ISO 19563:2017

Overview - ISO 19563:2017 (Theanine analysis by HPLC)

ISO 19563:2017 specifies a validated high-performance liquid chromatography (HPLC) method for the quantitative determination of theanine in tea (Camellia sinensis) and instant tea in solid form. The procedure covers sample preparation, optional polyamide clean-up to remove polyphenols, chromatographic conditions using a reversed-phase C18 column, and method performance characteristics (precision, repeatability, reproducibility). The method does not separate L- and D‑theanine; L‑theanine is the predominant enantiomer in tea.

Key technical topics and requirements

• Scope: Applicable to leaf tea and instant tea samples; results expressed per dry matter.
• Principle: Reversed-phase (RP) C18 column with UV detection at 210 nm; water and acetonitrile used as mobile phases.
• Reagents & standards: Grade 1 water (ISO 3696), HPLC‑grade acetonitrile, methanol, polyamide (Polyamide CC 6) for clean-up, and anhydrous L‑theanine standard (>99%).
• Calibration range: Working standards prepared in the 5 µg/ml to 200 µg/ml range (from a 1 mg/ml stock).
• Sample preparation:
  • Leaf tea: 1.00 g brewed in 100 ml boiling water for 5 min, cooled, made to volume and filtered (0.45 µm) or centrifuged.
  • Instant tea: 0.100 g dissolved in ≈50 °C water, sonicated, filtered or centrifuged.
• Optional clean-up: Polyamide column chromatography to retain polyphenols while eluting theanine - recommended where interfering peaks occur.
• Chromatography: Binary gradient-capable HPLC with thermostatted column compartment; suggested column (e.g., Phenomenex Aqua 250 × 4.6 mm or 2 mm equivalent). Flow rates: ~1.0 ml/min for 4.6 mm columns, ~0.25 ml/min for 2 mm columns.
• Method performance: Includes interlaboratory test data and stated repeatability/reproducibility criteria.

Practical applications and users

• Quality control in tea production and instant tea manufacturing - routine monitoring of theanine content to support flavor and product specifications.
• Food testing laboratories and regulatory bodies conducting compositional analysis or compliance testing.
• Research labs studying tea chemistry, sensory compounds (umami), or nutritional content of tea-derived products and supplements.
• Dietary supplement and novel food developers assessing theanine levels in ingredient sourcing and product formulation.

Related standards and references

• ISO 1839 (Tea - Sampling), ISO 1572 / ISO 1573 (sample preparation & moisture/dry matter), ISO 7513 / ISO 7516 (instant tea sampling and moisture). These referenced standards support representative sampling and dry-matter correction required by ISO 19563:2017.

Keywords: ISO 19563:2017, theanine analysis, HPLC, tea analysis, Camellia sinensis, L-theanine, polyamide clean-up, reversed-phase C18, UV 210 nm, instant tea.