Natural Food Supplements: Characterization using LC/MS – Ashwagandha, Turmeric and Ginger Examples

Importance of the Topic

Food supplements are widely used for health maintenance, but regulatory labeling typically reports ingredient amounts (e.g., root or extract weight) rather than the amounts of specific bioactive constituents. Accurate chemically selective characterization of supplements is essential for quality control, verification of label claims, safety assessment, and establishing dose–response relationships for active compounds. LC–MS methods provide the sensitivity, selectivity and structural information needed to profile and quantify characteristic phytochemicals in complex supplement matrices such as turmeric, ginger, boswellia and ashwagandha.

Objectives and Study Overview

The study developed and applied an LC–MS workflow to characterize and quantify key bioactive constituents in commercial natural food supplements. Target matrices included turmeric (Curcuma longa), ginger (Zingiber officinale), Boswellia serrata (boswellic acids) and ashwagandha (Withania somnifera). Goals were to (1) establish targeted MRM transitions and HRMS fragmentation data for identification, (2) validate calibration ranges and limits of quantitation sufficient to report compound mass per recommended serving, and (3) demonstrate the approach across a range of commercial dosage forms (capsules, tablets, softgels, gummies).

Methodology

Sample preparation:

• Capsules and tablets: 100 mg of powder (or a 100 mg tablet piece) extracted with 20 mL 50:50 water:methanol (v/v).
• Softgels: softgel ruptured by vortex/sonication in 30 mL water, then 10 mL methanol added.
• Gummies: sonicated in 100 mL water until dispersed, then 100 mL methanol added.
• All extracts: centrifugation at 4000 g for 5 min, filtration through 0.22 µm PTFE, and dilution in 50:50 water:methanol as required (10:1, 100:1 or 1000:1) prior to analysis.

Standards and calibration:

• Authentic standards used where available: 6‑Gingerol, 6‑Paradol, Curcumin, Boswellic Acid (generic), 3‑Acetyl‑11‑Keto‑Boswellic Acid (AKBA), 3‑Acetyl‑Boswellic Acid (ABA), Piperine and Withaferin A.
• Calibration range: 0.1 to 500 ng/mL in solvent standards; LOQs in solvent typically 0.1–1 ng/mL, enabling quantitation as low as ~0.005 mg per serving after back-calculation and correction for extraction/dilution.
• Relative calibration was applied for analyte classes lacking individual standards (e.g., using 6‑Gingerol as surrogate for other gingerols; 6‑Paradol for shogaol/paradol-type analytes; generic boswellic acid for some keto- and acetylated species).

Chromatography and MS conditions:

• UPLC system: ACQUITY UPLC I‑Class Plus with ACQUITY Premier BEH C18 column (1.7 µm, 2.1 × 50 mm) operated at 35 °C.
• Mobile phases: A = water with 2.5 mM ammonium acetate; B = methanol. Typical gradient for MRM: 5% B to 95% B over 0–10 min (gradient timings adjusted for QTof work).
• Mass spectrometers: Xevo TQ‑S micro triple quadrupole for targeted MRM quantitation; Xevo G3 QTof operated in MSe (data‑independent acquisition) for high‑resolution accurate mass and fragmentation data used for identification.
• Ionization: electrospray ionization (ESI) in both positive and negative modes according to compound class (piperine in positive mode; many phenolics and withanolides in negative mode).
• MRM optimization: precursor/product ions, cone voltages and collision energies optimized by infusion/injection experiments, with a full MRM parameter list available on request.

Main Results and Discussion

Identification and structural confirmation:

• HRMS (MSe) spectra from the QTof provided accurate mass and fragment ion information that enabled confident identification of target analytes in complex ashwagandha extracts and other matrices containing many natural-product constituents (withanolides, flavonoids, saponins).
• Typical fragment ions (example: m/z 122.0362 observed in a withaferin A spectrum) and retention time alignment supported assignments alongside MRM transitions.

Quantitation and variability between products:

• Calibration curves for representative analytes (e.g., Withaferin A, 6‑Gingerol, 6‑Paradol) showed good linear response across the studied range and enabled quantitation after dilution and correction for sample mass and serving size.
• Measured levels per declared serving demonstrated substantial variability between commercial products. Examples include:
• Ashwagandha products: withaferin A amounts ranged roughly from ~0.02 to ~0.96 mg/serving across samples tested, illustrating large inter‑product differences even among formulations labeled as standard extracts.
• Ginger products: 6‑Gingerol and 6‑Shogaol were measurable and varied by product; additional ginger homologues (8‑, 10‑, 12‑, 14‑gingerols and corresponding shogaols/paradols) were detected at lower concentrations or near LOQ.
• Turmeric products: curcuminoids (curcumin, demethoxycurcumin, bisdemethoxycurcumin) were quantifiable; products that included black pepper extract (Bioperine) contained measurable piperine which can affect bioavailability.
• Boswellia extracts: specific boswellic acids, including AKBA, ABA and KBA, were detected but generally constituted small fractions (ng to low µg levels per serving) relative to total extract mass, indicating variable enrichment of target actives.

Analytical performance and practical sensitivity:

• LOQs in solvent of 0.1–1 ng/mL translated to a capability of quantifying selected analytes down to approximately 0.005 mg (5 µg) per consumer serving after the sample workflow and dilution factors were applied.
• Relative quantitation strategies allowed reporting for analytes lacking certified standards, but introduce additional uncertainty compared with compound‑specific calibration.

Limitations highlighted:

• Incomplete availability of standards for all natural product constituents required surrogate calibration and limits absolute accuracy for some analytes.
• Potential matrix effects and extraction efficiency differences across dosage forms (gummies, softgels, tablets, encapsulated powders) can affect measured concentrations and require careful control or use of internal standards for robust quantitative comparability.

Benefits and Practical Applications of the Method

• Enables verification of label claims by converting measured analyte concentrations in extracts to mg/serving, providing actionable data for manufacturers, regulators and quality control laboratories.
• Combines targeted triple‑quad MRM quantitation with HRMS identification to balance sensitivity and structural confirmation, making it suitable for routine QC and for investigational work where unknowns require confirmation.
• Applicable to a variety of dosage forms with a simple, reproducible sample preparation workflow compatible with high throughput screening of commercial products.

Used Instrumentation

• Mass spectrometers: Waters Xevo TQ‑S micro (triple quadrupole) and Waters Xevo G3 QTof (high‑resolution QTof) operating in MRM and MSe modes, respectively.
• Chromatography: Waters ACQUITY UPLC I‑Class Plus; ACQUITY Premier BEH C18 column, 130 Å, 1.7 µm, 2.1 × 50 mm operated at 35 °C.
• Ionization: Electrospray ionization (positive and negative modes).

Future Trends and Potential Applications

• Expansion of authenticated standard libraries and certified reference materials for a broader range of phytochemicals to reduce reliance on surrogate calibration and improve quantitative accuracy.
• Increased use of high‑resolution MS and spectral libraries for non‑targeted screening and detection of adulterants, contaminants and novel metabolites introduced during formulation or storage.
• Development of standardized extraction and sample preparation protocols across dosage forms to reduce inter‑laboratory variability and support regulatory harmonization.
• Integration of quantitative chemical profiling with biological activity assays (bioassay‑guided fractionation) to better correlate measured constituent levels with expected physiological effects.
• Automation and informatics: adoption of automated sample prep, batch processing and data‑mining workflows to enable large‑scale marketplace surveillance and longitudinal quality studies.

Conclusion

The LC–MS workflow combining targeted triple‑quadrupole MRM quantitation and high‑resolution MSe structural confirmation provides a robust approach to characterize and quantify key bioactive constituents in turmeric, ginger, boswellia and ashwagandha supplements. The method achieves low limits of quantitation in solvent and—after appropriate extraction and dilution—permits reporting of analyte mass per serving down to low microgram levels. Results highlighted marked variability in active constituent content across commercial products and underscore the need for standardized analytical methods and expanded reference materials to support reliable label claims and consumer safety assessments.

Conflict of Interest

The authors of the original study are employees of Waters Corporation; the work was presented on behalf of the company.

Reference

1. Waters Corporation poster: Natural Food Supplements: Characterization using LC/MS — Ashwagandha, Turmeric and Ginger Examples. Authors: G. T. Fujimoto, L. E. Hatch, S. E. Dowd, N. E. Ellor, Waters Corporation, Milford, MA. Full MRM parameter list available from the authors upon request.
2. Mikulska, P.; et al. Withanolides and related natural products: chemistry and biological activity. Pharmaceutics. 2023 Mar 24;15(4):1057.