Journal of
MOLECULAR
STRUCTURE
ELSEVIER
Journal of Molecular Structure 348 (1995) 139-142
Assay of the active ingredient of an MRI contrast agent using Mid and Near
infrared spectroscopy and multivariate calibration
E. Harbo@,
I?. Klaeboea, C.J. Nielsen” and C.E. Sjogrenb
a Department of Chemistry, University of Oslo,
P.O. Box 1033 Blindern, N-0315 Oslo, Norway.
b Nycomed
Imaging AS, P.O. Box 4220 Torshov, N-0401 Oslo, Norway.
1. INTRODUCTION
Near Infrared (NIR) spectroscopy, combined with multivariate statistical calibration
methods, has rapidly gained acceptance as a quick and reliable way of quantitating properties
of a wide array of substances such as pharmaceuticals, petrochemicals, polymers and
foodstuffs [ 11. Rugged spectrometer designs and sampling probes based on optical fibres have
made NIR spectrometers the instrument of choice for many industrial applications. On the
other hand, the detailed molecular information contained in the ‘fingerprint’ region still
makes Mid-infrared (MIR) spectroscopy the preferred method for determining the identity
and morphology
of pharmaceutical substances. However, recent advances in Raman
spectroscopy may lead to a surge of interest for this particular technology in the industrial
environment [2,3].
In the present study both NIR and MIR spectroscopy have been investigated as possible
assay methods for OMNISCAN@ (Ny corned Imaging AS, Oslo, Norway), a non-ionic
paramagnetic contrast medium for magnetic resonance imaging (MRI). The contrast medium
is an aqueous solution of gadodiamide (GdDTPA-BMA), a gad o 1inium chelate of diethylene
triamine pentaacetic acid bis(methylamide). An excess of ligand is added in the form of the
corresponding calcium sodium chelate (caldiamide sodium) to prevent free gadolinium ions
to enter the solution by driving the dissociation equilibrium towards the gadolinium chelate.
2. MATERIALS AND METHODS
MIR spectra (4000-700 cm-‘, resolution 4 cm-l) were recorded on a Bruker IFS 66 FT-IR
spectrometer (Bruker GmbH, Germany) using a horizontal ATR unit (Graseby Specac, UK)
equipped with a 45’ ZnSe crystal. NIR spectra (2400-1200 nm) were recorded on a
Quantum 1200 Plus spectrometer (LT Industries, USA) using a fibre optic probe. The
concentration ranges investigated were 225345 mg/mL and 6-18 mg/mL for gadodiamide
and caldiamide sodium, respectively.
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140
Data treatment and calibration was carried out using the ‘Unscrambler’ software (CAM0
AS, Norway). Several methods for pretreatment
of the data (normalization
and
differentiation), as well as various multivariate calibration approaches, Principal component
regression (PCR) and Partial least squares regression (PLSl and PLS2), were tested in order
to arrive at the best possible calibration model. The calibration models were validated with
test sets consisting of spectra not contained in the calibration set. The long term precision
(reproducibility) of the MIR calibration models was furthermore investigated by predicting
the concentrations of a number of samples analyzed over an extended period of time. The
Root mean square error of prediction (RMSEP) obtained on this set of samples was used as
a measure of how well the calibration models performed.
3. RESULTS AND DISCUSSION
Since the two target compounds,
gadodiamide and caldiamide sodium,
differ only in the metal ion chelated
with the DTPA-BMA ligand, it is
obvious that only minor differences
are expected when spectra of the two
substances are compared. Fig. 1 shows
that this is indeed the case. The only
visible difference observed in the MIR
spectra of pure aqueous solutions of
the two compounds is a shift of the
band at oz. 1110 cm-‘. In the NIR
spectra no such differences could be
detected. This observation led us to
believe that the information content
of the MIR spectra would allow us to
predict
the
amounts
of both
components in a mixture. More subtle
spectral differences may of course be
present, both in the MIR and in the
NIR regions, but this information
may only be extracted and utilized for
calibration purposes by a multivariate
approach.
Absorbance
0.60 I4
1496
1303
1110
Wavenumberskm-1
Figure 1. Mid-infrared (MIR) spectra of aqueous
gadodiamide and caldiamide sodium.
3.1. NIR spectroscopy
The optimum calibration results with the NIR data set were obtained with a PLSl model
using four factors. No pretreatment was applied to the spectra. Fig. 2 shows a plot of the
predicted gadodiamide content versus known concentrations in the test set samples used to
validate the calibration model. Gadodiamide, which is the major constituent of the samples,
was successfully predicted with an RMSEP value of 4 mg/mL, i.e. 1.4% of the median of
the concentration range.
141
This estimate compares favourably with the reproducibility of the chromatographic test
method currently used as an assay of OMNISCAN@. However, the amount of caldiamide
sodium could not be predicted with sufficient precision by NIR spectroscopy. This is
interpreted as a failure of the broad overtone bands in the NIR region to reflect the subtle
differences between the two analytes.
In a mixture of two components, ideally two or three factors are expected to be
sufficient to model the structure in the data set. In the present case four factors were found
to give the optimum results, but a reasonable prediction was also obtained with two and
three factors (RMSEP values of ca. 6 mg/mL). However, since the NIR approach turned out
not to give a satisfactory quantitative calibration for caldiamide sodium, the attention was
focused on the MIR rather than a further optimization of the NIR procedure.
Predicted mg/mL (with 4 factors)
Predicted mg/mL (with 3 factors)
350
350
325
325
300
300
275
275
250
250
225
225 -i
225
250
275
300
325
350
Gadodiamide (known content) mg/mL
Figure 2. Gadodiamide content and
PLSl prediction with NIR data.
RMSEP 3.25
225
250
275
300
325
350
Gadodiamide (known content) mg/mL
Figure 3. Gadodiamide content and
PLSl prediction with MIR data.
3.2. Mid infrared spectroscopy
Figs. 3 and 4 show the quantitative results obtained with multivariate calibrations based
on MIR spectra. The plots show the predicted versus known concentrations in a set of
samples analyzed over an extended period of time. Optimum calibration results were
obtained by restricting the spectral region to the 1500-900 cm-’ ‘fingerprint’ range, followed
by normalisation and differentiation (first derivative) of the spectra. The results shown here
were obtained using PLSl calibration, but similar results were also obtained with PCR
models. In a discussion of the relative merits of the two calibration procedures [4] it is
claimed that the two approaches normally give similar results. However, a PLSl model may
in some cases have a better chance of leading to an acceptable model than the PCR
approach, particularly if the spectra (X-variables) contain a large variation irrelevant to the
modelling of the Y-variables (concentrations).
142
Using a PLSl model with three factors the gadodiamide content was predicted with an
RMSEP value of 3.3 mg/mL (Fig. 3), w h ich is somewhat better than the NIR results. An
important difference in the way the models were tested supports our conclusion: The MIR
prediction was carried out on a set of spectra
Predicted mg/mL (with 4 factors)
recorded over a period of several months, and
,,I
the RMSEP value thus obtained is an estimate
” . RMSEP 1.29
of the
expected
long
term
precision
(rtproducibility) of the MIR method. The
“.
14
spectra used to validate the NIR model, on the
other hand, were recorded on the same day.
12 Hence, the RMSEP value obtained with the
10 .
NIR data (4.0 mg/mL) is a measure of the
short term precision (repeatability) of the
’_
method, which with proper statistical control
6 .I
6
6
10
12
14
16
16
of the analytical procedure, is better than or
Caldiamide
Sodium
(known
content)
mg/mL
equal to the long term precision.
As opposed to the NIR models the amount
of the excipient, caldiamide sodium, could be Figure 4. Caldiamide sodium content
predicted with some degree of success by and PLSl prediction with MIR data.
models based on the MIR spectra. However,
the RMSEP value of 1.3 mg/mL obtained with
a four-factor PLSl model of the caldiamide sodium content (Fig. 4) corresponds to ca. 10%
of the median concentration value. This, clearly, does not represent a sufficiently high
precision for the MIR method to replace the titration method currently used to determine
the amount of caldiamide sodium in OMNISCAN@. The failure of the MIR calibration to
yield the desired result, despite the underlying spectral differences which were commented
upon initially (Fig. l), must be attributed to the large difference in concentration (a factor
of 20) between the two components in the mixtures studied here.
4. CONCLUSION
NIR and MIR spectroscopy combined with multivariate calibration were assessed as
potential quantitative methods for the determination of two, structurally very similar, metal
chelates in an aqueous mixture. Both methods gave a satisfactory prediction of the main
component, whereas the minor component could only be detected with MIR spectroscopy.
REFERENCES
1.
2.
3.
4.
D. A. Burns and E. W. Ciurczac (eds.), Handbook of Near-Infrared Analysis, Marcel
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B. Chase, Applied Spectroscopy 47 (1994) 14A.
F.J. Bergin and H.F. Shurvell, Applied Spectroscopy 43 (1989) 516.
H. R. Bjrarsvik and H. Martens, in Handbook of Near-Infrared Analysis,
D. A. Burns and E. W. Ciurczac (eds.), Marcel Dekker, New York, 1992.