Direct competitive enzyme-linked immunosorbent assay for the determination of the highly polar short-chain sulfophenyl carboxylates

Article abstract of DOI:10.1021/ac0502910

A direct enzyme-linked immunosorbent assay for the detection of the short-chain sulfophenylcarboxylic acids (SPCs), the main metabolites of the linear alkylbenzene-sulfonates, is reported. Six SPCs (2C₃, 2C ₄, 3C₄, 2C

Full text of DOI:10.1021/ac0502910

Anal. Chem. 2005, 77, 5283-5293
Direct Competitive Enzyme-Linked Immunosorbent
Assay for the Determination of the Highly Polar
Short-Chain Sulfophenyl Carboxylates
M.-Carmen Este´ vez, Roger Galve, Francisco Sa´ nchez-Baeza, and M.-Pilar Marco*
Applied Molecular Receptors Group (AMRg), Department of Biological Organic Chemistry, IIQAB-CSIC,
Jorge Girona, 18-26, 08034-Barcelona, Spain
Europe in 20002) and their broad use lead to important discharges
from industrial and domestic wastewater sites. Although an
efficient removal can take place in wastewater treatment plants
(close to 99% in some cases3), their residues are often detected
in surface waters, seawater, and sludge.1,4-6 LAS are considered
biodegradable products due to their short half-life.7 The limiting
step in the biodegradation pathway of LAS is the first oxidation
of the terminal methyl group of the alkyl chain (ω-oxidation) to
give long-chain sulfophenylcarboxylic acids (SPCs). Oxidations
of both terminal methyl groups give sulfophenyldicarboxylic acids
that have also been detected in the environment,8-10 although they
are much less common. The following consecutive â-oxidations
of the alkyl chain occurs much faster, leading to different short-
chain SPCs11,12 although less common processes of R-oxidation
have been also detected.13 Subsequent splitting of the aromatic
ring and loss of the sulfonate group occurs much slower and,
according to some authors,11 this would begin on SPCs with alkyl
chains of four to five atoms. Considering the complexity of the
LAS mixture and the above considerations, it can be assumed that
complex mixtures of positional isomers of SPCs with different
lengths of short alkyl chains are the final and more persistent
metabolites of LAS.
A direct enzyme-linked immunosorbent assay for the
detection of the short-chain sulfophenylcarboxylic acids
(SPCs), the main metabolites of the linear alkylbenzene-
sulfonates, is reported. Six SPCs (2C3, 2C4, 3C4, 2C5,
3C5, 3C6), differing in the length of the alkyl chain
(between C3 and C6) and in the position of the phenylsul-
fonic group versus the carboxylic group, have been
synthesized. Antibodies have been raised against a mix-
ture of the corresponding horseshoe crab hemocyanin
conjugates prepared by coupling the carboxylic acid to the
lysine amino acid residues. The immunoassay As115/
3C4-HRP achieves an IC50 value of 23 nM (6.67 µg L-1
)
and a detection limit of 0.85 nM (0.24 µg L-1), using as
standard analyte an equimolar mixture of the six SPCs.
The immunoassay has found to work better in media with
low or moderate ionic strength (4-30 mS cm-1). The
decrease in the detectability produced by the potential
formation of SPC salts with divalent cations such as Ca2+
can be prevented by lowering the pH of the assay medium
below the pKa value of the SPC carboxylic group and using
a buffer chelating with properties such as citrate buffer.
The assay can be considered specific for short-chain SPCs
since congeners with longer alkyl chains and other pol-
lutants containing sulfonic groups in their structure do
not interfere significantly in the assay. Preliminary experi-
ments addressed to evaluate the potential application of
this assay to environmental water samples demonstrate
the usefulness of the assay.
Whereas LAS can be found in aquatic emplacements and also
adsorbed in particulate matter and sediments, SPCs present a
higher polarity and are mainly found in solution. Thus, residues
(2) European Committee of Surfactants and their Organic Intermediates
(CESIO) 2001 CESIO News
5 http://www.cefic.be/files/Publications/
cesio•5.pdf.
(3) Trehy, M. L.; Gledhill, W. E.; Mieure, J. P.; Adamove, J. E.; Nielsen, A. M.;
Perkins, H. O.; Eckhoff, W. S. Environ. Toxicol. Chem. 1996, 15, 233-240.
(4) Matthijs, E.; Holt, M. S.; Kiewiet, A.; Rijs, G. B. J. Environ. Toxicol. Chem.
1999, 18, 2634-2644.
The use of surfactants has been widespread owing to their easy
and fast biodegradation substituting natural soaps. Linear alkyl-
benzenesulfonates (LAS) are anionic surfactants representing 40%
(5) Eichhorn, P.; Flavier, M. E.; Paje, M. L.; Knepper, T. P. Sci. Total Environ.
2001, 269, 75-85.
(6) Eichhorn, P.; Rodrigues, S. V.; Baumann, W.; Knepper, T. P. Sci. Total
Environ. 2002, 284, 123-134.
1
of the total surfactants consumed as active agents of detergent
formulations. The technical LAS consist of a complex mixture of
alkylbenzenesulfonates resulting from the combination of linear
alkyl chains of different size (usually ranging between 10 and 14
carbon atoms) and a varying number of positional isomers
differing in the location of the aromatic ring (see Figure 1). Their
high production volume (i.e., 434 × 103 metric tons in western
(7) Perales, J. A.; Manzano, M. A.; Sales, D.; Quiroga, J. M. Bull. Environ.
Contam. Toxicol. 1999, 63, 94-100.
(8) Di Corcia, A.; Capuani, L.; Casassa, F.; Marcomini, A.; Samperi, R. Environ.
Sci. Technol. 1999, 33, 4119-4125.
(9) Di Corcia, A.; Crescenzi, C.; Marcomini, A.; Samperi, R. Environ. Sci. Technol.
1999, 33, 4112-4118.
(10) Dong, W.; Eichhorn, P.; Radajewski, S.; Schleheck, D.; Denger, K.; Knepper,
T. P.; Murrell, J. C.; Cook, A. M. J. Appl. Microbiol. 2004, 96, 630-640.
(11) Scho¨berl, P. Tenside, Surfactants, Deterg. 1989, 26, 86-94.
(12) Divo, C.; Cardini, G. Tenside, Surfactants, Deterg. 1980, 17, 30-36.
(13) Leon, V. M.; Gomez-Parra, A.; Gonzalez-Mazo, E. Environ. Sci. Technol.
2004, 38, 2359-2367.
* To whom correspondence should be addressed. Phone: 93 4006171. Fax:
93 2045904. E-mail: mpmqob@iiqab.csic.es.
(1) Scott, M. J.; Jones, M. N. Biochim. Biophys. Acta 2000, 1508, 235-251.
10.1021/ac0502910 CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/15/2005
Analytical Chemistry, Vol. 77, No. 16, August 15, 2005 5283

Figure 1. LAS biodegradation pathway leading to the formation SPCs. Further degradation of the phenylsulfonic group occurs at lower rate.11,13,24
Attending to the composition of the LAS technical mixture regarding positional isomers, six short-chain SPC compounds have been proposed
as representatives of the final products.
have been detected in seawater,13 in groundwater,14 and in
drinking and surface waters6,15,16 usually at a low-microgram per
liter level although they can reach higher levels. The high levels
achieved in some areas, usually associated also with high levels
of LAS, are clear indications of deficiently treated or untreated
waters. The SPCs congeners found in higher concentrations are
those with a medium alkyl length (C7-C9),5,6,10,17 but the shortest
ones (eC5) have also been detected.3,17,18 SPCs can also be found
in aquatic organisms exposed to LAS.19,20 The widespread use of
LAS and their short half-life determine a continuous accumulation
of short-chain SPCs in the environment. The toxicity of SPCs is
not yet clear, but it seems to be lower than their parent compounds
and no evidence of estrogenic effects has been demonstrated.21
SPCs are basically analyzed by LC-MS,5,6,15,16 and less fre-
quently by GC/MS,3,18,22 after derivatization. The high polarity and
the complexity of the SPC mixture together with the lack of the
corresponding standards have become major drawbacks for their
analysis. Moreover, the whole analytic procedure involves extrac-
tion/purification steps where an important amount of the short-
chain SPC fraction can be lost due to their high polarity.15,16 These
aspects may have determined that usually higher concentration
values reported for the long-chain SPCs (usually between 7 and
11 atoms of carbon) while occurrence of short-chain SPCs (e5
µg L-1) is seldom reported. It also questions the fact that several
authors claim that SPCs with alkyl chains between four and eight
carbons could be the more persistent14 or the “key intermediates”
of LAS degradation.23,24
As an alternative, immunochemical techniques, running in
aqueous media and usually achieving sufficient detectability to
allow direct measurements of water samples, can be excellent
screening analytical methods for highly water soluble anions such
as SPCs. However, production of antibodies against an unknown
mixture of substances is challenging and uncertain. With this
objective, we have synthesized six positional and constitutional
isomers of SPCs possessing alkyl chain lengths between three
and six carbon atoms and used them as mixture to raise antibodies
and establish an enzyme-linked immunosorbent assay. To our
knowledge, this is the first time that an immunochemical method
for the analysis of short-chain SPCs is reported. Moreover, the
procedures to prepare short-chain SPC, described here also for
the first time, can be extremely useful to understand fate and
degradation of LAS.
EXPERIMENTAL SECTION
(A) Chemistry. Chemicals and Instruments. Thin-layer
chromatography (TLC) was performed on 0.25-mm precoated
silica gel 60 F254 aluminum sheets (Merck, Darmstadt, Germany).
Unless otherwise indicated, purification of the reaction mixtures
was accomplished by “flash” chromatography using silica gel as
1
stationary phase. H and 13C NMR spectra were obtained with a
Varian Unity-300 spectrometer (300 MHz for 1H and 75 MHz for
1
13C) or a Varian Inova 500 spectrometer (500 MHz for H and
125 MHz for 13C) (Varian Inc., Palo Alto, CA). Infrared (IR) spectra
were measured on a Bomen MB 120 FT-IR spectrophotometer
(Hartmann & Braun, Que´bec, Canada). Gas chromatography/
mass spectrometry (GC/MS) was performed on an MD-800
capillary gas chromatograph with an MS quadrupole detector
(Fisons Instruments, VG, Manchester, U.K.), and the data are
reported as m/z (relative intensity). The ion source temperature
was set at 200 °C, a 15 m × 0.25 mm i.d × 0.15 µm (film thickness)
DB-225 fused capillary column (J&W, Folsom, CA) was used; He
was the carrier gas employed at 1 mL min-1.The injector
temperature was set at 250 °C. Exact mass values of the
compounds was determined by the service of the Unity of Mass
(14) Field, J. A.; Leenheer, J. A.; Thorn, K. A.; Barber, L. B., II.; Rostad, C.;
Macalady, D. L.; Daniel, S. R. J. Contam. Hydrol. 1992, 9, 55-78.
(15) Gonzalez-Mazo, E.; Honing, M.; Barcelo, D.; Gomez-Parra, A. Environ. Sci.
Technol. 1997, 31, 504-510.
(16) Eichhorn, P.; Knepper, T. P.; Ventura, F.; Diaz, A. Water Res. 2002, 36,
2179-2186.
(17) Leon, V. M.; Saez, M.; Gonzalez-Mazo, E.; Gomez-Parra, A. Sci. Total
Environ. 2002, 288, 215-226.
(18) Ding, W.-H.; Tzing, S.-H.; Lo, J.-H. Chemosphere 1999, 38, 2597-2606.
(19) Tolls, J.; Haller, M.; Sijm, D. T. H. M. Anal. Chem. 1999, 71, 5242-5247.
(20) Tolls, J.; Haller, M.; Seinen, W.; Sijm, D. T. H. M. Environ. Sci. Technol.
2000, 34, 304-310.
(21) Navas, J. M.; Gonzalez-Mazo, E.; Wenzel, A.; Gomez-Parra, A.; Segner, H.
Mar. Pollut. Bull. 1999, 38, 880-884.
(23) Taylor, P. W.; Nickless, G. J. Chromatogr. 1979, 178, 259-269.
(22) Ding, W.-H.; Lo, J.-H.; Tzing, S.-H. J. Chromatogr., A 1998, 818, 270-279.
(24) Knepper, T. P.; Kruse, M. Tenside, Surfactants, Deterg. 2000, 37, 41-47.
5284 Analytical Chemistry, Vol. 77, No. 16, August 15, 2005

Figure 2. Schemes of the synthetic pathways followed for the preparation of the six short-chain R- and â-SPCs (2C3, 2C4, 2C5, 3C4, 3C5, 3C6)
selected as target analytes and of a ω-SPC (5C5).
Spectrometry of the Universidad de Santiago de Compostela
(Spain) by electrospray ionization mass spectrometry with a time-
of-flight analyzer (ESI-MS-TOF) using a Bruker Biotof II spec-
trometer. All the chemical reagents used for the synthesis of SPCs
were purchased from Aldrich Chemical Co. (Milwaukee, WI).
Following the usual nomenclature in the field, SPC are named as
XCY-SPC, where X is the position where the phenylsulfonic group
is located and Y indicates the length of the alkyl chain.
Synthesis of SPCs. Seven different short-chain SPCs (2C3,
2C4, 2C5, 3C4, 3C5, 3C6, 5C5) were synthesized by sulfonation
of the aromatic ring of the corresponding phenylcarboxylic acids
2 and 5-10 (see Figure 2). The acids 7-10 were obtained from
commercial sources. The acids 2, 5, and 6 were prepared as
described below. All spectroscopic and spectrometric data ob-
tained in the characterization of the haptens and the intermediates
are available as Supporting Information. The long linear 9C9-SPC
used as competitor in the development of the enzyme-linked
immunosorbent assay (ELISA) was previously synthesized.25
2-Phenylpentanoic 2. A mixture of phenylacetic acid (5 g,
36 mmol) in MeOH (70 mL) and few drops of concentrated H2-
SO4 were stirred at room temperature for 2 h until the total
disappearance of the starting reagent by TLC. The solvent was
evaporated, and the resulting crude was redissolved in Et2O and
washed with saturated NaHCO3 and saturated NaCl. The organic
phase was dried with MgSO4, filtered, and evaporated to obtain a
yellow oil corresponding to the methyl phenylacetate ester 1 (5.07
g, 92% yield) (see Supporting Information). NaH 95% (760 mg, 28
mmol). The ester 1 (4 g, 27 mmol) in anhydrous THF (10 mL)
was added to a suspension of NaH in anhydrous THF (15 mL)
placed in a dry round-bottom flask equipped with a refrigerant
and under argon atmosphere. Previously, the suspension had been
exhaustively washed with anhydrous pentane (3 × 15 mL).
Recently distilled 1-iodopropane was then added dropwise (2.9 g,
29.3 mmol), and the reaction mixture was heated to reflux for 15
h until the total disappearance of the starting material by TLC.
The mixture was slowly added to a solution of 1 N HCl and
extracted with Et2O. The organic phase was dried with MgSO4,
filtered, and evaporated to obtain a brown oil that was purified by
flash chromatography using a hexane/Et2O gradient as mobile
phase to finally isolate 3.6 g of the methyl 2-phenylpentanoate ester
(1a) (70% yield) (see Supporting Information). The methyl ester
(3 g, 15.6 mmol) obtained was hydrolyzed in MeOH (40 mL) with
1M KOH (31 mL) for 2h until no more ester was observed on
the TLC. The MeOH was then evaporated and the residue was
redissolved in 1M KOH. The aqueous solution was washed with
hexane, acidified with concentrated HCl to pH 1 and extracted
with Et2O. The organic phase was dried with MgSO4, filtered and
evaporated to dryness to finally obtain 2.41 g of the desired
26
product 2-phenylpentanoic 2 (86.7% yield) (see Supporting
Information).
(25) Ramon, J.; Galve, R.; Sanchez-Baeza, F.; Marco, M.-P. Submitted to Anal.
Chem.
Analytical Chemistry, Vol. 77, No. 16, August 15, 2005 5285

3-Phenylpentanoic Acid 5. Methyl diethylphosphonoacetate
(6.85 mL, 37 mmol) was added to a stirred suspension of 95%
NaH (940 mg, 37, mmol) in THF (40 mL) placed in a round-bottom
flask equipped with a refrigerant and under argon atmosphere.
The NaH had previously been washed with anhydrous pentane
(3 × 15 mL) and dried. Once the suspension was totally dissolved
and no more hydrogen formation was observed, propiophenone
(4.85 mL, 37 mmol) was added dropwise and the reaction was
heated to reflux for 5 h until no evolution was observed by TLC.
The mixture of the reaction was washed with saturated NaHCO3
and extracted with Et2O. The resultant organic phase was dried
with MgSO4, filtered, and evaporated to dryness to give a yellow
residue (6.7 g) consisting of a mixture of the propiophenone,
diethyl phosphate, and the Z/E isomers of the 3-phenyl-2-pentenoic
acid methyl ester. Hydrolysis of the ester was performed with 1
N KOH (65 mL) in MeOH (75 mL) at reflux for 20 h according to
TLC analysis. The resulting acids were extracted as described
above to obtain the desired Z/E 3-phenyl-2-pentenoic acid 3 (4.8
g, 67% yield) (see Supporting Information). Palladium on activated
carbon (5%, 1.36 g of Pd/C 10%) was added to a solution of 3 (4
g, 22.8 mmol) in absolute EtOH (115 mL). The suspension was
purged several times with vacuum/H2 cycles to remove the O2
present in the media and finally was kept under H2 at atmospheric
pressure. The reaction was stirred at room temperature overnight,
spectroscopic data of each SPC and the reaction yields are detailed
as Supporting Information.
(B) Immunochemistry. General Methods and Instru-
ments. The MALDI-TOF-MS used for analyzing the protein
conjugates was a Perspective BioSpectrometry Workstation pro-
vided with the software Voyager-DE-RP (version 4.03) developed
by Perspective Biosystems Inc. (Framingham, MA) and Grams/
386 (for Microsoft Windows, version 3.04, level III) developed by
Galactic Industries Corp. (Salem, NH). The pH and the conductiv-
ity of all buffers and solutions were measured with a pH meter
pH 540 GLP and a conductimeter LF 340, respectively (WTW,
Weilheim, Germany). Polystyrene microtiter plates were pur-
chased from Nunc (Maxisorp, Roskilde, Denmark). Washing steps
were performed on a SLY96 PW microplate washer (SLT Labin-
struments GmbH, Salzburg, Austria). Absorbances were read on
a SpectramaxPlus (Molecular Devices, Sunnyvale, CA). The
competitive curves were analyzed with a four-parameter logistic
equation using the software SoftmaxPro v2.6 (Molecular Devices)
and GraphPad Prism (GraphPad Sofware Inc., San Diego, CA).
Unless otherwise indicated, data presented correspond to the
average of at least two well replicates.
Chemicals and Immunochemicals. Proteins such as horse-
shoe crab hemocyanin (HCH), bovine serum albumin (BSA), and
horseradish peroxidase (HRP) were obtained from Sigma Chemi-
cal Co. (St. Louis, MO). The specific immunoreagents (antibodies
and protein and enzyme conjugates) were prepared as described
below. Standards of SPCs and the phenylcarboxylic acids used in
the cross-reactivity studies were synthesized as described before.
LAS standards were a gift from Petresa (Ca´diz, Spain). The other
sulfonated compounds were from Aldrich Chemical Co., Fluka
(Buchs, Switzerland), and Merck.
Buffers. Phosphate-buffered saline (PBS) is 0.01 M phosphate
buffer and 0.8% saline solution, pH 7.5. PBST is PBS with 0.05%
Tween 20. PBST (0.2T) is PBS with 0.2% Tween 20. Citrate (0.2T)
is a solution of 0.04 M sodium citrate with 0.2% Tween 20 at pH
4.5. Borate buffer is 0.2 M boric acid-sodium borate, pH 8.7.
Coating buffer is 0.05 M carbonate/bicarbonate buffer, pH 9.6.
Citrate buffer is a 0.04 M solution of sodium citrate, pH 5.5. The
substrate solution contains 0.01% 3,3′,5,5′-tetramethylbenzidine and
0.004% H2O2 in citrate buffer.
Preparation of the Immunogens and the BSA Homolo-
gous Antigens. Mixed Anhydride (MA) Method. The conjuga-
tion of 2C3-, 2C4-, 2C5-, 3C4-, 3C5-, and 3C6-SPC through their
carboxylic group to lysine residues of HCH and BSA was carried
out by the MA method following similar conditions previously
described28 by reacting tri-n-butylamine (23 µL, 96 µmol) and
isobutyl chloroformate (11.4 µL, 88 µmol) with a stirred solution
of the SPC (80 µmol) in anhydrous dimethylformamide (DMF,
400 µL) cooled to 4 °C in an ice bath. The solution was split in
two parts, reacted with HCH (20 mg) and BSA (20 mg) in 1.8 mL
of borate buffer, and left under stirring for 4 h at room temper-
ature. After purification of the conjugate by dyalisis, stock solutions
of 1 mg mL-1 were prepared in PBS buffer and stored in aliquots
at -40 °C. Working aliquots were stored at 4 °C in 10 mM PBS
at 1 mg mL-1. Hapten densities of the BSA conjugates were
determined by MALDI-TOF-MS by comparing the molecular
1
until the disappearance of the starting reagent followed by H
NMR. The catalyst was removed by filtration and the EtOH was
evaporated to dryness to obtain a solid corresponding to the
desired product 3-phenylpentanoic acid 5 (3.76 g, 93% yield) (see
Supporting Information).
3-Phenylhexanoic Acid 6. As described above for the
phenylcarboxylic acid 5, butyrophenone (4.9 mL, 34 mmol) was
reacted with methyl diethylphosphonoacetate (6.24 mL, 34 mmol)
followed by the hydrolysis of the ester to obtain a mixture of Z/E
isomers of 3-phenyl-2-hexenoic acid 4 (4.41 g, 64% yield) (see
Supporting Information). The double bond of 4 (3.5 g, 18.5 mmol)
was reduced using H2 with Pd as catalyst as described before to
obtain the desired product 3-phenylhexanoic acid 6 (3.46 g, 98%
yield) (see Supporting Information).
2C3, 2C4, 2C5, 3C4, 3C5, 3C6 and 5C5-SPC. Sulfonation
of the phenyl carboxylic acids 2 and 5-10 was performed
following a similar procedure as described by Sarrazin et al.27 The
corresponding phenylcarboxylic acids (6 mmol, 1 equiv) were
added to concentrated H2SO4 (2 mL, 36.7 mmol, 6.2 equiv) placed
in a round-bottom flask equipped with a refrigerant and previously
heated at 100 °C. The reaction mixture was stirred for 2 h and
then slowly poured into H2O (80 mL). The aqueous solution was
washed with Et2O (3 × 30 mL) and then neutralized with CaCO3
(6 g, 60 mmol). The solid formed was removed by filtration, and
the aqueous solution was evaporated under reduced pressure to
dryness to finally obtain the desired SPC as calcium salt ac-
companied by a certain amount of inorganic salts. Further
purification attempts by crystallization in different solvent mixtures
were unsuccessful, for which reason the purity was determined
1
by quantitative H NMR using p-cresol as internal standard. The
(26) Kinbara, K.; Kobayashi, Y.; Saigo, K. J. Chem. Soc., Perkin Trans. 2 1998,
8, 1767-1776.
(27) Sarrazin, L.; Arnoux, A.; Rebouillon, P. J. Chromatogr., A 1997, 760, 285-
(28) Ballesteros, B.; Barcelo´, D.; Camps, F.; Marco, M.-P. Anal. Chim. Acta 1997,
347, 139-147.
291.
5286 Analytical Chemistry, Vol. 77, No. 16, August 15, 2005

weight of the standard BSA and that of the conjugates.
maximal absorbance, B is the minimum absorbance, C is the
concentration producing 50% of the maximal absorbance, and D
is the slope at the inflection point of the sigmoid curve. Some
physicochemical data such as pH, conductivity, and the amount
of detergent of the media and also time-related parameters
affecting the final features of the assay were evaluated in the
chosen combination.
Preparation of the Enzyme Tracers (ET). Active Ester
Method. Haptens 2C3-, 2C4-, 2C5-, 3C4-, 3C5-, 3C6-, 5C5-, and
9C9-SPC were coupled to the lysine amino acid residues of HRP
through their carboxylic group using the active ester method as
previously described29 by reacting the haptens (10 µmol) dissolved
in anhydrous DMF (140 µL) with dicyclohexylcarbodiimide (50
µmol in 30 µL of DMF) and N-hydroxysuccinimide (25 µmol in
30 µL of DMF) and adding the active ester to a solution of HRP
(2 mg) in borate buffer (1.8 mL).
Hapten Density Analysis. Hapten densities of the BSA
conjugates were calculated by MALDI-TOF-MS by comparing the
molecular weights of the native proteins to that of the conjugates.
MALDI spectra were obtained by mixing 2 µL of the freshly
prepared matrix (trans-3,5-dimethoxy-4-hydroxycinnamic acid, 10
mg mL-1 in CH3CN/H2O 70:30, 0.1% TFA) with 2 µL of a solution
of the conjugates or proteins (5 mg mL-1 in MilliQ water). The
hapten density (δ hapten) was calculated according to the
following equation: {MW(conjugate) - MW(protein)}/MW-
(hapten).
Polyclonal Antisera. Three female New Zealand white rabbits
(115, 116, 117), weighting 1-2 kg were immunized with an
equimolar mixture of the six immunogens prepared (2C3, 2C4,
2C5, 3C4, 3C5 and 3C6 coupled to HCH, SPCMIX-HCH) following
the immunizing protocol described elsewhere.28 The evolution of
the antibody titer was assessed by measuring the binding of serial
dilutions of the antisera to a microtiter plate also coated with an
equimolar mixture of the BSA conjugates (SPCMIX-BSA). After
an acceptable antibody titer was observed, the animals were
exsanguinated and the blood was collected on Vacutainer tubes
provided with a serum separation gel. Antisera were obtained by
centrifugation and stored at -40 °C in the presence of 0.02% NaN3.
ELISA Development and Evaluation. Screening of the
Antisera (As) and Enzyme Tracers. The avidity of the antibodies
in the serum of each animal was determined using two-
dimensional checkerboard titration experiments by measuring the
binding of serial dilutions (1/500-1/32 000, 2 to 0.031 25 µg mL-1
in PBS) of the enzyme tracers 2C3-, 2C4-, 2C5-, 3C4-, 3C5-, 3C6-,
5C5-, and 9C9-HRP to microtiter plates coated with 12 different
dilutions of each sera (1/500-1/512 000 in coating buffer).
Optimal concentrations for enzyme tracer and antisera dilution
were chosen to produce absorbances around 0.7-1 units of
absorbance after 30 min at room temperature.
Optimized Direct ELISA. Microtiter plates were coated with
As115 in coating buffer (1/8000, 100 µL/well) covered with
adhesive plate sealers overnight at 4 °C. The following day, the
plates were washed (four times with PBST, 300 µL/well). An
equimolar mixture of the six SPCs (SPCMIX) was considered as
the standard analyte. The calibration curve (150 µM-2.56 pM
prepared in citrate (0.2T) was added to the plate (50 µL/well)
and was preincubated for 30 min. The enzyme tracer 3C4-HRP
(0.5 µg mL-1 in citrate (0.2T)) was then added to the plates (50
µL/well). After 30 min of competition, the plate was washed and
the plates were processed as described in the general protocol.
Cross-Reactivity Determinations. Stock solutions of each
SPC (6 mM in H2O or DMSO/MeOH) and several structurally
related compounds were prepared and stored at 4 °C. Standard
curves (150 µM-2.56 pM) were prepared in citrate (0.2T) by serial
dilution. Each IC50 was determined in the competitive experiments
following the optimized protocol described above. The cross-
reactivity (CR) values were calculated according to the equation
{IC50(SPCs)/IC50(cross-reactant)} × 100.
ELISA Matrix Effect Studies. Water samples from river, well,
and sea were collected from Aigu¨esTortes (Pyrinees), Barcelona,
and Pineda (Spain), respectively. Before measurements were
made, their conductivity and pH were measured and adjusted to
place the sample within the working conditions of the immunoas-
say by adding 5× citrate (0.2T). The matrixes were serially diluted
with citrate (0.2T) and used to prepare standard curves. The
parallelism of the sigmoidal curves was compared to that prepared
in the assay buffer in order to evaluate the extent of the
interferences caused by the matrix.
Accuracy Studies with Blind Spiked Samples. Different
blind spiked samples prepared in citrate buffer and in river and
well water samples were measured by the optimized ELISA at
different dilution factors (1/2, 1/4, and 1/8 in citrate 0.2T).
Analyses were done in duplicate. The correlation was evaluated
by establishing a linear regression between the spiked and the
measured values in the case of buffer samples.
General Direct ELISA Protocol. The plates were coated with
the As (100 µL/well in coating buffer) overnight at 4 °C covered
with adhesive plate sealers. The day after, the plates were washed
four times with PBST (300 µL/well) and the solutions of the
analyte (50 µL/well in PBST; only PBST for zero analyte) and
the ET (50 µL/well in PBST) were added and incubated for 30
min at room temperature. The plates were washed and the
substrate solution was added (100 µL/well) and incubated 30 min
protected from light at room temperature before the enzymatic
reaction was stopped by adding 4 N H2SO4 (50 µL/well). The
absorbances were read at 450 nm. The standard curve was fitted
to a four-parameter logistic equation according to the following
formula: Y ) {(A - B)/[1 - (C/x)D]} + B, where A is the
RESULTS AND DISCUSSION
The composition of LAS technical mixtures varies depending
on the manufacturing process. Usually, mixtures of alkylbenze-
nesulfonates with alkyl chains ranging between 10 and 14 carbon
units (i.e., Petresa S.A. C10, 12%; C11, 34%; C12, 30%; C13, 22%;
C14, 0.4%), each of them composed by positional isomers regard-
ing the position of the benzene ring, are obtained. The phenyl
group can be located randomly in any position with the exception
of the two terminal methyl groups, and the ratio of the different
positional isomers can also vary depending on the conditions and
reagents used in the production process.11 Attending to the
reported degradation pathway of these compounds,11 an initial
oxidation would usually occur at the more distal end of the longer
alkyl chain (in respect to the carbon atoms to the benzylic position,
even or odd number of carbon atoms) and the subsequent
(29) Gasco´n, J.; Oubin˜a, A.; Ballesteros, B.; Barcelo´, D.; Camps, F.; Marco, M.-
P.; Gonza´lez-Martinez, M.-A.; Morais, S.; Puchades, R.; Maquiera, A. Anal.
Chim. Acta 1997, 347, 149-162.
Analytical Chemistry, Vol. 77, No. 16, August 15, 2005 5287

â-oxidation steps would require two linear methylene groups.
Based on those assumptions, it has been proposed that a mixture
of short-chain SPC congeners having a total alkyl chain length
between three and six atoms of carbon (XC3-XC6) and possessing
the benzene ring in R (R-SPC) or â (â-SPC) position to the
phenylsulfonic group (2Cx and 3Cx, respectively) could be at the
end of this degradation sequence, before further breaking up the
aromatic ring and the sulfonate group. Therefore, 2C3-, 2C4-, 2C5-,
3C4-, 3C5-, and 3C6-SPCs have been considered as potential and
representative final degradation products of LAS (see Figure 1).
Determination of these short-chain SPCs by the usual analytical
methods is difficult due to their high polarity. In addition, the
unavailability of standards has also been one of the reasons
contributing to the lack of information regarding the presence of
these compounds in the environment. Thus, in this work, we first
focused on the synthetic preparation and structural characteriza-
tion of these analytes.
Table 1. Characteristics of the Best Immunoassays
Obtained in the Screening of As115, As116, and As117
with the Enzyme Tracersa
ET
IC50
IC50
As dilb ETc (µg mL-1) Amax Amin (nM) (µg L-1
)
slope
r2
115 1/12 2C3
1/16 2C4
1
0.719 0.112 758
1.016 0.026 632
1.162 0.004 754
1.328 0.001 205
1.383 0.165 738
1.410 0.059 1195
0.727 0.015 910
189
158
189
51
185
299
228
-0.83 0.9546
-0.73 0.9839
-0.64 0.9944
-0.52 0.9803
-1.67 0.8533
-0.96 0.9700
-0.794 0.9592
0.5
0.5
0.25
0.5
0.25
1
1/8 2C5
1/12 3C4
1/12 9C9
116 1/16 3C4
117 1/12 3C4
a Competitive assays using an equimolar mixture of SPCs as
standard analytes. The parameters were extracted from the four-
parameter equation used to fit the standard curves. Those assays with
IC50 values below ∼350 µg L-1 have been included. The concentration
(expressed in µg L-1) has been calculated by considering as molecular
weight the average of the six SPCs (MW ≈ 250). b Dilution factor of
the antiserum (As) (× 1000). c The enzyme tracers (ET) are HRP
conjugates.
Synthesis of Short-Chain SPCs. The six SPCs considered
were synthesized by sulfonation following described proce-
dures23,27,30 of the corresponding phenylcarboxylic acids following
the schemes presented in Figure 2. The preparation of 2C3-, 2C4-,
and 3C4-SPCs could easily be performed by direct sulfonation of
the corresponding commercially available phenylalkylcarboxylic
acids 7-9, using concentrated H2SO4 at 100 °C (see Figure 2,
scheme 3). Maintaining the temperature was crucial in order to
avoid polysubstituted byproducts and to favor the formation of
the para isomer. The isolation of the compound from the reaction
mixture was performed by preparing the corresponding Ca salts.27
The addition of CaCO3 produced the precipitation of CaSO4 salts
in aqueous media that were removed by filtration. 2C5-SPC was
obtained from phenylacetic acid, by alkylation of the benzylic
position of the methyl ester 1 with 1-iodopropane, followed by
hydrolysis of the ester and sulfonation of the aromatic ring (see
Figure 2, scheme 1). Finally, 3C5-SPC and 3C6-SPC were
prepared from propiophenon and butyrophenone, respectively,
through a Horner-Wadsworth-Emmons reaction using methyl
diethylphosphonoacetate leading to a mixture of the correspond-
ing Z/E isomers of the alkenes (3 and 4, Z/E ratio of 40:60
maximize recognition of the phenylsulfonate moiety. Each of the
short-chain SPCs (2C3, 2C4, 2C5, 3C4, 3C5, 3C6) was covalently
attached to lysine residues of the HCH by the mixed anhydride
method, and the conjugation reaction was verified by MALDI-
TOF-MS. Hapten densities between 20 and 25 were recorded for
the BSA conjugates, indicating a high degree of conjugation. The
immunogen consisted on an equimolar mixture of six SPC-HCH
conjugates (SPCMIX-HCH). Three rabbits where inoculated fol-
lowing immunization protocols established in our group. The
antibody titer of the serum of each animal was tested against
SPCMIX-BSA on an indirect ELISA monthly until no significant
increase of the titer was observed. The final antisera obtained from
each rabbit were named As115, As116, and As117.
The avidity of the antibodies for a battery of enzimatic tracers
(the short-chain SPCs, and two ω-SPCs 5C5- and 9C9-SPC coupled
to HRP conjugates) was evaluated by using noncompetitive direct
ELISAs. Hapten 5C5-SPC was prepared in one step by sulfonation
of 5-phenylpentanoic acid 10 (see Figure 2, scheme 3) following
the same procedure described before. The preparation of 9C9-
SPC is described elsewere.25 In general, As115 and As116 showed
broader recognition (all HRP tracers were recognized except
5C5-HRP) and higher antibody titers than As117 (only 2C3-,
2C4-, and 3C4-HRP tracers were recognized).
1
determined by H NMR) (see Figure 2, scheme 2). The double
bond was reduced with H2/Pd to obtain 5 and 6 and finally the
sulfonic group introduced using concentrated H2SO4 (see Figure
2, scheme 2).
Preparation of the Antibodies. Development of antibodies
with the ability to recognize several congeners of the same family
is challenging and risky. The strategy usually consists of using a
hapten that maximizes the common epitopes in the molecule. The
SPC structures possess two clearly different functional groups that
can be considered as strong antigenic determinants, the carboxylic
acid and the phenylsulfonate group. Under the physiological and
usual assay media, the sulfonate group will always exist in its
ionized form due to its extremely low pKa value. Previous
experience of our group25 already pointed at this last group as
one of the most important epitopes. Therefore, we took advantage
of the carboxylic group already present in the SPC molecules to
prepare the corresponding protein conjugates. The location of the
carboxylic group, further from the selected epitope, would
Competitive Direct ELISA. Those antisera/enzyme tracer
combinations showing acceptable titers were used to determine
the ability of the antibodies to recognize the analyte in competitive
assays. For this purpose, and keeping the idea of obtaining an
assay capable of recognizing not only one, but a broad family of
short alkyl chain SPCs, the mixture of the six SPCs used in the
immunization was also considered as a standard analyte by
preparing an equimolar mixture in MilliQ water. Following the
general protocol described in the Experimental Section, several
competitive assays were obtained, although in many cases the
IC50 values were higher than 500 µg L-1 (2000 nM), which were
considered not usable for environmental monitoring. Thus, Table
1 shows only the features for the competitive assays with IC50
below 350 µg L-1 (1400 nM). As it can be observed, As115 gave
the higher number of usable assays. In all cases, 3C4-HRP was
found to be the best enzyme tracer. At the light of the immu-
(30) Marcomini, A.; Di Corcia, A.; Samperi, R.; Capri, S. J. Chromatogr. 1993,
644, 59-71.
5288 Analytical Chemistry, Vol. 77, No. 16, August 15, 2005

Figure 4. Standard curves prepared using different buffer systems.
Except for the PBST at pH 7.5, the pH values of all the buffers were
adjusted to 4.5. Tween 20 was added to the solutions to have a total
content of detergent of 0.2%. â,â-DMG: â,â-dimethylglutaric acid.
Standard curves were prepared using two well replicates.
selected for further experiments. An improvement at higher
concentrations of detergent has also been observed in other
immunoassays for other high polar analytes such as the dealkyl-
ated hydroxytriazines.31 In contrast, this behavior is opposed to
the effect generally observed for ELISAs of nonpolar analytes,32,33
in which case the sensitivity improves at lower concentrations of
detergent. The increase of the maximum signal allowed diluting
further the immunoreagents (As115, 1/16 000 and 3C4-HRP,
0.125 µg mL-1). An increase of the ionic strength (from 4.3 to
115 mS cm-1, 2.5-100 mM in terms of PBS concentration and
MilliQ water, 0 mS cm-1) produced a clear negative effect on the
immunoassay detectability and in the maximum signal (see Figure
3B). In contrast, the assay performed very well in media with
conductivity values between 0 and 30 mS cm-1 although precision
was poorer when conductivity was at or below 4 mS cm-1
.
Attending to these results, further experiments were performed
in 10 mM PBS, which produces a conductivity of 16 mS cm-1
within the interval where the immunoassay parameters remain
constant. A broad degree of tolerance to media was found
regarding pH values of the assay buffer. The assay performed
very well within 4.5-10.5 units of pH, although the best detect-
ability was accomplished at acidic pH values. At pH 4.5, the
maximum absorbance of the assay was high and the IC50 was
much lower (see Figure 3C). Thus, whereas in PBST at pH 7.5
the IC50 was ∼145 nM (36 µg L-1), this value dropped down to 60
nM (15 µg L-1) setting the pH value at 4.5. Since this pH is outside
of the buffering interval of the PBS, other buffer systems, such
as citrate, acetate/acetic, â,â-dimethylglutaric acid/NaOH, and
succinic acid/NaOH, were evaluated, adjusting the pH at 4.5 and
maintaining the concentration of Tween 20 at 0.2%. The results
shown in Figure 4 demonstrate that, under these assay conditions,
only the citrate buffer and the PBS afforded acceptable calibration
curves. In fact, the detectability achieved using citrate buffer (pH
4.5, 0.2% Tween 20, conductivity 10 mS cm-1) was 23 nM (5.7 µg
Figure 3. Influence of several physicochemical parameters on the
SPCs immunoassay (As115/3C4-SPC-HRP) features. (A) Effect of
the concentration of Tween 20 in the competition buffer. (B) Effect of
the conductivity of the media. (C). Effect of the pH of the media. Left
axes show the maximum absorbance (Amax) and the quotient of the
Amax and the IC50 (Amax/IC50). The right axes show the values of IC50
expressed in µg L-1. The data shown have been extracted from the
four-parameter equation used to fit the standard curves. Standard
curves were prepared using two well replicates.
noassay parameters and of the reproducibility observed, immu-
noassay As115/3C4-HRP was chosen for further evaluation.
ELISA Evaluation. After evaluating different times (0-60
min), preincubating the analyte in the antibody-coated plate for
30 min, followed by a competitive step with the ET for 30 min,
was found to improve significantly performance of the assay in
terms of sensitivity without sacrificing the maximum signal of the
assay and without altering significantly other important features
such as the slope and the signal-to-noise (S/N) ratio (data not
shown). The concentration of detergent (Tween 20) produced a
clear influence on the assay parameters (see Figure 3A). Thus,
high concentrations of Tween 20 in the assay buffer diminished
the values of the IC50 (higher detectability) and increased the
maximum signal, whereas the slope and the signal-to-noise (S/
N) ratio remained unchanged. Thus, a concentration of 0.2% was
L
-1), significantly better than in PBS.
The significant improvement in the SPC recognition produced
after lowering the pH and changing the buffer system can be
explained by considering the type of immunogen used, the pKa
(31) Sanvicens, N.; Pichon, V.; Hennion, M. C.; Marco, M. P. J. Agric. Food Chem.
2003, 51, 156-164.
(32) Sanvicens, N.; Varela, B.; Marco, M.-P. J. Agric. Food Chem. 2003, 51, 3932-
3939.
(33) Manclus, J. J.; Montoya, A. J. Agric. Food Chem. 1996, 44, 4063-4070.
Analytical Chemistry, Vol. 77, No. 16, August 15, 2005 5289

Figure 5. Scheme showing the equilibria between the different SPC species potentially present in the aqueous media depending on the pH
(7.5 or 4.5) and on the buffer system used (PBS or citrate) and the effect of these on the IC50. Those conditions favoring (i.e, pH e pKaCOOH
,
chelating agents such as citrate) the presence of the SPCs as single free species can improve the immunoassay detectability.
Table 2. Working Conditions and Most Important
Features of the Optimized Immunoassay
As115/3C4-HRPa
As115 dilution
[3C4-HRP], µg mL-1
buffer
1/8000
0.5
citrate 40 mM, pH 4.5, 0.2% Tween
30 min
preincubation step
competition step
Amax
30 min
0.940 ( 0.132
0.055 ( 0.018
4.32 ( 1.65b
Amin
IC50, µg L-1
dynamic range, µg L-1
LOD, µg L-1
slope
0.48 ( 0.32 to 43 ( 14
0.15 ( 0.24
-0.619 ( 0.111
0.9793 ( 0.0115
R2
Figure 6. Standard curve for the SPCs immunoassay As115/3C4-
HRP obtained under the optimized conditions established in the
protocol described in the Experimental Section. The data presented
correspond to the average and standard deviation of 20 assays, run
on different days. The curves were prepared using two-well duplicates.
The features of the assay are summarized in Table 2.
a The parameters are extracted from the four-parameter equation
used to fit the standard curve. The data presented correspond to the
average and the standard deviation of 20 calibration curves run on
different days. Each curve was built using two-well replicates. The
concentrations (expressed as µg L-1) has been calculated by consider-
ing as molecular weight the average of the six SPCs (MW ≈ 250).
b Expressed in nM, the IC50 is 17.3 ( 6.6 and the LOD is 0.62 ( 0.49.
of a same molecule could also be formed, but seem more unlikely)
can be stabilized through the Ca2+ ions. A different situation might
be expected at pH 4.5, where the calcium cations could only form
dimers through the sulfonate groups (see Figure 5). The SPC
molecules are captured by the calcium ion more efficiently at pH
7.5 than at pH 4.5, hampered from interacting with the antibodies
as single free molecules, which could explain the improvement
of immunoassay detectability. The use of citrate buffer, with
recognized strong chelating properties for calcium ions, shifts the
equilibrium to the free form, allowing interaction with the antibody
and therefore improving even more the immunoassay detectability.
A final working protocol was established consisting of a
standard coating step of the antibodies on the microplate overnight
at 4 °C, a preincubation of the analyte with the antibody for 30
min, followed by 30 min of competition with the enzyme tracer.
The assay buffer used was 40 mM citrate buffer (0.2% Tween 20,
conductivity 10 mS cm-1, pH 4.5). These conditions as well as
the immunoassay features are summarized in Table 2. Figure 6
values, and the fact the SPC standards are calcium salts. In
general, antibody binding could be affected by the presence of
these ions in the media; however, for the particular case of the
SPCs, another explanation can be considered by contemplating
all these factors. According to the pKa values of the carboxylic
groups (∼4.5, for 3Cx SPCs and 4.15 for 2Cx SPCs), the
carboxylate is the predominant form of these compounds at
neutral pH, while at pH 4.5 the carboxylic acid form would be
more abundant. This neutralized form of the carboxylic group
mimics better the immunogen structure used to raise antibodies,
since the carboxylic group was blocked by the amide bonds
formed while coupling this molecule to the lysine amino acid
residues of the proteins. Moreover, as SPCs are calcium salts, at
pH 7.5, SPC symmetric (for both carboxylic or sulfonate groups
of two molecules) or asymmetric (with one carboxylic and one
sulfonate group) dimers (interactions with both anionic charges
5290 Analytical Chemistry, Vol. 77, No. 16, August 15, 2005

shows an example of the calibration curve obtained under these
conditions. A LOD of 0.82 nM (0.20 µg L-1, considering an average
molecular weight of 250) was reached, which is suitable to perform
studies of the levels of these products in real samples.
Table 3. Specificity of the Immunoassay As115/
3C4-HRP for Some Related Compoundsb
The specificity of the assay was first evaluated by regarding
individual recognition of the six SPCs used as immunogens (see
Table 3). Contrary to the expected similar recognition of the six
SPCs, the following sequence was observed: 3C6 > 3C5 . 2C5
≈ 3C4 > 2C4 > 2C3, which shows that the longer the alkyl chain
of the SPC, the better was its recognition, especially considering
the size of the alkyl group at the benzylic position. Moreover,
SPCs with the aromatic ring in the â position (3Cx) seem to be
better recognized that those with the phenyl group in R position.
(2Cx), which could be explained by a slightly greater steric
hindrance, caused by the protein with the distance to the
phenylsulfonic group smaller. Thus, except for 3C4-SPC, which
only cross-reacted 10% with respect to the equimolecular mixture
of the six SPCs, 3C5- and 3C6-SPCs, with the phenylsulfonic in
the â position were recognized at 198 and 84%. Thus, despite the
superior immunogenic properties of the phenylsulfonic group, the
antigenic value of the alkyl chain should not be underestimated.
The recognition decreased drastically for the less abundant
ω-SPCs, with the phenylsulfonic group in the terminal position.
Thus, 5C5-SPC was not recognized at all. The same is applicable
for long alkyl chain SPCs such as 9C9- and 12C12-SPCs (see Table
3). These data demonstrate that the ELISA developed can be
considered specific for short alkyl chain SPCs. The technical
mixture of LAS and p-ethylbenzenesulfonic acid were recognized
with cross-reactivity values around 6 and 5%, respectively; p-
toluensulfonic acid (p-TS) with only a methyl group in para
position was even less recognized. Other environmental pollutants
with sulfonic groups such as naphthalenesulfonates were tested,
and no significant recognition was observed in any case. Finally,
the corresponding alkylphenylcarboxylic acids, lacking the sul-
fonic group, were also evaluated but did not interfere in the assay,
indicating that the presence of the sulfonic group in the para
position is necessary for the recognition.
Immunoassay Matrix Effects. The application of this tech-
nique to measure real aqueous samples was preliminarily studied
regarding potential interferences caused by the different types of
water matrixes. For this purpose sea, river, and well water samples
were collected. The pH of these waters was in all the cases ∼7.5,
and the conductivity was very low for the river and well water
samples (0 and 2.1 mS cm-1, respectively) whereas the seawater
showed a higher value (55 mS cm-1). Standard curves were
prepared by spiking these samples with known concentrations of
the SPC standard mixture. As it can be observed in Figure 7, direct
analysis of those samples is not possible since a very different
behavior is observed when those curves were compared with the
ones prepared in the assay buffer. For the case of the seawater,
a diminution of the maximal absorbance and a decrease of the
assay detectability was observed. Buffering the samples by adding
20% 200 mM citrate buffer to adjust pH and conductivity readily
improved significantly immunoassay performance. The remarkable
effect of the buffer observed for the mountain spring clean river
water has been interpreted by a potential high content of calcium
or other bivalent cations in this water sample (sample was
collected from a karstic area). Further dilutions of these solutions
a 2C3, 2C4, 2C5, 3C4, 3C5, and 3C6 are the SPCs used as immunogens.
LAS, linear alkylbenzenesulfonates; p-TS, p-toluensulfonic acid; EBS, eth-
ylbenzenesulfonic acid; p-XS, p-xylenesulfonic acid; BDS, benzenedisulfonic
acid; 1-Napht, naphthalene-1-sulfonic acid; 2-Napht, naphthalene-1,5-disul-
fonic acid; 3-Napht, naphthalene-1,3-6-trisulfonic acid. b The percentage of
recognition has been expressed as cross-reactivity (CR%) according to the
expression [IC50(SPCs)/IC50(cross-reactant)] × 100.
Analytical Chemistry, Vol. 77, No. 16, August 15, 2005 5291

Figure 7. Matrix effect produced by sea, river, and well water samples on the SPC ELISA. The pH and the conductivity were adjusted to the
assay conditions by buffering with 200 mM citrate and maintaining the Tween 20 to a final concentration of 0.2%. Serial dilutions were done with
citrate (0.2T) buffer. Buffering the samples significantly improves immunoassay performance.
Table 4. Results from the Accuracy Studies
the number of environmental samples analyzed are being per-
formed in order to provide more objective data.
spiked concn (nM)
measd concn (nM)
% recovery
Buffer Samples
32.8 ( 2.7
30
60
120
300
109
115
122
78
CONCLUSIONS
69.5 ( 3.4
147.0 ( 18.7
233.6 ( 19.7
The analysis of highly polar substances such as the short-chain
(3-6 carbon units) SPCs by conventional techniques is trouble-
some due to the difficulties and low yields encountered during
the extraction and preconcentration steps. Moreover, there has
been a lack of SPC standards of these alkyl sizes and placement
of the phenylsulfonate group at the R and â positions in respect
of the carboxylic group. Consequently, environmental monitoring
of the shortest SPCs has been rarely reported. Immunochemical
techniques are not subject to these previous procedures since they
are performed in aqueous media and usually the detectability
achieved is sufficient to avoid preconcentration. For the first time,
we have reported here an immunochemical method for the rapid
detection and quantification of short-chain SPCs. Six congeners
(2C3-, 2C4-, 2C5-, 3C4-, 3C5-, and 3C6-SPC) have been synthe-
sized and used as standards and also prepared as an immunogen
that consists of an equimolar mixture of them. The direct ELISA
developed is specific for short-chain SPCs and can detect that
mixture with a limit of detection of 0.15 µg L-1 (0.6 nM). However,
due to the fact that not all SPC congeners synthesized have
identical immunogenic properties, these are not equally detected
by the antibodies raised. Thus, 3C6-SPC and 3C5-SPC are much
more recognized (198 and 84% cross-reactivity in respect to the
equimolar mixture) while 2C3-SPC is almost not detected (2%
cross-reactivity). Moreover, recognition is higher for the more
abundant short-chain R- and â-SPC, while the less probable
ω-SPCs are not recognized. The assay reported here is highly
tolerant to media with different pH values (pH 4-10), although
the best sensitivity levels are achieved in acidic media (pH 4.5).
It performs well in buffers with a moderate ionic strength (0-30
mS cm-1). It has been found that bivalent cations may interfere
with the assay and that this effect can be minimized by using a
buffer with chelating properties, such as the citrate buffer. Finally,
the preliminary matrix effect studies performed with natural water
samples indicates that, except for the seawater sample, small
dilution factors can be sufficient to avoid the interference.
Preliminary experiments that have been performed spiking natural
water samples indicate the potential of the method presented here
as a screening tool in environmental monitoring programs. Future
work will be directed to the validation of the assay with chro-
matographic methods.
Well Samples
7.72 ( 1.7
8
40
96
103
77
41.1 ( 1.7
200
153.6 ( 25.7
River Samples
1.85 ( 0.27
8.1 ( 0.9
1.6
8
40
115
101
109
43.6 ( 9.2
a The accuracy was evaluated by preparing blind spiked samples in
buffer and in natural water samples and measuring them with the
As115/3C4-HRP direct ELISA. Each value corresponds to the mean
of two replicates.
were carried out in 40 mM citrate (0.2T). For the case of the
seawater sample, it was necessary to further dilute more of the
sample with citrate (0.2T) in order to diminish the conductivity
of the media. However, after a 32 times dilution of the sample
(11.3 mS cm-1), the values of the IC50 (55 nM) were still slightly
higher than those obtained in buffer (20 nM), which indicates
the necessity to perform some type of sample treatment in order
to perform measurements without compromising the detectability
of the method. We must also consider the possibility that SPCs
from domestic effluents could be present in this sample since it
was collected close to an urban area. This fact has to be studied
in more detail on further experiments. In contrast, dilution of the
river and well water samples between 2 and 4 times with citrate
(0.2T) after buffering the samples was sufficient for direct analysis.
Immunoassay accuracy was preliminarily assessed by measur-
ing buffer and natural water samples spiked with the SPC mixture.
Results shown in Table 4 demonstrate that the analyses provided
good recovery values, between 96 and 115%, pointing to an
acceptable accuracy level. Only for those samples spiked at higher
concentration levels (>200 nM), the recoveries were lower but
still acceptable (>75%). This lower recovery could be related to a
higher number of dilution steps performed to place the samples
within the dynamic range of the ELISA; however, further experi-
ments have to be performed to prove this fact. More exhaustive
validation studies using chromatographic methods and increasing
5292 Analytical Chemistry, Vol. 77, No. 16, August 15, 2005

ACKNOWLEDGMENT
compounds described in the paper. This material is available free
of charge via the Internet at http://pubs.acs.org.
This work has been supported by the Ministry of Science and
Technology (Contract TEC2004-0121-E) and by the European
Community (IST-2003-508774). We thank PETRESA for providing
samples of the technical LAS.
Received for review February 17, 2005. Accepted June 16,
2005.
SUPPORTING INFORMATION AVAILABLE
Spectroscopic data (1H and 13C NMR, IR) and HRMS of the
AC0502910
Analytical Chemistry, Vol. 77, No. 16, August 15, 2005 5293