detailed information of various techniques used in bioanalysis of drugs from biological samples

1. Bio analysis of drugs from biological samples
Prepared by :
Yachita rajwadwala
(q.a.)
1

2. Index
topics S. No.
Definition – biological sample 3
Definition – bio analysis 5
Collection of sample 9
Pre treatment of biological sample 11
Essential concept for sample preparation 22
Objectives of sample preparation 23
Techniques of sample preparation 25
Solid phase extraction 26
Liquid liquid extraction 39
Membrane filtration 46
GTU question 55
2

3. What are biological samples?
 A biological specimen (also called a biospecimen) is a
biological laboratory specimen held by a
biorepository for research.
 Such a specimen would be taken by sampling so as to
be representative of any other specimen taken from
the source of the specimen.
 When biological specimens are stored, ideally they
remain equivalent to freshly-collected specimens for
the purposes of research.
 Human biological specimens are stored in a type of
biorepository called a biobank , and the science of
preserving biological specimens is most active in the
field of biobanking.
3

4. Examples :
 Blood
 Plasma
 Serum
 Tissue homogenates
 Urine
 Feces
 Gastric contents
 Saliva
 Breath
 CSF
 Seminal fluid
 sputum
 Bile
 Hair 4

5. What is bio analysis?
 Bio analysis is a sub-discipline of analytical
chemistry covering the quantitative measurement of
drugs and their metabolites in biological systems
like blood, plasma, serum, urine or tissue extracts
or
 Bio analysis in the pharmaceutical industry is to
provide a quantitative measure of the active drug
and/or its metabolite(s) for the purpose of
pharmacokinetics, toxicokinetics, bioequivalence and
exposure–response
(pharmacokinetics/pharmacodynamics studies).
5

6.  This technique is used very early in the drug
development process to provide support to drug
discovery programs on the metabolic fate and
pharmacokinetics of chemicals in living cells and in
animals
 Bio analysis also applies to drugs used for illicit
purposes, forensic investigations, anti-doping testing
in sports, and environmental concerns.
 Bio analytical assays to accurately and reliably
determine these drugs at lower concentrations.
 This has driven improvements in technology and
analytical methods.
6

7.  Bioanlaytical method consists of two main
components
 Sample preparation :
 extraction of the drug from the biological fluid
 a concentration step (usually included ) to enhance
the sensitivity of the method
 Detection method:
 Hyphenated techniques
o LC–MS (liquid chromatography–mass spectrometry)
o GC–MS (gas chromatography–mass spectrometry)
o LC–DAD (liquid chromatography–diode array
detection)
o CE–MS (capillary electrophoresis–mass
spectrometry)
7

8.  Chromatographic methods
o HPLC (high performance liquid chromatography)
o GC (gas chromatography)
o UPLC (ultra performance liquid chromatography)
o Supercritical fluid chromatography
 Steps
 The quantitative determination of the drugs and their
metabolites in Bioanalysis includes a number of steps
o Sample preparation
o Separation
o Identification, and
o Quantification of analytes
o Storage
8

9. Collection of sample
Sample Method
Blood By vein puncture either with a hypodermic
syringe
Plasma Blood with is centrifuged. Higher yield, few
problems of sensitivity
serum Blood without anticoagulant is centrifuged,
specially used in microbiological assays
Urine Easy to collect, large quantities,
concentrated, mainly used for the metabolic
studies
feces In aluminum foils and lyophilized, high
protein content, Difficult to handle
9

10.  After collection of sample , before sample
preparation pre-treatment of sample is necessary
because, few factors can affect the final
measurement ……
o The texture
o Chemical composition
o Degree of drug-protein binding
o Chemical stability and
o The types of interferences
10

11. PRE-TREATMENTOF BIOLOGICAL SAMPLE
 Various pretreatment steps are carried out like…
 Protein precipitation
 Solvent extraction to remove hydrophobic
compounds
 Lyophilisation
 Hydrolysis of conjugates
 Homogenization
 Chemical derivatization as a prelude to extraction
11

12. 1. Protein precipitation
 Biological samples such as plasma, feces and saliva
contain significant quantities of protein and it can
bind a drug easily.
 Acidic drugs bind more strongly to plasma proteins
than do neutral or basic drugs.
 Proteins, salts, lipids and other endogenous materials
can cause rapid deterioration of HPLC column
 For the analysis of the drugs in plasma when direct
injection of a sample onto a column is desired as
HPLC
Two types of protein precipitation:
1. Salting out: Ammonium sulphate is the salt usually
used for salting out, because of its high solubility
and high ionic strength (which is proportional to
12

13. the square of the charge on the ion, so that the ionic
strength of 1M (NH4)2SO4 is 3 times that of 1M
NaCl).
2. Solvent Precipitation: When large amounts of a
water-miscible solvent such as ethanol or acetone
are added to a protein solution, proteins precipitate
out.
 The conventional wisdom is that this is due to
decrease of the dielectric constant, which would
make interaction between charged groups on the
surface of proteins stronger.
 Protein denaturants frequently used
 Alcohol, Methanol and acetonitrile
 Heat,
 Trichloroacetic acid, Perchloric acid, Tungstic
acid. 13

14.  Methanol preferred because:
 Gives clear supernatant
 Prevents drug entrapment
 Gives flocculent precipitate
Procedure
 Addition of protein denaturant to a protein
containing sample kept in a centrifuge tube. A milky
white precipitate is obtained immediately.
 Centrifugation: At the speed of 3000 rpm for 10 to
20 minutes
Advantages:
 Fast & inexpensive
 Relatively clean samples obtained.
14

15.  Now protein precipitation plates are available, able to
remove the unwanted plasma proteins from plasma
fluid samples prior to analysis
 Protein precipitation plates can be used in a wide
range of aqueous and organic sample
 preparation including total drug analysis and sample
preparation prior to HPLC or LCMS
 Protein precipitation plates are compatible with
small volume of solvent
 Protein precipitation plate contains hydrophobic
PTFE membrane as a pre filter removes the
unwanted precipitated proteins prior to analysis.
 Traditionally in this method plasma is mixed with
protein precipitating agent and diluting solvent then
the whole mixture is vertex, mixed, centrifuge and
filter
15

16.  disadvantage
1. May increase the back pressure of the HPLC system
 Some components of plasma which are soluble in diluting
solvent that bound to stationary phase permanently that
will affect the column performance.
2. LYOPHILLISATION
 Effectively prepares biological samples for storage and
analysis
Mainly used for
 Large (excess ) volume biological samples
 Fecal samples
 Highly water soluble drugs
 Preparing dried extract by chemical derivatization
 Steam volatile inorganic compounds that co-vaporizes
with matrix materials
 Chemically or thermally unstable compounds
16

17. Procedure
 Freezing in a dry-ice acetone bath or in liquid
nitrogen
Solid samples: For feces or tissue freezing time:
1 to 10 mts.
Liquid samples: Frozen to form a thin shell ice
thereby maximizing surface area for evaporation.
 Placing the frozen sample then in a vacuum where
water and other volatile substance are removed by
vacuum sublimation
 Solubilizing the dried sample in a suitable solvent
and carrying out assay directly
17

18. 3. HYDROLYSIS OF CONJUGATES
 For drugs present as glucuronides conjugates or
sulfates
 The effect of the drug samples depends on the extent
of the biotransformation that occurs in body
 Isolation of the actual conjugates important
 But is usually lengthy because they are hydrophilic
and/or ionized at physiologic pH
 Thus, conjugates are not amenable to classic solvent
extraction techniques
 To overcome these problems, samples containing
either glucuronides acetals or sulfates esters are
usually pretreated using enzymatic or acid
hydrolysis.
18

19. Acid or enzymatic hydrolysis
 Hydrolysis products: unconjugated metabolites are
less hydrophilic than conjugates
 Therefore, can be extracted from the biologic matrix
 A nonspecific acid hydrolysis can be accomplished by
heating a biologic sample for 30 min at 90°-100°C in
2 to 5N HCl
 Upon cooling, the pH of the sample can be adjusted
to the desired level
 The metabolite are removed by the solvent
extraction
 Note: Stable conjugates sometimes require
hydrolysis in autoclave
19

20. 4. HOMOGENISATION
It is carried out
 For sample containing insoluble proteins such as muscle
or tissue: homogenization using 1N HCl is done
 For gelatinous sample such as seminal fluid or sputum:
liquefaction is achieved via sonication
 For solid sample such as feces: Homogenization can be
done with minimum amount of methanol.
5. CHEMICAL DERIVATIZATION
It is required
 For a drug or metabolite which is
 Chemically unstable in pH range necessary for
efficient solvent extraction or
 Not amendable to solvent extraction
20

21.  Method Of Chemical derivitazation:
 The substance is reacted with a suitable reagent to
form a stable derivative, successfully extracted in
terms of original drug concentration.
 Example: Hydralazine
 Unstable in the basic pH range
 Quantitative extraction from biological material with
organic solvent is difficult
 Converted to tetrazolo (1,5a) pthlazine by treating
Hydralazine present in a plasma sample with sodium
nitrite at acidic pH
 A stable derivative
 Thus, can be quantitatively extracted from plasma
and analysed in terms of hydralazine concentration
21

22. ESSENTIAL CONCEPTS FOR SAMPLE PREPARATION
 Influence of pH on ionization of sample
 Effects of Anticoagulants and Storage on Clot
Formation
 Evaluation of Matrix Effect
 Determination of Recovery
 Analyte recovery from a sample matrix (also called
extraction efficiency) is a comparison of the
analytical response from an amount of analytes
added to and extracted from the sample matrix (pre-
extraction spike) with that from a post-extraction
spike .
 The efficiency for an extraction should be 100% or
less. This determination is typically made at a
minimum of 3 concentrations. 22

23. OBJECTIES FOR SAMPLE PREPARATION
 Removal of unwanted matrix components
(primarily protein) (that may interfere with analyte
determination).
 Concentration of analyte (to meet the detection
limits of the analytical instrument.)
 Exchange of the solvent or solution in which the
analyte resides so that it is compatible with mobile
phase for injection into a chromatographic system
 Removal of selected analyte components if the
resolving power of the chromatographic column is
insufficient to separate all the components
completely
23

24.  Removal of material that could block the
chromatographic tubing or foul the interface of the
detector
 Dilution to reduce solvent strength or avoid solvent
incompatibility
 Solubilization of compounds to enable injection
under the initial chromatographic conditions
 Stabilization of analyte to avoid hydrolytic or
enzymatic degradation
24

25. GENEAL TECHNIQUES FOR SAMPLE PREPARATION
 Protein precipitation
 Filtration
 Protein removal by equilibrium dialysis or ultra-
filtration
 Liquid-liquid extraction (LLE)
 Solid-supported liquid-liquid extraction
 Solid-phase extraction (off-line)
 Solid-phase extraction (on-line)
 Turbulent flow chromatography
 Restricted access media
 Monolithic columns
 Immunoaffinity extraction
 Combinations of the above
25

26. Solid phase extraction (SPE)
 Most widely followed technique for sample
preparation in analysis of new pharmaceutical
compounds and metabolites in blood, serum and
urine.
 SPE is a more efficient separation process than LLE,
easily obtains a higher recovery of analyte by
employing a small plastic disposable column or
cartridge, often the barrel of a medical syringe
packed with 0.1 to 0.5 g of sorbent
 Solid Phase: XAD-2 Resin, Silica, Alumina, Charcoal,
Aluminum silicate.
 SPE evolved to be a powerful tool for isolation and
concentration of trace analysis in a variety of sample
matrices.
26

27. objectives
 to reduce the level interferences of, minimize the
final sample volume to maximize analyte sensitivity
and provide the analyte fraction in a solvent that is
compatible with the analytical measurement
techniques.
 As an added benefit, SPE serves as a filter to remove
sample particulates.
 The degree of enrichment achievable for a
particular sample is dependent upon:
a. The selectivity of the bonded phase for the
analyte
b. The relative strength of that interaction
27

28. mechanisms of SPE
 The separation mechanism is the function due to
the intermolecular interactions between analyte
and the functional group.
 The most common retention mechanisms in SPE
are based on van der Waals forces (“non-polar
interactions”), hydrogen bonding, dipole-dipole
forces (“polar” interactions) and cation-anion
interactions (“ionic” interactions) of the sorbent.
 Each sorbent offers a unique mix of these
properties which can be applied to a wide variety of
extraction problems. According to that mechanism
four general theory interactions exist:
28

29. 29

30.  Reversed phase
 Nonpolar - nonpolar interactions
 Van-der Waals or dispersion forces
 Solvate the bonded phase with six to ten holds up
volumes of methanol or acetonitrile.
 Flush the cartridge with six to ten hold up volumes of
water or buffer.
 Do not allow the cartridge to dry
 Load the sample dissolved in strongly polar solvents
into cartridge
 Elute unwanted components with a polar solvent
 Elute weakly held components of interest with a less
polar solvent
 Elute more tightly held components with
progressively more non- polar solvent
30

31.  On recovery of all components, discard the used
cartridge in an appropriate manner
 The materials that are used as reversed phases are
carbon-based media, polymer-based media, polymer-
coated and bonded silica media.
 alkyl- or aryl-bonded silicas (LC-18, ENVI-18, LC-
8,ENVI-8, LC-4, and LC-Ph) are in the reversed phase
category.
 Normal phase
 involve a polar analyte, a mid- to non-polar matrix
(e.g. acetone, chlorinated solvents and hexane) and a
polar stationary phase.
 Hydrophilic interactions – (Polar-polar interaction)
between polar functional groups of the analyte and
polar groups on the sorbent surface.
31

32.  Hydrogen bonding, dipole-dipole, induced dipole-
dipole and pi-pi.
 Conditioning the cartridge: with six to ten hold up
volumes of non-polar solvent, usually the sample
solvent
 Loading the sample into cartridge
 A non-polar solvent: Elutes unwanted components
 Elute the all components with interest of a polar
solvent
 On recovery of all components, discard the used
cartridge in an appropriate manner
 Polar-functionalized bonded silicas (LC-CN, LC-NH2,
and LC-Diol) and polar adsorption media(LC-Si, LC-
Florisil, ENVI-Florisil, and LC-Alumina) typically are
used
32

33. Ion Exchange
 Electrostatic attraction of charged group on
compound to a charged group on the Sorbent’s
surface.
 In this case, conditioning the cartridge with six to
ten hold up volumes of deionized water or buffer
 Load the sample dissolved in deionized water or
buffer into cartridge
 Elute unwanted weakly bound components with a
weak buffer
 Elute the first component of interest with a
stronger buffer (change the pH or ionic strength)
 Elute other components of interest with
progressively stronger buffers
 On recovery of all components, discard the used
cartridge in an appropriate manner
33

34.  Anionic (negatively charged) compounds can be
isolated on LC-SAX or LC-NH2 bonded silica
cartridges. Cationic (positively charged) compounds
are isolated by using LC-SCX or LC-WCX bonded
silica cartridges.
Steps of Solid Phase Extraction
 Pre treatment of sample - which includes dilution of
sample or pH adjustment, filtration to avoid the
blocking of the SPE cartridge and for better
adsorption.
 Conditioning of the cartridge - which is the main
step in case of reverse phase SPE cartridges.
Preconditioning is mainly done by solvent such as
methanol, acetonitrile, isopropyl alcohol or
tetrahydrofuran which is necessary to obtain
reproducible result. 34

35.  Loading the sample - Sample size must be scaled
to suit the size of the cartridge
 Wash - very important step in case of the sample
treatment by SPE. In this step a suitable solvent or
water mixture is passed through SPE bed to
remove the contaminants.
 Elution of fraction - in this a suitable solvent or
buffer is used to elute the analyte from the SPE
bed for analysis
 Characteristic features of SPE
- Complete flexibility
- Longer column lifetimes
- Powerful contaminant removal
- Greater recovery
- Better reproducibility
- More sensitivity
35

36.  Use SPE for Samples that
 Contain particulate matter causing system clogging
and high back-pressure
 Contain components that cause high background,
misleading peaks, and/or poor sensitivity
 Require clean-up, trace enrichment/concentration,
or purification
 Require sample matrix or solvent exchange
 Benefits of SPE
 Switch sample matrices to a form more compatible
with chromatographic analyses
 Concentrate analytes for increased sensitivity
 Remove interferences to simplify chromatography
and improve quantization
36

37.  Protect the analytical column from contaminants
 For amphoteric compounds that can’t be easily
extracted from water.
 Easier to automate
Comparison to LLE
 Faster sample preparation: average time cut by 2/3
 Less cost;
 less solvent and reagent consumption means less
hazardous waste
 Greater recoveries:
 minimum sample transfer
 Greater accuracy;
 No cross contamination
37

38.  Less sample handling;
 no emulsion problems
 Reduced harm to labile sample: minimum
evaporation
 Improved safety: due to reduced solvent/sample
exposure and glass ware
 Easy automation; simultaneous batch processing of
multi-samples
38

39. Liquid liquid extraction
 LLE is a technique used to separate analytes from
interferences in the sample matrix by partitioning
the analytes between two immiscible liquids, i.e
organic phase and the aqueous phase.
 Principle
 Partitioning or distribution of a drug between two
immiscible liquid phases can be expressed as
partition or distribution co-efficient ‘P’.
 Mathematically,
 Where P is the distribution constant, Co is the
concentration of the analyte in the organic phase, and
Caq is the concentration of the analyte in the aqueous
phase.
39

40.  To increase the value of P, several approaches may be
used:
 The organic solvent can be changed to increase
solubility of the analyte
 If the analyte is ionic or ionizable, its P may be
increased by suppressing its ionization to make it
more soluble in the organic phase.
 Metal ions can form a complex with hydrophobic
complexing agents.
 The salting out effect can be used to decrease
analytes concentration in the aqueous phase.
 Partition coefficient is usually constant for a
particular solute, temperature, and pair of
solvents used
 Corg >>Caq, thus P >>1. - Higher recoveries will be
obtained
40

41. Factors affecting distribution ratio
 Nature/Choice of extracting solvent
 Ratio of the volumes of the organic to aqueous phase
 pH of aqueous phase
 Ionic strength of aqueous phase
Choice of solvent
 Polarity / nature of solvent
 Solubility of drug in the solvent
 Density/viscosity
 Cost
 Toxicity
 Flammability
 Ease of handling
41

42. procedure
42

43. Advantages
 Very popular technique with wider & general
applicability
 Very simple
 Rapid
 Economical (relatively small cost factor per sample)
 Very clean extracts with good selectivity for the
target analytes.
 No Inorganic salts, (insoluble in the solvents
commonly used for LLE), proteins and water soluble
endogenous components
 As they remain behind in the aqueous phase along
with unwanted matrix materials.
 Relatively short time required for method
development (usually within two days)
43

44.  The extracted material can be re-dissolved in small
volumes (100µl to 500µl) there by extending the
sensitivity limits of limit assays.
 Possible to extract more than one sample
concurrently.
 Near quantitative recoveries (90% or better) of most
drugs can be obtained through multiple or
continuous extractions
 Provide potential benefits of
 extending lifetime of LC column,
 Minimizing the downtime of the mass
spectrometer caused by interface fouling.
44

45. Disadvantages
 Not applicable to all compounds.
 Very difficult in case of highly polar molecules,
although the use of an ion pairing reagent can
extend LLE to molecules of this type.
 Not very readily automatable.
 Formation of emulsions, difficult to break even by
Centrifugation or ultrasonification
 Can cause loss of analyte by occlusion within the
emulsion.
45

46. Membrane filtration
 In this technique, membranes may be used as a
sample preparation technique
 Alternative to SPE and/or LLE
 Two types - Porous
- Non-porous membranes
 Porous membranes:
 Sample pretreatment is based on the principle of size
exclusion to differentiate between substances
 In PMTs, the liquids on each side are physically
connected through pores.
 Transport through the membranes is based on size-
exclusion, i.e. Sufficiently small molecules can
permeate through the pores, whereas larger
molecules cannot.
46

47.  This can result in an efficient clean-up from large
matrix molecules but no distinction can be made
between small molecules
 The latter is only possible to some extent with
electro dialysis, for which an ion-exchange
membrane is used.
 Now, large molecules and molecules with a given
charge will be excluded.
 Non-porous membranes:
 based on the difference in partition coefficients of
substances, an actual extraction technique.
 Employ an organic or polymeric (solid or liquid)
layer, placed between two other liquid phases.
47

48.  The analyte must actually be extracted from the
donor phase, dissolve into the membrane in order to
be able to pass through, and then be released in the
acceptor phase
 Possible to separate molecules of similar size, yet
with different physicochemical properties.
NPMTs can be subdivided into four main groups
 Supported Liquid Membrane Extraction (SLME)
 Microporous Membrane Liquid-liquid Extraction
(MMLLE)
 Polymeric Membrane Extraction (PME)
 Membrane Extraction with a Sorbent Interface
(MESI).
48

49.  SLME is the most widely used non-porous membrane
technique ,but various applications of MMLLE, PME
and MESI have been reported as well.
 All NPMTs utilize a membrane unit
 two flow channels are formed
 One: Donor channel & the other: Acceptor channel.
 SLME (Supported Liquid Membrane Extraction)
 In principle, SLME utilizes a pH shift between the
donor phase, in which the analyte is charged, and the
acceptor phase, in which the analyte is protonated,
thus ensuring that no back-extraction in the
(organic) membrane can occur.
49

50.  MMLLE (Microporous Membrane Liquid-liquid Extraction)
 is performed with organic solvent as the acceptor
phase in the micropores of the organic membrane
and Can therefore be compared with a Single Liquid
Extraction.
 Mainly used for the analysis of hydrophobic
compounds that cannot be extracted from an organic
membrane into an aqueous acceptor solvent.
 PME (Polymeric Membrane Extraction)
 PME is similar to SLME, with the exception that a
polymeric membrane is used.Due to this
membrane, it is also possible to use organic solvent
in the donor and/or acceptor phase.
 However, the composition of the membrane is fixed.
50

51.  MESI (Membrane Extraction with a Sorbent Interface)
 MESI differs from the previous techniques in that a
solid polymeric membrane is used.
 Mainly developed for the combination with GC, thus
in order to use a gaseous acceptor phase while the
donor phase is aqueous or gaseous.
 MESI works best for the analysis of volatile and
relatively non-polar compounds.
 Most applications of MESI are in the environmental
field for the analysis of aqueous samples
 Both PMTs and NPMTs usually use the terms
efficiency and/or enrichment.
51

52.  Efficiency: the ratio between number of moles input
to the system during the extraction and the amount
collected in the acceptor. It can be directly measured
 Factors affecting Efficiency
 The composition of the donor phase
 The composition of the acceptor phase
 The membrane and
 The sample
 With high donor-flows, the efficiency is decreasing
due to incomplete diffusion of the analyte into the
acceptor phase
 Enrichment
 Accumulated amount of analyte in the acceptor phase
during a given time.
52

53.  The efficiency decreases with increasing donor flow.
But the enrichment is increasing with increasing
donor-flow.
The advantage of NPMTs in comparison to PMTs
 Higher degree of selectivity and High enrichment
 Relatively small solvent consumption
 The possibility of on-line coupling to various
analytical instruments, LC ,AAS (atomic absorption
spectrophotometry), electrochemical instruments
and flow-injection systems with UV detection
 Ease of automation makes SLME and MMLLE
attractive for bioanalysis. As with PMTs, protein
binding can decrease the extraction efficiency.
53

54.  Other critical factors: The short-term and the long-
term Stability of the membranes.
 Optimum Pressure difference to hold the organic
solvent in the pores of the hydrophobic membrane
and to prevent the acceptor phase from leaking into
the donor phase and vice versa. Chemical stability
may also be critical.
54

55. GTU QUESTIONS
 Enlist various sample preparation techniques in bio
analysis and explain SPE in detail.
 Explain the role & significance of bio analysis in
pharmacy. What are the major objectives to be
considered for bio analytical sample preparation.
 What are the biological samples? Give types &
collection methods for it.
 Write a note on protein precipitation method.
 Write a note on extraction technique of drug from
biological sample.
 Describe various parameters employed in bio
analytical and validation.
 Write a note on pre treatment of biological sample.
 Enumerate different biological method in pharma
analysis. 55

56. References
 Prabu S. Lakshamana and Suriyaprakash T.N.K;
Applied Biological Engineering-Principles and
Practice; 978-953-51-0412-4
56

57. Thank you
57