21

Extraction of
Drug from the Biological Matrix: A Review
S. Lakshmana Prabu1 and T. N. K. Suriyaprakash2
1Anna

University of Technology, Tiruchirappalli, Tamil Nadu,

2Periyar College of Pharmaceutical Sciences,
The Tamil Nadu Dr. M.G.R. Medical University, Chennai, Tamil Nadu,
India
1. Introduction
The assessment of therapeutic compliance is commonly done by either indirect method or
direct method. In indirect method, the assessments are done by indirect measurement of
parameters such as discussion with patients and pill counts at intervals during the treatment
which dont measure the drug concentration in a matrix such as blood or urine. In direct
method, the assessments which rely upon evidence provided by the patients or care giver on
the presumptive compliance based upon electronic medical event monitoring system and
this is based upon either the qualitative or quantitative measurement of the drug under
investigation in a biological matrix provided by the system.
A more objective assessment of patient compliance has its own limitations. The most widely
used matrix is plasma, but the major limitation with this approach is that it provides the
clinician with the concentration of drug within the systematic circulation at the time of
sample collection. This concentration is primarily related to the time interval between the
administration of the last dose and the sample collection time although other
pharmacokinetic parameters can also influence the concentration. Plasma samples should be
collected prior to the administration of the next dose. Plasma monitoring is a useful adjunct
for the assessment of therapeutic compliance (Williams, 1999).
Bioanalysis is a sub-discipline of analytical chemistry covering the quantitative measurement
drugs and their metabolites in biological systems. Bioanalysis 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 exposureresponse (pharmacokinetics
/pharmacodynamics studies). Bioanalysis also applies to drugs used for illicit purposes,
forensic investigations, anti-doping testing in sports, and environmental concerns.
Bioanalytical assays to accurately and reliably determine these drugs at lower concentrations.
This has driven improvements in technology and analytical methods.
Some techniques commonly used in bioanalytical studies include:

Hyphenated techniques

LCMS (liquid chromatographymass spectrometry)

GCMS (gas chromatographymass spectrometry)

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LCDAD (liquid chromatographydiode array detection)

CEMS (capillary electrophoresismass spectrometry)

Chromatographic methods

HPLC (high performance liquid chromatography)

GC (gas chromatography)

UPLC (ultra performance liquid chromatography)

Supercritical fluid chromatography

The area of bioanalysis can encompass a very broad range of assays which support the
clinical and nonclinical studies. Many factors influence the development of robust
bioanalytical methods for analyzing samples from clinical and nonclinical studies which
includes the matrices of interest, the range over which analytes need to be measured, the
number and structures of the analytes, the physicochemical properties of the analytes, and
their stability in the biological matrices from the time of sample draw to analysis also needs
to be measured.
Because biological samples are extremely complex matrices comprised of many components
that can interfere with good separations and or good mass spectrometer signals, sample
preparation is an important aspect of bioanalytical estimation. This is important whether
samples originate as tissue extracts, plasma, serum or urine. The question sometimes arises
as to whether serum or plasma should be collected for analysis. In general, from a
bioanalytical perspective, there are few advantages to choosing serum over plasma except
where chemical interferences in a given assay might require processing blood all the way to
serum.
Nearly all bioanalytical assays involve the use of an internal standard processed along with
the sample. The area ratio of analyte to internal standard is used in conjunction with a
standard curve to back calculate the concentration of analyte in the sample. The internal
standard is expected to behave similarly to the analyte with respect to extraction efficiency
across the range of concentration, which helps compensate for sample to sample differences
in sample preparation. Often, an analogue or similarly behaving compound is used as an
internal standard.
To produce adequate and precise results, certain criteria like minimal loss or degradation of
sample during blood collection, the appropriate sample cleanup and internal standard,
chromatographic conditions that minimize the interference and ion suppression, and
sufficient signal to noise to allow for reproducible peak integration.
However, an examination of the individual plasma samples could show one or more with
an unacceptable interference that is effectively washed out when the samples are pooled.
Thus, it is important to use several individual matrix samples when evaluating matrix
effects. If unacceptable matrix interferences are observed, it is necessary to further clean up
the sample to eliminate the interference.
A key component of the sample preparation is the emphasis on analyte stability which
needs to be carefully assessed from the time of sample drawn till the analysis is complete. It
is important to study the analytes stability in blood at the appropriate temperatures from
the time the sample is drawn until after centrifugation when plasma or serum are separated
and stored in a new vial. It is also important to ensure that the anticoagulant or other

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components of the blood collection do not interfere with the sample preparation (Destefano
& Takigiku, 2004).
Blood is the transporter of many vital substances and nutrients for the entire body and thus
contains many endogenous and exogenous compounds in different concentrations. Drug
determination in human plasma is often complicated by low concentrations (0.1 10ng mL-1
level). An extra problem posed by blood sample is the complex sample matrix due to
proteins, which can lead to protein binding of the analyte and by limited sample volumes
normally 0.5 1ml will be available for the determination. The magnitude of the challenge
of protein purification becomes clearer when one considers the mixture of macromolecules
present in the biological matrices. Hence, sample preparation is crucial in drug analysis
which includes both analyte pre-concentration and sample cleanup (Ho et al., 2002).
To produce meaningful information, an analysis must be performed on a sample whose
composition faithfully reflects that of the bulk of material from which it was taken.
Biological samples cannot normally be injected directly into the analyzing system without
sample preparation. Sample pretreatment is thus of utmost importance for the adequate
analysis of drugs. However, as sample pretreatment can be a time consuming process, this
can limit the sample throughput. The proper selectivity can be obtained during the sample
preparation, the separation and the detection. A major differentiation between the analyte of
interest and the other compounds is often made during the first step. Sensitivity is to a large
extent obtained by the detector. Thus, sample pretreatment is required for achieving
sufficient sensitivity and selectivity, whereas the time should be kept to a minimum in order
to obtain adequate speed. Therefore, there is a clear trend towards integration of sample
pretreatment with the separation and the detection. Numerous sample preparation
techniques have been developed for bioanalytical purpose. Sample preparation is a difficult
step, especially in clinical and environmental chemistry and generally involves filtration,
solid phase extraction with disposable cartridges, protein precipitation and desalting.
Sample preparation prior to chromatographic separation is performed to dissolve or dilute
the analyte in a suitable solvent, removing the interfering compounds and pre-concentrating
the analyte.
The principle objectives of sample preparation from biological matrix are;
a.
b.

Isolation of the analytes of interest from the interfering compounds

Dissolution of the analytes in a suitable solvent and pre-concentration.

In an analytical method sample preparation is followed by a separation and detection

procedure. In spite of the fact that sample preparation, in most of the analytical procedures,
takes 50-75% of the total time of the analysis, most technical innovations of the last 5 years
are related to separation and detection.
Extraction is one of humankinds oldest chemical operations. Extraction is the withdrawing
of an active agent or a waste substance from a solid or liquid mixture with a liquid solvent.
The solvent is not or only partially miscible with the solid or the liquid. By intensive contact
between analyte and the extraction medium this leads the analyte transfers from the solid or
liquid mixture into the extraction medium (extract). After thorough mixing the two phases
can be separated either by gravity or centrifugal forces (Gy, 1982; Arthur & Pawliszyn, 1990;
Zief & Kiser, 1990).

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2. Sampling
The sampling and sample preparation process begins at the point of collection and extends
to the measurement step. The proper collection of sample during the sample process (called
primary sampling), the transport of this representative sample from the point of collection to
the analytical laboratory, the proper selection of the laboratory sample itself (called
secondary sampling), and the sample preparation method used to convert the sample into a
form suitable for the measurement step can have a greater effect on the overall accuracy and
reliability of the results than the measurement itself.
Primary sampling is the process of selecting and collecting the sample to be analyzed. The
objective of sampling is a mass or volume reduction from the parent batch, which itself can
be homogeneous or heterogeneous. If the wrong sample is collected or the sample is not
collected properly, then all the further stages in the analysis become meaningless and the
resulting data are worthless. Unfortunately, sampling is sometimes left to people unskilled
in sampling methodology and is largely ignored in the education process, especially for
non-analytical chemists (Smith & James, 1981). Once the primary sample is taken, it must be
transported to the analytical laboratory without a physical or chemical change in its
characteristics. Even if a representative primary sample is taken, changes that can occur
during transport can present difficulties in the secondary sampling process. Preservation
techniques can be used to minimize changes between collection and analysis. Physical
changes such as adsorption, diffusion and volatilization as well as chemical changes such as
oxidation and microbiological degradation are minimized by proper preservation.
Common preservation techniques used between the point of collection and the point of
sample preparation in the laboratory are
-

Choice of appropriate sampling container

Addition of chemical stabilizers such as antioxidants and antibacterial agents
Freezing the sample to avoid thermal degradation
Adsorption on a solid phase

In secondary sampling, if the sample has made it to the laboratory, a representative subsample
must be taken. Statistical appropriate sampling procedures are applied to avoid discrimination,
which can further degrade analytical data. Clearly speeding up or automating the sample
preparation step will reduce analysis time and improve sample throughput (Keith, 1990).
Before sample preparation, solid or semisolid substances must be put into a finely divided
state. Procedures to perform this operation are usually physical methods, not chemical
methods. The reasons for putting the sample into a finely divided state are that finely
divided samples are more homogeneous, so secondary sampling may be carried out with
greater precision and accuracy, and they are more easily dissolved or extracted because of
their large surface-to-volume ratio.
2.1 Sample preparation
Sample preparation is necessary for at least two reasons:
a.
b.

To remove as many of the endogenous interferences from the analyte as possible

To enrich the sample with respect to the analyte, thus maximizing the sensitivity of the
system.

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It also serves to ensure that the injection matrix is compatible with the selected column and
mobile phase.
2.2 Goal and objectives of sample preparation
Two of the major goals of any sample pretreatment procedure are
-

Quantitative recovery
A minimum number of steps.

Successful sample preparation has a threefold objective.

-

In solution
Free from interfering matrix elements
At a concentration appropriate for detection and measurement

The most common approach in analyte separation involves a two phase system where
the analyte and interferences are distributed between the two phases. Distribution is
an equilibrium process and is reversible. If the sample is distributed between two
immiscible liquid phase, the techniques is called liquid-liquid extraction. If the sample is
distributed between a solid and a liquid phase, the technique is called liquid-solid
adsorption.
Often, when analysis involves the measurement of trace amounts of a substance, it is
desirable to increase the concentration of the analyte to a level where it can be measured
more easily. Concentration of an analyte can be accomplished by transferring it from a large
volume of phase to a smaller volume of phase. Separation can be carried out in a single
batch, in multiple batches or by continuous operation.
2.3 Types of samples
Sample matrices can be classified as organic and inorganic. Biological samples are a subset
of organic samples but often require different sample preparation procedures in order to
preserve biological integrity and activity. Compared to volatile compounds or solid,
liquid samples are much easier to prepare for analytical measurement because a
dissolution or an extraction step many not be involved. The major consideration for liquid
samples is the matrix interferences, the concentration of analyte, and compatibility with
the analytical techniques. When a sample is a solid, the sample pretreatment process can
be more complex. There are two specific cases: the entire sample is of interest and must be
solubilized or only a part of the solids is of interest and the analytes must be selectively
removed. If the solid is a soluble salt or drug tablet formulation, the only sample
preparation that may be required is finding a suitable solvent that will totally dissolve the
sample and the components of interest. If the sample matrix is insoluble in commonly
solvents but the analytes of interest can be removed or leached out, then sample
preparation can also be rather straightforward. In these cases, techniques such as
filtration, Soxhlet extraction, supercritical fluid extraction, ultrasonication or solid-liquid
extraction may be useful. If both the sample matrix and the sample analytes are not
soluble in common solvents, then more drastic measures may be needed.

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3. Physicochemical properties of drug and their extraction from biological

material (Chang 1977; Moore 1972; Barrow 1996; Wiltshire 2000)
3.1 Molecular phenomena for solubility and miscibility
To dissolve a drug, a solvent must break the bonds like ionic bond, hydrogen bond and Van
der Waals forces which inter links the compound to its neighbors and must not break
substantial intermolecular bonds of the solvent without replacing them with drug solvent
interaction. Because breaking of bonds is an endothermic process, requires energy and
causing an increasing in enthalpy. Similarly, if the gain in entropy from the dissolution of
two solvents in each other is insufficient to counteract any reduced amount of
intermolecular bonding, they will not be completely miscible.
3.2 Water miscibility and water immiscibility
Commonly alcohols can have hydrogen bonding with water and also dipole-dipole
interactions will aid miscibility. On the other hand, presence of alkyl groups will reduce the
solubility with water and the interaction may be by means of dispersive force.
Hydrocarbons are hydrophobic in nature, which dissolves the compounds by dispersive
forces. Whereas halogenated hydrocarbons are more polar and dissolve the compounds by
dispersive forces and dipolar interactions.
Hydrophilic groups, which are polar in nature, will encourage the solubility in water,
whereas C-C, C-H and C-X bonds are hydrophobic in nature will encourage the solubility in
organic solvents. Drug with several aromatic rings will have poor solubility in water due to
lack of hydrogen bonding with water and the strong intermolecular dispersive forces of the
solid drug will encourage the ready solubility in organic solvents.
3.3 Distribution coefficient
Drug which are in ionised forms are hydrophilic in nature than the unionized form because
of the hydration of the ions, therefore the ionized forms are difficulty to extract into organic
solvents whereas the unionized forms will dissolve in the organic solvents which can be
extracted into organic solvents.
3.4 Choice of solvent
Several factors are to be considered while choosing a solvent to extract a drug from the
matrix in addition to its powder to dissolve the required compounds which includes
selectivity, density, toxicity, volatility, reactivity, physical hazards and miscibility with
aqueous media.
-

Ethyl acetate is a powerful solvent for many organic compounds and will therefore
extract a considerable amount of endogenous material with the required drug.
If the drug is relatively non-polar, a more selective extraction could be obtained by
using a hydrocarbon solvent.
Halogenated hydrocarbons like chloroform and dichloromethane are excellent, volatile
solvents. However they are denser than water which makes them difficult to use for
analysis.

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Benzene is a useful solvent, reasonably volatile, inert and immiscible with water, but its
toxicity precludes its use.
Toluene has similar properties as a solvent to benzene is not particularly toxic, however
its boiling point is 111C and it is not really sufficient volatile for use as a solvent in bioanalysis.
Chloroform is an excellent solvent but reactivity with bases reduces its uses with basic
drugs that need to be extracted at high pH.
Di-isopropyl ether is less miscible with water than di-ethyl ether but is much more
likely to form explosive peroxides and is best avoided.
Diethyl ether is a good, volatile solvent but it is quite soluble (~4%) in water and
difficult to blow to complete dryness.

3.5 Mixed solvents

In some cases pure solvents will not be satisfactory for the extraction of the compound of
interest. Alcohols are excellent solvent but those with lower boiling points are too soluble in
water whereas less miscible one are having high boiling points, but the use of mixed
solvents containing alcohols can solve the problem. A 1:1 mixture of tetrahydrofuran and
dichloromethane is a powerful solvent for the extraction of polar compounds from aqueous
solutions.
3.6 Plasma proteins and emulsions
Presence of proteins can cause difficulties in extracting the drug from plasma. Emulsions are
often formed and partial precipitation can unclear the interface between the two layers. The
proteins can be precipitated by addition of 10-20% trichloroacetic acid or five volumes of a
water-miscible solvent like acetonitrile.
3.7 Role of pH for solvent extraction
Organic acids and bases are usually much less soluble in water than its salts. As a general
rule, extraction of bases into an organic solvent should be carried out at high pH usually
about 2 pH units above the pKa and extraction of acids carried out at low pH. If the drug is
reasonably non-polar base, it could be back extracted from the organic solvent into acid,
basified and re-extracted into the organic solvent and vice versa for the acidic drugs.

4. Sample pretreatment in different biological matrices (Horne, 1985; Christians

& Sewing, 1989; McDowall et al., 1989; Ingwersen, 1993; Krishnan & Ibraham
1994; Simmonds et al., 1994; Allanson et al., 1996; Plumb et al., 1997)
4.1 General concern with biological samples
Extraction of biological samples before injection into an HPLC system serves a number of
objectives.
-

Concentration
Clean-up
Prevention of clogging of analytical columns

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Elimination of protein binding

Elimination of enzymatic degradation of the analyte

4.2 Serum, plasma, and whole blood

Serum and plasma samples may not need to be pretreated for SPE. In many cases, however,
analytes such as drugs may be protein-bound, which reduces SPE recoveries. To disrupt
protein binding in these biological fluids, use of one of the following methods for reversed
phase or ion exchange SPE procedures.
-

Shift pH of the sample to extremes (pH<3 or pH>9) with acids or bases in the concentration
range of 0.1M or greater. Use the resulting supernatant as the sample for SPE
Precipitate the proteins using a polar solvent such as acetonitrile, methanol, or acetone
(two parts solvent per one part biological fluid is typical). After mixing and
centrifugation, remove the supernatant and dilute with water or an aqueous buffer for
the SPE procedure
To precipitate proteins, treat the biological fluid with acids or inorganic salts, such as
formic acid, perchloric acid, trichloroacetic acid, ammonium sulfate, sodium sulfate, or
zinc sulfate. The pH of the resulting supernatant may be adjusted prior to use for the
SPE procedure
Sonicate the biological fluid for 15 minutes, add water or buffer, centrifuge, and use the
supernatant for the SPE procedure

4.3 Urine
Urine samples may not require pretreatment for reversed phase or ion exchange SPE, but
often is diluted with water or a buffer of the appropriate pH prior to sample addition. In
some cases, acid hydrolysis (for basic compounds) or base hydrolysis (for acidic
compounds) is used to ensure that the compounds of interest are freely solvated in the urine
sample. Usually a strong acid (e.g. concentrated HCl) or base (e.g. 10M KOH) is added to
the urine. The urine is heated for 15- 20 minutes, then cooled and diluted with a buffer, and
the pH adjusted appropriately for the SPE procedure. Enzymatic hydrolysis that frees
bound compounds or drugs also may be used.
4.4 Solid samples
Solid samples ex. tissues, faeces are normally homogenized with a buffer or an organic
solvent, then remaining solids removed by centrifugation, and the diluted sample applied to
the cartridge.

5. Methods of extraction
The aim of the sample preparation process is to provide a suitable sample, usually for
chromatographic analysis, which will not contaminate the instrumentation and where the
concentration in the prepared sample is reflective of that found in the original. The method of
sample preparation selected is generally dictated by the analytical technique available and the
physical characteristics of the analytes under investigation (Watt et al., 2000). The two main

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sample preparation methods are matrix cleanup or direct injection. In a matrix cleanup
procedure, the aim is to remove as much endogenous material as possible from the drug sample.
Sample preparation is traditionally carried out (a) by liquid-liquid extraction, (b) solid-phase
extraction or (c) by precipitation of the plasma proteins, while the final analysis in most
cases is accomplished by liquid chromatography interfaced with mass spectrometry or
tandem mass spectrometry or capillary gas chromatography.
5.1 Liquid-Liquid Extraction (LLE) (Christian & OReilly, 1986; Harris, 1994; Majors &
Fogelman, 1993; Wells, 2003)
One of the most useful techniques for isolating desired components from a mixture is liquidliquid extraction (LLE). LLE is a method used for the separation of a mixture using two
immiscible solvents. In most LLEs, one of the phases is aqueous and the other is an
immiscible organic solvent. The concept like dissolves like works well in LLE. The ability
to separate compounds in a mixture using the technique of LLE depends upon how
differently the compounds of the sample mixture partition themselves between the two
immiscible solvents. Selective partitioning of the compound of interest into one of two
immiscible or partially miscible phases occurs by the proper choice of extraction of solvent.
In this technique sample is distributed in two phases in which one phase is immiscible to
other. LLE separates analytes from interferences by partitioning the sample between two
immiscible liquids or phases. First, the component mixture is dissolved in a suitable solvent
and a second solvent that is immiscible with the first solvent is added. Next, the contents are
thoroughly mixed (shaking) and the two immiscible solvents allowed separating into layers.
The less dense solvent will be the upper layer, while the more dense solvent will be the
lower layer. The components of the initial mixture will be distributed amongst the two
immiscible solvents as determined by their partition coefficient. The relative solubility that a
compound has in two given solvents can provide an estimation of the extent to which a
compound will be partitioned between them. A compound that is more soluble in the less
dense solvent will preferentially reside in the upper layer. Conversely, a compound more
soluble in the more dense solvent will preferentially reside in the lower layer. Lastly, the
two immiscible layers are separated, transferred and the component in that solvent is
isolated. Generally after extraction hydrophilic compounds are seen in the polar aqueous
phase and hydrophobic compounds are found mainly in the organic solvents. Analyte is
extracted into the organic phase are easily recovered by evaporation of the solvent, the
residue reconstituted with a small volume of an appropriate solvent preferably mobile
phase while analyte extracted in to the aqueous phase can be directly injected into a RP
column. LLE technique is simple, rapid is relative cost effective per sample as compared to
other techniques and near quantitative recoveries (90%) of most drugs can be obtained by
multiple continuous extraction (Fig.1).
Several useful equations can help illustrate the extraction process. The Nernst distribution
law states that any neutral species will distribute between two immiscible solvents so that
the ratio of the concentration remains constant.
KD = Co/Caq
Where KD 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.

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Fig. 1. Gravity Filtration Setup

To increase the value of KD, 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 KD may be increased by suppressing its ionization
to make it more soluble in the organic phase. It can also be extracted into the organic
phase by the formation of an ion pair through the addition of a hydrophobic counterion.
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.

If the KD value is unfavorable, addition extraction may be required for better solute
recovery. In this case, a fresh portion of immiscible solvent is added to extract additional
solute. Normally, the two extracts are combined. Generally, for a given volume of solvent,
multiple extractions are more efficient in removing a solute quantitatively than a single
extraction. Sometimes, back extractions can be used to achieve a more complete sample
cleanup.

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If KD is very low or the sample volume is high, it becomes nearly impossible to carryout
multiple simple extractions in a reasonable volume. Also, if the extraction rate is slow, it
may take a long time for equilibrium to be established. In these cases, continuous liquidliquid extraction is used, where pure solvent is recycled through the aqueous phase.
Benefit of this technique is that, with a judicious choice of solvents and pH, very clean
extracts can be obtained with good selectivity for the targeted analyte. The drug is extracted
from the aqueous phase to the organic phase. One point of note is that LLE system unlike
solid-phase systems, are more likely to give consistent results year after years, as there is
usually less batch to batch variation with solvents.
The extraction of drug from the aqueous phase is mainly depends on the following factors:
-

Solubility of analyte in the organic solvent

Polarity of the organic solvent
pH of the aqueous phase

In some cases there is a possibility that interferences may present in the extracted sample. In
that case back liquid-liquid extraction can be performed, this gives a clear extracts. Here two
times organic solvent is used for the extraction of analyte from the matrix. Often, however, it
is not possible to find the optimum condition that provides both high recovery and purity of
the analyte in one extraction step. Low recoveries may require further extraction to achieve
acceptable value. About the purity it may require second extraction procedure with different
solvent or pH of the aqueous phase. Each successive extraction increase the analytical time,
also the resulting large volume of extraction solvent must be evaporated to recover the
product. If extraction requires many steps, techniques such as Craig Counter Current
distribution can be used to increase recovery and purity. However this technique increases
the cost and time of the analysis.
5.1.1 Selection of the solvent
There are also practical concerns when choosing extraction solvents. As mentioned
previously, the two solvents must be immiscible. The properties of an ideal solvent is that it
should withdraw the active agent from a mixture by liquid-liquid extraction are
-

Selectivity: Only the active agent has to be extracted and no further substances which
mean that a high selectivity is required.
Capacity: To reduce the amount of necessary solvent, the capacity of the solvent has to
be high.
Miscibility: To achieve simple regeneration of the solvent the miscibility of solvent and
primary solvent has to be low.
Difference in density: After extraction, the two phases have to be separated in a
separator and for this a high positive difference in density is required.
Optimal surface tension: low low amount of energy for dispersing required; if
surface tension < 1 mN/m stable emulsions are produced; > 50 mN/m high
amount of energy for dispersing and high tendency to coalesce.
Recovery: The solvent has to be separated from the extract phase easily to produce
solvent free active agents.

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Corrosion: If the solvent is corrosive prices for construction increases.

Low price
No or low toxicity and not highly flammable
Flame temperature: 25C higher than operating temperature
Vapour pressure: To prevent loss of solvent by evaporation a low vapour pressure at
operating temperature is required.
Viscosity: A low viscosity of the solvent leads to low pressure drop and good heat and
mass transfer.
Chemical and thermal stability
Environmentally acceptable or easily recoverable
Convenient specific gravity
Suitable volatility
High chemical stability and inertness
Not prone to form an emulsion
Dissolves the neutral but not the ionized form of the analyte

The stoichiometric ratio of the analyte in the organic phase compared to that in the aqueous
phase is known as the distribution ratio D. Ideally, this ratio should approach 100% in order
to minimize the losses through the effects of small changes in sample composition,
temperature and pH. Reproducibility also increases with increasing extraction efficiency,
although a consistent low recovery may be acceptable if an internal standard is used to
compensate for changes in efficiency.
5.1.2 Extraction under basic and acidic conditions
As mentioned above, the ability to separate compounds of a mixture using liquid-liquid
extraction procedures depends upon the relative solubility that each compound has in the
two immiscible solvents. A change in the pH of the solvent (the addition of acid or base) can
change the solubility of an organic compound in a solvent considerably.
Liquid/liquid extraction is the most common technique used to separate a desired organic
product from a biological matrix. The technique works well if your target compound is
more soluble in one of two immiscible solvents. Extraction usually involves shaking a
solution that contains the target with an immiscible solvent in which the desired substance
is more soluble than it is in the starting solution. Upon standing, the solvents form two
layers that can be separated. The extraction may have to be repeated several times to effect
complete separation.
In general liquid-liquid extractions can separate four different classes of compounds:
a.
b.
c.
d.

Organic bases: Any organic amine can be extracted from an organic solvent with a
strong acid such as 1M hydrochloric acid
Strong acids: Carboxylic acids can be extracted from an organic solvent with a weak
base such as 1M sodium bicarbonate
Weak acids: Phenols can be extracted from an organic solvent with a strong base such
as 1M sodium hydroxide
Non-polar compounds stay in the organic layer

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5.1.3 Disadvantages
-

Large solvent consumption is needed for extraction of drug.

LLE is time consuming process when compare to other methods.
LLE require an evaporation step prior to analysis to remove excess of organic solvent.
LLE technique is not a suitable one for the estimation of several analytes.
Emulsion formation may be possible when two immiscible phases were used in the
extraction procedure.

5.2 Solid Phase Extraction (Zwir Ferenc & Biziuk, 2006; James, 2000; Krishnan &
Abraham, 1994; Moors et al., 1994; Plumb et al., 1997; Arthur & Pawliszyn, 1990; Zief
& Kiser, 1990; MacDonald & Bouvier, 1995; Wells, 2003: Scheurer & Moore, 1992)
Since the 70s SPE has become a common and effective technique for extracting analytes
from complex samples. Solid phase extraction is the very popular technique currently
available for rapid and selective sample preparation. Many sample preparation methods
today rely on solid-phase extractions, an advantage being that SPE is amenable to
automation and parallel processing. SPE evolved to be a powerful tool for isolation and
concentration of trace analysis in a variety of sample matrices. The versatility of SPE allows
use of this technique for many purposes, such as purification and trace enrichment (Rawa et
al., 2003).
The objectives of SPE are to reduce the level of interferences, 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.
b.

The selectivity of the bonded phase for the analyte

The relative strength of that interaction

SPE prepares multiple samples in parallel (typically 12-24) and uses relatively low quantities
of solvents and the procedures can be readily automated. As the name implies the principle
of SPE is an extraction technique similar to that of LLE, involving a partitioning of solutes
between two phases. However, instead of two immiscible liquid phases, as in LLE, SPE
involves partitioning between a liquid (sample matrix or solvent with analytes) and a solid
(sorbent) phase (Table 1).
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 which is commonly RP material (C18silica). The components of interest may either preferentially adsorbed to the solid, or they
may remain in the second non-solid phase. Once equilibrium has been reached, the two
phases are physically separated by decanting, filtration, centrifugation or a similar process.
If the desired analyte is adsorbed on the solid phase, they can be selectively desorbed by
washing with an appropriate solvent. If the component of interest remains in a liquid phase,
they can be recovered via concentration, evaporation and or recrystallization. When SPE is
performed in this single step equilibrium batch mode, it will be similar to LLE, where the
solid sorbent simply replaces one of the immiscible liquids. By passing a liquid or gas

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through the uniform solid bed, the liquid solid phase extraction technique becomes a form
of column chromatography, now commonly called phase extraction that is governed by
liquid chromatographic principle.

Polarity
Nonpolar

Polar

Strong Reversed
Phase

Weak Reversed
Phase

Weak Normal
Phase

Strong Normal
Phase

Solvent

Miscible in
Water?

Hexane

No

Isooctane
Carbon
tetrachloride
Chloroform
Methylene
Chloride
Tetrahydrofuran
Diethly ether
Ethyl acetate
Acetone
Acetonitrile Yes
Isopropanol
Methanol
Water

No

Yes
No
Poorly
Yes
Yes
Yes
Yes
Yes

Acetic Acid

Yes

No
No
No

Table 1. Characteristics of Solvents Commonly Used in SPE

Liquid samples are added to the cartridge and wash solvent is selected to either strongly
retain or un-retain the analyte. Interferences are eluted or washed from the cartridge, even
as the analyte is strongly retained, so as to minimize the presence of interferences in the final
analyte fraction, the analyte is then eluted with a strong elution solvent, collected and either
injected directly or evaporated to dryness followed by dilution with the HPLC mobile
phase. Conversely, when interferences are strongly held in the cartridge, the analyte can be
collected for the further treatment. Advantages of SPE over LLE include a more complete
extraction of the analyte, more efficient separation of interferences from analyte, reduced
organic solvent consumption, easier collection of the total analyte fraction, more convenient
manual procedures, better removal of particulates and more easy automation.
5.2.1 Mechanism of Solid Phase Extraction process (Font et al., 1993; Sabik et al.,
2000)
The ideal process is to create a digital chromatography system on the SPE cartridge such
that the analyte is at first fully bound, then the interferences are completely eluted, and then
the analyte is entirely eluted; it is either all on or all off the cartridge. The separation
mechanism is the function due to the intermolecular interactions between analyte and the
functional groups of the sorbent.

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Fig. 2. Partitioning of drugs towards aqueous and organic solvent

The selection of an appropriate SPE extraction sorbent depends on understanding the
mechanism(s) of interaction between the sorbent and analyte of interest. That understanding
in turn depends on knowledge of the hydrophobic, polar and inorganic properties of both
the solute and the sorbent. 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).
Each sorbent offers a unique mix of these properties which can be applied to a wide variety
of extraction problems (Fig 2). Four general theory interactions exist (Yu et al., 2004):
a.

Reversed phase involves a polar or moderately polar sample matrix (mobile phase) and
a non-polar stationary phase. The analyte of interest is typically mid- to non- polar.

Retention occurs via non-polar interaction between carbon-hydrogen bonds of the analyte
and carbon-hydrogen bonds of the sorbent function groups due to Van der Waals or
dispersion forces.
The materials that are used as reversed phases are carbon-based media, polymer-based
media, polymer-coated and bonded silica media.
Carbon-based media consist of graphitic, non-porous carbon with a high attraction for
organic polar and non-polar compounds from both polar and non-polar matrices. Retention
of analytes is based primarily on the analytes structure, rather than on interactions of
functional groups on the analyte with the sorbent surface.
Polymer-based sorbents are styrene/divivinylbenzene materials. It is used for retaining
hydrophobic compounds which contain some hydrophilic functionality, especially
aromatics.
Polymer-coated and bonded silica media is hydrophobic-bonded silica that is coated with a
hydrophilic polymer. The pores in the polymer allow small, hydrophobic organic

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compounds of interest (e.g. drugs) to reach the bonded silica surface, while large interfering
compounds (e.g. proteins) are shielded from the bonded silica by the polymer and are
flushed through the SPE tube.
Several SPE materials, such as the alkyl- or aryl-bonded silicas (LC-18, ENVI-18, LC-8,
ENVI-8, LC-4, and LC-Ph) are in the reversed phase category.
b.

Normal phase involve a polar analyte, a mid- to non-polar matrix (e.g. acetone,
chlorinated solvents and hexane) and a polar stationary phase. Retention of an analyte
under normal phase conditions is primarily due to interactions between polar
functional groups of the analyte and polar groups on the sorbent surface. Ex. Hydrogen
bonding, dipole-dipole, induced dipole-dipole and pi-pi.

These phases can offer a highly selective extraction procedure capable of separating
molecules with very similar structures. The main drawback is that the analyte must be
loaded onto the sorbent in a relatively non-polar organic solvent such as hexane.
Polar-functionalized bonded silicas (e.g. LC-CN, LC-NH2, and LC-Diol), and polar
adsorption media (LC-Si, LC-Florisil, ENVI-Florisil, and LC-Alumina) typically are used
under normal phase conditions.
c.

The bonded silicas have short alkyl chains with polar functional groups bonded to the
surface. These silicas, because of their polar functional groups, are much more
hydrophilic relatively to the bonded reversed phase silicas.

The bonded silicas LC-CN, LC-NH2, and LC-Diol - have short alkyl chains with polar
functional groups bonded to the surface.
d.

Ion exchange SPE can be used for compounds that are in a solution. Anionic
(negatively charged) compounds can be isolated on an aliphatic quaternary amine
group that is bonded to the silica surface. Cationic (positively charged) compounds are
isolated by using the silica with aliphatic sulfonic acid groups that are bonded to the
surface.

Biofluids can usually be applied directly to ion-exchange sorbents following dilution of the
sample with water or a buffer, and possibly adjustment of pH. However, elution from
strong ion-exchange sorbents can be a problem as high ionic strength or extremes of pH,
may be required which may affect analyte stability or further processing of sample.
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.

5.2.2 General properties of bonded silica sorbents

Although other materials are available ex. polymeric resins and alumina, the vast majority
of SPE extractions are carried out by using bonded silica materials similar to those used in
HPLC columns except the particle size and diameter. Bonded silica materials are rigid and
do not shrink or swell like polystyrene-based resins in different solvents. Use of too high
flow rate when processing cartridges may affect retention of analytes, particularly during
the sample loading and elution steps. Potentially the capacity of the cartridge will be
affected by all the components from the sample not only the analytes of interest.

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5.2.3 Steps of Solid Phase Extraction

Generally following steps are followed for developing the method for extracting the analyte
from plasma (Fig 3).
a.
b.

c.

d.

e.

Pretreatment 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.
Without this step, a highly aqueous solvent cannot penetrate the pores and wet the
surface. Thus, only a small fraction of the surface area is available for interaction with
the analyte. For the same reason, it is important not to let the cartridge dry out between
the salvation step and addition of the sample.
Loading the sample - Sample size must be scaled to suit the size of the cartridge bed. A
typical reverse phase cartridge may have capacity for up to 100 mg of very strongly
retained substances.
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.

(a)

(b)

(c)

(d)

(e)

Fig. 3. Five steps of SPE: (a) selection of tube, (b) conditioning of tube, (c) addition of sample,
(d) washing and (e) elution
5.2.4 A strategy for method development for plasma samples
-

Rationale
Practical consideration
Pretreatment of samples and cartridges
Screening sorbents and pH values
Optimizing the washing procedure
Optimizing the elution procedure
Final optimization and simplification
Strategies for removing persistent interferences

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5.2.5 Developing SPE methods

In developing a SPE method, the properties of the isolate, sorbent and the matrix should be
considered. The mechanisms of retention and the various secondary interactions may occur,
and that a selective extraction from biofluid also requires the sorbent not to extract a large
number of unknown compounds from the matrix. The best conditions are not easily
predicted and the method needs to be developed experimentally in combination with the
analytical method being used.
The following stages are recommended for method development.
a.
b.

c.

d.

Consider physic-chemical properties of analyte, nature of matrix and known

chromatographic properties of analytes and the possible interaction with SPE sorbents.
Screen a range of cartridges (ex. C18, C8, C2, Ph, CBA, SCX, SAX, PRS, NH2, DEA, CH, Si
and polymeric sorbent) under simple conditions (ex. from aqueous buffer solutions)
looking for good retention of the analyte. If radiolabelled analyte is available this can
conveniently be used to track analyte in such screening experiments. This experiment
should not only identify likely cartridges for use in the assay, but should be used to try
to confirm possible mechanism of interaction between the analyte and sorbent.
Select a more limited number of sorbents and examine conditions for loading/wash/elution
(consider if pH control is needed, possible strength (% organic solvent) of wash solvents).
Try the extraction from bio-fluid and select sorbent for final development.
Final development of extraction conditions and chromatographic analysis. Consider the
robustness of the assay when finalizing extraction conditions. Ex. Do not select a wash
solvent where a small change in composition could lead to elution of analyte from the
cartridge.

The choice of different cartridges, manufactures and formats is becoming so extensive that it
can appear almost overwhelming. It is generally better to build experience with a more
limited set of sorbents, perhaps concentrating on cartridges from only one manufacturer.
Also concentrate on investigating the use of cartridges with different functional groups (i.e.
test C18, C8, C2, Ph, CBA, SCX, SAX, PRS, NH2, DEA, CH, Si, etc.), and those that use a
contrasting mechanism to the analytical separation.
5.2.6 Characteristic features of SPE
-

Complete flexibility
Longer column lifetimes
Powerful contaminant removal
Greater recovery
Better reproducibility
More sensitivity

5.2.7 Advantages of SPE over LLE

-

In SPE by choosing selective adsorbent analyte can be driven completely by adsorption

and desorption
In single stage LLE each extraction step equivalent to one chromatographic plate on the
other hand by SPE in single step one can generate 10-50 plates

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Higher plate numbers in SPE leads to higher recoveries and purer of the analyte as
compared to LLE
For extracting more than one component from a mixture of component of different
desorption solvent are required in case of SPE. To achieve similar results with LLE, one
must perform several liquid extractions
SPE is less time consuming and not tedious as compare to LLE

5.2.8 Limitation
-

Depending on the nature of the analyte, SPE may not always be the method of choice,
and liquid-liquid extraction may be a more viable solution.

6. Protein precipitation method (Backes, 2000; Wells, 2003)

This method is least one in bioanalytical. This is a very simple technique for extraction of the
analyte from the matrix. If protein binding is suspected, then protein precipitation prior to
sample extraction may be considered. Reagents to evaluate include perchloric,
trichloroacetic and tungstic acids, and organic solvents such as acetonitrile or methanol.
With all of these it is necessary to bear in mind the ability of the analytes and the matrix
requirements of the extraction procedures. If protein binding is believed to be through a
covalent linkage, then there is very little change of breaking it since this is the strongest of
the intermolecular forces.
The main requirement for this technique is that the analyte should be freely soluble into
reconstituting solvent. Preparation of sample through protein precipitation achieves
separation by conversion of soluble proteins to an insoluble state by salting out or by
addition of water miscible precipitation solvent or organic solvents such as acetone, ethanol,
acetonitrile or methanol. Ideally, precipitation results in both concentration and purification,
and is often used early in the sequence of downstream purification, reducing the volume
and increasing the purity of the protein prior to any chromatography steps. In addition,
precipitating agents can be chosen that provide a more stable product than found in the
soluble form.
Proteins might stick to each other through one of three forces: electrostatic, hydrophobic,
and van der Waals. The last one is difficult to distinguish from hydrophobic and operates
over only in a very short range. Electrostatic forces operate at long range, but between like
molecules are repulsive rather than attractive, so molecules have the same charge and repel
each other.
Proteins are made insoluble by altering their surface properties, charge characteristics or
changing the solvent characteristics; but changing the solvent characteristics being
preferred. Greater the initial concentration of the desired protein, greater is the efficiency of
precipitation; proteins are least soluble as its isoelectric point (pI) ranges from 4 - 10. The
selection of a buffer at or near the pI of the protein is recommended. However some proteins
may denature at their pI and above the pI. The solubility of a protein increases with the
addition of salt and reaches a maximum after which there is a rapid linear decrease in
solubility. There are several methods to reduce the solubility of proteins, which are ionic
precipitation ex. ammonium sulphate, sodium chloride; metal ions ex. Cu+2, Zn+2 and Fe+2;

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non-ionic polymers ex. polyethylene glycol; organic solvents ex. ethanol, acetone; tannic
acids, heparin, dextran sulphates, cationic polyelectrolytes ex. protamines; short chain fatty
acids ex. caprylic acid; trichloroacetic, lecithins ex. concanavalin A and group specific dyes
ex. procion blue. The use of temperature, pH or organic solvents can lead to denaturation
and should be performed with care to minimize any decrease in yield or activity.
6.1 Type of protein precipitation
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 the square of the charge on the
ion, so that the ionic strength of 1M (NH4)2SO4 is 3 times that of 1M NaCl). Neither ion
associates much with proteins, which is good since such association usually destabilizes
proteins. Its solubility changes little with temperature, it is cheap, and the density of even a
concentrated solution is less than that of protein, so that protein can be centrifuged down
from concentrated solutions.
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 interactions
between charged groups on the surface of proteins stronger. Water miscible solvents
associates with water much more strongly than do proteins, so that its real effect is to
dehydrate protein surfaces, which then associate by van der Waals forces, at least if they are
isoelectric or reasonably close to it. Removal of water molecules from around charged
groups would also deshield them and allow charge interactions to occur more strongly, if
there are areas of opposite charge on the surfaces of two proteins.
In practice, solvent precipitation is usually performed at low temperature. The condition for
the protein is at 0C and the solvent colder, -20C in an ice-salt bath, because proteins tend
to denature at higher temperatures though if sufficient control can be achieved and your
protein is more stable than others, this can be selective and achieve greater purification.
Solvent precipitation can be done with polyethylene glycol at concentrations between 5 and
15%. It probably works the same way, by competing with the protein for water, but is less
likely to inactivate the protein and does not require such low temperatures, but it tends to
give an oily precipitate.
Commonly the sample is centrifuged at high speed for sufficient time, all the precipitated
components of plasma will be settled at the bottom and clear supernatant liquid will be
separated out. The obtained supernatant liquid can be injected directly into the HPLC or it
can be evaporated and reconstituted with the mobile phase and further clean up of the
sample can be carried out by using micro centrifuge at very high speed.
6.2 Advantage
-

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/MS

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Protein precipitation plates are compatible with small volume of solvent

Protein precipitation plate contains hydrophobic PTFE membrane as a prefilter 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
By using the new protein precipitate filter plate, precipitating solvent is added first
followed by the plasma sample. This method does not require any mixing. Generally
these plates are fitted to 96 well extraction plates. This new process showed 90%
removal of plasma proteins when compare to the old method 60-65%

6.3 Disadvantage
-

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.

7. Solid Phase Microextraction (SPME) (Pawliszyn et al., 1997; Pawliszyn,

1995, 1998, 1999, 2003; Wercinski 1999)
Miniaturization of sorbent technology and the concomitant decrease in solvent purchase,
exposure and disposal has also taken a further giant step with the development of SPME.
Solid-Phase Microextraction (SPME) is a very simple and efficient, solventless sample
preparation method. SPME integrates sampling, extraction, concentration and sample
introduction into a single solvent-free step. In this technique a fused silica fiber coated with
polyacrylate, polydimethylsiloxane, carbowax or other modified bonded phase is placed in
contact with a sample, exposed to the vapour above a sample (solid, liquid) or placed in the
stream of a gaseous sample to isolate the analyte and concentrate analytes into a range of
coating materials. After extraction, the fibers are transferred with the help of syringe like
handling device to analytical instrument like gas chromatography (GC) and GC/mass
spectrometry (GC/MS) for separation and quantification of the target analyte. In such a
manner SPME has been used frequently to analyze volatile and semi-volatile compounds
and may be used to purge and trap procedures. The method saves preparation time and
disposal costs and can improve detection limits. The most interesting aspect of this
technology involves the ability to use the exposed fiber as extraction and sample delivery
device. Phases for SPME are available in a range of polarities and properties for analyses of
volatile and semi-volatile compounds as well as application to extraction of analytes from
liquid samples. One of the limitations of SPME is the low capacity of the fiber and the
perturbation of equilibria that can occur in the presence of sample components or analytes
at very high concentration versus those of lesser concentration. Dilution of the sample can
overcome some of these problems but not all, as limits of detetion for trace analytes are
compromised. Formaldehyde, Trition X-100, Phenylurea, pesticides and amphetamines are
some of the analytes which are successfully extracted using SPME.
7.1 SPME basics (Vas & Vekey, 2004)
The concept of SPME may have been derived from the idea of an immersed GC capillary
column. The SPME apparatus looks like modified syringe containing a fiber holder and a
fiber assembly, the latter containing a 12 cm long retractable SPME fiber. The SPME fiber

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itself is a thin fused-silica optical fiber, coated with a thin polymer film conventionally used
as a coating material in chromatography. In SPME the amount of extraction solvent is very
small compared to the sample volume. As a result, exhaustive removal of analytes to the
extracting phase does not occur; rather equilibrium is reached between the sample matrix
and the extracting phase. In practical, the extracting phase is a polymeric organic phase
commonly poly(dimethylsiloxane) and polyacrylate which is permanently attached to rod.
The rod consists of an optical fiber made of fused silica, which is chemically inert. A
polymer layer is used to protect the fiber against breakage. Poly(dimethylsiloxane) behaves
as a liquid, which results in rapid extraction compared to polyacrylate, which is a solid.
When the coated fiber is placed into an aqueous matrix the analyte is transferred from the
matrix into the coating. The extraction is considered to be complete when the analyte has
reached an equilibrium distribution between the matrix and fiber coating. The amount of
extracted analyte is independent of the volume of the sample. Therefore, there is no need to
collect a defined amount of sample prior to analysis. Thus, the fiber can be exposed directly
to the ambient air, water, production stream, etc., and the amount of extracted analyte will
correspond directly to its concentration in the matrix.
7.2 Principle modes of SPME
7.2.1 Direct extraction
In the direct-extraction mode, the coated fiber is inserted directly into the sample, and
analytes are extracted directly from the sample matrix to the extraction phase. For gaseous
samples, natural air convections and high diffusion coefficients are typically sufficient to
facilitate rapid equilibration. For aqueous matrices, more efficient agitation techniques are
required, such as forced flow, rapid fiber or vial movement, stirring or sonication.
7.2.2 Headspace configuration
In the headspace mode, the vapor above the bulk matrix is sampled. Thus, analytes must be
relatively volatile in order to be transported from the bulk matrix to the fiber coating.
7.2.3 Advantages
Sampling of the headspace protects the fiber coating from damage by hostile matrices, such
as those at very high or low pH, or those with large molecules, such as proteins which tend
to foul the coating.
7.2.4 Types of extraction in SPME
-

Fiber extraction
In-tube extraction
Stir BAR sorptive extraction (SBSE)

7.2.5 Advantage of SPME

-

In SPME volatile, semi-volatile and non-volatile organic and inorganic analytes can be
used for analyzed

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During desorbtion of the analyte, the polymeric phase is cleaned and therefore ready
for reuse. The absence of solvent in SPME is an important feature, as it is not only
environmentally friendly but makes the separation faster
Important feature of SPME is its small size, which is convenient for designing portable
devices for field work
Solvent free environment, fast extraction, convenient automation and easy hyphenation
with analytical instrument

8. Matrix Solid-Phase Dispersion (MSPD) (Barker et al., 1989, 1993; Walker et

al., 1993)
Matrix solid phase dispersion is a sample preparation technique for use with solid sample
matrices. MSPD is a microscale extraction technique, typically using less than 1g of sample
and low volumes of solvents. It has been estimated to reduce solvent use by up to 98% and
sample turnaround time by 90%.
Conventional extraction of organic analytes from tissue usually begins with a
homogenization of a small amount of sample tissue with bulk bonded silica based sorbent in
a pestle and mortar. The mechanical shearing forces produced by the grinding process
disrupt the structure of the tissue, dispersing the sample over the surface of the support
sorbent by hydrophilic and hydrophobic interaction which produces the mixture to become
semi-dry and free-flowing, and a homogenous blend of sample. The bound solvent in the
sorbent will aid complete sample disruption during the sample blending process and the
sample disperses over the surface of the bonded phase-support material to provide a new
mixed phase for isolating analytes from various sample matrices. The blend is then
transferred into a pre-fitted SPE cartridge and elution of interference compounds and
analytes of interest. This technique has recently been applied, using acid alumina, to extract
the organic analyte. However, MSPD procedure needs longer analytical time generally and
its limit of determination (LOD) is limited.
In method development using MSPD sorbents, the following points are to be considered
-

Sample pre-treatment
Interference elution
Analyte elution
Sample clean up

9. Supercritical fluid extraction (Mohamed & Mansorri, 2002; Antero, 2000)

Supercritical fluid extraction is becoming a very popular technique for the removal of
non-polar to moderately polar analytes from solid matrices. Supercritical fluids (SCFs)
are increasingly replacing the organic solvents that are used in industrial purification
and recrystallization operations because of regulatory and environmental pressures on
hydrocarbon and ozone-depleting emissions. SCF processes eliminate the use of organic
solvents, so it has attracted much attention in the industrial sectors like
pharmaceuticals, medical products and nutraceuticals. Pharmaceutical chemists have
found SCFs useful for extraction of drug materials from tablet formulation and tissue
samples.

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Supercritical fluids exhibit a liquid-like density, while their viscosity and diffusivity remain
between gas and liquid values. The recovery of a supercritical solvent after extraction can be
carried out relatively simply by reducing the pressure and evaporating the solvent. Above
the critical temperature the liquid phase will not appear even the pressure is increased. The
compressibility of a supercritical fluid just above the critical temperature is large compared
to the compressibility of ordinary liquids. A small change in the pressure or temperature of
a supercritical fluid generally causes a large change in its density. The unique property of a
supercritical fluid is that its solvating power can be tuned by changing either its
temperature or pressure. The density of a supercritical fluid increases with pressure and
becomes liquid-like, the viscosity and diffusivity remain between liquid-like and gas-like
values. Additionally, supercritical fluids exhibit almost zero surface tension, which allows
facile penetration into microporous materials leads to more efficient extraction of the analyte
than the organic solvents. Carbon dioxide is a relatively good supercritical solvent will
dissolve many relatively volatile polar compounds. In the presence of small amounts of
polar co-solvents like water and short-chain alcohols to the bulk, the carbon dioxide gas can
enhance the solubility of polar, non-volatile solutes in supercritical carbon dioxide.
Supercritical fluids can be used to extract analytes from samples.
The main advantages of using supercritical fluids for extractions is that they are
inexpensive, extract the analytes faster and more environmental friendly than organic
solvents. For these reasons supercritical fluid CO2 is the reagent widely used as the
supercritical solvent.
9.1 Advantages
-

SCFs have solvating powers similar to organic solvents, with higher diffusivity, lower
viscosity and lower surface tension
The solvating power can be changed by changing the pressure or temperature for
effective extraction
Separation of analytes from solvent is fast and easy
Polarity can be changed by using co-solvent leads to have more selective separation of
the analyte
Products are free from residual solvents
SCFs are generally cheap, simple and safe
Disposal costs are less
SCF fluids can be recycled

10. Column switching (Falco et al., 1993, 1994; Henion et al., 1998; Lee & Kim
2011)
Column switching techniques afford an interesting and creative form of sample preparation.
This approach depends on the selectivity of appropriately chosen HPLC stationary phase to
retain and separate the analyte of interest while allowing unretained components to be
eliminated from the column. The benefits of this technique include total automation and
quantitative transfer of targeted compounds within the column switching system. In the
heart cut mode a narrow retention time region containing a desired components is cut from
the chromatogram and transferred onto another HPLC column for further separation. In this

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instance, quantitative transfer of the components without adsorptive or degradative losses

can be assured.
10.1 Advantages
It is capable of having increased selectivity by the judicious choice of two or more HPLC
stationary phases. A limitation of column switching system is that sample throughout will
likely not be as high as for other sample clean up methods. A second limitation of the
column switching approaches includes restricted sample enrichment because of the limited
amount of original, untreated, crude sample that can be loaded onto the first column of the
HPLC separation. These limitations can be overcome by combining online SPE and a
column switching system. In this method the recovery is more but very expensive one.

11. Future directions

Bio-equivalency is an important one for the Pharmaceutical Formulations especially in the
Pharmaceutical Regulatory Market. The availability of methodology for the extraction
procedure of the interested analyte coupled along with the analytical method for the
quantification of the interested analyte in GC-MS/MS or LC-MS/MS will greatly facilitate
future studies to rigorously establish a bio-equivalency of the Pharmaceutical formulations
in the Pharmaceutical regulatory market.

12. Conclusion
The subject area of bioanalysis is a sub-discipline of analytical chemistry which covers a
very broad range of assays like quantitative measurement of drugs and their metabolites in
various biological systems like whole blood, plasma, serum, urine, faeces and tissues. The
biological samples cannot be analyzed directly into the analytical system for the
quantification of the interested analyte. Estimation of the analyte in the biological matrix can
be done after the isolation of the interested analyte from the interferences in the biological
sample. Isolation and estimation of the analyte is based on the sample preparation and
extraction of the analyte from the biological matrix. The process adopted for the isolation of
the interested analyte like sample preparation procedure and isolation steps must ensure the
stability of the analyte until the completion of the analytical estimation. Bioanalysis 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
exposureresponse (pharmacokinetics/pharmacodynamics) studies. In this chapter, various
extraction methods of the analyte from the biological matrix have been described with its
advantages and disadvantages.

13. References
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Applied Biological Engineering - Principles and Practice

Edited by Dr. Ganesh R. Naik

ISBN 978-953-51-0412-4
Hard cover, 662 pages
Publisher InTech

Published online 23, March, 2012

Published in print edition March, 2012

Biological engineering is a field of engineering in which the emphasis is on life and life-sustaining systems.
Biological engineering is an emerging discipline that encompasses engineering theory and practice connected
to and derived from the science of biology. The most important trend in biological engineering is the dynamic
range of scales at which biotechnology is now able to integrate with biological processes. An explosion in
micro/nanoscale technology is allowing the manufacture of nanoparticles for drug delivery into cells,
miniaturized implantable microsensors for medical diagnostics, and micro-engineered robots for on-board
tissue repairs. This book aims to provide an updated overview of the recent developments in biological
engineering from diverse aspects and various applications in clinical and experimental research.

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