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CHROMATOGRAPHIC TECHNIQUES
Presented by;
Mahboob Alam
M. Phil (Analytical chemistry)
Mpc 225
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Liquid chromatography–tandem
mass spectrometry detection of
diclofenac and related compounds
in water samples
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Received: 24 June 2017,
Accepted: 17 January 2018,
Available online: 18 January 2018
Published in Journal of Chromatography A
Impact Factor: 3.72
Liquid chromatography–tandem mass spectrometry detection of 4
diclofenac and related compounds in water samples

Stephan Schmidt, Holger Hoffmann, Leif-alexander Garbe, Rudolf J. Schneider.

 BAM federal institute for materials research and testing, department of

analytical chemistry; reference materials, Richard willstätter-str. 11,12489 berlin,
Germany
 Technische universität berlin, straße des 17. Juni 135, 10623 berlin, Germany
 Humboldt-universität zu berlin, department of chemistry, brook-taylor-str.2, 12489 berlin,
Germany
 Hochschule neubrandenburg, department of agroscience and food science, brodaer str. 2,
17033 Neubrandenburg, Germany
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Key words:
 Diclofenac
 Solid-phase extraction
 Sewage treatment plant
 Cyclization
 Sample treatment
 HPLC-MS
 Introduction 6
 Diclofenac (DCF) is a common environmental contaminants
found in surface waters and drinking water. It is used medicine.
 Diclofenac directed orally is excreted to 65-70% with the urine and
20-30% via faeces mainly in the form of its metabolites.
It is also heavily used is a treatment for any kind of muscular
pain.
 Only 6% of DCF are taken up from the ointment over the skin, the
reminder is washed off.
 Excessive use and incomplete removal in wastewater treatment
plants (21-40%) result in up to 75% of the DCF consumed entering the
water cycle and effect on the food chain.
 DCF is harmful to many species. Microgram per litre concentrations
already activate toxic effect in liver and kidney.
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 European concentrations show in average of 0.7µg/L with maximum of
11µg/L. In ground or drinking water , the concentrations reach 1-7 ng/L.
 Determination was performed by LC-MS , GC-MS.
 Metabolites and degradation products are present in wastewater which
may have a higher toxicity than DCF itself.
 Due to the continuous input from the treatment plants would be constant
concentration exists in surface waters. This is one of the reasons, why
diclofenac was included in the watch list of the EU water Framework
Directive.
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• The main metabolite of DCF is 4-hydroxydiclofenac (4-OH-DCF) was
present in much higher concentration than DCF. From cyclization
reaction DCF can undergo various conditions, the resulting is DCF
lactam. Another active ingredient is aceclofenac (ACF), which is the
glycolic acid ester of diclofenac.
• A common sample preparation is pre-concentration by means of
solid-phase extraction (SPE).
• Organic acids present in the samples, cyclization takes place under
acidic conditions.
• DCF metabolites cyclize in a sample pre-treatment step for gas
chromatography.
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HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC)
 High-performance liquid chromatography (sometimes referred to as high-pressure liquid
chromatography), is a chromatographic technique used to separate a mixture of compounds
with identifying, quantifying or purifying the individual components of the mixture.
 HPLC is just one type of liquid chromatography, meaning the mobile phase is a liquid.
 It is called high pressure b/c operational pressures are significantly higher (50–350 bar)
 HPLC columns are made with smaller adsorbent particles e.g., Silica, polymers, etc (2–50
μm average particle size). This gives HPLC superior resolving power (the ability to
distinguish between compounds) when separating mixtures.
 Various detectors are in common use, such as uv/vis, photodiode array (pda) and mass
spectrometers.
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Tandem mass spectrometry
 Tandem mass spectrometry, also known as MS/MS or MS2,
is a technique in Instrumental Analysis where two or
more mass spectrometers are coupled together using an
additional reaction step to increase their abilities
to analyse chemical samples.
 The molecules of a given sample are ionized and the first
spectrometer (designated MS1) separates these ions by
their mass-to-charge ratio. Ions of a particular m/z-ratio
coming from MS1 are selected and then made to split into
smaller fragment ions.
 These fragments are then introduced into the second mass
spectrometer (MS2), which in turn separates the fragments
by their m/z-ratio and detects them. The fragmentation step
makes it possible to identify and separate ions that have very
similar m/z-ratios in regular mass spectrometers.
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Experimental
 Reagents and Materials

 Diclofenac sodium salt, aceclofenac, 13C6 labeled diclofenac salt, 4-hydroxydiclofenac were used.
 For HPLC the following solvent compounds were used:
Ammonium acetate (NH4Ac), glacial acetic acid (AcOH), methanol (MeOH), folded filters, Strata TM-X
33µm
polymeric Reversed Phase SPE cartridges and ultrapure water.
 An agilent 1260 infinity LC system with a binary pump, degasser, autosampler, column heater and UV
detector
was used.
 The mobile phases were ultra-pure water with 0.1% (v/v) acetic acid (A) and MeOH with 0.1%
(v/v)acetic acid (B). The flow rate was 350 L/min and the column heater temperature was 55◦C.
 The injection volume was 10 µL.
 The quantification was performed by triple quad mass spectrometer. 13
 The parameters used to produce fragment ions in selected reaction
monitoring mode (SRM) and collision energies (CE) are given in Table 1.
 SPE was performed with an Auto Trace TM SPE workstation. 14
Firstly, Strata TM-X cartridges were washed with 10 ml MeOH,
equilibrated with 10 ml ultrapure water, followed by loading the
respective sample volume of 1000 ml. The flow rate for each step
was 10 ml/min. Afterwards, the cartridges were dried by flushing
N2 (20 psi) for 15 min and the adsorbates were eluted with 10 ml
MeOH.
 The eluate was concentrated in a flow of N2 to less than 1 ml. The
concentrate was filled up with ultrapure water to 1.0 ml and
filtrated through a syringe glass fibre filter (no.7-8808). After
filtration, the samples were stored at 4 ◦C.
Result and discussion 15
 Fig1 show exemplarity chromatograms
obtained of DCF and related
substances.
 DCF have a retention time of 16.59
min. Experimental, two mass
transitions can be used for detection
and quantification
 For measurements of isotopically
labelled DCF, 4-OH-DCF or ACF ,
analogous fragmentations were
determined.
 4-OH-DCF has a retention time of
14.98 min, ACF of 16.81 min.
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 The fragmentation is shown in
fig. 2. It leads to protonated
fragments with molecular weights
of 250 da and 214 da.
 The molecular weight of
protonated DCF is 296 Da.

Fig. 2. Relevant DCF fragmentations.

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The fragment With a mass of 214 da is

the same as in fig. 2. For the fragment
with a mass of 151 da only one fragment
is possible, which is shown in fig 3.

Fig. 3. Postulated fragment of DCF-lactam with a mass of 151 Da.

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As can be seen in fig. 4 it was possible to obtain linear
calibrations for all compounds with HPLC-MS/MS,
which allows the quantification of the analytes in
environmental samples. The calibration functions are
listed in table 2.

Fig. 4LC-MS/MS calibrations for DCF, DCF-lactam, 4-OH-DCF and ACF

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Conclusion
 By means of HPLC-MS/MS the concentrations of diclofenac(DCF), as well as
its two major metabolites 4’-hydroxydiclofenacand diclofenac-lactam have
been determined.
 While in other European waters, concentration of 4-hydroxydiclofenac higher
than diclofenac itself have been detected.
 In berlin waters diclofenac was found in higher concentrations than its
metabolites. With diclofenac-lactam a photodegradation product of DCF was
quantified in European waters for the first time. This may be the result of
possible cyclization during sample preparation.
 Aceclofenac, a related drug, the glyoxylic acid ester of DCF, could only be
quantified in certain WWTP samples.
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