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Short Communication

Multi-residue method for the analysis ofpharmaceutical compounds in sewagesludge, compost and sediments bysonication-assisted extraction and LCdetermination

A method for the simultaneous determination of 16 pharmaceutical compounds in threetypes of sewage sludge (primary, secondary and anaerobically digested dehydrated

sludge), compost and sediment samples is described. Pharmaceutical compounds eval-

uated were nonsteroidal anti-inflammatory drugs (acetaminophen, diclofenac, ibuprofen,

ketoprofen, naproxen and salicylic acid), antibiotics (sulfamethoxazole and trimethoprim),an anti-epileptic drug (carbamazepine), a b-blocker (propranolol), a nervous stimulant

(caffeine), estrogens (17a-ethinylestradiol, 17b-estradiol, estriol and estrone) and lipid

regulators (clofibric acid, metabolite of clofibrate and gemfibrozil). The method is based

on the ultrasonic-assisted extraction, clean-up by SPE and analytical determination by

HPLC with diode array and fluorescence detectors. The best extraction recoveries were

achieved in a three-step extraction procedure with methanol and acetone as extractionsolvents. Extraction recoveries of several pharmaceutical compounds as caffeine were

highly dependent on the type of sample evaluated. The applicability of the method was

tested by analyzing primary, secondary and anaerobically digested dehydrated sludge,

compost and sediment samples from Seville (Southern Spain). Ten of the sixteen phar-maceutical compounds were detected in sludge samples and five in compost and sedi-

ment samples. The highest concentration levels were recorded for ibuprofen in sewage

samples, whereas salicylic acid and 17a-ethinylestradiol were detected in all of the

samples analyzed.

Keywords: Compost / HPLC / Pharmaceutically active compound / Sediment /

SludgeDOI 10.1002/jssc.200900873

1 Introduction

The presence of pharmaceutical compounds in wastewater

has been widely described in the last years. Nevertheless,

there is scarce information about their concentration levels

in sewage sludge generated and in river sediments affected

by wastewater discharges. The knowledge about the concen-tration levels in the different stages of sewage sludge is

necessary: first, because one of the main disposal options forthe generated sewage sludge is its application to soils. That

disposal option is recommended whenever possible in the

third draft of the future European Union Directive on Sludge[1], but in such a way as to minimize the risk of negative

effects. Second, to achieve a full assessment of the removal

processes of pharmaceutical compounds alongside sludge

treatment. Nevertheless, in the last years, the pollutantsmore widely studied in sewage sludge continue to be the

priority pollutants, such as heavy metals [2] and several

selected organic compounds [3], whereas the presence ofpharmaceutical compounds, some of them with endocrine

disruption properties, has been slightly studied. Moreover,

the knowledge about the concentration levels in riversediments is necessary to estimate the persistence and

environmental risk of these pollutants. Methods reported for

the determination of pharmaceutical compounds in sewage

sludge or river sediments are based on pressurized liquid

extraction (PLE) [48], ultrasonic solvent extraction (USE)[812] and microwave-assisted extraction (MAE) [13] and

most of them have just been developed and validated for a

specific sample matrix (Table 1). The aim of this study was to

Julia MartnJuan Luis SantosIrene AparicioEsteban Alonso

Department of AnalyticalChemistry, University of Seville,

Seville, Spain

Received December 27, 2009

Revised March 19, 2010

Accepted March 30, 2010

Abbreviations: DAD, diode array detector; Fl, fluorescence;

MAE,microwave-assisted extraction; PLE,pressurized liquid

extraction; USE, ultrasonic solvent extraction

Correspondence: Dr. Esteban Alonso, Department of Analytical

Chemistry, University of Seville, C/Virgen de Africa, 7, 41011

Seville, Spain

E-mail:[email protected]

Fax: 134-9-5428-2777

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2010, 33, 176017661760


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Table

1.

Characteristicsofthemeth

odsdescribedintheliteratureforthedeterminationoftheselectedpharmaceuticalcompoundsinsolidenvironmentalsamp

les

Th

erapeu

ticgroup

Samp

le

Extrac

tion

tec

hn

ique

Samp

le

amoun

t(g)

Extrac

tionso

lven

ts

Tota

lso

lven

t

volu

me

(mL)

Ana

lytica

l

de

term

ina

tion

Recoveries

(%)

LOQ

(mg

/kg

dm

)

Re

ferences

An

ti-i

nfl

amma

tory

drugs,an

tibiotics,

b-b

loc

ker,nervous

stim

ula

nts

,lipid

regu

lators

Prima

ryslu

dge

Secon

daryslu

dge

Diges

ted

slu

dge

PLE

1

Wa

ter/me

thano

l(2

:1v/v)

22

LC/MS

-MS

36.8

130

33.5

122

29.2

102

0.4489

.2

0.4489

.2

2.1096

.3

[4]

An

tibiotics

Secon

daryslu

dge

Diges

ted

slu

dge

PLE

0.2

Wa

ter/me

thano

l(1

:1v/v)

22

LC/MS

-MS

51

64

14

15

[5]

An

ti-i

nfl

amma

tory

drugs,an

tibiotics,

an

ti-e

pile

ptics,

b-b

loc

ker,nervous

stim

ula

nt,

lipid

regu

lators

Diges

ted

slu

dge

Soil

PLE

1 2.7

Wa

ter/me

thano

l(1

:1v/v)

53

LC/MS

45

120

50

110

23

50a)

,

0.8

120a

)

[6]

An

ti-i

nfl

amma

tory

drugs,

an

tibiotics,b

-blo

cke

r,

nervousst

imu

lan

ts,

lipid

regu

lators

Sludg

e

PLE

5

Ph

osp

horicac

id50m

M/

me

thano

l(1

:1v/v)

40

LC/MS

72

109

14

32

[7]

An

ti-i

nfl

amma

tory

drugs,

an

tibiotics,an

ti-e

pile

ptics,

b-b

loc

ker,nervousst

imu

lan

t,

lipid

regu

lators

Prima

ryslu

dge

Diges

ted

slu

dge

PLE

an

dUSE

Wa

ter/me

thano

l(9

:1v/v)

95

LC/MS

-MS

70

115

[8]

An

ti-i

nfl

amma

tory

drugs,

an

ti-e

pile

ptics,

lipid

regu

lators

Secon

daryslu

dge

Diges

ted

slu

dge

USE

0.5

Me

thano

l(412m

L)

Ace

tone

(212m

L)

10

LC/MS

-MS

52

72

43

76

20

50

[9]

An

tibiotics

Diges

ted

slu

dge

USE

2

Ph

osp

ha

teb

uff

erp

H6

(10m

L);

Wa

ter/me

thano

l(75:25v/v)

,

5%TEA(10m

L)

20

LC/MS

-MS

64

71

100

1100

[10]

An

ti-i

nfl

amma

tory

drugs,

an

tibiotics

Sedim

en

t

USE

50

Ace

tone

/ace

ticac

id(20:1v/v);

ethylace

tateorme

thano

l;

ace

tone;e

thylace

tate

180

LC/MS

-MS

47

107

0.4

20

[11]

Estrogens

Secon

daryslu

dge

Diges

ted

slu

dge

Sedim

en

t

USE

0.5

0.5

5

Me

thano

l(413m

L)

Ace

tone

(313m

L)

13

GC/MS

-MS

83

119

94

117

105118

24 24 0.2

0.4

[12]

An

ti-i

nfl

amma

tory

drugs

Sedim

en

t

MAE

5

Me

thano

l/ace

tone

(1:1v/v)

40

GC/MS

46

87

30

80a)

[13]

a)LODs.

J. Sep. Sci. 2010, 33, 17601766 Liquid Chromatography 1761



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develop an analytical method, reliable and affordable, for

the simultaneous determination of 16 pharmaceuticalcompounds from seven therapeutic groups in different

environmental matrices: sewage sludge from different

sludge stages (primary, secondary and anaerobically digested

dehydrated sludge), compost and river sediments. For our

study, we chose sample extraction by USE because of itslower cost with respect to the other techniques reported forthe extraction of pharmaceuticals from solid environmental

samples, PLE and MAE. Moreover, lower degradation

processes occur in USE than in PLE as extraction is carried

out at room temperature instead of at high temperatures ofaround 1001C reported in PLE methods and, usually,

precisions higher than in MAE are achieved.

2 Materials and methods

2.1 Chemicals and reagents

HPLC-grade water, acetonitrile and methanol were

purchased from Romil (Barcelona, Spain). Hexane, ethylacetate, acetone (HPLC grade) and sulphuric acid of

analytical grade were obtained from Panreac (Barcelona,

Spain). Potassium dihydrogen phosphate of analytical grade

was purchased from Scharlau (Barcelona, Spain). Acetami-

nophen, carbamazepine, diclofenac, ketoprofen, naproxenand salicylic acid (97100% purity) were purchased from

Sigma-Aldrich (Steinheim, Germany). Caffeine was

obtained from Merck (Darmstadt, Germany). Clofibric acid,

gemfibrozil, ibuprofen, propranolol hydrochloride, 17a-

ethinylestradiol, 17b-estradiol, estriol, estrone, sulfamethox-

azole and trimethoprim (99% purity) were purchased fromDr. Ehrenstorfer (Augsburg, Germany). SPE cartridges,

packed with 60 mg of Oasis HLB, were purchased from

Waters (Milford, MA, USA).

Stock solutions of each pharmaceutically activecompound at a concentration level of 1000 mg/L were

prepared in methanol and stored at 41C. Calibration solutions,

in the concentration range from 0.01 to 5 mg/L, were

prepared by diluting the stock standard solutions in methanol.

2.2 Sewage treatment plants and sampling

Sludge samples were collected from a wastewater treatmentplant sited in Seville (Southern of Spain) where primarysludge is obtained by settling fresh sludge from the primary

clarifier; secondary sludge is obtained by flotation of fresh

sludge from the biological reactor and digested sludge is

obtained by anaerobic digestion of primary and secondary

sludge mixture. Digested sludge is dehydrated by centrifu-

gation and composted in dynamic batteries with thermallycontrolled aeration facilitated by turning. Sediment samples

were collected from the surface of Guadiamar River (Seville,

Southern Spain), downstream from the discharge points of

several wastewater treatment plants. Each sample was

obtained by mixing the aliquots collected using a gripping

device from at least five sampling sites across the river. Twoliters of each liquid sample (primary and secondary sludge)

and 1 kg of each solid sample (anaerobically digested

dehydrated sludge, compost and sediment) were collected.

All types of samples were lyophilized in a Cryodos-50

lyophilizer (Telstar, Terrasa, Spain), sieved (particle sizeo100 mm), stored in amber glass bottles and maintained at301C until analysis.

2.3 Sample treatment

Aliquots of 1.00 g of primary or secondary sludge, 1.50 g of

digested sludge or 2.00 g of compost or sediment samples

were successively extracted with 5 and 2 mL of methanoland 2 mL of acetone. In each extraction step, sample was

vigorously shaken during 30 s, ultrasonicated for 15 minand centrifuged at 4000 rpm for 20 min. The supernatants

obtained from each extraction step were combined andevaporated to 0.2 mL under a nitrogen stream. The extract

was diluted to 250 mL with deionized water acidified to pH 2with sulphuric acid and subjected to the cleanup procedure

reported by Camacho et al . [14] except that 6 mL of

water/methanol (95:5 v/v) were used for cartridge rinsing

and the evaporated extract was dissolved in 150 mL ofmethanol.

2.4 Liquid chromatography

Chromatographic analysis was performed on an Agilent

1200 series HPLC (Agilent, USA) equipped with anultraviolet diode array detector (DAD) and a rapid scan

fluorescence (Fl) detector connected on line. Separation was

carried out using a ZORBAX Eclipse XDB-C18

(150 mm 4.6 mm id, 5 mm) cartridge column (Agilent)protected by an XDB C-18 (4 mm 4 mm id, 5 mm) guard

column (Agilent) and thermostated at 301C. Chromato-

graphic analysis was carried out at a flow rate of 1.2 mL/min

by gradient elution with acetonitrile and a 25 mM potassium

dihydrogen phosphate solution, using a wavelength

program according to Camacho et al. [14]. The compoundswere identified by comparison of their retention times and

UV and Fl spectra with those in the standard solution

chromatograms. Peak areas were used for quantification.Fluorescent compounds were quantified using peak areasfrom Fl chromatograms instead from DAD chromatograms

because of the higher sensitivity and selectivity achieved.

3 Results and discussion

3.1 Extraction optimization

The effects of the extraction solvent (acetone, ethyl acetate

and methanol) and time of sonication (5, 10 and 15 min)

J. Sep. Sci. 2010, 33, 176017661762 J. Martn et al.



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were evaluated to obtain the optimal extraction conditions

for the studied pharmaceutically active compounds.Aliquots of 2.0 g of sediment samples, which are the less

contaminated samples, were spiked in triplicate with the

studied pharmaceutically active compounds at a concentra-

tion level of 300 mg/kg. Spiked and blank samples were

successively extracted by sonication for 15 min using 5, 2and 2 mL of the tested solvent. Recoveries obtained areshown in Fig. 1. In spite that most of the compounds were

better extracted with methanol, the presence of interfering

compounds in the chromatograms was more significant

than when acetone was used as an extraction solvent. Inorder to reduce the presence of matrix components, the use

of two aliquots of 5 and 2 mL methanol and 2 mL of acetone

was tested and selected for sample extraction.

Shorter sonication times (5 and 10 min) were testedusing the three-step extraction procedure with 5 and 2 mL of

methanol and 2 mL of acetone. At sonication times of 5 min,a significant decrease of extraction recovery of most of the

compounds was observed to the point that two of them,ibuprofen and salicylic acid, were not extracted (Fig. 1).

Similar extraction recoveries were obtained with sonication

for 10 and 15 min except for ibuprofen, ketoprofen, trime-

thoprim and propranolol which were more efficientlyextracted at longer sonication times. Finally, extraction

times of 15 min were selected.

3.2 Method validation

The method was validated for each type of matrix in terms

of recovery, precision and LOQs. Acetaminophen was poorly

extracted from all the sample matrices with recoveries lower

than 15% (Table 2) and could not be determined with themethod developed. Several of the pharmaceuticals were

more efficiently extracted from some types of samples than

from the others. The differences in the extraction recoveries

between pharmaceutical compounds and sample matricesare consistent with those reported by Radjenovic et al. in

2009 [4]. The proposed method significantly increases theextraction recoveries of the method developed for

Radjenovic et al. for the determination of pharmaceuticalcompounds in primary, secondary and digested and

dehydrated sludge which, to the best of our knowledge, is

Figure 1. Influence of extrac-

tion solvent and sonication

time in recovery efficiencies

from sediment samples

(n53). (Aep, anti-epilectic

drugs; bbl, b-blocker; Ns,

nervous stimulant; Lrg, lipid

regulators).




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Table 2. Average recoveries (R, n53) and LOQ of each compound in primary, secondary, anaerobically digested dehydrated sludge,

compost and sediment samplesa)

Therapeutic group Pharmaceutical

compound

Primary sludge Secondary sludge Digested sludge Compost Sediment

R LOQ R LOQ R LOQ R LOQ R LOQ

(%) (mg/kg

dm)

(%) (mg/kg

dm)

(%) (mg/kg

dm)

(%) (mg/kg

dm)

(%) (mg/kg

dm)

Anti-inflammatory

drugs

Acetaminophen o15 83.5 o15 184 o15 109 o15 70.6 o15 30.3

Diclofenac 85.8 100 86.9 58.6 41.6 3.69 92.4 5.31 72.9 3.63

Ibuprofen 93.6 355 100 277 115 192 99.9 166 88.3 187

Ketoprofen 89.5 18.7 96.2 11.4 63.8 10.5 91.7 7.72 84.1 5.04

Naproxen 84.3 4.72 90.6 5.94 79.7 7.53 106 0.40 103 2.57

Salicylic acid 61.5 5.85 75.1 4.79 41.3 5.81 88.6 2.03 42.2 2.05

Antibiotics Sulfamethoxazole 61.9 26.8 52.4 31.5 41.1 170 58.2 10.5 58.4 10.9

Trimethoprim 73.5 205 47.8 315 106 95.3 81.0 13.8 79.2 8.01

Anti-epileptic drug Carbamazepine 102 14.8 89.0 5.89 110 1.39 95.2 1.81 95.1 1.11

b-Blocker Propranolol 93.4 3.21 97.0 3.09 79.6 2.51 90.6 1.66 90.3 1.66

Nervous stimulant Caffeine 67.7 22.2 50.0 66.1 41.1 45.4 84.6 2.84 102 1.49

Estrogens 17a-Ethinylestradiol 105 3.28 89.8 3.84 100 2.30 87.1 1.99 93.5 1.8517b-Estradiol 106 3.54 94.5 3.97 94.9 2.63 83.9 2.24 98.8 1.90

Estriol 107 3.22 96.9 3.56 86.3 2.66 89.3 1.94 87.6 1.97

Estrone 65.1 30.4 85.9 23.0 88.8 14.8 96.3 10.2 99.7 9.93

Lipid regulators Clofibric acid 89.2 94.0 94.1 89.3 60.0 93.5 103 40.7 69.4 36.4

Gemfibrozil 68.5 360 70.4 350 66.0 249 108 114 69.8 68.4

a) dm: dry matter.

Figure 2. HPLC-DAD (A) and Fl (B) chroma-

tograms of a compost sample spiked with

the pharmaceutically active compounds at a

concentration level of 300 mg/kg dm each.




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the only method described in the literature for the

determination of pharmaceutical compounds in severaltypes of sewage sludge. The precision of the method,

estimated as RSD, varied in the range from 0.1 to 23% in

sewage sludge, which are consistent with the values

reported by Radjenovic et al. (0.529.6%) for sewage samples

[4], and in the range from 0.6 to 9.1% in compost andsediment samples. Linearity was observed in the concentra-tion range studied with the coefficients of correlation

greater than 0.992 for all the pharmaceuticals analyzed.

LOQs of each pharmaceutical compound in each type

of sample evaluated are listed in Table 2. Chromatogramsof a compost sample spiked with the pharmaceutical

compounds at a concentration level of 300mg/kg and a

blank compost sample are shown in Figs. 2 and 3,

respectively.The proposed method is based on the sonication-assis-

ted extraction which requires equipment commonly avail-able in most of the laboratories and requires smaller solvent

extraction volume, 9 mL, than the others (10100 mL) [413](Table 1). Only one method uses similar sample amounts

[13], extraction solvent volume and the same extractiontechnique but lower recoveries are reported, the method has

been validated only for secondary and digested sludge and

fewer therapeutic groups were considered. Similar recov-

eries to those reported by other authors [413] were obtained

in spite that smaller solvent volumes were used in theextraction step.

3.3 Application to sewage sludge, compost and

sediment samples

The optimized method was applied to the determination of

the concentration levels of the pharmaceutically active

compounds in primary, secondary, anaerobically digested

dehydrated sludge, compost and sediment samples fromSeville city (Table 3). Concentrations were consistent with

the scarce information reported about pharmaceutical

concentration levels in solid environmental samples [4, 9].

The compounds most frequently detected were salicylic acidand 17a-ethinylestradiol, followed by naproxen, carbamaze-

pine and 17b-estradiol. These compounds were detected atconcentration levels in the range of 10203 mg/kg dm which

are similar to those reported by Barron et al. in Ireland [6]and Okuda et al. in Japan [8]. The highest concentration

levels were found for ibuprofen as in the study of 20pharmaceutical compounds in sewage sludge reported by

Radjenovic et al. [4], but concentration levels measured were

significantly higher than those reported by Radjenovic et al.

Figure 3. HPLC-DAD (A) and Fl

(B) chromatograms of a blank

compost sample.




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Caffeine was quantified only in the primary sludge and

sediment samples. Other compounds detected in some of

the types of samples analyzed were gemfibrozil, propranolol

and estriol.

4 Concluding remarks

The developed method allows the simultaneous extractionand determination of 16 pharmaceutical compounds

belonging to seven therapeutic groups in complex solid

environmental matrices: sludge from different treatment

stages, compost and river sediment samples. The method

has been validated for the determination of the pharmaceu-tical compounds in each of the five matrices evaluated.

Quite different recovery efficiencies were obtained for some

compounds from one type of sample to another what

remarks the necessity of validating the method in each typeof environmental matrix. The proposed method is, to the

best of our knowledge, the first methodology developedand validated for the determination of pharmaceutical

compounds from different therapeutic groups in different

types of sewage sludge and in river sediments. This fact

makes the proposed method a useful tool to properlyevaluate the removal rates of pharmaceutical compounds

alongside sludge treatments and to estimate at which degree

the discharges of pharmaceutical compounds from waste-

water treatment plants affect river sediments.

The authors thank the financial support of this work from

the Ministerio de Educacion y Ciencia, Spain (Project no.

CGL2007-62281).

The authors have declared no conflict of interest.

5 References

[1] Working Document on Sludge, Third Draft, 27 April

2000. European Union, Brussels, Belgium.

[2] Alonso, E., Aparicio, I., Santos, J. L., Villar, P., Santos,

A., Waste Manage. 2009, 29, 418424.

[3] Abad, E., Martnez, K., Planas, C., Palacios, O.,

Caixach, J., Rivera, J., Chemosphere 2005, 61,

13581369.

[4] Radjenovic, J., Jelic, A., Petrovic, M., Barcelo, D., Anal.Bioanal. Chem. 2009, 393, 16851695.

[5] Go bel, A., Thomsen, A., McArdell, C. S., Alder, A. C.,

Giger, W., TheiX, N., Lo ffler, D., Ternes, T. A., J. Chro-

matogr. A 2005, 1085, 179189.

[6] Barron, L., Tobin, J., Paull, B.,J. Environ. Monit. 2008,

10, 353361.

[7] Nieto, A., Borrull, F., Pocurull, E., Marce, R. M., J. Sep.

Sci. 2007, 30, 979984.

[8] Okuda, T., Yamashita, N., Tanaka, H., Matsukawa, H.,

Tanabe, K., Environ. Int. 2009, 35, 815820.

[9] Ternes, T. A., Bonerz, M., Herrmann, N., Lo ffler, D.,

Keller, E., Lacida, B. B., Alder, A. C., J. Chromatogr. A

2005,1067, 213223.

[10] Lindberg, R. H., Wennberg, P., Johansson, M. I., Tysk-

lind, M., Andersson, B. A. V., Environ. Sci. Technol.

2005,39, 34213429.

[11] Lo ffler, D., Ternes, T. A., J. Chromatogr. A 2003, 1021,

133144.

[12] Antonic, J., Heath, E., Anal. Bioanal. Chem. 2007, 387,

13371342.

[13] Ternes, T. A., Andersen, H., Gilberg, D., Bonerz, M.,

Anal. Chem. 2002, 74, 34983504.

[14] Camacho, D., Martn, J., Santos, J. L., Aparicio, I.,

Alonso, E., J. Sep. Sci. 2009, 32, 30643073.

Table 3. Mean concentration levels of pharmaceutically active compounds in primary, secondary, anaerobically digested dehydrated

sludge, compost and river sediment samples from Seville ( n53)

Therapeutic group Pharmaceutical compound Mean concentration (mg/kg)

Primary sludge Secondary sludge Digested sludge Compost Sediment

Anti-inflammatory drugs Diclofenac o

LOQ o

LOQ o

LOQ o

LOQ o

LOQIbuprofen 3519 1322 5096 oLOQ oLOQ

Ketoprofen oLOQ oLOQ oLOQ oLOQ oLOQ

Naproxen 43.1 29.1 14.9 oLOQ 11.2

Salicylic acid 128 54.7 37.4 22.1 9.49

Antibiotics Sulfamethoxazole oLOQ oLOQ oLOQ oLOQ oLOQ

Trimethoprim oLOQ oLOQ oLOQ oLOQ oLOQ

Anti-epileptic drug Carbamazepine 21.1 128 50.7 11.0 oLOD

b-Blocker Propranolol 3.69 oLOQ oLOQ oLOQ 3.37

Nervous stimulant Caffeine 83.1 oLOQ oLOQ oLOQ 7.21

Estrogens 17a-Ethinylestradiol 27.8 13.6 63.5 94.9 48.1

17b-Estradiol 11.9 82.3 203 91.7 oLOQ

Estriol 33.9 oLOQ 35.6 4.85 oLOQ

Estrone oLOQ oLOQ oLOQ oLOQ oLOQ

Lipid regulators Clofibric acid oLOQ oLOQ oLOQ oLOQ oLOQ

Gemfibrozil 374 oLOQ oLOQ oLOQ oLOQ



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