Advanced Bioanalytical Method Development and Sample Analysis Service

LC Systems

• 11 x Waters Acquity UPLC (1 with 2D technology)
• 4 x Agilent 1290 Infinity 2D uHPLC
• 2 x Agilent 1200 uHPLC

GC Systems

• 1 x Waters Atmospheric Pressure Gas Chromatography (APGC)

UV and Radiochem Detectors

• 2 x Acquity UPLC PDA detectors
• 1 x Agilent DAD
• 1 x LabLogic β-RAM^® radiochemical detector

Mass Spectrometers

• 11 x Waters Xevo^® TQ-MS
• 4 x Applied Biosystems Sciex QTRAP^® 5500
• 2 x Applied Biosystems Sciex TripleTOF^® 6600
• 1 x Waters Xevo^® G2-S QTof
• 2 x Agilent 6410
• 1 x Waters QDa

All our LC-MS/MS systems are protected by an uninterrupted power supply system ensuring power input fluctuations are smoothed out/reduced and the systems are supported by a limited battery backup power supply in case of mains power failure.

Cyprotex has expertise in a wide range of bioanalytical techniques including:

• sensitive small molecule detection and quantification
• high resolution metabolite ID studies
• peptide biotherapeutic bioanalysis

Advanced bioanalytical method development service offering

How would you approach a bioanalytical method development study?

Cyprotex feel that it is important to work closely with clients for these studies. It is vital to identify specific customer requirements or issues prior to initiating the work. For example, the customer may have a need to detect compounds at a particular level for future routine studies or they may be aware of specific issues such as light sensitivity or stability. It is beneficial to have as much information as possible from the customer regarding the compound e.g., structure, physicochemical properties or previously developed methods. This will help us to identify the optimal conditions with respect to chromatographic columns and mobile phase gradients.

Method development is performed in 4 phases. The client will be consulted at each stage of the process.

1. MS/MS Optimization – If the preferred method of detection is mass spectrometry, initially, the compound is optimized using either ESI (electrospray) or APCI (atmospheric pressure chemical ionization) in both positive and negative modes. The purpose of this step is to characterize the precursor and product transition parameters required for sample analysis.
2. Chromatographic Method Development – The compound is assessed using established Cyprotex chromatographic methods based on C18 reverse phase chromatography. Calibration standards are prepared in a ‘matrix free’ environment to assess the sensitivity and linearity of the compound under the established conditions. If the initial investigation identifies that the existing Cyprotex chromatographic method is not suitable for the compound then a specific bioanalytical method is developed. Depending on the results obtained in the initial investigation and the structural and physicochemical nature of the compound, a number of different column chemistries (e.g., phenyl, HILIC or C8) and mobile phases can be assessed. Alternatively, should simple 1D chromatography not provide sufficient selectivity, a number of 2D methods can be employed to generate a more robust methodology. Once an appropriate method has been developed, matrix free calibration standards are used to assess the sensitivity of the compound response at this stage.
3. Sample Preparation – For small molecule analysis in plasma, protein precipitation is the most convenient and cost effective method of sample preparation. Therefore, protein precipitation is assessed for its suitability as an initial assessment. Urine is diluted with high purity water and injected directly onto the LC-MS/MS system. Alternative matrices can be investigated. Two calibration standards (low and high concentrations) are selected to assess recovery. If the initial investigation identifies that protein precipitation is not a suitable method for sample preparation then alternative methods (e.g., SPE, SLE, PL depletion etc.) may be investigated. The recovery does not need to be 100% but the extent of the recovery of an analyte should be consistent, linear, precise and reproducible. For peptides and other biotherapeutics a more structured sample preparation approach is adopted in consultation with the client.
4. Linearity in Matrix – Once a suitable extraction method is identified, calibration standards are prepared in the relevant matrix and analysed to confirm linearity. QC samples are analyzed at low, medium and high concentrations to check the validity of the standard curve.

Describe the advanced bioanalytical method development procedure.

This may evaluate linearity and sensitivity, accuracy and precision, inter and intra batch variability, selectivity, matrix effects and stability. The process can be customized depending on specific customer requirements.

How are data reported to the customer?

For the method development studies, details of the protocol for the bioanalytical method along with any supporting data generated during the development process is presented as a written report.

Example reports are available on request.

Do you offer this service to GLP?

Although these studies are not performed to GLP they are able to follow the relevant aspects of the current regulatory guidance;

FDA Guidance for Industry for Bioanalytical Method Validation (May 2018)².

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EMA Guideline on Bioanalytical Method Validation which came into effect in February 2012³.

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¹ Pandey S, et al. (2010) Bioanalysis in drug discovery and development. Pharm Methods 1(1); 14-24
² FDA Guidance for Industry for Bioanalytical Method Validation (May 2018)
³ EMA Guideline on Bioanalytical Method Validation (Feb 2012)