HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY - HPLC
High performance liquid chromatography is a powerful tool in
analysis. This page looks at how it is carried out and shows how it
uses the same principles as in thin layer chromatography and
column chromatography.
Note: It is important to read the introductory page about thin
layer chromatography before you continue with this one particularly the part about how thin layer chromatography
works. High performance liquid chromatography works on the
same basic principle. HPLC is essentially an adaptation
ofcolumn chromatography - so it might be a good idea to have
a (very quick) look at that as well.
Use the BACK button on your browser to return quickly to this
page.

Carrying out HPLC

Introduction
High performance liquid chromatography is basically a highly
improved form of column chromatography. Instead of a solvent
being allowed to drip through a column under gravity, it is forced
through under high pressures of up to 400 atmospheres. That
makes it much faster.
It also allows you to use a very much smaller particle size for the
column packing material which gives a much greater surface area
for interactions between the stationary phase and the molecules
flowing past it. This allows a much better separation of the

components of the mixture.

The other major improvement over column chromatography
concerns the detection methods which can be used. These
methods are highly automated and extremely sensitive.

The column and the solvent

Confusingly, there are two variants in use in HPLC depending on
the relative polarity of the solvent and the stationary phase.
Normal phase HPLC
This is essentially just the same as you will already have read
about in thin layer chromatography or column chromatography.
Although it is described as "normal", it isn't the most commonly
used form of HPLC.
The column is filled with tiny silica particles, and the solvent is nonpolar - hexane, for example. A typical column has an internal
diameter of 4.6 mm (and may be less than that), and a length of
150 to 250 mm.
Polar compounds in the mixture being passed through the column
will stick longer to the polar silica than non-polar compounds will.
The non-polar ones will therefore pass more quickly through the
column.
Reversed phase HPLC
In this case, the column size is the same, but the silica is modified
to make it non-polar by attaching long hydrocarbon chains to its
surface - typically with either 8 or 18 carbon atoms in them. A polar
solvent is used - for example, a mixture of water and an alcohol
such as methanol.
In this case, there will be a strong attraction between the polar

solvent and polar molecules in the mixture being passed through

the column. There won't be as much attraction between the
hydrocarbon chains attached to the silica (the stationary phase)
and the polar molecules in the solution. Polar molecules in the
mixture will therefore spend most of their time moving with the
solvent.
Non-polar compounds in the mixture will tend to form attractions
with the hydrocarbon groups because of van der Waals dispersion
forces. They will also be less soluble in the solvent because of the
need to break hydrogen bonds as they squeeze in between the
water or methanol molecules, for example. They therefore spend
less time in solution in the solvent and this will slow them down on
their way through the column.
That means that now it is the polar molecules that will travel
through the column more quickly.
Reversed phase HPLC is the most commonly used form of HPLC.
Note: I have been a bit careful about how I have described the
attractions of the non-polar molecules to the surface of the
stationary phase. In particular, I have avoided the use of the
word "adsorpion". Adsorption is when a molecule sticks to the
surface of a solid. Especially if you had small molecules in your
mixture, some could get in between the long C18 chains to give
what is essentially a solution.
You could therefore say that non-polar molecules were more
soluble in the hydrocarbon on the surface of the silica than they
are in the polar solvent - and so spend more time in this
alternative "solvent". Where a solute divides itself between two
different solvents because it is more soluble in one than the
other, we call it partition.
So is this adsorption or partition? You could argue it both ways!
Be prepared to find it described as either.

Looking at the whole process

A flow scheme for HPLC

Injection of the sample

Injection of the sample is entirely automated, and you wouldn't be
expected to know how this is done at this introductory level.
Because of the pressures involved, it is not the same as in gas
chromatography (if you have already studied that).

Retention time
The time taken for a particular compound to travel through the
column to the detector is known as its retention time. This time is
measured from the time at which the sample is injected to the point
at which the display shows a maximum peak height for that
compound.
Different compounds have different retention times. For a particular
compound, the retention time will vary depending on:

the pressure used (because that affects the flow rate of the

solvent)

the nature of the stationary phase (not only what material it

is made of, but also particle size)

the exact composition of the solvent

the temperature of the column

That means that conditions have to be carefully controlled if you are

using retention times as a way of identifying compounds.

The detector
There are several ways of detecting when a substance has passed
through the column. A common method which is easy to explain
uses ultra-violet absorption.
Many organic compounds absorb UV light of various wavelengths.
If you have a beam of UV light shining through the stream of liquid
coming out of the column, and a UV detector on the opposite side
of the stream, you can get a direct reading of how much of the light
is absorbed.
The amount of light absorbed will depend on the amount of a
particular compound that is passing through the beam at the time.

You might wonder why the solvents used don't absorb UV light.
They do! But different compounds absorb most strongly in different

parts of the UV spectrum.

Methanol, for example, absorbs at wavelengths below 205 nm, and
water below 190 nm. If you were using a methanol-water mixture as
the solvent, you would therefore have to use a wavelength greater
than 205 nm to avoid false readings from the solvent.
Note: If you are interested, there is a whole section aboutUVvisible spectroscopy on the site. This explores the question of
the absorption of UV and visible light by organic compounds in
some detail.

Interpreting the output from the detector

The output will be recorded as a series of peaks - each one
representing a compound in the mixture passing through the
detector and absorbing UV light. As long as you were careful to
control the conditions on the column, you could use the retention
times to help to identify the compounds present - provided, of
course, that you (or somebody else) had already measured them
for pure samples of the various compounds under those identical
conditions.
But you can also use the peaks as a way of measuring the
quantities of the compounds present. Let's suppose that you are
interested in a particular compound, X.
If you injected a solution containing a known amount of pure X into
the machine, not only could you record its retention time, but you
could also relate the amount of X to the peak that was formed.
The area under the peak is proportional to the amount of X which
has passed the detector, and this area can be calculated
automatically by the computer linked to the display. The area it
would measure is shown in green in the (very simplified) diagram.

If the solution of X was less concentrated, the area under the peak
would be less - although the retention time will still be the same.
For example:

This means that it is possible to calibrate the machine so that it can

be used to find how much of a substance is present - even in very
small quantities.
Be careful, though! If you had two different substances in the
mixture (X and Y) could you say anything about their relative
amounts? Not if you were using UV absorption as your detection
method.

In the diagram, the area under the peak for Y is less than that for X.
That may be because there is less Y than X, but it could equally
well be because Y absorbs UV light at the wavelength you are
using less than X does. There might be large quantities of Y
present, but if it only absorbed weakly, it would only give a small
peak.
Note: If you want lots more detail about HPLC you could
explore the site operated by Waters Corporation - a supplier of
HPLC equipment.
You will also find a useful industry training video which talks
through the whole process by following this link.

Linking to other sites is always a little bit hazardous because

sites change. If you find that either of these links don't work,
please contact me via the address on the About this sitepage.

Coupling HPLC to a mass spectrometer

This is where it gets really clever! When the detector is showing a
peak, some of what is passing through the detector at that time can
be diverted to a mass spectrometer. There it will give a
fragmentation pattern which can be compared against a computer
database of known patterns. That means that the identity of a huge
range of compounds can be found without having to know their
retention times.

THIN LAYER CHROMATOGRAPHY

This page is an introduction to chromatography using thin layer

chromatography as an example. Although if you are a beginner you
may be more familiar with paper chromatography, thin layer
chromatography is equally easy to describe and more
straightforward to explain.
Note: I'm taking a simple view of the way that thin layer
chromatography works in terms of adsorption (see below)
which should be adequate for students doing courses for 16 18 year olds. The reality is more complicated and the
explanation will vary depending on what sort of solvent or
solvent mixture you are using. Some similar problems are
discussed on the page about paper chromatography, but I am
unwilling to do the same thing on this page which is intended
as a fairly gentle introduction to chromatography.

Carrying out thin layer chromatography

Background
Chromatography is used to separate mixtures of substances into
their components. All forms of chromatography work on the same
principle.
They all have a stationary phase (a solid, or a liquid supported on
a solid) and a mobile phase (a liquid or a gas). The mobile phase
flows through the stationary phase and carries the components of
the mixture with it. Different components travel at different rates.
We'll look at the reasons for this further down the page.
Thin layer chromatography is done exactly as it says - using a thin,
uniform layer of silica gel or alumina coated onto a piece of glass,
metal or rigid plastic.
The silica gel (or the alumina) is the stationary phase. The
stationary phase for thin layer chromatography also often contains

a substance which fluoresces in UV light - for reasons you will see

later. The mobile phase is a suitable liquid solvent or mixture of
solvents.

Producing the chromatogram

We'll start with a very simple case - just trying to show that a
particular dye is in fact a mixture of simpler dyes.

Note: The chromatography plate will in fact be pure white - not

pale grey. I'm forced to show it as off-white because of the way
I construct the diagrams. Anything I draw as pure white allows
the background colour of the page to show through.

A pencil line is drawn near the bottom of the plate and a small drop
of a solution of the dye mixture is placed on it. Any labelling on the
plate to show the original position of the drop must also be in
pencil. If any of this was done in ink, dyes from the ink would also
move as the chromatogram developed.

When the spot of mixture is dry, the plate is stood in a shallow layer
of solvent in a covered beaker. It is important that the solvent level
is below the line with the spot on it.
The reason for covering the beaker is to make sure that the
atmosphere in the beaker is saturated with solvent vapour. To help
this, the beaker is often lined with some filter paper soaked in
solvent. Saturating the atmosphere in the beaker with vapour stops
the solvent from evaporating as it rises up the plate.
As the solvent slowly travels up the plate, the different components
of the dye mixture travel at different rates and the mixture is
separated into different coloured spots.

The diagram shows the plate after the solvent has moved about
half way up it.
The solvent is allowed to rise until it almost reaches the top of the
plate. That will give the maximum separation of the dye
components for this particular combination of solvent and
stationary phase.

Measuring Rf values
If all you wanted to know is how many different dyes made up the
mixture, you could just stop there. However, measurements are
often taken from the plate in order to help identify the compounds
present. These measurements are the distance travelled by the
solvent, and the distance travelled by individual spots.
When the solvent front gets close to the top of the plate, the plate is
removed from the beaker and the position of the solvent is marked
with another line before it has a chance to evaporate.
These measurements are then taken:

The Rf value for each dye is then worked out using the formula:

For example, if the red component travelled 1.7 cm from the base
line while the solvent had travelled 5.0 cm, then the R f value for the
red dye is:

If you could repeat this experiment under exactly the same

conditions, then the Rf values for each dye would always be the
same. For example, the Rf value for the red dye would always be
0.34. However, if anything changes (the temperature, the exact
composition of the solvent, and so on), that is no longer true. You
have to bear this in mind if you want to use this technique to identify
a particular dye. We'll look at how you can use thin layer
chromatography for analysis further down the page.

What if the substances you are interested in are colourless?

There are two simple ways of getting around this problem.
Using fluorescence
You may remember that I mentioned that the stationary phase on a
thin layer plate often has a substance added to it which will
fluoresce when exposed to UV light. That means that if you shine
UV light on it, it will glow.
That glow is masked at the position where the spots are on the final
chromatogram - even if those spots are invisible to the eye. That
means that if you shine UV light on the plate, it will all glow apart
from where the spots are. The spots show up as darker patches.

While the UV is still shining on the plate, you obviously have to

mark the positions of the spots by drawing a pencil circle around
them. As soon as you switch off the UV source, the spots will
disappear again.

Showing the spots up chemically

In some cases, it may be possible to make the spots visible by
reacting them with something which produces a coloured product.
A good example of this is in chromatograms produced from amino
acid mixtures.
The chromatogram is allowed to dry and is then sprayed with a
solution of ninhydrin. Ninhydrin reacts with amino acids to give
coloured compounds, mainly brown or purple.

In another method, the chromatogram is again allowed to dry and

then placed in an enclosed container (such as another beaker
covered with a watch glass) along with a few iodine crystals.
The iodine vapour in the container may either react with the spots
on the chromatogram, or simply stick more to the spots than to the
rest of the plate. Either way, the substances you are interested in
may show up as brownish spots.

Using thin layer chromatography to identify compounds

Suppose you had a mixture of amino acids and wanted to find out
which particular amino acids the mixture contained. For simplicity
we'll assume that you know the mixture can only possibly contain
five of the common amino acids.
A small drop of the mixture is placed on the base line of the thin
layer plate, and similar small spots of the known amino acids are
placed alongside it. The plate is then stood in a suitable solvent
and left to develop as before. In the diagram, the mixture is M, and
the known amino acids are labelled 1 to 5.
The left-hand diagram shows the plate after the solvent front has
almost reached the top. The spots are still invisible. The second

diagram shows what it might look like after spraying with ninhydrin.

There is no need to measure the Rf values because you can easily

compare the spots in the mixture with those of the known amino
acids - both from their positions and their colours.
In this example, the mixture contains the amino acids labelled as 1,
4 and 5.
And what if the mixture contained amino acids other than the ones
we have used for comparison? There would be spots in the mixture
which didn't match those from the known amino acids. You would
have to re-run the experiment using other amino acids for
comparison.

How does thin layer chromatography work?

The stationary phase - silica gel
Silica gel is a form of silicon dioxide (silica). The silicon atoms are
joined via oxygen atoms in a giant covalent structure. However, at
the surface of the silica gel, the silicon atoms are attached to -OH

groups.
Note: If you aren't sure about it, you will find one
possiblestructure of silicon dioxide towards the bottom of the
page you will get to by following this link.
Use the BACK button on your browser to return quickly to this
page.

So, at the surface of the silica gel you have Si-O-H bonds instead
of Si-O-Si bonds. The diagram shows a small part of the silica
surface.

The surface of the silica gel is very polar and, because of the -OH
groups, can form hydrogen bonds with suitable compounds around
it as well as van der Waals dispersion forces and dipole-dipole
attractions.
Note: If you aren't sure about hydrogen bonds and van der
Waals forces follow this link to the page about hydrogen
bonding. You will find a further link to van der Waals forces at
the bottom of that page.

Use the BACK button on your browser to return quickly to this

page.

The other commonly used stationary phase is alumina - aluminium

oxide. The aluminium atoms on the surface of this also have -OH
groups attached. Anything we say about silica gel therefore applies
equally to alumina.

What separates the compounds as a chromatogram develops?

As the solvent begins to soak up the plate, it first dissolves the
compounds in the spot that you have put on the base line. The
compounds present will then tend to get carried up the
chromatography plate as the solvent continues to move upwards.
How fast the compounds get carried up the plate depends on two
things:

How soluble the compound is in the solvent. This will

depend on how much attraction there is between the
molecules of the compound and those of the solvent.

How much the compound sticks to the stationary phase - the

silica gel, for example. This will depend on how much
attraction there is between the molecules of the compound
and the silica gel.

Suppose the original spot contained two compounds - one of which

can form hydrogen bonds, and one of which can only take part in
weaker van der Waals interactions.
The one which can hydrogen bond will stick to the surface of the
silica gel more firmly than the other one. We say that one
isadsorbed more strongly than the other. Adsorption is the name

given to one substance forming some sort of bonds to the surface

of another one.
Adsorption isn't permanent - there is a constant movement of a
molecule between being adsorbed onto the silica gel surface and
going back into solution in the solvent.
Obviously the compound can only travel up the plate during the
time that it is dissolved in the solvent. While it is adsorbed on the
silica gel, it is temporarily stopped - the solvent is moving on
without it. That means that the more strongly a compound is
adsorbed, the less distance it can travel up the plate.
In the example we started with, the compound which can hydrogen
bond will adsorb more strongly than the one dependent on van der
Waals interactions, and so won't travel so far up the plate.
What if both components of the mixture can hydrogen bond?
It is very unlikely that both will hydrogen bond to exactly the same
extent, and be soluble in the solvent to exactly the same extent. It
isn't just the attraction of the compound for the silica gel which
matters. Attractions between the compound and the solvent are
also important - they will affect how easily the compound is pulled
back into solution away from the surface of the silica.
However, it may be that the compounds don't separate out very well
when you make the chromatogram. In that case, changing the
solvent may well help - including perhaps changing the pH of the
solvent.
This is to some extent just a matter of trial and error - if one solvent
or solvent mixture doesn't work very well, you try another one. (Or,
more likely, given the level you are probably working at, someone
else has already done all the hard work for you, and you just use
the solvent mixture you are given and everything will work
perfectly!)

A simple and specific method for the analysis of codeine phosphate, chlorpheniramine maleate, phenylephrine
hydrochloride and acetaminophen in pharmaceutical dosage forms was developed. The procedure consists of direct
application of diluted liquid dosage forms and the solutions of solid dosage forms on silica gel plates. The mobile phase for
development consisted of n-butanol-methanol-toluene-water and acetic acid. The separated components were measured
quantitatively by densitometry. Linearity, reproducibility and percentage recovery of active ingredients from dosage forms
were calculated.

Recent

updates

on

Bhandari1, Anil

Monika

Bhandari2, Aakanksha

Jodhpur

Lachoo Memorial College of Science and Technology, Jodhpur, Rajasthan, India

College

of

Date of Web Publication

Pharmacy,

codeine

Jodhpur

National

University,

Bhandari2

Jodhpur,

Rajasthan,

16-May-2011

Correspondence

Address:

Monika
Jodhpur

India

Bhandari
College

of

Pharmacy,

Jodhpur

National

University,

Jodhpur,

Rajasthan

India

DOI: 10.4103/2229-4708.81082
PMID: 23781422

Abstract
Alleviation of pain is a major objective in medicine to increase the quality of life. Analgesics are agents that relieve
pain by elevating the pain threshold without disturbing consciousness or altering other sensory modalities. Opium is
an isoquinoline alkaloid obtained from poppy plant Papaver somniferum (Papaveraceae). Codeine is an alkaloid
prepared from opium or morphine by methylation. Codeine is used as a central analgesic, sedative, hypontic,
antinonciceptive, antiperistaltic, and is also recommended in tuberculosis and insomnia due to incessant coughing.
The literature information relate mostly to the determination of codeine active components using Gas
chromatography (GC), Capillary electrophoresis, Thin layer chromatography, High-performance thin layer
chromatography, UV-Vis Spectrophotometry, High-performance liquid chromatography and GC in combination with
Mass spectroscopy. This contribution provides a comprehensive review of its analytical and pharmacologic profile of
codeine.

Keywords: Analgesic, Codeine, Isoquinoline alkaloid, HPLC, HPTLC Introduction

How to cite this article:

Bhandari M, Bhandari A, Bhandari A. Recent updates on codeine. Pharm Methods 2011;2:3-8

How to cite this URL:

Bhandari M, Bhandari A, Bhandari A. Recent updates on codeine. Pharm Methods [serial online] 2011 [cited 2014
Apr 19];2:3-8. Available from: http://www.phmethods.org/text.asp?2011/2/1/3/81082

Introduction

Codeine (methyl morphine) is a strong mono acidic base and laevorotatory. It effloresces slowly in dry air and is
affected by light. The chemical name of codeine phosphate is 7,8-didehydro-4,5alpha-epoxy-3-methoxy-17methylmorphinan-6alpha-olphosphate (1:1) (salt) hemihydrate and has the empirical formula of
C 18 H 21 NO 3H 3 PO 41/2H 2 O. [1] Codeine phosphate contains not less than 98.5 percent and not more than 101.0
percent of C 18 H 21 NO 3 , H 3 PO 4 calculated on the dried basis. [2] Codeine molecular weight is 406.4. Each soluble
tablet contains 30 mg (0.074 mmol) or 60 mg (0.15 mmol) of codeine phosphate. These tablets also contain lactose
and sucrose. Soluble tablets of codeine phosphate are freely soluble in water. [1]

Mechanism of Action

Codeine is a weak narcotic pain reliever and cough suppressant similar to morphine and hydrocodone. A small
amount of codeine is converted to morphine in the body. Like morphine, codeine binds to receptors in the brain
(opioid receptors) that are important for transmitting the sensation of pain throughout the body and brain. Codeine
increases tolerance to pain, decreasing discomfort but the pain still is apparent to the patient. In addition to reducing
pain, codeine also causes sedation, drowsiness, and depressed breathing. Codeine frequently is combined with
acetaminophen (Tylenol) or aspirin for more effective pain relief. [3]
Analytical and pharmacologic profile of codeine
Presence and formation of codeine and morphine in the male Sprague-Dawley rat
Endogenous codeine and morphine were identified in rat brain by immunologic determination following highperformance liquid chromatography (HPLC). To demonstrate the occurrence of a biosynthetic pathway to morphine in
mammals similar to that used by the poppy plant, (+)-salutaridine, (-)-thebaine, and (-)-codeine were administered to
rats intravenously. These compounds which are intermediates in the synthesis of morphine in Papaver
somniferum caused a marked increase in the codeine and morphine levels in rat tissues. This provides evidence for a
biosynthetic pathway to morphine in mammalians. [4]
Codeine in plasma with fluorescence detection
Determination of codeine in human plasma was based on HPLC for separation and the natural fluorescence of
codeine for detection. Codeine was extracted from alkalinized plasma with a mixture of hexane and dichloromethane
and the extract was further purified and chromatographed. In this method, a stock solution of codeine was prepared
by dissolving the amount of codeine phosphate equivalent to 1 mg of codeine in 10 mL of methanol. The stock
solution was further diluted with an equivolume mixture of methanol and water to yield a solution containing 1 g of
codeine per milliliter. This solution was used for supplementary drug-free samples of plasma. A stock solution and a

working solution of internal standard were prepared in the same way.

Solutions: Phosphate buffer solution (50 mmol/L, pH 8), was prepared by dissolving 7 g of monobasic sodium
phosphate in 1 L of water and adjusting the pH to 8 with a 1 mol/L solution of NaOH.
Procedure: Pipet 2 mL of plasma into a 15 mL test tube and add 200 ng of the internal standard. Alkalinize the
plasma with 2 mL of the 50 mmol/L phosphate buffer solution and extract twice with 6 mL portions of
hexane/dichloromethane (2/1 by vol) by manually shaking the mixture for 2 min then centrifuging. Combine the
organic extracts and wash with 1 mL of NaOH, 50 mmol/L to remove any potentially interfering substances. Transfer
the extract to a 15 mL conical test tube and evaporate under a gentle stream of nitrogen to about 1 mL. Wash down
the inside wall of the test tube with 1 to 2 mL of methanol and evaporate the entire contents of the test tube to
dryness under nitrogen. Dissolve the residue in 200 L of HPLC mobile phase . Place the test tube in an ultrasonic
bath for 30 s, then vigorously vortex-mix. Inject a 50 L aliquot onto the chromatographic column.
Chromatographic conditions: The mobile phase was composed of methanol/water (21/79 by vol), containing 1.5 g of
phosphoric acid per liter. Flow rate was set at 2 mL/min and columns were operated at ambient temperature. Column
pressure was maintained between 1000 to 2000 psi (6.89 10 6 to 13.7 10 6 Pa). The recorder was used at a chart
speed of 5 mm/min.
The method can be used for routine assay of codeine at concentrations of 10 g/L or greater in human plasma. As
little as 4 g/L can be detected. Coefficients of variation for the assay of codeine in the concentration range of 10-100
g/L were 2.2%-7.4% (n = 6). This method is used to establish a concentration/time profile for plasma from a human
volunteer after a 60 mg oral dose of codeine sulfate. [5]
Determination of morphine and codeine in plasma
A cheap simple and rapid extraction procedure followed by a UV HPLC assay was described for the simultaneous
determination of morphine and codeine in plasma. This method was based on the extraction of these opiates from
plasma using reversed phase (solid phase) extraction columns followed by HPLC with UV detection at 240 nm. The
extraction step provides, respectively, 85% and 80% recovery for morphine and codeine. The response of the
detection system was linear for both molecules in the studied range from 50 to 750 ng/mL. No other drugs have been
found to interfere with the assay. This method offers a quick, cost-effective, and reliable procedure for specifically
determining morphine and codeine from a small sample volume. [6]
Undeclared codeine in antiasthmatic Chinese proprietary medicine
A rapid and specific liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) method was applied
to confirm the presence of codeine by selected reaction monitoring. Codeine was extracted from the capsules by
dissolving in sodium dihydrogen phosphate buffer (10 mM, pH = 2.2) and ethanol then made alkaline (pH = 9) and
extracted using chloroform. The amount of codeine in AsthmaWan was found to be 61.8 g/capsule [relative standard
deviation (RSD) = 7.9%, n = 9]. Excellent resolution was obtained despite the complexity of the product, which
claimed to contain at least nine herbal ingredients, none of which will give rise to codeine. As a further confirmation
method, LC-MS-MS was accurate and specific. [7]
Analysis of codeine and its metabolites in human plasma
The resolving power of high pressure liquid chromatography has been combined with the sensitivity of
electrochemical oxidation to develop a method for determination of codeine and its metabolites, morphine and
norcodeine, in plasma. Plasma samples containing Internal Standard (dihydromorphinone) were extracted at pH 8.9
into a 2/98 v/v butanol/methyl tertiary butyl ether organic solvent system and back extracted into 25 mM phosphate
buffer pH 2.8. The optimal recovery was greater than 90% for codeine and 70% for morphine and norcodeine.
Reverse phase chromatography (5 m phenyl column) with detection by electrochemical oxidation at +1.2 V vs
Ag/AgCl was utilized. The method was sensitive, specific, and precise. This method was used to establish a
concentration-time profile for plasma codeine and morphine from a human volunteer after a 60 mg oral dose of

codeine phosphate. No measurable concentration of norcodeine was found in the plasma. There was no reported
method to simultaneously quantitate codeine and its O-demethylated and N-demethylated metabolites in plasma.
Radioimmunoassay (RIA) can be used for quantization of codeine and morphine but the assay was not specific due
to cross-reaction with the other opiates. Procedures such as GS and thin-layer chromatography (TLC) have also
been reported to be used in forensic toxicology studies. [8]
Rapid extraction of codeine and morphine in whole blood
A rapid and efficient procedure was described for the extraction and analysis of codeine and morphine in whole
blood. Red blood cells were fragmented by sonication and the blood sample was extracted by passing through a
bonded silica column (Bond Elute , Jone chromatography). The adsorbed drugs were washed and eluted followed
by HPLC analysis. Recoveries were between 95% and 100% at 5 ng/mL concentrations. [9]
Capillary electrophoresis chemiluminescence detection
A simple and robust capillary electrophoresis chemiluminescence detection system for the determination of codeine,
6-methoxycodeine, and thebaine was described based on the reaction of these analytes with chemically generated
tris(2,2A-bipyridyl)ruthenium(iii) prepared in sulfuric acid (0.05 M). The reagent solution was contained in a glass
detection cell, which also held both the capillary and the cathode. The resultant chemiluminescence was monitored
directly using a photomultiplier tube mounted flush against the base of the detection cell. The RSDs of the migration
times and the peak areas for the three analytes ranged from 2.2% to 2.5% and 1.9% to 4.6%, respectively. [10]
Simultaneous determination of paracetamol and codeine phosphate in combined tablets dosage form
Two simple, accurate, and reproducible UV spectrophotometric techniques have been developed for the
simultaneous determination of paracetamol and codeine phosphate in combined tablets. The first one was based on
the measurement of first-order derivative amplitudes at zero-crossing points 263.5 and 218.4 nm for the assay of
paracetamol (5.0-25.0 mg/L) and codeine phosphate (1.25-10.0 mg/L), respectively. The second one uses the
amplitudes of ratio spectra first-order derivatives at 255.3 and 221.4 nm to quantify paracetamol (5.0-30.0 mg/L) and
codeine phosphate (0.625-10.0 mg/L), respectively. Both the techniques have been extensively validated and
compared with an official HPLC method showing their applicability in the routine analysis. [11]
Codeine and caffeine in codeine-aspirin-phenacetin-caffeine tablets
A procedure was reported for determining the codeine and caffeine content of individual codeine-aspirin-phenacetincaffeine tablets. Codeine was determined fluorometrically; after extraction into dilute sulfuric acid, caffeine was
extracted from a chloroform solution of the remaining ingredients with phosphoric acid and determined by UV
spectroscopy. Average recoveries with a synthetic mixture were 99.7% and 98.9% for codeine and caffeine,
respectively. Assay results were reported for codeine [1-65 mg (1/65 1 grain/tablet] and caffeine [32 mg 1/2
grain)/tablet] in several different commercial samples. [12]
Estimation of uncertainty for measuring codeine phosphate tablets formulation
Analytical results represent a very important part in a quality control program. Uncertainty estimation was an
important step in method validation. The objective of this article was to study the uncertainty of measurement
estimation in the quantitative determination of codeine phosphate from pharmaceutical formulations using UV-Vis
spectrophotometry. The uncertainty estimation was performed using the Ishikawa diagram. The estimation of
uncertainty components proved to be a good way for the experimental model to obtain low contribution of uncertainty
to the analytical result. [13]
Determination of propyphenazone, paracetamol, caffeine, and codeine phosphate with thin-layer
chromatography
The conditions have been examined for the determination of propyphenazone, paracetamol, caffeine, and codeine

phosphate in caffetin tablets with preparative TLC. The separation of propyphenazone, paracetamol, and caffeine
was performed by use of a mobile phase chloroform-acetone-ammonium hydroxide (25%) in volume ratios of 8:2:0.1.
Codeine phosphate was separated from the other components with chloroform-ethanol in the volume ratio of 8:2 as a
mobile phase. The location of spots on the chromatogram was detected with UV lamp on 254 nm. Every component
was eluted from the adsorbent with a solution of 0.1 mol/L hydrochloric acid in ultrasonic bath for 5 min. The solutions
were filtered and the absorbency was measured at 240 nm for propyphenazone, 245 nm, for paracetamol, 273 nm for
caffeine, and 285 nm for codeine phosphate. The results show that the applied method was fast, reproducible, and
suitable for routine analysis. [14]
Simultaneous determination of acetaminophen and codeine
First derivative UV spectrophotometry has been used for the simultaneous determination of acetaminophen and
codeine. Acetaminophen has been assayed by measuring the first derivative absorbances at 263.4 nm and codeine
at 251.2 nm. The concentrations of acetaminophen and codeine have been calculated without interference of each
other. The procedure was simple and rapid and provides accurate and precise results. [15]
Simultaneous quantification of morphine and codeine in poppy capsules
TLC-UV densitometric and GS-MS detection (GC-MSD) methods were developed for simultaneous quantification of
morphine and codeine in poppy capsules (P. somniferum). Morphine and codeine were isolated by extraction with
chloroform:isopropanol (3:1, v/v) at pH = 8.5 and by solid-phase extraction on Snap-Cap cartridges at pH = 8.5. The
TLC-UV densitometric quantification was performed by external standard method on silica gel plates using ethyl
acetate:toluene:methanol:ammonia (68:17:10:5, v/v) as developing solvent and UV detection at 275 nm. For the GCMSD analysis, the drugs were derivatized with acetic anhydride:pyridine (1:1, v/v) and separated on a 30 m HP5
capillary column. The quantification was performed using nalorphine as internal standard. [16]
Identification of codeine
A new spray reagent, a 1% (w/v) aqueous solution of ferric chloride, and a 1% (w/v) acidified alcoholic solution of
2,2-dipyridyl has been developed for the detection and identification of heroin (diacetylmorphine). A red spot was
observed for heroin when the high-performance TLC (HPTLC) plate was sprayed with the reagent and heated at
100C. Similar spots were observed for morphine, codeine, and thebaine (opiates containing the phenanthrene
nucleus). It did not react with papavarine and narcotine (opiates containing the isoquinoline nucleus). The reagent
was specific, sensitive, and can be used for the detection and identification of heroin in forensic samples. [17]
Antistress activity
P. somniferum produces secondary metabolites, which have important roles in self-defense processes in plant
biochemistry and in allelochemistry. By performing experiments to determine how irregular stress effects changed the
alkaloid content of poppies we have shown that different types of stress affect the quantities of alkaloids. P.
somniferum plants were grown for 2 months from seeds in quartz-sand (natural light, temperature 24C-28C, in
Knopf"s nutritive solution). In this work, the alkaloids in poppies were subjected to two kinds of stress-mycotoxin and
drought. The amounts of alkaloids were measured by different separation and detection procedures TLC and HPTLC
with fluorescence detection and HPLC. HPLC proved superior for identification and approximate estimation of the
morphine alkaloids but the effects of stress on poppy plants can be detected by use of either method. Drought stress
resulted in higher levels of the alkaloids, whereas mycotoxin stress did not result in significant difference. [18]
Chronic cancer pain control
The improved pain control provided by regular dosing of opioid analgesics in patients with severe cancer pain has
been well established. This randomized double-blinded study was designed to evaluate the efficacy of controlledrelease codeine (Codeine Contin) in patients with cancer pain and to estimate its dose equivalence to a standard
combination of acetaminophen plus codeine. Twenty-four patients with at least moderate cancer pain were
randomized to codeine contin 100, 200, or 300 mg every 12 h or acetaminophen plus codeine (600 mg/60 mg) every

6 h. On days 1 and 4 of dosing, pain intensity and pain relief were assessed hourly for 12 h. The sum of pain intensity
differences from baseline and the total pain relief scores demonstrated a dose-response relationship for codeine
contin on days 1 and 4 that was statistically significant on day 1 and suggested a greater analgesic efficacy on day 4
compared with day 1. Codeine contin 150 mg every 12 h was estimated to be equianalgesic to acetaminophen plus
codeine (600 mg/60 mg) given every 6 h. Because a similar equivalence was also demonstrated from analysis of
adverse event data, it is concluded that codeine contin 150 mg produces analgesia, and a side-effect profile similar to
a 40% lower dose of codeine provided by the combination. These results indicate the need for studies of scheduled
every 12-h dosing of codeine contin in comparison with "as needed" dosing of combination preparations in patients
with cancer pain. [19]
Hepatotoxic activity
The administration of codeine to freshly isolated rat hepatocytes resulted in cytotoxicity characterized by a dose- and
time-dependent leakage of lactate dehydrogenase (LDH) out of the cells. Cytochrome P-450 content and NADPH
levels were not changed. Induction and inhibition studies of several potential pathways of codeine biotransformation
were carried out to determine if codeine must be metabolized to a reactive intermediate to elicit these hepatotoxic
effects. Codeine hepatotoxicity as measured by LDH release was not changed after induction of cytochrome P-450
by phenobarbital and was decreased after cytochrome P-448 induction by B-naphthoflavone. However, codeine
hepatotoxicity was inhibited when an inhibitor of cytochrome P-450 metabolism, metyrapone, was added. Inhibition of
the other major hepatic oxidative enzyme system, flavin adenine dinucleotide (FAD)-containing monooxygenase,
increased the cytotoxicity of codeine. Inhibition of alcohol dehydrogenase had no effect on codeine hepatotoxicity.
These results indicate that codeine hepatotoxicity was caused by a cytochrome P-450-generated intermediate of
codeine, whereas FAD-containing monooxygenase may metabolize codeine to a nontoxic intermediate. [20]
Antinociceptive activity
Centrally administered codeine glucuronide has been shown to exhibit antinociceptive properties with decreased
immunosuppressive effects compared with codeine. In this study, codeine-6-glucuronide was administered to rats
and its analgesic effect was compared with that of codeine. The concentrations of codeine and its metabolites in
plasma and brain were also determined at the peak response time after administration of each compound. Receptorbinding studies with rat brain homogenates and affinity profiles were also determined. Intravenous administration of
codeine-6-glucuronide resulted in approximately 60% of the analgesic response elicited by codeine itself. Analysis of
plasma and brain showed that codeine-6-glucuronide was relatively stable in vivo with only small amounts of
morphine-6-glucuronide being detected in addition to unchanged codeine-6-glucuronide. The receptor affinity of
codeine-6-glucuronide was similar to that of codeine. It was concluded that intravenously administered codeine 6glucuronide possesses analgesic activity similar to that of codeine and may have clinical benefit in the treatment of
pain. [21]
Antitussive activity
Codeine 10, 20, and 50 mg/kg dose-dependently depressed the coughs caused by larynx stimulation. The antitussive
failed to depress the cough caused by stimulation to the tracheal bifurcation although a large dose (50 mg/g)
significantly depressed the cough. In capsaicin-treated guinea pigs codeine at 20 mg/g significantly depressed the
cough caused by stimulation to the tracheal bifurcation. The present results suggest that cough caused by
mechanical stimulation to the larynx might be more sensitive to codeine treatment than cough caused by stimulation
to the bifurcation of trachea. It was suggested that coughs caused by mechanical stimulation to both sites might
consist of at least two components as regards their pharmacologic nature. [22]

Conclusion

From time immemorial, codeine has been widely used as curative agent for a variety of ailments. The codeine
possesses significant central analgesic activity so it becomes very popular drug to relieve the pain in patient suffering
from the cancer and in head trauma. It also recommended in incessant coughing. Quite a significant amount of work
has been done on the analytical evaluation and indications of codeine. Recent evidence relates that it is a
combination of four components: propyphenazone, paracetamol, caffeine, and codeine phosphate; in this
combination caffeine and codeine phosphate increase the analgesic effect of the paracetamol and the
propyphenazone synergistically.

References

1.

Available from: http://www.rxlist.com/codeine_phosphate-drug.htm. [Last accessed on 2010 Aug 05].

2.

Anonymous,

3.

Available from: http://www.medicinenet.com/codeine/article.htm. [Last accessed on 2010 Aug 05].

4.

Donnerer J, Kazuhiro Oka, Arnold Brossit, Ricet KC, Sydney Spector. Presence and formation of codeine and
morphine
in
the
rat.
Neurobiology
1986;83:4566-7.

5.

Tslna IW, Marian Fass, Jennifer A, Debban, Matin SB. Liquid Chromatography of Codeine in Plasma with
Fluorescence
Detection.
Clinical
Chemistry
1982;28:1137-9.

6.

Michel Freiermuth, Jean-Claude Plasse. Determination of morphine and codeine in plasma by HPLC following
solid phase extraction, Journal of Pharmaceutical and Biomedical Analysis. 1997;15:759-64.

7.

Liu SY, Woo SO, Holmes MJ, Koh HL. LC and LC-MS-MS analyses of undeclared codeine in antiasthmatic
Chinese
proprietary
medicine.
J
Pharm
Biomed
Anal
2000;22:481-6.
[PUBMED] [FULLTEXT]

8.

Jaymin Shaha, Mason WD. Analysis of Codeine and its Metabolites in Human Plasma by HPLC with
Electrochemical
Detection,
Informa
World
Taylor
and
Francis
1987;20:881-93.

9.

Tebbett R. Rapid extraction of codeine and morphine in whole blood for HPLC analysis, Chromatographia
1987;23:377-8.

10.

Barnett NW, Hindson BJ, Lewis SW, Purcell SD. Determination of codeine, 6-methoxycodeine and thebaine
using capillary electrophoresis with tris (2,2A-bipyridyl) ruthenium (ii) chemiluminescence detection, Anal
Commun
1998;35:321-4.

11.

Duong Thi Thuy An, Vu Dang Hoang. Simultaneous determination of paracetamol and codeine phosphate in
combined tablets by first-order derivative and ratio spectra first-order derivative UV spectrophotometry. Asian J

Indian

Pharmacopoeia,

Pharmacopoeial

commission,

Ghaziabad.

2007;2:344.

Research

Chem

2009;2:143-7.

12.

Theron J. Individual tablet analysis for codeine and caffeine in codeine-aspirin-phenacetin-caffeine tablets. J
Pharmac
Sci
2006;62:1500-02.

13.

Diaconua I, Aboul-Eneinbc HY, Bunaciud AA, Ion G. Estimation of uncertainty for measuring codeine phosphate
tablets formulation using UV-VIS spectrophotometry, Informa World Taylor and Francis 2010;43:1207-16.

14.

Dimitrovskat A, Trajkovic-Jolevskat S, Nancovskat A, Ilievska M. Determination of propyphenazone,

paracetamol, caffeine and codeine phosphate with thin layer chromatography. Bulletin Chemists Technologists
Macedonia
1995;14:39-41.

15.

Hanaee J. Simultaneous determination of acetaminophen and codeine in pharmaceutical preparations by

derivative
spectrophotometry.
Pharmaceutica
Acta
Helvetiae
1997;72:239-41.

16.

Popa DS, Oprean R, Curea E, Preda N. TLC-UV densitometric and GC-MSD methods for simultaneous
quantification of morphine and codeine in poppy capsules. J Pharm Biomed Anal 1998;18:645-50.
[PUBMED] [FULLTEXT]

17.

Dongre VG, Kamble VW. HPTLC detection and identification of heroin (diacetylmorphine) in forensic samples
part
III.
J
planar
chromatography
2007;16:458-60.

18.

Szabo B, Lakatos A, Koszegi T, Botz L. HPTLC and HPLC determination of alkaloids in poppies subjected to
stress.
J
Planar
Chromatography
modern
TLC
2003;16:293-7.

19.

Chary S, Goughnour BR, Moulin DE, Thorpe WR, Harsanyi Z, Darke AC. The dose response relationship of
controlled-release codeine (CodeineContin) in chronic cancer pain. J Pain Symptom Management 1994;9:36371.

20.

Ellington SP, Rose GM. Codeine-mediated hepatotoxicity in isolated rat hepatocytes, Toxicol Appl Pharmacol
1987;90:156-65.

21.

V Srinivasan, Wielbo D, Tebbett IR. Analgesic effects of codeine-6-glucuronide after intravenous administration.
Dep
European
J
Pain
1997;1:185-90.

22.

Takahama K, Wakuda I, Fukushima H, Isohama Y, Kai H, Miyata T. Differential effect of codeine on coughs
caused by mechanical stimulation of two different sites in the airway of guinea pigs. Euro J Pharmacology
1997;329:93-7.