Biomedical Engineering Reference
In-Depth Information
recovery or extract insolubility or nonideal chromatography conditions such as
matrix effects or an incompatibility with some combination of mobile phase, needle
wash, or sample reconstitution solvent. The systematic approach to troubleshoot an
inconsistent IS response is to perform intra- and intervial injection. If the inter and
intravial injections are very precise, it clearly indicates that the inconsistent IS
response is related to extraction. While this simple experiment does not conclu-
sively identify the actual source of the erratic internal standard behavior, it can at
least isolate it to one of two areas for further investigation.
This strategy can be demonstrated clearly in the following example. This is a
proprietary assay for the quantitation of two analytes (parent analyte and its sulfox-
ide metabolite) in human urine using two stable labeled internal standards. Because
of the extremely high linear range (5.00-100 mg/mL), a simple dilution of urine was
utilized for sample preparation. The initially developed method utilized normal
phase liquid chromatography [MonoChrom
®
Diol LC column (2.0 × 50 mm, 5 m m)]
and a single premixed mobile phase (2 % MeOH in MeCN with 0.1 % Formic acid)
under isocratic conditions. The internal standard response for the metabolite is
extremely imprecise (Fig.
9
). A quick intravial injection experiment outlined above
was conducted to identify that the primary source of the imprecision was due to
chromatographic condition. Because normal phase chromatography was used, the
more polar sulfoxide metabolite eluted before the parent analyte and was the only
analyte affected, suggesting that some early eluting interferences from the urine
(not phospholipids in this case) might be causing an undesired matrix effect.
Although an alternative and more selective extraction would likely solve this issue,
it was determined that the simple dilution procedure was still the most desirable
option due to the high concentration range. Thus, the final solution was to add a
switching valve into the LC program in which the column was backflushed with
strong organic solvent (100 % MeOH) at a high flow rate (1.0 mL/min) after the two
analytes had eluted. Using the new conditions, the same sample batch was injected
and a much improved internal standard response was obtained for the sulfoxide
metabolite (Fig.
10
) .
5
Method Validation
Regulated method validation must be conducted according to Method Validation
(MV) SOP which is typically written following FDA guidance and white papers for
regulated bioanalysis validation. The MV SOP describes various validation tests to
verify that a method is reliable and reproducible using calibration standards (STD)
and quality control (QC) samples. For a formal MV, a Principle Investigator (PI) or
Study Director (SD) is assigned for assuring that the validation tests meet the accep-
tance criteria and are adequate for the analysis of study samples. A Study Protocol
(SP) has to be written and approved
a priori
to initialing validation. Minimally, the
experiments, reference materials and calibration curve range(s) and statistical tests
