8
METHOD DEVELOPMENT
Rosario LoBrutto
8.1 INTRODUCTION
The primary focus of this chapter in on general approaches and considerations
toward development of high-performance liquid chromatography (HPLC)
methods for separation of pharmaceutical compounds, which may be applied
within the various functions in the drug development continuum. It is very
important to understand the aim of analysis and the requirements for a par-
ticular method to be developed. The aim of analysis of each HPLC method
may vary for each developmental area in the drug development process and
specific examples are given in Section 8.2.
General method development considerations that apply to all reversed-
phase methods are discussed in Section 8.3. These include properties of the
analyte, detector, mobile phase, stationary phase, and gradient considerations.
Building upon this knowledge, strategies for method development for target
analytes in which the structure is known and not known are given as general
guidelines. This material is reinforced with several method development case
studies emphasizing the approaches used and the shortcomings that were
encountered during the method development continuum. Also, a method
development flow chart for gradient separations is provided in Section 8.5.6
(Figure 8-37) which can be used as an excellent starting point for the devel-
opment of HPLC methods.
Also, throughout this chapter we focus on analytical challenges a pharma-
ceutical scientist encounters during method development; these include speed
347
HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto
Copyright © 2007 by John Wiley & Sons, Inc.
of separation, sample preparation, extraction issues, solution stability,
detection sensitivity (effect of pH), and separations for structurally similar
species.

the purity of the final compound and to avoid any undesired secondary reac-
tions that may carry forward in the downstream chemistry. Starting materials
can be classified as raw materials or key raw materials. The latter are raw mate-
rials in which some part of the raw material structure is incorporated into the
final structure of the drug substance. For the raw materials, usually an identi-
fication test and concentration of the reagent suffices although the purity of
these materials is sometimes also deemed as a necessity. However, for key raw
materials the purity of this reagent must be known. The analysis of the key
raw materials depends on the nature of the substance (i.e., volatility) and a
348 METHOD DEVELOPMENT
variety of methods could be used such as GC and HPLC.To ensure the quality
of the key raw materials, the level and type of impurities present in these mate-
rials need to be determined and appropriate specifications need to be set.
These can be determined by running a small-scale synthesis (i.e., front run)
with the key raw material; and if an intermediate with an acceptable purity is
obtained in the next step of the synthesis, the level and type of impurities
present in the key raw materials can be considered acceptable. Note that the
impurities may carry forward in the downstream chemistry and/or may react
to form new synthetic by-products. If these synthetic by-products are carried
forward to the final API, these impurities must be qualified in the appropri-
ate toxicological studies if they are above a certain level (also, any potential
genetoxic impurities must be identified and well-controlled with sensitive ana-
lytical methods). Different lots from the same manufacturer and/or lots of key
raw materials from different manufacturers are usually tested.
In the following example, the importance of determining the quality of the
key raw material is highlighted. Trimethoprim (TMP) (2-4-diamino-5-(3,4,5-
trimethoxybenzyl)pyrimidine) is an antibacterial, folic acid antagonist that is
usually used with sulfamethoxazole as a treatment for urinary tract infections.
Shown in Figure 8-1 is one synthetic scheme of TMP [1]. The starting mater-
ial that is used is 3,4,5-trimethoxy benzaldehyde (a key raw material). Depend-

In general, the detection and identification of impurities present in API and
formulations play an integral role in the drug development process and
methods need to be developed to adequately resolve these species and quan-
titate them. The International Conference on Harmonization (ICH) [3] has
published a guidelines on impurities in new drug substances and new drug
products (see Table 8-1). The acceptable limit of the impurities in drug sub-
stances is dependent upon the maximum daily dose and the qualification
threshold; however, lower thresholds are sometimes deemed necessary if the
TYPES OF METHODS 351
Figure 8-3. Trimethoprim (API) and two potential synthetic by-products. (Reprinted
from reference 2, with permission.)
TABLE 8-1. Reporting Thresholds of Impurities [3]
Maximum Reporting Identification Qualification
Daily Dose
a
Threshold
b,c
Threshold
c
Threshold
c
≤2 g/day 0.05% 0.10% or 1.0mg/day intake 0.15% or 1.0mg/day intake
(whichever is lower) (whichever is lower)
>2 g/day 0.03% 0.05% 0.05%
a
The amount of drug substance administered per day.
b
Higher reporting thresholds should be scientifically justified.
c
Lower thresholds can be appropriate if the impurity is unusually toxic.

mized and HPLC is used as a tool to monitor the reaction. It must be deter-
mined how long the reaction should proceed in order to form the desired
intermediate in good yield and for how long the intermediate is stable in solu-
tion prior to going to the next step (hold point stability).
These reaction monitoring analyses should be fast because the reaction
time scales may be in the order of minutes to hours. In-line flow injection
analysis or spectroscopic methods are sometimes used to monitor reactions
(reaction conversion) that are on the minute time scale and for reactions that
involve hazardous materials because by the time the samples are analyzed by
an off-line chromatographic method, the reaction has gone to completion
and/or undesired by-products may have been formed. However, off-line
HPLC may still be needed to determine the purity of the desired intermedi-
ate present in the reaction solvent before proceeding to the next step of the
reaction, and fast HPLC methods can be employed. If the reaction conversion
is along the time scale of an hour or greater, fast HPLC methods are used
implementing nonporous stationary-phase materials, monolithic columns, and
columns packed with sub-2 µm particles (uPLC, xPLC, fPLC) (more informa-
tion on fast HPLC methods is provided in Chapter 17). It is advantageous to
have short methods to analyze these reaction conversion samples. The in-
process samples may have to be diluted with an appropriate solvent to quench
the reaction and to have the desired components within the linear range of
the chromatographic method.The diluent must be chosen such that it does not
react with the components in the mixture. Also, all blank system suitability
and standards samples should be run on the chromatographic system prior to
injection of the reaction sample to ensure that the HPLC system is working
properly and that no downtime in the reporting of the results to the process
TYPES OF METHODS 353
Figure 8-5. Reaction monitoring of converting the starting material to intermediate.
chemist and/process engineer. For the more conservative chromatographer,
two HPLC systems can be set up in the event that an instrument breaks down

these layers is usually checked as well to ensure that the proper amount of
acid or base has been added to the reaction mixture either to quench the reac-
tion or to drive the desired product into the organic layer.
8.2.2.3 Purity of Intermediates. Determining the purity of the desired
product in the organic layers is important to ensure that an adequate number
of aqueous washes removed the unwanted by-products. This organic layer may
be carried forward to the next step without any further isolation. However, if
the intermediate will be isolated, then the purity and weight percent of the
isolated intermediate needs to be determined to ensure mass balance and
determine overall yield of the reaction. The purity of the intermediates needs
to be evaluated in order to determine if synthetic by-products generated in a
354 METHOD DEVELOPMENT