Annals of West University of Timisoara
Series of Chemistry 19 (1) (2010) 83-90
DERIVATIZATION OF ASPIRIN FOR GAS
CHROMATOGRAPHIC ANALYSIS
R. C. Gr oza, I. Ciucanu*
West University of Timişoara, Faculty of Chemistry, Biology, Geography, Department
of Chemistry, Pestalozzi, 16, Timişoara, 300115, ROMANIA
*Corresponding author: e-mail: [email protected]
Received: 20 May 2010
Modified 26 May 2010
Accepted 2 June 2010
SUMMARY
Acetylsalicylic acid (ASA), also known as aspirin, is widely used as an analgesic,
anti-inflammatory and antithrombotic drug. The most commonly used methods for the
determination of ASA include spectrophotometric, fluorimetric and chromatographic
methods. The lowest detection limit has been reached using gas chromatography. In this
case, a derivatization of ASA is needed prior to analysis. ASA can be methylated,
trimethylsilylated, or converted to fluorinated derivatives. This work presents for the first
time the optimal conditions in the trimethylsilylation process with one of the most used
reagents for this operation, trimethylchlorosilane.
Keywords: aspirin, acetylsalicylic acid, derivatization, gas chromatography,
trimethylchlorosilane, hexamethyldisilazane, pyridine, trimethylsilylation
INTRODUCTION
The analgesics industry appeared at the end of the 19th century. This type of drugs
became very popular due to the fact that they were very efficient, didn’t cause addiction and
could be purchased without a medical prescription. From the earlier used analgesics, aspirin
is one of the few that is still in use [1].
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GROZA R. C., CIUCANU I.*
COOH
O CO CH3
Figure 1. Aspirin’s chemical structure
Aspirin (Figure 1) is part of a class of chemical compounds called salicylates,
which derive from salicylic acid. Aspirin has the chemical structure of salicylic acid with
the phenolic group esterified with acetic acid. ASA hydrolyzes in the presence of water to
form salicylic and acetic acids, being the most stable at pH = 2.5 (25°C). The hydrolysis of
aspirin implies the use of a nonprotic solvent like 1,4-dioxane in order to inhibit this
process. At 25°C, aspirin’s solubility in dioxane is 3.680M and it increases with
temperature, being 4.659 at 40°C. Also, in carrying out the analysis, the samples of aspirin
must be kept in absence of water as much as possible [2].
The most commonly used methods for aspirin’s analysis include
spectrophotometric, fluorimetric and chromatographic methods. Spectrophotometric
methods imply chemical reactions in which the analyte forms a colored compound. This
method is used for biological probes. Hydrolyzed aspirin forms a purple colored complex
with Fe3+ ions in a weak acidic media. Unhydrolyzed aspirin in the human body will be
calculated as the difference between the administrated and the determined amount. The
absorbance of the colored solution is measured at 540 nm. The disadvantage of this
spectrophotometric method is that it has a high detection limit, approximately 50 mg/L and
it doesn’t differentiate between some of aspirin’s metabolites.
Fluorimetric methods imply the formation of a chemical combination that emits
fluorescence. Aspirin forms highly fluorescence compounds with lanthanides like terbium
or with EDTA. The detection limit is approximately 1 mg/L and it doesn’t differentiate
between aspirin’s metabolites [3-5].
Chromatographic methods are used for the determination of aspirin because of
their low detection limit, ease of work and because they can differentiate between
metabolites. Gas chromatography has the lowest detection limit, approximately 10 μg/L.
This method implies a derivatization step prior to analysis. By replacing the active hydrogen
we can obtain a compound with low polarity and a good volatility for gas chromatography,
an increase in thermal stability and overall a better behavior in columns [4-6].
Aspirin can be derivatized by methylation, by forming fluorinated compounds and
by trimethylsilylation. This work presents for the first time the optimal conditions in the
trimethylsilylation process with one of the most used reagents for this operation,
trimethylchlorosilane (TMCS). Derivatization of aspirin was performed with TMCS in the
absence and in the presence of a proton capturing agent such as hexamethyldisilazane
(HMDS) and pyridine. A weak base helps improve the process of trimethylsilylation by
capturing the released protons from aspirin’s carboxylic group [7-12].
84
DERIVATIZATION OF ASPIRIN FOR GAS CHROMATOGRAPHIC ANALYSIS
MATERIALS
AND
METHODS
Materials
Pure aspirin (>99.95%) was obtained from Sinteza Oradea S.A.. Dioxane
(>99.5%), anisaldehyde (>99%), trimethylchlorosilane (>99.5%), hexamethyldisilazane
(>98.5) and pyridine (>99%) were obtained from MERCK.
Apparatus
Analysis was performed with a Carlo Erba Instruments model HRGC 3000 MEGA
SERIES gas chromatograph. The capillary column was an DB-XLB (30 m x 0.25 mm x
0.25 μm film thickness) from J&W Scientific INC (USA) with hydrogen as carrier gas at
0.8 mL/min. The injector temperature was set at 220°C with injection volumes of
approximately 1.2 μL. Detection was performed with a flame ionization detector with the
temperature set at 220°C. The flame uses a mixture of air and hydrogen, both at 25 mL/min.
The oven temperature was set at 150°C, above the boiling point of underivatized aspirin
(140°C with decomposition).
Derivatization method
Derivatization was performed in 4 different reaction conditions: TMCS alone,
TMCS in the presence of pyridine, TMCS in the presence of HMDS and TMCS in the
presence of both HMDS and pyridine. TMCS is one of the first and most used derivatization
agents for carboxylic acids. Pyridine is a weak base that helps improve the derivatization
process by acting as a proton capturing agent, neutralizing the chlorhydric acid formed in
the reaction between aspirin and TMCS. HMDS is a derivatization agent that also acts as a
proton capturing agent. Anisaldehyde was chosen as internal standard due to the fact that it
has good volatility for gas chromatography and it doesn’t react with the derivatization
agents used in this work. Septum vials of 3 and 15 mL were used in order to prevent loses
caused by volatile substances.
The derivatization process was performed in an excess of trimethylsilyl groups to
aspirin of 11:1. Pyridine was also added in an excess of 11:1. Therefore, 0.5 mL solution
would contain, depending on the reaction conditions, 1.25 mg ASA, 0.1 μL anisaldehyde, 10
μL TMCS (equivalent to 3.3 μL TMCS + 5.5 μL HMDS which maintains the same amount
of trimethylsilyl groups) and 6.1 μL pyridine.
The effect of a weak base in the process of trimethylsilylation was studied by
determining the quantitative variation in time for aspirin’s trimethylsilyl derivative (ASATMS). For each reaction condition, 9 injections were made into the gas chromatograph at
approximately 10 minutes between them [7-12].
85
GROZA R. C., CIUCANU I.*
RESULTS
In Figure 2 it can be seen that by using HMDS, a precipitate (NH4Cl) is formed in
the system while in the absence of HMDS the solution remains clear. The formed precipitate
can occlude the microsyringe needle.
TMCS
TMCS + pyridine
TMCS + HMDS
TMCS + HMDS + pyridine
Figure 2. Solutions after derivatization for the four different conditions used
Figure 3 shows the gas chromatogram obtained for derivatization with TMCS in
the presence of HMDS and pyridine. Figure 4 shows the detailed gas chromatogram for the
same reaction condition.
To study the effect of the weak base and to be able to compare the peak areas
obtained for ASA-TMS, the peak areas obtained for the internal standard were equalized to
100 area units. This operation is necessary because the volumes of the injected samples are
not reproducible.
86
DERIVATIZATION OF ASPIRIN FOR GAS CHROMATOGRAPHIC ANALYSIS
Figure 3. Gas chromatogram for derivatization with TMCS in the presence of HMDS and pyridine
Figure 4. Detailed gas chromatogram for derivatization with TMCS in the presence of HMDS and
pyridine
Figure 5 shows the quantitative variation in time of ASA-TMS for each reaction
condition.
87
GROZA R. C., CIUCANU I.*
Figure 5. Quantitative variation of ASA-TMS for each reaction condition
DISCUSSION
In the reaction between aspirin and TMCS, chlorhydric acid is formed. The
reaction takes place through the nucleophilic attack of oxygen from aspirin’s carboxylic
group against the silicon atom from TMCS. The derivatization process is disturbed by the
presence of HCl because the esteric bonds CO-O-Si and CO-O-C in aspirin’s derivative can
hydrolyze.
Pyridine, being a weak base, neutralizes the chlorhydric acid. HMDS acts as a
derivatizing agent and also as a proton capturing agent. The ammonia formed in the reaction
between ASA and HMDS neutralizes the chlorhydric acid formed in the reaction between
ASA and TMCS, resulting in NH4Cl which precipitates.
O
O
C OH
+
H3C OCO
CH3
Cl
Si CH3
CH3
ASA
H3C CH3
dC O .......Si .........Cl
H
CH3
H3C OCO
O
+
N
pyridine
N (+)
H
Cl
(-)
CH3
C O Si CH3
- Cl
+
-H
CH3
H3C OCO
ASA-TMS
TMCS
+H
+ Cl
88
#
d+
DERIVATIZATION OF ASPIRIN FOR GAS CHROMATOGRAPHIC ANALYSIS
COOH
2
OCO CH3
+
H3C
CH3
H3C Si NH Si CH3
CH3
H3C
COO-Si(CH3)3
2
HCl
+ NH3
+
NH3
ASA-TMS
HMDS
ASA
OCO CH3
NH4Cl
Figure 6. Chemical reactions describing the derivatization process of aspirin
In Figure 5, graph a) was obtained by derivatizing aspirin with TMCS. Graph b)
was obtained by derivatizing aspirin with TMCS in the presence of pyridine. Pyridine
retains the chlorhydric acid, thus preventing the hydrolysis of ASA-TMS. In this conditions
there is an increase of about 3 times the quantity of ASA-TMS in graph b) compared to
graph a).
Graph c) was obtained by derivatizing aspirin with TMCS in the presence of
HMDS which retains the chlorhydric acid, generating NH4Cl, but also acts as a derivatizing
agent. The ammonia generated in the system is a stronger base than pyridine (pKb for
ammonia is 4.75 and pKb for pyridine is 8.79). That explains why derivatization in the
presence of HMDS generates more ASA-TMS than in the presence of pyridine.
Graph d) was obtained by derivatizing aspirin with TMCS in the presence of
HMDS and pyridine. In this case, the resulting hydrochloric acid is retained as NH4Cl and
also by pyridine. Over all, the amount of ASA-TMS is considerably higher when
introducing HMDS in the system instead of pyridine, but the best result are obtained when
using both of them.
CONCLUSION
1.
2.
3.
4.
Aspirin is a widely used analgesic, anti-inflammatory and antithrombotic drug.
Aspirin is mostly analysed by spectrophotometric, fluorimetric and
chromatographic methods, but the lowest detection limits were obtained using gas
chromatography.
In order to analyze aspirin by gas chromatography, a derivatization step is needed
prior to analysis. This can be realized by methylation, by forming fluorinated
compounds or by trimethylsilylation.
When derivatizing aspirin with TMCS, one of the most used derivatization agents
for the trimethylsilylation of carboxylic acids, the process is affected by the released
protons. Introducing a weak base will improve the process.
89
GROZA R. C., CIUCANU I.*
5.
In this work, four different reaction conditions for the trimethylsilylation of aspirin
were presented. The best results were obtained by using TMCS in the presence of
HMDS and pyridine.
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