Results and discussion
Figure 8: Validation set. Standard error of prediction (SEP) = 0.1216 mg. The N – 1 phenylephrine (PE) samples (10 mg PE–HCl,
0 mg chlorpheniramine maleate [CPM]) were tested in the validation (but not included in the SEP) and predicted results near
zero, demonstrating specificity for CPM. HPLC is high-performance liquid chromatography, NIR is near infrared.
Figure 3 shows the raw diffuse transmission NIR spectra from the calibration set. By taking the second derivative of the spectra,
as shown in Figure 4, the baseline was normalized and the spectral features were enhanced. The derivative was smoothed with
a segment of 10 and a gap of 0.
Partial least squares (PLS) regression was used to develop the prediction model, which uses principal-component analysis and
is a variation of principle-component regression. The correct number of principal components or factors was determined by
the Vision software supplied with the NIR instrument by determining where the predicted residual error sum of squares (PRESS)
reaches a minimum (6). Figure 5 shows the PRESS plot for the CPM PLS model selecting seven factors.
Figure 6 plots the PLS loadings that show where CPM concentration is highly correlated with spectral data. The structure of
chlorpheniramine is superimposed on the plot. Aromatic C–H stretching is evident in the correlation plot at 1140 nm. The resulting
CPM model had an R
value of 0.9811 and a standard error of calibration (SEC) of 0.0739 mg. The "one-left-out" cross-validation demonstrated
good predictability with a standard error of cross validation (SECV) of 0.0901 mg.
Figure 9: Predicted residual error sum of squares (PRESS) plot of partial least squares factors used to predict phenylephrine–HCl
concentration using three factors.
Figure 7 shows the NIR predicted CPM level versus the HPLC values. Twenty-five percent of the total tablets scanned were randomly
left out of the calibration for prediction model validation. Figure 8 shows the NIR predictions of the validation set versus
the HPLC values for the CPM active ingredient. Although the model did not include zero-level CPM, the N-1 PE samples were tested in the validation. These samples contained an average of 8.95 mg PE–HCl and no CPM. The N-1 PE samples predicted at 0.0 mg CPM, thus demonstrating the specificity of the model for CPM. The standard error of prediction
(SEP) was 0.1216 mg for the validation samples, not including the N-1 PE samples. The relative standard deviation (RSD) for the HPLC results for N-1 CPM tablets was 11.95%, and they were not used in the model.
Figure 10: Partial least squares loadings showing where phenylephrine–HCl is highly correlated with spectral data. The structure
of phenylephrine is superimposed.
Figure 9 is a plot of the PRESS leading to a model with three factors for PE–HCl. Figure 10 plots the PLS loadings spectra
that show where PE–HCl concentration is highly correlated with spectral data. The structure of PE is superimposed. Although
O–H and N–H bonds are present in the structure, the aromatic C–H stretch is dominant at 1140 nm, which is shown in blue as
the first factor (7).
Figure 11: Calibration set. R2 = 0.9427, standard error of calibration = 0.3682 mg, and standard error of cross validation = 0.3807 mg. HPLC is high-performance
liquid chromatography, NIR is near infrared, and PE is phenylephrine.
The resulting model had an R
value of 0.9427 and an SEC of 0.3682 mg. The SECV value was 0.3807 mg. Figure 11 shows the NIR-predicted PE–HCl level versus
the HPLC values. Figure 12 shows the NIR predictions of the validation set versus the HPLC values for the PE–HCl active. Although
the model did not include zero-level PE–HCl, the N-1 CPM samples were tested in the validation. These samples contained an average of 3.72 mg CPM and no PE–HCl (the target
was 4.0 mg CPM and 0.0 mg PE–HCl). They predicted slightly less than 0 mg for the N-1 samples because the spectra were distinctly different, as shown in Figure 3. The prediction near 0 mg demonstrated that
the model is unique for PE–HCl. The SEP was 0.4758 mg for the validation samples (not including the N-1 samples). The RSD for the HPLC results for N-1 PE–HCl tablets was 8.15%, and they were not used in the model.
Figure 12: Validation set. Standard error of prediction = 0.4758 mg. The N –1 chlorpheniramine maleate (CPM) samples (0 mg
phenylephrine [PE]–HCl, 4 mg CPM) were tested in the validation. The model predicted results were slightly below zero, demonstrating
specificity for PE–HCl. HPLC is high-performance liquid chromatography, and NIR is near infrared.
Table II shows the repeatability results for a separate set of T-5 label claim tablets nominally containing 4.0 mg CPM and
10.0 mg PE–HCl. The standard deviation for predicting the CPM active level of these five measurements of the same tablet was
0.0104 mg. The standard deviation of the repeatability for the same tablet for nominal 10.0 PE–HCl was 0.0129 mg (see Table
Table II: Chlorpheniramine maleate (CPM) repeatability.