ments for Pharmaceuticals for Human Use (ICH) guidelines.
These guidelines outline reporting, identification and qualification
requirements for impurities in a drug, specifically advising on
those impurities potentially arising from degradation products of
the drug substance, ranging from catalyst byproducts in manufacturing to interactions with packaging materials. Furthermore,
the ICH guidelines propose threshold values for the control and
quantitation of impurities based on the maximum daily dose of a
substance in the final product.
In regard to manufacturing, the ICH recommends the use of less
toxic solvents in the manufacturing process, in case of solvent integration into the drug, and sets pharmaceutical limits for residual
solvents in drug products. By providing a consistent global policy
for limiting impurities both qualitatively and quantitatively in drug
products and ingredients, these guidelines can help direct how
current approaches can be upgraded to exceed recommendations.
However, if current approaches overlook compounds due to method inadequacies, the guidelines cannot be met, and analysis could
result in potential inclusion of impurities that affect the efficacy
and safety of the drug, as well as the health of the patient.
More comprehensive analysis
In order to gain insight into how current methods might be
improved for quality drug assessment, researchers compared
the commonly used UHPLC-UV/Vis approach to an alternative
detection method, charged aerosol detection (CAD), in evaluating
impurities in the chemotherapy drug paclitaxel. Applying CAD
to impurity detection could help overcome some of the obstacles
met with UV/Vis detection, since CAD response does not require a
chromophore for detection and can provide a uniform response independent of chemical structure. It was hypothesized that applying
both detection methods could utilize the strengths of each, providing a more comprehensive profile and more universal response
across all types of compounds.
In this study, calibration measurements of paclitaxel and two
known compounds associated with the drug were assessed. When
testing impurities for pharmaceutical applications, calibration standards are useful to quantify and confirm the identity of a known
compound. However, calibration standards in early drug discovery are not always available, creating additional challenges with
certain analytical methods. To accommodate this, CAD was used
to allow for a single calibration and enable comparison of known
compounds to unknown impurities.
A stock solution of paclitaxel and the two related compounds
was generated for use as a calibration standard measurement. A
series of dilutions based on the stock solution were analyzed in
three consecutive runs with blank injection runs between each
dilution to reduce carry over. This generated a single calibration
curve, where all three standards displayed similar linearity. Highly
similar linear calibration is key to ensure proper quantitation of
known compounds as well as accurate relative quantitation of un-
known impurities. Detector response was also evaluated with UV
detection and CAD to determine the relative differences in each
method. The inherent sensitivity in UV detector response to chem-
ical structure was highlighted in the results, in which data showed
a variable response among the three analytes measured at similar
concentrations. CAD response in these circumstances remained
consistent across the three analytes, indicating an independent
response from chemical structure. Given the linearity of calibra-
tion and uniform response, these measurements demonstrated the
ability of the UHPLC and CAD system to apply directly to drug
assessment and comparison of unknown compounds in a sample
to a single calibration.
Since CAD response is known to be affected by mobile phase
composition, an inverse gradient compensation was implemented
to assess the ability for uniform response across a typical drug
gradient elution. This turned out to be an important addition for
CAD response, as results without gradient compensation demon-
strated an underestimation of compounds prior to active phar-
maceutical ingredient (API) detection and an overestimation of
compounds after the API due to the changing organic composition
within a gradient. However, with gradient compensation, a more
uniform response of detected analytes over the course of the gradi-
ent elution is noticeable, which minimizes quantitation errors.
Comparison of CAD with inverse gradient compensation to
UV response suggests that a combination of both methods offers
a more universal response and the most comprehensive evaluation
of a sample, detecting and allowing measurement of all impurities
and related compounds present in the sample.
UV detection can identify volatile compounds in a sample
unidentified by CAD, while CAD can detect compounds lacking
a chromophore that cannot be determined by UV detection. By
combining these two detection methods with UHPLC, improved
identification and measurement of pharmaceutical samples overcomes challenges inherent to each, as well as confidently meets
ICH guidelines to ensure the safety of a drug.
The future of impurities testing
Pharmaceuticals, such as paclitaxel, are analyzed to ensure
that the level of impurities in the final drug product are at a safe
and consistent level by accurately measuring the compounds and
implementing removal processes downstream from the analysis.
Additional testing of the combined approach on different drug
samples would further qualify UHPLC-UV-CAD as a valid method
for impurity analysis.
Analytical methods applied to detect impurities within a therapeutic drug, for cancer or other diseases, should have both the
selectivity to separate impurities from each other and the sensitivity
to quantify low levels of analytes. Given that single methods each
have their own unique obstacles that make them less effective to use
alone, combining and supplementing methods with complementary
approaches can overcome individual challenges to ensure that a
complete analysis for impurities can be performed on any drug.
Combining multiple high performance techniques can improve the overall
view of drug components and impurities.