A novel methodology of in-sample calibration curves (ISCC) using multiple isotopologue reaction monitoring (MIRM) of multiple naturally occurring isotopologue transitions of a stable isotopically labeled (SIL) analyte for instant liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis of biomarkers, biotherapeutics, and small-molecule compounds is proposed and demonstrated for the first time. The theoretical isotopic abundances of the SIL analyte in its MIRM channels can be accurately calculated based on the isotopic distributions of its daughter ion and neutral loss. The isotopic abundances in these MIRM channels can also be accurately measured with a triple quadrupole mass spectrometer. By spiking a known amount of a SIL analyte into each study sample, an ISCC can be established based on the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) in the selected MIRM channels of the SIL analyte and the measured MS/MS peak areas in the corresponding MIRM channels in each individual study sample. The analyte concentration of each study sample can then be calculated individually with the ISCC instantly without using an external calibration curve. The MIRM− ISCC−LC-MS/MS methodology was evaluated and demonstrated in this work with the examples of quantitation of a protein biomarker in human and monkey serum processed with immunocapture and trypsin digestion; three surrogate peptides in trypsin-digested human colon tissue homogenates; and a small-molecule drug in human and rat plasma extracted with liquid− liquid extraction. The potential applications of the MIRM−ISCC−LC-MS/MS methodology in quantitative proteomics, clinical laboratories, and other areas are also discussed in this paper. Without the need for using external calibration curves, this novel MIRM−ISCC−LC-MS/MS methodology can provide accurate and reliable bioanalysis in many potential applications, especially for cases where authentic matrices for external calibration curves are not available.
Preparation of multisample
external calibration curves and dilution
of study samples are critical steps in bioanalytical sample processing
for quantitative liquid chromatography–tandem mass spectrometry
(LC-MS/MS) based bioanalysis of small-molecule compounds, biotherapeutics,
and biomarkers, but they can be time-consuming and prone to error.
It is highly desired to simplify or eliminate these two steps in order
to improve the assay throughput and robustness. While multisample
external calibration curve preparation using authentic matrices can
be eliminated with a previously reported in-sample calibration curve
(ISCC) approach using multiple isotopologue reaction monitoring (MIRM)
of a stable isotopically labeled (SIL) analyte, dilution of study
samples is still inevitable due to limited LC-MS/MS assay ranges.
In this work, a one-sample multipoint external calibration curve and
isotope sample dilution, both using MIRM of an analyte, for quantitative
LC-MS/MS based bioanalysis are proposed and demonstrated. By spiking
a known amount of an analyte into one blank authentic matrix sample,
a one-sample multipoint external calibration curve in an authentic
matrix can be established on the basis of the relationship between
the calculated theoretical isotopic abundances (analyte concentration
equivalents) and the MS/MS responses in the corresponding MIRM channels.
This one-sample multipoint external calibration curve can be used
in the same way as the traditional multisample external calibration
curve for quantitative LC-MS/MS-based bioanalysis. As isotopic abundance
in each MIRM channel can be calculated and measured accurately, isotope
sample dilution can be achieved by simply monitoring one or a few
of the MIRM channels of the analyte in addition to the most abundant
MIRM channel for study samples. While the most abundant MIRM channel
(isotopic abundance of 100%) is used for the quantitation of samples
having concentrations within the assay calibration curve range, less
abundant MIRM channels (isotopic abundance of IA%) can be used for
the quantitation of samples having concentrations beyond the assay
upper limit of quantitation (ULOQ), resulting in isotope dilution
factors (IDF) of 100%/IA%. The approaches of one-sample multipoint
external calibration curve and isotope sample dilution were evaluated
and demonstrated in this work with an example of the quantitation
of daclatasvir in human plasma extracted with liquid–liquid
extraction. Using these approaches together with the MIRM-ISCC methodology,
accurate and reliable LC-MS/MS bioanalysis can be achieved without
the need of preparation of multisample external calibration curve
and dilution of study samples.
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