Efficient sustainable online SPE-UHPLC workflow for the determination of bitter acids in hops

Efficient sustainable online SPE‑UHPLC workflow for determination of bitter acids in hops

Significance of the topic

Bitter acids (α‑acids: humulones; β‑acids: lupulones) are the principal contributors to beer bitterness and are critical quality attributes for hop raw material selection and recipe design. Reliable, fast and environmentally responsible analytical workflows for profiling these compounds are essential for breweries, hop producers, and analytical laboratories to control product consistency, optimize bittering efficiency (IBU), and minimize environmental impact from sample preparation.

Goals and overview of the study

The study aims to develop and validate an efficient, automated and greener method for determination of α‑ and β‑bitter acids in hops by coupling online solid‑phase extraction (SPE) with UHPLC (Vanquish Online SPE UHPLC). Key objectives were to reduce manual sample preparation time and solvent/plastic waste, achieve baseline separation of critical isomeric pairs when possible, and deliver robust quantification for routine profiling of different hop varieties.

Methodology and sample preparation

• Extraction: 100 mg ground hop sample extracted with 5 mL of 80% ethanol (green solvent) using 45 min ultrasonic treatment at room temperature, followed by centrifugation; supernatant aliquoted and stored at −20 °C. Filtration was avoided by a two‑step centrifugation (4,700 g then 11,180 g).
• Calibration: ICE‑4 international hop standard (known composition of α/β acids) prepared as 1.139 mg/mL stock in acetonitrile and serially diluted to create a four‑point calibration (100, 75, 50, 25%).
• Chromatographic strategy: Online SPE (Hypersil GOLD C8, 20 × 2.1 mm, 5 µm) used for cleanup and concentration prior to analytical separation on a Hypersil GOLD C18 Selectivity column (1.9 µm, 100 × 2.1 mm). Mobile phases were 0.1% formic acid in water (A) and in acetonitrile (B).
• Key operational choices: short SPE cycle (≈2 min), low total solvent consumption (<8 mL per run for A and B combined), and minimized single‑use cartridge waste by integrating SPE inline with the UHPLC.

Instrumentation used

• Thermo Scientific Vanquish Flex Online SPE UHPLC System (System Base Vanquish Duo, Dual Pump F, Split Sampler FT, Column Compartment H, Variable Wavelength Detector F).
• Online SPE column: Hypersil GOLD C8, 5 µm, 20 × 2.1 mm.
• Analytical column: Hypersil GOLD C18 Selectivity, 1.9 µm, 100 × 2.1 mm.
• Detector: UV at 270 nm (semi‑micro flow cell 2.5 µL); data collected with Chromeleon 7.3.2 CDS.
• Auxiliary: Viper fittings for low dead volume; autosampler at 5 °C; injection volumes: 1 µL for hop extracts, 5 µL for standards.

Main results and discussion

• Method performance: Calibration curves from ICE‑4 dilutions showed excellent linearity (R² > 0.995) and high precision (peak area RSDs typically <0.5% across tested levels).
• Chromatography: Optimized C18 selectivity column shortened runtime to 15 min and resolved critical isomeric pairs sufficiently for reporting. Reported resolutions included humulone vs. adhumulone (R = 2.29) and lupulone vs. adlupulone (R = 1.06). Where complete baseline separation is not required by industry practice, quantification of summed pairs (humulone+adhumulone and lupulone+adlupulone) follows ASBC recommendations.
• Throughput and sustainability: The automated online SPE step reduced sample preparation time dramatically (≈2 min SPE vs. 30–60 min for offline SPE), lowered solvent use (<8 mL total A+B per analysis), and eliminated disposable SPE cartridges, reducing plastic waste.
• Application to hop varieties: Four hop cultivars (Sultana, Barbe Rouge, Azacca, Sabro) were profiled. α‑acids were consistently higher than β‑acids. Reported α‑acid content ranged approximately from 5.8% (Barbe Rouge) to 12.5% (Sultana). Cohumulone and humulone+adhumulone were the dominant components with cultivar‑specific fingerprints; measured single‑sample repeatability (n=6) showed SDs generally <0.5%.

Benefits and practical applications

• Operational efficiency: Integration of online SPE with UHPLC automates cleanup, shortens turnaround, increases sample throughput, and reduces operator variability.
• Cost and waste reduction: Lower solvent consumption and elimination of single‑use SPE cartridges reduce consumable costs and environmental footprint.
• Analytical quality: Sufficient chromatographic resolution and validated linearity/precision support reliable quantification for quality control, raw material specification, and R&D applications in brewing and hop breeding.

Future trends and possibilities

• Further miniaturization and UHPLC‑MS coupling could increase sensitivity and enable detection of minor prenylated flavonoids and degradation products while preserving the green SPE workflow.
• Method extension to beer matrices: adapting online SPE to monitor transformation of hop α‑acids into iso‑α‑acids during wort boiling/fermentation would support process control and product consistency.
• Automation and digital integration: Combining fast online SPE‑UHPLC with laboratory information management systems (LIMS) and chemometric tools would facilitate high‑throughput profiling and variety classification.
• Greener solvents and lifecycle assessment: Continued optimization of extraction solvents and full environmental impact assessments (including energy and consumables) will strengthen sustainability claims.

Conclusion

The online SPE‑UHPLC method presented demonstrates a rapid, reproducible and more sustainable approach to quantify hop bitter acids. Using 80% ethanol extraction and inline SPE (Hypersil GOLD C8) coupled to a Hypersil GOLD C18 selectivity analytical column yields robust calibration (R² > 0.995), low RSD (<0.5%), short runtimes (15 min), and markedly reduced solvent and plastic waste. The workflow is well suited for routine QC of hop materials and supports industry practice of reporting summed isomeric pairs where appropriate.

References

1. Marques SP, Owen RW, da Silva AMA, Neto MLA, Trevisan MTS. QuEChERS extraction for quantitation of bitter acids and xanthohumol in hops by HPLC‑UV. Food Chemistry. 2022;388:132964.
2. Duarte LM, Aredes RS, Amorim TL, de Carvalho Marques FF, de Oliveira MAL. Determination of α‑ and β‑acids in hops by liquid chromatography or electromigration techniques: A critical review. Food Chemistry. 2022;397:133671.
3. Heidorn M. Monitor the brewing process with LC‑transformation of hop α‑acids into beer iso‑α‑acids. Thermo Fisher Scientific Application Brief 155. 2016.