Determination of acidic drugs in sewage water by gas chromatography–mass spectrometry as tert.-butyldimethylsilyl derivatives

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Abstract

A procedure is described for the determination of five acidic non-steroidal anti-inflammatory pharmaceuticals (ibuprofen, naproxen, ketoprofen, tolfenamic acid and diclofenac) in sewage water. The analytical method involves the concentration of water samples using a solid-phase extraction polymeric sorbent, functionalized with N-vinylpyrrolidone. Analytes were eluted with ethyl acetate, derivatized using N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) and analyzed by GC–MS. Influence of time, temperature and volume of MTBSTFA in the yield of the derivatization step were studied in detail using a factorial central composite design. Quantification limits of the analytical procedure for 500 ml of sewage water ranged from 20 to 50 ng/l. Recoveries from 90 to 115% were found for sewage water samples spiked with the studied compounds at the low ng/ml level. Results obtained for real samples show the presence of ibuprofen and naproxen in both influent and effluent of a sewage water treatment plant.

Introduction

In the last few years there has been a growing interest in the role of different groups of pharmaceutical compounds such as anti-inflammatories, lipid regulators, psychiatric and antiepileptic drugs in the aquatic environment. In developed countries, annual prescriptions of some of these compounds can achieve several hundreds of tons [1]. Human excretion of the original drugs (as free or conjugated species) and of their metabolites have caused their presence in the influent of sewage treatment plants. Furthermore, a number of studies have shown that some acidic pharmaceutical compounds are not totally eliminated in sewage treatment plants, therefore, they can reach surface and groundwaters [1], [2], [3]. Concentrations of anti-inflammatory pharmaceuticals such as diclofenac and ibuprofen in the range of several hundreds of ng/l have been found in different European rivers [1], [4], [5], [6]. Obviously, these levels are much lower than those used in medical applications, and their possible ecotoxicological effects are still unknown [3], [6]; however, these compounds must be classified as environmentally relevant.

Quantitative evaluation of the fate of NSAI drugs (NSAIDs)in the aquatic environment, proper risk assessment and improvement of the efficiency of sewage treatment plants need sensitive and reliable analytical methods. Generally, procedures used for the analysis of acidic pharmaceuticals, such as NSAIDs, in aqueous samples are based on an enrichment step followed by the chromatographic determination of the analytes, usually with mass spectrometric detection. Sample pre-concentration is normally performed using solid-phase extraction after pH adjustment to 2–3. Reversed-phase sorbents such as C18 [7], polymeric materials (e.g. LiChrolut EN) [8] and also functionalized polymers such as the Oasis HLB sorbent [5] (polydivinylbenzene-co-N-vinylpyrrolidone) are currently used. Typically, 500 ml of sample are concentrated in case of wastewater, and up to 1000 ml for river and groundwater.

Regarding the determination step, recently HPLC–MS has been successfully employed in the analysis of acidic drugs in water samples [6], [9]. However, GC–MS is by far the most often used technique; probably, because of the widespread and availability of GC–MS systems in environmental laboratories, and also because of possible problems related with signal suppression in HPLC–MS, when extracts from real samples are analyzed. Gas chromatographic separations of NSAIDs can be performed only after derivatization of the native compounds to less polar species. The carboxylic group of these drugs can be converted into their methyl ester derivative using diazomethane [4], [5], [7]. The yield of the reaction is usually excellent; however, because of some drawbacks of the process such as the high toxicity of diazomethane, its low stability and the need to be generated in situ, some alternatives to their use have been proposed in the literature. Zwiener et al. [8] described an on-line method which allows the methylation of several NSAIDs in the hot injector of a gas chromatograph using trimethylsulfonium hydroxide. Sacher et al. [10] derivatized several acidic drugs containing carboxylic groups using a solution of pentafluorobenzyl bromide in cyclohexane; the reaction was performed at 100 °C for 2 h after dryness evaporation of the sample extract. Several silylation reagents have been widely used as alternatives to diazomethane for the derivatization of pesticides and drugs, containing phenolic, carboxylic or amide groups in environmental and biological samples, respectively [11], [12], [13], [14]; however, only one publication reports the use of bis(trimethylsilyl)trifluoroacetamide (BSTFA), for the analysis of NSAIDs in organic extracts of water samples [15].

The aim of this paper was the optimization of a GC–MS method for the analysis of NSAIDs in sewage water, based on their derivatization using a commercial silylation reagent, which serves as alternative to the use of diazomethane. N-Methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) was preferred to trimethylsilyl derivatization because of the greater thermal and hydrolytic stability of the tert.-butyldimethylsilyl derivatives, added to their higher molecular mass that improves chromatographic separation and MS detection [16]. The previous solid-phase extraction (SPE) step was carried out using Oasis HLB cartridges, which exhibit both hydrophilic and lipophilic retention characteristics. Elution of analytes from the sorbent material was performed with solvents compatible with their further silylation, thus evaporation of the extract to dryness was not necessary. Influence of experimental parameters such as time, temperature and volume of MTBSTFA in the efficiency of the derivatization reaction were also evaluated using an experimental design. This study was performed with extracts of spiked real sewage water samples. In this way, matrix influence on the derivatization step was taken into account. The developed method was applied to the analysis of NSAIDs in 24 h composite water samples taken in the inlet and the outlet streams of a sewage treatment plant.

Section snippets

Reagents

HPLC-grade methanol and ethyl acetate were supplied by Merck (Darmstadt, Germany). Pesticide grade n-hexane was also purchased from Merck. PCB-30 (2,4,6-trichlorobiphenyl) was obtained from Dr Ehrensdorfer (Augsburg, Germany); ibuprofen, naproxen, ketoprofen, tolfenamic acid, diclofenac and meclofenamic acid were purchased from Aldrich (Milwaukee, WI, USA). MTBSTFA was also obtained from Aldrich in 1 ml ampoules. Individual stock solutions of NSAIDs were prepared in methanol. Diluted standards

Derivatization reaction

Normally, the weakest point of GC methods applied to the analysis of acidic compounds is the derivatization step, therefore in this study it was optimized in detail:

Conclusions

An analytical procedure for the determination of five NSAIDs in water samples by GC–MS was developed. The use of MTBSTFA to silylate the studied acidic compounds was a valuable alternative to other derivatization procedures based mainly in the use of diazomethane. The method was applied to the analysis of sewage water samples and it could be extended to cleaner samples such as surface and pre-potable water due to the large breakthrough volume of the SPE sorbent. Analysis of non spiked sewage

Acknowledgements

Financial support from the Spanish DGICT (proyect REN 2000-0984HIP) is acknowledged. JC and JBQ acknowledge their doctoral grants from the regional government (Xunta de Galicia) and the Spanish Ministry of Education, respectively. M. Carballa and Aquagest are thanked for the supply of sewage water samples.

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