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Hernandez,
P. Campns
R. Herraez
C. Molins Legua
, Facultad de Qumica
Abstract
The generalised H-point standard addition method (GHPSAM) is proposed for isolating the analytical signal of an analyte
from the signal of an unknown sample. Samples containing two and three coeluting compounds have been analysed. The
accuracy of the predictions depends on the shape of the analyte and interferent spectra but not on the degree of
chromatographic overlap. This methodology involves the location of linear intervals for the unknown interference spectrum
from the spectrum of the sample. Once the linear interval has been found the selection of three wavelengths within the
interval will allow the cancellation of the signal of the unknown interferent. The method has been applied to the
determination of diuretics, amphetamines and phenols in water. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Generalised H-point standard addition method; Detection, LC; Diuretics; Phenols; Amphetamines
1. Introduction
The physical separation of the components of a
mixture provided by chromatographic techniques
allows the analyst the simultaneous determination of
several analytes in complex matrices. Use of highefficiency columns has become commonplace due to
their commercial availablity. In spite of the use of
these columns when analysing real mixtures of great
complexity, some unresolved peaks may appear.
Several methods have been developed for the de*Corresponding author. Tel.: 134-6-3983-002; fax: 134-6-3864436.
Falco)
tection of overlapped peaks [14] since the occurrence of overlapped peaks may be much more
prevalent than expected [5]. When overlapping peaks
are detected the mobile phase and the column
characteristics must be adjusted to improve resolution. However, when a large number of compounds
are present in the sample, it may be impossible to
resolve all the peaks under the same chromatographic conditions. On the other hand, the optimising
process is a tedious task that increases the analysis
time.
In the literature there are a number of reports
where numerical methods are used to resolve overlapping peaks. Among these methods are self-modelling curve resolution (SMCR), generalised rank
0021-9673 / 99 / $ see front matter 1999 Elsevier Science B.V. All rights reserved.
PII: S0021-9673( 99 )00636-6
362
(1)
l2 2 l1
l3 2 l 2
p 5 ]]; q 5 ]]
l3 2 l1
l3 2 l 1
(2)
(3)
363
Fig. 1. Spectral features for the X analyte (bumetanide), the Y interferent (ethacrynic acid) and the S sample in the wavelength interval
selected as linear for the interferent.
(4)
0
0
where A X1
, A 0X2 and A X3
are the absorbances of the
analyte in the sample at the wavelengths 1, 2 and 3,
respectively. eX1 , eX2 and eX3 are the molar absorption coefficients of the analyte at the wavelengths 1,
2 and 3, respectively [20]. The abscissa of the
H-point includes the qDA S( 2,1) 2pDA S( 3,2 ) increment
and then cancels the interferent contribution to the
signal. This increment value is only related with
364
Fig. 2. Graphic representation of the lines qDA S( 2,1 ) (h) and pDA S( 3,2 ) (s) for the different amounts of added analyte.
(5)
(6)
(7)
(8)
(9)
(10)
A 99
Y 50
(11)
C 0X 2 C RX R0
R0
A S99 2 A X 5 ]]]
AX
C RX
365
(15)
(12)
3. Experimental
(13)
(14)
3.1. Apparatus
A Hewlett-Packard Model 1040A liquid chromatograph, equipped with a diode array detector linked to
a data system (Hewlett-Packard Chemstation) was
used for data acquisition and storage. The system
was coupled to a quaternary pump (Hewlett-Packard,
1050 Series) with a 25-ml sample loop injector for
diuretics and amphetamines and a 100-ml sample
loop injector for phenols.
The detector (Hewlett-Packard, 1100 series) was
set to collect a spectrum every 640 ms (over the
range 200400 nm and every 4 nm). All the assays
were done at ambient temperature.
For the analysis of diuretics (mixtures of acetazolamideamiloride and ethacrynic acidbumetanide)
the range of measured wavelengths was set to 222
302 nm since the analysis of these drugs in biological fluids are, generally, carried out in this zone.
The mixtures of phenols and amphetamines were
measured in the ranges 222398 and 272600 nm,
respectively. All the spectra were obtained at the
maximum of the peaks of the chromatograms.
366
3.2. Reagents
All reagents were of analytical grade. Methanol
(Scharlau, Barcelona, Spain) and acetonitrile (J.T.
Baker, Deventer, The Netherlands) were of HPLC
grade.
The following reagents were used: amiloride
hydrochloride (ICI-Pharma, Pontevedra, Spain), acet
azolamide (Cynamid Iberica,
Spain), amphetamine
sulphate (Sigma, St. Louis, MO, USA), methamphetamine hydrochloride (Sigma), b-phenylethylamine hydrochloride (Sigma), 1,2-naphtoquinone-4sulphonic acid sodium salt (Sigma), bumetanide
(Boehringer Ingelheim), ethacrynic acid (Sigma),
phenol, o-cresol and m-cresol (Merck, Darmstadt,
Germany) and 2-chlorophenol (Aldrich, Steinheim,
Germany).
Sodium hydroxide and sodium chloride (Panreac,
Barcelona, Spain), phosphoric acid (Probus,
Badalona, Spain), sodium dihydrogenphosphate
monohydrate (Merck), amonium hydroxide (Probus)
hydrochloric acid (Probus), acetate trihydrate (Probus), acetic acid (Probus) sodium hydrogencarbonate
(Probus) and propylamine hydrochloride (Fluka,
Buchs, Switzerland) were also used. Water was
distilled, deionized and filtered in 0.45-mm nylon
membranes (Teknokroma, Barcelona, Spain).
367
368
Fig. 3. Chromatogram at 275 nm of a mixture of 150 ppm of amiloride and 50 ppm acetazolamide, respectively. Insets: Spectra obtained at
the top of the peaks and location of the linear interval for the interferent in the determination of acetazolamide (the samples contained 50,
100, 150, 200 and 250 ppm of acetazolamide and 100 ppm of amiloride).
the determination of 2-chlorophenol, gives as possible linear intervals: 244255, 254264 and 266
284 nm. The comparison of the regression values for
the first and second derivatives suggested that the
real one was 266271 nm. The equations of the
regressions (A S9 2 A X9 ) vs. A X9 , and (A S99 2 A X99 ) vs. A X99
for this interval were: (A 9S 2 A 9X )50.01210.4554A X9 ,
99 )5 20.000810.46A X99 ,
r 2 50.9996 and (A 99
S 2AX
r 2 50.992. It can be seen that no significant differences between both slopes exists and that the intercept value for the second derivative regression is
nearly zero.
Bumetanide was determined in samples that also
contained ethacrynic acid as unknown interference.
Fig. 6 shows the plot of A S99 /e 0 (e 0 calculated from a
369
Fig. 4. Chromatogram at 275 nm of a mixture of 10 ppm of amphetamine, 10 ppm of methamphetamine and 10 ppm of b-phenylethylamine
(in order of elution). Inset: location of the linear interval for the interferent in the determination of methamphetamine.
370
Fig. 5. Chromatogram at 220 nm of a mixture of m-cresol, o-cresol and 2-chlorophenol (in order of elution) at the concentrations: 3.6, 4.8
and 6 ppm, respectively. Inset the recorded spectra at the top of each peak.
371
Fig. 6. Location of the linear interval for the interferent in the determination of bumetanide in presence of ethacrynic acid. The samples
contained 50, 100, 150, 200 and 250 ppm of bumetanide and 100 ppm of ethacrynic acid.
Table 1
Calculated values for the analyte concentration (mean6standard deviation) in the mixtures of acetazolamide and amiloride a
True acetazolamide
(ppm)
True amiloride
(ppm)
Measured acetazolamide
(mean6SD) (ppm)
Error
Measured amiloride
(mean6SD) (ppm)
Error
50
100
150
200
50
50
50
100
100
100
100
100
150
200
46.560.2
9761
15261
20461
27.0
23.0
1.3
2.0
24.6
23.2
0.6
95.460.4
145.260.5
198.960.2
Every result is the mean of seven predictions. Error5(mean measured value2true value)3100 / true value.
372
Fig. 7. Chromatogram at 275 nm of a mixture of ethacrynic acid and bumetanide. Absorbance ratio at 254 / 266 nm (s) and at 286 / 266 nm
(h) for a solution of composition: 50 ppm of bumetanide and 50 ppm of ethacrynic acid.
373
Table 2
Calculated values for bumetanide (mean6standard deviation) in the mixtures of bumetanide and ethacrynic acid (every value is the mean of
three predictions)
True bumetanide a
(ppm)
Measured bumetanide
(mean6SD) (ppm)
Error
50
100
150
200
50
100
150
200
250
50
100
150
200
250
50
50
50
50
50
50
50
50
50
100
100
100
100
100
49.660.5
10461
143.560.4
202.760.7
43.060.7
104.460.3
160.260.3
188.460.7
24361
5161
104.160.9
148.460.8
21362
23561
21.3
4.0
4.3
1.4
214.0
4.4
6.8
25.8
22.8
2.0
4.1
21.1
211.5
26.0
5. Conclusions
The usefulness of the GHPSAM has been proved
for the resolution of overlapped chromatographic
peaks when the interference is unknown. In contrast
with other proposed methods for chromatography the
results obtained do not depend on the degree of
Acknowledgements
The authors are grateful to the DGICYT (Project
No. PB 94-0984) for its financial support.
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