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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.
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MS/MS Optimisation - Initially, the compound is optimised on the mass spectrometer using both ESI (electrospray) and APCI (atmospheric pressure chemical ionisation) in both positive and negative modes. The purpose of this step is to characterise the precursor and product transition parameters required for sample analysis.
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Chromatographic Method Development – The compound is assessed in clean solution using existing Cyprotex chromatographic methods using a Waters HSS T3 2.1 x 50 mm 1.8 µm column and a gradient consisting of either, 10mM ammonium formate and 0.1% formic acid in methanol and water, or 10mM ammonium acetate in methanol and water. Calibration standards in clean solution are prepared at this stage to assess the sensitivity of the compound response. 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 may be assessed e.g., phenyl, HILIC or C8 columns. Similarly, different gradient profiles and mobile phases may be investigated. Calibration standards in clean solution are used to assess the sensitivity of the compound response at this stage.
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Sample Preparation – For 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 solid phase extraction (SPE) 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.
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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 analysed at low, medium and high concentrations to check the validity of the standard curve.
Describe the bioanalytical method validation procedure.
Cyprotex offer both a partial or full validation service.
The partial validation typically evaluates linearity and sensitivity, accuracy and precision and selectivity. The full validation typically evaluates linearity and sensitivity, accuracy and precision, inter and intra batch variability, selectivity including matrix effects and stability. The validation process can be customised 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.
For the method validation studies, a written report is returned detailing the bioanalytical method plus validation data.
Example reports are available on request.
Do you offer this service to GLP?
Although these studies are not performed to GLP they do follow the current FDA Guidance for Industry for Bioanalytical Method Validation published in May 2001.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070107.pdf
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