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FOR BIOANALYSIS
Wahyu Utami, M.Sc., PhD, Apt
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Bioanalysis
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Sampling
• Sample is • Sample
Concentration
Compatibility
Cleanliness
• Target
analyte(s) not matrix
not compatible components
concentrated with or will interfere
enough for would be with the
quantitative harmful to analysis
detection. your
chromatogra
phic system
Goals of Sample Preparation
• Minimize Risk
• Minimise interference from biological matrices, such as protein,
peptides, lipids, inorganic salts, or surfactants from the formulations
of compound.
• Eliminate sample to sample variability (more reproducible
quantitation, more robust assays)
• Decrease assay variability
• Increased sensitivity
• Sample concentration
• Removal of interferences
• Cleaner Samples
• Increased instrument uptime
• Improve method robustness
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SAMPLE PREPARATION
METHOD
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Less selective
• Dilution
• Filtration
• Basic particulate removal from ALL kinds of samples
• Useful when additional step of lipid content removal is needed
• Protein Precipitation (PPT)
• Liquid-Liquid Extraction (LLE)
• Straightforward sample preparation technique
• Useful for in-house or commercial extraction
• Solid supported liquid extraction (SLE)
More selective
• Increased productivity using liquid/liquid extraction principle and the
concept of automation
• Ideal for aqueous sample or samples
• Solid-Phase Extraction (SPE)
• Ultra-clean sample preparation for analysis when high selectivity and
sensitivity are required
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Filtration
• Basic sample prep method for ALL kinds of samples
• Can be the 1st choice of sample prep or 2nd secondary step
• Mechanical filtration for visible interference removal
• – Syringe filters
• – Syringeless filters
• – Agilent Captiva (cartridge and 96-well plate formats)
• – Agilent Captiva ND (cartridge and 96-well plate formats)
• Mechanical filtration + extraction by sorbent for lipid removal
• – Agilent Captiva ND Lipids (cartridge and 96-well plate
formats)
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Liquid-Liquid Extraction
Disadvantages
easily removed • Large volumes of
• Short method organics
development time • Difficult to
• Low cost automate
• Flexible for a • Variable results
variety of sample • Expensive, clean
types glassware
• Easy to perform • Emulsion formation
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• Filtration
Cleanliness
• Dilute and shoot (guard columns or retention
gaps)
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Typical Process
1. The simplest method which meets the assay needs is
usually chosen
• PPT or LLE are common starting points
2. For challenging assays (low detection limits, closely
related endogenous constituents, inhalation products,
peptides, etc) SPE may be first choice
3. Exact technique chosen will depend on outcome of
study and how much risk can be tolerated