Simultaneous Quantitation and Discovery analysis: Combining targeted and untargeted metabolomics on Orbitrap mass spectrometers

Significance of the topic

The simultaneous integration of targeted and untargeted metabolomics addresses a central bottleneck in metabolic research: the trade-off between precise, quantitative measurement of predefined analytes and broad, discovery-driven profiling of unknown metabolites. By combining high-resolution mass spectrometry with sophisticated acquisition and data-analysis workflows, laboratories can produce hypothesis-driven quantitative data while retaining the ability to discover and annotate novel compounds from the same sample injection. This has direct practical relevance for clinical biomarker work, nutritional studies, microbiome research, metabolic engineering and any setting where limited sample material, throughput, or retrospective mining capability are important.

Goals and study overview

This technical note introduces the SQUAD (Simultaneous Quantitation and Discovery) workflow developed for Thermo Scientific Orbitrap platforms. Objectives are: to describe experiment design and sample preparation for SQUAD, to outline acquisition strategies across Orbitrap Exploris, Tribrid, and Astral systems, to present data-analysis pathways for both targeted and untargeted outputs, and to demonstrate the approach with a case study using NIST SRM 1950 plasma spiked with isotope-labeled amino and organic acids.

Methodology

SQUAD combines two parallel aims in a single LC–MS injection: (1) targeted quantitation of a predefined compound list using calibration curves and internal standards; and (2) untargeted discovery by collecting high-resolution MS1 and data-dependent MSn for structural annotation. Key method components include:

• Quantitation strategies: preferred—absolute quantitation with dilution-series authentic standards plus isotopically labeled internal standards (IS); alternatives—calibration curves without IS or single-point semi-quantitative approaches (lower accuracy).
• Sample preparation: typical plasma workflow uses 80% methanol (v/v) for protein precipitation and extraction. Samples are thawed cold, mixed with IS, diluted with methanol (reported sample:methanol ratio 2:8), centrifuged at low temperature, supernatant dried under N2 and reconstituted in a chromatography-compatible solvent.
• Chromatography: example method used a Hypersil GOLD C18 column (2.1 × 150 mm, 1.9 µm) on a Vanquish Horizon UHPLC with a water/formic acid (0.1%) and methanol/formic acid (0.1%) gradient; small injection volumes (2 µL) and elevated column temperature (45 °C) were used for speed and peak shape optimization.
• Quality control: rigorous QC strategy with processing blanks, pooled biological QC samples, system suitability tests (SST), and isotopically labeled IS to monitor extraction recovery and normalize ionization variability. Blanks are run to check contamination; pooled QCs interspersed through batches monitor drift and support AcquireX-type fragmentation strategies.

Instrumentation used

The SQUAD workflows were developed and demonstrated on multiple Thermo Scientific platforms with associated instrumentation and software:

• Mass spectrometers: Orbitrap Exploris 240, Orbitrap IQ‑X Tribrid (also referred to as Tribrid), and Orbitrap Astral.
• Chromatography: Thermo Scientific Vanquish Horizon UHPLC and Hypersil GOLD C18 Selectivity column.
• Specialized acquisition and workflows: AcquireX intelligent data acquisition, Real-Time Library Search (RTLS), tSIM/tMS2 and PRM/PRM-like experiments on HRAM instruments.
• Software: TraceFinder (targeted quantitation; case study used v5.1), Compound Discoverer (untargeted workflows; case study used v3.3), mzCloud spectral library and mzVault for local libraries.

Main results and discussion

The case study (NIST SRM 1950 plasma spiked with isotope-labeled amino and organic acids across 1 femtomole to 2.5 millimole) demonstrates the practical performance trade-offs and strengths of each platform and the SQUAD strategy:

• Orbitrap Exploris (MS1‑based SQUAD with polarity switching): high-resolution MS1 quantitation with broad metabolome coverage enabled by fast polarity switching and HRAM. Reported MS1 limits: LLOQ ~50 femtomoles and LLOD ~25 femtomoles (five orders of magnitude dynamic range). tSIM/tMS2 options allow sensitivity gains for targeted analytes.
• Orbitrap Tribrid / linear ion trap quantitation: linear ion trap offers enhanced sensitivity for targeted MSn quantitation with reported LLOQ ~5 femtomoles and LLOD ~0.5 femtomoles, and an extended dynamic range (~six orders of magnitude). Tribrid systems support RTLS-guided acquisition and multi-fragmentation library creation (HCD, CID, etc.) for confident annotation.
• Orbitrap Astral: combines Orbitrap MS1 HRAM with a fast, sensitive Astral analyzer for HRAM DDA MS2. Reported performance improvements include ≥90% fragmentation efficiency, an LLOQ of 1 femtomole and an LLOD reported down to 5 attomoles for phenylalanine in plasma with a six-order-of-magnitude dynamic range, plus markedly faster analysis—LC gradients can be shortened up to threefold without loss of MS1/MS2 quality. A 5‑minute gradient on Astral produced ~25% more annotated compounds with MS2 compared to a 15‑minute Orbitrap‑Orbitrap run.

These results show that SQUAD permits sensitive, absolute quantitation of targeted analytes while simultaneously collecting deep MSn information for unknown annotation. AcquireX and RTLS strategies maximize MS2 coverage from pooled QC samples and enable decision-based triggering that focuses MSn acquisition on informative features, preserving sample and instrument time.

Benefits and practical applications

Primary advantages of SQUAD workflows:

• Single-injection dual outcome: generates quantitative data for known targets and discovery data for unknowns from the same sample, conserving precious sample and avoiding re-preparation artifacts.
• Retrospective mining: HRAM MS1 data enable later post hoc interrogation of features without re-running samples.
• Improved throughput: advanced analyzers (Astral) and fast polarity switching (Exploris) permit shorter gradients and higher sample throughput while maintaining annotation depth.
• Robust quantitation: inclusion of calibration series and isotopically labeled IS supports accurate absolute quantitation and cross-lab comparability when applicable.

Applied contexts include clinical biomarker discovery and monitoring, nutritional metabolomics, microbiome metabolite mapping (e.g., bile acids), and metabolic engineering to quantify pathway substrates/products while discovering unexpected metabolic changes.

Data analysis workflow

SQUAD uses a two-pronged software pipeline tuned to the dual objectives:

• TraceFinder: focused on the targeted quantitation workflow—construct compound databases, build Master Methods (MS1 or tMS2 quantitation strategies), generate calibration models, batch-process samples, and produce standardized reports. Smart integration, flagging and calibration tools streamline high-throughput targeted analyses.
• Compound Discoverer: untargeted processing—feature detection, elemental composition by HRAM isotopic patterns, automated MSn library searches (mzCloud), predicted fragmentation annotation, and statistical analyses (univariate and multivariate, volcano plots, PCA, PLS‑DA, heatmaps). Workflow nodes are drag‑and‑drop, with templates and the Study/Analysis Wizard for rapid setup.
• RTLS and mzVault/mzCloud integration: create MS2 libraries from standards or pooled QC runs, then use RTLS to trigger MSn only when spectral matches or criteria are met, increasing efficiency of informative fragmentation collection.

Future trends and possibilities for application

Expected directions and opportunities from the SQUAD concept include:

• Tighter integration of intelligent acquisition (RTLS, AcquireX) with curated spectral libraries to further reduce instrument time while improving annotation confidence.
• Broader adoption of high-speed HRAM detectors (e.g., Astral‑style architectures) to enable routine sub‑5‑minute discovery-enabled workflows with retained quantitation performance.
• Improved community spectral libraries and standardized QC materials to increase reproducibility and cross‑laboratory comparability for SQUAD datasets.
• Deeper coupling of SQUAD outputs with automated cheminformatics and pathway-mapping tools to accelerate biological interpretation and biomarker translation.

Conclusion

SQUAD provides a practical, instrument‑aware framework for simultaneous targeted quantitation and untargeted discovery on modern Orbitrap platforms. By combining rigorous calibration and IS strategies for absolute quantitation with HRAM MS1 and intelligent MSn acquisition for discovery, SQUAD enables efficient, high‑value metabolomics workflows that conserve samples, increase throughput, and expand the breadth of biological insight obtainable from single injections. Choice of instrumentation and acquisition parameters allows tailoring sensitivity, dynamic range and throughput to study needs.

References

The technical note references and resources used or mentioned in the study include:

• Thermo Scientific Orbitrap Exploris, Orbitrap Tribrid (IQ‑X), and Orbitrap Astral mass spectrometer platforms (product-specific application notes and technical materials).
• Thermo Scientific AcquireX intelligent data acquisition workflow and Real‑Time Library Search (RTLS) methods.
• TraceFinder Software (example version: 5.1) and Compound Discoverer Software (example version: 3.3).
• mzCloud mass spectral library and mzVault library management.
• Thermo Scientific Pierce Small Molecule System Suitability Standard (Part No. A51740) and Hypersil GOLD C18 Selectivity HPLC Column (Part No. 25002‑052130).
• Case study materials: NIST SRM 1950 plasma, isotope-labeled amino acids and organic acids from Cambridge Isotope Laboratories and Sigma‑Aldrich.