Alternative Proteins Testing Application Compendium

Significance of the Topic

Alternative proteins and functional food ingredients such as amino acids, vitamins, isoflavones, sugars, and organic acids are critical targets for food quality, safety, and nutrition analysis. Comprehensive analytical strategies enable manufacturers and regulators to ensure authenticity, label compliance, and consumer health benefits. Advances in chromatographic and mass spectrometric instrumentation now allow for highly sensitive, high-throughput, and robust testing of both target and nontarget component profiles.

Objectives and Study Overview

This compendium presents several workflows for the analysis of key analyte classes in alternative proteins and related food matrices:

• Nontargeted LC/Q-TOF profiling and chemometric analysis of taste and flavor compounds in animal meats and plant-based analogs.
• Automated precolumn derivatization and HPLC-FLD of amino acids in beverages by in-loop OPA/FMOC reactions.
• Simultaneous LC/MS/MS quantitation of 14 water-soluble B vitamins in dietary supplements.
• LC/MS/MS measurement of water-soluble vitamins and metabolites via HILIC separation at varied pH levels.
• Reversed-phase LC/MS/MS quantitation of isoflavones in soybean cultivars.
• Simultaneous HPLC-RID/UV analysis of sugars and low-calorie sweeteners in beverages.
• GC/FID comparison of WAX UI and FFAP columns for underivatized volatile organic acids and free fatty acids.

Instrumentation and Methodologies

• High-resolution LC/Q-TOF MS for nontargeted profiling, combined with PCA, heat maps, and hierarchical clustering in MassHunter and MPP software.
• 1260 Infinity II Prime LC with Fluorescence Detector and in-loop automated derivatization to separate 23 amino acids in 9 min.
• 1290 Infinity II LC coupled to 6470 triple quadrupole MS for dynamic MRM of B vitamins, using isotopic internal or standard addition calibration.
• HILIC separation on Poroshell 120 HILIC-Z columns with mobile phase pH optimization and surface-passivation strategies (phosphoric acid washes and deactivator additive) for vitamin metabolites.
• Reversed-phase LC/MS/MS on ZORBAX Eclipse Plus C18 for isoflavone separation under gradient conditions.
• Isocratic HPLC-RID in acetonitrile/ammonium acetate mobile phase for simultaneous sugar (fructose, glucose, sucrose, maltose, lactose) and sweetener (acesulfame, saccharin, cyclamate, aspartame) analysis.
• GC/FID with J&W DB-FATWAX UI, DB-FFAP, and standard wax columns for direct injection of aqueous organic acids and fatty acids, testing peak shape, inertness, and retention time stability over repeated injections.

Main Results and Discussion

• Nontargeted LC/Q-TOF profiling distinguished plant-based and animal meats, revealing distinct compound clusters associated with umami, bitter, and fatty acid profiles, guiding ingredient selection.
• Automated in-loop amino acid derivatization achieved <1% RSD in peak areas and LODs down to 0.225 pmol/µL for 23 amino acids, demonstrated in soft drink and wine matrices.
• Simultaneous detection of 14 B vitamins showed excellent linearity (R²>0.99), LODs as low as 0.225 ng/mL, and compliance with supplement label claims by matrix-matched correction or standard addition.
• HILIC-MS/MS vitamin metabolite separation improved with mid-pH mobile phases; phosphorylated metabolites required phosphoric acid washes and deactivator additives or PEEK-lined columns for peak shape and inertness.
• Soybean isoflavone quantitation by LC/MS/MS achieved LODs between 0.7 and 6.7 ppb, linear ranges up to 5 ppm, and <10% RSD for field samples, showing genetic cultivar differences.
• Single-run HPLC-RID/UV method quantified sugars and sweeteners in beverages, matching nutrition labels and literature values, with LOQs <11 µg/mL for most analytes.
• DB-FATWAX UI columns provided superior inertness and reproducibility for underivatized short-chain organic acids and free fatty acids in repeated aqueous injections, outperforming modified FFAP and other commercial WAX phases.

Practical Benefits and Applications

These validated workflows support food and beverage R&D, QA/QC labs, allergen and authenticators, and regulatory compliance by providing:

• Higher throughput via multiplexed and combined analyte methods.
• Improved data quality and quantitation accuracy through isotope-labeled standards, matrix-matched calibration, and surface-passivation.
• Reduced sample preparation time by eliminating derivatization or using automated derivatization.
• Enhanced compound identification with high-resolution data and advanced chemometrics.

Future Trends and Opportunities

Emerging trends include integration of high-resolution and ion mobility spectrometry for deeper profiling, machine learning-driven data analysis for rapid screening, microflow and chip-based separations for reduced solvent usage, and automated multi-omics workflows coupling metabolomics with proteomics for holistic food quality assessments.

Conclusion

Advanced LC/MS, HILIC, HPLC-FLD, HPLC-RID, and GC methodologies provide robust, sensitive, and efficient analysis of alternative proteins, vitamins, amino acids, isoflavones, sugars, sweeteners, and organic acids. By leveraging high-resolution instrumentation, automated workflows, tailored columns, and data-driven analytics, laboratories can meet the growing demands of food innovation, safety, and regulatory compliance.