Instruments and Capabilities

Agilent  GC-MSD

In the era of metabolomics, GC-MS has resurged and has increasingly gained importance. GC-MS is devoid of the notorious problem of ion suppression/ion enhancement that limit the use of LC-electrospray MS for such applications.

The assay established on iC42’s GC-MS system include fatty acid profiling, profiling of HDL lipids, metabolomic profiles in plasma, urine and cell extracts, labeled glucose and glycerin analyses and labeled nucleotides after H2O2 exposure.  iC42 has developed a unique 1H-NMR/ GC-MS metabolomics strategy that is based on the measurement of a sample using 1H-NMR spectroscopy, and since a non-destructive technology, subsequent derivatization of this sample and analysis by GC-MS.

This strategy combines the strength of both technologies and since there is only little overlap in the metabolite spectra detected, complementary metabolite information covering wider spectra in terms of metabolite structures and concentration ranges. This instrument is equipped with the AMDIS (Automated Mass Spectral Deconvolution and Identification System) software and the latest version of the NIST metabolite database. 

Agilent 1100/ 12XX Series HPLC systems

The standard set up of our HPLCs is as 2D-LC systems with high-flow online extraction and peak focusing. This allows for a high degree of automation and usually reduces sample preparation to a simple one-step procedure consisting of protein precipitation and simultaneous addition of the internal standard. Most of our assays are based on 96-well sample plates. The combination with online extraction not only leads to automation, but also decreases intra-assay variability and total imprecision due the elimination of multiple manual pipetting steps. It also reduces the chance of random errors such as switching of sample vials.

HTC PAL Leap Autosamplers

This technology allows iC42 to have complete control automated sample injections. Workstations can handle large volumes and since more or less laboratory pipetting robots enable us to provide a variety of tasks. In our experience the HTC Leap autosampler is the autosampler with the least carry over due to extensive needle and loop washing capabilities. Another advantage is the number of cooled sample trays that are of importance in the age of high-throughput LC-MS/MS analysis.

Thermo Scientific Exactive Orbitrap with UPLC & Turbulent Flow Inline Extraction System

The discovery platform of choice for comprehensive protein and metabolite identification, characterization and quantitation.  The orbitrap technology routinely delivers the highest-resolution and mass accuracy needs with a mass accuracy of typically 1 ppm and better and thus clearly outperforms time-of-flight mass spectrometers. 

Combined with superior dynamic range and unsurpassed sensitivity, Orbitrap platforms are the only technology capable of providing all four benefits at the same time, with out compromise. One of the reasons is a trap that has a very high capacity. In our hands the Exactive mass spectrometer has the same sensitivity as low to mid-end triple stage quadrupoles while recording full scan spectra at a 100,000 resolution.

At iC42, this instrument is used for studies assessing blood and urine samples for yet unknown metabolites (e.g. in pediatric patients), for drug metabolism studies, and for toxicology and drug screens. One of the major advantages is that, in contrast to quadrupoles that will only detect what the quadrupoles are focused on, full high-resolution spectra are recorded that allow for re-evaluation and re-analysis later, should for example a metabolite or an at the time unknown drug be found later and the question is if this was already present in a patient’s sample.

Quadrupoles typically would require re-extraction and re-analysis of a sample, raising concern about the sample’s long-term and freeze-thaw stabilities. One of the strengths of the orbitrap is its potential to discover unknowns and to identify those based on the exact molecular mass, a feature that is of great potential in toxicology and drug screening.

Agilent LC/MSD Trap XCT Ultra (several)

iC42’s high-performance ion trap mass spectrometer allows our scientists the to identify 60 to 80 percent more peptides than preceding models in proteomics applications, such as protein identification and biomarker discovery. The Agilent 1100 Series LC/MSD Trap XCT Ultra offers significantly improved scan cycle times, enabling collection of mass spectra at rates up to three times faster than its predecessors.

iC42 uses these instruments in combination with nano HLPC and chip sources for proteomics and in combination with an online extraction HPLC system (2D-LC) for the structural identification of drug metabolites and for the structural identification of compounds synthesized by our medical chemists. The high sensitivity of an iontrap’s full scan mode has been used to identify drug metabolite structures directly in patients’ blood.

Thermo Electron DELTA V advantage

Considered the next generation in isotope ratio MS this technology combines the highest sensitivity with the versatility needed for iC42’s advanced clinical research and development projects. The system has an automated combustion chamber for the analysis of doubly labeled water as a measure of energy expenditure and for the analysis of 13CO2 in breath samples.

Varian INOVA NMR 600MHz spectrometer

NMR-based chemical shift imaging technologies have extensively been used for monitoring metabolic changes in vitro, ex vivo and non-invasively in vivo. NMR is non-destructive, highly discriminatory and can quantify compounds in rather crude samples without the requirement for extensive sample clean up.

iC42 metabolomics studies typically include an 1H-NMR-based metabolite screen as a first step. The instrument is also used for structural identification of metabolites, drug isomers, degradation products and newly synthesized drugs and their intermediates.

Agilent Mass Selective Detector

This instrument is a robust and highly sensitive mass spectrometer that is typically preferable if the specificity of a more complex tandem mass spectrometer is not required.

At iC42, this instrument is typically used for the development of sample preparation strategies, chromatography, the quantification of drug metabolites (in vitro enzyme kinetics, clinical trials and, as LC-UV-MS system, for the establishing purity of isolated metabolites). In addition, this instrument is used for targeted pathway assays such as the quantification of high energy phosphates.

Agilent TOF

This technology provides accurate mass analyses for profiling, identifying, characterizing and quantifying low molecular-weight compounds.  This system delivers exceptional MS analyses that support demanding applications such as proteomics, metabolomics, impurity testing, and product degradation studies. At iC42 largely replaced by the more high-resolution orbitrap, this instrument is valuable for LC-MS metabolomics studies and the analysis and quantification of >20 amino acids in different matrices.

MDS Sciex API 4000 (several) 

The API 4000 LC/MS/MS System is a triple mass spectrometry system that iC42 uses clinical samples.  This system comes with a mass range of 5-3,000 Daltons and a mass accuracy of 0.01%. These instrument typically allow for the quantification of drugs with lower limits of quantification between 25-100 pg/mL depending on the compound. As these instruments typically are the backbone of most quantitative industry bioanalytical laboratories, at iC42 they are used for clinical therapeutic drug monitoring, most pharmacokinetic studies, the analysis of drug from dried blood spots and for the quantification of endogenous compounds and molecular markers such as vitamin Ds.

MDS Sciex API 5000 (several) 

Clinical research organizations require advanced tools for critical safety and with the improved sensitivity provided by this LC/MS/MS system, iC42 has the ability to assay a drug candidate, its metabolites or its contaminants.  These systems are considered the next generation platform for bioanalytical research and typically has a sensitivity >10-fold better than those of the API4000 instruments. The API5000 allows us to reach lower limits of quantification that are in the realm that previously could only be reached with radio immunoassays. Thus this instrument has been used for replacing several immuno assays with more specific and robust assays. A representative example us our assay to quantify isoprostanes in the plasma and urine of healthy individuals and patients. Isoprostanes are stable markers of oxidative stress and the lower limit of quantitation in plasma is 2.5 pg/mL. In combination with a sophisticated, yet robust and automated sample preparation strategy including online high-flow extraction and peak focusing, we have achieved lower limits of detections as low as 330 fg/mL for sirolimus derivatives in whole blood of patients after implantation of drug-eluting stents.

MDS Sciex API 5500 QTRAP 

The combination of best-in-class sensitivity and unsurpassed speed allows our scientists to analyse a comprehensive range of compounds in a shorter period of time than it currently takes with existing triple quadrupole systems.  This robust system delivers the most accurate quantitative results at the industry’s fastest scan speeds to identify the greatest number of target analytes at the lowest concentrations in complex samples.

This new level of speed and sensitivity enables a greater amount of data to be acquired from an experiment, which leads to greater quantitative accuracy and lower limits of detection, compared to competitive triple quadrupole instruments.  The end result is faster analysis than what was previously possible, enabling scientists to measure a greater number of compounds in a single pass. Equipped with nano HPLC and alternatively with an Advion source, this instrument is used for discovery proteomics (MUDpit), quantitative proteomics SILAC and iTRAQ labeling technologies,  as well as for the quantification of peptides and proteins. In combination with the orbitrap instrument, it is used as a second and confirmatory strategy for our toxicology, drug and WADA drug of abuse screening assays. The instrument is fully equipped with proteomics and biomarker software and databases.

Waters Acquity UPLC-MS/MS 

The Waters Acquity features the highest levels of tandem quadrupole MS selectivity, robustness, speed and accuracy.  This instrument is usually used for high troughput assays that do not require ultimate sensitivity, but the specificity of an MS/MS system. Examples for iC42 assays set up on this instrument are an assay to quantify the immunosuppressant mycophenolic acid and all its metabolites including its potentially toxic acyl-glucuronide metabolite and an assay to quantify metabolite kidney dysfunction markers (glucose, creatinine, hippurate, trimethylamine oxide, citrate and succinate) in urine.