relevant in vitro assay for evaluation of a photogenotoxic potential in skin, was developed using ex vivo human skin.
Advantages of the human skin model are:
- best mimicking the human situation
- target organ for photocarcinogenicity
- relevant barrier properties of the stratum corneum
- metabolic capacity
Human skin is obtained from surgery. Circular membranes are cultured in an air-liquid interface and exposed to test chemicals for 1 h, either via the medium or the topical site of the skin, followed by UV exposure (5 J.cm-2 UVA, 0.19 J.cm-2 UVB) and preparation of cell suspensions for comet analysis. Predictivity of the photo-comet assay was evaluated using 6 compounds (fluoroquinolones) with varying degrees of photosafety liability. Sparfloxacin, lomefloxacin, and ciprofloxacin (known photocarcinogenic potential) clearly demonstrated a dose-related increase in %tail DNA in the presence of UV, while the non-photocarcinogenic compounds levofloxacin, gemifloxacin and gatifloxacin did not. The %tail DNA was not increased in the absence of UV. In parallel, the same compounds were evaluated in the in vitro photo-comet with L5178Y cells and an in vivo skin photo-micronucleus assay with rats, and compared with results of ex vivo human skin to develop a tiered testing strategy for photogenotoxicity assessment. The results with human and rat skin were comparable, while the in vitro photo-comet appeared to be more sensitive.
Results thus far indicate that the human skin model can be a relevant alternative for photogenotoxicity evaluation of compounds that reach the skin, such as personal care products and pharmaceuticals.

Monitoring Matrix EffectsM in Biological Samples Utilizing Dual Scan MRM Mode Mass Spectrometry
Paul D Rainville, Joanne Mather, Robert S Plumb, Jing Lin (Waters Corporation, Milford, MA)

The process of developing LC/MS methodology for the purpose of quantitative assays for medicines and their associated metabolites is a daunting challenge. Human and animal biofluids such as urine, plasma and bile vary in components and complexity. Additionally, endogenous analytes generated by a subject’s age, gender and medication regimen can further interfere, contributing to the complexity in quantitative bioassay development and validation. Chromatographic techniques that utilize sub 2 micron particles have shown significant contributions to the area of quantitative bioanalysis. This is due to the increased chromatographic efficiency and subsequent generation of sharp chromatographic peaks, often 2 – 3 seconds at base, resolving analytes of interest from interfering matrix components.
However, even under the best chromatographic performance co-elution can occur. Therefore implementing an approach whereby qualitative MS scan data obtained from the matrix is simultaneously acquired with quantitative MRM MS data can aid in the monitoring of potential interfering compounds ensuring assay robustness and reproducibility. Furthermore, the fact that scan data is obtained simultaneously with the MRM data means data mining can be performed at a later time if questions arise as to the presence of analytes not targeted in the original analysis.
Here we present the utilization of simultaneous acquisition of MS scan and MRM data coupled with liquid chromatography utilizing sub 2 micron particle columns as a method development tool for quantitative bioanalytical applications

The CMA 64 IView microdialysis Catheter
Noud Grimberg (Aurora Borealis Control B.V., Postbus 2, 7760 AA Schoonebeek)

The CMA 64 IView microdialysis Catheter is a sterile, single use device intended for intravenous monitoring through a standard peripheral vein catheter (PVC) up to 72 hours. The clean samples and possibility for frequent sampling makes it ideal for research.

Small substances diffuse into the IView catheter reflecting accurate concentrations in blood. It is perfused with a physiologic solution, containing the anticoagulant dalteparin sodium, to avoid clotting

Accuracy of incurred sample results, the final validation challenge in quantitative bioanalysis ?
Theo de Boer, Marjan Hagenouw, Rudy Francke and Jaap Wieling (Xendo Drug Development B.V., Hanzeplein 1, NL-9713 GZ Groningen, The Netherlands)

Since incurred sample reproducibility validation has found a common ground in today’s bioanalysis, a final challenge remains relatively unattended sofar, i.e. accuracy of incurred sample results. One of the main reasons for this is the disability to obtain or prepare a quality control sample that represents the study samples, caused by the phenomenon that each individual sample from the same and from different subjects is unique in its composition and hence its behaviour in a bioanalytical method.
We propose a methodology that, similar to incurred sample reproducibility determinations, utilises study samples to determine the accuracy of the results obtained for them, by applying the standard addition approach and then determine the spike recovery.
We have set up such an experiment for a bioequivalence study for alendronate. Therefore, a relatively complex urine method involving derivatisation and solid-phase extraction with LC-MS/MS quantitation with stable isotope labelled alendronate as the internal standard was developed and validated prior to the study sample analyses.
Subsequently study samples at three time points from several subjects were analysed in singlicate. In two separate batches selected samples were repeated in duplicate after additional spiking with analyte at two levels. These batches were executed by two different technicians to include inter-analyst variation
The results enabled us to both evaluate incurred sample reproducibility but also spike recovery and spike recovery repeatability. The spike recovery was then considered to be a measure of incurred sample accuracy.
In general, the results for this approach looked very promising. For the samples investigated by this approach generally the spike recovery was 80-120%, with repeatability being less than 4% and reproducibility less than 15% for all samples.
For this method applied to this study we showed that the spike recovery approach to determine accuracy of incurred sample results performed satisfactory.
Thereby a proposed methodology is obtained to determine the accuracy of incurred sample results, that may be used by the bioanalytical community to serve as a basis for further discussions. We challenge others to build up experience with this approach in order to be able to exchange experiences and further tune the approach.

Application of Dried Blood Spot sampling within clinical trials: Feasibility study of pardoprunox, a potent anti-Parkinson Drug
O.A.M. Brockhoff¹, P.H. van Amsterdam¹, J. Thomson², C. Troupe³ (1. Solvay Pharmaceuticals, Global Bioanalytics Department, Weesp, The Netherlands 2. Aptuit Inc, Riccarton, Scotland, 3. Quintiles Laboratories, Marietta, GA, USA)

To assess efficacy and safety of new drugs in development numerous trails in patients are conducted. Often during these trails blood samples are collected to determine drug and metabolite levels and/or biomarkers. Blood sampling, plasma or serum generation, logistics and interim storage can be challenging and costly factors.
Dried blood sampling technique has been around for more then 40 years and is widely used in newborn screening for the detection of inherited metabolic diseases. As the technique could ease the sampling process in patient trails and especially largely reduce the costs of sample logistics and storage there may be great advantages to implement it. Obviously the analytes should still be measurable with the same performance characteristics as in the ‘classical plasma tube’ case.

To explore the possibilities of working with bloodspots, a feasibility study has been done at Aptuit. The primary goal was to investigate blood spot analyses of pardoprunox and its metabolites and to investigate stability at room temperature. A key prerequisite was: limits of quantification must be comparable to the bioanalytical method validated in human plasma.