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Background: 3-N-trimethylaminopropionic acid (β-alanine betaine) and trimethylamine (TMA) are important nitrogenous compounds that perform fundamental roles in biological pathways throughout all kingdoms of life; however, yet their simultaneous determination method is hardly reported. Methods: Capillary electrophoresis method for the simultaneous determination of TMA and β-alanine betaine in microbial culture and plant samples was developed. To increase the sensitivity, TMA and β-alanine betaine in the samples were first derivatized with bromophenacyl bromide and then analyzed by capillary electrophoresis under low pH. Results: The derivatization was found to be practically useful for the elimination of interfering substances from plant and microbial extracts, as well as giving well resolved peaks for the analytes (β-alanine betaine esters and TMA salt). Analytical features of the developed method showed its respectable performance in terms of linearity (r > 0.99), precision (relative standard deviation (RSD) < 5%), and detection limits (0.01 mM). Conclusion: The developed method allows the quantitative determination of TMA and β-alanine betaine in complex biological samples and assists to study biosynthetic and degradation pathways of these important compounds. Keywords: β-alanine betaine; Capillary electrophoresis; Microbial culture; Plant leaves; Trimethylamine Background accumulated in many plants in response to various envir- In nature, many plants, bacteria, and marine algae accu- onmental stresses, various members of the highly stress- mulate quaternary ammonium compounds in response tolerant plant family Plumbaginaceae accumulate β-alanine to various environmental stresses such as flooding, freez- betaine instead of glycine betaine (Hanson et al. 1994). ing, heating, drought, and salinity (Rhodes and Hanson β-Alanine betaine synthesis is not controlled by choline 1993; Gorham 1995). These compounds form a structur- availability, because it is derived from β-alanine by three- ally heterogeneous class of compounds with a unifying step methylation (Rathinasabapathi et al. 2000). Distinct character of a polar and fully methyl substituted nitrogen from glycine betaine synthesis, β-alanine betaine synthesis atom, creating a permanent positive charge on the N moi- does not require oxygen, and therefore, it was proposed to ety (Rhodes and Hanson 1993). Of them, glycine betaine, be suitable for osmoprotection under saline and hypoxic 3-N-trimethylaminopropionic acid (β-alanine betaine), and conditions (Hanson et al. 1994; Rathinasabapathi et al. proline betaine are known to be the most effective osmo- 2000). Consequently, β-Alanine betaine appears to be ef- protectants and are widely distributed in the biosphere fective over a broader ecological spectrum than glycine (Yancey 2005). Although glycine betaine is extensively betaine (Rhodes and Hanson 1993). In soil microorganisms, our recent reports revealed that β-alanine betaine was accumulated as an intermediate me- * Correspondence: email@example.com Department of Food Science and Technology, Faculty of Agriculture, tabolite in the degradation pathway of homocholine by University of Khartoum, Shambat 13314, Sudan 2 members of the genera Arthrobacter, Rhodococcus,and Arid Land Research Center, Tottori University, 1390 Hamasaka, Tottori Pseudomonas (Mohamed Ahmed et al. 2009a, b; Mohamed 680-0001, Japan Full list of author information is available at the end of the article © 2014 Mohamed Ahmed et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mohamed Ahmed et al. Journal of Analytical Science and Technology 2014, 5:38 Page 2 of 8 http://www.jast-journal.com/content/5/1/38 Ahmed et al. 2010; Mohamed Ahmed et al. 2014). The po- acid (1 g, 6.5 mM) and KHCO (1.3 g, 13 mM) in 20 ml of tential role of β-alanine betaine in plants' and microorgan- methanol. The mixture was stirred overnight at room isms' tolerance to salinity and hypoxia makes its synthetic temperature and then decanted. Thereafter, the liquid pathway an interesting target for metabolic engineering. phase was concentrated, and the residue was extracted However, the estimation methods of this interesting me- using 15 ml of mixed solvent (acetonitrile/methanol = 10:1, tabolite are still scarce. v/v). The combined extracts were dried under a nitrogen Trimethylamine (TMA) is a volatile low molecular stream to give β-alanine betaine as a colorless powder weight tertiary aliphatic amine that has been recognized (1.2 g, 63.2%). The structure and purity of β-alanine widely in many animal and plant tissues and is one of betaine were confirmed using proton nuclear magnetic the degradation products of nitrogenous organic mater- resonance ( H NMR) and capillary electrophoresis. ial such as quaternary ammonium compounds such as Unless otherwise specified, all other reagents were of choline and homocholine (Craciun and Balskus 2012; analytical grade and were from either Wako (Wako Pure Mohamed Ahmed et al. 2010). Commonly, the amount Chemical Industries Ltd, Tokyo, Japan) or Sigma (St. of TMA is a useful indicator of spoilage in fresh and Louis, MO, USA). lightly preserved seafood as it increases during the break- down of seafood, such as fish and shrimp (Dalgaard 2006; Extract preparation from microbial samples Ghaly et al. 2010). In medical diagnosis, an increase in the Homocholine-degrading strains were isolated from the concentration of TMA in the breath of patients can be soil samples obtained from different locations at Tottori used as a sign of viremic disease (Siminhoff et al. 1977). University and around Tottori City, Japan. The bacterial Therefore, detection of TMA is of high interests in many strains were cultivated for 24 h at 30°C on 75 ml of basal fields such environmental protection, food industry, and homocholine liquid media containing 20 mM homocho- medical diagnosis. However, one of the challenging as- line as a sole source of carbon, nitrogen, and energy. pects of the analysis of β-alanine betaine and TMA lies in The cells were harvested at the exponential phase by their lack of useful chromospheres, and their chemical centrifugation at 10,000 × g for 20 min at 4°C. The structures have permanently charged groups that prevent supernatant was collected and preserved at −20°C until gas chromatographic separation in their intact forms. In used for detection of β-alanine betaine and TMA. The the past, analyses of β-alanine betaine and TMA relied on harvested bacterial cells were washed three times with sa- qualitative or semi-quantitative colorimetric tests that line solution (8.5 g/l KCl), and re-suspended in 50 mM employed either thin-layer chromatography and Dragen- potassium phosphate buffer (pH 7.5). The resting cell re- dorff's reagent or reaction with picric acid to form colored action was started by the addition of homocholine complex (Blunden et al. 1981; Grieve and Grattan 1983). (20 mM) to the cell suspension. The suspension was incu- Since these methods are limited in their sensitivity, select- bated on a shaker at 120 rpm and 30°C. At appropriate ivity and quantitative accuracy, and ability to assay beta- time intervals (30 min, 1 h, 2 h, 3 h, and 6 h), aliquots of ines and TMA in one sample, knowledge of the identities the cell suspension were withdrawn and boiled for 3 to and absolute concentrations of β-alanine betaine and 5 min to stop the reaction. These extracts were preserved TMA in biological materials remained inadequate. Re- at −20°C until used for sample derivatization. cently, capillary electrophoresis has been applied in many different fields because of its extremely high resolution, its speed, and its applicability to a wide range of molecules Extract preparation from plant samples whether they are charged or uncharged, or of low or high Plant (Limonium suffruticosum, Phragmites australis,and molecular weight (Shintani and Polonsky 1997). In the Elaeagnus oxycarpa) leaf samples, at productive stage, present work, capillary electrophoresis method under were collected from an area around Aiding Lake in the low pH (Nishimura et al. 2001; Zhang et al. 2002) was Turpan Basin, Xinjiang, China, in August 2010. The area effectively improved for simultaneous determination of of the study site is about 10,000 m (100 m × 100 m), and β-alanine betaine and TMA in both plant leaves and mi- three plots (10 m × 10 m) were established randomly. The crobial culture samples. samples were collected from five plants of each species and carefully washed with water. The samples were dried Methods in oven at 85°C for 48 h, ground to fine power, and then Materials brought to Arid Land Research Center, Tottori University, β-alanine betaine was synthesized by N-methylation of Japan, for analysis. For extract preparation, about 100 mg dimethylaminopropionic acid (Tokyo Kasei Kogyo Co. Ltd, of powdered samples were added to 1.5 ml water, mixed Tokyo, Japan) with methyl iodide as described previously in a plastic tube, incubated at 75°C for 20 min, and then (Mohamed Ahmed et al. 2010). Briefly, 4 ml of methyl iod- centrifuged at 15,000 × g for 10 min. These samples were ide was added to a suspension of dimethylaminopropionic preserved at −20°C until used for sample derivatization. Mohamed Ahmed et al. Journal of Analytical Science and Technology 2014, 5:38 Page 3 of 8 http://www.jast-journal.com/content/5/1/38 Sample derivatization Results and discussion One of the challenging aspects of analysis of β-alanine Sample derivatization betaine and TMA lies in their lack of useful chromo- In the literature, it has been established that 4-bromophenacyl phores and thus could not be detected in ultraviolet- bromide reagent reacts with quaternary ammonium com- visible (UV/vis) light range. To overcome this limitation, pounds and can accurately be used for their quantification the samples were derivatized with 4-bromphencyl bro- (Gorham et al. 1982). We have adapted and modified this mide before analysis with capillary electrophoresis. Este- derivatization method for the determination of β-alanine rification was carried out following the methods of betaine and TMA in plant and microbial samples by capil- Nishimura et al. (2001) with some modifications. Briefly, lary electrophoresis. Since β-alanine betaine and TMA 0.1 ml of the sample extract and/or authentic standards lack of useful chromophores that lead to the inability of β-alanine betaine and TMA were placed in a micro- for detection at the UV/vis range, in the current work, tube and mixed with 0.05 ml of buffer solution (100 mM they were derivatized with 4-bromophenacyl bromide to KH PO /distilled water/acetonitrile = 1:1:4). To the mix- form β-alanine betaine-ester and TMA-salt (Figure 1). 2 4 ture, 0.3 ml of 4-bromophenacyl bromide (20 mg/ml in These reaction products showed a maximum absorption acetonitrile) was added. The tube was capped and heated at 262 nm, which was within the range 214 to 266 nm re- at 90°C for 90 min. The reaction mixture was evaporated ported previously for various betaine esters (Gorham et al. to dryness with a centrifugal evaporator (CVE-200D; 1982; Zhang et al. 2002). Determination of betaine as the Tokyo Rikakikai, Tokyo, Japan). The residue was dissolved 4-bromophenacyl ester has previously been reported to be in 300 μl of 50 mM sodium phosphate buffer (pH 3.0), exceptionally sensitive and specific (Gorham et al. 1982). mixed well, and centrifuged at 10,000 × g for 20 min at 4°C. Generally, absolute acetonitrile is usually used as solvent The supernatants, which contained ester and salt of the of 4-bromophenacyl for reaction with betaines (Gorham metabolites β-alanine betaine and TMA, were filtered using et al. 1982) giving a rapid esterification reaction at 45-μm filter (Millex Millipore, Billerica, MA, USA) to re- neutral or slightly alkaline pH levels. Nevertheless, in move the micro-particles that might block the flow through the current work, the reaction was carried out in less the capillary tube. The filtered samples were then analyzed than 80% acetonitrile solution that gave an average yield by capillary electrophoresis. of 4-bromophenacyl esters and salts of more than 70%. To avoid the chemical decomposition of β-alanine beta- Capillary electrophoresis analysis ine through C-N bond cleavage that may lead to the Capillary electrophoresis analysis was conducted using a production of trimethylamine and acrylate under alka- capillary electrophoresis system model Photal CAPI-3300 line condition (Gorham et al. 1982; Zhang et al. 2002), (Otsuka Electronics. Co. Ltd., Osaka, Japan) equipped with the derivatization condition was optimized and carried a fused silica capillary of 75-μm i.d. with a total length of out at slightly acidic conditions (pH 5.6) in the reaction 80 cm (effective length of 68 cm). Before starting the ana- mixture that contain potassium dihydrogen phosphate/ lysis, the capillary was conditioned with 0.1 M NaOH for distilled water/acetonitrile (1:1:4 v/v). Interestingly, these 5 min followed by conditioning with distilled water for conditions resulted in efficient detection and quantifica- 3 min and electrolyte buffer for 3 min (50 mM sodium tion of β-alanine betaine in its intact form without de- phosphate buffer, pH 3.0). Between each run, the capillary composition as shown from the derivatized authentic was flushed with distilled water (1 min) and electrolyte standard of β-alanine betaine (Figure 2). In the mean- buffer (3 min). The temperature of the capillary was set at while, TMA was also effectively detected and quantified 25°C and then the samples and/or the authentic standards after derivatization under the same conditions. Thus, (β-alanine betaine and TMA) were injected hydrostatically the derivatization protocol of the current study could (25 mm, 60 s). During the run and in order to avoid sam- strikingly be used for simultaneous determination of ple carry-over into the electrophoresis buffer, the capillary both β-alanine betaine and TMA in plant, food, and was dipped twice in distilled water and washing buffer microbial samples. In many previous reports, various (same electrophoresis buffer that set in other tubes). The betaines were esterified with 4-bromophenacyl bromide applied potential was 20 kV, and the peaks of TMA-salt in the presence of a potassium bicarbonate/potassium and β-alanine betaine-ester were monitored at 262 nm. dihydrogen phosphate/acetonitrile (1:1:4 v/v)(Zhang et al. 1997; Nishimura et al. 2001; Zhang et al. 2002). Statistical analyses Under these conditions, peak of trimethylamine salt Statistical analyses were performed with the SPSS v. 18.0 derived from the breakdown of β-alanine betaine software (SPSS Inc., Chicago, IL, USA). One-factor was observed in the capillary electropherogram ANOVA was performed to identify statistically signifi- as confirmed by using authentic trimethylamine cant differences among treatments, followed by Tukey's (Nishimura et al. 2001; Zhang et al. 2002). During HSD test (P ≤ 0.05). derivatization with 4-bromophenacyl bromide, the Mohamed Ahmed et al. Journal of Analytical Science and Technology 2014, 5:38 Page 4 of 8 http://www.jast-journal.com/content/5/1/38 Figure 1 Reaction of β-alanine betaine and trimethylamine (TMA) with 4-bromophenacyl bromide and formation of β-alanine betaine-ester and TMA-salt. compound dimethylsulfoniopropionate (DMSP), which completely avoided by omitting potassium bicarbonate structurally resembles β-alanine betaine, was also degraded from the derivatization buffer. to dimethylsulfide (DMS) and acrylate under an alkaline condition (Gorham et al. 1982; Zhang et al. 2005). Zhang Repeatability, linearity, and detection limit of TMA and et al. (2005) also reported that they kept the decompos- β-alanine betaine ition of DMSP as minimum by lowering the pH of the The repeatability of the proposed method was explored esterification reaction mixture to 4.0 by using 18- by five consecutive runs of separate authentic standards Crown-6 instead of potassium carbonate. In the current (2.5 mM) of TMA and β-alanine betaine. The relative study, the decomposition of β-alanine betaine and con- standard deviation (RSD) for migration time of TMA sequently the production of the trimethylamine were and β-alanine betaine was 0.09% and 0.16%, respectively. Figure 2 Authentic standard of TMA salt and β-alanine betaine ester detected by capillary electrophoresis. About 3.2 mM of β-alanine betaine and TMA were esterified with 4-bromophenacyl bromide as described in the ‘Methods’ section. The samples were injected hydrostatically (25 mm, 60 s) at 25°C. The applied potential was 20 kV, and the peaks were monitored at 262 nm. Mohamed Ahmed et al. Journal of Analytical Science and Technology 2014, 5:38 Page 5 of 8 http://www.jast-journal.com/content/5/1/38 While, the RSD for peak area of the analytes TMA and peak area. They stated that these figures are acceptable β-alanine betaine was 4.24% and 2.57%, respectively. in many applications; however, the percentage of RSD These values were in general agreement with those re- could be improved by including an internal standard ported previously for TMA and glycine betaine when an- within the samples. Linearity was investigated using the alyzed by capillary electrophoresis (Timm and Jørgensen stock solution containing either TMA or β-alanine beta- 2002; Zhang et al. 2002). However, lower percentage of ine, which was serially diluted. Then, eight concentra- RSD of glycine betaine, choline, and TMA analyzed by tions (0.05 to 32 mM) of the analytes were analyzed in ion exchange chromatography with non-suppressed con- triplicate, and the calibration curves were constructed by ductivity detection method was recently reported (Zhang plotting the peak area versus the concentration (mM) of and Zhu 2007). To determine the day-to-day repeatabil- each analyte. The results showed that the suggested pro- ity of the analysis, the samples were analyzed by capillary cedure produced highly linear calibration curves (Figure 3) electrophoresis for three consecutive days, performed as with the correlation coefficients of 0.9933 and 0.9997 for sequence of five runs each day. The RSD of the migra- TMA and β-alanine betaine, respectively. The linearity tion time was 2.45% and 1.96% for TMA and β-alanine range of β-alanine betaine in the current study is greater betaine, respectively, whereas that of the peak area was than the range 0.05 to 5.0 mM reported previously 3.49% and 3.74% for TMA and β-alanine betaine, re- for many betaines analyzed by capillary electrophoresis spectively. In their analysis of day-to-day repeatability of (Zhang et al. 2002). Moreover, the linearity range of capillary electrophoresis method for estimation of vari- TMA in the current study is also greater than the range ous amines, Timm and Jørgensen (2002) reported a RSD 0.25 to 10.0 μg/ml reported previously for the analysis of less than 3% for migration time and less than 10% for of TMA in water samples by liquid chromatography Figure 3 Linearity curves of β-alanine betaine and trimethylamine as estimated by the developed capillary electrophoresis method. Trimethylamine (upper panel); β-alanine betaine (lower panel). Mohamed Ahmed et al. Journal of Analytical Science and Technology 2014, 5:38 Page 6 of 8 http://www.jast-journal.com/content/5/1/38 Figure 4 Capillary electrophoresis chromatogram of the intermediate metabolites of homocholine biodegradation by soil microorganism. The culture filtrate samples were esterified with 4-bromophenacyl bromide as described in the ‘Methods’ section. The samples were injected hydrostatically (25 mm, 60 s) at 25°C. The applied potential was 20 kV, and the peaks were monitored at 262 nm. The chromatogram showed the generated metabolite peaks from the degraded homocholine by the isolated strains alongside with authentic standard peaks of trimethylamine (TMA) and β-alanine betaine (β-AB). (Chafer-Pericas et al. 2004). The good linearity range of ratio was observed. The limit of detection of both com- the current methods is a good indication for its applic- pounds was found to be 0.01 mM (100 μM). Similarly, ability to accurately estimate these analytes in various the detection limit of glycine betaine estimated by low samples including plants, microbial, food, and clinical pH capillary electrophoresis method was reported to be samples. The limit of detection was calculated as the 0.01 mM (Nishimura et al. 2001; Zhang et al. 2002). More- concentration that produced a signal-to-noise ratio of 3 over, slightly lower detection limit (0.005 mM) for DMSP- and was estimated by analyzing solution of decreasing ester by low pH capillary electrophoresis has also been concentration of TMA and β-alanine betaine until this reported (Zhang et al. 2005). On the other hand, a lower detection limit (50 ng/ml) of TMA in water samples ana- lyzed by liquid chromatography has also been reported Table 1 Trimethylamine and β-alanine betaine content in (Chafer-Pericas et al. 2004). However, the detection limit bacterial culture and plant samples of many amines including trimethylamine as analyzed by Samples β-alanine Trimethylamine capillary electrophoresis with indirect UV-detection mode betaine was reported to be 0.01 mM (Timm and Jørgensen 2002). Bacterial culture (mmol/l) Generally, the detection limits of both compounds by the Arthrobacter sp. strain E5 5.52 9.22 proposed method in the current study well agreed with Pseudomonas sp. strain A9 4.62 41.48 those of the previously reported detection methods for in- Rhodococcus sp. strain A2 3.89 30.93 dividual compounds. Strikingly, in the current method, both compounds could simultaneously be determined Rhodococcus sp. strain A4 4.38 23.96 even if they exist in relatively lower concentrations. Al- Plant (μmol/g DW) though, the proposed method may be suitable for most Limonium suffruticosum 65.97 28.93 applications concerning the determination of TMA and Phragmites australis 64.44 26.96 β-alanine betaine in plants, microbial, environmental, and Elaeagnus oxycarpa (200 mM NaCl) 16.29 n.d. food samples, methods with extremely lower detection DW, dry weight; n.d., not detected. limits for these analytes might still be required. Mohamed Ahmed et al. Journal of Analytical Science and Technology 2014, 5:38 Page 7 of 8 http://www.jast-journal.com/content/5/1/38 Application of the method to microbial and plant samples of TMA in all microbial culture samples was higher com- To illustrate an application of the developed method, pared to the quantity of β-alanine betaine. This is an ideal three samples of either microbial cultures or dry cell reac- phenomenon because these strains were observed to tion mixtures or plant leaves were prepared as described cleave C/N pond of β-alanine betaine and rapidly use the in the ‘Methods’ section. The intermediate metabolites in resulted carbon chain as source of carbon with the release the microbial culture filtrate of different microorganisms of TMA as a major metabolite (Mohamed Ahmed et al. grown on homocholine as the sole source of carbon and 2009a, b; Mohamed Ahmed et al. 2010; Mohamed Ahmed nitrogen was analyzed by the developed method. During et al. 2014). To check the accuracy of this method, the the consumption of homocholine by the growing cell cul- TMA concentration in the culture filtrate and intact cell tures of the isolated strains of the genera Arthrobacter, reaction products of the isolated strains was analyzed by Pseudomonas,and Rhodococcus (Mohamed Ahmed et al. using picric acid-based colorimetric method (Dyer 2009a, b; Mohamed Ahmed et al. 2010), there were a con- 1945). The results showed no significant differences current formation and accumulation of some soluble me- from that obtained by the developed capillary electro- tabolites identified as trimethylamine (peak 3, TMA) and phoresis method. To expand the applicability of the de- β-alanine betaine (peak 4, β-AB) as detected by capillary veloped method, the plant leaves that were obtained electrophoresis method (Figure 4). Under the optimized from Xinjiang, China, in August 2010 were also pre- derivatization conditions of the developed method, the pared and analyzed by this method as described in the TMA-salt and β-alanine betaine-ester were successfully ‘Methods’ section. The results again showed clear peaks separated and detected with very clear and sharp peaks of TMA and β-alanine betaine in the leaf samples of many using a UV detector at 262 nm. The amounts of these me- of plants growing on saline soil in this area (Figure 5). tabolites were successfully estimated (Table 1) by calculat- These osmotolerant plants accumulated sufficient amounts ing the area of each peak. It can be seen that the quantity of nitrogenous compounds to cope with these osmotic Figure 5 Capillary electrophoresis chromatogram of the osmolytes in the leaves of Limonium suffruticosum grown in saline soil. Plant leaf samples were esterified with 4-bromophenacyl bromide as described in the ‘Methods’ section. The samples were injected hydrostatically (25 mm, 60 s) at 25°C. The applied potential was 20 kV, and the peaks were monitored at 262 nm. The chromatogram showed the accumulated osmolytes in the plant samples alongside with authentic standard peaks of trimethylamine (TMA) and β-alanine betaine (β-AB). Mohamed Ahmed et al. Journal of Analytical Science and Technology 2014, 5:38 Page 8 of 8 http://www.jast-journal.com/content/5/1/38 stresses. Of these nitrogenous compounds, considerable Dyer WJ (1945) Amines in fish muscles 1. Colorimetric determination of trimethylamine as the picrate salt. J Fish Res Board Can 6(5):351–358 amounts of both TMA and β-alanine betaine were quanti- Ghaly AE, Dave D, Budge S, Brooks MS (2010) Fish spoilage mechanisms and tatively estimated using the developed method (Table 1). preservation techniques: review. Amer J Appl Sci 7:859–877 Similarly, the concentration of β-alanine betaine in many Gorham J (1995) Betaines in higher plants—biosynthesis and role in stress metabolisms. In: Wallsgrove RM (ed) Amino acids and their derivatives in plants of the family Plumbaginaceae was found in the higher plants. Cambridge University Press, Cambridge, England, pp 173–203 range of 1 to 147 μg/g DW, which was estimated by either Gorham J, McDonnell E, Wyn Jones RG (1982) Determination of betaines as TLC and autoradiography (Rathinasabapathi et al. 2000) or ultraviolet-absorbing esters. Anal Chim Acta 138:277–283 Grieve CM, Grattan SR (1983) Rapid assay for determination of water soluble H NMR (Baysalfurtana et al. 2013). Collectively, the above quaternary ammonium compounds. Plant Soil 70:303–307 findings clearly demonstrated the suitability of the de- Hanson AD, Rathinasabapathi B, Rivoal J, Burnet M, Dillon MO, Gage DA (1994) veloped method for the simultaneous detection and Osmoprotective compounds in the Plumbaginaceae: a natural experiment in metabolic engineering of stress tolerance. Proc Natl Acad Sci USA quantification of these analytes in microbial culture and 91:306–310 plant samples. Although it is not tested in the current Mohamed Ahmed IA, Jiro A, Ichiyanagi T, Sakuno E, Mori N (2009a) Isolation and study, the developed method could efficiently be used characterization of 3-N-trimethylamino-1-propanol degrading Rhodococcus sp. strain A2. FEMS Microbiol Lett 296(2):219–225 for the estimation of TMA and β-alanine betaine in Mohamed Ahmed IA, Jiro A, Ichiyanagi T, Sakuno E, Mori N (2009b) Isolation and both food and feed samples. characterization of 3-N-trimethylamino-1-propanol degrading Arthrobacter sp. strain E5. Res J Microbiol 4(2):49–58 Mohamed Ahmed IA, Jiro A, Ichiyanagi T, Sakuno E, Mori N (2010) Isolation and Conclusion characterization of homocholine degrading Pseudomonas sp. strain A9 and A capillary electrophoresis method for the simultaneous B9b. World J Microbiol Biotechnol 26(8):1455–1464 determination of TMA and β-alanine betaine was devel- Mohamed Ahmed IA, Eltayeb MM, Arima J, Mori N, Yamanaka N, Taniguchi T (2014) Screening for enzymatic activities in the degradation pathway of oped. The method described here has generally wide de- homocholine by soil microorganisms. Australian J Bas Appl Sci 8(2):222–233 tection range suitable for analysis of TMA and β-alanine Nishimura N, Zhang J, Abo M, Okubo A, Yamazaki S (2001) Application of betaine in microbial and plant samples. The advantages capillary electrophoresis to the simultaneous determination of betaines in plants. Anal Sci J 17:103–106 of the current method are its low cost, low detection Rathinasabapathi B, Sigua C, Ho J, Gage DA (2000) Osmoprotectant β-alanine limit, simple operation, rapid, and high sensitivity. betaine synthesis in the Plumbaginaceae: S-adenosyl-L-methionine dependent N methylation of β-alanine to its betaine via N-methyl and Competing interests N, N-dimethyl β-alanines. Physiol Plant 109:225–231 The authors declare that they have no competing interests. Rhodes D, Hanson AD (1993) Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu Rev Plant Physiol Plant Mol Biol 44:357–384 Authors' contributions Shintani H, Polonsky J (1997) Handbook of capillary electrophoresis applications. IAMA has performed most of the experimental and analytical work and Blackie Academic & Professional, London prepared the draft of the manuscript. AM has performed the plant-related Siminhoff ML, Burke JF, Saukkonen JJ, Ordinario AT, Doty R, Dunn S (1977) parts of the experimental work with the direct help from IAMA. The Biochemical profile of uremic breath. The New Engl J Med 297:132–135 guidelines and supervision of this work was provided by NM, NY, and TT. Timm M, Jørgensen BM (2002) Simultaneous determination of ammonia, All authors read and approved the final manuscript. dimethylamine, trimethylamine and trimethylamine-N-oxide in fish extracts by capillary electrophoresis with indirect UV-detection. Food Chem Acknowledgements 76(4):509–518 Financial assistance from the Ministry of Education, Culture, Sports, Science, Yancey PH (2005) Organic osmolytes as compatible, metabolic and counteracting and Technology of Japan in the form of a scholarship for the first and cytoprotectants in high osmolarity and other stresses. J Exp Biol 208:2819–2830 second authors is gratefully acknowledged. Zhang J, Zhu Y (2007) Determination of betaine, choline and trimethylamine in feed additive by ion-exchange liquid chromatography/non-suppressed Author details conductivity detection. J Chromatogr A 1170:114–117 Department of Food Science and Technology, Faculty of Agriculture, Zhang J, Okubo A, Yamazaki S (1997) Determination of betaines in plants by University of Khartoum, Shambat 13314, Sudan. Arid Land Research Center, low-pH capillary electrophoresis as their phenacyl esters. Bunseki Kagaku Tottori University, 1390 Hamasaka, Tottori 680-0001, Japan. School of (in Japanese) 46:275–279 Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Zhang J, Nishimura N, Okubo A, Yamazaki S (2002) Development of an analytical Tottori University, Koyama, Tottori 680-8553, Japan. method for the determination of betaines in higher plants by capillary electrophoresis at low pH. Phytochem Anal 13:189–194 Received: 3 June 2014 Accepted: 24 July 2014 Zhang J, Nagahama T, Abo M, Okubo A, Yamazaki S (2005) Capillary electrophoretic analysis of dimethylsulfoniopropionate in sugarcane and marine algal extracts. Talanta 66:244–248 References Baysalfurtana G, Duman H, Tipirdamaz R (2013) Seasonal changes of inorganic doi:10.1186/s40543-014-0038-x and organic osmolyte content in three endemic Limonium species of Lake Cite this article as: Mohamed Ahmed et al.: Simultaneous determination Tuz (Turkey). Turk J Bot 37:455–463 of β-alanine betaine and trimethylamine in bacterial culture and plant Blunden G, El Barouni MM, Gordon SM, McLean WFH, Rogers DJ (1981) samples by capillary electrophoresis. Journal of Analytical Science and Extractions, purification and characterization of Dragendorff-positive Technology 2014 5:38. compounds from some British marine algae. 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"Journal of Analytical Science and Technology" – Springer Journals
Published: Dec 1, 2014
Keywords: Analytical Chemistry; Characterization and Evaluation of Materials; Monitoring/Environmental Analysis
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