On-line derivatization into precolumns for the determination of drugs by liquid chromatography and column switching: determination of amphetamines in urine

Article abstract of DOI:10.1021/ac9505076

A chromatographic system for the on-line derivatization of drugs using column switching is described. The system uses a 20 mm × 2.1 mm i.d. precolumn packed with a unmodified ODS stationary phase. This column is used for sample cleanup and enrichment of t

Full text of DOI:10.1021/ac9505076

Anal. Chem. 1996, 68, 734-739
On-Line Derivatization into Precolumns for the
Determination of Drugs by Liquid Chromatography
and Column Switching: Determination of
Amphetamines in Urine
Rosa Herra´ ez-Herna´ ndez, Pilar Campı´ns-Falco´ ,* and Adela Sevillano-Cabeza
Department of Analytical Chemistry, University of Vale`ncia, Dr. Moliner 50, 46100 Burjassot, Vale`ncia, Spain
umn packings and integrated into the LC system. Since the choice
of pore size of the solid support (or percentage of cross-linking
when using a polymer network) will effect size exclusion, thus
enabling derivatization of small molecules but hindering the large
ones, these precolumns can be used for sample cleanup. At the
same time or somewhat later, the reagent part of the solid-phase
material can be made to react with the adsorbed analytes. This
methodology has been successfully applied to the derivatization
of a variety of drugs.8 However, the synthesis of the solid-phase
reagent is often difficult, and periodic regeneration of the reagent
may be required to obtain high yield, fewer side products, and
reproducible results. In addition, these reagents have a limited
stability in aqueous solutions, especially at elevated temperatures
and pH, which are often required to obtain satisfactory reaction
rates.4,7
A chromatographic system for the on-line derivatization
of drugs using column switching is described. The system
uses a 2 0 mm × 2 .1 mm i.d. precolumn packed with a
unmodified ODS stationary phase. This column is used
for sample cleanup and enrichment of the analytes. Next,
the trapped analytes are derivatized by injection of the
derivatization reagent into the precolumn. Finally, the
derivatives are transferred to the analytical column for
their separation under reversed-phase conditions. The
influence of several parameters such as the reaction time,
the amount of derivatization reagent, or the system design
has been studied using some amphetamines as model
compounds and three derivatization reagents: sodium
1,2-naphthoquinone-4-sulfonate, o-phthaldialdehyde, and
9 -fluorenylmethyl chloroformate. The potential of the
described approach is illustrated by determining amphet-
amine and methamphetamine in untreated urine at ambi-
ent temperature.
In a previous study we demonstrated the possibility of
performing derivatization with sodium 1,2-naphthoquinone-4-
sulfonate (NQS) into C18 solid-phase extraction cartridges.9 The
described procedure was applied to the determination of amphet-
amine in pharmaceuticals and in urine samples.
In the determination of traces of analytes in biological fluids
by column liquid chromatography (LC), two types of problems
are commonly encountered. First, sample conditioning is required
before injection into the LC system. Otherwise, column clogging
and rapid deterioration in column performance (caused by
irreversible adsorption of the proteins) occur, resulting in a limited
lifetime of the column. Second, since analyte concentration is
usually low, some kind of enrichment and/ or derivatization is often
required to improve analyte detectability.
Many strategies have been developed to overcome laborious
sample cleanup, most of them based on precolumn techniques.
In this way, sample cleanup and analytical separation can be
performed on-line in the same system. However, on-line deriva-
tization of anlaytes is not so widespread, probably because of the
inherent difficulties of incorporating a chemical reaction into the
flow scheme of a liquid chromatograph. This difficulties mainly
concern the compatibility of the derivatization reagent and
derivatized products with the mobile phase used for separation.1,2
Ideally, sample cleanup and derivatization should be integrated
in the same process. In this respect, one of the most attractive
approaches proposed is based on immobilizing detection-sensitive
reagents on solid supports (mainly silica, alumina, and organic
polymer based).3-7 Solid-phase reagents can be used as precol-
In this work, we describe a very simple procedure for the on-
line derivatization of drugs based on the employment of precol-
umns packed with a conventional ODS stationary phase. The
precolumn is used to purify the sample and concentrate the
analytes and, next, to retain the derivatization agent and/ or the
derivatized analytes. Finally, the derivatives are transferred to
the analytical column by means of a switching arrangement. This
method offers some advantages over derivatization into solid-phase
extraction cartridges. First, sample handling is minimized since
untreated samples are directly injected into the system. Second,
the analytes are transferred to the analytical column for separation
immediately after derivatization, which avoids stability problems.10
Moreover, parameters affecting the reaction rates, especially the
reaction time, can be better controlled. This is very important if
the derivatization reaction exhibits slow kinetics but suitable
sensitivity is achieved without quantitative conversion.
The described system has been applied to the derivatization
of three amphetamines, the primary amines â-phenylethylamine
(4) Gao, C.-X.; Chou, T.-Y.; Krull, I. S. Anal. Chem. 1 9 8 9 , 61, 1538.
(5) Bourque, A. J.; Krull, I. S. J. Chromatogr. 1 9 9 1 , 537, 123.
(6) Zhou, F.-X.; Krull, I. S.; Feibush, B. J. Chromatogr. 1 9 9 2 , 609, 103.
(7) Zhou, F.-X.; Krull, I. S.; Feibush, B. J. Chromatogr. 1 9 9 3 , 648, 357.
(8) Krull, I. S.; Szulc, M. E.; Bourque, A. J.; Zhou, F.-X.; Yu, J.; Strong, R. J.
Chromatogr. 1 9 9 4 , 659, 19.
(1) Turnell, D. C.; Cooper, J. D. H. J. Chromatogr. 1 9 8 9 , 492, 59.
(2) Krull, I. S.; Deyl, Z.; Lingeman, H. J. Chromatogr. 1 9 9 4 , 659, 1.
(3) Gao, C.-X.; Krull, I. S. BioChromatography 1 9 8 9 , 4, 222.
(9) Camp´ıns-Falco´, P.; Molins-Legua, C.; Sevillano-Cabeza, A.; Kohlman, M. J.
Chromatogr., submitted.
(10) Farrel, B. M.; Jefferies, T. M. J. Chromatogr. 1 9 8 3 , 272, 111.
734 Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
0003-2700/96/0368-0734$12.00/0 © 1996 American Chemical Society

Table 1. Summary of the Chromatographic Conditions Used for the Separation of Amphetamine Derivatives
elution program
mobile-phase comptn (%)
derivtzn
reagent
analytical column
time (min)
acetonitrile
aq solventa
flow (mL/ min)
1
detection
NQS
Hypersil ODS C18, 5 µm, 250 mm × 4 mm i.d.
0
2.5
3.5
8
40
50
70
70
40
50
70
70
40
50
70
100
60
50
30
30
60
50
30
30
60
50
30
0
UV 280 nm
OPA
LiChrospher 100 RP 18, 5 µm 125 mm × 4 mm i.d.
LiChrospher 100 RP 18, 5 µm 125 mm × 4 mm i.d.
0
2.5
5
10
0
2.5
5
0.8
1.5
fluorescence
λexc ) 345 nm
λem ) 445 nm
FMOC
fluorescence
λexc ) 264 nm
λem ) 313 nm
10
a
Propylamine hydrochloride (0.5%) in water (v/ v) for the NQS and OPA methods; water for the FMOC method.
and amphetamine and the secondary amine methamphetamine,
because most UV and fluorescent methods proposed for the
analysis of these drugs involve their chemical transformation.11
We have evaluated the possibility of extending this technique to
different derivatization reagents: NQS, o-phthaldialdehyde (OPA),
and 9-fluorenylmethyl chloroformate (FMOC), which have been
widely used for the off-line derivatization of primary and secondary
amines (only primary amines when using OPA.)2,11-14 Since the
determination of amphetamine and methamphetamine is of great
interest in toxicologic and forensic fields, the potential of the
described approach has been tested by analyzing these drugs in
urine at their therapeutic concentrations.
Derivatization Reagents. Concentrations of the derivatization
reagents were selected from the literature,12-15 the ratio of
concentration of derivatization reagent to the concentration of analyte
in the samples being in the 60-300 range. NQS solutions (0.5%,
w/ v) were prepared daily by dissolving the pure compound in
water. OPA solutions were prepared by dissolving 0.05 g of the
pure compound in 0.5 mL of methanol and 0.1 mL of 2-mercap-
toethanol. This solution was further diluted to 10 mL with to 15
mM boric acid solution. The boric acid solution was adjusted to
pH 10 with 10% NaOH. This reagent is stable at least for 3 days
when protected from light and stored at 2 °C. FMOC solutions
(20 mM) were prepared by dissolving the pure compound in
acetonitrile.
EXPERIMENTAL SECTION
For the NQS and FMOC determinations, a 4% bicarbonate
buffer (adjusted to pH 10 with 10% NaOH) was also used.
Columns and Mobile P hases. The precolumn (20 mm ×
2.1 mm i.d.) was dry-packed with a Hypersil ODS-C18 (30 µm) or
a SynChropack C18 (30-70 µm) stationary phase (Hewlett-
Packard). Purified water was used for washing the precolumn in
both sample cleanup and derivatization steps. A Hypersil ODS-
C18, 250 mm × 4 mm i.d. (5 µm) (Hewlett-Packard) or a
LiChrospher 100 PR 18, 125 mm × 4 m i.d. (5 µm) (Merck,
Darmstat, Germany) column was used as an analytical column.
Chromatographic conditions used for separation of the amphet-
amine derivatives are summarized in Table 1.
In all instances, the mobile phases were prepared daily, filtered
with a nylon membrane, (0.45 µm; Teknokroma, Barcelona,
Spain), and degassed with helium before use.
Off-Line Derivatization. For the NQS method, 0.5 mL of
reagent and 0.5 mL of buffer were added to 1 mL of sample. After
a given reaction time, the mixture was subjected to liquid-liquid
extractions with three 2 mL aliquots of n-hexane-ethyl acetate
(1:1). The organic phase was evaporated to dryness by heating
at 80 °C, and the residue was reconstituted in 2 mL of acetonitrile-
water (1:1).
Apparatus. The chromatographic system used consisted of
two quaternary pumps (Hewlett-Packard, 1050 Series, Palo Alto,
CA), an automatic sample injector (Hewlett-Packard, 1050 series),
and two high-pressure six-port valves (Rheodyne Model 7000). A
diode array (Hewlett-Packard, 1040 series) or a fluorescence
(Hewlett-Packard, 1046 series) detector linked to a data system
(Hewlett-Packard HPLC Chem Station, Dos Series) was used for
data acquisition and storage.
Reagents. All the reagents were of analytical grade. Aceto-
nitrile, ethyl acetate (Scharlau, Barcelona, Spain), and n-hexane
(Panreac, Barcelona, Spain) were of HPLC grade. Sodium 1,2-
naphthoquinone-4-sulfonate, â-phenylethylamine hydrochloride,
methamphetamine hydrochloride, and amphetamine sulfate were
obtained from Sigma (St. Louis, MO). o-Phthaldialdehyde, and
propylamine hydrochloride (Fluka, Buchs, Switzerland), 9-fluo-
renylmethyl chloroformate (Aldrich, Steinheim, Germany), sodium
hydrogen carbonate (Probus, Badalona, Spain), sodium hydroxide,
boric acid (Panreac), and 2-mercaptoethanol (Merck, Hoenbrunn,
Germany) were also used.
P reparation of Solutions. Amphetamine Solutions. Stock
solutions (1000 µg/ mL) of each compound were prepared in
water. Working solutions were prepared from the stock solutions
by dilution with water. These solutions were stored in the dark
at 2 °C.
For the OPA method, 0.5 mL of sample was added to 0.25 mL
of water and 0.25 mL of derivatization reagent; for derivatizations
with FMOC, 0.5 mL of sample was added to 0.25 mL of carbonate
buffer and 0.25 mL of FMOC reagent. For the OPA and FMOC
methods, 1 mL of acetonitrile was added to the reaction mixture
after the reaction time.
(11) Camp´ıns-Falco´, P.; Sevillano-Cabeza, A.; Molins-Legua, C. J. Liq. Chromatogr.
1 9 9 4 , 17, 731.
(12) Kinberger, B. J. Chromatogr. 1 9 8 1 , 213, 166.
(13) Maeder, G.; Pelletier, M.; Haerdi, W. J. Chromatogr. 1 9 9 2 , 593, 9.
(14) Camp´ıns-Falco´, P.; Bosch-Reig, F.; Sevillano-Cabeza, A.; Molins-Legua, C.
Anal. Chim. Acta 1 9 9 4 , 287, 41.
(15) Camp´ıns-Falco´, P.; Molins-Legua, C.; Herra´ez-Herna´ndez, R.; Sevillano-
Cabeza, A. J. Chromatogr. 1 9 9 5 , 663, 235.
Analytical Chemistry, Vol. 68, No. 5, March 1, 1996 735

Figure 1. Schematic representation of the system used for the on-
line cleanup plus derivatization of amphetamines.
In all instances, derivatizations were carried out at ambient
temperature and a 50 µL aliquot of the final solutions was directly
injected into the analytical column. Each sample was assayed in
triplicate.
On-Line derivatization. The setup used for the on-line
derivatization of the samples is shown in Figure 1. A 50 mL
aliquot of sample was injected into the precolumn; next, the
precolumn was flushed with 2 mL of water (at a flow rate of 1
mL/ min) for sample cleanup. Meanwhile, the autosampler was
programmed to take variable volumes of derivatization reagent
and 25 µL of buffer (this latter solution only for the NQS and
FMOC methods) from different vials and to mix them in the
needle. The reagent mixture was injected into the precolumn
immediately after sample cleanup. After injection, pump 1 was
stopped for a given period of time (reaction time). For the NQS
method, pump 1 was activated after the reaction time to eliminate
the excess of reagent by flushing the precolumn with water (at a
flow rate of 1 mL/ min). Finally, V1 was rotated, so the derivatives
were transferred to the analytical column by means of the eluent
from pump 2.
When urine samples were derivatized with NQS or OPA, the
precolumn was flushed after the analytical separation with 1 mL
of ethyl acetate, 2 mL of n-hexane, and again 1 mL of ethyl acetate.
In that case, an additional valve (V2) was incorporated to the
system, so the eluent was sent directly to waste.
At the end of each assay, valves were turned to the original
positions to regenerate and reequilibrate both the precolumn and
the analytical column. Rotation of the valves was manually
performed.
Recovery Studies. The efficiency of the on-line sample
cleanup and derivatization steps was calculated by comparing the
peak areas obtained for the injection of 50 µL of standard solution
samples (10 µg/ mL) in the described system, with the values
obtained for direct injections of the off-line derivatized extracts
containing the equivalent amount of analytes. Each concentration
was assayed in triplicate.
Urine Samples. Untreated urine samples were spiked with
amphetamine or methamphetamine reproducing concentrations
in the 0.4-4.0 and 0.5-10.0 µg/ mL intervals, respectively.
Volumes of 1 mL of these samples were placed into glass injection
vials, and 50 µL was injected onto the chromatographic system.
Figure 2. UV chromatograms obtained for aqueous solutions of
amphetamines after off-line (a) and on-line (b) derivatization with NQS.
Fluorescence chromatograms obtained for aqueous solutions of
amphetamines after off-line (c) and on-line (d) derivatization with OPA,
and after off-line (e) and on-line (f) derivatization with FMOC.
Concentration of each compound, 10 µg/mL; reaction time, 10 min;
derivatization reagent volume used for the on-line derivatization, 25
µL. (â-PEA ) â-phenylethylamine; AMP ) amphetamine; MET )
methamphetamine). For experimental details, see text.
and derivatizations are performed off-line. These peaks are most
probably due to partial hydrolysation of the derivatization reagents
at pH 10, which is necessary to obtain satisfactory derivatization
rates. Hydrolysis of derivatization reagents is one of the most
important obstacles for derivatization in aqueous solutions,
particularly at low analyte concentrations.7,8,13,16 As can be seen
in Figure 2, this effect is more important when the reaction is
carried out in the precolumn. In that case, additional peaks are
observed, even when pure water is processed; the number and
intensity of these peaks depend on the stationary phase used in
the precolumn. Thus, they are most probably due to the presence
of impurities in the packing material or to degradation of the
packing itself at those basic pH.
RESULTS AND DISCUSSION
On-Line vs Off-Line Derivatization. The chromatograms
obtained for an aqueous mixture of the tested compounds after
off-line and on-line derivatization are compared in Figure 2. As
can be see in this figure, peak broadening introduced by the
described setup is negligible.
The efficiency of the derivatization is also dependent on the
packing material. This is illustrated in Table 2, which shows the
Various unknown peaks are observed in the chromatograms
when analyzing blank (water) and standard amphetamine solutions
(16) Kwakman, P. J. M.; Koelewijn, H.; Kool, I.; Brinkman, U. A. Th.; De Jong,
G. J. J. Chromatogr. 1 9 9 0 , 511, 155.
736 Analytical Chemistry, Vol. 68, No. 5, March 1, 1996

a mixture of amphetamines (concentration of each compound, 10
µg/ mL) were comparable to those obtained for solutions contain-
ing a single drug. This means that the three amphetamines can
be simultaneously quantified.
Table 2. Recoveries of Amphetamines from Aqueous
Solutions after On-Line Cleanup plus Derivatization
Using Hypersil and SynChropack Packingsa
recovery (n ) 3) (%)
Derivatization with OP A. OPA reacts rapidly at ambient
temperature with amphetamine (Figure 3c), and its derivative can
be transferred to the analytical column immediately after injection
of the reagent, resulting in a very rapid procedure. No significant
differences in the responses of this compound were observed for
volumes of reagent in the 10-75 µL interval (Figure 3d). In order
to minimize peak responses corresponding to hydrolysation
byproducts, a reagent volume of 15 µL was selected for urine
studies.
Hypersil packing
AMP MET
SynChropack packing
â-PEA AMP MET
17 ( 2
26 ( 3
derivtzn
reagent â-PEA
NQS
OPA
FMOC
97 ( 7 103 ( 6 27 ( 5
129 ( 7 76 ( 1
96 ( 4 98 ( 3 96.6 ( 0.2 11.3 ( 0.8 11 ( 5 21 ( 2
12 ( 3 12 ( 1
31 ( 2
a
Concentration of analytes, 10 µg/ mL; reaction time, 10 min;
derivatization reagent volume used for the on-line derivatization, 25
µL. (â-PEA ) â-phenylethylamine; AMP ) amphetamine; MET )
methamphetamine).
Derivatization with FMOC. The reaction with FMOC is also
very rapid, and there are no significant differences in the reaction
rates within the 0-10 min reaction time interval (Figure 3e).
However, recoveries lower than expected were observed for
FMOC volumes higher than 20 µL (see Figure 3f). This can
explained by losses of the amphetamines and/ or their derivatives
by breakthrough, due to the presence of acetonitrile in the
derivatization reagent solution. At the present concentration (20
mM), FMOC is partially soluble in water. However, in order to
avoid precipitation in the needle of the autosampler during dilution
with the buffer, FMOC solutions were prepared in acetonitrile.
Satisfactory results are obtained by injecting 15 µL of reagent. As
for the NQS method, the three amphetamines tested can be
simultaneously quantified.
Analysis of Urine Samples. On the basis of the above
studies, the determination of amphetamine and methamphetamine
in urine at their therapeutical levels was performed, using a
precolumn packed with a Hypersil material. Optimal conditions
for each derivatization reagent are summarized in Table 3.
For the NQS and (by minor extension) for the OPA methods,
the responses drastically decrease after a few injections, when
urine samples instead of water solutions are processed. In our
experience, the same solid-phase cartridge or precolumn can be
repeatedly used for trapping amphetamines in urine prior to their
off-line derivatization. Therefore, the decrease in the reaction rates
can be explained by the irreversible adsorption of urinary
components to the precolumn, which makes the retention of the
derivatization agent and/ or the retention of the derivatives more
difficult. To overcome this problem, several eluents of different
polarities were tested for cleaning of the precolumn after every
urine injection. Best results were obtained by using acetate-n-
hexane. Therefore, pump 2 was programmed to deliver 1 mL of
ethyl acetate, 2 mL of n-hexane, and again 1 mL of ethyl acetate
ethyl (at a flow rate of 1 mL/ min) after the analytical separation.
In order to protect the analytical column from the desorbed
impurities, an additional valve (V2 in Figure 1) was incorporated
in the setup, so the washing eleuent was sent directly to waste.
Under the described configuration, the reequilibration of the
analytical column after processing a sample takes a few minutes,
but it is carried out during cleanup and derivatization of the next
one. For the FMOC method, cleaning of the precolumn with 1
mL of acetonitrile provided reproducible responses, so the final
procedure can be simplified.
percentage of drugs transformed for the two packings evaluated
relative to the amount of drug transformed in the off-line
procedure. Best results are obtained for the Hypersil packing,
which exhibits the highest loading capacity. Similar behavior was
observed when derivatizations were performed off-line in ODS
solid-phase extraction cartridges (data not shown). ODS packings
have been successfully used for the selective retention of un-
derivatized amphetamines.15 Therefore, the poor recoveries
observed are most probably due to the low retention of the
derivatization reagent and/ or the derivatives formed. For the OPA
method, the recovery of â-phenylethylamine in a Hypersil material
is unacceptable high. This can be explained by the interference
produced by an hydrolysation byproduct. Therefore, a Synchro-
pack stationary phase should be used for the determination of
this compound.
Following these results, in further experiments a Hypersil
packing was used, and the OPA method was only applied to the
derivatization of amphetamine. The time of reaction and the
derivatization reagent volume were optimized for the on-line
procedure. The results of these studies are shown in Figure 3.
Derivatization with NQS. Total conversion of amphetamines
into their NQS derivatives usually involves high temperatures and
reaction times.14 However, we observed that at a pH of ∼10,
reaction rates are satisfactory at ambient temperatures.9 Stable
responses are obtained for â-phenylethylamine and amphetamine
when reaction times equal or higher than 10 min are used (Figure
3a). The secondary amine methamphetamine exhibits reaction
rates lower than those obtained for â-phenylethylamine and
amphetamine (primary amines); however, if reaction times equal
or higher than 10 min are used, the sensitivity observed is
satisfactory for most applications concerning to the determination
of this compound.11
The excess of NQS must be eliminated before linking the
precolumn to the analytical column. Otherwise severe interfer-
ence with interesting compounds is observed. Indeed, analyte
responses can be increased by increasing the volumes of NQS
injected (Figure 3b). However, reagent volumes higher than 25
µL are not recommended, because in such a case, the flushing
stage must be prolonged to eliminate the excess of reagent. For
reagent volumes in the 10-25 µL interval, satisfactory chromato-
grams were obtained when the precolumn was flushed with 5 mL
of water (at a flow rate of 1 mL/ min). A reaction time of 10 min
in combination with a derivatization reagent volume of 25 µL was
selected as the best option for derivatization of interesting
compounds. Under these conditions, the responses obtained for
In Table 4 are shown relevant analytical data of the described
procedures. Although rotation of the valves was manually
performed, the reproducibility achieved is comparable to that of
most other LC methods involving on-line derivatizations.4 How-
Analytical Chemistry, Vol. 68, No. 5, March 1, 1996 737

Figure 3. Effects of the reaction time and derivatization reagent volume on peak areas: (a, c, e) effect of the reaction time for NQS, OPA, and
FMOC methods, respectively; (b, d, f) effect of the reagent volume for NQS, OPA, and FMOC methods, respectively. (â-PEA )
â-phenylethylamine; AMP ) amphetamine; MET ) methamphetamine). For experimental details, see text.
ever, the on-line to off-line responses obtained for methamphet-
amine in the NQS method are unacceptable low. This is in
agreement with the fact that slow kinetic reactions of secondary
amines into solid supports provide lower rates compared with the
analogous solution derivatization.4,9 In this instance, the recovery
can be improved by extending the reaction time or by performing
the derivatization at higher temperatures.
Utility of the System. The main advantage over previously
published procedures is the possibility of performing sample
cleanup, derivatization, and elimination of the excess of deriva-
tizing agent (if necessary) within the same system and using
conventional ODS packings. In the described assay, the limiting
step is the chromatographic separation. However, separation of
a sample can be performed during sample cleanup plus derivati-
zation of the next one. Although untreated samples are directly
injected, both the precolumn and the analytical column are
effectively equilibrated after an analysis. In all instances, at least
40 samples can be processed without replacing the precolumn.
Best sensitivity is achieved with FMOC. In addition, the
procedure can be simplified when this latter reagent is used, and
amphetamine and methamphetamine can be quantified in a single
run, the total analysis time being 12 min. Therefore, FMOC would
738 Analytical Chemistry, Vol. 68, No. 5, March 1, 1996

Table 3. Conditions of the On-Line Cleanup plus Derivatization of Amphetamines in Urine
derivtzn
reagent
Sample
cleanup
Vderivtzn reagent
(µL)
Vbuffer
(µL)
reaction time
(min)
cleaning of the
precolumn after derivtzn
cleaning of the
precolumn after anal. sepn
NQS
2 mL of water
2 mL of water
2 mL of water
25
15
15
25
10
5 mL of water
1 mL of ethyl acetate
2 mL of n-hexane
1 mL of ethyl acetate
1 mL of ethyl acetate
2 mL of n-hexane
OPA
0
1 mL of ethyl acetate
1 mL of acetonitrile
FMOC
25
0
Table 4. Analytical Data for the On-Line Determination of Amphetamine and Methamphetamine in Urine
linearity precision (%)
derivtzn
reagent
recovery
intraday
interday
limit of detection
(ng/ mL)
total anal.
time (min)
analyte
AMP
(n ) 3) (%)
y ) a + bx
Sxy
n
(n ) 6)
(n ) 15)
NQS
101 ( 3
31 ( 3
a ) -14.5 ( 3.6
b ) 73.7 ( 1.5
a ) -0.7 ( 3.0
b ) 19.4 ( 0.5
a ) -6.7 ( 4.6
b ) 79.8 ( 2.0
a ) 3.2 ( 1.1
b ) 15.2 ( 0.5
a ) 15.1 ( 1.3
b ) 15.0 ( 0.2
6.55
12
3
4
7
8
8
8
25
50
10
1
35
MET
AMP
AMP
MET
6.10
8.46
2.07
2.48
11
11
12
12
5
6
7
4
OPA
77 ( 5
20
12
FMOC
98 ( 3
86.4 ( 0.2
1
a
AMP ) amphetamine; MET ) methamphetamine. b Determined at half of highest concentration in tested range.
In principle, the described methodology could be used for the
formation of diastereomeric derivatives, for example, by using a
homochiral thiol in the OPA method or a chiral active chlorofor-
mate. Coupling of a chiral column to the derivatization precolumn
could be also possible. Both possibilities are currently under
investigation in our laboratory.
CONCLUSIONS
This study illustrates the potential of conventional ODS
precolumns for the on-line sample cleanup and derivatization of
solutes in the analysis of biological samples prior to LC analysis.
The described approach can be used with different derivatization
reagents, but operating conditions should be optimized according
to the kinetics of each reaction. Efficiency of the derivatization
mainly depends on a proper selection of the precolumn stationary
phase, the major limitation being the presence of hydrolysation
byproducts.
Figure 4. Fluorescence chromatograms obtained for blank (a) and
spiked with amphetamine and methamphetamine (b) urine samples.
Concentration of analytes: amphetamine, 2.5 µg/mL; methamphet-
amine, 5.0 µg/mL. (AMP ) amphetamine; MET ) methamphet-
amine). For experimental details, see text.
Compared with off-line derivatization methods, the described
setup facilitates the analysis and provides better reproducibility,
since no manipulation of the samples is involved. In contrast to
solid-phase reagents, an excess of derivatization reagent is
necessary; however, consumption of reagents can be reduced by
using higher reaction times or higher tempratures. The described
approach is fully automatable, only requiring electrically controlled
switching valves.
be the reagent of choice for routine determinations of amphet-
amine and methamphetamine in urine. As an example, typical
chromatograms obtained for blank and spiked urine samples
derivatized with FMOC are shown in Figure 4.
Under normal conditions, methamphetamine is partially dem-
ethylated to amphetamine, the methamphetamine-to-amphetamine
ratio being generally between 3 and 10 in urine samples.17-20 About
30-40% of amphetamine is excreted unchanged in urine.17 Since
amphetamine and methamphetamine are the predominant forms
in urine, the present method can be considered suitable for most
applications concerning these compounds.
ACKNOWLEDGMENT
The authors are grateful to the ClCyT for financial support
received for the realization of Project SAF 95-0586.
On the other hand, enantiomeric analysis of amines is of
interest since the pharmacological activities of the isomers differ.
Received for review May 25, 1995. Accepted December 4,
1995.X
(17) Beckett, A. H.; Rowland, M. Nature 1 9 6 4 , 204, 1203.
(18) Shinichi, S.; Takako, I.; Tetsukichi, N. J. Chromatogr. 1 9 8 3 , 267, 381.
(19) Tsuchihashi, H.; Nakajima, K.; Nishikawa, M.; Shiomi, K.; Takahashi, S. J.
Chromatogr. 1 9 8 9 , 467, 227.
AC9505076
(20) Binder, S. R.; Regalia, M.; Biaggi-McEachern, M.; Mazhar, M. J. Chromatogr.
1 9 8 9 , 473, 325.
X Abstract published in Advance ACS Abstracts, January 15, 1996.
Analytical Chemistry, Vol. 68, No. 5, March 1, 1996 739