Also the spectral homogeneity of the peak of interest must be taken into
consideration.
Diode array spectra at least three points across the peak should
be taken to ensure the peak is spectrally homogenous see Figure 8-14. If the
peak is not spectrally homogenous, the overlay of the spectra will show
368 METHOD DEVELOPMENT
Figure 8-13. Optimal wavelength selection for API and related impurities.
Figure 8-14. Determination of peak homogeneity: Diode array detection (DAD).
(Reprinted from reference 10, with permission.)
distinct differences (see Figure 8-15). However, even if the diode array spectra
do overlay, this does not absolutely ensure that the peak does not contain any
coeluting impurities, because the impurities could have similar diode array
spectra and/or if there is a low level of a coeluting species with a different
diode array spectrum, it may not be determined by this approach. In these
cases, MS detection needs to be employed to ensure MS spectral homogene-
ity. MS spectra are taken across the peak and the MS spectra across the peak
should not show the presence of any other coeluting species of different
masses. This does not absolutely ascertain that the peak is homogeneous since
isomers of the same compound will have the same [M + H] and is indistin-
guishable from the parent compound. Also, the impurity that may be coelut-
ing may not have an appreciable ionization efficiency at the particular
mobile-phase and mass spectrometric conditions.
An example of where using diode array detection may not be helpful is
shown in Figure 8-16. Note that for this reaction mixture (convergent synthe-
sis) the desired product 1 has the same diode array spectra as synthetic pre-
cursors 2 and 3. If these two synthetic precursors had coeluted with 1, they
would not have been able to be deconvoluted. This stresses the importance
of running LC-MS in a parallel to diode array studies during method
development.
8.4.3 Solution Stability and Sample Preparation
It should be determined if the drug substance being analyzed is stable in solu-

of caution here is that filter studies should be performed to ensure that no
adsorption of the compound on the filter is observed. This is particularly the
case with protein and peptide samples. Note that for proteins and peptides the
impact of centrifugation (speed and time) must be investigated because this
may lead to increased aggregate formation. Also, for protein and peptides the
initial concentration of the sample could also have an impact on the concen-
tration gradient of the sample in the centrifuge tube, and the concentration of
the top, middle, and bottom portions should be assessed.
The effectiveness of the syringe filters is largely determined by their ability
to remove contaminants/insoluble components without leaching undesirable
artifacts (i.e., extractables) into the filtrate. Extractables are often the result
of inappropriate material construction and improper handling of the device
during the manufacturing process. Particular attention should be paid to
potential extractables from the membrane and housing material. The sample
preparation procedure should be adequately described in the respective ana-
lytical method that is applied to a real in-process sample or a dosage form for
subsequent HPLC analysis. The analytical procedure must specify the manu-
facturer, type of filter, and pore size of the filter media. Also, it must be known
if the particular filter type is compatible with the type of analyte, organic
solvents, and pH of the solution to be filtered.
The following procedure may be used to determine if there is any absorp-
tion on the filter.A stock solution is prepared at the target concentration. One
aliquot of the stock solution is centrifuged, and other aliquots from the cen-
trifuged stock solution are filtered through the desired filters (pre-wet with
5 mL of diluent) and the results compared. If any additional peaks are observed
in the filtered samples, then the diluent must be filtered to determine if a leach-
able component is coming from the syringe filter housing/filter. In Figure 8-17
a solid oral dosage form was prepared at 1 mg/mL concentration. The initial
stock solution was centrifuged (no filter) and two additional samples from the
centrifuged solution were filtered with a nylon filter and a cellulose filter. The

% S
rel
0.3% 0.0% 1.4% 0.0% 1.4%
Centrifuged samples
1 99.1 0.080 0.080 0.025 0.042
2 98.9 0.081 0.080 0.025 0.042
3 100.0 0.078 0.078 0.025 0.042
Mean 99.3 0.080 0.079 0.025 0.042
%S
rel
0.6%
1.9% 1.5% 0.0% 0.0%
adequate and does not cause any specific absorption of the active and/or
impurities.
Other considerations for sample preparation include incorporation of
methanol in the sample preparation scheme, especially if a second dilution is
used (check for sample reactivity). The impact on peak shape (diluent/mobile
phase mismatch for components with k < 2) should also be considered. Sample
preparation usually constitutes approximately 70% of solvent usage, and
incorporating methanol for routine sample preparation can lead to reduction
in solvent costs.
8.4.4 Choice of Stationary Phase
Ideally for a reversed-phase separations, the retention factors (k) for all com-
ponents in a sample should lie between 1 and 10 to achieve separation in a
reasonable time. For a given stationary phase the k of a particular component
can be controlled by changing the solvent composition of the mobile phase.
However, the impact of eluent composition will depend on the type of sta-
tionary phase and the nature of the components in the mixture. In reversed-
phase HPLC the most common solvent mixtures are: water and acetonitrile,
water and methanol, and water and THF. The elution strength increases as the