this presentation contains all the preparation techniques for the preparation of biological matrix on academic and high throughput laboratories.

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

20. Alternative strategies

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!!