In-Sample Calibration Curve Using Multiple Isotopologue Reaction Monitoring of a Stable Isotopically Labeled Analyte for Instant LC-MS/MS Bioanalysis and Quantitative Proteomics

Abstract

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.

Eliminating Preparation of Multisample External Calibration Curves and Dilution of Study Samples Using the Multiple Isotopologue Reaction Monitoring (MIRM) Technique in Quantitative LC-MS/MS Bioanalysis

“…15 N 4 13 C 2 -Daclatasvir was used as the assay SIL-IS for the multisample external calibration curves and one-sample multipoint external calibration curves. It was also used as an SIL analyte to construct ISCCs for each sample as reported previously . The samples were extracted with liquid–liquid extraction using a Janus Mini liquid handler (PerkinElmer, Waltham, MA).…”

“…This methodology was initially used to support several microdosing absolute bioavailability studies by properly arranging stable isotopic labeling plans (number and positions of labeling) for both intravenous drugs and assay internal standards (IS) in order to mitigate the isotopic interferences in LC-MS/MS assays . Recently, by using this approach, we successfully proposed and demonstrated a novel methodology to construct an in-sample calibration curve (ISCC) in every study sample on the basis of the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) and the measured MS/MS peak areas in the corresponding MIRM channels for instant LC-MS/MS bioanalysis . With the advantages of the eliminated need to use authentic matrices and multisample external calibration curves, simplified workflow, and improved throughput, it is expected that the MIRM-ISCC-LC-MS/MS methodology will be gradually adopted in targeted quantitative proteomics and bioanalysis of biomarkers, as well as pharmacokinetic (PK) and toxicokinetic (TK) sample analysis in drug discovery and development.…”

“…In this paper, we propose two applications, both using the MIRM technique, to eliminate the steps for preparation of multisample external calibration curves and dilution of study samples, respectively. The first application is proposed to eliminate the need for preparation of multisample external calibration curves by constructing one-sample multipoint external calibration curves using the MIRM technique (Figure ).…”

Design of a Quantitative LC-MS Method for Residual Toxins Adenylate Cyclase Toxin (ACT), Dermonecrotic Toxin (DNT) and Tracheal Cytotoxin (TCT) in Bordetella pertussis Vaccines

“…The MRM workflow (Figure 1) combined MS-based protein quantitation using surrogate AQUA peptide standards for ACT and DNT, with traditional small molecule quantitation using an external standard curve for TCT. Performance metrics were re-assessed in the MRM method using multiple isotopologue reaction monitoring (MIRM) [34]. The mass resolution setting for the 1st quadrupole (Q1) was adjusted from unit mass resolution with full width at half maximum set to 0.75 (FWHM = 0.75) to a custom MIRM setting (FHWM = 0.5).…”

Matrix Matching in Quantitative Bioanalysis by Lc–MS/MS a Dream or a Reality?

“…In realizing the impracticality of exact matrix matching, Gu et al [12] recently reported a novel in-sample calibration curve (ISCC) methodology. In this approach, multiple naturally occurring isotopologs of stable isotopically labeled IS spiked in the sample at known concentration is used to build the calibration curve in each sample.…”