Determination of Acidic Pharmaceuticals
in the Surface Water by GC-MS
Badrinath Dhakal
Department of Chemistry
Eastern Michigan University
December-16, 2009
Abstract
Gas chromatography-mass spectrometry (GC-MS) with solid phase extraction (SPE) was
employed to identify the acidic pharmaceuticals in the surface water of Huron River. A small
peak was observed for the ibuprofen in the sample water which was confirmed by comparing
its mass spectra with the reference from the library database. Acetylsalicylic acid and naproxen
were not detected in the sample. A new technique of derivatization was developed for this
purpose in which phenyltrimethylammonium hydroxide was added to the methanol extract of
the pharmaceutical compounds just before the injection and derivatization took place at the
injection port.
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1. Introduction
The use of drugs has as long history as the human being came to exist. Increasing
population demands the rapid use of drugs for many purposes. More than 3,000 chemicals are
being used in manufacturing drugs1. Pharmaceuticals are the chemical substances synthesized
to produce specific physiological response in human, animals and plants. Because of this their
presence in environment cannot be overlooked. Nearly 100 organic wastewater contaminants
(OWCs) along with pharmaceuticals have been reported in the various streams and rivers of
USA2. According to the EPA report, 74 pharmaceuticals have been identified using HPLC-MS/MS
technique. More than 80 % of the pharmaceuticals consumed remains unused and goes to the
environment. In environment, these compounds may undergo certain changes and would pose
a more serious threat. There are complex pathways of transporting these pharmaceuticals from
manufacturing to the surface water. Effluent from municipal wastewater treatments plants are
not only identified as a source of organic wastewater contaminants but also have been
reported to have estrogenic properties3. The major sources of pharmaceuticals in the surface
water are human, animals and industries.
Pharmaceuticals have been reported in trace amount in the surface water in various
parts of the globe. The analysis of these compounds in the water resources is important mainly
to ensure the safety of drinking water, to be prepared for its consequences in the future and to
have a documented data. But, it is very difficult to find the accurate data about these
substances because of the lack of suitable technique. The development of a method for this
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kind of analysis seems to be quite intricate due to various reasons: analytes are present in trace
amount; they are of diverse polarities and functionalities; they undergo certain changes in
mixtures, and they cause high matrix effect.
The most effective method for this is high performance liquid chromatography with
mass spectrometry/mass spectrometry (HPLC-MS/MS)4. This method is the one developed and
recommended by Environmental Protection Agency (EPA). Though this technique is sensitive, it
is difficult to develop the method and is also expensive to apply. In contrast, the gas
chromatography with mass spectrometry (GC-MS) is easy to develop and not so expensive to
apply. In the present work, GC-MS with solid phase extraction was developed to determine the
acidic pharmaceuticals particularly aspirin, ibuprofen and naproxen in the surface water. In this
method, the sample water was filtered and the acidic pharmaceuticals were extracted by using
preconditioned SPE cartridge. The extract was dried and redissolved in methanol. A new
derivatizing agent, phenyltrimethylammonium hydroxide, was explored to obtain volatile
derivatives of these compounds. The derivatization was carried out at the GC injection port
instead of incubating the extract for a longer time. The resulting chromatograms and mass
spectra were interpreted to identify these compounds. For the validity of the method, some
standard samples were also used.
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2. Experimental
2.1. Reagents and materials
All the reagents used in this experiment were of analytical grade. Solid- phase extraction
was performed using Accu Bond SPE ODS-C18 cartridges, 100 mg/mL, Agilent Technologies.
VWR brand Glass microfiber filter 691 was used to filter the sample water before extraction.
The methanol, ethyl acetate and acetone were of chromatographic purity.
Phenyltrimethylammonium hydroxide (5% in methanol), TCI America was used as a
derivatizing agent. Ibuprofen (FW 206.28) and Naproxen (FW 230.26) from Spectrum Chemical
MFG. Corp. and acetylsalicylic acid (MW 180.15)
from MP Biomedical , LLC were used as
standards. Varian Chromo pack CP-3800 Gas chromatography and Varian Saturn 2000 GC/MS
were used. The sample water was taken from the surface of Huron River. The control solution
was prepared by spiking 500µl solution of standard (2mg/mL) in 500mL of deionized water.
2.2. Preparation of sample and standards
Five hundred milliliter of water sample was filtered through Glass microfiber filter. The
pH of the water was adjusted at 2 with 3.5M hydrochloric acid solution. The extraction of acidic
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pharmaceuticals was previously reported to be optimum at this pH. The SPE cartridge was
conditioned successively with 3mL 50:50 (v/v) ethyl acetate-acetone, 3mL methanol, and 3mL
deionized water adjusted to the pH 2 and the sample water was loaded to the SPE cartridge
under vacuum. After sample loading, cartridge was washed with 40:60 (v/v) methanol-water
and dried under vacuum for one hour. The acidic pharmaceuticals were then eluted with 5 mL
ethyl acetate-acetone and the extract was evaporated to dryness under a gentle stream of
nitrogen
and
redissolved
in
50µL
methanol.
A
30µL
derivatizing
agent
(phenyltrimethylammonium hydroxide) was added to the extract. The use of N,O-bis
(trimethylsilyl)trifluoroacetamide (BSTFA) was reported to be effective derivatizing agent for
the acidic pharmaceuticals5. Analysis was also carried out using BSTFA as derivatizing agent but
it did not work even in the standard samples.
Ibuprofen (Spectrum chemicals), acetylsalicylic acid (MP Biomedical) and naproxen
(Spectrum chemicals) were used to prepare standard solution. The standard solutions of
different concentrations (1ppm -100ppm) were prepared using this acidic mixture. The control
sample was prepared by spiking 500µL of standard solution into 500mL of deionized water.
2.3. GC-MS Analysis of Pharmaceuticals
GC-MS analysis was performed with Varian Chromo pack CP-3800, Varian Saturn 2000
equipped with 30m long and 0.25 mm internal diameter capillary column with 0.25 µm thick
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film coating and 5% phenol, Polydimethylsiloxane (PDMS) as stationary phase. A two micro liter
sample was injected in split less mode at 250 0c with a flow rate of 1.3mL/min. The oven
temperature was maintained at 700c for 2 minutes and programmed at 100 per minute to a
final temperature of 250 0c. The GC-MS analysis of standard sample was also carried out in the
same way.
3. Result and discussion
The chromatograms obtained for all the standards are shown in fig.1. All the three
compounds were detected in the concentrated standard (2mg/mL) but only ibuprofen and
naproxen were identified in the standard samples of concentration range of 1- 100ppm. These
peaks were confirmed by comparing their mass spectra with standard spectra stored in the
library for these compounds.
Fig.1 Chromatograms of the standard samples.
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The mass spectra of naproxen and ibuprofen obtained in standards are shown in Fig. 2
and Fig.3 respectively. The base peak for methyl ester of naproxen at 185 and molecular ion
peak for its methyl ester at 245 are clearly observed in the mass spectra. Similarly the peak at a
retention time of 17.9 min is very close to the literature value. The base peak at 161 and
molecular ion peak at 220 correspond to the ibuprofen. The peak for ibuprofen was obtained at
a retention time of 12.6 minute which is
exactly the same reported in literature albeit
retention time alone is not enough to identify a substance in chromatography.
The chromatogram for the Huron River water is shown in fig.4 (lower). Only one peak
was identified which is ibuprofen. Acetylsalicylic acid and naproxen were not detected in the
sample water.
Fig 2. Mass spectra of naproxen.
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Fig.3 Mass spectra of Ibuprofen.
Fig.4 Chromatograms of sample water (lower).
The information about retention time, base peak and molecular ion peak for the analytes
are shown in the Table 1.
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Table 1. Experimentally observed parameters
Aspirin
Ibuprofen
Naproxen
Retention time
11
12.6
17.9
Base peak
-
161
185
Molecular ion peak
-
220
245
Conclusion
Only ibuprofen was detected in the sample water. The performance of the technique
was tested by running standard as well as spiked samples. Ibuprofen and naproxen were clearly
detected in a concentration range of 1-100ppm while acetylsalicylic acid was not detected even
in 100ppm standard solution. Aspirin might be decomposed or was not derivatized properly in a
lower concentration.
Aspirin has been detected as low as 0.6ng per liter and naproxen and
ibuprofen in the concentration range 0.9ng/L and 0.4ng/L respectively1. BSTFA was reported to
be an effective derivatizing agent but it did not work here5.
Further study is necessary to investigate the effectiveness of SPE as well as this
derivatization technique to both qualitative and quantitative determination of these
pharmaceuticals in the environmental samples.
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References
1. Togola A.; Budzinski H. , Anal. Bioanal. Chem. (2007) 388:624.
2. U.S. Geological Survey (1999-2000), Environmental Science and Technology / VOL.36,
NO.6, 2002.
3. Kolpin D.; Skopec M.; Meyer M.; Furlong E.; Zaugg S. Science of the Total Environment
328(2004)119.
4. http://www.epa.gov/waterscience/methods/method/files/1694.pdf
5. Budzinski H.; Togola A; Devier MH; Labadie P. Anal. Bioanal. Chem. (2007) 388:624.
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