Sample preparation techniques for biological sample
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The document discusses various sample preparation techniques for biological fluids in analytical chemistry, emphasizing the need for such processes due to the complex nature of biological matrices that can contaminate instruments. Techniques covered include protein precipitation, liquid-liquid extraction, solid-phase extraction, and alternatives like dialysis and affinity sorbent extraction, each with distinct advantages and disadvantages. It highlights modern laboratory practices, particularly the utilization of 96-well plates for high-throughput sample processing.
Sample preparation techniques for biological sample
- 1. SAMPLE PREPARATION TECHNIQUES FOR BIOLOGICAL FLUID Presented by: Arun Agarwal (CSIR-JRF) ID: 53253 Supervisor: Dr. Wahajuddin (Principal scientist) Division: Pharmaceutics and pharmacokinetics
- 2. Contents Introduction Why sample preparation is required? Main sample preparation techniques Protein Precipitation Liquid-liquid extraction Solid phase extraction Dilute and shoot technique Alternative techniques Dialysis Ultrafiltration Affinity solvent extraction Summary
- 3. Introduction Biological fluids are complex in nature so they cannot be injected directly to the analytical instrument for analysis of drug substance. For example, biological fluids such as plasma, serum, cerebrospinal fluid consists of complex matrix components such as: phospholipids, proteins etc. These matrix components can contaminate the instrument.
- 4. Why sample preparation is required? Suppression ion Effect on ionization of analyte due to same elution time of matrix component and analyte False identification of analyte Similar molecular mass and retention time of matrix components and target analyte. Bio-fluids can contaminate the analytical instrument. Due to broad range of protein, protein presence can clog LC or GC column. Compatibility of biological samples with column is necessary such as: only organic sample can be give to GC column.
- 5. Main Sample Preparation Technique
- 6. Protein precipitation technique (PPT) PPT is intended to remove protein from plasma or serum leaving target analyte in the aqueous medium of the plasma sample only. Figure 1: Principle of protein precipitation
- 7. Approaches to PPT Organic solvents It acts by decreasing the dielectric constant of plasma protein which increases the electrostatic interaction. It also displaces water molecule associated with hydrophobic region of protein. Acids It forms insoluble salts with the positively charged amino groups of the plasma protein at pH value below the isoelectric point and this causes precipitation. Metal ions and salts they are also used for precipitation but less often
- 8. Blanchard, J., 1981. Evaluation of the relative efficacy of various techniques for deproteinizing plasma samples prior to high-performance liquid chromatographic analysis. Journal of Chromatography B: Biomedical Sciences and Applications, 226(2), pp.455-460. Frequently used precipitant
- 9. Advantages and disadvantages of PPT technique Advantages of PPT method Rapid procedure Require minimal equipment Per sample cost is low Disadvantages of PPT method Limited sample clean up except proteins other exogenous and endogenous component remain in the supernatant No pre-concentration of target analyte Precipitant dilute the plasma which leads to lower concentration of target analyte in supernatant as compare to the original plasma sample
- 10. Protein Precipitation in Modern High- Throughput Laboratories Sample numbers are high in modern bioanalytical laboratories. So they use multi-well plate (most commonly 96 well plate) It comprises of two portion: Top portion is PPT plate Bottom portion is collection plate PPT plate contain 0.2µm pore size filter Figure 2: 96 well protein precipitation
- 11. Liquid-liquid extraction (LLE) It is intended to transfer target analytes from the biological fluid to an organic solvent. LLE is most applicable to compounds of low polarity. The extraction solvent should be immiscible with water, and form a two- phase system with the sample. Figure 3: Principle of liquid–liquid extraction
- 12. Fundamentals of LLE For neutral analyte choice of extraction solvent is having high partition ratio i.e. KD. For acid analytes acidification of sample is required. for basic analytes alkalization of sample is required.
- 13. Solvents for LLE When analyte molecules are extracted and dissolved into an organic solvent in LLE, favorable interactions take place between the analyte molecules and the solvent molecules. These molecular interaction are as follows: Hydrophobic interaction Between two nonpolar molecules Example: Aliphatic hydrocarbon in hexane Dispersion interaction Between a polar and nonpolar molecule Ex. Aromatic solvent and p-xylene Dipole interaction Between two molecules with permanent dipole moment Ex. Nitrile and alcohol Hydrogen bonding interaction Between hydrogen donor and hydrogen acceptor Ex. Carboxylic acid and diethyl ether Figure 4: Overview of molecular interactions in liquid–liquid extraction
- 14. Frequently used liquid–liquid extraction solvents and their physiochemical properties
- 15. LLE in Modern High-Throughput Laboratories This is performed in 96 well plates. The major benefit of this is that 96 samples can be extracted simultaneously, and this of course increases the sample throughput dramatically. The most popular format of 96- well LLE is supported liquid extraction (SLE). Figure 5: Principle of supported liquid extraction
- 16. Solid phase extraction (SPE) SPE is frequently used for sample preparation of biological fluids like plasma, serum, and urine. It includes following steps: Conditioning of column Sample loading Washing of the column Addition of eluent SPE is normally a very efficient extraction method, and extraction recoveries are close to 100%. Figure 6: Principle of solid-phase extraction
- 17. Stationary phase based solid phase extraction SPE type Principle Stationary phase used Reverse phase Hydrophobic interaction Octadecyl C18, octyl C8, etyl C2, cyclohexyl, cynopropyl. Ion exchange Ionic interaction Sulfonylpropyl, Carboxymethyl, Trimethylaminopropyl, Diethylaminopropyl Mix-mode hydrophobic interactions and ionic interactions Polymeric-based stationary phase Normal phase Dipole interaction Silica, diol, cynopropyl, aminopropyl
- 18. SPE in Modern High-Throughput Laboratories In modern high-throughput laboratories, SPE is often performed in 96-well format. The great advantage of SPE in 96-well format is that 96 samples can be extracted in parallel. Equipment for SPE in the 96-well format can be purchased from several different manufacturers. Figure 7: solid-phase extraction 96-well plate
- 19. Dilute and shoot (DAS) DAS is only a dilution of the sample with a proper solvent. In other words, compounds are neither isolated nor removed from the sample. Easy sample preparation and omission of time-consuming extractions are factors that favor the use of DAS. DAS is more frequently used in urine sample preparation compared to plasma and whole blood sample preparation. Figure 8: Dilute and shoot
- 21. Dialysis Separation based on the molecular weight of analyte. Two compartment are separated by semi-permeable membrane. Principle is based on the filtration not extraction. Molecular weight cutoff (MWCO) typically is between 0.1-100 kDa. Equilibrium dialysis is a specific dialysis setup used to determine the drug– protein binding in plasma. Figure 8: principle of dialysis
- 22. Ultrafiltration Ultrafiltration is another way of removing particles from a solution using a semi-permeable membrane. This process is similar as dialysis in ultrafiltration the MWCO normally is between 1-1000 kDa. Ultrafiltration may, as dialysis, be performed using both flat membranes and hollow fibers. This technique also used for the determination of drug-protein binding in early drug development.
- 23. Affinity Sorbent Extraction Affinity-based sorbent extraction is based on selective interaction between sorbent material and analyte. There are different kinds of selective sorbents based on the affinity principle such as: Antigen-Antibody interaction Molecularly imprinted polymers (MIPs) Aptamers
- 24. Antigen-antibody interaction The most widespread affinity principle is the use of sorbent material involving antigen–antibody interaction. antibodies are normally covalently bound to an appropriate sorbent forming a so-called immunosorbent. Antibodies are synthesized by initiating an immune response in an animal toward the analyte (antigen). Figure 9: Immunosorbent image
- 25. Molecularly imprinted polymers (MIPs) MIPs are synthetic polymers with specific cavities for the target analyte. They are sometimes called synthetic antibodies, and their main advantages compared to conventional antibodies are with respect to their preparation, which is easy, inexpensive, and rapid. They are produced by bulk polymerization method. MIPs are not fully commercialized due to irregular particle size which raise problem to pack SPE column.
- 26. Aptamers Aptamers are short (≤110 bp) synthetic single-stranded oligonucleotides (DNA or RNA). They are prepared by combining a random pool of oligonucleotide sequences from a library with the analyte in question, The whole process of aptamer selection and production is called SELEX (systematic evolution of ligands by exponential enrichment). It is an automatable process, and reasonable amounts of highly specific aptamers for the desired analyte can be produced. The oligosorbent is produced by linkage of the aptamer via a spacer to a solid support (e.g., silica, sepharose, or agarose) or to magnetic particles. Figure 10: Oligosorbent
- 27. Summary Characteristic PPT LLE SPE DAS Cost Low Medium High Lest Equipment required Less number More than PPT Greater than LLE Least Technical assessment Less Medium High Least Sample Least Higher than PPT Highest Limited to urine
- 28. Characteristic Antigen-antibody MIPs aptamers Cost Expensive Inexpensive Inexpensive Time Time consuming Faster than antibody Fastest Animal requirement Yes No No
- 29. Reference Hansen, S.H. and Pedersen-Bjergaard, S. eds., 2015. Bioanalysis of pharmaceuticals: sample preparation, separation techniques and mass spectrometry. John Wiley & Sons.
- 30. Thank you!!