Determination of anti-inflammatory drugs in water samples

by in situ derivatization, solid phase microextraction and
gas chromatography – mass spectrometry
Lilia Araujo, Johana Wild, Noreiva Villa, Nuris Camargo,
Dalia Cubillan, Avismelsi Prieto*
Laboratory of Analytical Chemistry and Electrochemistry, Faculty of Engineering,
University of Zulia, P O Box 4011-A-526, Maracaibo, Venezuela.

A new analytical method is presented for the determination of non-steroidal acidic antiinflammatory drugs in water samples. These compounds are used as anti-inflammatory,
antipyretic and analgesic drugs in human health care and in veterinary applications. The
analytical procedure involves in situ derivatization of analytes to their methyl esters
with dimethyl sulphate, headspace sampling using solid-phase microextraction
(SPME), and gas chromatography-mass spectrometry (GC-MS). Optimization of
derivatization, pH, ionic strength, extraction time, SPME fibre and extraction
temperature are described. Methyl esters were extracted with a fused-silica fibre coated
with 100 µm polydimethylsiloxane. Response was linealy dependent on concentration
in the range 0.1 – 10.0 ng/mL. Detection limits are achieved at the level of 0.3 – 2.9
ng/L. Derivatization – SPME/GC-MS analysis yielded good precision (RSD between
7.9 and 14.3 %). The method was validated by analysis of spiked matrix samples.
Key words: non-steroidal acidic anti-inflammatory drugs, derivatization, solid-phase
microextraction, gas chromatography-mass spectrometry.

*Corresponding author. Fax: 0058-261-7598736; e-mail:

solvent-free. sample clean-up or the use of organic solvents. Generally. the analytes are 2 . Due to their biological activity. Suitable methods exist for a quantitative measurement of NSAIDs in water samples. Non-steroidal acidic anti-inflammatory drugs (NSAIDs) are among the group of pharmaceutical compounds most often used to treat human and animal illnesses. but in general the preconcentration step is extensive.0 L). The complete effects on aquatic organisms and humans are not well understood. prior to the determination of acidic drugs in water. Since its introduction. SPME requires less sample volume than solid phase extraction or liquid liquid extraction. SPME has gained popularity as a simple.5 and 2. On the other hand GC analysis of acidic drugs is complicated because a derivatization step is necessary prior to analysis. micellar electrokinetic capillary chromatography (MEKC) and capillary electrophoresis [7-12]. There is a need for fast and simple methods to supply these analysis. These compounds are excreted unchanged or as active metabolites in high percentages and continuously discharged into domestic wastewaters. reliable and flexible tool for the sampling of a variety of volatile and semivolatile compounds. Solid phase microextraction (SPME) is a two-step process conducive to the simultaneous extraction and preconcentration of organic compounds. Introduction Pharmaceuticals used for humans and animals have been identified as emerging environmental pollutants [1-2]. SPME is suitable for analysis of drugs residues in water. using gas chromatography-mass spectrometry (GC-MS). Incomplete removal during wastewaters biological treatments has caused their presence into surface waters at concentrations from ng/L to µg/L level [ 3-5]. their determination in the environment is important [6]. requires large sample volumes (between 0. high performance liquid chromatography-mass spectrometry (HPLC-MS).1.

However few studies consider the option of performing the derivatization directly in the water matrix. Tetrabutylammonium hydrogen sulfate (TBA-HSO4) was obtained from Fluka.1. Experimental 2. 2. and meclofenamic acid) in water samples using headspace SPME/GC-MS is proposed. flufenamic acid. MO. mefenamic acid. tolfenamic acid and meclofenamic acid were supplied by Sigma (St. naproxen.sigma-aldrich. By in situ methylation. Two types of fibre. 100 m polydimethylsiloxane and 85 m polyacrylate were obtained from the same manufacturer and conditioned before use 3 . tolfenamic acid. Materials and reagents All reagents were of analytical-reagent grade unless stated otherwise. Working solutions were obtained by appropriated dilutions with Milli-Q water. USA).transferred to an organic matrix where derivatization takes place. To date. Water was purified with a Milli-Q plus system (Millipore). Ibuprofen. A manual fibre holder for SPME was purchased from Supelco (www. flufenamic acid. NSAIDs have not been investigated by direct derivatization-SPME procedure. The analytical procedure involves in situ derivatization of acidic drugs to their methyl esters with dimethyl sulfate. which may improve the extraction into the SPME fibre. Louis. a new method for analysis of six non steroidal acidic antiinflammatory drugs (ibuprofen. mefenamic acid. naproxen. in the SPME fibre coating or in the GC injector port [13-16]. The derivatization reagent dimethyl sulfate (DMS) was purchased from Riedel-de Haen. A stock standard soultion of 1000 µg/mL of each compound was prepared in basic Milli-Q In this paper. SPME allows the derivatization of the analytes to take place in the sample matrix. the acidic drugs are converted to less polar methyl esters.

2. 2. 3.295 for flufenamic acid. the vial was closed. The injector temperature was set at 250 C and the transfer line temperature was 280 C. 185-244 for naproxen. Temperature was held at 250 C for 4.0. 2. After addition of the ion-pairing reagent (TBA-HSO4. 7.88 g) were then added and the sample was agitated with a 1. 60 µL).999 %) at a flow rate of 1 mL/min. A silanized narrow-bore injector liner (0.. and mass spectrometer detector model Agilent Technologies 5973 was used. 208-275 for tolfenamic acid and 242-309 for meclofenamic acid.d. The following m/z values were acquired in the electron impact ionization mode by single ion monitoring and used for quantification of the analytes: 161. Instrumentation A Agilent Technologies 6890N GC system equipped with a split-splitless injector for the HP-5MS fused silica capillary column (30 m x 0. then heated to 250 C at a heating rate of 30 C/min.0 mol/L. 15 µL of derivatization reagent (DMS) was then injected through the septum and the vial was immersed in a temperature-controlled oil bath.220 for ibuprofen. The carrier gas was helium (purity 99.0 mL) and Na2SO4 (2.5x10-4 M.5 min.3 Procedure 6. The mass spectrometer detector was tuned by maximum sensitivity autotune. After 10 min at 65 ± 2 4 .25 m film thickness). 263.75 mm id) for the SPME injections was installed and the fiber was inserted into this injector using the splitless mode with the split closed for 3 min. 223-255 for mefenamic recommended by the manufacturer. Phosphate buffer solution (pH 6.25 mm i. 0.8 cm long PTFE-coated stir bar. 2. The oven temperature was held at 50 C for 3 min.0 mL of standard solution or sample was placed in a 14 mL screw-cap glass vial. The Statgraphics software package was used for the regression analysis (linear model).

Optimization of the microextraction with in situ derivatization It has been reported that DMS reacts with haloacetic acids and chlorophenoxy acid herbicides in water to produce the corresponding methyl ester [17-18]. the PDMS fibre was exposed to the headspace above the aqueous solution for 45 min. which activate the analytes during derivatization. 5 . increases esterification yields and thus improves the sensitivity of the procedure. experiments were performed in which DMS and the ion-pair agent TBA-HSO 4 was added to aqueous solutions containing each of the six test compounds.1. In this work. After the reaction. Next. Results and discussion 3. The more adequate fibre was found by comparing the extraction behavior on two commercial SPME fibres. After the extraction the fibre was directly exposed to the hot injector of the GC for 5 min and the chromatogram was registered. preliminary experiments were carried out to optimise the main parameters affecting both derivatization and SPME of the analytes investigated. 3. The samples were agitated with a magnetic stirring bar at 500 rpm during both derivatization and extraction. the solution was extracted with headspace SPME and analysed by means of GC-MS. 100 m polydimethylsiloxane polydimethylsiloxane and 85 m polyacrylate. Detectable yields of methyl esters were achieved for the analytes and identified on the basis of their mass spectra. fibre exhibited the highest extraction performance and was chosen for the rest of experiments. Calibration graphs in Milli-Q water were thus constructed using solutions of the analytes of known concentrations. The addition of ion-pairing agents. In these studies Milli-Q water samples spiked with the appropriate amount of the standard solution were used.ºC.

Figure 1 The effect of temperature was monitored by extracting samples of 10 ng/mL of NSAIDs in the range of 40 . We investigated the effect of the TBA-HSO4 volume on the amount of NSAIDs extracted fron the sample onto the PDMS fibre. As a compromise. 2 shows a clear increase in the amount of analytes adsorbed when the temperature increases. 1. 65 C was chosen as the optimum temperature for the derivatization and SPME extraction of all the methyl derivatives of NSAIDs. However. The TBA-HSO 4 volume profile was 6 . a range of 5-100 µL of DMS volumes were tested at 65 C. A 0. As shown in the Fig.0.0) was selected to obtain an adequate buffering capacity. Increasing the volume of DMS in the range 5-60 µL improved slight the yield of methyl esters while the response decreased at higher amounts of DMS. 100 µm PDMS fibre was too fragile at DMS volume higher than 15 µL and it could only be used for a limited number of experiments. For this reason 15 µL was used for the rest of experiments. At higher pH.75 M concentration of the phosphate buffer (pH 6. From these results it was decided to adjust the pH of water samples to 6.80 C. a decrease was observed in the response. there was a decrease in the amount of NSAIDs extracted except for mefenamic acid. Fig. when the temperature exceeded 70 C.The optimum pH was determined in the range between 3 and 10. no significant effect was observed in the range of 3 . However. Figure 2 In order to set an optimal volume of the derivatization reagent necessary for complete esterification of NSAIDs.7. tolfenamic acid and meclofenamic acid.

No signals were obtained when a 5 min desorption time was chosen. blanks were run after extractions of 50 ng/ mL of NSAIDs. The role of the ionic strength of the matrix was investigated using sodium chloride and sodium sulfate. The amounts tested for a sample volume of 8 mL varied from 0 to 2. Figure 3 Extraction time profiles were studied extracting samples of 10 ng/mL of NSAIDs and monitoring the GC area counts as a function of exposure time. 3). The results obtained show an strong increment in the signal for all analytes when the NaCl or Na 2SO4 amount increases. the partitioning from the aqueous solution to the headspace is improved. For quantitative analysis it is not necessary for the analytes to have reached equilibrium. and thus.88 g of Na2SO4 was selected to obtain an adequate salting-out effect. aqueous solubility decreases with increasing ionic strength. The addition of 60 µL of TBA-HSO4 ensured the maximum response for NSAIDs methyl esters.88 g of NaCl or Na 2SO4. An extraction time of 45 min was selected as a compromise between analyte response and time analysis. Equilibrium was not attained even after 90 min. Thus. 2. To study the carryover effect. 7 . The highest peak area response was achieved using Na2SO4. but only for sufficient loading onto the fibre and reproducible extraction time [19]. For many organic analytes.studied by monitoring the GC area counts as a function of TBA-HSO 4 volume. which ensured a complete desorption of methyl esters. The effect of the ion-pairing agent on the yield of the methylation is shown by the increase in the peak area obtained with the use of TBA-HSO 4 (Fig.

A S/N of 10 was applied for the calculation of the quantification limit.2 Application and Validation of the Proposed Method Calibration graphs for Milli-Q water samples treated according to the procedure described previously. Precisions ranged from 7. SPME-derivatization-GC/MS or SPE- HPLC/MS [11. 23-24]. Absolute response was lower with wastewater samples than with Milli-Q water. The results for the intercept (a). which should be satisfactory for determining the NSAIDs in water matrix.D.10.3.D(b)] (%) are summarised in Table 1. Two replicates were used for each of six prepared standards to obtain the calibration graphs.S. slope (b). The detection limit was calculated by comparing the signal–to-noise ratio (S/N) of the lowest detectable concentration to a S/N = 3. SPE-derivatization-GC/MS. The detection limits are in accordance with other methods based on SPE-MEKC. This might be due to the presence of additional organic and inorganic compounds that may interfer with diffusion and volatilization of methyl esters decreasing the 8 . are linear for the concentration range 0. The precision was measured by performing 8 independent determinations. The detection limits found were between 0. This range agreed with environmental levels in water currently reported in the literature for these compounds [20-22]. (Table 1).1 . monitored using SIM mode.S.9 ng/L ( Table 1). 14. Table 1 The optimum headspace SPME sampling conditions for Milli-Q water were applied to the tap water and wastewater matrices. regression coeficient (r) and linearity [1 .3 and 2.R.9 to 14.0 ng/mL.3 % R.

We did not find NSAIDs above our detection limit. a 100 m polydimetilsiloxane fibre. mefenamic acid. Samples were fortified with different levels of NSAIDs. In view of it simplicity and sensitivity. samples were diluted 3:1 with buffer solution.0 % and no interfering peaks were observed for the blank samples. Non-equilibrium conditions were adopted in order to reduce the extraction time. The recoveries were greater than 76. pH 6.0 ng/mL of each analyte is depicted in Fig. Sensitive responses were obtained using 15 µL of DMS. 9 . and meclofenamic acid in water samples is presented. External calibration was used in the evaluation of NSAIDs in tap water and standard addition calibration was used in wastewater because matrix effects were observed. flufenamic acid. Table 2 shows results and a representative chromatogram of wastewater samples spiked with 1.88 g Na2SO4. Conclusions A simple and practical GC-MS method in combination with in situ derivatization headspace SPME for the determination of the non steroidal acidic anti-inflammatory drugs ibuprofen. the present method is applicable for the quantification of residues of NSAIDSs studied in tap water and wastewater samples. naproxen. Figure 4 4.0 and 65C in combination. tolfenamic acid.performance extraction by headspace SPME. 4. To improve the extraction yield. We tried to find NSAIDs in tap water and wastewater samples. Matrix effects can be avoided with the use of the standard addition method for quantitation. 45 min extraction time. 2. Acknowledgements The authors thank the CONDES-LUZ for providing support for this work. 60 µL of TBA-HSO4.

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452 -117.0 ng/mL 7. FLU: flufenamic acid.6 11.3 14.4 10.D.4 2.S.4 9.1 ng/mL 1.7 10.6 98.8 9.9 14.3 1.6 98.4 5. 12 .7 1.9x106 2.5x106 1.4x106 1.6 11.998 0.S.6 10.2 3. NAP: naproxen.2 14.9 9.0 4.2 13.102 Intercept (a) -152.2x106 1.1 0.3 97.9 2.0 12.9 8.4 9.%). TOL: tolfenamic acid.1 11.3 0. MEF: mefenamic acid.0 1.416 Slope (b) 2.Table 1 Analytical parameters IBU FLU Analytes* NAP MEF TOL MEC 296.999 0.8x106 1.994 Linearity [1 .991 0.D(b)] (%) Detection limit (ng/L) 99.6 2.7 13.8 1.4 10.0x106 Correlation coefficient 0.2 11.996 0. MEC: meclofenamic acid.486 102.4 *IBU: ibuprofen. n = 8 0.9 98.997 0..5 10.2 12.3 ng/mL 11.390 -345.R.5 ng/mL 11.012 215.9 Quantification limit (ng/L) Repetibility (R.3 10.7 97.7 11.

08 ± 0.67 90.10 82.57 ± 0.0 4.83 ± 0.3 Mefenamic Tap Water 0.01 88.0 2.11 116. Figures captions Figure 1.09 0.0 3.8 Tolfenamic Tap Water 0.0 Wastewater 0.40 92.56 ± 0.5 0.76 ± 0.8 Flufenamic Tap Water 0.09 0.46 ± 0.5 0.56 86. Effect of sample pH for in situ methylation head space SPME of the six NSAIDs.01 100.08 ± 0.0 3.0 Wastewater 0.69 ± 0.5 Meclofena Tap Water 0.0 3.0 4.5 0.0 4.02 133.0 3.46 ± 0.02 107.55 86.37 91.75 ± 0.05 76.08 ± 0.14 ± 0.09 114.8 acid 3.5 mic acid 3.  "% Recovery" refers to the NSAIDs concentrations determined rather than the actual percent of analytes extracted by the SPME analysis.41 ± 0.58 ± 0.47 ± 0.5 0.0 4.48 86.0 3.6 Wastewater 0.5 0. Analyte Sample Spiked Found* % Recovery (ng/mL) (ng/mL) Ibuprofen Tap Water 0.0 4.48 ± 0.0 2. 13 .0 3.Table 2 Results of assays to check the accuracy of the proposed method for NSAIDs in spiked top water and wastewater samples.5 0.5 Naproxen Tap Water 0.33 94.09 ± 0.01 77.0 2.0 2.5 * Average value  standard deviation of five determinations.9 3.32 92.70 92.0 Wastewater 0.76 ± 0.7 Wastewater 0.59 78.09 0.0 3.12 ± 0.02 88.0 3.07 ± 0.06 96.41 102.09 0.97 ± 0.63 ± 0.12 112.39 99.0 4.3 Wastewater 0.10 ± 0.3 acid 3.0 2.8 acid 3.09 0.09 0.38 ± 0.

Influence of amount of 7. 6: meclofenamic acid. 5: tolfenamic acid.Figure 2. 4: mefenamic acid. Concentration of ibuprofen is 5 ng/mL.5x10-4 M TBA-HSO4 added on NSAIDs reaction yields to the corresponding methyl esters. 1: ibuprofen. the scale is shown on the right. Effect of temperature on the extraction of 10 ng/mL of NSAIDs with polydimetilsiloxane fibre.0 ng/mL of each analyte. For ibuprofen. the scale is shown on the right. Figure 3. Typical chromatogram obtained in the SIM mode of a wastewater sample spiked with 1. 2: flufenamic acid. 14 . Figure 4. 3: naproxen. For ibuprofen.

0E+07 Area 4.Ibuprofen Naproxen Tolfenamic acid Flufenamic acid Mefenamic acid Meclofenamic acid 6.0E+07 0.0E+00 3 5 7 9 11 pH Figure 1 15 .0E+07 2.

Figure 2 16 .

0E+07 4.Flufenamic acid Mefenamic acid Meclofenamic acid 1.0E+00 0.0E+07 0 50 100 150 200 250 µL of TBA-HSO4 added Figure 3 17 .0E+00 300 Area 8.5E+07 1.0E+07 5.2E+08 Naproxen Tolfenamic acid Ibuprofen 1.0E+06 0.

Figure 4 18 .