Determination of anti-inflammatory drugs and estrogens in water by HPLC with UV detection

J. Sep. Sci. 2007, 30, 985 – 991 A. Stafiej et al. 985

Anna Stafiej1

Krystyna Pyrzynska1

Fiona Regan2

1Department of Chemistry,Warsaw University, Warsaw,Poland

2School of Chemical Science,Dublin City University,Glasnevin, Dublin, Ireland

Original Paper

Determination of anti-inflammatory drugs andestrogens in water by HPLC with UV detection

An analytical method based on LC and UV detection has been developed for thedetermination of anti-inflammatory compounds and estrogens in water samples.The drugs investigated were diclofenac, ketoprofen, ibuprofen, naproxen, clofibricacid, estriol, 17b-estradiol, estrone and ethynylestradiol. The detection limits werein the range of 6–74 lg/L and 0.041–0.16 mg/L for acidic pharmaceuticals and estro-gens, respectively, using narrow-bore C18 analytical column. Analyte enrichmentfrom water samples was achieved by SPE procedure using polymeric Strata-X cartrid-ges. Average recoveries obtained from 2.5 L of surface water sample were in therange of 77–98%.

Keywords: Acidic drugs / Estrogens / Water analysis /

Received: October 20, 2006; revised: February 7, 2007; accepted: February 7, 2007

DOI 10.1002/jssc.200600433

1 Introduction

The focus of environmental research has been recentlyextended beyond classical pollutants such as dioxins,PCBs and pesticides to pharmaceuticals, steroids, hor-mones and personal care products. Prescription and salesof these compounds have shown continuous growth inrecent years and they are continuously released into theenvironment through human activities. Environmentalproblems may arise when excreted drugs (and those dis-posed of unused) enter water as the parent compound ora metabolite, via sewage effluent from domestic dwell-ings and hospitals or from landfill leachates. Urban orindustrial discharges introduce these compounds intothe environment and their presence in various environ-mental compartments has recently been reported [1–4].Clofibric acid, for example, is known to be present in riv-ers, but also in ground and tap water [5]. This compoundis an active metabolite of drugs used as blood lipid regu-lators (95–99% excretion in the urine). Thus, there is con-siderable interest in developing analytical methods fordetermining these compounds in environmental sam-ples at low levels.

GC with mass spectrometric detection (GC-MS) is oneof the most common techniques [6–8]. However, some ofthese compounds cannot be determined by GC withoutderivatization, because they are thermally unstable,

polar or ionic. Derivatization step makes the sample pre-paration laborious and time-consuming, increases thepossibility of contamination and errors, and may lead todegradation of labile compounds. HPLC-MS technique isa good alternative for determining these polar com-pounds in aqueous samples [3, 9–11]. One drawback ofthe electrospray MS technique during measurements ofenvironmental samples is the signal suppression effect,which has a negative influence on the reproducibilityand accuracy of the analyses [12]. Besides, these instru-ments are still very expensive and consequently notwidely distributed. Application of HPLC system with UVdetection and appropriate preconcentration step allowsto develop simple and sensitive procedure for the anal-ysis of pharmaceuticals. Moreover, an efficient sampleclean-up utilizing SPE is advisable before its injection inthe chromatographic system.

The intention of this work was the optimization of sam-ple pretreatment procedure for chromatographic deter-mination of five selected analgestic compounds that canbe purchased without medical prescription – diclofenac,ketoprofen, ibuprofen, naproxen, clofibric acid – andfour hormones such as 17b-estradiol (E2), estriol (E3),estrone (E1) and ethynylestradiol (EE2). The natural sexhormone E2 and its metabolites (E1 and E3) as well as syn-thetic steroid EE are mainly excreted in the urine of mam-mals. Their contribution to estrogenicity of wastewatertreatment plants has been already identified [13]. Themain objectives were to develop a simple and efficientHPLC procedure with UV detection for simultaneousdetermination of two groups of drugs (anti-inflammatorycompounds and estrogens) from one injection in rela-tively short time and to evaluate various solid sorbents for

Correspondence: Dr. Krystyna Pyrzynska, Department of Chem-istry, Warsaw University, Pasteura 1, 02-093 Warsaw, PolandE-mail: [email protected]: +4822 8223532

Abbreviations: ACN, acetonitrile; E1, estrone; E2, 17b-estradiol;E3, estriol; SPE, solid phase extraction

i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

986 A. Stafiej et al. J. Sep. Sci. 2007, 30, 985 – 991

their ability to extract analytes from a large volume ofwater. C18 BondElut and polymeric materials ENV BondE-lut as well as Strata-X were included in the study. Based onthe results of the recovery studies, a method for the extrac-tion of 2.5 L surface water and determination of targetcompounds by HPLC-UV was proposed.

2 Reagents and methods

2.1 Reagents

All the pharmaceuticals studied (diclofenac, ketoprofen,ibuprofen, naproxen and clofibric acid) were supplied by

Aldrich. Estrone, estriol and 17b-estradiol hemihydratewere purchased from Sigma-Aldrich (Laborchemikalien,Seelze) and ethynylestradiol from Fluka Buchs (Switzer-land). Their chemical structures, use and origin are pre-sented in Table 1.

Individual standard solutions of these compoundswere prepared in methanol and stored in the dark at 48C,prior to use. High-purity water was taken from a Milli-Qwater system (Millipore). ACN for chromatography fromLAB-SCAN Ltd (Dublin, Ireland) was applied for the pre-paration of HPLC eluents, which were passed through a0.45 lm membrane filter.


Table 1. Names, chemical structures, use and origin of tested compounds

Compound Structure Use/origin

Clofibric acid Metabolite of lipid regulator

Diclofenac Antiphlogistic

Estriol (E3) Steroid

17b-Estradiol (E2) Steroid

Estrone (E1) Steroid

Ethynylestradiol (EE2) Steroid

Ibuprofen Analgestic/anti-inflammatory

Ketoprofen Anti-inflammatory

Naproxen Analgestic/anti-inflammatory

J. Sep. Sci. 2007, 30, 985 – 991 Liquid Chromatography 987

2.2 HPLC analysis

The separations were carried out by an HPLC system (Agi-lent Technologies 1050 series), which consisted of auto-sampler, quaternary pump, detector and degasser. Theanalytical column was a Zorbax Eclipse XDB-C18(15064.6 mm2, 5 lm) from Agilent. Its dead time for iso-cratic elution (2.2 min) was determined by injection ofuracil. The mobile phase consisted of 6.9 mmol/L aceticacid adjusted to pH 4.0 (by NaOH) with 35% v/v ACN. Itwas delivered isocratically at 1 mL/min. For gradient elu-tion, ACN was selected as phase A and ACN/6.9 mmol/Lacetic acid at pH 4.0 (30:70%, v/v) as phase B. Elution wasperformed by increasing the content of organic modifierfrom 3% (18 min), then 12% (22 min) to 40% (40 min). Inboth the modes of elution, the injected volume was 20 lLand UV detection was performed at 230 nm. The concen-trations of the analytes were calculated by measuringthe peak area for each compound and comparing it withthose obtained from a standard solution.

In part of the experiments, Zorbax Eclipse XDB-C18 col-umn (15062.1 mm, 5 lm) was used. Using a flow rate of0.35 mL/min, the optimized mobile phase was a gradientof ACN (A) and 6.9 mmol/L ammonium acetate adjustedto pH 4.0 with 25% v/v ACN (B) in the following form:1:99, 8 min; 17:83, 20 min and 60:40 for 50 min. Injectedvolume was 10 lL.

2.3 Enrichment of water samples

An SPE vacuum station (Varian) was used. SPE was carriedout with C18 BondElut (200 mg/3 mL) and ENV BondElut(500 mg/6 mL) – both from Varian and Strata-X (200 mg/6 mL) from Phenomenex. Prior to extraction, all the car-tridges were conditioned with 5 mL of methanol fol-lowed by 5 mL of high-purity water.

For the optimization of the SPE procedure (choice ofstationary phase, pH of the sample), a sample volume of2000 mL was employed, whereas the analysis of naturalwater samples by HPLC-UV was performed using a samplevolume of 2500 mL. The samples were brought to pH 2.0with concentrated hydrochloric acid and passed troughthe SPE cartridges with a flow rate of 20 mL/min. Thetested compounds were eluted using 10 mL of ACN. Theextract was brought to dryness in a nitrogen stream andredissolved in 2 mL of ACN.

Natural water samples were collected from the riverSword, near Dublin and filtered through a 0.45 lm filter.Then the samples were brought to pH 2.0 with hydrochlo-ric acid and passed through a 200 mg Strata-X cartridge.Extractions were done within 48 h after collection.

3 Results and discussion3.1 Chromatographic separation

For the LC separation of acidic pharmaceuticals RP octa-decyl silica is most frequently used. However, the disso-

ciation of these compounds should be suppressed as faras possible. For this purpose, the mobile phase should beacidified, buffered or should contain an ion-pair compo-nent. In our work, isocratic elution followed by UV detec-tion (220–280 nm) were performed as they are more sim-ple methods.

A study to select the adequate composition of themobile phase (pH and organic modifier concentration)was performed using ACN–acetate mixture (40:60% v/v).An increase in the pH of the eluent reduces the retentionof investigated pharmaceuticals, particularly for ibupro-fen and diclofenac (Fig. 1). These compounds are rela-tively strong acids (pKa 4.2–4.4), almost completely disso-ciated in water and are not retained by RP C18 in theionised state. In order to increase the retention capacity,pH of the eluent should be lower than pKa of the analytes,thus pH 4 seems to be the most suitable.

Figure 2 shows the effect of ACN content in the mobilephase on the retention of acidic pharmaceuticals. AsACN content increased at constant pH, the value of reten-tion factor for the more hydrophobic compoundsdecreased. The increase of ACN content increases thestrength of eluent and reduces hydrophobic interactionbetween analyte and the stationary phase. Retentiontimes of the more hydrophobic herbicides were lowenough (18 min) with the mobile phase containing ACN/acetate solution (45:55% v/v), however, poor resolution ofketoprofen and naproxen peaks was observed. Mobilephase with 40% of ACN allowed the chromatographicseparation with total resolution in a time of 26 min.Similar retention times of acidic pharmaceuticals (clofib-ric acid, naproxen, diclofenac and ibuprofen) wereobtained with mobile phase of ammonium acetate atpH 5.5 but with methanol gradient [9]. The wavelengthused for detection and quantification of tested pharma-ceuticals was set to 220 nm as this allows to reach much


Figure 1. Effect of the mobile phase pH on the retention oftested pharmaceuticals. Eluent: ACN/acetate (40:60% v/v).


better sensitivity, particularly for ibuprofen peak, andgave a better S/N.

For liquid chromatographic separation of estrogens,usually the mobile phase with methanol [14] or ACN [15,16] as an organic modifier has been used. These com-pounds have a very weak acidic character (pKa values10.3–10.7). Thus, their retention behaviour does notdepend on pH of the mobile phase, as it was observed foracidic pharmaceutics. Using the optimal chromato-graphic conditions established earlier for separation oftested pharmaceutical, the retention behaviour of estro-gens was checked. With mobile phase containing ACN/acetate (40:60% v/v) at pH 4, no acceptable resolution ofketoprofen and E2 peaks was achieved. Much better sep-aration for these compounds was obtained with a littlelower content of ACN (35%). These conditions allowed anacceptable separation of five acidic pharmaceuticals andfour hormones (Fig. 3A). This method is the first to be pro-posed that enables the simultaneous determination ofthese compounds from one injection. LOD was estimatedby decreasing the concentration of the analytes down tothe smallest detectable peaks and then this concentra-tion was multiplied by three. LOD values were in therange of 0.02–0.3 lg/mL and of 0.2–0.5 lg/mL for acidicpharmaceuticals (the highest for ibuprofen) and hor-mones, respectively.

To increase the speed of separation, gradient elution(as described in Section 2) was examined increasing theeluent strength. Good separation quality and peakshapes were achieved for these nine compounds with atotal run time of 35 min (Fig. 3B). Due to the shift of thebaseline at 220 nm, the wavelength for UV detection wasset at 230 nm.

When developing new HPLC methods, suitable forquantitative routine applications, analysts often tend touse conventional columns with standard internal dia-

meter, e. g., 4.6 mm, generally providing good separa-tions. Reduction of the column diameter can increasethe efficiency (and consequently, the resolution) andreduce analysis time. Narrow-bore columns may also bemore amenable for ICP-MS detection as the amount ofbuffer salt and organic modifier constituents in themobile phase is reduced considerably [11, 16]. Retentionof nine tested compounds on the chromatographic col-umn with 2.1 mm (id) and a gradient with ACN andammonium acetate at pH 4.0 was similar to this obtainedfor the wider inner diameter (4.6 mm), however, consid-erably higher sensitivity was achieved using a narrow-bore column (Table 2). The peaks for estriol and clofibricacid were broad and higher LOD was calculated for theseanalytes.

3.2 Comparison of tested SPE materials

To meet the objectives for the monitoring of these com-pounds in natural water and wastewater, a preliminarypreconcentration step is necessary. Several types of sor-bents have been reported for isolation and enrichmentof acidic pharmaceuticals [1, 3, 6, 9, 11 and 17] and hor-mones [4, 14, 16 and 18], however, the efficiencies of theproposed procedures are rarely compared. Generally,polymeric sorbents (a variety of hyper-crosslinked poly-styrene-divinylbenzene with different degrees of linkage,porosity and surface area) have a higher adsorption capa-city than C18 sorbent for polar analytes and a broader pHstability range. Additional advantage of polymeric sor-bents include selective p–p interaction with analytescontaining aromatic rings. Oasis HLB sorbent is based ona macroporous copolymer made from two monomers,the divinylbenzene and the N-vinylpyrrolidone, whichexhibits both hydrophilic and liphophilic retention char-acteristics [19].

The performance of C18 BondElut, Lichrolut ENV (com-posed of an ethylvinylbenzene-divinylbenzene copoly-mer) and polymeric Strata-X (crosslinked polystyrene-


Figure 2. Effect of ACN content in the mobile phase on theretention factor of tested pharmaceuticals.

Table 2. LOD (S/N = 3) for tested compounds using chroma-tographic columns with different diameters (gradient elutionwithout preconcentration)

Compound LOD (mg/L)

4.6 mm 2.1 mm

Estriol 0.24 0.63Clofibric acid 0.06 0.17Ketoprofen 0.35 0.1317b-Estradiol 0.44 0.16Naproxen 0.02 0.006Ethynylestradiol 0.51 0.088Estrone 0.33 0.041Diclofenac 0.29 0.03Ibuprofen 0.77 0.074


divinylbenzene) for preconcentration of the tested phar-maceuticals was evaluated by spiking 2.0 L of deionisedwater with 15 lg/L of each compound. The enrichmenton hydrophobic sorbents requires acidic conditions,thus, the pH of the samples was adjusted to 2 (withhydrochloric acid). For comparison, the recovery studieswere also conducted at neutral pH, where acidic pharma-ceuticals are present in their respective ionized forms.The obtained results are presented in Table 3.

The recovery of the investigated hormones, generally,does not depend on sample pH and it is almost quantita-tive for all the tested sorbents. Among these compounds,estriol exhibits the lowest affinity, particularly for silica

gel C18. For this compound extraction recovery of about75% was obtained using the polymer Oasis HLB [4]. Wei-gel et al. [6] have reported similar results for preconcen-tration of estrone and 17b-estradiol with different poly-meric sorbents. More complete retention of investigatedpharmaceuticals was obtained, as it was expected, atlower pH. Recovery of acidic pharmaceuticals from acidi-fied samples exceeded 88% for all the sorbents. Even clo-fibric acid, the most hydrophilic compound, was almostquantitatively recovered. Application of PS-DVB sorbentswith high surface areas and copolymers composed ofboth liphophilic and hydrophobic monomers gave muchlower recoveries for clofibric acid [6].


Figure 3. Chromatogram obtained for the mixture of pharmaceuticals (3 mg/L) and hormones (4.5 mg/L) with UV detection at230 nm, Zorbax Eclipse XDB-C18 (15064.6 mm2, 5 lm) column, flow rate 1 mL/min; (A) isocratic elution; (B) gradient elution.Peaks: (1) E3; (2) clofibric acid; (3) ketoprofen; (4) E2; (5) naproxen; (6) EE2; (7); (8) E1; (9) diclofenac and (10) ibuprofen.


Most manufacturers recommended elution volumes ofabout 5 mL for 200 mg cartridges. In order to assure acomplete elution of all the analytes, the cartridges wereeluted with 10 mL of ACN. No additional recoveries wereobserved for larger volume of eluent. Employing thecombination of SPE and HPLC analysis, a sample pretreat-ment procedure was developed allowing the enrichmentof water sample by a factor of 1000. The LOD (S/N = 3) fordetermination of tested compounds (C18, 15064.6 mm2

analytical column) with preliminary preconcentrationstep was in the range of 0.03–0.9 and 0.3–0.6 lg/L foranalgestics compounds and hormones, respectively.These results are better than those reported previouslyfor HPLC-MS analysis [9, 10].

The matrix effect was evaluated by performing recov-ery experiments with river water samples spiked at the7.5, 15 and 30 lg/L levels. For this experiments polymericPS-DVB sorbent Strata-X was applied and unspiked sam-ple was used as control. As can be seen from the data pre-sented in Table 4 good recoveries of the tested com-pounds were obtained, even at the highest concentrationof the analytes. It suggested that the capacity of theapplied sorbents is sufficient. Figure 4 shows a chromato-gram obtained by extraction of surface water samplespiked at 7.5 lg/L level. At low wavelength detection as itwas used in this study, usually the interferences fromhumic substances show up in the beginning of the chro-matogram as a broad hump that causes difficulties for


Table 3. Mean recoveriesa) of tested compounds using different sorbents (n = 3)

Compound C18 Strata X ENV

Acidicb) Neutralc) Acidic Neutral Acidic Neutral

Estriol 62 60 81 75 80 77Clofibric acid 92 0 92 35 99 45Ketoprofen 101 56 98 97 99 10217b-Estradiol 100 96 98 95 99 101Naproxen 97 27 89 93 97 99Ethynylestradiol 95 92 102 91 99 100Estrone 92 93 88 92 103 99Diclofenac 88 62 90 81 89 83Ibuprofen 94 72 93 93 103 104

a) 2.0 L of deionised water was spiked with 15 lg/L of each compound.b) Sample acidified to pH 2.0.c) At neutral sample pH without acidification.

Figure 4. Chromatogram obtained by extraction (Strata-X) of surface water sample spiked at 7.5 lg/L level of each analyte.Chromatographic conditions and identification of peaks as in Fig. 3.


the reliable quantification. The clean-up procedure couldreduce the amount of coextracted substances, however,but not to the extent desirable for ESI technique in MSdetection. Therefore, for each individual sample signalsuppression was calculated and quantification resultswere corrected [4].

4 Concluding remarks

The developed analytical method, reliable and inexpen-sive, enables the simultaneous determination of someacidic pharmaceuticals (diclofenac, ketoprofen, ibupro-fen, naproxen, and clofibric acid) and estrogens (17b-estradiol, estriol, estrone and ethynylestradiol) in nat-ural water samples. It is being adaptable also for fielduse. After extraction, the light-weight cartridge can beshipped to the laboratory for analyte desorption.

The presented methodology can help to evaluate theenvironmental risk of these compounds. Although thepharmaceutical contribution to the whole toxicity islow, they can be considered as traces of toxicity [20]. The

monitoring of drug residues is certainly an issue for thefuture in order to avoid possible residues in water sys-tems used for drinking water supply.

5 References

[1] Tauxe-Wuersch, A., De Alencastro, L. F., Grandjean, D., Tarradel-las, J., Water Res. 2005, 39, 1761 – 1772.

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Table 4. Recoveriesa) of tested compounds from river watersamples using polymeric Strata-X sorbent

Compound Mean recovery (%)

7.5 lg/L 15 lg/L 30 lg/L

Estriol 101Clofibric acid 95 98 94Ketoprofen 98 110 7917b-Estradiol 99 102 79Naproxen 93 104 77Ethynylestradiol 96 93 93Estrone 97 93 78Diclofenac 102 96 79Ibuprofen 101 100 85

a) 2.5 L of sample after acidification to pH 2 (n = 3).

Determination of anti-inflammatory drugs and estrogens in water by HPLC with UV detection

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