American Journal of Anal yt ical Chemistry, 2011, 2, 863-870
doi:10.4236/ajac.2011.28099 Published Online December 2011 (
Copyright © 2011 SciRes. AJAC
Optimization of Analytical Conditions to Determine
Steroids and Pharmaceuticals Drugs in Water Samples
Using Solid Phase-Extraction and HPLC
Ramiro Vallejo-Rodríguez1, Alberto Lopez-Lopez1, Hugo Saldarriaga-Noreña2,
Mario Murillo-Tovar1, Leonel Hernández-Mena1
1Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ);
Normalistas 800, Colinas de la Normal, Guadalajara, México
2Facultad de Ciencias Químicas, Universidad Autónoma de Coahuila, República Ote., Saltillo, México
Received August 22, 2011; revised October 4, 2011; accepted October 18, 2011
Two reliable methods were optimized to determine two steroids (17
-Estradiol and 17
and two pharmaceutical drugs (ibuprofen and naproxen) using Solid-Phase Extraction (SPE) for sample
preparation and High Performance Liquid Chromatography (HPLC) for analysis. SPE (C18) conditions were
evaluated varying elution solvent volume, pH conditions and sample mass in the cartridge and reduction
techniques of the extract. The efficiency of the analytical methods was evaluated by spiking ultrapure water
samples with compounds at three and four levels of concentration for steroids and pharmaceutical drugs, re-
spectively. The recoveries were independent (P > 0.05) of added mass of target analytes with a repeatability
lower than 6.5% for steroids and 12.1% for pharmaceutical compounds. The recovery factor (coefficient of
variation, CV) was higher than 83% for steroids (CV < 3.8%) and >93% for pharmaceuticals (CV < 5.2%).
The optimized analytical method was applied for the evaluation of a steroid degradation test using ozone,
finding that the estimated limit of detection is sufficient to determine the residual mass (μg·L–1) of
17β-Estradiol after the experiment.
Keywords: Steroids, Pharmaceuticals Drugs, Solid-Phase Extraction, HPLC-DAD
1. Introduction
The ever increasing use of synthetic steroids and phar-
maceutical products both in humans as well as in ani-
mals is becoming a new environmental issue to such
an extent that the interest in collecting information
regarding the origin of these substances in the envi-
ronment and their possible effects on humans and
ecological systems has increased [1,2]. So far, the
generalized use of oral contraceptives formulated with
steroids capable of inducing estrogenic responses in
fishes at low concentrations such as 1 μg·L–1 has
raised concern among the scientific community due to
the potentially dangerous conesquences on aquatic
media [3,4].
The environment, specifically aquatic, presents di-
verse pathways for contamination by pharmaceuticals
and steroids. Such compounds enter the sewage sys-
tem through urine and feces and finally end up in
wastewater treatment plants (WWTPs) or are dis-
charged directly into bodies of surface water. The uses
of residual sludge from WWTPs that contain estrogens
in agriculture are another source of contamination in
surface waters through run-offs [5,6]. Also, the
WWTPs’ effluents that originate from the pharmaceu-
tical industry are an important source responsible for
pharmaceuticals present in the environment [7]. The
low efficiencies of the WWTPs to degrade two classes
of compounds due to their persistence and also efflu-
ents discharged from urban and industrial sources
without any previous treatment result in the presence
of natural and synthetic estrogens in bodies of sur-
face water [6,8].
Pharmaceutical drugs occurring in wastewaters or
WWTP effluents have been reported in several research
papers in concentration levels of μg·L–1 for naproxen and
ibuprofen [9,10]. For steroids, specifically natural ones
and the synthetic steroids considered to be the most
powerful estrogenic compounds, several studies have
also reported very low environmental concentrations on
the order of μg·L–1 [11-13]. Since these substances rep-
resent a potential danger for human health and because
of their environmental levels, it is important to monitor
both pharmaceuticals as well as steroids in water. There-
fore, robust and reliable analytical methods are required
which will allow estimating the presence of such com-
pounds in water samples [10,14,15]. Although several
authors have determined steroid concentration in water
by using gas chromatography coupled with mass spec-
trometry (GC-MS), using solid-phase extraction as sam-
ple preparation method [16,17], HPLC coupled to mass
spectrometry (HPLC-MS) has also been used by other
authors [11,18-20]. It is important to highlight that
these techniques are robust, but at the same time costly,
which does not allow many laboratories to acquire such
infrastructure to develop similar methodologies. Con-
sidering the fact that most of them have High-Perfor-
mance Liquid Chromatographs with Diode-Array De-
tection (HPLC-DAD), the implementation of analytical
methods to determine pharmaceuticals and steroids in
water by using this available analytical infrastructure is
then justified. Before chromatography gas or liquid
analysis, sample preparation techniques must be care-
fully selected and optimized because the low concen-
tration of steroids and pharmaceutical drugs [14] can
make the detection difficult in environmental samples
but the determination is becoming even more challeng-
ing when target analytes are degraded and then their
concentration are as low that analytical signal become
undetectable reliably in treated effluent. The solid-
phase extraction (SPE) is one of the alternatives more
frequently used with this purpose [21], since it isolates
the analytes from liquid sample and then these are con-
centrated [28]. In spite of there are so many reports on
the analysis of steroids and pharmaceutically drugs
[7,10,11,14], these are more as a guide or alternative
method, therefore their conditions still must be op-
timized before be applied on samples and a step imple-
mentation and evaluation of quality analytical parame-
ters in laboratory is necessary.
The objective of this work was the optimization of
analytical conditions for the extraction and determination
of two steroids and two pharmaceutical compounds by
SPE and subsequent analysis by HPLC-DAD, in water
ultrapure samples, with the aim to evaluate the efficiency
of their chemical degradation by using ozone processes.
2. Material and Methods
2.1. Chemicals and Materials
The compounds utilized in this work were selected ac-
cording to their commercial demand, frequency of use,
and presence in bodies of water. These were two ster-
oids: one natural, 17β-Estradiol (E2), and one synthetic,
17α-Ethinylestradiol (EE2) [12,13]; and two anti-infla-
mmatory pharmaceuticals, naproxen and ibuprofen [9,
10,15]. The standards of compounds (purity in paren-
theses) of E2 (98%), EE2 (98%), naproxen and ibupro-
fen (98%), were purchased as powders from Sigma-
Aldrich (St. Louis, MO). The stock standard and further
working solutions were prepared in methanol. The sol-
vents, HPLC grade, were obtained as follows: acetoni-
trile (ACN) was from Tedia (Fairfield, OH), methanol
(MeOH) from J.T. Baker (Phillipsburg, NJ), ethyl ace-
tate from Burdick & Jackson (Muskego, MI), and ul-
trapure water was obtained directly in our laboratory
from an ultrapure water system (Nanopure, Barnestead).
The packed syringe barrel with 500 mg of C18 Strata
(dp 55 μm) SPE was acquired from Phenomenex (Tor-
rance, CA). The potassium phosphate monobasic
(KH2PO4) with 99% purity was acquired from Sigma
(St. Louis, MO).
2.2. Preparation of Samples and Standard
Solution Stock
The samples were prepared by spiking ultrapure water
with a known amount of the selected compounds. The
optimization and adjustment of conditions procedure
were carried out at only one concentration level, while
evaluation of method was done at different levels; three
for steroids and four for pharmaceuticals compounds,
which were based on the levels reported in the literature
[14,15]. Although, only one type of stationary phase was
used, the samples spiked with steroids and pharmaceuti-
cals were processed separately because these compounds
have different chemical properties. The standard stock
solution of 17β-Estradiol (E2) and 17α-Ethinylestradiol
(EE2) (2500 µg·mL–1) was prepared by dissolving 125
mg of both compounds in 50 mL of methanol in a volu-
metric flask. The working standard solution (0.5 - 50
µg·mL–1) was prepared by diluting the stock solution
with methanol. For naproxen and ibuprofen (500
µg·mL–1) the stock solution was prepared by dissolving
25 mg of both compounds. The working standard solu-
tions for naproxen (0.1 - 5.0 µg·mL–1) and ibuprofen (1.0
- 250 µg·mL–1) were prepared from the stock solutions
with methanol.
Copyright © 2011 SciRes. AJAC
2.3. Optimization and Evaluation of Analytical
2.3.1. Conditioning of Solid Phase
The C18 cartridges were conditioned before the extraction
step using methanol, ethyl acetate, acetonitrile and water.
For each kind of compound a different sequence was
used. For steroids extraction, conditioning was done by
passing 8 mL of acetonitrile through of the phase, then 7
mL of methanol and finally 5 mL of water. For the
pharmaceuticals, 3 mL of ethyl acetate, and then 3 mL of
methanol and 3 mL of water were used. Both sequence
of solvents were based on previous work [14,15], respec-
2.3.2. Efficiency of the Eluting and Volume Solvent
The capacity and the effectiveness of elution solvent
from the C18 cartridge were tested by using the selected
solvents. In separate experiments, both solvents were
spiked with known amounts of the compounds and were
passed by gravity through a column that was precondi-
tioned. Later, seven aliquots of 2 mL were collected and
then analyzed separately to know total recovery and
volume necessary for the elution step. For steroids a so-
lution of 5 mg·L–1 was used and for pharmaceuticals so-
lutions of 8.3 mg·L–1 (naproxen) and 83.3 mg·L–1 (ibu-
profen) were used. The amounts of compounds that were
selected were based on environmental levels previously
reported [14,15].
2.3.3. Solid Phase Extraction Conditions
The spiked ultrapure water samples were passed through
the conditioned cartridges placed in a Manifold Varian
VAC ELUT-20, which was connected to a vacuum pump
with a pressure and vacuum controller. For steroids the
flow rate was 3.5 mL·min–1 and a vacuum pressure of 3
in Hg, while for the pharmaceuticals these were 4.2
mL·min–1 and 4.5 in Hg. Before the elution step, the car-
tridges were dried under vacuum (5 in Hg) for approxi-
mately twenty minutes to remove all water because these
molecules could make the elution defaulted and yield
low recoveries of compound from the stationary phase.
The elution of the steroids was done with two aliquots (5
mL) of ACN and for pharmaceuticals it was carried out
with three aliquots (1 mL) of ethyl acetate and combined
in an amber bottle.
2.3.4. Effect of the pH on the Solid Phase Extraction
of Pharmaceutical Compounds
The efficiency of extraction for the pharmaceuticals was
evaluated at neutral (pH = 7.0) and at acid conditions
(pH = 4.5) with the objective to know the effect on re-
covery. Both pH of the water sample and that used for
conditioning of the stationary phase was fixed with a
buffer solution (KH2PO4 50 mM).
2.3.5. Efficiency of the Reduction Technique
Two different methods were used to evaluate the effi-
ciency of the reduction technique. One method consisted of
subjecting the extracts to a gentle stream of chroma-
tographic nitrogen gas and for the other the extracts were
reduced using a Büchi R-210 rotary evaporator (30 rpm) at
40˚C, with vacuum pressure (5 in Hg). All reduced extracts
were adjusted to 1 mL.
2.4. Evaluation of the Optimized Analytical Con-
To check recoveries, linearity, precision and limit of de-
tection (LOD) of the optimized methods, known amounts of
each compound at three different concentration levels were
added to ultrapure water (1 L). Therefore, three concentra-
tions levels were used for both steroids (12.5, 25, 50
µg·mL–1), and fourth levels for naproxen (2.5, 6.25, 12.5, 25
µg·mL–1) and ibuprofen (25, 62.5, 125, 250 µg·mL–1). Each
spiked level was assayed in duplicate using optimized con-
ditions in each step of the analytical method.
2.5. Chromatography Analysis
2.5.1. Apparatus and Conditions
The determination for all compounds was performed on
HPLC-DAD Varian ProStar 7725 equipped with Varian
ProStar 230 DAD detector. Varian (Galaxy soft-
ware was used to record the chromatograms and to cal-
culate the chromatographic parameters. The mobile
phase for steroids separation was prepared by mixing
acetonitrile and ultrapure water in a gradient elution. For
pharmaceuticals, this was prepared by mixing methanol
and 50 mM potassium dihydrogen phosphate buffer. The
mobile phases were filtered trough 0.45 µm nylon filters
(Millipore) and degassed before use. The chroma-
tographic separations were performed using LiChrospher
100 RP-18, 5 µm, 250 mm × 4.6 mm i.d. column (Agilent
Technologies, Waldbronn, Germany) for all compounds,
eluted with the mobile phase at the flow rate of 1.0
mL·min–1. The measurements were made with an injec-
tion volume of 100 µL for steroids and 20 µL for phar-
maceuticals. The detection was carried out with a Varian
ProStar 230 (Walnut Creek, CA); diode-array detector
(DAD); using the maximum wave-length (λmax) of 197
nm for steroids and 220 and 230 nm for ibuprofen and
naproxen, respectively (Figure 1).
2.5.2. Linearity
The linearity of the HPLC-DAD analysis was checked
Copyright © 2011 SciRes. AJAC
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Elution gradient
% A% B
50 100 0
52 100 0
% A% B% C
00 1585
4.50 1585
12.5 20080
18 25 075
27 45 055
45 45 055
Figure 1. Chromatograms obtained by HPLC analysis of two standard mixtures of a) 50 μg·mL–1 of E2 and EE2 eluted with A: acetonitrile
and B: ultrapure water (18 M); and b) 25 μg·mL–1 of naproxen and 250 μg·mL–1of ibuprofen elute with A: acetonitrile, B: methanol and C:
Buffer solution 50 mM KH2PO4.
by analyzing seven solutions in the range of 1.0 - 50
µg·mL–1 for steroids and 0.5 - 50 µg·mL–1 and 0.5 - 250
µg·mL–1 for naproxen and ibuprofen, respectively. Each
concentration was analyzed in triplicate. Calibration
curves for each compound were generated by plotting the
analyte peak area against theoretical concentration of the
analytes. The linear regression coefficient (r) for each
compound were higher than 0.999.
2.5.3. Limits of Detection and Quantification
Limits of detection (LOD) and quantification (LOQ)
were determined in accordance with Mitra [28]. The
LODs were 0.65 μg·mL–1 and 0.57 μg·mL–1 for EE2 and
E2, respectively, and LOQ values for EE2 and E2 were
2.16 µg·mL–1 and 1.89 µg·mL–1, respectively. Similarly,
for pharmaceutical compounds the LODs were 0.20
μg·mL–1 (naproxen) and 0.89 μg·mL–1 (ibuprofen) and
LOQs were 0.65 μg·mL–1 (naproxen) 2.97 μg·mL–1 (ibu-
profen), respectively.
2.5.4. Specificity
The specificity was evaluated for interference at the re-
tention times of E2, EE2, naproxen and ibuprofen. Lack
of interfering peaks in the spiked water sample with all
compounds at the retention times of the four compounds
was taken as an indication of the specificity of the meth-
ods (Figure 1).
2.6. Application of Method
The analytical method was applied to evaluate the effi-
ciency of degradation of E2 by using ozone. The tests
were carried out in batch reactors adapted with 100 mL
flasks, in analyte concentrations prepared in ultrapure
water of 0.7 μM with ozone doses of 1.3 μM from stock
solutions. The assays were carried out in triplicate at pH
= 6.0 and temperature (21˚C) [23]. The ozone was gener-
ated by a Pacific Ozone G11 ozone generator (Benicia,
California, USA) and ultrapure water saturation by bub-
bling into a Pyrex glass reactor of 2 L per batch at 20˚C
and pH = 6, obtaining stock solutions saturated with
ozone, 10 mg·L–1 (0.20 mM). Ozone concentrations and
residual ozone were measured using the indigo colori-
metric method [24]. One aliquot from stock solution (500
μL) was taken and added to the flask that contained the
E2; later, the flask was vigorously agitated during 3 s;
after one minute of reaction, the residual ozone was zero.
3. Results and Discussion
3.1. Elution Conditions
Initially, it was found that the compounds were eluted
efficiently from the stationary solid phase (0.5 g of C18)
with recoveries of 90% for E2 and 82% for EE2 when 10
mL of ACN were used; and 94% of naproxen and 85%
of ibuprofen with 3 mL of ethyl acetate. Those results
were obtained using standard solutions that contained 50
μg of each one of the steroids, and 8.3 μg and 83.3 μg of
naproxen and ibuprofen, respectively.
3.2. Effect of pH on Extraction Procedure
The results showed that the recoveries of pharmaceuti-
cal compounds increased with a reduction in pH; with
pH equal to 7.0 the recoveries for naproxen and ibu-
profen were 15% and 24%, respectively. While at a pH
equal to 4.5 the recoveries for both compounds in-
creased until 59% (naproxen) and 58% (ibuprofen).
This increase in the efficiency is probably due the re-
ducing pH value providing the non-protonated form of
the pharmaceuticals drug, increasing its interaction with
the stationary phase. However, the phase (C18) with less
available silanols is stabilized when lower pH values
are used, due to the fact that it is less susceptible to
hydrolysis [21] as used here. So, the selective retaining
and desorption of the pharmaceuticals drugs were sig-
nificantly improved as has been reported in other re-
search works [15,22]. This modification was considered
in carrying out the rest of the experiments and in opti-
mizing the method of extraction by means of SPE for
those compounds.
3.3. Selection of Reduction Technique
The reduction of the eluted extract from the cartridges
was carried out in two ways; by means of a gentle stream
of chromatographic nitrogen and by using a rotary
evaporator to evaluate the effect of the reduction tech-
nique over the efficiency of recovery of the analytes.
Both techniques were tested with the same amount of
fortification mass (Table 1). The recovery was obtained
by comparing the response of the mass in the reduced
extract with respect to the standard reference. The results
showed that the recoveries of the steroids and pharma-
ceuticals increased when the rotary evaporator was used
(Table 1). In the evaporator, the higher recoveries were
Table 1. Efficiency of recovery with the two reduction tech-
niques of the extract.
Compounds% R (CV) with N2 %R (CV) with Rotary evaporator
aE2 72.6 (2.4) 89.5 (6.5)
bEE2 67.7 (0.1) 80.1 (4.3)
cIbuprofen23.9 (28.4) 94.0 (2.1)
dNaproxen15.1 (67.3) 93.0 (2.7)
Fortified samples used with a,b50 μg· L–1 , c250 μg·L–1 and d25 μg·L–1; %R:
percentage of recovery; CV: coefficient of variation (n = 2). N2: nitrogen.
likely due to the greater control over the conditions of
reduction. The lower recoveries, performed by means of
the gentle flow of chromatographic nitrogen gas, were
likely because a very fast flow was used which produced
a significant co-evaporation of compounds and solvents
[25], however further experiments should be performed
to demonstrate these results more reliably. Regardless,
the reduction using the rotary evaporator presents the
advantage of being faster and cheaper than the technique
using a gentle flow of nitrogen, because solvents with
high vapor pressure, such as acetonitrile and ethyl acetate,
require longer times for evaporating, which increases the
cost due to the use of larger volumes of nitrogen chro-
matographic gas.
3.4. Assessment of Optimized Conditions
3.4.1. Linearity
The linearity of the proposed spiked range was also
evaluated plotting the observed response or absorbance,
after the treatment of the sample, against that of the dif-
ferent added mass. The correlation coefficients (r) were
greater than 0.993 for all the analyzed compounds (P <
0.05) (Table 2).
3.4.2. Recoveries
The optimized method of extraction in the solid phase
was applied to spiked water samples with different con-
centrations of steroids and pharmaceuticals in order to
know the behavior of recovery regarding the amount of
analyte present in the sample. Each spiked level was as-
sayed in duplicate and the recovery was calculated by
comparing the response in the tested sample with the
dissolution of the reference standard. First, it was dem-
onstrated that the extraction efficiencies (Table 2) were
independent of the loaded mass of analyte (ANOVA test
P > 0.05). Therefore, a recovery factor >83% for steroids
and >94% for pharmaceuticals would be applied for ad-
justing the concentration found in real samples within the
evaluated range. The recoveries of the steroids are lower
than those reported by López de Alda and Barceló [14],
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while for pharmaceuticals, they are in accordance with
the average recoveries reported by Santos et al. [15]
(98% naproxen and 89% ibuprofen). Both are satisfacy
since the variations introduced by each one of the stages
are considered during the treatment of the sample.
3.4.3. Precision
This parameter for quality of the method was represented
by the coefficient of variation (CV) and it was evaluated
as repeatability for each level tested and as reproducibil-
ity throughout the levels (n = 6 and 8). For steroids the
reproducibility obtained was 3.8% (E2) and 3.3% (EE2),
while for the pharmaceuticals the values were 5.2%
(ibuprofen) and 3.2% (naproxen) (Table 2).
3.4.4. Limits of Detection and Quantification of
Limits of detection (LOD) and quantification (LOQ) of
method were determined in accordance with Mitra [28]
and considering each step of samples preparation (ex-
traction, elution, reduction). The LODs for steroids were
1.24 μg· L–1 (EE2) and 4.21 μg·L–1 (E2) and LOQS were
4.13 μg·L–1 (EE2) and 14.04 μg·L–1 (E2). Similarly, for
pharmaceutical compounds the LODs were 18.6 μg·L–1
(naproxen) and 1.48 μg·L–1 (ibuprofen) and LOQs were
62.7 μg·L–1 (naproxen) 4.93 μg·L–1 (ibuprofen), respec-
3.5. Degradation Efficiency of Steroid
The residual concentration was compared to the initial
one and it was used to determine the efficiency of deg-
radation of the E2. The results (Table 3) indicate that the
process degrades more than 98% of the steroid, which
are similar results to those reported by Huber et al. [23],
Deborde et al. [26], and Guedes-Maniero et al. [27].
Also, it was found that the determination of the residual
mass (6.72 to 17.8 μg) of the steroid in the treated sam-
ple (1 L) was favored by the application of the method
under optimal conditions, due fundamentally to the fact
that the extraction and concentration stages facilitated
obtaining the compound at levels that are above the limit
of detection method (1.24 μg·L–1), contrary to the case
where it would have been directly injected. Besides, the
high efficiency of the method of extraction and the cor-
rection of the final result by the recovery of the method
provide higher reliability for the determination, since in
lower recoveries the probability of identifying and quan-
tifying the compound could diminish especially if it is
found in lower concentrations and in environmental
samples, where there are more interferences.
Table 2. Extraction efficiency of steroids and pharmaceuticals at different fortification levels under optimized conditions and
linear regression obtained to estimate the recovery factors.
Steroids Pharmaceuticals
2 EE2 Ibuprofen Naproxen
(μg·L–1) %R (CV) %R (CV) (μg·L–1) %R (CV) (μg·L–1) %R (CV)
12.5 82.0 (4.1) 90.1 (4.6) 25 88.0 (2.4) 2.5 72.9 (5.2)
25 89.1 (0.8) 91.2 (1.0) 62.5 95.3 (12.1) 6.25 102.3 (4.5)
50 89.5 (6.5) 80.1 (4.3) 125 85.0 (3.4) 12.5 82.4 (1.0)
250 94.0 (2.7) 25 93.0 (2.1)
r 0.999 0.996 r 0.993 0.997
m ± sd 0.969 ± 0.010 0.832 ± 0.032 m ± sd 0.932 ± 0.042 0.938 ± 0.026
b ± sd –54.6 ± 7.7 22.2 ± 18.1 b –2.7 ± 4.5 –1.1 ± 1.4
%R: recovery percentage, CV: coefficient of variation (n=2), r: correlation coefficient, m: slope or recovery factor, sd: standard desviation, b:intercept.
Table 3. Results of the degradation of steroid E2 to evaluate the recovery applying the analytical method.
Experiment [E2]a, final (μg·L–1)b,c Degradation (%) CV (%) Total CV (%)
1 6.72 96.6 12.9 4.7
2 17.84 91.1 1.1
2] initial = 200 μg·L–1; bcorrected by recovery factor, 93%; and cconcentration factor, 10/1000.
Copyright © 2011 SciRes. AJAC
4. Conclusions
Finally, two efficient and reliable methods were opti-
mized and applied for the determination of two steroids
and two pharmaceuticals in water samples by SPE and
HPLC-DAD analysis. The recoveries obtained after
sample treatment were 83 % (EE2) and 97 % (E2) for the
steroids and 93 % (ibuprofen) and 94 % (naproxen) for
The estimated detection limits suggested that it is pos-
sible to treat similar concentrations to that of environ-
mental samples (μg·L–1), with recoveries that do not de-
pend on the amount of compounds into the mass levels
assayed. Therefore, the SPE procedure ensures that lower
concentrations of steroids and pharmaceuticals are de-
tected, which is important because it facilitates reaching
the detection levels provided by the HPLC-DAD. These
methods are presented as an accessible alternative, sim-
ple and economic, since the cost of reactants and materi-
als is lower than those of GCMS techniques and the
availability of the chemical analysis systems is actually
possible in most of the research laboratories. Addition-
ally, the applicability of the analytical methods for the
determination of residual mass of steroids after a degra-
dation treatment with ozone was demonstrated.
5. Acknowledgements
The authors would like to thank CONACYT (Consejo
Nacional de Ciencia y Tecnología) for the financial aid
granted during this research (Project: CONACYT-
CB-84425). Thanks also to Sandra Bravo (CIATEJ) for
the technical support.
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