J S S
ISSN 1615-9306 · JSSCCJ 40 (3) 597–820 (2017) · Vol. 40 · No. 3 · February 2017 · D 10609
JOURNAL OF
SEPARATION SCIENCE
Methods Chromatography · Electroseparation
3 17 VOLUME
40
www.jss-journal.com
Applications Biomedicine · Foods · Environment
JSSC_40_3_cover.indd 1
13/02/17 4:38 PM
Received: 30 July 2016
Revised: 30 October 2016
Accepted: 9 November 2016
DOI: 10.1002/jssc.201600852
RESEARCH ARTICLE
Simultaneous analysis of ten low-molecular-mass organic acids in the tricarboxylic acid cycle and photorespiration pathway in Thalassiosira pseudonana at different growth stages Mengwei Ye1,3 Xiaokai Wu3
Lijing Zhang2 Juanjuan Chen1
1 Key Laboratory of Applied Marine Biotechnology, Ningbo University, Chinese Ministry of Education, Ningbo, P.R. China 2 Zhejiang
Pharmaceutical College, Ningbo, P.R.
Panpan Xu1
Runtao Zhang1
Chengxu Zhou3
Jilin Xu1
Xiaojun Yan3
A method using high-performance liquid chromatography coupled with tandem mass spectrometry was developed for the simultaneous determination of organic acids in microalgae. o-Benzylhydroxylamine was used to derivatize the analytes, and stable isotope-labeled compounds were used as internal standards for
China
precise quantification. The proposed method was evaluated in terms of linearity, recovery, matrix effect,
3 Collaborative
Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University, Ningbo, P.R. China
sensitivity, and precision. Linear calibration curves with correlation coefficients >0.99 were obtained over
Correspondence Jilin Xu, Key Laboratory of Applied Marine Biotechnology, Ningbo University, Chinese Ministry of Education, Ningbo, Zhejiang 315211, P. R. China. E-mail:
[email protected] Xiaojun Yan, Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy aquaculture Aquaculture, Ningbo University, Ningbo, Zhejiang 315211, P. R. China. E-mail:
[email protected]
isocitric acid, 2–200 ng mL−1 for citric acid, 100–10000 ng mL−1 for cis-aconitic acid, and 1–100 ng
the concentration range of 0.4–40 ng/mL for glycolic acid, 0.1–10 ng/mL for malic acid and oxaloacetic acid, 0.02–2 ng/mL for succinic acid and glyoxylic acid, 4–400 ng/mL for fumaric acid, 20–2000 ng/mL for mL−1 for α-ketoglutaric acid. Analyte recoveries were between 80.2 and 115.1%, and the matrix effect was minimal. Low limits of detection (0.003–1 ng/mL) and limits of quantification (0.01–5 ng/mL) were obtained except cis-aconitic acid. Variations in reproducibility for standard solution at three different concentrations levels were 100, there is a positive matrix effect with signal enhancement; and if the recovery (%) < 100, there is a negative matrix effect with signal suppression. Supporting Information Table S5 shows mild matrix effects for all organic acids as the values were closed to 100. Malic acid,
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TABLE 1
Calibration data, LODs, and LOQs for organic acids Calibration data
Analytes
LOD (ng/mL)
LOQ (ng/mL)
Linearity range (ng/mL)
Equation
R2
Glycolic acid
0.025
0.1
0.4–40
y = 0.0052 + 0.0003x
0.9911
Malic acid
0.01
0.02
0.1–10
y = 2.5715 + 0.4556x
0.9903
Succinic acid
0.003
0.01
0.02–2
y = 3.7827 + 18.0728x
0.9921
Fumaric acid
0.75
1
4–400
y = 0.0422 + 0.0172x
0.9907
Isocitric acid
1
5
20–2000
y = 0.0092 + 0.0034x
0.9935
Glyoxylic acid
0.005
0.01
0.02–2
y = 0.0034 + 0.0009x
0.9919
Citric acid
0.05
0.15
2–200
y = 0.034 + 0.0090x
0.9902
Oxaloacetic acid
0.01
Cis-aconitic acid α-Ketoglutaric acid TABLE 2
0.1–10
y = 0.6724 + 0.4666x
0.9922
100–10000
y = 0.0018 + 0.0056x
0.9923
1–100
y = 0.4037 + 0.1975x
0.9936
0.03
25
50
0.02
0.05
Recovery and precision obtained for organic acids Recovery (%)
Analytes
1a) 91.3 ± 4.5
Glycolic acid
2b) 92.6 ± 6.9
Intraday precision, RSD%
Interday precision, RSD%
3c)
1
2
3
1
2
3
106.2 ± 4.8
1.32
0.43
0.69
1.35
0.83
0.71
95.9 ± 1.7
94.2 ± 2.8
93.7 ± 3.3
1.87
3.45
2.15
1.76
3.12
2.34
Succinic acid
103.2 ± 1.8
104.5 ± 4.6
110.9 ± 3.2
4.32
2.16
3.14
2.21
2.56
0.93
Fumaric acid
110.4 ± 6.2
106.7 ± 5.5
92.5 ± 2.2
5.47
2.56
3.72
2.43
1.69
2.51
Isocitric acid
107.9 ± 2.6
115.1 ± 3.2
112.5 ± 3.2
5.79
5.56
6.85
3.79
4.16
2.53
95.1 ± 3.9
96.1 ± 3.3
80.2 ± 3.2
5.78
1.78
4.11
4.19
3.65
4.03
Malic acid
Glyoxylic acid
95.6 ± 2.0
93.5 ± 2.9
92.5 ± 3.9
7.89
7.43
6.42
5.99
7.44
8.81
Oxaloacetic acid
106.1 ± 2.8
106.3 ± 4.8
86.1 ± 4.9
4.35
6.01
1.53
4.71
3.22
4.90
Cis-aconitic acid
97.7 ± 2.9
96.8 ± 4.1
92.3 ± 3.4
4.51
2.8
2.23
4.67
3.78
2.07
α-Ketoglutaric acid
85.7 ± 3.0
88.0 ± 3.8
87.6 ± 4.7
2.02
2.78
1.56
3.49
2.06
1.50
Citric acid
a) Low concentration. b) Intermediate concentration. c) High concentration.
succinic acid, fumaric acid, citric acid and oxaloacetic acid showed enhancement effects with recoveries ranging from 101 to 107%, whereas glycolic acid, isocitric acid, glyoxylic acid, cis-aconitic acid and α-ketoglutaric acid showed suppressive effects with recoveries below 100%. Therefore, the use of stable isotope-labeled internal standards contributed to the minimization of matrix effects for the accurate quantitative analysis of organic acids in T. pseudonana. 3.5.3
Sensitivity
The high concentrations of organic acids made derivatization rapid and complete, but it influenced the separation and detection in the HPLC–ESI-MS/MS. Therefore, the optimal concentration of organic acids for detection needed to be evaluated. Under optimal conditions, LOD and LOQ were defined as the concentrations of standard compounds generating an S/N of 3 and 10, respectively. The measured values ranged from 0.003 ng/mL for succinic acid to 25 ng/mL for cisacoitic acid for detection limit and from 0.01 to 50 ng/mL for the quantification limit. A complete listing of these values is
provided in Table 1. It was observed that the organic acids containing C=C bonds had low sensitivity when compared with other organic acids, such as cis-aconitic acid and fumaric acid. We hypothesized that the chemical stability of the double bond or its poor ionization efficiency resulted in their higher LOD values. 3.5.4
Precision
The precision of proposed method was investigated as intraday and interday RSD values at three different concentration levels (Supporting Information Table S4). To evaluate intraday RSD, a mixture of standard solution in T. pseudonana sample was analyzed in triplicate within one day. For interday RSD, standard mixtures of organic acids in T. pseudonana sample were prepared and analyzed in triplicates per day for three consecutive days. As shown in Table 2, the result indicated that the intraday precision values varied from 0.43 to 7.89%, and the interday precision values varied from 0.71 to 8.81%, demonstrating satisfactory reproducibility for all organic acids.
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FIGURE 4
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The changes of organic acids content in T. pseudonana samples that were harvested at four different culture phases
3.6 The changes of the ten low molecular mass organic acids in T. pseudonana at different growth stages For precise quantification, samples were spiked with mixed isotope internal standards. Unfortunately, because of technical limitations, it was difficult to synthesize individual isotope standard for all organic acids. An alternative approach was to use compounds whose chemical properties are close to them. Tan et al. used 2-hydroxyglutarate-d4 as an internal standard to quantify malic acid and oxaloacetic acid [39]. In this work, we used 2-α-ketoglutaric acid-113 C for analyzing oxaloacetic acid, because its chemical
structure was similar to oxaloacetic acid, which included two carboxylic acid groups and one carbonyl group. This substitution also reduced cost because of the same reagent could be used to quantify α-ketoglutaric acid. Another group utilized 13 C-malic acid for the quantification of oxaloacetic acid, citric acid, cis-aconitic acid, and malic acid, and 13 C-succinic acid for the quantification of succinic acid and fumaric acid [36]. In contrast, it seems that the quantitative result became more precise and more accurate with the use of the corresponding isotope internal standards for each organic acid instead of analogues, although the total analysis cost would be higher. It is important to note that the sample was diluted before
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analysis, since the dilution of sample could reduce the interference greatly and yield a good signal in the mass spectrometer. The organic acids were identified in the sample by comparing the retention times and molecular masses with standard substances. The concentration of unlabeled organic acids could be calculated according to the peak abundance ratios of the labeled and unlabeled organic acids [51]. The developed method was applied to analyze the levels of various carboxylic acids during four growth stages of T. pseudonana. As shown in Fig. 4, an increasing trend was observed during the adaptation phase for malic acid and fumaric acid, whereas a significant decreasing trend occurred at the stationary phases. The opposite trend of change was observed, in which the citric acid and succinic acid concentration increased, and the oxaloacetic acid and isocitric acid concentrations decreased during the whole course of growth. The content of glyoxylic acid and glycolic acid gradually decreased, whereas the levels of glycolic acid produced fluctuated during the adaptation phase. In addition, citric acid and fumaric acid were found at high levels in the T. pseudonana samples, contributing >70% of all acids during each growth period. The cis-aconitic acid could not be detected in the sample although it was likely present in the sample, albeit in low concentrations. As previously reported, the variation in organic acid content appears to be correlated with photosynthesis [52, 53]. The TCA cycle has a crucial function in photosynthetic sucrose synthesis and cell growth by providing adenosine triphosphate and various carbon skeletons [54]. Therefore, the highest content of organic acids in the TCA cycle occurred in the exponential phase. The exception was oxaloacetic acid, which was easily hydrolyzed to pyruvate because of its instability at acidic and basic pH or high temperature [42,43,55]. The stationary phase brought about a decreased rate of photosynthesis and enzymatic activity. This may account for the observation that some organic acids decreased or became invariable during this phase. Further studies are necessary to clarify the reason why succinic acid and citric acid increased in this phase. Glyoxylic acid and glycolic acid are inhibitors of photosynthesis [56], although the concentration was lower than others. We believe that lower levels of these inhibitory carboxylic acid help to fix more carbon by photosynthesis for plant growth in the exponential phase. We were able to determine the changes of low molecular mass organic acids in microalgae, study how their levels interact with the growth state, and provide reference value for high yields of microalgae culture.
4
CO NC LU D I NG R E M A R K S
In this work, we developed a reliable method to simultaneously analyze many low molecular mass organic acids in
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T. pseudonana by o-BHA derivatization and HPLC–ESIMS/MS analysis. The analysis was carried out following an easy extraction procedure, a simple derivatization, and a rapid chromatographic separation and analyte detection. The use of stable isotope-labeled compounds as internal standards effectively improved accuracy. Validation experiments demonstrated that this approach had good linearity and sensitivity as well as sufficient accuracy and precision, and the matrix effect was minimal. This method successfully quantified nine organic acids, and the changes of organic acids at different growth stage indicated that their levels were related to the growth stage. ACKNOWLEDGMENTS
This research was supported by the National Natural Science Foundation of China (31172448); National Sparking Plan Project of China (2014GA720002); Project of Ministry of Education, China (20133305130001); Zhejiang Natural Science Foundation, China (LY15C190004), Ningbo Science and Technology Research Projects, China (2014C10005); Ningbo Marine Algae Biotechnology Team, China (2011B81007), and partly sponsored by K. C. Wong Magna Fund in Ningbo University. REFERENCES 1. Lopez-Bucio, J., Nieto-Jacobo, M. F., Ramırez-Rodrıguez, V., HerreraEstrella, L., Organic acid metabolism in plants: From adaptive physiology to transgenic varieties for cultivation in extreme soils. Plant Sci. 2000, 160, 1–13. 2. Ma, J. F., Role of organic acids in detoxification of aluminum in higher plants. Plant and Cell Physiol. 2000, 41, 383–390. 3. Abadía, J., López-Millán, A.-F., Rombolà, A., Abadía, A., Organic acids and Fe deficiency: A review. Plant Soil. 2002, 241, 75–86. 4. Vance, C. P., Uhde-Stone, C., Allan, D. L., Phosphorus acquisition and use: Critical adaptations by plants for securing a nonrenewable resource. New Phytol. 2003, 157, 423–447. 5. Zhang, F., Ma, J., Cao, Y., Phosphorus deficiency enhances root exudation of low-molecular weight organic acids and utilization of sparingly soluble inorganic phosphates by radish (Raghanus satiuvs L.) and rape (Brassica napus L.) plants. Plant Soil. 1997, 196, 261–264. 6. Rigobello-Masini, M., Penteado, J. C. P., Tiba, M., Study of photorespiration in marine microalgae through the determination of glycolic acid using hydrophilic interaction liquid chromatography. J. Sep. Sci. 2012, 35, 20–28. 7. Aliyev, J. A., Photosynthesis, photorespiration and productivity of wheat and soybean genotypes. Physiol Plant. 2012, 145, 369–383. 8. Kloos, D., Lingeman, H., Mayboroda, O., Deelder, A., Niessen, W., Giera, M., Analysis of biologically-active, endogenous carboxylic acids based on chromatography-mass spectrometry. TrAC Trends Anal. Chem. 2014, 61, 17– 28. 9. Visentin, M., Pietrogrande, M C., Determination of polar organic compounds in atmospheric aerosols by gas chromatography with ion trap tandem mass spectrometry. J. Sep. Sci. 2014, 37, 1561–1569. 10. Arnetoli, M., Montegrossi, G., Buccianti, A., Gonnelli, C., Determination of organic acids in plants of Silene paradoxa L. by HPLC. J. Agric. Food Chem. 2008, 56, 789–795. 11. Erro, J., Zamarreno, A. M., Yvin, J.-C., Garcia-Mina, J. M., Determination of organic acids in tissues and exudates of maize, lupin, and chickpea by
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S U P P O RTI NG I N FO R M AT I O N
Additional Supporting Information may be found online in the supporting information tab for this article. How to cite this article: Ye M, Zhang L, Xu P, Zhang R, Xu J, Wu X, Chen J, Zhou C, Yan X. (2017). Simultaneous analysis of ten low-molecular-mass organic acids in the tricarboxylic acid cycle and photorespiration pathway in Thalassiosira pseudonana at different growth stages. J. Sep. Sci., 40, 635–645. DOI: 10.1002/jssc.201600852
