Chemistry Reference
In-Depth Information
falling out of favor, and is not used by the ICH, but is instead addressed in guideline
Q2 (R1) [4] under the discussion of intermediate precision (Section 4.3.2.2, within-
laboratory variations: different days, analysts, equipment, etc.) and reproducibility
(Section 4.3.2.3, between-laboratory variations from collaborative studies).
4.3.3 S
PecIfIcIty
Specificity is the ability to measure accurately and specifically the analyte of inter-
est in the presence of other components that may be expected to be present in the
sample. It takes into account the degree of interference from other active ingredients,
excipients, impurities, degradation products, etc. Specificity in a method ensures that
a peak's response is due to a single component (no peak overlaps). Specificity for a
given analyte is commonly measured and documented by resolution, plate number
(efficiency), and tailing factor.
For identification purposes, specificity is demonstrated by either the ability to dis-
criminate between other compounds in the sample or by comparison to known refer-
ence materials. For assay and impurity tests, specificity can be shown by the resolution
of the two most closely eluted compounds. These compounds usually are the major
component or active ingredient and a closely eluted impurity. If impurities
are
avail-
able, it must be demonstrated that the assay is unaffected by the presence of spiked
materials (impurities or excipients). If the impurities
are not
available, the test results
must be compared to a second, well-characterized procedure. For assay, the two results
are compared directly; and for impurity tests, the impurity profiles are compared.
Comparison of test results will vary with the particular method, but may include visual
comparison as well as retention times, peak areas (or heights), peak shape, etc.
Starting with the publication of
USP 24
, and as a direct result of the ICH process,
it is now recommended that a peak-purity test based on photodiode array (PDA)
detection or mass spectrometry (MS) be used to demonstrate specificity in chro-
matographic analyses. Modern PDA technology is a powerful tool to evaluate speci-
ficity [11]. PDA detectors can collect spectra across a range of wavelengths at each
data point collected across a peak, and through software processes, each spectrum
can be compared to determine peak purity. Used in this manner, PDA detectors
today can distinguish minute spectral and chromatographic differences not readily
observed by simple overlay comparisons, even at low levels as shown in Figure 4.4.
More information on using PDA detectors to evaluate specificity using spectral con-
trast techniques can be found in Chapter 7, Section 7.3.1.4.
PDA detectors can be limited in the evaluation of peak purity on occasion by a
lack of UV response, as well as by the noise of the system and the relative concentra-
tions of interfering substances. Also, the more similar the spectra are, and the lower
the relative absorbances, the more difficult it is to distinguish co-eluted compounds.
MS detection overcomes many of these limitations of the PDA, and in many labo-
ratories it has become the detection method of choice for method validation. MS
can provide unequivocal peak purity information, exact mass, and structural and
quantitative information. The combination of both PDA and MS in a single HPLC
instrument can provide valuable orthogonal information to help ensure that interfer-
ences are not overlooked during method validation.
