5 research outputs found
Selecting the Correct Weighting Factors for Linear and Quadratic Calibration Curves with Least-Squares Regression Algorithm in Bioanalytical LC-MS/MS Assays and Impacts of Using Incorrect Weighting Factors on Curve Stability, Data Quality, and Assay Performance
A simple procedure for selecting
the correct weighting factors
for linear and quadratic calibration curves with least-squares regression
algorithm in bioanalytical LC-MS/MS assays is reported. The correct
weighting factor is determined by the relationship between the standard
deviation of instrument responses (σ) and the concentrations
(<i>x</i>). The weighting factor of 1, 1/<i>x</i>, or 1/<i>x</i><sup>2</sup> should
be selected if, over the entire concentration range, σ
is a constant, σ<sup>2</sup> is proportional to <i>x</i>, or σ is proportional to <i>x</i>, respectively.
For the first time, we demonstrated with detailed scientific reasoning,
solid historical data, and convincing justification that 1/<i>x</i><sup>2</sup> should always be used as the weighting factor
for all bioanalytical LC-MS/MS assays. The impacts of using incorrect
weighting factors on curve stability, data quality, and assay performance
were thoroughly investigated. It was found that the most stable curve
could be obtained when the correct weighting factor was used, whereas
other curves using incorrect weighting factors were unstable. It was
also found that there was a very insignificant impact on the concentrations
reported with calibration curves using incorrect weighting factors
as the concentrations were always reported with the passing curves
which actually overlapped with or were very close to the curves using
the correct weighting factor. However, the use of incorrect weighting
factors did impact the assay performance significantly. Finally, the
difference between the weighting factors of 1/<i>x</i><sup>2</sup> and 1/<i>y</i><sup>2</sup> was discussed. All of
the findings can be generalized and applied into other quantitative
analysis techniques using calibration curves with weighted least-squares
regression algorithm
Practical and Efficient Strategy for Evaluating Oral Absolute Bioavailability with an Intravenous Microdose of a Stable Isotopically-Labeled Drug Using a Selected Reaction Monitoring Mass Spectrometry Assay
A strategy of using selected reaction monitoring (SRM)
mass spectrometry
for evaluating oral absolute bioavailability with concurrent intravenous
(IV) microdosing a stable isotopically labeled (SIL) drug was developed
and validated. First, the isotopic contribution to SRM (ICSRM) of
the proposed SIL drug and SIL internal standard (IS) was theoretically
calculated to guide their chemical synthesis. Second, the lack of
an isotope effect on drug exposure was evaluated in a monkey study
by IV dosing a mixture of the SIL and the unlabeled drugs. Third,
after the SIL drug (100 μg) was concurrently IV dosed to humans,
at <i>T</i><sub>max</sub> of an oral therapeutic dose of
the unlabeled drug, both drugs in plasma specimens were simultaneously
quantified by a sensitive and accurate SRM assay. This strategy significantly
improves bioanalytical data quality and saves time, costs, and resources
by avoiding a traditional absolute bioavailability study or the newer
approach of microdoses of a radio-microtracer measured by accelerator
mass spectrometry
Calculation and Mitigation of Isotopic Interferences in Liquid Chromatography–Mass Spectrometry/Mass Spectrometry Assays and Its Application in Supporting Microdose Absolute Bioavailability Studies
A methodology for the accurate calculation and mitigation
of isotopic
interferences in liquid chromatography–mass spectrometry/mass
spectrometry (LC–MS/MS) assays and its application in supporting
microdose absolute bioavailability studies are reported for the first
time. For simplicity, this calculation methodology and the strategy
to minimize the isotopic interference are demonstrated using a simple
molecule entity, then applied to actual development drugs. The exact
isotopic interferences calculated with this methodology were often
much less than the traditionally used, overestimated isotopic interferences
simply based on the molecular isotope abundance. One application of
the methodology is the selection of a stable isotopically labeled internal standard (SIL-IS) for an LC–MS/MS
bioanalytical assay. The second application is the selection of an
SIL analogue for use in intravenous (IV) microdosing for the determination
of absolute bioavailability. In the case of microdosing, the traditional
approach of calculating isotopic interferences can result in selecting
a labeling scheme that overlabels the IV-dosed drug or leads to incorrect
conclusions on the feasibility of using an SIL drug and analysis by
LC–MS/MS. The methodology presented here can guide the synthesis
by accurately calculating the isotopic interferences when labeling
at different positions, using different selective reaction monitoring
(SRM) transitions or adding more labeling positions. This methodology
has been successfully applied to the selection of the labeled IV-dosed
drugs for use in two microdose absolute bioavailability studies, before
initiating the chemical synthesis. With this methodology, significant
time and cost saving can be achieved in supporting microdose absolute
bioavailability studies with stable labeled drugs
Fully Validated LC-MS/MS Assay for the Simultaneous Quantitation of Coadministered Therapeutic Antibodies in Cynomolgus Monkey Serum
An LC-MS/MS assay was developed and
fully validated for the simultaneous
quantitation of two coadministered human monoclonal antibodies (mAbs),
mAb-A and mAb-B of IgG4 subclass, in monkey serum. The total serum
proteins were digested with trypsin at 50 °C for 30 min after
methanol denaturation and precipitation, dithiothreitol reduction,
and iodoacetamide alkylation. The tryptic peptides were chromatographically
separated with a C18 column (2.1 × 100 mm, 1.7 μm) with
mobile phases of 0.1% formic acid in water and acetonitrile. Four
peptides, a unique peptide for each mAb and two confirmatory peptides
from different antibody domains, were simultaneously quantified by
LC-MS/MS in the multiple reaction-monitoring mode. Stable isotopically
labeled peptides with flanking amino acids on C- and N-terminals were
used as internal standards to minimize the variability during sample
processing and detection. The LC-MS/MS assay showed lower limit of
quantitation (LLOQ) at 5 μg/mL for mAb-A and 25 μg/mL
for mAb-B. The intra- and interassay precision (%CV) was within 10.0%
and 8.1%, respectively, and the accuracy (%Dev) was within ±5.4%
for all the peptides. Other validation parameters, including sensitivity,
selectivity, dilution linearity, processing recovery and matrix effect,
autosampler carryover, run size, stability, and data reproducibility,
were all evaluated. The confirmatory peptides played a critical role
in confirming quantitation accuracy and the integrity of the drugs
in the study samples. The robustness of the LC-MS/MS assay and the
data agreement with the ligand binding data demonstrated that LC-MS/MS
is a reliable and complementary approach for the quantitation of coadministered
antibody drugs
Selective Reaction Monitoring of Negative Electrospray Ionization Acetate Adduct Ions for the Bioanalysis of Dapagliflozin in Clinical Studies
Dapagliflozin
(Farxiga), alone, or in the fixed dose combination
with metformin (Xigduo), is an orally active, highly selective, reversible
inhibitor of sodium-glucose cotransporter type 2 (SGLT2) that is marketed
in United States, Europe, and many other countries for the treatment
of type 2 diabetes mellitus. Here we report a liquid chromatography–tandem
mass spectrometry (LC–MS/MS) bioanalytical assay of dapagliflozin
in human plasma. A lower limit of quantitation (LLOQ) at 0.2 ng/mL
with 50 μL of plasma was obtained, which reflects a 5-fold improvement
of the overall assay sensitivity in comparison to the previous most
sensitive assay using the same mass spectrometry instrumentation.
In this new assay, acetate adduct ions in negative electrospray ionization
mode were used as the precursor ions for selective reaction monitoring
(SRM) detection. Sample preparation procedures and LC conditions were
further developed to enhance the column life span and achieve the
separation of dapagliflozin from potential interferences, especially
its epimers. The assay also quantifies dapagliflozin’s major
systemic circulating glucuronide metabolite, BMS-801576, concentrations
in human plasma. The assay was successfully transferred to contract
research organizations (CROs), validated, and implemented for the
sample analysis of pediatric and other critical clinical studies.
This assay can be widely used for bioanalytical support of future
clinical studies for the newly approved drug Farxiga or any combination
therapy containing dapagliflozin