Analysis of Ornithine in Bunashimeji (Beech mushroom) Using Triple Quadrupole LC/MS/MS

Significance of the Topic

Ornithine is a non-essential amino acid known for supporting liver function and promoting recovery from fatigue, improving sleep quality, and stimulating growth hormone secretion. Its abundance in certain foods, such as shijimi clams and mushrooms, makes it a valuable nutritional marker. Accurate quantification of ornithine can inform both food science research and quality control in food production.

Objectives and Study Overview

This study aims to develop and validate a high-sensitivity, rapid LC-MS/MS method for quantifying ornithine in bunashimeji (beech mushroom) following the Japanese Agricultural Standard JAS 0016. The work demonstrates the capabilities of the Shimadzu LCMS-8060NX system for routine analysis without requiring derivatization.

Methodology and Instrumentation

Sample Preparation:

• Removal of hard stem tips and homogenization.
• Extraction with dilute hydrochloric acid and adjustment to constant volume.
• Centrifugation and filtration of the supernatant.
• 100-fold dilution and addition of theanine as an internal standard.

LC-MS/MS Conditions:

• Column: Shim-pack GIST Amide (100 mm × 2.1 mm, 3.0 µm).
• Mobile phase: A = 0.3% formic acid in water; B = acetonitrile with 0.3% formic acid; gradient 90% B to 50% B between 0–5 min, hold 5–8 min, return to 90% B by 8.01 min; total run 13 min.
• Flow rate: 0.30 mL/min; column temperature: 40 °C; injection volume: 0.5 µL.
• Ionization: ESI positive; MRM transitions: ornithine m/z 133→70 (CE −22 V), theanine m/z 175→84 (CE −21 V).

Used Instrumentation

• Shimadzu LCMS-8060NX triple quadrupole mass spectrometer.
• Shim-pack GIST Amide analytical column.

Key Results and Discussion

The method achieved baseline separation of ornithine and the internal standard within 5 and 3.5 minutes, respectively. Calibration was linear over 20–2000 ng/mL with R² > 0.9998. Spike-and-recovery tests at 50 ng/mL yielded recoveries between 97% and 103%. Quality control samples at 100 ng/mL maintained accuracy within ±10% across pre-, mid-, and post-run checks.
Analysis of five commercial bunashimeji samples revealed ornithine concentrations of 779–2198 mg/kg, corresponding to approximately 7–14 times the levels typically found in shijimi clams.

Benefits and Practical Applications

• Highly sensitive detection of ornithine without chemical derivatization.
• Rapid analysis (13-minute run time) suitable for high-throughput laboratories.
• Robust quantitation with excellent linearity and recovery, supporting food quality control and research.

Future Trends and Opportunities

• Expanded application of rapid LC-MS/MS assays to other nutrients and bioactive compounds in various food matrices.
• Integration of automated sample preparation to increase throughput and reduce manual labor.
• Development of miniaturized and portable MS platforms for on-site food analysis.
• Use of high-resolution mass spectrometry for broader metabolomic profiling in mushrooms and related foods.

Conclusion

The validated LCMS-8060NX method aligns with JAS 0016 standards, offering a sensitive, precise, and efficient approach for measuring ornithine in bunashimeji mushrooms. Results confirm that bunashimeji is a superior dietary source of ornithine compared to shijimi clams, highlighting its potential nutritional value.

Reference

• Ministry of Agriculture, Forestry and Fisheries (2021). JAS Notification No. 445: Determination of ornithine in mushrooms (Hypsizygus marmoreus) by high-performance liquid chromatography.
• FAMIC. Commentary on JAS 0016: Determination of ornithine in mushrooms (Hypsizygus marmoreus).