Hydroquinone-based derivatization reagents for the quantitation of amines using electrochemical detection

Article abstract of DOI:10.1021/ac981236c

Two new reagents, NDTE (2,5-dihydroxyphenylacetic acid, 2,5-bis-tetrahydropyranyl etherp-nitrophenyl ester) and HLTE (homogentisic γ-lactone tetrahydropyranyl ether), are described for the chemical derivatization of primary and/or secondary amines to form an electrochemically active product. These reagents undergo reaction with the aforementioned analytes to form a product possessing the hydroquinone moiety, thus allowing for reversible electrochemical detection at mild oxidation potentials. The reactivity of each reagent was demonstrated by using N-ethylbenzylamine (EBzA) and the dipeptide isoleucine leucine methyl ester as model analytes. The investigation included the isolation and identification of the intermediates and final products from derivatization of EBzA. These isolated standards were subsequently characterized with respect to electrochemical properties by means of cyclic voltammetry. In LC-EC experiments, the concentration limit of detection (CLOD) of the purified EBzA product was determined to be 5 nM (100 fmol) at a detection potential of +200 mV vs Ag/AgCl ([Cl^-] = 3 M). The CLOD values obtained by LC-EC after derivatization of aqueous solutions of EBzA and Ile-Leu-OMe with NDTE were 25 nM (250 fmol) and 250 nM (2.5 pmol), respectively.

Full text of DOI:10.1021/ac981236c

Anal. Chem. 1999, 71, 2221-2230
Hydroquinone-Based Derivatization Reagents for
the Quantitation of Amines Using Electrochemical
Detection
Mark J. Rose,†,§ Susan M. Lunte,†,‡,§ Robert G. Carlson,‡,§ and John F. Stobaugh*,†,‡,§
Department of Pharmaceutical Chemistry, Department of Chemistry, and Center for Bioanalytical Research,
The University of Kansas, Lawrence, Kansas 66047
of detection in the low femtomole region were frequently achieved.
These analytes represented an excellent technique-problem
match due to their inherent electrochemical activity and, notwith-
standing the numerous advances in analytical methodologies and
instrumentation, at present LC-EC usually remains the method
of choice for the aforementioned substances.
Two new reagents, NDTE (2 ,5 -dihydroxyphenylacetic
acid, 2 ,5 -bis-tetrahydropyranyl ether p-nitrophenyl ester)
and HLTE (homogentisic γ-lactone tetrahydropyranyl
ether), are described for the chemical derivatization of
primary and/ or secondary amines to form an electro-
chemically active product. These reagents undergo reac-
tion with the aforementioned analytes to form a product
possessing the hydroquinone moiety, thus allowing for
reversible electrochemical detection at mild oxidation
potentials. The reactivity of each reagent was demon-
strated by using N-ethylbenzylamine (EBzA) and the
dipeptide isoleucine leucine methyl ester as model ana-
lytes. The investigation included the isolation and iden-
tification of the intermediates and final products from
derivatization of EBzA. These isolated standards were
subsequently characterized with respect to electrochemi-
cal properties by means of cyclic voltammetry. In LC-EC
experiments, the concentration limit of detection (CLOD)
of the purified EBzA product was determined to be 5 nM
(1 0 0 fmol) at a detection potential of +2 0 0 mV vs Ag/
AgCl ([Cl-] ) 3 M). The CLOD values obtained by LC-EC
after derivatization of aqueous solutions of EBzA and Ile-
Leu-OMe with NDTE were 2 5 nM (2 5 0 fmol) and 2 5 0
nM (2 .5 pmol), respectively.
While some substances exhibit suitable electrochemical reac-
tivity and thus are amenable to determination at trace concentra-
tions by LC-EC, there are unfortunately a large number of
important analytes (e.g., various drug substances, their associated
metabolites, amino acids, peptides, etc.) that are not amenable to
high sensitivity detection techniques such as fluorescence and
electrochemistry in their native state. For molecules bearing
reactive functional groups, traditionally the analyst has often
turned to the selective chemical modification of the analytical
target to form a product that is highly detectable. A classic
example of this approach is the amino acid analyzer,7 an instru-
ment that is simply the combination of ion-exchange LC followed
by postcolumn reaction detection, wherein the separated amino
acids undergo transformation to a colored product.
Analysts have continued to devise numerous chemistries for
the derivatization of many functional groups, with the overall goal
usually being the enhancement of analyte detection. Many
references to the original descriptions of such research can be
found in a series of books devoted to sample preparation and
derivatization techniques for use in LC.8-14 In this regard, it
appears that the vast majority of reagents developed for application
in LC are intended to form a product 3 possessing efficient
For the past two decades instrumentation has been available
for the routine application of liquid chromatography (LC) with
electrochemical (EC) detection. With the advent, growth, and now
widespread practice of LC-EC, this technology provides the
bioanalytical chemist with a valuable tool for the sensitive and
selective determination of numerous substances present in biof-
luids and tissues. Among the first described applications of LC-
EC were methods for the determination of various endogenously
occurring catecholeamines and indoleamines,1-6 where mass limits
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Frei, R. W., Lawrence, J. F., Eds.; Plenum Press: New York, 1981.
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Continuous Flow Techniques; Frei, R. W., Lawrence, J. F., Eds.; Plenum
Press: New York, 1982.
† Department of Pharmaceutical Chemistry.
‡ Department of Chemistry.
(10) Reaction Detection in Liquid Chromatography; Krull, I. S., Ed.; Marcel
Decker: New York, 1986.
§ Center of Bioanalytical Research.
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10.1021/ac981236c CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/30/1999
Analytical Chemistry, Vol. 71, No. 11, June 1, 1999 2221

fluorescence. As examples the following reagents: fluorescam-
ine,15 dansyl chloride (DNS),16 7-fluoro-4-nitrobenzo-2-oxa-1,3-
diazole (NBD-F),17 o-phthalaldehyde (OPA),18-20 and naphthalene-
2,3-dicarboxaldehyde (NDA),21-23 all of which are useful for the
derivatization of analytes possessing primary (DNS, NBD-F, OPA,
and NDA applicable) or secondary (DNS and NBD-F applicable)
amine functional groups, were all initially described in applications
employing fluorescence detection.
is often led to the use of laser-induced fluorescence (LIF);
however, in most cases this is a situation where one has a
miniature separation system that is burdened with a substantially
larger detection system. Because electrochemical detection is a
reaction-detection process dependent on analyte diffusion and
because of the ease of electrode miniaturization, this technique
actually benefits from down-sizing. There are now numerous
examples where miniaturized LC systems30-33 and capillary
electrophoresis systems34-42 have been successful when operated
using EC detection. With regard to µ-chip scale instruments,
several groups43-46 have described the fabrication of separation-
detection systems involving LIF detection, but recent publications
are now describing the use of EC detection in association with a
µ-chip/ [µ-plate separation systems.47,48 Thus, with the advent and
continued progress in the development of these miniaturized
separation systems, there is clearly the need for the development
of reagents designed for use with EC detection.
On the basis of these considerations, the objective of the
present research was the design and development of analytical
derivatization reagents capable of imparting electrochemical
activity upon analytes bearing primary or secondary amine
functional groups. Because of documented detection sensitivity,
ease of oxidation while maintaining stability in acid media, and
oxidation-reduction reversibility, the hydroquinone (HQ) moiety
was selected as the reagent substructure intended to convey EC
activity to the analytical target. Herein we describe our initial
strategies regarding reagent design and results obtained in the
development of the candidate reagents intended to achieve this
goal.
In contrast the systematic development of derivatization
reagents intended to impart electrochemical activity to analytes
appears to have been much more modest. This is highlighted by
the fact that after the introduction of OPA and NDA as primary
amine specific fluorogenic reagents, other researchers subse-
quently reported the electrogenic nature of these reagents.24-26
Thus, while perhaps not true in every case, it does appear that
the majority of reagents available to convey EC activity upon an
analyte were not purposefully designed for said purpose.
In using derivatization reactions for detection enhancement,
one is frequently faced with situations where more than one
functional group is kinetically competent to participate in the
proposed derivatization reaction. When these situations are
encountered, one can optimize the derivatization conditions to
exhaustively label all available sites, thus overcoming the issue
of forming multiple products; however, in the case of fluorescence
detection there is the distinct possibility of interactions leading
to fluorescence quenching and thus a substantially diminished
detection sensitivity.22,27 In contrast, investigations have clearly
demonstrated that, in the case of electrochemical derivatization,
chemical modification of multiple sites simply results in enhanced
detectability.28,29
Current trends in bioanalytical chemistry are clearly mvoing
toward miniaturization of sample size requirements and as a result
the proportional down-sizing of the separation systems. As regards
fluorescence detection, since this technique is optically based,
these miniaturized separation systems naturally lead to the use
of diminished optical path length detection cells, leading to a
compromise in concentration detection limits. To compensate, one
EXPERIMENTAL SECTION
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2222 Analytical Chemistry, Vol. 71, No. 11, June 1, 1999

Spectroflow 783 programmable UV absorbance detector, and a
Bioanalytical Systems, Inc. LC4B dual channel amperometric
detector (carbon disk, Ag/ AgCl reference([CIi] ) 3 M), 0.002
in. gasket). Reversed-phase LC was conducted using an in-house
packed column (150 mm × 4.6 mm i.d.; ODS Hypersil, 5-µm
particles). Preparative LC experiments were performed using a
Whatman Magnum 9 (Partisil 10, ODS-2), 50-cm semipreparative
column. Mobile-phase pH was determined using an Accumet pH
meter 910 that was calibrated using pH 4 and pH 7 buffer
standards obtained from Fisher Scientific, Inc. A Houston Instru-
ments Omniscribe strip chart recorder was used to record
chromatogram peak heights. Derivatization reaction temperatures
were maintained using a Pierce Reacti-Therm heating module.
Derivatizations were carried out in both 4- and 1-mL vials with
silicon rubber/ Teflon septum to enable head space evacuation
when applicable. Preparative normal phase chromatography was
performed using a Fluid Metering Inc. pump (model IP-5Y) and
an Isco Instrumentation Co. UV absorbance monitor (model 1840).
1H NMR, IR, and mass spectra were obtained on a GE QE 300
Plus NMR, a Perkin Elmer 1420 ratio recording infrared spectro-
photometer, and a Nermag R10-10 quadrupole GC/ MS system
with SPECTRAL 30 data system, respectively. Voltammetry
experiments analysis were performed using a Cypress Systems,
model CS-1087 potentiostat, version 6.1/ 2V equipped with a carbon
disc electrode (4-mm diameter), Ag/ AgCl reference electrode (RE-
4) and a platinum counter electrode, all from Bioanalytical Systems
Inc.
Stock Solutions. Reagent stock solutions were prepared by
weighing the required mass of each reagent followed by dissolu-
tion in acetonitrile. The resulting concentration of the stock
solutions was NDTE, 40 mM and HLTE, 200 mM. The acylation
catalyst, 1-methylimidazole, was used in all cases with the
appropriate amount being added to the reagent stock solutions
by volumetric pipette. The catalyst concentrations in the NDTE
and HLTE solutions were 40 and 100 mM, respectively. Amino
acid and dipeptide stock solutions were prepared by measurement
on a Cahn microbalance and then transfer into a solution of either
acetonitrile or 50:50 acetonitrile/ water. Subsequent dilutions for
standard curves were prepared by dilution of the initial stock
solution using class A volumetric pipettes and volumetric flasks.
N-Ethylbenzylamine stock solutions were prepared volumetrically
using density at 25 °C and dilution of this initial stock for standard
curve construction. Molar concentrations quoted for limit of
detection experiments reflect the concentration of analyte in
solution during derivatization step 1.
Derivatization P rocedures. Reagent stock solutions were
added to equivalent volumes of a known concentration of the
amine to be derivatized in 50% acetonitrile and 50% water for NDTE
derivatizations and 100% acetonitrile for HLTE derivatizations. Step
1 (Scheme 1) was allowed to proceed for a minimum of 30 min at
25 °C for NDTE, and 24 h at 50 °C for HLTE unless otherwise
specified. HLTE derivatizations were performed under an argon
head-space when noted. After step 1 (Scheme 1), the reaction
mixture for both reagents was diluted 1:1 with aqueous phosphoric
acid (pH 1.2, step 2) and allowed to sit at room temperature for
a minimum of 1 h unless otherwise specified. The reaction
mixtures were filtered and analyzed for derivatized amine by direct
injection into the RPLC system.
HP LC Mobile P hases. Mobile phases for LC were prepared
by mixing the appropriate ratio of acetonitrile with phosphate
buffer (v/ v) and then filtering under vacuum through a 0.2-µm
Nylon 66 filter. The mobile phase was varied from 20 to 35%
acetonitrile depending on the retention and resolution of the
analytes involved. The phosphate buffer component was prepared
by dissolution of Na2HPO4 (0.05 M) and dropwise titration to pH
7.0 with concentrated phosphoric acid.
Synthetic P reparations. Homogentisic γ-Lactone Tetrahydro-
pyranyl Ether (HLTE, I). The precursor lactone, 2,5-dihydrox-
yphenylacetic acid γ-lactone (1033.7 mg, 7 mmol) and 3,4-dihyro-
2H-pyran (883 mg, 10.5 mmol) were added to methylene chloride
(50 mL) containing pyridinum p-toluenesulfonate (PPTS) catalyst
(179.8 mg) prepared as described in a previously published
method.49 The reaction mixture was stirred for 24 h at ambient
temperature. Upon completion (TLC) the solution was diluted with
ethyl ether (50 mL) and washed with half-saturated NaCl solution
(100 mL). The ether fraction was evaporated under reduced
pressure (water aspirator) using a rotary evaporator with slight
heating (40 °C) to afford a red oil containing the product and 2,5-
dihydroxyphenylacetic acid γ-lactone as an impurity. Final puri-
fication was accomplished using low pressure preparatory chro-
matography (silica gel 60, particle size 63-200 µm.; glass column,
20 in. × 9/ 16 in.; solvent, 40:60 ethyl acetate/ hexane; flow rate,
5.5 mL/ min; Detection, UV at 254 nM). The product, a diastero-
Chemicals and Solvents. The reagent chemicals, 2,5-dihy-
droxyphenylacetic acid (98%), 2,5- dihydroxyphenylacetic γ-lactone
(97%), p-nitrophenol (99%), N-ethylbenzylamine (97%), 1-meth-
ylimidazole (99%), and 1-(3-dimethylaminopropyl)-3-ethylcarbodi-
imide HCl (+98%) were obtained from Aldrich Chemical Co. and
were used as received. N-ethylbenzylamine was purified by
vacuum distillation prior to use in trace analysis experiments. The
dipeptide, Ile-Leu methyl ester was obtained from Bachem
Biosciences Inc. and was used as received. Methylene chloride,
hexane, ethyl acetate, and acetonitrile were HPLC grade solvents
(99.9%) obtained from Fisher Scientific, Inc. Methylene chloride
was distilled from calcium hydride and stored over 4 Å molecular
sieves (Union Carbide Corp.) All other solvents were used as
received. Water was purified with bulk carbon and mixed-bed
deionization cartridges and then passed through a Millipore
MILLI-Q water system to a resistance of 18 Ω/ cm. The silica gel
60 used in preparative scale chromatography was obtained from
EM Science Inc.
Electrochemical Characterization of the Reagents, Inter-
mediates, and P roducts. The cyclic voltammetry scans were
obtained in 50:50 acetonitrile/ phosphate buffer (0.1 M, pH 7.0).
Cyclic scans were performed from -500 mV to +1000 mV to -500
mV (vs Ag/ AgCl, [Cl-] ) 3 M) on the various compounds (1 mM
concentrations) at a scan rate of 100 mV/ s. The carbon electrode
was polished using 5-µm alumina before each run. Standards of
the derivatization product of N-ethylbenzylamine were generated
from both the NDTE or HLTE intermediates (2 mM solutions in
acetonitrile) in situ by exposure to an equivalent volume of
phosphoric acid (pH 2.0). After acid cleavage of the THP
protecting group(s), the solution was brought to pH 7.0 with 0.1
M NaOH for voltammetry measurements.
(49) Miyashita, M.; Yoshikoshi, A.; Grieco, P. A. J. Org. Chem. 1 9 8 3 , 48, 3465-
3471.
Analytical Chemistry, Vol. 71, No. 11, June 1, 1999 2223

Scheme 1. Two General Pathways Examined for the Derivatization of Primary or Secondary Aminesa
a Path a homogentisic γ-lactone tetrahydropyranyl ether (HLTE, I); path b p-nitrophenyl 2,5-dihydroxyphenylacetate, bis-tetrahydro-
pyranyl ether (NDTE, IV).
meric mixture, was a faintly colored yellow oil which crystallized
upon standing at room temperature to form similarly colored
crystals (yield 77%, mp 64-65 °C. IR (neat) 2960, 1820, 1460, 1220,
2,5-Dihydroxyphenylacetic Acid, Bis-tetrahydropyranyl Ether.
Crude 2,5-dihydroxyphenylacetic acid methyl ester (1200 mg, 7
mmol) containing p-toluenesulphonic acid as an impurity was
added to degassed 3,4-dihydro-2H-pyran (150 mL) and stirred at
room temperature for 4 h. The reaction mixture was then diluted
with ethyl ether (150 mL) and washed with aqueous 1 N NaOH
(2 × 100 mL). The organic layer was dried (Na2SO4) and
evaporated under reduced pressure to afford a yellow oil (2500
mg), which was subsequently dissolved in methanol (160 mL)
followed by the addition of water (40 mL). The pH (pH paper) of
this mixture was raised to 14 with solid NaOH and allowed to stir
overnight. After stirring, additional water (100 mL) was added and
the mixture acidified to pH 4 by dropwise addition of concentrated
phosphoric acid. The aqueous solution was extracted with
chloroform (2 × 100 mL). The chloroform solution was dried (Na2-
SO4) and evaporated under reduced pressure to afford 2,5-
dihydroxyphenylacetic acid bis-tetrahydropyranyl ether as a yellow
1
1120, 1050, 970, 870, 815 and 640 cm-1. H NMR (CDCl3, 300
MHz) 7.0 (s, 1H), 6.95 (d, 2H), 5.3 (t, 1H), 3.85 (t, 1H), 3.7 (s,
2H), 3.5 (m, 1H), 1.8 (d, 2H), 1.6 (m,4H). High resolution mass
spectrum (FAB+), calculated MW: 234.0891, determined MW:
234.0887.
N,N-Benzylethyl-2,5-dihydroxyphenylacetamide 5-tetrahydropyra-
nyl ether (VI). Homogentisic γ-lactone tetrahydropyranyl ether
(I) (0.21 g, 0.0009 M) was added to N-ethylbenzylamine (0.6 g,
0.0045 mol) in 20 mL of dry methylene chloride. The reaction
was stirred at 25 °C for 2 days. The product was isolated from
starting materials using reverse phase semipreparatory chroma-
tography (Partisil 10 ODS-2; column dimensions mm, 500 × 9
mm i.d.; mobile phase, 50:50 water/ acetonitrile; flow rate, 4.0 mL/
min; Detection, UV at 254 nM). The product, a mixture of two
diastereomers, was a light yellow oil that solidified over 24 h. IR
1
semisolid. H NMR (CDCl3, 300 MHz) 7.0 (d, 1H), 6.88 (d, 2H),
(neat) 2950, 2780, 1770, 1460, 1350, 1200, 1115, 865, and 810 cm-1
.
5.27 (m, 4H), 4.9 (m, 4H), 3.9 (m, 2H), 2.2-1.8-1.5 (m, 12H).
p-Nitrophenyl 2,5-Dihydroxyphenylacetate Bis-tetrahydropyranyl
Ether (NDTE, IV). 2,5-dihydroxyphenylacetic acid bis-tetrahy-
dropyranyl ether (2.40 g, 7 mmol) and p-nitrophenol (1.390 g, 10
mmol) were dissolved in pyridine (15 mL) and cooled to ice
temperature followed by the additional of 1-(3-dimethylaminopro-
pyl)-3-ethylcarbodiimide HCl (1.91 g, 10 mmol). The reaction was
stirred under nitrogen for 36 h, slowly coming to room temper-
ature over the first 12 h. Pyridine was evaporated under vacuum
and the product was dissolved in ethyl acetate (100 mL). This
was extracted with water (2 × 100 mL) and 1.0 M sodium
bicarbonate (2 × 100 mL). The organic layer was dried and
evaporated under vacuum to afford a red oil. This was dissolved
in acetone (20 mL) and filtered through silica gel 60 to remove
the red color. The gel was rinsed with additional acetone (60 mL)
to elute any retained NDTE. The acetone was evaporated leaving
1H NMR 7.3 (m, 5H), 6.9 (m, 3H), 5.2 (s, 2H), 4.65 (s, 2H) 1.95
(m, 2H), 1.8 (m, 2H),1.6 (m, 2H), 1.18 (q, 3H). Mass spectrum
(CI mode, NH3) m/ e 370 (M + 1).
Methyl 2,5-Dihydroxyphenylacetate. The precursor carboxylic
acid, 2,5-dihydroxyphenylacetic acid (1.10 g, 6.5 mmol) together
with p-toluenesulphonic acid (200 mg, 1 mmol) was dissolved in
methanol (150 mL) and refluxed for 4 h under argon. The
methanol was evaporated under high vacuum using a rotary
evaporator leaving a brownish solid (1400 mg) composed of 2,5-
dihydroxyphenylacetic acid methyl ester and p-toluenesulphonic
acid. A small amount of this solid was extracted into chloroform
for identification. The chloroform was evaporated under high
vacuum leaving a white solid consisting of the product. Isolated
1
product mp 116-117 °C, H NMR(acetone-d6, 300 MHz) 7.82 (s,
1H), 7.7 (s, 1H), 6.6 (m, 3H), 3.5 (s, 2H), 3.56 (s, 3H).
2224 Analytical Chemistry, Vol. 71, No. 11, June 1, 1999

a light orange oil, which was a mixture of one major and three
minor products separable by low pressure preparatory chroma-
tography (silica gel 60; particle size, 63-200 µm; glass column,
20 × 9/ 16 in. i.d.; solvent, 40:60 ethyl acetate/ hexane; flow rate,
5.5 mL/ min; detection, UV at 254 nM). The major product peak
was collected and the solvent evaporated to leave NDTE as a white
solid (mp 114-117 °C). H NMR (CDCl3, 300 MHz) 8.2 (d, 2H),
7.25 (d, 2H), 7.05 (d, 1H), 7.0 (m, 2H), 5.3 (m, 2H), 3.85 (m, 4H),
3.6 (m, 2H), 1.9-1.85-1.6 (m, 12H).
1) to form amide intermediates (II, V). Acidification (step 2)
causes hydrolytic cleavage of the tetrahydropyranyl (THP)-
protecting group(s) to release the hydroquinone moiety (III) and
thus achieve incorporation of the HGA template into the analytical
product for EC detection. As shown by the reaction pathways,
HLTE and NDTE differ in acyl-group activation, the intermediates
formed, and native physico-chemical properties, but ultimately
produce the same product. For both reagents, the following
chemical aspects are of importance. Initially, the HQ moiety of
each reagent is protected against autooxidation at the basic
conditions required for amine acylation (step 1) due to the
presence of either one (HLTE) or two (NDTE) THP-protecting
1 2 groups. Subsequently, acidification of the reaction mixture
(step 2) serves to demask the HQ moiety in an acidic environment
where liability to autooxidation is prevented prior to chromatog-
raphy and detection.
1
N,N-Benzylethyl-2,5-dihydroxyphenylacetamide Bis-tetrahydropy-
ranyl Ether (V, R1 ) C2H5, R2 )CH2C6H5). 2,5-Dihydroxypheny-
lacetic acid bis-tetrahydropyranyl ether (600 mg, 1.7 mmol) and
N-benzylethylamine (675 mg, 5 mmol) were added to pyridine
(15 mL). The solution was cooled to 0 °C and to this was added
1-(3-dimethylaminopropyl) 3-ethylcarbodiimide HCl (957 mg, 5
mmol). The reaction stirred under nitrogen for 24 h, slowly
coming to room temperature over the first 12 h. The pyridine was
evaporated under vacuum. The red product was dissolved in ethyl
acetate (100 mL), washed with water (2 × 100 mL) and 1 N
sodium bicarbonate (2 × 100 mL). The solvent was dried (MgSO4)
and removed using a rotary evaporator under high vacuum,
leaving a brown red oil (178 mg, 0.4 mmol). The oil was purified
using low pressure preparatory chromatography (silica gel 60;
particle size, 63-200 µm; glass column, 20 in. × 9/ 16 in. i.d.;
solvent, 40:60 ethyl acetate/ hexane; flow rate, 5.5 mL/ min;
detection, UV at 254 nM). Further purification was accomplished
using reverse phase preparatory chromatography (Partisil 10 ODS-
2; 500 mm × 9 mm i.d.; mobile phase, 30:70 water/ acetonitrile;
flow rate, 4.0 mL/ min; detection, UV at 254 nM). 1H NMR (CDCl3,
300 MHz) 7.25 (m, 5H), 7.14 (m, 1H), 7.04 (d, 1H), 7.0 (s, 1H),
5.27 (m, 2H), 4.0 (q, 2H), 3.9 (m, 4H), 3.75 (m, 2H), 3.55 (m,
2H), 1.6-1.8-1.9 (m, 12H), 1.1 (t, 3H). Mass spectrum (FAB+)
m/ e 453 (M).
EC Characterization of HLTE, Intermediates, and P rod-
ucts. HLTE, the first hydroquinone-labeling reagent studied in
these experiments, demonstrates electrogenic properties when
used as an amine derivatization reagent as it exhibits no significant
electrochemical activity (Scheme 2, I) when examined by cyclic
voltammetry (CV). Therefore, when used in LC applications with
EC detection, excess HLTE would not appear as a chromato-
graphic interference. Additionally, the cyclic voltammograms of
Scheme 2 illustrate the redox properties of the isolated intermedi-
ate and final product of HLTE reaction with N-ethylbenzylamine
(EBzA) as well as the major hydrolysis product (VII) obtained in
acid media. The HLTE reagent shows no significant oxidative
electrochemical activity in either its native (I) or deprotected form
(VII) until approximately +550 mV. Both the phenolic intermedi-
ate (VI) and the de-protected HQ product (VIII) possess electro-
chemical activity at oxidation potentials lower than those observed
for the unreacted reagent. The intermediate (VI) as shown in
Scheme 3, is electrochemically active due to the EC-mediated
cleavage of the THP group. The final product of the derivatization
reaction (VIII) was shown by cyclic voltammetry to undergo a
chemically reversible oxidation with a peak potential (Epa) that is
substantially lower (+0.200 V versus +0.600 V) than that observed
for native hydroquinone analyzed under equivalent experimental
conditions.
RESULTS AND DISCUSSION
Reagent Design and Anticipated P roperties. As noted
earlier, the goal of this research was the design of reagents
capable of conveying the HQ moiety to a primary or secondary
amine-bearing analyte. This requires the introduction of a func-
tional group that will ideally undergo rapid reaction with a
primary/ secondary amine to form a stable linkage between the
reagent and analyte. From an operational standpoint, one has to
conduct the reaction in mildly basic aqueous (organic-aqueous)
media. Based on these criteria, one can narrow the derivatization
process to a relatively few functional group transformations, with
the following having been considered in the present research:
isothiocyanate, activated carboxylic acid, and activated carbamate.
Although precedence exists for the use of each of these derivative
forming groups, after consideration of the overall goal in conjunc-
tion with availability of starting materials and ease of preparation,
2,5-dihydroxyphenylacetic acid (homogentisic acid, HGA) was
selected as the template on which to base systematic reagent
development. The structures presented in Scheme 1 (HLTE and
NDTE) represent two differing approaches envisioned as modi-
fications of the HGA template whereby the overall goal could be
achieved.
Electrochemical data for cyclic voltammetry performed on
isolated standards of VII and VIII are summarized in Table 1 along
with data for HGA and hydroquinone obtained under the same
conditions. The high oxidative detection potential observed for
VII at the end of the derivatization reaction is dependent on the
lactone ring remaining closed. Though not observed to open at
pH 7 during the 3-h time frame of the cyclic voltammetry
experiments, in other experiments the lactone ring was observed
to slowly open under hydrolytic .conditions. Ring opening was
observed in 50:50 mixtures of acetonitrile/ buffer at high pH (96%
in 6 h at pH 8) and also at low pH (14% after 24 h at pH 1, 8.5%
after 24 hours at pH 2). Due to the ring-opening reaction, the
reagent loses some of its electrogenic characteristics as it is slowly
hydrolyzed to HGA. Interestingly, as shown in Table 1, homogen-
tisic acid, though more easily oxidized than the closed lactone,
still possesses a peak oxidation potential which is approximately
+150 mV greater than the HLTE-EBzA product (VIII). Even after
deprotection (THP group removal) and hydrolysis of the lactone
The chemical pathways for amine transformation using HLTE
(I) and NDTE (IV) are shown in Scheme 1. As shown, the
reagents first acylate amine analytes in mildly basic media (step
Analytical Chemistry, Vol. 71, No. 11, June 1, 1999 2225

Scheme 2. Components Present during the Derivatization of N-Ethylbenzylamine with HLTE and the
Corresponding Cyclic Voltammograms at pH 7 of Each As Isolated 1 mM Standardsa
a See Experimental Section for a detailed description and parameters.
Scheme 3. Nonreversible Removal of the HLTE Masking Group As Mediated by Oxidative
Electrochemistry and Subsequent Reversible Reaction of the Liberated Hydroquinonea
a Example is specifically shown for HLTE reaction with N-ethylbenzylamine.
Table 1. Cyclic Voltammetry Parameters for the
Intermediate (VI) and Final Product (VIII) in the
Derivatization of N-Ethylbenzylamine Derivatized
Using HLTE and for Homogentisic Acid and
reversed-phase (RP) LC with EC detection (RPLC-EC). Figure 1
shows an analysis by RPLC-EC using dual electrode detection of
a pure synthetic standard of VIII at a high and a low concentration
near the limit of detection. The chromatograms show the series
electrochemical detector output for oxidation (upstream, +300
mV) of VIII to form the quinone (IX) and the subsequent
reduction (downstream, -300 mV) of the quinone to the initial
HQ-form (Scheme 3). Chromatogram (b), near the limit of
detection, represents 1 pmol injected (20 µL injection, 50 nM
solution). The mass limit of detection (MLOD) for this compound
was found to be 100 fmol (S/ N ) 4.5 at +200 mV and S/ N ) 3
at +300 mV) at the upstream oxidative electrode (20 µL injection,
5 nM solution).
Hydroquinone Analyzed under Equivalent Conditions
Epa
ipa
Epc
ipc
E°′
∆E
analyte
(mV) (µA) (mV) (µA) (mV) (mV)
hydroquinone
homogentisic acid +361
VI
+318
29.4
21.4
15.1
23.5
-240
-246
-211
-148
23.1
10.9
10.5
18.9
+78
+115
+428
+56
558
607
850
352
+639
+204
VIII
to the open form, the resulting HGA is still somewhat electrogenic
since it is clearly more difficult to oxidize than the analytical
derivative (VIII).
LC-EC of Isolated HLTE Intermediates/ P roducts. The low
oxidation potentials and chemical reversibility observed in free
solution voltammetry for VIII are predictive of the low limits of
detection that can potentially be obtained for this compound using
The low limit of detection observed for VIII demonstrates the
usefulness of forming amine derivatives bearing the HQ moiety
when determinations at trace concentrations are required. Figure
2 shows the hydrodynamic voltammogram for the oxidative
detection of VIII using RPLC-EC at apparent pH 7. The current
2226 Analytical Chemistry, Vol. 71, No. 11, June 1, 1999

Table 2. Analytes and Concentration Ranges over
Which Linear Calibration Plots Were Attained in the
Derivatization of N-Ethylbenzylamine and the
Dipeptide, Ile-Leu-OMe, with Each Reagent
derivatization
reagent
number concentration correlation
analyte
standards range (µM)
(r2)
HLTE
HLTE
HLTE
NDTE
NDTE
benzylethylamine
benzylethylaminea
H-Ile-Leu-OMea
benzylethylamine
H-Ile-Leu-OMe
9
8
9
11
6
10 000-50
1000-5
0.998
0.994
0.992
1.000
1.000
1000-2
25-0.025
25-0.250
a
Performed under an argon headspace.
additional selectivity for easily oxidized analyte interferences that
do not participate in a similarly mild reduction. In addition, the
mild detection potentials that could be used for the detection of
hydroquinone-labeled analytes (+200 to +400 mV) offered sig-
nificantly decreased electrode-fouling and decreased electrode
equilibration time when compared to assays performed using
higher potentials.
HLTE Reactivity Issues. With the preceding data in hand,
research efforts were next directed at the utilization of HLTE in
an actual derivatization reaction, i.e., excess HLTE was used to
derivatize aqueous solutions spiked with poorly detectable model
amines. Table 2 lists the calibration curves generated for the HLTE
derivatization and RPLC-EC determination of two model amines,
EBzA and the dipeptide methyl ester, Ile-Leu-OMe. Sample
chromatograms from the EBzA derivatization are shown in Figure
3. The lower limit of quantitation obtained for EBzA was a pre-
derivatization concentration limit of detection (CLOD) of 5 µM
(20 µL injection), representing a MLOD of 50 pmol. This is a 500-
fold greater MLOD than that obtained for the pure standard of
derivatized EBzA (VIII), which represents the lowest limit of
detection that should be obtainable for a real sample in the limit
of 100% derivatization efficiency and the generation of no chemical
noise.
Figure 1. Isolated standard of the N-ethylbenzylamine derivative
(VIII) analyzed using RP-LCEC in the dual detection mode (oxidation
followed by reduction), (a) 5 nmol injected, (b) 1 pmol injected.
The inability to reach these expected detection limits with
HLTE reagent was due to a low yield of the product and the
formation of unknown substances during the derivatization reac-
tion, which were chromatographic interferences. Step 2 in the
derivatization procedure was shown to proceed in a quantitative,
side product free fashion in under 30 min at 25 °C when starting
with the pure intermediate (VI). No additional side products were
observed to form in this step due to presence of spiked excess
HLTE reagent, its precursors, or excess amine. On the basis of
these observations, step 1 was implicated as the cause of both
the low product yield (2-5% observed for EBzA using the
procedure detailed in the experimental section) and the generation
of interfering side products. These data are highly suggestive that
a major limitation of HLTE in the derivatization of amines is its
poor acylation potential. This could be partially rectified using
higher reaction temperatures and longer reaction times in step
1, but even with these aggressive measures quantitative yields of
product were never fully achieved. As an example of the slow
reactivity of HLTE, the yield for the derivatization of EBzA (1 mM)
with a 100-fold excess of HLTE in acetonitrile was still incomplete
after 48 h at 75 °C (80% yield). The high temperatures and
extended reaction times required to generate adequate product
Figure 2. Hydrodynamic voltammogram for N,N-benzylethyl-2,5-
dihydroxyphenylacetamide (VIII) generated by RPLC-EC; 20 µL
injection of a 500 µM solution (see Experimental Section for details).
limiting plateau is reached at approximately +350 mV with less
than an order of magnitude decrease in signal when the detection
potential is lowered to +100 mV (175 nA f 75 nA). The low
oxidation-potential observed for HGA-labeled analytes allows for
selective detection against numerous compounds requiring higher
potentials and the ease of the reverse reduction process provides
Analytical Chemistry, Vol. 71, No. 11, June 1, 1999 2227

tively. Electrochemically detectable byproducts increase in relation
to the presence of molecular oxygen (compare Figure 4a to b to
c), and in the case of 100% oxygen headspace, there is a sharp
decrease in derivatized product response due to electrode fouling
from these oxidative byproducts (Figure 4c).
In summary, it was observed that the undesirable byproducts
and associated electrode fouling were minimized by the exclusion
of molecular oxygen from the reaction solution, but these
byproducts were never fully eliminated. In any case, the use of
reagents requiring the total exclusion of molecular oxygen are
highly undesirable and impractical when large numbers of samples
require derivatization. Thus, based on the information gleamed
from these studies, it appeared that the next logical aspect of
reagent design would be to enhance the reactivity (acylation
potential) of the reagent so that a mild and rapid first step would
be possible and this presumably would minimize the problem of
oxygen related impurities that seemed to be associated with the
aggressive conditions required in step 1 when HLTE was used
as the derivatization reagent.
NDTE-HLTE Comparisons. NDTE (Scheme 1, IV), an
alternate HGA-based reagent was developed in response to the
limitations observed with HLTE. The rational for the design of
the NDTE structure was the enhancement of acylation potential
so that step 1 would be quantitative and rapidly proceed under
relatively mild conditions, thus circumventing the formation of
the unwanted byproducts previously observed with HLTE. Acy-
lation ability was enhanced by the use of the p-nitrophenol active
ester as an alternative to the lactone ring of HLTE. The remaining
phenolic hydroxyl group was protected by forming an additional
THP group, thus preventing any pre-oxidation from occurring in
step 1 where it is necessary to use basic media.
Figure 3. RP-LCEC determination using oxidative and reductive
detection in series. The test solutions were as follows: (a) 250 µM
solution of N-ethylbenzylamine in acetonitrile derivatized with HLTE,
(b) 25 pM solution of N-ethylbenzylamine derivatized with HLTE. The
injection volume was 20 µL in each case.
A side by side comparison of the relative reactivity of NDTE
and HLTE toward the model amine Ile-Leu-OMe is shown in
Figure 5, along with separate derivatizations of blank solutions.
Reaction time, temperature, and molar concentrations of both
reagents and the analyte were the same for all experiments. NDTE
is faster reacting and provides a 20-50-fold improvement in
product yield in a significantly shorter time. For this direct
comparison, step 1 acylation for both reagents was performed at
50 °C for 16 h, conditions required for the HLTE reagent to
provide useful product yields. As will be seen in the following
section, the NDTE reagent provides a similar yield of derivatized
product to that shown in substantially less time; however, it was
necessary to conduct the experiment in this fashion to appreciate
the lack of side products formed with NDTE as compared to
HLTE.
NDTE-Reaction Kinetics with Model Analytes. Figure 6
shows the reaction kinetics at 25 °C for the derivatization of a
trace concentration of EBzA using NDTE. Acylation (step 1) and
deprotection (step 2) are each shown to reach maximal yield in
under 30 minutes for a total derivatization time at 25 °C of 1 h.
The reaction yield was 95.1% when compared with the pure
standard (VIII). Based on reaction rate, yield, and the mild
reaction conditions, NDTE is clearly a superior reagent for
primary/ secondary amine derivatizations as compared to HLTE.
A negative aspect of NDTE is that it is nonelectrogenic as the
excess reagent is converted to electrochemically active HGA
during the derivatization procedure. From a practical standpoint,
Figure 4. RPLC-EC chromatograms of identical HLTE derivatiza-
tions of 500 µM N-ethylbenzylamine solutions (2 mL) in the presence
of variable headspace (2 mL) oxygen levels: (a) 100% argon
headspace, (b) 21% oxygen headspace, (c) 100% oxygen headspace.
yields during step 1 are accompanied by a correspondingly high
yield of what appear to be oxidative byproducts that both interfere
with the derivatized analyte during trace quantitation and are also
the source of electrode fouling. Figure 4 shows RPLC-EC chro-
matograms where identical concentrations of EBzA were reacted
with HLTE in a sealed vial having a headspace consisting of 0%
(argon), 21 (air), and 100% oxygen, Figure 4a, b, and c, respec-
2228 Analytical Chemistry, Vol. 71, No. 11, June 1, 1999

Figure 7. RPLC-EC determination using series oxidation and
reduction performed on (a) a solution of 62.5 nM N-ethylbenzylamine
in 75% acetonitrile and 25% water derivatized with NDTE, (b) a blank
solution of 75% acetonitrile and 25% water derivatized with NDTE.
marized in Table 2. Figure 7a shows the oxidative/ reductive
chromatograms for a single RPLC-EC determination of EBzA
derivatized with NDTE at a prederivatization concentration of 62.5
nM or 625 fmol injected (1:1 acid dilution in step 2, 20 µL
injection). The CLOD for EBzA was determined to be 25 nM
(MLOD, 250 fmol injected, S/ N ) 3/ 1 at +300 mV). This MLOD
is very similar to that obtained for the synthetically prepared
standard of HGA-labeled EBzA (VIII), indicating that the deriva-
tization procedure used and the performance of the reagent are
now optimal. Figure 7b shows the results from a derivatization
blank illustrating the minimal interferences resulting from the
NDTE reagent.
Figure 5. RPLC-EC chromatograms resulting from oxidative detec-
tion performed on (a) 50 pLM solution of isoleucine leucine methyl
ester in acetonitrile derivatized with NDTE, (b) 50 µM solution of
isoleucine leucine methyl ester in acetonitrile derivatized with HLTE;
(c) blank of acetonitrile derivatized using NDTE, (d) a blank of
acetonitrile derivatized using HLTE.
Experience gained in derivatizing various amines with NDTE
using the herein described derivatization procedure have estab-
lished CLODs usually falling between 25 and 250 nM, which is
similar to the data presented in Table 2. The higher limit of
detection for Ile-Leu-OMe is likely related to the fact that
N-terminal isoleucine is slowly reactive to acylation type reac-
tions,50 and in data not presented (other amino acid and peptide
analytes), it has been generally observed that detection limits
similar to the 25 nM limit obtained with EBzA are normally
attained.
CONCLUSIONS
Figure 6. Time to maximal yield for step 1 (9) and step 2 (b) in
the derivatization of N-ethylbenzylamine (50 µM) using NDTE reagent
at 25 °C (see Experimental Section for detailed derivatization
parameters).
The concepts that served as the basis for the design of HLTE
and NDTE have proven to be valuable and represent significant
steps toward the rational development of reagents intended to
impart EC activity upon various amine analytes. The results of
the present investigation will serve as a basis for the design of
future reagents where one would seek to take advantage of the
positive observations of the present work, i.e., the diminishment
of EC activity that results from phenolic group lactonization and
the need to have a mild acylation step to ensure high chemical
yield and minimal reagent related byproducts. Of note, despite
the carboxylic acid group of HGA is fully ionized at pH 7, which
results in minimal RPLC retention of the polar HGA. Accordingly
in the determination of EBzA (Figure 7) the excess reagent was
not a chromatographic interference.
For comparative purposes, NDTE was used for the determi-
nation of EBzA and Ile-Leu-OME as was previously done using
HLTE. The resulting calibration data for each reagent is sum-
(50) Rutter, W. J.; Santi, D. V. U.S. Patent 5,010,175, 1991.
Analytical Chemistry, Vol. 71, No. 11, June 1, 1999 2229

the lack of electrogenicity of NDTE, this did not prove to be a
substantial limitation primarily due to the highly polar nature of
HGA which results in essentially void-volume elution in RPLC
applications. Notwithstanding this result, an obvious next step in
EC reagent development would be the incorporation of structural
features that provide for a reagent exhibiting a high degree of
electrogenicity. In this regard, perhaps one option would be the
placement of a structural element that would have the effect of
promoting lactonization of the reagent hydrolysis product (HGA
or a related aromatic phenolic carboxylic acid). Such structural
modifications have proven to serve this function in similar
molecules.51 Finally, it can be concluded that the development
and application of reagents such as NDTE, and perhaps more
advanced versions that possess electrogenicity, will become
increasingly important as miniaturization in analytical chemistry
continues to place limitations and demands on detection technolo-
gies.
ACKNOWLEDGMENT
This research was supported by the National Cancer Institute
Training Grant (CA-09242), The Pharmaceutical Research and
Manufacturers Association, Proctor & Gamble, and the Center
for Bioanalytical Research at the University of Kansas. We thank
the University of Kansas Mass Spectrometry Facility for assistance
in identification of the various compounds prepared in the course
of this investigation.
Received for review November 10, 1998. Accepted March
22, 1999.
(51) Hillary, P. S.; Cohen, L. A. J. Org. Chem. 1 9 8 3 , 48, 3465-3471.
AC981236C
2230 Analytical Chemistry, Vol. 71, No. 11, June 1, 1999