Bioanalytical sample preparation

Bioanalytical Samples Preparation
Presented by: Mr. Ashok Choudhary
Bioanalytical Quality Monitor
Bioanalytical Research Department

INTRODUCTION
• Life Cycle of Bioanalytical Samples:
• Sample Collection
• Sample Management
• Sample Preparation
• Sample Analysis

Sample Preparation
Bioanalytical Sample Preparation:
The bioanalytical sample preparation is step in the bioanalytical process typically consists of an
extraction procedure that results in the isolation and enrichment of components (analyte/metabo
lite) of interest from a biological matrix. (e.g. Plasma, Urine, Skin, Saliva, etc).
 Objective
Sample preparation / treatment is essential step of chromatographic analysis and intended for:-
• Clean up by Remove unwanted sample matrix components
• Improve Detection Limits
• Improve Specificity
• Improve Reproducibility
• Improve Recovery
• Improve Instrument life
• Reduce backpressure and LC system fouling

Why is sample preparation required?
• Current Sample = Unsuitable for further analysis!!!… Why?
• Too dirty- Contains other sample matrix components that interfere with the analysis.
• Too dilute- Analyte(s) not concentrated enough for quantitative detection.
• Present sample matrix incompatible with or harmful to the chromatographic column/ syste
m.

Why is sample preparation especially
important in Bioanalysis?
Due to many types of sample matrices encountered:
• Plasma- Proteins, lipids, and other endogenous macromolecules
• Urine- Contains uric acid and many nitrogenous base products
• Serum
• Bile- Bilirubin, Cholesterol, Fatty acids
• Tissue Homogenates
• Saliva
• Seminal Fluid
• Others

Sample Preparation
 Pre-requisite
Sample Preparation required
• Time, it’s time consuming process
• Patience, tedious techniques/procedures
• Costly Chemicals, Reagents, Solvents and Consumables.
• Significant amount of man power
• Costly instrumentation for automated sample preparation.

Sample Preparation
 Precautions
• Lab should be equipped with all necessary contingency arrangements.
• Personnel should perfectly be aware of Good Laboratory Practices (GLP) an
d all relevant Standard Operating Procedures (SOP) to be followed while sa
mple Preparation.
• Personnel should be trained, and with all required safety measurements.
• Handle biological matrices with taking proper care to avoid any health hazar
d.
• Don’t rush while preparing samples as samples are precious.

Sample Preparation
 Classification of Sample Preparation Techniques:
1. Protein Precipitation (PPT)
2. Liquid-Liquid Extraction (LLE)
3. Solid Phase Extraction (SPE)
4. Hybrid Extraction Techniques i.e. Combination of two or more tec
hniques.
e.g. PPT and SPE or PPT and LLE.

Sample Preparation
 Selection of the technique
While selecting the method, following criteria’s should be notice
d..
• Composition of matrix
• Chemical structure of analyte
• Lower Limit of Quantitation (LLOQ)
• Specificity
• Reproducibility
• Recovery
• Time required for sample preparation

Sample Preparation
1. Protein Precipitation (PPT):
Principle: Denaturing of proteins
 Protein precipitation is the method of denaturing of the proteins
which can be done by:
I Changing the pH of the medium
II Using Organic Solvents (Denaturing agents)
III Using Salts (Denaturing agents)

Sample Preparation
Protein Precipitation
How it happens… ?
• The denaturing process causes disabling of protein’s ability of binding to analyte
molecules.
• Analytes are typically released from the proteins and remained in the supernatant
liquid.

Sample Preparation
 Methods of Precipitation
I Changing the pH of the medium
Reagents: Perchloric Acid (PCA),
Trichloroacetic acid (TCA),
Trifluoroacetic acid (TFA), etc.
How it happens… ?
• At low pH, proteins have net Positive (+) charge.
• At high pH, proteins have net negative (-) charge.
• At Isoelectric pH proteins are having zero net charge which leads to insolubility
and it helps in precipitation.

Sample Preparation
II Addition of organic solvents
Organic solvents: Methanol,
Acetonitrile,
Chloroform,
Acetone, etc.
How it happens… ?
• By adding organic Solvent the protein losses their structure and this leads to release of binded
drug from the proteins in the added organic solvent.
• Decrease the di-electric constant of the medium, leads to insolubility thus precipitates.
• High affinity for the hydrophobic surfaces of the protein leads to denaturing of proteins.

Sample Preparation
III Salt induced precipitation
Salts: Citrates,
Phosphates,
Sulphates,
Acetates, etc.
How it happens… ?
• At low concentrations, solubility of proteins increases.
• At high concentration, solubility of proteins drop sharply, thus precipitates.

Sample Preparation
Protein Precipitation
Steps involved:
Sample (With ISTD)
Addition of denaturing agent(s) to the sample
Vortex
Centrifugation
Phase separation (Supernatant Transfer)
Analysis

Sample Preparation
 Protein Precipitation
Advantages
• Less steps involved, easy to implement
• Simple and time saving process.
• Excellent Reproducibility
• No evaporation, in most of the cases
• Cost effective (Less usage of Manpower or chemicals or (Consumables)

Sample Preparation
 Protein Precipitation
Disadvantages
• In-complete clean-up
• Ineffective at removing salts and lipids
• Massive ion suppression, in case of LC/MS/MS analysis
• Low specificity
• Low recovery
• Low sensitivity

Sample Preparation
2. Liquid-Liquid Extraction (LLE):
Liquid–liquid extraction is a method to separate compounds, based on their rela
tive solubilities in two different immiscible liquids, usually aqueous and an org
anic solvent.
“Selective extraction of intended analyte present in liquid sam
ple through immiscible organic solvent”
Principle: Partitioning coefficient and Differential Solubility.

Sample Preparation
Liquid-Liquid Extraction
How it Happens ?
• LLE based on Differential Solubility and Partitioning equilibrium.
• LLE required to immiscible phases, in most of the cases one aqueous and second organic
phase, both phases must be immiscible.
• Analyte can be removed from matrix selectively by choosing a suitable Extraction Solvent
and Buffering (pH adjustment of sample to get analyte in unionized form) of sample, if re
quired.

Sample Preparation
In short
“LLE provides efficient removal of analyte with desired specif
icity /selectivity required for intended bioanalysis”

Sample Preparation
 Log P = 1 10:1 (Organic: Aqueous)
 Log P = 0 1:1 (Organic: Aqueous)
 Log P = -1 1:10 (Organic: Aqueous)
• Log P value is more than 1 indicates Drug is present in Unionized form, as Solubilit
y in the Non Polar Solvent (prefer LLE Extraction method).
• Log P value is Less than 1 indicates Drug is having more Solubility in the Aqueous
Phase (Water/Plasma) (prefer SPE Extraction method).

Sample Preparation
 LLE can be carried out by using any one of the methods mentioned
below:
I. Single Step LLE
II. Double Extraction
III. Back Extraction

Sample Preparation
 Selection of Extraction Solvent
Selection of extraction solvent depends on:
 Analyte Characteristics
• Structure
• pKa
• Solubility
 Solvent Characteristics
• Miscibility • Inertness
• Purity • Volatility
• Efficiency • Viscosity
• Polarity • Density

Sample Preparation
Commonly used Extraction Solvents
• Tertiary-Butyl Methyl Ether (TBME)
• Dichloromethane (DCM)
• Ethyl Acetate
• Diethyl Ether
• Hexane etc.
Any solvent as individual or in combination with any other suitable solvent
can be used as an extraction solvent.

Sample Preparation
Solvent Polarity Index:
Solvent Polarity Index Direction
Hexane 0.1
Cyclohexane 0.2
Toluene 2.4
Tertiary-Butyl Methyl
Ether (TBME)
2.4
Diethyl Ether 2.8
Dichloromethane 3.1
Isopropyl Alcohol 3.9
Tetrahydrofuran 4.0
Chloroform 4.1
Ethyl Acetate 4.4
Methanol 5.1
Acetonitrile 5.8
Dimethyl Sulfoxide 7.2
Water 10.2

Sample Preparation
I. Single Step LLE
•This is a simple and most commonly used
LLE method
•In this, extraction is carried out in single step
Steps involved:
Sample (With ISTD)
Addition of buffering agent(s), if required
Addition of Extraction solvent
Extraction (Use Vortex shaker or Extractor)
Centrifugation
Phase separation
Evaporation
Reconstitution
Analysis

Sample Preparation
II. Double Extraction
• This method is used to improve the Re
covery of analyte.
• In this, extraction is carried out by usi
ng more than single step, extraction ste
ps can be increased as per specific req
uirement.
Steps involved:
Sample (With ISTD)
Centrifugation
Phase separation
Addition of Extraction solvent (2 – 3 times)
Evaporation
Reconstitution
Analysis

Sample Preparation
III. Back Extraction
• This method is used to improve the Specificity
of extraction, means; back extraction can provid
e cleaner sample.
• This, in general extraction is carried out by usin
g single step LLE, but after phase separation, ap
propriate back extraction agent in general acid
(i.e. 0.1N HCl or 0.1N H2SO4 etc.) can be used
to remove unwanted material from the sample.
 Steps involved:
Sample (With ISTD)
Centrifugation
Phase separation
Addition of Back Extraction agent, Vortex
Centrifugation
Phase separation, partial evaporation, if required
Analysis

Sample Preparation
Advantages
• Specific
• Sensitive
• Reproducible
• Reasonably, Good Recovery
• Increase no. of injections
• Improve column life

Sample Preparation
 Disadvantages
• Washing/Cleaning, is difficult to incorporate.
• Tedious process, difficult to implement and reproduce, needs expertise.
• Emulsion formation is potential problem.
• Time consuming, involves so many tedious steps.
• Needs significant amount of manpower, higher volumes of costly organic sol-vents and costly
consumables to take care of compatibility issues.
• Reproducibility may be a problem in terms of Area, in some cases Area Ratio also.
• Health hazards, personal safety is a question.

Sample Preparation
3. Solid phase extraction (SPE):
Solid-phase extraction is an extractive technique by which compounds that are
dissolved or suspended in a liquid mixture are separated from other compounds i
n the mixture according to their physical and chemical properties.
“Selective retardation of analyte using solid sorbent under
specific conditions”
Principle: Adsorption (Absorption on surface)

Sample Preparation
 Objective
Solid phase extraction is intended for
• Clean up
• Class fractionation
• Improve Detection Limits, Concentrate the analyte
• Improve Reproducibility

Sample Preparation
 Solid phase extraction
 How it Happens ?
• SPE is based on the selective Adsorption mechanism.
• If the targeted analytes are adsorbed on the solid phase, they can selectively be re
moved / eluted by using an appropriate Elution Solvent (solvent /solvent mixture).
Elution : Removal of analyte from adsorbent

Sample Preparation
 Features of Solid sorbent
• C18 is the most hydrophobic silica based sorbent
• 40µ particle size, 60 A° pore size
• rigid silica particles
• Fast equilibration
• Medical grade polypropylene tubes
• 20µ polypropylene frits

Sample Preparation
Types of Solid Phase Extraction:
1.) Normal Phase (Polar) Extraction
2.) Reverse Phase (Non-Polar) Extraction
3.) Ion Exchange Extraction

Sample Preparation
1.) Normal Phase (Polar) extraction The stationary bed is strongly polar (Hydrophilic)
in nature and elution Solution is non-polar (Hydrophobic).
e.g.:- C8, C18 Silica Bonded StrataX Cartridges, Generik H2P-40 is made of polymer of d
ivinyl benzene (DVB) Orochem.
2.) Reverse Phase (Non-polar) extraction The stationary bed is non-polar (Hydrophobi
c) in nature, while the elution Solution is a polar (Hydrophilic), such as mixtures of water
and methanol or acetonitrile.
e.g.:- C18, tC18, C8, CN, Diol, HLB, Porapak RDX, NH2
3.) Ion exchange extraction The stationary bed has an ionically charged surface of oppo
site charge to the sample ions (cation, anion, and covalent). This technique is used almost
exclusively with ionic or ionizable samples. The stronger charge on the sample, the stron
ger it will be attracted to the ionic surface and, thus, the longer it will take to elute (extra
ct).
e.g.:- SCX, WCX, SAX, WAX (NH2, Accell™ Plus QMA, Accell Plus CM)

Sample Preparation
Types of Cartridges (Solid Phase Sorbents):

Sample Preparation
Extraction Cartridge:
• Oasis sorbents are available in 5 different SPE chemi
stries, providing a range of options for method develop
ment.
• The Oasis HLB Sorbent is a macroporous co-polymer
made from a balanced ratio of two monomers, the lipo
philic Divinylbenzene and the hydrophilic N-vinylpyrr
olidone.
• It provides reversed-phase capability with a special
“polar hook” for enhanced capture of polar analytes an
d excellent wettability.
Oasis MCX:- Mixed-Mode Cation-Exchange
Oasis MAX:- Mixed-Mode Anion-Exchange
Oasis WCX:- Mixed-Mode Weak Cation-Exchange
Oasis WAX:- Mixed-Mode Weak Anion-Exchange
Oasis HLB Back bone

Sample Preparation
Hydrophilic Lipophilic Balance (HLB):

Sample Preparation
 Solid Phase Extraction
• Steps Involved
Sample (With ISTD)
Addition of buffering agent(s), if required, Vortex
Conditioning
Sample Application
Rinsing / Washing
Drying
Elution
General Steps involving in SPE

Sample Preparation
1. Conditioning:
Solvent is passed through the SPE material to wet the bonded functional groups => ensures consistent
interaction.
Conditioning Solvents
Methanol, Acetonitrile, small volumes of DCM, t-BME or any other organic solvent, Water, Buffer.
All SPE tubes are required to be conditioned with appropriate solvents prior to sample application. Condition is
must to:
• Activate the sites of stationary phase bed.
• Avoid drying and keep the sites in arising position.
• Remove any dust, moisture or any other process contaminants.
• Remove the excess of Conditioning solvent prior to sample application.

Sample Preparation
Sample Loaded on conditioned cartridge and Non-conditioned cartridge:

Sample Preparation
Sample Pre-treatment (Equilibrium):
Sorbent/ phase is treated with a solution that is similar (in polarity, pH, etc.) to
the sample matrix => maximizes retention.
• It involves:
Transfer of recommended sample volume into intended sample tube.
Addition of appropriate volume of Internal Standard to the sample, vortex.
Addition of appropriate volume of suitable buffering reagent to the sample,
vortex.

Sample Preparation
2. Sample Application (Loading):
• Apply sample from the top of the cartridge at a slow flow rate without any break,
take care that no sample drop should remain on the inner wall of the cartridges.
• Slow rate is necessary to allow analyte to interact with adsorbent thus to achieve the
retention of analytes because of forming temporary weak bonding with sorbent
components.

Sample Preparation
3. Rinsing or Washing:
Selectively remove unwanted interferences co-extracted with the analyte without prematurely
eluting analytes of interest.
Rinsing or Washing solvents – assists to achieve or improve specificity.
Water
Buffers, different pHs
It is intended for the:
• Removal of matrix components or other interferences by washing the cartridge with relative
weak, dilute solvent or solvent mixtures or buffers.
• Interferences that are weakly retained than the analytes are drained out from the cartridge .

Sample Preparation
4. Drying:
• Drying can be done by applying appropriate vacuum for recommended time
period with help of Vacuum pump.
•Recommended drying time is 2-3 minutes.
• Removal of excess washing solvents / buffers to:-
Avoid any interaction / precipitation possibility.
Avoid any blockage of cartridges due to air bubble formation
during elution.

Sample Preparation
5. Elution:
Removing analytes of interest with a solvent that overcomes the primary and secondary retention
interactions b/w sorbent and analytes of interest.
Elution Solvents:
Methanol, Acetonitrile, acidic or alkaline MeOH or MeCN, small volumes of DCM, t-BME or any
other organic solvent in combination with MeOH or MeCN or any other suitable mixture.
 Passing of strong solvent through cartridge at a slow flow rate thus allowing more soak time on the
packing to reach maximum extraction efficiency.
 Cleavage of weak bonds formed between analyte and sorbent.
 Usage of multiple small volumes to improve elution.

Sample Preparation
Advantages
• Simple
• Sensitive
• Highly Specific
• Excellent Reproducibility
• Excellent Recovery
• Reduced organic solvent consumption
• Easy Automation, faster
• Less Health Hazards

Sample Preparation
Disadvantages
• Needs usage of costly cartridges and solvents
• Needs Expertise
• Prone to contamination, as so many steps are involved
• Batch to batch Variability of SPE Cartridges (manufacturing defect) may lead to
poor Reproducibility
• Tedious process
• Time consuming
• Irreversible adsorption of some analytes on SPE Cartridges may lead to poor
recovery
• Needs perfect optimization of each step, that may need more development time.

Sample Preparation
4. Hybrid Extraction
“Selective extraction of analyte by using the combination of two or m
ore extraction techniques”

Sample Preparation
 Objective
Hybrid Extraction is intended for
• Clean up
• Improve Detection Limits

Sample Preparation
 Hybrid Extraction
Advantages
• Specific
• Sensitive
• Reproducible

Sample Preparation
 Hybrid Extraction
Disadvantages
• Needs usage of costly chemicals and consumables
• Needs Expertise
• Prone to contamination, as so many steps are involved
• Prone to Variability, as so many steps are involved, in case of insufficient expertise
• Tedious process
• Time consuming
• Needs perfect optimization of each step, that may need more development time.

Sample Preparation
Conclusions And Outlook:
Sample preparation (extraction) is a key component of successful bioanalytical sample
analysis. Extraction, purification, and pre-concentration techniques are particularly
relevant for the quantification of trace analytes in complex sample bio-matrixes.

End of The PresentationEnd of The Presentation

Bioanalytical sample preparation

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