A standard, analytical protocol for the quantitation of non-structural carbohydrates in seagrasses that permits inter-laboratory comparison

Non-structural carbohydrate (NSC) content provides a quantitative measure of seagrass response to environmental stress and recovery. A review of the literature shows that numerous analytical methods are used to measure seagrass NSC. Colorimetric methods are most commonly used; however, these methodologies lack precision and provide no insights with respect to individual monosaccharides released by hydrolysis. We determined experimentally that assays by high performance liquid chromatograph (HPLC) are more precise and capable of estimating carbohydrates to within 1.1% of expected carbohydrate levels, compared to a greater than 15.0% deviation by colorimetric assays (phenol-sulfuric acid). Furthermore, solvents used for soluble sugar extraction (ethanol/water, methanol/water and water) and starch gelatinization (hydrochloric acid and sodium hydroxide) result in significantly different NSC estimations. Thus, estimation of seagrass NSC by different analytical protocols are non-comparable. We present an optimized 5-step protocol for the quantitation of seagrass soluble NSC and starch using HPLC that identifies individual mono- and oligosaccharides. Creating and using a standardised protocol for NSC quantitation will create a more cogent bio-indicator that enables comparisons of estimates between studies. Additionally, quantitation of individual sugars by HPLC will enable detailed investigation of seagrass metabolic responses to environmental stress.