the flow rate (mL/min) to determine the time (in minutes) needed. Therefore,
the lower the flow rate, the longer the equilibration time.
Some typical equilibration times for various column dimensions are shown
in Table 8-7; however, these should only be used as a guide. If complete equi-
libration is not achieved, early eluting components may show differences in
retention from run to run. An experiment could be run such that three dif-
ferent methods could be run with different equilibration times. For example,
if a 15-cm × 4.6-mm i.d. column and a flow rate of 1mL/min was used, then
the equilibration times for the three methods would be 5 min (3 CV), 9min (5
CV), and 11 min (6 CV) equilibration times, respectively. If the retention of
the early eluting components are consistent (less than 1% variation in reten-
tion time) in all three methods, then the lowest equilibration time could be
used. However, if the early eluting components show greater variation in their
retention time with the 5-min equilibration time compared to the methods
with the 9- and 11-min equilibration time, then an equilibration time of greater
than 5 min is warranted. Optimization of the optimal equilibration time is
required for reproducible methods.
Other considerations include differences in dwell volumes from the differ-
ent HPLC systems.The dwell volume should be determined for all the systems
in the laboratory and based on these determinations, this should be factored
into the calculation of the equilibration time. For example, if the maximum
dwell volume of all the systems in a particular laboratory to which the method
is transferred to is 2 mL and you are running on an instrument at 1 mL/min
that has a dwell volume of 1 mL, then you should add an extra minute of equi-
libration time. This becomes extremely important during method transfers
where the instruments in the receiving laboratory may be different.
8.5 METHOD DEVELOPMENT APPROACHES
8.5.1 If Analyte Structure Is Known
Determine if analytes are acidic, basic, or neutral. This will allow the
chromatographer to choose a pH such that the analyte is being analyzed
METHOD DEVELOPMENT APPROACHES 385

should be on the order of 5–20 µL and the concentration of the analyte should
be 0.5–1 mg/mL. This corresponds to approximately 5–20 µg injected on
column. On the other hand, for neutral analytes higher analyte loading
such as 50–100 µg maybe used since nonideal interactions with the stationary
phase are less prevalent. Note that for ionizable compounds, especially
basic compounds when analyzed in their ionized state, higher mass on column
may lead to mass overload of “hot spots” on the bonded phase and poor peak
efficiencies may be observed. Try not to load more than 10 µg on column
for basic compounds. Usually greater loading capacity is obtained for basic
compounds when they are analyzed in their neutral state. Note that for
columns with larger inner diameters such as 4.6 mm, larger sample loads may
be acceptable.
Once the probe gradient is run, check the diode array purity; and if LC-MS
is available, run as well to check for peak homogeneity. If you have any known
precursors or impurities, run them as well to ensure resolution from the main
component and to make sure they are adequately retained. The main analyte
should elute between k 2–5. If the main component elutes at k = 2–5 and is
spectrally pure and the impurities all elute k > 1, the method is complete. If
the retention factor of the impurities is below 1, then an isocratic hold at the
initial organic composition should be implemented until the minor component
(impurity) elutes k > 1 and then a linear gradient can be implemented. The
method could be further optimized by increasing the flow rate as long as the
backpressure limitation of the system has not been reached. A general rule of
thumb is that the backpressure should not exceed 85% of the maximum back-
pressure for a particular HPLC system.
If resolution is not achieved between a critical pair, the use of a shallower
gradient can be investigated. If that does not increase the resolution, then a
longer column (15-cm column, packed with 3-µm particles of the same sta-
tionary phase type) should be used with a reduced flow rate of 0.7 mL/min
(due to backpressure limitations).

shifts in the hydro-organic media.
8.5.2 If Method Is Being Developed for Separation of Active and
Unknown Component
Define the criteria for the method such as the LOQ, maximum run time, wave-
length detection, and so on. Look at the structure of the target analyte (esti-
mate pK
a
) or use ACD (advanced chemistry development) and determine the
best pH to run the method. Try to use shorter columns for gradient scouting
experiments (5 cm × 4.6mm ) packed with 3-µm columns or use a high-
pressure system (max pressure 15,000 psi) with 10-cm × 2.1-mm, 1.7-µm parti-
cles. Use 35–45°C as starting temperature. If pH scouting studies are needed,
run a probe linear gradient using 0.2 v/v% phosphoric acid on a short column
(5-cm × 4.6-cm column) to determine the isocratic conditions for the
pH studies. Run pH studies isocratically to determine the desired pH region
to understand the behavior of the impurities in the analyte mixture. The
desired pH region of the aqueous phase is the pH region where the retention
of the components in the mixture do not significantly change their retention
as a function of the pH of the aqueous phase. Track impurities using diode
array if possible. Run a linear gradient at a pH within the desired pH region
and hold at high organic concentration on 5-cm × 4.6-mm column. If
you obtain sufficient resolution, then you are finished. If you need more
METHOD DEVELOPMENT APPROACHES 387
resolution, then use a 15-cm × 3-mm i.d. column. If resolution is obtained,
then you are finished. If desired resolution/selectivity is not obtained, then
screen different organic modifiers/different stationary phase types. Note
that the separation of the critical pair may be obtained on an alternate
stationary phase that offers additional selectivity. In addition to the weak
dispersive types of interaction that are available on a C8 or C18 phase,
phenyl phases may provide additional interactions such as π–π-type interac-

•
Carbohydrates
•
Proteins and peptides
The DryLab model utilized in Waters AMDS has additional requirements:The
number of sample components should not exceed 12; peak area% should be
greater than 1%. These requirements are necessary to achieve greater predic-
tion accuracy only. Any discrepancies could be corrected manually in DryLab
using the data entry screen by manually entering the retention of the compo-
nents from the scouting runs (to assign the peaks with a certain number).
DryLab has been used for the method development of model drug candidates
388 METHOD DEVELOPMENT
and their degradation products, by optimization of temperature and gradient
slope, and the historical review on the milestones and concepts in the devel-
opment of DryLab software is given in references 20–23.
8.5.3 Defining System Suitability
System suitability parameters with their respective acceptance criteria
should be a requirement for any method. This will provide an added level
of confidence that the correct mobile phase, temperature, flow rate, and
column were used and will ensure the system performance (pump and
detector). This usually includes (at a minimum) a requirement for injection
precision, sensitivity, standard accuracy (if for an assay method), and retention
time of the target analyte. Sometimes, a resolution requirement is added for
a critical pair, along with criteria for efficiency and tailing factor (especially
if a known impurity elutes on the tail of the target analyte). This is added
to ensure that the column performance is adequate to achieve the desired
separation.
System suitability requirements for retention time, efficiency, resolution,
and tailing factor are set based on prior method challenging experiments and
prior method development experience. This is a dynamic process; and as the

Tailing factor (5% peak height) for peak A ≤ 1.5
•
Rt for peak A must be 12.0 ± 1.3 min
•
Rt for peak B must be 21 ± 1.0 min
•
The S/N of the LOQ solution (0.05%) for both actives A and B must be
≥10:1
In the second example, if it is known that a potential degradation product can
occur and will elute close to the active, a resolution requirement should be set
for this critical pair.When trying to set a resolution requirement between crit-
ical pairs of impurities, standard samples containing the critical pair should be
readily available. However, standard samples may not be available with all
critical impurities so the standard may be spiked with authentic impurities. If
authentic impurities are not available or are in limited quantity, then the drug
substance may be degraded in solution using mild stress conditions to produce
a decomposition product or products that can be used to define a resolution
requirement for a critical pair. The mild stress conditions should produce
decomposition products in situ in a fast time scale. In the following example
in Figure 8-23, the drug substance was stressed with 3% hydrogen peroxide
for 1 hr at 25°C and 80°C to generate impurity A. At 80°C, suitable degrada-
tion was obtained to determine the resolution requirement between impurity
A and the active B (target analyte). This requirement was set because it was
postulated that this drug substance could be readily oxidized. Indeed in
solid state stability studies, minor amounts of the impurity A (oxidized impu-
rity) were observed under accelerated conditions (40°C/75% RH, 3 months).
390 METHOD DEVELOPMENT
Figure 8-22. Waters XBridge 150- × 3.0-mm, 3.5-µm C18 column. Column temperature
40°C [(A) 90%: 20 mM ammonium phosphate buffer: 10% MeCN, (B) 100% MeCN].
Gradient: 10% A to 85% B over 38 min. Flow: 0.6 mL/min.

working at pH values at or near the pK
a
values of the API will lead to sepa-
rations that may not be robust and (b) what influence the pH has on the inher-
ent retention of intermediate compound A and related synthetic by-products.
These experiments could be conducted as an exercise to further understand
the effect of pH on the retention of the species in the sample of interest since
the synthetic by-products may have different ionizable functionalities then the
parent compound (intermediate).
METHOD DEVELOPMENT APPROACHES 391
Figure 8-23. In situ degradation for generation of system suitability solution.
8.5.4.1 Gradient Screening. An initial method development was performed
using a Phenomenex Luna C18 (2) column with acetonitrile as the organic
mobile-phase component, and the aqueous portion was a 10 mM ammonium
monohydrogen phosphate buffer adjusted to pH 2 with phosphoric acid. Ini-
tially, a linear gradient was used from 60% to 80% MeCN with a hold at 80%
MeCN for 10 minutes. An early eluting component was observed close to the
void volume using this probe gradient. Also, no peaks were seen to elute
during the 80% MeCN isocratic hold. Therefore, a new gradient method
(shown in Figure 8-24) with an initial isocratic hold to retain the more polar
species and removal of the latter isocratic hold at 80% MeCN was used. The
new method employed an isocratic hold at 50% MeCN for 5 min, and then a
linear gradient was run from 50% MeCN to 80% MeCN from 5 to 25 minutes.
Note that a 150- × 4.6-mm column was used, but a 150- × 3.0-mm could have
been easily used with proper adjustment of the flow rate.
8.5.4.2 pH Screening Study. Once the probe gradient method is selected, a
pH study can be conducted. The pH study in gradient mode was carried out
using 10 mM ammonium monohydrogenphosphate as a buffer. The
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