UHPLC-MS/MS Triple Quadrupole Analysis of Anthocyanin Metabolites in Human Plasma Using Protein Precipitation and Solid Phase Extraction For Determination of Uptake from Food

Importance of the topic

Anthocyanins are bioactive flavonoid pigments abundant in fruits and vegetables with potential health benefits including cardioprotection, anti-inflammation, and metabolic regulation. Measuring their metabolites in human plasma supports nutritional research, pharmacokinetics, and dietary intervention studies. Reliable quantification in complex biological matrices is challenged by low concentration levels, matrix effects, and compound instability under non-acidic conditions.

Study objectives and overview

This study aimed to develop and validate an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for five anthocyanin compounds and metabolites in human plasma following strawberry ingestion. A comparison between protein precipitation (PPT) and solid phase extraction (SPE) sample preparation techniques was conducted to assess recovery, precision, and sensitivity. Plasma samples were collected at baseline (0 h), 2 h, and 3 h after consumption of a beverage containing 40 g freeze-dried strawberry powder.

Methods and instrumentation

The PPT procedure added acidified acetonitrile (1% formic acid) to plasma, followed by cold incubation, centrifugation, evaporation under nitrogen, and reconstitution in weak organic mobile phase. The SPE protocol used Agilent Bond Elut Plexa cartridges conditioned with acidified solvents, sample loading, aqueous wash, elution with acidified methanol, nitrogen drying, and reconstitution. An Agilent 1290 Infinity UHPLC system coupled to an Agilent 6460 Triple Quadrupole MS with electrospray ionization and Jet Stream technology performed MRM analysis. Chromatographic separation used a Poroshell C18 column (2.1×150 mm, 2.7 μm) at 30 °C under a 22-min gradient (5–90% acetonitrile with 1% formic acid).

Key results and discussion

SPE extracts appeared visually cleaner and provided higher recoveries (60.8–121.1%) versus PPT (4.2–18.4%). Method linearity (R2>0.9999), limits of detection (0.03–0.50 ng/mL), and quantification (0.10–1.50 ng/mL) were established using matrix-matched calibration standards. Peak plasma concentrations occurred at 2 h post-dose. SPE yielded significantly greater pelargonidin-3-O-glucoside and pelargonidin glucuronide levels compared to PPT (p<0.05). Chromatograms demonstrated baseline resolution of target analytes, with distinct MRM transitions separating isobaric compounds.

Benefits and practical applications

The validated SPE-UHPLC-MS/MS workflow enhances sensitivity, reproducibility, and accuracy for trace anthocyanin metabolites in human plasma. Its high throughput and robustness support clinical nutrition trials, biomarker discovery, and quality control in food science. The method accommodates low sample volumes and minimizes matrix interference, meeting regulatory and research requirements.

Future trends and opportunities

Integration of high-resolution mass spectrometry may enable identification of novel phase II metabolites and conjugates. Automation of SPE and microfluidic sample preparation could improve throughput and reduce solvent consumption. Advances in data processing and machine learning will enhance spectrum interpretation and metabolite profiling in large cohorts.

Conclusion

This study demonstrates that SPE coupled with UHPLC-MS/MS on a triple quadrupole platform provides a reliable, sensitive, and reproducible approach for quantifying anthocyanin metabolites in human plasma. SPE outperforms PPT in recovery and extract cleanliness, enabling accurate pharmacokinetic characterization of dietary flavonoids.

References

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