Chapter 17 Analysis of Betaines from Marine Algae Using LC-MS-MS Shawna L. MacKinnon and Cheryl Craft Abstract Betaines are a class of quaternary ammonium compounds found in marine algae that can act as osmolytes and/or affect gene expression, and therefore improve plant tolerance to stresses such as temperature extremes, drought, and salinity when applied to agricultural crops. In humans, glycine betaine acts as a methyl donor and has been shown to protect internal organs, improve vascular risk factors, and enhance sport performance. Here we describe a sensitive LC-MS-MS method for the baseline separation and quantification of four betaines found in algae, namely, glycine betaine, δ-aminovaleric acid betaine, γ-aminobutyric acid betaine, and laminine. Key words Glycine betaine, δ-aminovaleric acid betaine, γ-aminobutyric acid betaine, Laminine, LC-MS-MS analysis, Macroalgae
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Introduction Historically, brown seaweeds, such as Ascophyllum nodosum, have been applied directly to the soil, either freshly harvested or as dried ground seaweed meal, primarily as a soil conditioner or source of organic matter [1]. More recently, aqueous and alkaline extracts prepared from a variety of commercially available seaweeds have been used in agriculture and horticulture systems as foliar sprays, soil drenches, or often a combination of both. The beneficial effects of seaweed extract application on crop performance have been attributed to a variety of constituents, including betaines [2]. These naturally occurring quaternary ammonium compounds can act as osmolytes and/or affect gene expression, therefore improving plant tolerance to stresses such as temperature extremes, drought, and salinity [3–8]. For over 50 years glycine betaine has been added to animal feeds as a nutritional supplement [9]. In the sports medicine arena glycine betaine has been found to enhance sport performance (ergogenics) by improving muscle endurance and increasing quality of repetitions performed [10]. The bioactivity of glycine betaine
Dagmar B. Stengel and Solène Connan (eds.), Natural Products From Marine Algae: Methods and Protocols, Methods in Molecular Biology, vol. 1308, DOI 10.1007/978-1-4939-2684-8_17, © Springer Science+Business Media New York 2015
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is associated with its ability to act as a methyl donor in the methionine cycle primarily in the human kidney and liver [9]. Other beneficial effects on human health include helping to protect cells and organs from osmolytic stress and improving heart health by improving vascular risk factors [9]. Over 13 betaine analogs have been identified in Chlorophyta (green), Heterokontophyta (brown), and Rhodophyta (red) seaweeds using thin layer chromatography and NMR techniques [11]. The isolated yield of each betaine ranged from 0 to 2 % per algal dry weight with the lowest betaine levels being reported in the Phaeophyceae. Seaweed biomass and commercial extracts prepared from Ascophyllym nodosum, and Fucus and Laminaria species have been found to contain glycine betaine, γ-aminobutyric acid betaine, δ-aminovaleric acid betaine, and laminine [12, 13]. Early analytical methods aimed at the quantification of betaines in plants, seaweeds, or commercial seaweed extracts included the use of thin layer chromatography [14], thin layer electrophoresis [15], and nonspecific precipitation reagents [16, 17]. The development of an HPLC-based approach to betaine analysis has been ongoing since the late 1970s [18]. Betaines do not fluoresce appreciably and can only be detected at low UV wavelengths (190–220 nm) as they lack a strong chromophoric moiety [19]. Attempts at increasing the detection limits of betaines using derivatization reagents have given unsatisfactory results because of the lack of reactivity of the betaines towards some reagents [20]. Proton magnetic resonance (1H NMR) has also been developed as a method of betaine analysis for seaweed extracts and plant material [21] but has the limitation that it cannot be used to quantify the levels of betaines that are present as minor components [12]. The LC-MS-MS method described herein quantifies betaines in seaweeds and commercial seaweed extract products using a rapid sample preparation and cleanup protocol. It utilizes the sensitivity and selectivity of LC-MS-MS making it a method that is suitable for the routine analysis of glycine betaine, γ-aminobutyric acid betaine, δ-aminovaleric acid betaine, and laminine.
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Materials and Instrumentation
2.1 Solvents and Standards
1. Ultrapure water is prepared by purifying distilled water to attain a resistance of 18 MΩ at 25 °C with a TOC of
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Introduction Historically, brown seaweeds, such as Ascophyllum nodosum, have been applied directly to the soil, either freshly harvested or as dried ground seaweed meal, primarily as a soil conditioner or source of organic matter [1]. More recently, aqueous and alkaline extracts prepared from a variety of commercially available seaweeds have been used in agriculture and horticulture systems as foliar sprays, soil drenches, or often a combination of both. The beneficial effects of seaweed extract application on crop performance have been attributed to a variety of constituents, including betaines [2]. These naturally occurring quaternary ammonium compounds can act as osmolytes and/or affect gene expression, therefore improving plant tolerance to stresses such as temperature extremes, drought, and salinity [3–8]. For over 50 years glycine betaine has been added to animal feeds as a nutritional supplement [9]. In the sports medicine arena glycine betaine has been found to enhance sport performance (ergogenics) by improving muscle endurance and increasing quality of repetitions performed [10]. The bioactivity of glycine betaine
Dagmar B. Stengel and Solène Connan (eds.), Natural Products From Marine Algae: Methods and Protocols, Methods in Molecular Biology, vol. 1308, DOI 10.1007/978-1-4939-2684-8_17, © Springer Science+Business Media New York 2015
267
268
Shawna L. MacKinnon and Cheryl Craft
is associated with its ability to act as a methyl donor in the methionine cycle primarily in the human kidney and liver [9]. Other beneficial effects on human health include helping to protect cells and organs from osmolytic stress and improving heart health by improving vascular risk factors [9]. Over 13 betaine analogs have been identified in Chlorophyta (green), Heterokontophyta (brown), and Rhodophyta (red) seaweeds using thin layer chromatography and NMR techniques [11]. The isolated yield of each betaine ranged from 0 to 2 % per algal dry weight with the lowest betaine levels being reported in the Phaeophyceae. Seaweed biomass and commercial extracts prepared from Ascophyllym nodosum, and Fucus and Laminaria species have been found to contain glycine betaine, γ-aminobutyric acid betaine, δ-aminovaleric acid betaine, and laminine [12, 13]. Early analytical methods aimed at the quantification of betaines in plants, seaweeds, or commercial seaweed extracts included the use of thin layer chromatography [14], thin layer electrophoresis [15], and nonspecific precipitation reagents [16, 17]. The development of an HPLC-based approach to betaine analysis has been ongoing since the late 1970s [18]. Betaines do not fluoresce appreciably and can only be detected at low UV wavelengths (190–220 nm) as they lack a strong chromophoric moiety [19]. Attempts at increasing the detection limits of betaines using derivatization reagents have given unsatisfactory results because of the lack of reactivity of the betaines towards some reagents [20]. Proton magnetic resonance (1H NMR) has also been developed as a method of betaine analysis for seaweed extracts and plant material [21] but has the limitation that it cannot be used to quantify the levels of betaines that are present as minor components [12]. The LC-MS-MS method described herein quantifies betaines in seaweeds and commercial seaweed extract products using a rapid sample preparation and cleanup protocol. It utilizes the sensitivity and selectivity of LC-MS-MS making it a method that is suitable for the routine analysis of glycine betaine, γ-aminobutyric acid betaine, δ-aminovaleric acid betaine, and laminine.
2
Materials and Instrumentation
2.1 Solvents and Standards
1. Ultrapure water is prepared by purifying distilled water to attain a resistance of 18 MΩ at 25 °C with a TOC of
