# Determination of Free Amino Acids in Three Species of Duckweed (Lemnaceae)

Authors: Xiaolong Zhang; Hongyan Chen; Dawei Wu; Wei Gu; Xiao Sun; Jian Chen; Qinan Wu
Journal: Journal of Food Quality
 (2018)

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## Abstract

In this study, a fast, simple, precise, and sensitive hydrophilic interaction liquid chromatography (HILIC) method was established for simultaneous determination of free amino acids in three different varieties of duckweed includingSpirodela polyrhiza (L.) Schleid., Landoltia punctata (G. Mey.) Les & D. J. Crawford, and Lemna aequinoctialis Welwitsch by ultrahigh performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Method validation was processed in terms of linearity, precision, stability, repeatability, and accuracy as well as limits of detection and quantification. The developed method was applied for quantification of 59 batches of samples. Then chemometric analysis was used to evaluate different duckweeds by principle component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). The results demonstrated that there was no significant difference in FAAs’ profile among three varieties of duckweed.


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## Body

## 1. Introduction

Amino acids are kinds of nitrogenous components that play vital and diverse roles in metabolism and have attracted significant attention in food, feedstuff, and alimentary supplements. Amino acids are basic units and important ingredients of proteins and involved in the progress of biosynthesis for glycoprotein, porphyrins, neurotransmitters, polyamines, and nitric oxide [1–5]. Nutritional studies show that amino acids can modulate gene expression and enhance the growth of skeletal muscle and small intestine [6].The family of Lemnaceae colloquially known as duckweed has been consumed as human food since long [7]. Duckweeds have the striking capacity of explosive reproduction [8]. They can grow almost everywhere with appropriate temperature and nutrition in water. But their greatest potential is to produce large quantities of protein-rich biomass that is a promising food resource for humans [9] and is suitable for feeding a wide range of animals including fish, poultry, and cattle [10–12]. The contents of free amino acids (FAAs) are very important for the evaluation of the protein-rich food. Fresh duckweed contains a large amount of water with moisture content of 86%–94%. But for dried samples, their protein concentration ranges from 25% to 40% with much lower fibrous material than that of land forage [13]. The habitat influences the content of proteins and fibers dramatically. Content of amino acids and protein always depend on their species pattern and growth environment, such as sunshine, nutrition media, and their growth pattern: gregarious or solitary. Under high nutritional conditions, more proteins and fewer fibers are accumulated in duckweed and vice versa. The amino acid type of duckweed is very close to that of animals and thus could be used by animals efficiently [7, 14]. The first limited amino acid (lysine) is similar to soybean and is more superior to that in other food such as sorghum and maize.There are several methods reported in literature for analysis of free amino acids. In general, the FAAs are extracted with solvents water, formic acid, hydrochloric acid, or ethanol, followed by filtration or centrifugation before analysis [15–17]. Several techniques have been described for detection of free amino acids including ion-exchange chromatography [18], precolumn derivatization followed by reversed high-performance liquid chromatography coupled with diode array or fluorescence detector [19, 20], gas chromatography, mass spectrometry, and capillary electrophoresis [21]. In recent years, determination of FAAs using ultrahigh performance liquid chromatography hyphenated to mass spectrometry has gained great notice because of the merit of selective separation for the polar underivatized analytes under HILIC condition with acetonitrile-dominated mobile phase [22–25].In present study, a feasible and reliable UPLC-QTRAP-MS/MS method was developed and validated for quantitative analysis of 24 underivatized FAAs simultaneously. Multivariate statistical analysis was employed to assess the differences in the profiles of FAAs among three species of duckweed. This simple and fast analytical method would be used for quality control of duckweeds.


## 2. Materials and Methods

### 2.1. Reagents and Materials

59 batches of duckweed were collected from different regions in China as shown in Table1. The samples were identified and classified by PCR [26–28], and the number of populations was as follows: 25 populations from species Spirodela polyrhiza, 16 populations from species Landoltia punctata (its another previously invalid name is Spirodela oligorrhiza (Kurz) Hegelm.), and 18 populations from Lemna aequinoctialis. Fresh materials were washed by tap water, dried in sunshine, and pulverized, and the powder was screened through 60-mesh sieve. Reference standards of tyrosine (Tyr), alanine (Ala), glutamic acid (Glu), phenylalanine (Phe), histidine (His), isoleucine (Ile), aspartic acid (Asp), lysine (Lys), glycine (Gly), valine (Val), citrulline (Cit), cystine (Cys2), proline (Pro), tryptophan (Trp), leucine (Leu), arginine (Arg), cysteine (Cys), hydroxylproline (Hpro), methionine (Met), threonine (Thr), asparagine (Asn), and serine (Ser) were purchased from National Institutes for Food and Drug Control (Beijing, China) with the purity more than 98%. Reference standards of γ-aminobutyric (GABA) and glutamine (Gln) with the purity more than 98% were obtained from Aladdin Industrial Corporation (Shanghai, China). Acetonitrile (HPLC grade) was purchased from Merk (Darmstadt, Germany). Formic acid and ammonium formate (HPLC grade) were provided by Mreda Technology Incorporation (USA). Deionized water used for analysis procedure was produced by a Milli-Q Academic ultrapure water system (Millipore, Bedford, MA, USA).Table 1
Information of duckweeds from different regions.
Sample
Species
Site
SP1-7
Spirodela polyrhiza
Yangzhou City of Jiangsu Province
SP8-9
Spirodela polyrhiza
Yancheng City of Jiangsu Province
SP10-11
Spirodela polyrhiza
Nanjing City of Jiangsu Province
SP12-13
Spirodela polyrhiza
Huaian City of Jiangsu Province
SP14-17
Spirodela polyrhiza
Lianyungang City of Jiangsu Province
SP18-19
Spirodela polyrhiza
Linyi City of Shandong Province
SP20-21
Spirodela polyrhiza
Hefei City of Anhui Province
SP22-23
Spirodela polyrhiza
Hangzhou City of Jiangsu Province
SP24-25
Spirodela polyrhiza
Baoding City of Hebei Province
LP1-11
Landoltia punctata
Yangzhou City of Jiangsu province
LP12-16
Landoltia punctata
Huaian City of Jiangsu province
LA1-3
Lemna aequinoctialis
Yangzhou City of Jiangsu Province
LA4-8
Lemna aequinoctialis
Yancheng City of Jiangsu Province
LA9-14
Lemna aequinoctialis
Nanjing City of Jiangsu Province
LA15-16
Lemna aequinoctialis
Lianyungang City of Jiangsu Province
LA17-18
Lemna aequinoctialis
Linyi City of Shandong Province


### 2.2. Instrumentation and Chromatographic and Mass Conditions

Chromatographic analysis was performed on a Shimadzu LC-30 ultrahigh performance liquid chromatography system (Shimadzu, Japan), which consisted of a communication bus module (CMB-20A), a vacuum degasser, binary gradient pumps (LC-30AD), an autosampler (SIL-30A), and a column oven (CTO-30A) coupled with an AB Sciex QTRAP 5500 (AB SCIEX, USA). A Waters XBridge Amide column (2.1 mm × 150 mm, 2.5μm, Waters, USA) was used for chromatographic separation. The binary mobile phase was composed of acetonitrile (A) and 10 mM ammonium formate with 0.2% (v/v) formic acid (B) at a flow rate of 0.4 mL/min. The linear gradient elution was carried out as follows: 0–5 min, 90%–85.5% A; 5–11 min, 85.5%–54% A; 11-12 min, 54%–10% A; 12–14 min, 10% A; 14-15 min, 10%–90% A; 15–20 min, and 90% A. The reequilibration time was 4 min with a total running time of 20 min. The column compartment was kept at 20°C, while the autosampler trial was maintained at 15°C, and injection volume was 2 μl. The needle was washed with mixtures of acetonitrile and water.The mass spectrometry assay was performed on a triple quadruple mass spectrometer equipped with an electrospray ionization source. The spectra were recorded under positive-ion type with the multiple reaction monitoring (MRM) mode. The capillary voltage was 5500 V, and desolvation gas temperature was 550°C. The curtain gas was 35 psi with both nebulizer and drying gas of 55 psi. Nitrogen was used as source gas with purity more than 95% and collision gas with purity over 99.999%. The 24 components of amino acids were optimized in the tuning mode with mass only to obtain the mass of the precursor and product ion, the best declustering potential (DP), and collision energy (CE), respectively. The details are shown in Table2. The entrance potential (EP) and collision cell exit potential (CXP) were set as default value. The typical LC-QTRAP-MS/MS chromatograms are shown in Figure 1.Table 2
The precursor ions, product ions, declustering potential, and collision energy of amino acids.
Number
Compound
Precursor ion
Product ion
DP (V)
CE (V)
1
Tyr
182.0
91.1
60
37
2
Ala
90.0
44.1
50
14
3
Glu
148.1
84.0
60
21
4
Phe
166.1
120.1
60
19
5
His
156.1
110.3
80
20
6
Ile
132.1
86.1
50
14
7
Asp
134.1
74.0
50
19
8
Lys
147.0
84.1
60
23
9
Gly
76.1
29.9
40
16
10
Val
118.1
71.9
50
15
11
Cit
176.2
70.0
60
31
12
GABA
104.1
86.8
50
14
13
Cys2
241.1
73.9
80
40
14
Pro
116.0
70.1
60
22
15
Trp
205.1
146.0
50
24
16
Leu
132.1
86.1
50
14
17
Arg
175.2
70.2
90
30
18
Cys
122.1
59.0
40
32
19
Hpro
132.1
86.1
60
20
20
Met
150.1
103.9
60
14
21
Thr
120.1
74.1
40
14
22
Asn
133.1
74.0
60
21
23
Gln
147.0
84.1
60
23
24
Ser
106.0
60.1
60
15Figure 1
Typical MRM chromatograms of the 24 amino acids.


### 2.3. Preparation of Reference Compound Solution

Stock solutions of individual standards were made by dissolving accurately weighed components in 0.5% formic acid at a concentration of 0.2 mg/mL approximately and stored at 4°C before use. The mixed working solutions of all the standards were diluted with 0.5% formic acid solution to a series of appropriate concentrations immediately before analysis and used to attain the calibration curves.


### 2.4. Preparation of Sample Solution

Each sample powder was weighed 0.2 g accurately and transferred into a 25 mL stainless tube with 6 stainless beads with a diameter of 0.6 mm. Then, the sample was extracted in a high-throughput tissue-grinding apparatus with vibrating frequency of 70 kHz for 120 s with 15 mL 0.5% formic acid. The sample solution was centrifuged at 15000 rpm for 10 min, and then the supernatant was screened through a 0.22μm polytetrafluoroethylene membrane filter and stored in a glass bottle at 4°C for later LC-MS/MS analysis.


### 2.5. Method Validation

For each reference substance, the calibration curve was confirmed by linear regression of the peak area versus concentration. The limits of detection (LOD) and limits of quantification (LOQ) were determined by diluting the standard solution till the signal-to-noise ratios were about 3 and 10, respectively. Precision was determined by intraday and interday variability. The intraday variability was conducted by determining the same standard solution in six replicates on the same day. The interday variability was conducted by determining the same solution for three consecutive days. The relative standard deviation (RSD) values were calculated to denote the precision. Stability of the sample solution was analysed by peak areas of analytes at 0, 2, 4, 8, 12, and 24 h at room temperature. To evaluate the repeatability of the developed method, the same sample was analysed in six replicates and variations were expressed as RSD. The recovery test was performed to assess the accuracy of the method. For the test, a known amount of the 24 standard components was spiked into a certain amount (0.1 g) of sample. Then, the spiked sample was extracted and analysed as described. The recovery rate was calculated by using the following formula: recovery (%) = (measured amount − original amount)/spiked amount  × 100%. Six replicates were performed for the recovery test.


### 2.6. Data Analysis

The quantitative analyses of acquired data were processed by MultiQuant 3.0.2 (AB SCIEX, USA). Multivariate statistical analysis including PCA and OPLS-DA was used to classify the sample. SIMCA-P 14.1 (Umetrics AB, Sweden) was used to perform statistical analysis for the data of 59 batches of duckweed.


### 2.7. Analysis of Minerals and Total Proteins

Twenty-four minerals of each environment water where duckweed lived were quantified by ICP-MS. Total nitrogen and phosphorus of the water body were measured according to the alkaline potassium persulfate digestion method GB11894-89 and the ammonium molybdate spectrophotometric method GB11893-89 published by the State Bureau of Technological Supervision of China. Total proteins of each duckweed were determined by the Kjeldahl method. Each sample of duckweeds was hydrolyzed in acidic conditions, and then total amino acids were quantified by the HPLC-MSMS method mentioned above.


## 2.1. Reagents and Materials

59 batches of duckweed were collected from different regions in China as shown in Table1. The samples were identified and classified by PCR [26–28], and the number of populations was as follows: 25 populations from species Spirodela polyrhiza, 16 populations from species Landoltia punctata (its another previously invalid name is Spirodela oligorrhiza (Kurz) Hegelm.), and 18 populations from Lemna aequinoctialis. Fresh materials were washed by tap water, dried in sunshine, and pulverized, and the powder was screened through 60-mesh sieve. Reference standards of tyrosine (Tyr), alanine (Ala), glutamic acid (Glu), phenylalanine (Phe), histidine (His), isoleucine (Ile), aspartic acid (Asp), lysine (Lys), glycine (Gly), valine (Val), citrulline (Cit), cystine (Cys2), proline (Pro), tryptophan (Trp), leucine (Leu), arginine (Arg), cysteine (Cys), hydroxylproline (Hpro), methionine (Met), threonine (Thr), asparagine (Asn), and serine (Ser) were purchased from National Institutes for Food and Drug Control (Beijing, China) with the purity more than 98%. Reference standards of γ-aminobutyric (GABA) and glutamine (Gln) with the purity more than 98% were obtained from Aladdin Industrial Corporation (Shanghai, China). Acetonitrile (HPLC grade) was purchased from Merk (Darmstadt, Germany). Formic acid and ammonium formate (HPLC grade) were provided by Mreda Technology Incorporation (USA). Deionized water used for analysis procedure was produced by a Milli-Q Academic ultrapure water system (Millipore, Bedford, MA, USA).Table 1
Information of duckweeds from different regions.
Sample
Species
Site
SP1-7
Spirodela polyrhiza
Yangzhou City of Jiangsu Province
SP8-9
Spirodela polyrhiza
Yancheng City of Jiangsu Province
SP10-11
Spirodela polyrhiza
Nanjing City of Jiangsu Province
SP12-13
Spirodela polyrhiza
Huaian City of Jiangsu Province
SP14-17
Spirodela polyrhiza
Lianyungang City of Jiangsu Province
SP18-19
Spirodela polyrhiza
Linyi City of Shandong Province
SP20-21
Spirodela polyrhiza
Hefei City of Anhui Province
SP22-23
Spirodela polyrhiza
Hangzhou City of Jiangsu Province
SP24-25
Spirodela polyrhiza
Baoding City of Hebei Province
LP1-11
Landoltia punctata
Yangzhou City of Jiangsu province
LP12-16
Landoltia punctata
Huaian City of Jiangsu province
LA1-3
Lemna aequinoctialis
Yangzhou City of Jiangsu Province
LA4-8
Lemna aequinoctialis
Yancheng City of Jiangsu Province
LA9-14
Lemna aequinoctialis
Nanjing City of Jiangsu Province
LA15-16
Lemna aequinoctialis
Lianyungang City of Jiangsu Province
LA17-18
Lemna aequinoctialis
Linyi City of Shandong Province


## 2.2. Instrumentation and Chromatographic and Mass Conditions

Chromatographic analysis was performed on a Shimadzu LC-30 ultrahigh performance liquid chromatography system (Shimadzu, Japan), which consisted of a communication bus module (CMB-20A), a vacuum degasser, binary gradient pumps (LC-30AD), an autosampler (SIL-30A), and a column oven (CTO-30A) coupled with an AB Sciex QTRAP 5500 (AB SCIEX, USA). A Waters XBridge Amide column (2.1 mm × 150 mm, 2.5μm, Waters, USA) was used for chromatographic separation. The binary mobile phase was composed of acetonitrile (A) and 10 mM ammonium formate with 0.2% (v/v) formic acid (B) at a flow rate of 0.4 mL/min. The linear gradient elution was carried out as follows: 0–5 min, 90%–85.5% A; 5–11 min, 85.5%–54% A; 11-12 min, 54%–10% A; 12–14 min, 10% A; 14-15 min, 10%–90% A; 15–20 min, and 90% A. The reequilibration time was 4 min with a total running time of 20 min. The column compartment was kept at 20°C, while the autosampler trial was maintained at 15°C, and injection volume was 2 μl. The needle was washed with mixtures of acetonitrile and water.The mass spectrometry assay was performed on a triple quadruple mass spectrometer equipped with an electrospray ionization source. The spectra were recorded under positive-ion type with the multiple reaction monitoring (MRM) mode. The capillary voltage was 5500 V, and desolvation gas temperature was 550°C. The curtain gas was 35 psi with both nebulizer and drying gas of 55 psi. Nitrogen was used as source gas with purity more than 95% and collision gas with purity over 99.999%. The 24 components of amino acids were optimized in the tuning mode with mass only to obtain the mass of the precursor and product ion, the best declustering potential (DP), and collision energy (CE), respectively. The details are shown in Table2. The entrance potential (EP) and collision cell exit potential (CXP) were set as default value. The typical LC-QTRAP-MS/MS chromatograms are shown in Figure 1.Table 2
The precursor ions, product ions, declustering potential, and collision energy of amino acids.
Number
Compound
Precursor ion
Product ion
DP (V)
CE (V)
1
Tyr
182.0
91.1
60
37
2
Ala
90.0
44.1
50
14
3
Glu
148.1
84.0
60
21
4
Phe
166.1
120.1
60
19
5
His
156.1
110.3
80
20
6
Ile
132.1
86.1
50
14
7
Asp
134.1
74.0
50
19
8
Lys
147.0
84.1
60
23
9
Gly
76.1
29.9
40
16
10
Val
118.1
71.9
50
15
11
Cit
176.2
70.0
60
31
12
GABA
104.1
86.8
50
14
13
Cys2
241.1
73.9
80
40
14
Pro
116.0
70.1
60
22
15
Trp
205.1
146.0
50
24
16
Leu
132.1
86.1
50
14
17
Arg
175.2
70.2
90
30
18
Cys
122.1
59.0
40
32
19
Hpro
132.1
86.1
60
20
20
Met
150.1
103.9
60
14
21
Thr
120.1
74.1
40
14
22
Asn
133.1
74.0
60
21
23
Gln
147.0
84.1
60
23
24
Ser
106.0
60.1
60
15Figure 1
Typical MRM chromatograms of the 24 amino acids.


## 2.3. Preparation of Reference Compound Solution

Stock solutions of individual standards were made by dissolving accurately weighed components in 0.5% formic acid at a concentration of 0.2 mg/mL approximately and stored at 4°C before use. The mixed working solutions of all the standards were diluted with 0.5% formic acid solution to a series of appropriate concentrations immediately before analysis and used to attain the calibration curves.


## 2.4. Preparation of Sample Solution

Each sample powder was weighed 0.2 g accurately and transferred into a 25 mL stainless tube with 6 stainless beads with a diameter of 0.6 mm. Then, the sample was extracted in a high-throughput tissue-grinding apparatus with vibrating frequency of 70 kHz for 120 s with 15 mL 0.5% formic acid. The sample solution was centrifuged at 15000 rpm for 10 min, and then the supernatant was screened through a 0.22μm polytetrafluoroethylene membrane filter and stored in a glass bottle at 4°C for later LC-MS/MS analysis.


## 2.5. Method Validation

For each reference substance, the calibration curve was confirmed by linear regression of the peak area versus concentration. The limits of detection (LOD) and limits of quantification (LOQ) were determined by diluting the standard solution till the signal-to-noise ratios were about 3 and 10, respectively. Precision was determined by intraday and interday variability. The intraday variability was conducted by determining the same standard solution in six replicates on the same day. The interday variability was conducted by determining the same solution for three consecutive days. The relative standard deviation (RSD) values were calculated to denote the precision. Stability of the sample solution was analysed by peak areas of analytes at 0, 2, 4, 8, 12, and 24 h at room temperature. To evaluate the repeatability of the developed method, the same sample was analysed in six replicates and variations were expressed as RSD. The recovery test was performed to assess the accuracy of the method. For the test, a known amount of the 24 standard components was spiked into a certain amount (0.1 g) of sample. Then, the spiked sample was extracted and analysed as described. The recovery rate was calculated by using the following formula: recovery (%) = (measured amount − original amount)/spiked amount  × 100%. Six replicates were performed for the recovery test.


## 2.6. Data Analysis

The quantitative analyses of acquired data were processed by MultiQuant 3.0.2 (AB SCIEX, USA). Multivariate statistical analysis including PCA and OPLS-DA was used to classify the sample. SIMCA-P 14.1 (Umetrics AB, Sweden) was used to perform statistical analysis for the data of 59 batches of duckweed.


## 2.7. Analysis of Minerals and Total Proteins

Twenty-four minerals of each environment water where duckweed lived were quantified by ICP-MS. Total nitrogen and phosphorus of the water body were measured according to the alkaline potassium persulfate digestion method GB11894-89 and the ammonium molybdate spectrophotometric method GB11893-89 published by the State Bureau of Technological Supervision of China. Total proteins of each duckweed were determined by the Kjeldahl method. Each sample of duckweeds was hydrolyzed in acidic conditions, and then total amino acids were quantified by the HPLC-MSMS method mentioned above.


## 3. Results and Discussion

### 3.1. Optimization of Extraction Procedure

Free amino acids can dissolve in methanol, ethanol, water, diluted acid, and so on. In order to acquire an efficient extraction strategy of amino acids from duckweed, variables of the extraction process were investigated. The solvents (methanol, ethanol, water, 0.1% formic acid, and 0.5% formic acid), methods (heating reflux, ultrasonication, and tissue-grinding), and time (20, 30, and 40 min for ultrasonication; 60, 90, and 120 s for tissue-grinding) were studied. The results showed that the amount of amino acids had no significant difference between the ultrasonication time of 30 min in 0.5% formic acid and tissue-grinding time of 120 s in 0.5% formic acid. The amounts of extraction by other methods were not more than that by these two modalities. From the viewpoint of working efficiency, the tissue-grinding method was much more time-saving than the ultrasonication method by the extracting time of 120 s versus 30 min.


### 3.2. Optimization of UPLC Condition

Chromatographic conditions were optimized to attain a satisfactory separation for amino acid especially for isomeric molecules. Columns (Waters XBridge Amide, 2.5μm, 2.1 × 150 mm and Agilent Poroshell 120 HILIC, 2.7 μm, 2.1 × 150 mm), column temperature (20, 25, 30, 35, and 40°C), mobile phase (acetonitrile-0.1% formic acid, acetonitrile-0.2% formic acid, acetonitrile with 0.2% formic acid-0.2% formic acid, and acetonitrile-10 mM ammonium formate with 0.2% formic acid), and different kinds of gradient elution were investigated.The results demonstrated that the peak shape of amino acids became good when the concentration of formic acid rose, but retention time would be shortened significantly especially if some formic acid was complemented into the acetonitrile. The buffers such as ammonium formate increased the resolution degrees of amino acids. Better retention times and separations were observed by conducting analysis on Waters Amide column than on Agilent Poroshell 120 HILIC column, and the best chromatograms were gained with temperature controlled at 20°C. Especially for the isomers of leucine and isoleucine, baseline separation was successfully achieved when using the mobile phase consisting of acetonitrile-10 mM ammonium formate with 0.2% formic acid. Under other abovementioned conditions, these two isomeric amino acids were not well separated reciprocally.


### 3.3. Optimization of MS/MS Conditions

Primary experiments were conducted to acquire the best mass spectrometry conditions of amino acids individually. For this purpose, each reference component of amino acids with the concentration of approximate 100 ng/mL was delivered into the mass spectrometer directly via a syringe pump with the velocity of 7μL/min. While tuning the standard dissolved in water, most response of amino acids was very low when the sodium-adducted ion, [M+Na]+, was relatively high. On the contrary, when dissolving the standard in 0.1% or higher concentration of formic acid solution, the response of protonated molecule, [M+H]+, was dramatically higher and the metal-adducted ion was barely observed. Consequently, precursor ion (Q1), product ion (Q3), declustering potential (DP), and collision energy (CE) of each amino acid were acquired and optimized.


### 3.4. Method Validation

As shown in Table3, the calibration curves of twenty-four amino acids showed good linear regression (all correlation coefficients > 0.995) within a wide range of concentration. The LOD and LOQ of each analyte with the signal-to-noise ratio (S/N) of 3 and 10 were in the range of 0.37–1.94 ng/mL and 0.74–4.49 ng/mL, respectively. The RSD values of intraday and interday precision are summarized in Table 4. From the results, the present method was found to be precise with intraday variability less than 3.14% and interday variability less than 4.27%. The sample solutions were stable within 24 h with stability RSD < 3.82%. The RSD values of repeatability were 0.52%–3.86%. The recoveries of 24 amino acids ranged from 95.27%–107.51% with RSD < 4.03%.Table 3
Calibration curves, LODs, and LOQs for the 24 compounds.
Number
Compound
Calibration curve
R
2
Linear range (ng/mL)
LOQ (ng/mL)
LOD (ng/mL)
1
Tyr
y = 6534.1x + 6063.4
0.9984
1.16–5780
0.92
0.46
2
Ala
y = 7168.9x + 4932.8
0.9969
5.30–5300
3.71
1.24
3
Glu
y = 12796x + 47334
0.9960
6.41–2564
4.49
1.50
4
Phe
y = 59157 x + 42917
0.9965
0.99–992.0
0.79
0.40
5
His
y = 20263x + 247917
0.9962
5.57–5570
3.90
1.30
6
Ile
y = 70857x + 46587
0.9957
1.07–1074
0.86
0.43
7
Asp
y = 7084.7x + 86152
0.9963
5.43–5430
3.80
1.27
8
Lys
y = 4305.1x + 17648
0.9980
5.53–5530
3.87
1.94
9
Gly
y = 1063.0x + 3407.3
0.9990
0.94–4680
0.75
0.37
10
Val
y = 31972x + 35728
0.9962
1.11–1112
0.89
0.44
11
Cit
y = 8520.8x + 9811.0
0.9980
1.01–5070
0.81
0.41
12
GABA
y = 18495x + 14623
0.9981
1.10–5490
0.88
0.44
13
Cys2
y = 2389.7x + 3146.6
0.9978
1.09–5440
0.87
0.44
14
Pro
y = 51204x + 58539
0.9981
1.02–510.0
0.82
0.41
15
Trp
y = 19030x + 4465.3
0.9977
1.24–2476
0.99
0.50
16
Leu
y = 56715x + 40047
0.9974
1.01–1012
0.81
0.40
17
Arg
y = 11505x + 84828
0.9974
5.96–5960
4.17
1.39
18
Cys
y = 2496.6x + 1017.5
0.9988
1.20–6012
0.96
0.48
19
Hpro
y = 15783x + 3968.1
0.9977
0.95–1892
0.76
0.38
20
Met
y = 8626.4x + 1545.9
0.9984
1.05–5250
0.84
0.42
21
Thr
y = 8807.8x + 19073
0.9957
4.46–4460
3.12
1.56
22
Asn
y = 4939.9x + 22367
0.9967
1.20–5980
0.96
0.48
23
Gln
y = 7733.9x + 8338.4
0.9961
0.93–4650
0.74
0.37
24
Ser
y = 7731.3x + 54859
0.9977
5.09–5090
3.56
1.19Table 4
Precision, stability, repeatability, and accuracy of the investigated analytes (n=6).
Number
Compound
Precision
Stability RSD (%)
Repeatability RSD (%)
Accuracy
Intraday RSD (%)
Interday RSD (%)
Recovery (%)
RSD (%)
1
Tyr
1.36
1.85
0.94
3.05
98.56
1.33
2
Ala
2.60
3.31
3.47
3.09
102.49
2.18
3
Glu
2.07
2.56
2.10
2.11
106.94
3.76
4
Phe
0.66
1.03
2.60
1.66
97.23
4.03
5
His
1.07
3.30
1.58
1.17
98.32
2.20
6
Ile
0.97
3.21
3.82
0.71
99.09
3.57
7
Asp
1.96
3.98
2.42
1.84
97.86
2.36
8
Lys
2.70
3.51
1.44
0.99
102.30
3.02
9
Gly
1.66
2.65
3.43
3.74
104.48
3.07
10
Val
2.42
2.89
3.30
0.83
107.51
1.52
11
Cit
1.47
1.88
3.12
3.86
99.14
2.21
12
GABA
3.14
2.32
2.27
0.82
102.54
3.41
13
Cys2
2.53
4.27
3.24
1.49
96.60
1.27
14
Pro
1.21
3.39
1.09
2.09
101.94
3.20
15
Trp
0.37
3.93
1.36
1.39
99.11
3.46
16
Leu
1.50
3.90
3.40
1.50
95.27
3.61
17
Arg
2.72
4.15
3.72
1.45
102.37
1.79
18
Cys
2.82
3.63
1.16
0.53
103.65
3.82
19
Hpro
3.05
2.23
0.87
2.38
97.55
3.91
20
Met
1.57
3.02
1.26
0.52
104.89
2.14
21
Thr
2.11
2.13
1.77
3.01
95.97
3.00
22
Asn
2.93
2.85
1.48
3.68
105.35
2.95
23
Gln
0.97
1.87
1.22
2.72
102.33
2.16
24
Ser
2.71
2.86
0.98
1.48
97.44
1.88


### 3.5. Quantitative Analysis for Samples

The developed UPLC-MS/MS method was applied for simultaneous determination of 24 free amino acids in 59 batches of three species of duckweed. All the contents are displayed in Table5. The results showed that the total amount of amino acids varied dramatically among each sample from different regions and species. Total content of AAs in SP25 from Baoding City of Hebei Province reached as high as 1899.50 μg/g, which was much more than that in SP17 of 166.05 μg/g from Lianyungang City of Jiangsu Province. For each amino acid, the content also varied significantly even in the same sample. Coincidentally, the contents of free amino acids that include sulfur in their molecules were much less than others, including cysteine, cystine, and methionine with the concentration of 0.24, 4.25, and 4.20 μg/g in maximum, respectively. Especially for cysteine, it was not found in all of Spirodela polyrhiza and only detected in several samples of other two species. The putative reason was that the thiol in cysteine was not stable and easily oxidized. But in general, there was no significant difference observed in the total content of all FAAs among different species.Table 5
Contents of 24 free amino acids, total amino acids, total proteins (μg/g), and ratio of free to total amino acids (%) in three species of duckweed.
Sample
Tyr
Ala
Glu
Phe
His
Ile
Asp
Lys
Gly
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
SP1
3.16
6259.48
0.0505
6.95
10680.18
0.0651
25.27
16774.31
0.1506
0.44
9597.56
0.0046
1.52
4255.26
0.0357
0.38
8532.07
0.0045
9.31
11169.49
0.0834
11.95
7164.09
0.1668
3.33
8764.89
0.0380
SP2
6.73
6161.25
0.1092
65.17
10089.59
0.6459
54.61
25366.04
0.2153
0.95
10729.73
0.0089
4.43
3888.26
0.1139
1.35
10094.12
0.0134
56.68
15862.58
0.3573
21.28
10090.45
0.2109
8.38
8695.46
0.0964
SP3
39.72
6115.87
0.6495
173.11
9881.22
1.7519
96.43
15463.41
0.6236
7.97
8667.06
0.0920
22.26
3824.22
0.5821
11.90
7226.86
0.1647
69.12
12387.34
0.5580
112.90
8153.11
1.3847
128.27
6293.65
2.0381
SP4
29.21
6251.45
0.4673
157.69
12424.62
1.2692
49.52
22986.51
0.2154
5.01
10467.29
0.0479
32.80
3569.79
0.9188
11.14
8392.28
0.1327
170.28
17175.49
0.9914
137.48
10225.20
1.3445
78.17
9626.14
0.8121
SP5
5.58
6257.00
0.0892
29.04
13866.45
0.2094
34.93
23518.46
0.1485
0.86
11427.08
0.0075
6.41
4312.95
0.1486
0.96
9753.24
0.0098
16.39
18654.70
0.0879
21.25
11094.76
0.1915
5.53
10174.01
0.0544
SP6
27.42
6245.68
0.4390
273.35
12005.84
2.2768
49.09
18787.83
0.2613
2.55
10284.72
0.0248
45.46
3424.75
1.3274
13.55
8465.53
0.1601
132.84
15752.94
0.8433
139.27
8608.52
1.6178
77.36
7010.22
1.1035
SP7
39.61
5884.11
0.6732
229.20
12123.87
1.8905
71.86
18146.81
0.3960
4.90
8585.01
0.0571
46.17
4088.80
1.1292
11.22
7231.96
0.1551
94.84
14833.85
0.6393
230.52
7636.34
3.0187
65.35
5417.04
1.2064
SP8
5.71
6124.81
0.0932
38.00
9734.57
0.3904
53.14
17640.33
0.3012
1.17
8527.84
0.0137
4.56
3751.52
0.1216
1.40
7266.84
0.0193
42.82
14315.75
0.2991
22.77
7392.06
0.3080
5.63
7832.50
0.0719
SP9
13.90
6054.66
0.2296
120.58
9953.18
1.2115
70.75
16620.79
0.4257
3.68
8384.54
0.0439
18.01
3496.99
0.5150
5.25
6745.54
0.0778
94.10
11606.55
0.8107
73.99
6323.41
1.1701
21.63
7899.86
0.2738
SP10
16.70
6097.17
0.2739
118.61
13272.16
0.8937
74.72
25451.33
0.2936
3.72
11056.99
0.0336
28.19
4110.01
0.6859
4.41
9671.14
0.0456
36.11
17757.11
0.2034
70.81
8546.75
0.8285
25.55
7505.72
0.3404
SP11
23.10
6004.10
0.3847
205.08
11155.38
1.8384
67.31
26088.53
0.2580
10.26
9883.88
0.1038
61.35
3238.60
1.8943
14.23
8891.25
0.1600
56.14
15601.46
0.3598
133.66
8386.70
1.5937
87.43
9088.41
0.9620
SP12
9.38
6127.68
0.1531
93.06
9760.92
0.9534
51.58
26516.14
0.1945
7.60
7997.37
0.0950
31.87
3300.60
0.9656
4.09
9260.26
0.0442
225.20
12413.46
1.8142
74.86
5684.24
1.3170
24.76
6908.86
0.3584
SP13
93.61
6130.06
1.5271
195.69
8956.55
2.1849
52.85
16377.44
0.3227
23.33
7668.17
0.3042
46.78
2889.26
1.6191
14.85
7650.07
0.1941
4.76
10136.24
0.0470
135.91
6635.82
2.0481
73.19
5663.18
1.2924
SP14
19.98
6120.49
0.3264
161.86
13337.15
1.2136
96.09
15114.77
0.6357
5.94
10678.41
0.0556
26.03
4189.64
0.6213
6.56
9933.71
0.0660
29.20
13225.32
0.2208
102.55
11141.92
0.9204
34.50
7778.37
0.4435
SP15
1.13
6057.44
0.0187
19.99
13617.28
0.1468
17.01
21045.25
0.0808
0.62
11056.19
0.0056
9.77
4213.32
0.2319
0.30
8452.34
0.0035
0.95
12630.34
0.0075
17.70
8018.34
0.2207
6.64
6872.17
0.0966
SP16
61.29
5884.22
1.0416
280.92
12551.36
2.2382
46.82
20775.77
0.2254
19.27
10311.88
0.1869
54.60
3881.89
1.4065
20.18
9290.92
0.2172
112.81
13791.37
0.8180
158.24
7293.79
2.1695
52.41
8307.13
0.6309
SP17
0.89
5938.26
0.0150
23.96
10170.30
0.2356
46.90
15656.17
0.2996
0.42
10562.87
0.0040
2.49
5358.17
0.0465
1.11
10201.54
0.0109
2.95
12263.36
0.0241
13.08
8069.24
0.1621
4.44
5669.36
0.0783
SP18
12.28
5985.32
0.2052
157.75
13673.70
1.1537
81.27
15272.53
0.5321
15.58
10449.06
0.1491
28.93
4949.42
0.5845
10.04
9948.89
0.1009
85.38
20641.51
0.4136
79.39
11463.15
0.6926
29.19
6848.95
0.4262
SP19
45.66
5897.55
0.7742
182.98
12217.80
1.4977
87.43
20888.93
0.4185
28.84
10128.19
0.2847
48.02
4231.77
1.1347
20.85
9161.44
0.2276
10.22
14574.99
0.0701
83.12
9060.52
0.9174
34.17
6664.53
0.5127
SP20
28.03
5860.01
0.4783
241.91
9302.34
2.6005
46.17
14837.19
0.3112
23.18
7603.97
0.3048
64.10
3206.91
1.9988
14.80
6774.94
0.2185
109.03
11544.71
0.9444
164.87
6737.28
2.4471
73.15
6715.81
1.0892
SP21
2.75
6264.80
0.0439
55.55
10989.25
0.5055
91.79
22493.65
0.4081
0.84
9615.10
0.0087
23.80
4257.07
0.5591
1.00
8364.75
0.0120
—
11442.81
—
31.30
6932.76
0.4515
9.46
6910.06
0.1369
SP22
7.95
5839.15
0.1361
29.20
10343.17
0.2823
22.86
16692.99
0.1369
1.07
8957.29
0.0119
5.17
3585.28
0.1442
1.06
8011.79
0.0132
0.91
13590.39
0.0067
15.15
6361.56
0.2381
11.21
7059.65
0.1588
SP23
6.86
6265.35
0.1095
68.62
11510.74
0.5961
100.28
20064.52
0.4998
2.53
9584.25
0.0264
32.22
3573.51
0.9016
1.84
7753.50
0.0237
33.31
15126.56
0.2202
42.69
7622.22
0.5601
11.21
8763.05
0.1279
SP24
2.86
6318.22
0.0453
121.06
11508.52
1.0519
133.69
16946.16
0.7889
1.99
9477.13
0.0210
25.81
4474.44
0.5768
2.27
7926.91
0.0286
47.91
15776.45
0.3037
30.02
7939.55
0.3781
10.04
8867.42
0.1132
SP25
43.24
5967.19
0.7246
261.74
11024.75
2.3741
53.23
21609.69
0.2463
16.89
9293.80
0.1817
64.22
4222.87
1.5208
20.92
9560.51
0.2188
162.59
18624.39
0.8730
209.44
7299.84
2.8691
63.50
7917.96
0.8020
LP1
15.93
5971.26
0.2668
66.50
12151.90
0.5472
30.75
24958.79
0.1232
11.80
10826.10
0.1090
35.08
4061.19
0.8638
4.88
9319.60
0.0524
108.30
14896.35
0.7270
47.16
8613.79
0.5475
14.76
9583.97
0.1540
LP2
38.97
5921.55
0.6581
140.81
11584.07
1.2155
63.83
21969.13
0.2905
15.15
9814.43
0.1544
30.41
4647.49
0.6543
2.61
8750.97
0.0298
59.96
16728.80
0.3584
134.72
7842.84
1.7177
30.64
6109.14
0.5015
LP3
10.25
6287.19
0.1630
65.63
11013.29
0.5959
55.16
18366.66
0.3003
2.58
10992.07
0.0235
16.14
3302.54
0.4887
0.65
8567.75
0.0076
28.45
14101.74
0.2017
40.15
6593.31
0.6090
7.10
4632.59
0.1533
LP4
47.76
6004.07
0.7955
180.48
11148.05
1.6189
83.06
18093.89
0.4590
7.65
9312.27
0.0821
48.49
3515.42
1.3794
9.18
8187.52
0.1121
103.28
15513.40
0.6657
94.68
8007.43
1.1824
74.60
8640.35
0.8634
LP5
74.45
6011.02
1.2386
318.14
9785.11
3.2513
50.05
16830.48
0.2974
17.43
8490.88
0.2053
38.14
3571.09
1.0680
11.66
7900.11
0.1476
218.70
13106.01
1.6687
129.60
8308.91
1.5598
44.93
5574.15
0.8060
LP6
22.44
6404.35
0.3504
187.93
9320.51
2.0163
51.11
19093.97
0.2677
10.76
10746.47
0.1001
17.66
4145.39
0.4260
4.29
8189.38
0.0524
137.49
13879.01
0.9906
57.61
6486.39
0.8882
13.94
6180.53
0.2255
LP7
17.40
6413.54
0.2713
167.19
10950.61
1.5268
73.42
20616.00
0.3561
10.75
9606.73
0.1119
20.94
3464.43
0.6044
2.23
8717.93
0.0256
207.21
15567.27
1.3311
51.72
8761.56
0.5903
14.60
6213.59
0.2350
LP8
35.02
6285.44
0.5572
139.27
11569.10
1.2038
72.38
18381.09
0.3938
18.51
10138.80
0.1826
28.90
4434.85
0.6517
7.46
9558.72
0.0780
73.30
15357.08
0.4773
78.65
7886.32
0.9973
24.80
8792.99
0.2820
LP9
41.17
5951.60
0.6917
149.55
11075.37
1.3503
48.55
22021.01
0.2205
9.10
11619.61
0.0783
52.62
3058.17
1.7206
12.36
8587.29
0.1439
182.44
15444.00
1.1813
202.10
10583.78
1.9095
176.76
6207.16
2.8477
LP10
23.74
6012.91
0.3948
187.15
10077.16
1.8572
57.20
22847.31
0.2504
3.20
10674.83
0.0300
11.62
3881.87
0.2993
6.47
10514.94
0.0615
48.04
19845.70
0.2421
111.09
8283.89
1.3410
107.73
7557.95
1.4254
LP11
14.86
6110.90
0.2432
94.14
9256.17
1.0171
16.70
26967.52
0.0619
5.60
7780.40
0.0720
7.52
3541.02
0.2124
3.81
7300.09
0.0522
6.45
13678.56
0.0472
75.66
7596.11
0.9960
46.74
6276.56
0.7447
LP12
8.06
6259.52
0.1288
25.80
10729.35
0.2405
11.61
28848.73
0.0402
1.45
9609.21
0.0151
4.31
4095.51
0.1052
0.93
8635.84
0.0108
2.44
11847.35
0.0206
29.23
8736.23
0.3346
7.00
7434.59
0.0942
LP13
33.36
6270.49
0.5320
274.21
10059.65
2.7258
85.36
26195.21
0.3259
5.22
11114.42
0.0470
44.10
3500.71
1.2597
8.74
9771.06
0.0894
78.86
17064.92
0.4621
148.06
8026.46
1.8446
92.39
7807.71
1.1833
LP14
29.35
6070.40
0.4835
160.58
13818.32
1.1621
57.00
27399.97
0.2080
5.18
12682.81
0.0408
38.41
5535.27
0.6939
9.59
11826.29
0.0811
139.52
14718.19
0.9479
158.90
7779.67
2.0425
85.79
7101.48
1.2081
LP15
14.08
6310.67
0.2231
108.54
12226.01
0.8878
11.43
20415.24
0.0560
4.29
10235.21
0.0419
6.83
4786.65
0.1427
3.49
10163.51
0.0343
4.42
16813.25
0.0263
70.82
8454.76
0.8376
57.87
8826.59
0.6556
LP16
38.10
6308.99
0.6039
224.42
10920.13
2.0551
58.19
18915.27
0.3076
12.98
9252.57
0.1403
25.58
3501.54
0.7305
6.06
8361.97
0.0725
187.80
14243.86
1.3185
79.64
8381.28
0.9502
24.49
7349.44
0.3332
LA1
39.53
5844.15
0.6764
153.40
11080.16
1.3845
53.94
17900.19
0.3013
5.26
8940.22
0.0588
38.98
3436.65
1.1342
10.44
7739.17
0.1349
92.27
11396.06
0.8097
116.20
8514.88
1.3647
16.63
8366.72
0.1988
LA2
7.06
6163.58
0.1145
110.58
11359.90
0.9734
38.25
21707.93
0.1762
8.65
9105.55
0.0950
45.78
3419.43
1.3388
10.95
9140.90
0.1198
84.67
16077.78
0.5266
87.09
8669.05
1.0046
14.22
7375.81
0.1928
LA3
49.51
6127.32
0.8080
189.37
10480.08
1.8070
47.31
23969.23
0.1974
23.46
8251.42
0.2843
73.17
3213.54
2.2769
30.13
8053.11
0.3741
190.71
18067.61
1.0555
206.67
10157.90
2.0346
118.69
11012.41
1.0778
LA4
9.45
6231.43
0.1517
101.15
10764.28
0.9397
43.93
18445.78
0.2382
6.83
9106.47
0.0750
5.48
3968.13
0.1381
2.07
7183.48
0.0288
14.98
11095.78
0.1350
44.31
8400.33
0.5275
18.02
5093.33
0.3538
LA5
8.21
6468.09
0.1269
101.70
10782.93
0.9432
49.41
17857.46
0.2767
4.67
9378.50
0.0498
27.88
4320.50
0.6453
7.67
8732.91
0.0878
245.22
11348.94
2.1607
101.42
9642.82
1.0518
59.10
7589.10
0.7787
LA6
95.03
6441.94
1.4752
53.56
10969.89
0.4882
45.63
19113.36
0.2387
34.05
8826.04
0.3858
22.67
3432.16
0.6605
15.73
7873.91
0.1998
194.49
13395.53
1.4519
203.37
9453.82
2.1512
165.17
5220.68
3.1638
LA7
43.57
6390.61
0.6818
136.48
9697.57
1.4074
44.59
17227.16
0.2588
26.85
10617.54
0.2529
53.25
3342.84
1.5930
8.20
8078.65
0.1015
99.41
13308.10
0.7470
156.91
8444.81
1.8581
124.79
6172.25
2.0218
LA8
25.51
5969.99
0.4273
159.29
9551.72
1.6677
21.69
22493.72
0.0964
7.29
12500.69
0.0583
28.21
5068.31
0.5566
12.52
10500.36
0.1192
2.23
19936.15
0.0112
66.00
11282.00
0.5850
75.19
11732.39
0.6409
LA9
3.26
6188.23
0.0527
19.10
14974.86
0.1275
71.65
23929.16
0.2994
0.84
12303.21
0.0068
3.65
4847.99
0.0753
0.36
10895.91
0.0033
2.47
21371.14
0.0116
18.43
10548.00
0.1747
7.33
7698.82
0.0952
LA10
29.15
5884.38
0.4954
95.43
11229.43
0.8498
89.66
20489.06
0.4376
16.96
9051.65
0.1874
21.61
3412.09
0.6333
16.06
8721.17
0.1841
2.82
17303.92
0.0163
74.14
8057.43
0.9201
27.50
5058.68
0.5436
LA11
10.88
6259.53
0.1738
40.56
13131.47
0.3089
121.99
22020.10
0.5540
2.48
10555.63
0.0235
5.92
4847.60
0.1221
0.48
9784.56
0.0049
7.09
16593.97
0.0427
26.86
7655.07
0.3509
7.73
7284.80
0.1061
LA12
48.02
6403.75
0.7499
149.63
9735.88
1.5369
62.74
14671.39
0.4276
20.20
8380.51
0.2410
56.05
3384.94
1.6559
5.41
6896.77
0.0784
91.38
11321.11
0.8072
109.18
5802.35
1.8817
46.13
7209.85
0.6398
LA13
22.08
5983.25
0.3690
77.34
12359.55
0.6258
49.89
24426.48
0.2042
13.64
10650.97
0.1281
31.43
4286.46
0.7332
3.68
8877.22
0.0415
184.36
14885.17
1.2385
77.19
10677.05
0.7230
23.24
10012.96
0.2321
LA14
29.06
6197.60
0.4689
219.41
12097.18
1.8137
68.78
22967.64
0.2995
7.41
10651.10
0.0696
33.00
4079.19
0.8090
10.50
8376.22
0.1254
21.77
16404.09
0.1327
122.70
8502.69
1.4431
92.02
9152.03
1.0055
LA15
28.90
6409.17
0.4509
11.61
12035.67
0.0965
43.15
25371.86
0.1701
5.48
11118.01
0.0493
35.81
3597.27
0.9955
6.72
6464.97
0.1039
159.51
12763.92
1.2497
120.12
7008.78
1.7139
55.67
5676.13
0.9808
LA16
20.91
6243.38
0.3349
95.37
11719.51
0.8138
41.47
21759.15
0.1906
2.48
11669.90
0.0213
27.93
5249.47
0.5321
5.48
10752.39
0.0510
113.55
14828.06
0.7658
123.52
9586.25
1.2885
34.37
10915.62
0.3149
LA17
33.89
6029.51
0.5621
12.57
12627.27
0.0995
65.10
23746.45
0.2741
13.69
10538.29
0.1299
34.97
4468.75
0.7825
7.61
9975.36
0.0763
117.69
13313.53
0.8840
122.84
9248.08
1.3283
36.44
8620.93
0.4227
LA18
29.56
6046.27
0.4889
137.19
10245.05
1.3391
54.86
18325.38
0.2994
10.74
8928.25
0.1203
48.27
3846.06
1.2551
6.44
7467.00
0.0862
91.91
12882.36
0.7135
125.43
7366.73
1.7027
36.87
4820.33
0.7649
Sample
Val
Cit
GABA
Cys2
Pro
Trp
Leu
Arg
Cys
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
SP1
4.42
6012.65
0.0735
2.49
4673.88
0.0533
3.46
3353.11
0.1032
—
11525.08
—
4.15
6607.74
0.0628
0.24
3637.74
0.0066
1.37
12211.14
0.0112
34.88
9489.73
0.3676
—
5538.05
—
SP2
15.20
10648.71
0.1427
6.07
3955.21
0.1535
21.27
4028.12
0.5280
—
13123.45
—
12.90
6341.28
0.2034
0.51
6868.79
0.0074
4.08
11230.44
0.0363
87.38
8979.88
0.9731
—
8675.28
—
SP3
15.36
6327.29
0.2428
24.59
4218.39
0.5829
83.54
2906.04
2.8747
0.67
9520.68
0.0070
27.55
7911.24
0.3482
2.67
6256.52
0.0427
33.86
9531.17
0.3553
68.39
8667.37
0.7891
—
3752.42
—
SP4
16.09
10136.97
0.1587
9.45
4525.17
0.2088
112.07
1860.38
6.0240
0.71
8713.26
0.0081
22.64
9816.87
0.2306
3.14
6886.81
0.0456
25.37
11900.95
0.2132
41.42
7830.13
0.5290
—
5020.00
—
SP5
12.81
12270.45
0.1044
3.68
5960.38
0.0617
9.31
2163.00
0.4304
—
11194.54
—
5.92
8973.82
0.0660
0.39
6741.47
0.0058
3.03
17710.97
0.0171
75.96
11624.78
0.6534
—
8152.58
—
SP6
18.63
6921.90
0.2691
54.08
6475.87
0.8351
110.08
2010.87
5.4742
0.26
9803.15
0.0027
28.19
7126.12
0.3956
4.73
3570.11
0.1325
26.75
15586.82
0.1716
43.86
5689.69
0.7709
—
3789.97
—
SP7
17.07
9259.39
0.1844
29.75
4140.35
0.7185
103.95
2825.91
3.6785
2.41
9778.44
0.0246
24.62
8226.72
0.2993
3.95
4856.66
0.0813
27.66
10861.08
0.2547
34.40
4988.56
0.6896
—
3471.13
—
SP8
15.86
7296.00
0.2174
3.13
4702.29
0.0666
20.48
1682.03
1.2176
—
6139.57
—
10.52
6680.26
0.1575
0.56
4883.67
0.0115
4.38
9640.58
0.0454
64.28
4886.46
1.3155
—
3120.50
—
SP9
37.20
6514.46
0.5710
10.18
5254.55
0.1937
57.02
2501.97
2.2790
0.14
10056.41
0.0014
15.77
5340.61
0.2953
1.20
4253.18
0.0282
14.42
11713.99
0.1231
124.64
5090.33
2.4486
—
3897.56
—
SP10
38.29
9908.44
0.3864
13.50
7105.23
0.1900
57.85
1953.05
2.9620
0.26
10687.68
0.0024
19.03
7381.05
0.2578
1.17
7036.56
0.0166
13.77
14235.69
0.0967
108.51
4268.65
2.5420
—
8653.16
—
SP11
21.90
10089.65
0.2171
8.30
5747.51
0.1444
108.20
3155.71
3.4287
0.79
9550.72
0.0083
24.66
5804.81
0.4248
1.94
7077.68
0.0274
26.89
14118.06
0.1905
67.72
5222.67
1.2967
—
6165.20
—
SP12
36.91
5104.20
0.7231
31.74
3267.50
0.9714
56.19
3092.80
1.8168
0.58
8821.94
0.0066
11.01
6511.44
0.1691
2.35
4185.92
0.0561
10.46
8633.77
0.1212
99.42
6433.17
1.5454
—
2940.84
—
SP13
27.76
5423.78
0.5118
27.14
3816.49
0.7111
82.12
2484.15
3.3058
0.99
6828.14
0.0145
22.06
7524.55
0.2932
11.42
3149.92
0.3625
31.25
8945.92
0.3493
73.82
5010.51
1.4733
—
6503.52
—
SP14
44.12
10896.32
0.4049
11.57
6328.11
0.1828
70.23
4220.23
1.6641
0.28
9165.62
0.0031
21.63
8005.88
0.2702
2.44
4732.57
0.0516
18.29
13035.53
0.1403
65.62
6826.41
0.9613
—
8677.59
—
SP15
6.17
7444.35
0.0829
5.45
3756.96
0.1451
4.23
3324.82
0.1272
—
10198.26
—
14.42
5416.47
0.2662
—
5143.26
—
1.80
14814.40
0.0122
30.19
5059.02
0.5968
—
6694.08
—
SP16
26.04
6396.60
0.4071
36.82
3762.26
0.9787
134.46
3870.97
3.4735
0.72
8704.16
0.0083
27.10
7353.81
0.3685
15.39
7545.48
0.2040
30.79
12588.03
0.2446
43.32
6483.46
0.6682
—
4369.57
—
SP17
9.24
8969.61
0.1030
4.12
3703.39
0.1112
2.34
3148.92
0.0743
—
8001.88
—
14.78
8403.61
0.1759
—
4014.56
—
5.35
10274.36
0.0521
9.96
4876.50
0.2042
—
6360.07
—
SP18
26.19
12386.71
0.2114
26.35
5209.87
0.5058
98.88
3534.87
2.7973
0.12
12402.18
0.0010
17.21
9211.57
0.1868
2.08
7798.78
0.0267
22.77
17831.20
0.1277
108.35
4662.91
2.3237
—
7665.04
—
SP19
25.16
8312.52
0.3027
8.51
4849.89
0.1755
73.42
1868.93
3.9285
0.28
8968.53
0.0031
17.64
8754.89
0.2015
3.68
5290.70
0.0696
16.70
11607.35
0.1439
80.47
7406.94
1.0864
—
7063.71
—
SP20
25.85
8395.03
0.3079
17.22
4586.93
0.3754
131.28
2873.62
4.5685
0.40
8894.24
0.0045
18.50
4472.47
0.4136
9.86
3888.62
0.2536
24.40
8829.37
0.2764
42.54
4662.51
0.9124
—
4657.46
—
SP21
11.27
5982.04
0.1884
24.90
4366.50
0.5703
10.82
3551.90
0.3046
0.24
8933.72
0.0027
28.86
6002.68
0.4808
0.15
6460.55
0.0023
3.89
12300.36
0.0316
55.98
5963.67
0.9387
—
5955.22
—
SP22
10.06
9469.47
0.1062
5.09
5545.62
0.0918
1.90
1902.37
0.0999
—
10023.81
—
18.22
5611.46
0.3247
0.14
3261.78
0.0043
9.76
12591.58
0.0775
18.52
8217.10
0.2254
—
6884.34
—
SP23
19.80
6941.68
0.2852
35.46
4560.09
0.7776
10.96
2519.87
0.4349
0.31
11078.52
0.0028
25.31
7474.12
0.3386
0.55
4776.94
0.0115
5.34
9323.61
0.0573
75.70
8379.33
0.9034
—
4296.33
—
SP24
24.68
7991.34
0.3088
29.19
6319.28
0.4619
4.94
3668.10
0.1347
0.09
10178.72
0.0009
23.29
5293.61
0.4400
0.60
4551.39
0.0132
3.83
14205.93
0.0270
58.04
6170.55
0.9406
—
5644.77
—
SP25
28.45
7459.65
0.3814
46.15
5088.78
0.9069
163.48
3897.18
4.1948
1.16
11215.85
0.0103
24.67
7584.57
0.3253
7.63
7080.31
0.1078
35.40
14162.74
0.2500
74.34
9656.72
0.7698
—
6166.09
—
LP1
21.36
7244.74
0.2948
5.16
4405.39
0.1171
58.37
2407.83
2.4242
0.13
13065.90
0.0010
11.76
8651.28
0.1359
3.62
5123.83
0.0707
13.93
10764.87
0.1294
30.24
8884.37
0.3404
—
4324.94
—
LP2
14.46
7373.76
0.1961
16.36
4874.65
0.3356
40.10
2138.03
1.8756
0.77
7988.91
0.0096
14.86
8851.40
0.1679
14.60
3817.53
0.3824
15.17
13357.73
0.1136
26.71
9701.11
0.2753
—
7977.29
—
LP3
9.83
8734.76
0.1125
60.38
3744.80
1.6124
14.14
2099.63
0.6735
—
9725.45
—
18.43
4540.45
0.4059
0.45
4728.65
0.0095
3.52
9822.04
0.0358
74.16
5810.06
1.2764
—
3530.69
—
LP4
35.80
9919.45
0.3609
3.79
3526.80
0.1075
122.04
2438.49
5.0047
0.37
9387.49
0.0039
18.34
8296.58
0.2211
1.14
5349.26
0.0213
22.21
9157.07
0.2425
48.84
8460.67
0.5773
—
6861.56
—
LP5
35.99
8871.65
0.4057
11.90
4587.62
0.2594
133.05
3159.85
4.2106
4.21
7298.47
0.0577
25.23
5061.04
0.4985
4.34
5510.02
0.0788
31.89
11224.14
0.2841
45.88
6939.12
0.6612
—
3854.06
—
LP6
24.78
7215.09
0.3434
9.73
4416.05
0.2203
91.48
2092.89
4.3710
2.32
7641.78
0.0304
14.71
6655.83
0.2210
5.76
3078.31
0.1871
13.10
10225.91
0.1281
77.03
6842.51
1.1258
—
3272.62
—
LP7
18.41
6396.21
0.2878
10.84
4275.51
0.2535
64.21
2197.40
2.9221
4.25
11232.05
0.0378
9.15
8097.10
0.1130
4.71
3289.73
0.1432
11.93
9991.64
0.1194
30.66
7921.66
0.3870
0.14
6607.63
0.0021
LP8
23.88
7401.70
0.3226
16.38
4758.26
0.3442
69.10
3565.33
1.9381
0.55
8739.92
0.0063
14.73
8982.82
0.1640
2.82
4541.13
0.0621
24.29
16149.82
0.1504
31.42
6643.33
0.4730
—
5673.14
—
LP9
22.74
8090.74
0.2811
32.17
6784.16
0.4742
95.67
3874.88
2.4690
0.34
11756.40
0.0029
25.48
9895.35
0.2575
3.59
4755.18
0.0755
30.55
11213.15
0.2724
118.53
7876.17
1.5049
0.22
4743.85
0.0046
LP10
44.70
10435.87
0.4283
15.47
3862.27
0.4005
82.77
2085.51
3.9688
0.95
13573.29
0.0070
21.56
8888.77
0.2426
0.77
7378.31
0.0104
16.31
14411.77
0.1132
63.57
9539.07
0.6664
0.14
8866.38
0.0016
LP11
30.18
7118.23
0.4240
10.12
3840.01
0.2635
38.62
2188.18
1.7649
0.39
9579.85
0.0041
14.31
6477.73
0.2209
0.56
4389.22
0.0128
9.34
10905.75
0.0856
74.49
5696.30
1.3077
—
5334.87
—
LP12
10.67
9073.21
0.1176
9.45
4795.94
0.1970
6.74
2798.07
0.2409
—
9754.10
—
10.14
5687.18
0.1783
0.22
6220.07
0.0035
2.99
11120.12
0.0269
89.48
9822.46
0.9110
—
7787.85
—
LP13
24.43
7986.97
0.3059
10.20
5208.39
0.1958
105.99
3587.82
2.9542
1.19
10899.43
0.0109
25.32
9578.40
0.2643
2.01
6255.14
0.0321
24.85
10572.72
0.2350
76.46
7230.87
1.0574
—
8350.79
—
LP14
21.85
12895.61
0.1694
10.61
6247.35
0.1698
114.88
3385.86
3.3929
0.65
12941.50
0.0050
24.21
10165.28
0.2382
0.42
5801.66
0.0072
24.35
12767.46
0.1907
38.76
9267.96
0.4182
0.10
4749.52
0.0021
LP15
30.04
10600.31
0.2834
8.02
6112.10
0.1312
39.61
2063.22
1.9198
0.26
10874.29
0.0024
14.42
6109.32
0.2360
0.63
7137.55
0.0088
7.68
12394.41
0.0620
71.29
6514.00
1.0944
—
4351.01
—
LP16
26.39
5728.98
0.4606
10.83
4203.58
0.2576
96.25
3176.63
3.0299
3.59
6782.15
0.0529
16.36
8733.17
0.1873
4.94
6147.21
0.0804
18.97
9376.15
0.2023
51.19
8498.90
0.6023
0.14
5213.83
0.0027
LA1
45.00
5702.67
0.7891
13.87
3437.80
0.4035
97.36
3259.71
2.9868
0.34
9934.14
0.0034
16.88
5375.18
0.3140
5.39
5649.12
0.0954
25.80
11813.05
0.2184
46.47
7148.37
0.6501
—
6609.48
—
LA2
19.62
10334.80
0.1898
28.58
3747.09
0.7627
81.64
2390.45
3.4153
0.86
7845.80
0.0110
12.38
8095.98
0.1529
2.57
5265.52
0.0488
23.74
15177.45
0.1564
33.03
7150.11
0.4620
—
5064.86
—
LA3
39.08
11531.59
0.3389
11.17
7658.73
0.1458
96.78
3420.33
2.8296
0.34
9648.03
0.0035
25.22
6740.71
0.3741
14.39
6990.99
0.2058
28.29
16947.54
0.1669
35.87
11157.91
0.3215
0.12
5317.49
0.0023
LA4
30.49
9094.55
0.3353
5.75
3337.54
0.1723
59.10
1960.88
3.0140
—
7030.26
—
21.76
7834.00
0.2778
1.38
6225.95
0.0222
12.30
8279.16
0.1486
67.99
6933.19
0.9806
—
5458.32
—
LA5
41.41
10636.04
0.3893
53.62
5018.49
1.0684
47.11
3686.56
1.2779
1.53
7468.47
0.0205
28.74
6068.81
0.4736
1.29
4893.15
0.0264
22.91
11517.82
0.1989
104.70
5679.74
1.8434
—
6326.99
—
LA6
26.86
10152.01
0.2646
10.02
5879.06
0.1704
109.90
3477.43
3.1604
0.66
10000.06
0.0066
23.06
5135.21
0.4491
7.12
3736.03
0.1906
33.26
15229.07
0.2184
38.54
6481.81
0.5946
0.24
5289.57
0.0045
LA7
37.39
5299.21
0.7056
7.21
4582.47
0.1573
137.64
3164.85
4.3490
—
6820.33
—
25.78
4508.55
0.5718
2.81
5062.03
0.0555
30.16
14035.50
0.2149
37.11
8557.89
0.4336
—
6719.24
—
LA8
33.07
10470.63
0.3158
14.37
6157.76
0.2334
26.46
2514.35
1.0524
—
12359.55
—
27.27
8147.42
0.3347
3.27
7846.61
0.0417
18.76
12842.68
0.1461
88.93
8060.89
1.1032
—
4516.77
—
LA9
3.40
12345.60
0.0275
9.98
5675.92
0.1758
2.04
4742.21
0.0430
—
15127.20
—
6.76
10585.00
0.0639
0.29
8961.94
0.0032
1.34
12900.12
0.0104
13.42
10716.38
0.1252
—
5820.67
—
LA10
32.37
8506.23
0.3805
15.12
4869.46
0.3105
40.05
3515.47
1.1393
0.11
11225.08
0.0010
15.86
4962.73
0.3196
1.40
6261.69
0.0224
8.88
9143.75
0.0971
86.86
5400.65
1.6083
—
7815.34
—
LA11
5.06
11187.35
0.0452
16.69
6130.94
0.2722
2.35
2533.92
0.0927
0.36
13012.18
0.0028
5.43
10083.36
0.0539
0.97
5607.42
0.0173
1.87
15543.78
0.0120
7.40
7751.63
0.0955
—
4413.68
—
LA12
38.36
6567.71
0.5841
37.15
4331.21
0.8577
73.93
1638.83
4.5111
0.13
10001.78
0.0013
19.64
5219.17
0.3763
1.98
3334.61
0.0594
16.66
13924.60
0.1196
43.90
4477.46
0.9805
—
3708.31
—
LA13
33.49
9334.49
0.3588
21.25
5427.96
0.3915
57.89
3900.56
1.4841
0.15
11927.09
0.0013
18.94
7570.98
0.2502
1.52
3832.52
0.0397
14.66
14801.24
0.0990
130.27
9104.06
1.4309
—
4437.40
—
LA14
48.22
9862.78
0.4889
10.25
5078.48
0.2018
68.84
1820.89
3.7806
0.18
12167.89
0.0015
20.49
9331.57
0.2196
3.12
6487.16
0.0481
27.88
15343.15
0.1817
73.66
5754.34
1.2801
—
4845.05
—
LA15
33.62
4870.39
0.6903
38.52
4318.57
0.8920
46.08
2538.52
1.8152
—
8893.47
—
18.06
5895.62
0.3063
0.58
3366.22
0.0172
18.67
7744.33
0.2411
97.99
6807.78
1.4394
0.19
6361.27
0.0030
LA16
28.73
10676.60
0.2691
17.16
5956.75
0.2881
30.34
4234.41
0.7165
0.10
10936.85
0.0009
20.90
7579.89
0.2757
1.06
6621.76
0.0160
17.03
15351.32
0.1109
39.65
7199.84
0.5507
0.16
4666.63
0.0034
LA17
25.33
11534.16
0.2196
8.43
5093.69
0.1655
57.74
3692.35
1.5638
0.41
10297.60
0.0040
12.50
7491.18
0.1669
4.43
3719.53
0.1191
16.02
17696.48
0.0905
32.56
9782.67
0.3328
—
8946.44
—
LA18
24.44
9269.41
0.2637
7.67
3801.25
0.2018
56.82
3179.11
1.7873
0.60
9490.75
0.0063
15.74
6342.64
0.2482
2.92
3811.54
0.0766
15.43
11215.99
0.1376
37.22
8358.76
0.4453
—
3946.18
—
Sample
Hpro
Met
Thr
Asn
Gln
Ser
Total free amino acids
Total proteins (%)
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
SP1
0.34
12275.50
0.0028
—
2102.26
—
12.78
7963.72
0.1605
39.82
3834.80
1.0384
1.04
1971.45
0.0528
4.76
6466.09
0.0736
172.07
16.19
SP2
0.84
11585.53
0.0073
—
1927.32
—
38.01
7239.98
0.5250
215.37
4986.79
4.3188
3.20
2096.01
0.1527
22.43
5929.40
0.3783
646.85
18.88
SP3
10.37
9975.53
0.1040
4.20
1384.33
0.3034
157.92
4945.98
3.1929
133.70
3711.29
3.6025
68.98
1951.23
3.5352
144.02
5954.58
2.4186
1437.51
15.19
SP4
3.50
12741.33
0.0275
—
1958.62
—
168.24
8709.13
1.9318
139.13
4253.91
3.2706
143.70
2036.62
7.0558
163.42
5609.49
2.9133
1520.16
17.82
SP5
0.48
15829.31
0.0030
—
2151.19
—
34.91
8355.57
0.4178
258.84
4912.45
5.2691
3.06
2256.83
0.1356
16.69
10234.90
0.1631
546.01
20.46
SP6
6.53
12371.35
0.0528
—
1608.64
—
185.93
5726.31
3.2469
131.14
4602.59
2.8493
53.20
2164.53
2.4578
144.62
8041.81
1.7984
1568.88
17.51
SP7
3.37
12423.61
0.0271
0.10
1470.47
0.0068
168.48
4848.42
3.4749
137.82
3978.83
3.4638
115.64
2145.57
5.3897
153.92
8601.82
1.7894
1616.80
15.59
SP8
0.83
9760.19
0.0085
—
1320.98
—
36.00
5251.98
0.6855
243.18
3721.89
6.5338
4.33
2074.31
0.2087
23.02
7285.14
0.3160
601.76
14.41
SP9
2.33
10641.34
0.0219
0.10
1927.64
0.0052
101.08
6224.18
1.6240
287.73
3643.59
7.8969
22.11
2123.14
1.0414
80.72
7300.64
1.1057
1176.53
14.43
SP10
5.44
15070.02
0.0361
0.26
2233.47
0.0116
111.26
9030.91
1.2320
295.67
4158.01
7.1109
19.66
2820.23
0.6971
98.55
9877.31
0.9977
1162.05
19.14
SP11
11.23
13641.99
0.0823
0.28
1744.92
0.0160
176.59
7985.90
2.2113
140.18
4655.28
3.0112
144.55
2112.77
6.8417
179.12
6088.37
2.9420
1570.89
17.01
SP12
12.01
11162.90
0.1076
0.24
1881.32
0.0128
125.79
4403.32
2.8567
137.25
3369.96
4.0727
41.76
1600.68
2.6089
57.70
6605.01
0.8736
1145.82
14.13
SP13
2.39
10615.66
0.0225
3.51
1382.26
0.2539
228.15
6391.62
3.5695
165.25
4095.51
4.0349
269.51
1696.44
15.8868
168.80
6708.25
2.5163
1755.14
13.75
SP14
1.44
13205.06
0.0109
—
2393.23
—
132.66
8976.95
1.4778
149.56
4798.05
3.1171
125.79
2465.90
5.1012
129.14
7298.41
1.7694
1255.50
19.06
SP15
6.10
10366.51
0.0588
—
1814.22
—
15.94
7685.12
0.2074
22.02
4676.65
0.4708
6.45
2382.45
0.2707
7.22
5548.49
0.1301
194.11
15.85
SP16
7.67
12903.86
0.0594
0.23
2509.21
0.0092
85.06
5550.87
1.5324
142.06
4329.68
3.2811
176.62
3056.34
5.7788
145.74
5866.53
2.4843
1678.57
18.29
SP17
2.03
10626.57
0.0191
—
1794.68
—
10.28
5329.55
0.1929
3.24
3731.24
0.0868
2.10
2828.19
0.0743
6.37
4657.92
0.1368
166.05
15.26
SP18
3.88
12874.45
0.0301
0.25
2623.84
0.0095
124.83
7649.77
1.6318
148.44
4745.86
3.1278
123.11
3038.81
4.0513
102.24
8464.54
1.2079
1304.51
19.91
SP19
6.64
13923.42
0.0477
0.39
1611.21
0.0242
116.76
6748.38
1.7302
49.92
4539.22
1.0997
45.19
3232.29
1.3981
77.95
5525.06
1.4108
1063.99
17.50
SP20
1.26
8623.89
0.0146
0.17
1655.87
0.0103
184.54
4751.82
3.8836
152.52
3938.78
3.8723
120.92
2150.51
5.6229
176.26
7450.83
2.3656
1670.95
13.61
SP21
10.27
10862.57
0.0945
—
1789.85
—
19.55
7093.47
0.2756
38.03
4126.30
0.9216
7.72
2908.79
0.2654
11.94
6041.00
0.1976
460.11
16.55
SP22
3.58
11476.96
0.0312
0.36
1441.11
0.0250
11.15
5896.68
0.1891
3.63
3801.35
0.0955
8.88
2075.29
0.4279
6.66
7009.63
0.0950
192.55
15.30
SP23
6.80
11931.55
0.0570
—
1557.75
—
48.22
5634.77
0.8558
90.90
3869.18
2.3493
14.04
1927.16
0.7285
18.66
8290.65
0.2251
651.61
16.73
SP24
6.27
10593.16
0.0592
0.12
2127.69
0.0056
64.57
5101.21
1.2658
104.24
3837.96
2.7160
14.64
2823.06
0.5186
24.13
9012.82
0.2677
734.27
16.87
SP25
1.86
13824.44
0.0135
0.44
2716.54
0.0162
186.66
4842.06
3.8550
146.65
4187.28
3.5023
97.55
2771.87
3.5193
189.30
8595.86
2.2022
1899.50
19.76
LP1
0.47
14631.38
0.0032
1.18
1948.75
0.0606
65.83
6231.55
1.0564
95.95
4013.49
2.3907
86.11
3138.12
2.7440
85.73
5753.40
1.4901
814.99
18.47
LP2
0.64
12206.18
0.0052
1.85
2297.38
0.0805
78.50
5838.37
1.3446
145.43
4899.76
2.9681
52.18
2104.26
2.4797
73.92
6212.65
1.1898
1012.65
17.73
LP3
4.52
8885.71
0.0509
0.13
1858.23
0.0070
41.09
5789.60
0.7097
124.04
2882.55
4.3031
10.24
2456.95
0.4168
10.18
6286.97
0.1619
597.23
13.95
LP4
5.45
10112.04
0.0539
—
1630.58
—
163.52
7525.00
2.1730
143.69
3758.46
3.8231
51.80
1875.09
2.7625
160.80
7731.94
2.0797
1426.96
16.05
LP5
1.71
9028.04
0.0189
—
1621.37
—
152.81
4615.56
3.3108
134.30
3260.74
4.1187
112.30
1704.64
6.5879
122.16
4651.12
2.6265
1718.87
15.03
LP6
0.71
11053.96
0.0064
—
1379.86
—
85.21
6409.94
1.3293
270.66
3875.46
6.9839
136.75
1528.04
8.9494
72.78
6453.61
1.1277
1308.25
13.94
LP7
0.41
10437.44
0.0039
0.99
1616.03
0.0613
48.37
6713.60
0.7205
199.42
3616.48
5.5142
71.18
2426.57
2.9334
73.61
5273.31
1.3959
1113.74
16.11
LP8
0.60
12028.82
0.0050
2.77
2130.37
0.1300
64.52
8264.66
0.7807
93.71
4102.87
2.2840
128.62
3030.50
4.2442
77.21
8764.09
0.8810
1028.91
17.52
LP9
2.85
15395.17
0.0185
1.09
2611.93
0.0417
95.31
6079.19
1.5678
135.68
5800.10
2.3393
21.02
2847.50
0.7382
160.76
6714.80
2.3941
1620.64
20.09
LP10
3.15
12510.19
0.0252
0.19
2294.16
0.0083
139.99
5986.95
2.3383
316.13
4149.54
7.6184
20.34
2755.14
0.7383
126.41
9249.73
1.3666
1408.67
18.88
LP11
2.02
11304.83
0.0179
0.44
1616.02
0.0272
92.06
7087.95
1.2988
100.62
3134.85
3.2097
9.67
2255.64
0.4287
81.26
7479.28
1.0865
735.57
14.11
LP12
1.26
11500.95
0.0110
0.34
1721.63
0.0197
24.77
6419.44
0.3859
25.52
4463.29
0.5718
3.93
2645.29
0.1486
15.06
7598.99
0.1982
291.40
16.46
LP13
6.41
13163.18
0.0487
0.30
2716.29
0.0110
172.44
9432.07
1.8282
143.41
4225.29
3.3941
134.72
2932.22
4.5945
169.75
8497.59
1.9976
1667.79
19.79
LP14
3.64
16225.57
0.0224
0.17
2793.92
0.0061
175.47
6457.65
2.7172
140.65
5052.79
2.7836
118.28
3874.56
3.0527
171.21
9352.55
1.8306
1529.56
21.15
LP15
1.70
11614.17
0.0146
0.09
2501.01
0.0036
89.92
6615.18
1.3593
269.56
3725.94
7.2347
3.34
2877.91
0.1161
82.11
8616.92
0.9529
900.44
18.46
LP16
0.94
11631.20
0.0081
0.99
1651.61
0.0599
95.46
5189.08
1.8396
201.46
3964.71
5.0813
106.74
2083.97
5.1220
89.52
5649.83
1.5845
1381.04
15.81
LA1
0.75
10525.37
0.0071
0.33
1722.41
0.0192
119.80
5747.08
2.0845
146.08
4354.39
3.3548
117.79
2828.86
4.1639
99.93
8354.17
1.1962
1262.44
15.27
LA2
0.71
11050.46
0.0064
2.14
2075.76
0.1031
88.95
5755.14
1.5456
114.22
4714.20
2.4229
113.30
2590.48
4.3737
128.49
8446.54
1.5212
1057.50
16.42
LA3
1.58
14292.88
0.0111
—
2387.79
—
85.96
9803.09
0.8769
129.12
5651.48
2.2847
172.44
2107.17
8.1835
147.16
11408.14
1.2900
1716.54
20.72
LA4
0.78
11457.33
0.0068
—
2109.40
—
75.39
7598.12
0.9922
177.20
4225.71
4.1934
34.18
2341.13
1.4600
45.77
7073.90
0.6470
778.32
15.58
LA5
6.18
11098.92
0.0557
0.60
1527.50
0.0393
127.09
6426.35
1.9776
196.17
4798.09
4.0885
29.66
2500.29
1.1863
66.00
7750.15
0.8516
1332.28
16.58
LA6
1.69
11256.40
0.0150
0.14
1485.02
0.0094
92.57
6651.83
1.3916
129.99
4328.83
3.0029
86.79
1722.57
5.0384
157.13
8406.98
1.8690
1547.66
16.04
LA7
2.07
11307.16
0.0183
—
1577.93
—
92.43
4656.66
1.9849
145.34
4362.67
3.3314
135.71
1877.25
7.2292
170.21
8033.47
2.1188
1517.92
14.71
LA8
10.79
15918.39
0.0678
0.19
2259.56
0.0084
117.78
7883.83
1.4939
154.15
5792.38
2.6613
216.67
2621.75
8.2643
61.37
7941.33
0.7728
1171.00
21.30
LA9
0.76
14178.74
0.0054
0.14
2618.77
0.0053
6.51
6538.81
0.0996
7.87
4624.51
0.1702
2.68
2321.32
0.1155
6.76
10561.97
0.0640
189.02
21.46
LA10
1.44
11088.93
0.0130
0.26
1806.72
0.0144
62.25
6942.43
0.8967
53.97
4802.60
1.1238
47.46
2041.76
2.3245
36.17
5403.37
0.6694
775.54
16.41
LA11
0.84
15220.71
0.0055
1.65
2528.91
0.0652
8.60
7038.75
0.1222
1.31
5236.37
0.0250
4.91
2429.40
0.2021
7.80
6092.24
0.1280
289.21
19.05
LA12
2.55
9164.65
0.0278
0.18
1894.44
0.0095
174.89
4404.00
3.9712
138.27
3675.47
3.7620
72.48
1770.28
4.0943
85.28
5128.63
1.6628
1294.14
14.54
LA13
2.16
13906.13
0.0155
—
2514.70
—
116.52
7070.17
1.6481
294.15
3964.62
7.4194
42.77
2727.93
1.5679
65.75
7050.95
0.9325
1282.38
18.31
LA14
9.08
13760.74
0.0660
0.60
2297.56
0.0261
164.90
9014.10
1.8294
131.85
5045.39
2.6133
63.90
2704.59
2.3627
144.27
5601.90
2.5754
1371.90
17.81
LA15
3.69
8467.71
0.0436
0.30
1826.09
0.0164
135.18
5450.03
2.4804
62.94
3497.92
1.7994
46.02
1747.54
2.6334
51.56
4656.58
1.1073
1020.37
13.89
LA16
2.78
16217.53
0.0171
0.29
2603.75
0.0111
97.73
8455.20
1.1559
22.61
5055.17
0.4473
18.70
2206.25
0.8476
48.71
9026.29
0.5396
811.03
20.42
LA17
0.68
12202.21
0.0056
2.23
2011.22
0.1109
81.41
5966.49
1.3645
207.86
3770.32
5.5131
55.84
3327.23
1.6783
76.89
7660.33
1.0037
1027.14
18.44
LA18
0.55
9875.86
0.0056
2.42
1754.15
0.1380
76.82
5468.75
1.4047
143.75
3704.63
3.8803
52.67
1760.52
2.9917
90.61
6192.83
1.4631
1068.96
14.93
—, analytes were not detected.Twenty-six minerals, total proteins, and the content of amino acids after hydrolysis are shown in Tables5 and 6. From the tables, it could be found that the total protein contents in 59 samples ranged from 13.61% to 21.46%. The relative contents of amino acids such as Lys, Ala, Ser, Thr, GABA, Gln, and Asn were all a little higher than other components based on the average value of each species among three different varieties of duckweed. The contents of Cys, Cys2, and Met were higher after hydrolysis. Obviously, it could be seen that the contents of free amino acids in duckweed were much lower than that of protein-bound amino acids. Moreover, the data showed that the contents of free amino acids in duckweed were negatively correlated with binding amino acids.Table 6
Contents of 26 minerals in living water of three species of duckweed (μg/L).
Sample
Ti
V
Cr
Mn
Co
Ni
Cu
Zn
As
Se
Sr
Mo
Cd
Sn
Sb
Ba
Tl
Pb
SP1
22.831
2.900
2.178
47.015
0.181
1.860
1.061
90.405
1.530
1.578
261.614
2.840
0.007
0.087
0.440
96.840
0.003
0.058
SP2
27.277
1.017
2.922
36.990
0.177
1.648
0.806
80.395
2.091
1.288
199.006
3.275
0.006
0.007
0.516
60.613
0.003
0.001
SP3
35.621
1.915
1.838
41.295
0.162
2.899
1.500
56.437
1.127
1.983
172.960
3.017
0.016
0.168
0.386
70.196
0.002
0.024
SP4
22.183
3.099
1.687
55.683
0.214
1.767
1.690
63.717
3.164
1.935
178.985
0.407
0.014
0.168
0.487
77.659
0.001
0.041
SP5
45.839
3.162
1.413
24.431
0.189
2.829
0.615
71.741
0.920
1.861
275.418
2.211
0.011
0.098
0.542
63.126
0.004
0.074
SP6
41.446
1.783
2.371
2.110
0.240
2.157
2.805
87.665
2.146
1.464
193.120
0.577
0.002
0.121
0.536
39.704
0.004
0.046
SP7
37.099
2.423
2.068
13.993
0.219
2.583
1.424
88.936
2.601
1.747
165.967
1.562
0.010
0.095
0.316
69.916
0.003
0.050
SP8
28.377
2.251
2.276
54.028
0.197
1.709
2.941
46.581
2.262
2.139
249.180
2.345
0.015
0.125
0.359
110.809
0.004
0.047
SP9
17.084
1.069
2.452
40.559
0.192
3.140
1.247
29.974
2.770
1.664
163.694
2.659
0.015
0.139
0.637
60.895
0.004
0.018
SP10
37.166
3.316
1.705
35.254
0.184
1.793
2.040
78.451
1.247
1.407
174.872
0.632
0.002
0.137
0.720
86.360
—
0.003
SP11
24.163
1.033
1.368
60.738
0.190
3.261
2.894
66.981
1.135
2.291
207.084
1.840
0.003
0.160
0.488
117.603
0.004
0.046
SP12
38.630
2.597
1.146
53.768
0.252
2.550
1.505
76.263
1.772
2.126
180.679
0.626
0.003
0.124
0.628
142.280
0.001
0.062
SP13
39.728
2.347
1.577
26.733
0.173
3.173
2.882
75.098
1.628
1.112
225.261
0.323
0.012
0.106
0.550
95.230
0.001
0.001
SP14
39.483
2.488
2.539
3.742
0.249
2.137
1.427
55.271
1.670
1.581
154.375
3.214
0.019
0.135
0.338
119.138
0.003
0.043
SP15
19.629
2.734
2.326
22.405
0.198
3.153
1.427
55.631
1.175
2.065
247.855
2.928
0.011
0.011
0.633
136.536
0.004
0.030
SP16
34.619
1.068
2.064
52.250
0.163
2.169
3.017
54.488
2.321
1.985
253.553
2.155
0.002
0.120
0.377
68.487
0.001
0.022
SP17
28.672
3.244
2.192
31.414
0.159
1.880
2.871
91.946
1.444
1.287
175.165
1.721
0.018
0.138
0.669
91.883
0.004
0.030
SP18
25.018
1.789
2.378
45.984
0.183
2.570
2.365
24.786
1.877
2.146
194.164
3.450
0.007
0.049
0.318
95.469
0.002
0.057
SP19
22.802
2.136
2.199
53.284
0.212
2.704
1.054
85.613
0.913
2.073
269.684
3.174
0.014
0.043
0.423
111.149
0.003
0.004
SP20
40.638
1.682
1.746
14.621
0.180
1.611
2.705
73.740
3.221
2.055
225.646
3.023
0.013
0.159
0.450
137.841
0.004
0.019
SP21
23.513
1.413
2.811
12.908
0.236
1.959
0.607
22.616
2.711
1.170
227.681
3.187
0.005
0.037
0.205
62.615
0.003
0.016
SP22
32.010
1.755
2.461
56.750
0.202
2.515
1.114
80.116
1.210
2.322
223.754
1.075
0.015
0.155
0.573
96.469
0.002
0.012
SP23
17.817
2.259
2.391
43.452
0.219
1.903
0.700
88.007
3.277
1.855
155.450
2.793
0.014
0.164
0.311
82.020
0.002
0.010
SP24
24.306
2.384
1.841
32.935
0.253
1.896
3.027
63.243
1.975
1.109
214.325
2.979
0.008
0.044
0.642
47.431
0.003
0.061
SP25
30.842
2.878
1.693
29.377
0.254
2.786
1.811
69.276
1.082
1.349
218.438
0.896
0.011
0.125
0.474
126.036
0.001
0.032
LP1
37.974
1.441
2.756
42.898
0.251
1.828
2.325
46.010
2.655
2.264
162.610
0.298
0.004
0.049
0.368
106.287
0.002
0.055
LP2
25.601
3.144
1.914
10.035
0.173
2.425
2.422
53.173
1.031
1.740
211.860
0.256
0.017
0.153
0.596
73.995
0.002
0.001
LP3
40.576
2.424
1.579
36.158
0.203
2.635
1.373
32.781
1.839
1.363
234.793
1.450
0.001
0.145
0.697
107.956
0.001
0.032
LP4
29.919
1.451
2.468
31.060
0.239
3.221
2.253
43.918
2.873
1.754
193.086
2.154
0.013
0.027
0.249
36.637
0.001
0.015
LP5
43.240
2.466
2.331
46.991
0.209
3.218
1.577
26.153
1.994
2.211
234.265
1.895
0.010
0.017
0.224
84.449
0.004
0.006
LP6
29.925
2.796
2.933
11.764
0.248
2.974
2.174
43.896
2.394
1.784
247.178
3.487
0.012
0.017
0.336
125.785
0.003
0.016
LP7
28.783
1.090
2.067
41.129
0.244
2.388
1.514
76.640
1.923
1.886
157.760
2.486
0.003
0.031
0.241
87.240
0.004
0.053
LP8
45.076
3.030
1.857
7.724
0.247
2.328
2.152
87.415
2.455
2.044
225.691
2.976
0.011
0.009
0.607
111.718
0.003
0.023
LP9
20.651
2.339
2.731
42.403
0.257
1.681
1.012
89.960
1.066
1.855
210.521
2.237
0.007
0.052
0.424
32.520
—
0.033
LP10
23.769
1.646
1.288
52.927
0.243
2.319
2.950
93.752
2.929
1.882
217.655
2.496
0.017
0.165
0.196
135.624
0.003
0.051
LP11
32.364
2.560
3.084
33.335
0.208
3.219
2.180
82.812
1.451
1.918
269.769
3.250
0.019
0.008
0.406
66.278
0.003
0.051
LP12
24.918
1.470
2.999
37.041
0.243
1.893
1.928
95.305
2.415
1.698
224.103
1.906
0.008
0.041
0.570
64.033
0.004
0.008
LP13
33.468
1.704
2.451
45.575
0.176
2.507
1.882
38.837
2.524
1.580
187.087
1.245
0.003
0.133
0.285
44.075
0.003
0.030
LP14
28.614
2.426
1.879
39.614
0.209
3.272
1.963
74.622
2.875
1.962
261.572
2.514
0.013
0.136
0.287
100.138
0.002
0.048
LP15
35.617
2.088
2.116
42.214
0.162
2.284
1.641
39.037
1.412
1.185
244.588
0.489
0.007
0.158
0.512
110.953
0.003
0.032
LP16
32.522
2.845
1.545
41.087
0.186
2.100
1.860
94.105
3.060
1.921
165.053
1.398
0.015
0.008
0.451
69.796
0.002
0.066
LA1
36.451
2.711
1.271
38.156
0.233
2.893
0.909
65.183
3.041
1.926
194.455
3.342
0.005
0.055
0.262
51.502
0.002
0.023
LA2
43.394
2.188
2.067
17.427
0.165
2.206
1.564
58.425
3.159
1.247
254.469
1.789
0.006
0.146
0.280
33.628
0.003
0.052
LA3
24.311
1.120
2.917
35.712
0.247
2.359
2.087
59.228
3.345
2.092
247.432
1.778
0.010
0.108
0.362
105.945
—
0.069
LA4
26.794
1.110
1.710
13.192
0.232
1.909
1.963
25.002
3.351
1.618
198.614
1.801
—
0.011
0.452
33.107
0.003
0.024
LA5
39.248
2.722
1.585
24.608
0.203
2.755
2.186
68.836
1.896
1.634
279.990
3.413
0.016
0.037
0.213
37.247
0.004
0.077
LA6
20.981
2.024
1.392
25.368
0.248
2.878
2.542
45.526
1.863
1.095
215.126
3.019
0.009
0.148
0.651
117.017
0.003
0.010
LA7
30.279
1.148
2.330
62.335
0.231
2.772
2.791
44.135
2.816
1.556
187.777
3.290
0.012
0.151
0.436
36.940
0.003
0.041
LA8
39.053
0.971
3.084
15.630
0.243
3.097
1.738
42.571
2.567
1.411
231.825
2.281
0.010
0.013
0.499
33.670
0.003
0.053
LA9
41.198
2.925
2.254
52.257
0.230
2.490
2.132
95.196
2.872
2.220
170.239
1.302
0.002
0.132
0.395
80.576
0.003
0.015
LA10
16.833
1.384
2.832
34.567
0.188
2.044
1.951
62.570
0.922
1.571
169.715
0.952
0.001
0.110
0.496
81.418
0.002
0.044
LA11
38.564
2.900
3.171
30.344
0.257
2.860
2.083
37.280
1.155
1.073
172.207
1.959
0.008
0.117
0.433
108.524
0.002
0.014
LA12
43.364
1.975
2.442
7.660
0.253
1.986
0.775
85.359
2.247
1.143
237.191
0.673
0.009
0.155
0.201
41.025
—
0.002
LA13
30.181
3.229
1.146
53.644
0.230
2.341
2.091
77.183
0.842
2.028
177.614
2.395
0.009
0.105
0.689
97.023
0.001
0.022
LA14
31.940
3.492
1.508
64.537
0.181
2.947
2.883
24.336
3.049
2.137
243.782
1.989
0.017
0.117
0.272
125.192
0.003
0.021
LA15
22.170
2.327
3.077
51.812
0.175
3.050
0.998
60.176
2.860
1.716
206.597
0.281
0.006
0.058
0.397
108.215
—
0.025
LA16
42.638
1.570
1.978
49.228
0.258
1.959
0.924
88.563
2.498
1.683
201.422
3.242
0.014
0.029
0.295
80.373
0.003
0.075
LA17
39.670
1.249
2.650
27.755
0.259
2.112
2.250
55.219
2.830
1.217
154.053
1.978
0.006
0.099
0.561
116.452
—
0.047
LA18
23.423
2.528
2.932
10.179
0.199
2.743
2.953
66.269
3.199
1.527
209.326
3.075
0.016
0.116
0.306
79.441
0.001
0.040
Sample
Na
Mg
Al
K
Ca
Fe
N
P
Total
SP1
410579.062
209771.718
18661.431
178472.536
591631.101
334974.476
4.303
0.914
1744628.970
SP2
546214.809
232797.639
9209.284
416708.514
386703.829
349989.801
2.389
0.675
1942044.979
SP3
384732.341
302374.639
16864.219
351597.651
645593.344
330485.336
18.804
5.589
2032063.470
SP4
763978.060
217006.139
24174.116
515345.319
525036.011
165789.077
19.687
4.182
2211765.493
SP5
498927.575
304758.187
29743.190
617594.946
663524.773
200323.584
25.750
23.839
2315416.327
SP6
568688.625
219822.814
31062.346
305039.982
486491.766
202085.631
25.562
22.681
1813617.704
SP7
815621.400
203217.299
32660.784
559562.408
566642.730
171755.702
2.565
1.507
2349855.405
SP8
704283.730
188285.004
23602.584
315772.349
624642.806
439955.497
23.372
6.689
2297077.677
SP9
876213.262
151574.474
27629.879
516294.381
409003.501
35506.241
18.458
4.258
2016572.669
SP10
873643.979
294027.085
21164.307
737471.818
383002.078
37324.576
33.550
19.033
2347111.719
SP11
491391.823
256934.016
10147.423
610308.309
636084.667
367347.644
31.694
25.205
2372762.062
SP12
796497.255
269227.175
17999.811
414658.818
512138.780
171064.616
16.646
2.703
2182110.814
SP13
366808.463
156147.673
11576.232
471660.374
756208.109
347515.518
11.647
4.202
2110408.155
SP14
512150.165
243500.636
31493.169
641312.090
499270.353
208734.109
11.077
1.602
2136861.051
SP15
533557.790
280721.134
16614.646
267728.355
378226.451
306162.902
13.593
4.564
1783528.184
SP16
644403.624
276057.049
21110.856
521532.157
583524.045
59176.189
11.942
25.264
2106319.987
SP17
751229.025
173163.933
10495.020
625004.125
472688.229
345116.023
8.458
5.204
2378144.753
SP18
522140.520
176639.957
29371.612
557932.109
690911.336
157124.877
29.906
10.221
2134563.150
SP19
579415.759
238453.094
30677.565
439717.245
686006.551
382672.972
10.823
3.662
2357515.153
SP20
476281.317
182915.790
9070.049
325811.742
373586.252
432553.750
34.616
21.891
1800784.763
SP21
760676.992
188574.381
12126.295
629919.272
484913.883
230880.412
26.927
9.833
2307491.687
SP22
709408.940
239417.173
10783.138
619462.993
550226.213
88795.927
20.170
9.877
2218626.941
SP23
513303.754
213899.504
26441.948
351694.267
732144.084
326876.462
34.159
26.699
2164823.520
SP24
824465.276
238490.190
31283.397
717257.458
679154.325
297001.594
14.031
10.796
2788075.528
SP25
815267.536
215764.623
30718.721
536395.881
508251.297
371174.761
24.650
2.494
2478087.325
LP1
897950.747
158686.979
27871.364
590366.194
696960.175
327232.239
17.263
7.080
2699502.116
LP2
531566.916
294929.433
14640.021
570417.870
648189.841
273059.464
15.536
1.239
2333208.859
LP3
858626.936
266231.149
32272.481
727712.714
480257.660
348345.090
12.319
1.894
2713926.251
LP4
431098.253
221743.162
34467.794
311381.975
785570.359
339960.811
26.584
17.225
2124617.501
LP5
818404.736
237360.411
25820.309
683695.992
566015.946
406722.563
20.219
7.521
2738498.958
LP6
614726.783
203772.151
23626.598
170198.351
715325.262
132978.487
8.402
1.447
1861115.202
LP7
498622.492
200069.361
34249.218
182114.487
450498.128
213156.897
26.337
12.664
1579155.065
LP8
407734.217
308322.609
12414.630
462642.984
726996.054
273473.025
23.952
9.428
2192112.264
LP9
591923.425
196758.141
20795.603
169080.807
379447.395
244832.850
24.638
15.719
1603288.325
LP10
506188.280
278745.249
26183.977
548614.526
619327.368
71487.282
27.250
10.475
2051124.319
LP11
484360.416
252740.828
27267.497
394747.762
420528.173
411590.345
15.427
3.462
1991756.825
LP12
755545.368
290158.455
18713.693
269669.218
653034.467
303936.794
26.120
0.889
2291545.587
LP13
644431.270
171083.444
14922.153
701260.007
799614.153
241821.499
27.383
10.915
2573534.387
LP14
855511.785
226105.300
11812.507
382201.190
657443.790
421778.147
4.108
0.257
2555378.850
LP15
840333.425
261183.216
19461.126
468066.062
568653.040
96502.984
17.469
12.422
2254714.244
LP16
662138.232
247763.574
23217.103
653430.420
481555.835
52632.672
16.299
0.242
2121172.397
LA1
542867.038
291631.774
16101.327
208393.119
534546.718
287585.245
20.487
11.217
1881559.346
LA2
768502.457
174767.313
34293.763
538598.282
767009.154
130620.652
24.093
8.098
2414246.025
LA3
836100.485
232547.859
8383.317
197659.044
475008.913
70420.274
26.668
18.771
1820654.452
LA4
554969.006
231606.445
11451.728
365538.304
505163.079
269818.459
11.932
3.908
1938873.753
LA5
794677.014
293809.893
30832.050
293530.490
510493.154
262663.280
12.498
6.307
2186491.358
LA6
704360.729
287183.610
31019.483
189142.179
384371.287
327789.933
12.239
4.852
1924324.212
LA7
894339.623
204701.453
27985.214
281212.437
595254.900
371594.312
24.934
18.350
2375510.268
LA8
427283.433
184599.086
23700.009
211637.098
596714.072
325251.967
22.097
1.616
1769588.097
LA9
879491.821
180076.383
12682.543
611024.725
416252.729
426535.290
22.887
10.851
2526553.669
LA10
712373.467
297199.560
9966.649
218385.532
678770.388
129164.596
32.940
4.216
2046274.948
LA11
625486.302
275066.946
25810.603
496586.040
470812.328
438093.869
14.927
1.233
2332275.197
LA12
796469.226
199565.531
26532.869
616737.846
538184.180
286481.065
25.413
16.087
2464438.679
LA13
587462.535
234893.095
16533.152
573600.610
441381.833
215218.753
5.375
3.154
2069549.281
LA14
598797.362
200101.308
32285.037
179408.885
737688.278
321911.868
10.937
7.808
2070719.888
LA15
775176.535
182419.544
9875.652
375172.731
686178.073
146549.529
20.767
18.862
2175875.636
LA16
486462.514
269482.486
19514.461
382028.835
385846.710
419515.606
25.566
4.690
1963357.619
LA17
397975.248
220647.104
20225.722
404790.872
406870.509
69049.196
25.782
16.448
1520009.288
LA18
852308.317
145491.528
11612.476
499143.838
570244.949
84828.689
23.929
16.569
2164078.568
—, analytes were not detected.The elements with higher content in the samples were sodium, calcium, potassium, iron, magnesium, and aluminum. Their concentration ranges were 366808.463–897950.747, 373586.252–799614.153, 169080.807–737471.818, 35506.241–439955.497, 145491.528–308322.609, and 8383.317–34467.794μg/L, respectively. The concentration ranges of strontium, barium, zinc, manganese and titanium were 154.053–279.990, 32.520–142.280, 22.616–95.305, 2.110–64.537, 16.833–45.839 μg/L. The concentrations of vanadium, chromium, cobalt, nickel, copper, arsenic, selenium, molybdenum, cadmium, tin, antimony, thallium, and lead are very low. It ranged from 0.0 to 3.492 μg/L. Evidently, the contents of macroelements in the samples were higher while they contained much lower trace elements.


### 3.6. Statistical Analysis

As one of the most important multivariate analysis techniques, PCA was employed to evaluate the variation among three species of duckweed. PCA is an unsupervised pattern recognition method without prior information of the data set and retained maximum variance of multidimensional data while reducing its dimensionality into two- or three-dimension [29–31]. Furthermore, all of the data information about amino acids was subjected to a supervised method, OPLS-DA [32], to get more information about different duckweeds. All the raw data were processed by normalization and Pareto scaling (Par) before modeling. Three dimensions were established in both the PCA and OPLS-DA models based on the content of free amino acids and the ratio of free to total amino acids with R2X (cum) value of 0.628, 0.596 and 0.758, 0.676, respectively. As shown in Figures 2–5, both PCA and OPLS-DA methods demonstrated that Spirodela polyrhiza, Landoltia punctata, and Lemna aequinoctialis were clustered together with each other in the score plot. There was no remarkable difference in different duckweeds.Figure 2
Three-dimensional score plot of principle component analysis (PCA) based on the content of free amino acids.Figure 3
Three-dimensional score plot of orthogonal partial least square-discriminant analysis (OPLS-DA) based on the content of free amino acids.Figure 4
Three-dimensional score plot of principle component analysis (PCA) based on the ratio of free to total amino acids.Figure 5
Three-dimensional score plot of orthogonal partial least squares-discriminant analysis (OPLS-DA) based on the ratio of free to total amino acids.


## 3.1. Optimization of Extraction Procedure

Free amino acids can dissolve in methanol, ethanol, water, diluted acid, and so on. In order to acquire an efficient extraction strategy of amino acids from duckweed, variables of the extraction process were investigated. The solvents (methanol, ethanol, water, 0.1% formic acid, and 0.5% formic acid), methods (heating reflux, ultrasonication, and tissue-grinding), and time (20, 30, and 40 min for ultrasonication; 60, 90, and 120 s for tissue-grinding) were studied. The results showed that the amount of amino acids had no significant difference between the ultrasonication time of 30 min in 0.5% formic acid and tissue-grinding time of 120 s in 0.5% formic acid. The amounts of extraction by other methods were not more than that by these two modalities. From the viewpoint of working efficiency, the tissue-grinding method was much more time-saving than the ultrasonication method by the extracting time of 120 s versus 30 min.


## 3.2. Optimization of UPLC Condition

Chromatographic conditions were optimized to attain a satisfactory separation for amino acid especially for isomeric molecules. Columns (Waters XBridge Amide, 2.5μm, 2.1 × 150 mm and Agilent Poroshell 120 HILIC, 2.7 μm, 2.1 × 150 mm), column temperature (20, 25, 30, 35, and 40°C), mobile phase (acetonitrile-0.1% formic acid, acetonitrile-0.2% formic acid, acetonitrile with 0.2% formic acid-0.2% formic acid, and acetonitrile-10 mM ammonium formate with 0.2% formic acid), and different kinds of gradient elution were investigated.The results demonstrated that the peak shape of amino acids became good when the concentration of formic acid rose, but retention time would be shortened significantly especially if some formic acid was complemented into the acetonitrile. The buffers such as ammonium formate increased the resolution degrees of amino acids. Better retention times and separations were observed by conducting analysis on Waters Amide column than on Agilent Poroshell 120 HILIC column, and the best chromatograms were gained with temperature controlled at 20°C. Especially for the isomers of leucine and isoleucine, baseline separation was successfully achieved when using the mobile phase consisting of acetonitrile-10 mM ammonium formate with 0.2% formic acid. Under other abovementioned conditions, these two isomeric amino acids were not well separated reciprocally.


## 3.3. Optimization of MS/MS Conditions

Primary experiments were conducted to acquire the best mass spectrometry conditions of amino acids individually. For this purpose, each reference component of amino acids with the concentration of approximate 100 ng/mL was delivered into the mass spectrometer directly via a syringe pump with the velocity of 7μL/min. While tuning the standard dissolved in water, most response of amino acids was very low when the sodium-adducted ion, [M+Na]+, was relatively high. On the contrary, when dissolving the standard in 0.1% or higher concentration of formic acid solution, the response of protonated molecule, [M+H]+, was dramatically higher and the metal-adducted ion was barely observed. Consequently, precursor ion (Q1), product ion (Q3), declustering potential (DP), and collision energy (CE) of each amino acid were acquired and optimized.


## 3.4. Method Validation

As shown in Table3, the calibration curves of twenty-four amino acids showed good linear regression (all correlation coefficients > 0.995) within a wide range of concentration. The LOD and LOQ of each analyte with the signal-to-noise ratio (S/N) of 3 and 10 were in the range of 0.37–1.94 ng/mL and 0.74–4.49 ng/mL, respectively. The RSD values of intraday and interday precision are summarized in Table 4. From the results, the present method was found to be precise with intraday variability less than 3.14% and interday variability less than 4.27%. The sample solutions were stable within 24 h with stability RSD < 3.82%. The RSD values of repeatability were 0.52%–3.86%. The recoveries of 24 amino acids ranged from 95.27%–107.51% with RSD < 4.03%.Table 3
Calibration curves, LODs, and LOQs for the 24 compounds.
Number
Compound
Calibration curve
R
2
Linear range (ng/mL)
LOQ (ng/mL)
LOD (ng/mL)
1
Tyr
y = 6534.1x + 6063.4
0.9984
1.16–5780
0.92
0.46
2
Ala
y = 7168.9x + 4932.8
0.9969
5.30–5300
3.71
1.24
3
Glu
y = 12796x + 47334
0.9960
6.41–2564
4.49
1.50
4
Phe
y = 59157 x + 42917
0.9965
0.99–992.0
0.79
0.40
5
His
y = 20263x + 247917
0.9962
5.57–5570
3.90
1.30
6
Ile
y = 70857x + 46587
0.9957
1.07–1074
0.86
0.43
7
Asp
y = 7084.7x + 86152
0.9963
5.43–5430
3.80
1.27
8
Lys
y = 4305.1x + 17648
0.9980
5.53–5530
3.87
1.94
9
Gly
y = 1063.0x + 3407.3
0.9990
0.94–4680
0.75
0.37
10
Val
y = 31972x + 35728
0.9962
1.11–1112
0.89
0.44
11
Cit
y = 8520.8x + 9811.0
0.9980
1.01–5070
0.81
0.41
12
GABA
y = 18495x + 14623
0.9981
1.10–5490
0.88
0.44
13
Cys2
y = 2389.7x + 3146.6
0.9978
1.09–5440
0.87
0.44
14
Pro
y = 51204x + 58539
0.9981
1.02–510.0
0.82
0.41
15
Trp
y = 19030x + 4465.3
0.9977
1.24–2476
0.99
0.50
16
Leu
y = 56715x + 40047
0.9974
1.01–1012
0.81
0.40
17
Arg
y = 11505x + 84828
0.9974
5.96–5960
4.17
1.39
18
Cys
y = 2496.6x + 1017.5
0.9988
1.20–6012
0.96
0.48
19
Hpro
y = 15783x + 3968.1
0.9977
0.95–1892
0.76
0.38
20
Met
y = 8626.4x + 1545.9
0.9984
1.05–5250
0.84
0.42
21
Thr
y = 8807.8x + 19073
0.9957
4.46–4460
3.12
1.56
22
Asn
y = 4939.9x + 22367
0.9967
1.20–5980
0.96
0.48
23
Gln
y = 7733.9x + 8338.4
0.9961
0.93–4650
0.74
0.37
24
Ser
y = 7731.3x + 54859
0.9977
5.09–5090
3.56
1.19Table 4
Precision, stability, repeatability, and accuracy of the investigated analytes (n=6).
Number
Compound
Precision
Stability RSD (%)
Repeatability RSD (%)
Accuracy
Intraday RSD (%)
Interday RSD (%)
Recovery (%)
RSD (%)
1
Tyr
1.36
1.85
0.94
3.05
98.56
1.33
2
Ala
2.60
3.31
3.47
3.09
102.49
2.18
3
Glu
2.07
2.56
2.10
2.11
106.94
3.76
4
Phe
0.66
1.03
2.60
1.66
97.23
4.03
5
His
1.07
3.30
1.58
1.17
98.32
2.20
6
Ile
0.97
3.21
3.82
0.71
99.09
3.57
7
Asp
1.96
3.98
2.42
1.84
97.86
2.36
8
Lys
2.70
3.51
1.44
0.99
102.30
3.02
9
Gly
1.66
2.65
3.43
3.74
104.48
3.07
10
Val
2.42
2.89
3.30
0.83
107.51
1.52
11
Cit
1.47
1.88
3.12
3.86
99.14
2.21
12
GABA
3.14
2.32
2.27
0.82
102.54
3.41
13
Cys2
2.53
4.27
3.24
1.49
96.60
1.27
14
Pro
1.21
3.39
1.09
2.09
101.94
3.20
15
Trp
0.37
3.93
1.36
1.39
99.11
3.46
16
Leu
1.50
3.90
3.40
1.50
95.27
3.61
17
Arg
2.72
4.15
3.72
1.45
102.37
1.79
18
Cys
2.82
3.63
1.16
0.53
103.65
3.82
19
Hpro
3.05
2.23
0.87
2.38
97.55
3.91
20
Met
1.57
3.02
1.26
0.52
104.89
2.14
21
Thr
2.11
2.13
1.77
3.01
95.97
3.00
22
Asn
2.93
2.85
1.48
3.68
105.35
2.95
23
Gln
0.97
1.87
1.22
2.72
102.33
2.16
24
Ser
2.71
2.86
0.98
1.48
97.44
1.88


## 3.5. Quantitative Analysis for Samples

The developed UPLC-MS/MS method was applied for simultaneous determination of 24 free amino acids in 59 batches of three species of duckweed. All the contents are displayed in Table5. The results showed that the total amount of amino acids varied dramatically among each sample from different regions and species. Total content of AAs in SP25 from Baoding City of Hebei Province reached as high as 1899.50 μg/g, which was much more than that in SP17 of 166.05 μg/g from Lianyungang City of Jiangsu Province. For each amino acid, the content also varied significantly even in the same sample. Coincidentally, the contents of free amino acids that include sulfur in their molecules were much less than others, including cysteine, cystine, and methionine with the concentration of 0.24, 4.25, and 4.20 μg/g in maximum, respectively. Especially for cysteine, it was not found in all of Spirodela polyrhiza and only detected in several samples of other two species. The putative reason was that the thiol in cysteine was not stable and easily oxidized. But in general, there was no significant difference observed in the total content of all FAAs among different species.Table 5
Contents of 24 free amino acids, total amino acids, total proteins (μg/g), and ratio of free to total amino acids (%) in three species of duckweed.
Sample
Tyr
Ala
Glu
Phe
His
Ile
Asp
Lys
Gly
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
SP1
3.16
6259.48
0.0505
6.95
10680.18
0.0651
25.27
16774.31
0.1506
0.44
9597.56
0.0046
1.52
4255.26
0.0357
0.38
8532.07
0.0045
9.31
11169.49
0.0834
11.95
7164.09
0.1668
3.33
8764.89
0.0380
SP2
6.73
6161.25
0.1092
65.17
10089.59
0.6459
54.61
25366.04
0.2153
0.95
10729.73
0.0089
4.43
3888.26
0.1139
1.35
10094.12
0.0134
56.68
15862.58
0.3573
21.28
10090.45
0.2109
8.38
8695.46
0.0964
SP3
39.72
6115.87
0.6495
173.11
9881.22
1.7519
96.43
15463.41
0.6236
7.97
8667.06
0.0920
22.26
3824.22
0.5821
11.90
7226.86
0.1647
69.12
12387.34
0.5580
112.90
8153.11
1.3847
128.27
6293.65
2.0381
SP4
29.21
6251.45
0.4673
157.69
12424.62
1.2692
49.52
22986.51
0.2154
5.01
10467.29
0.0479
32.80
3569.79
0.9188
11.14
8392.28
0.1327
170.28
17175.49
0.9914
137.48
10225.20
1.3445
78.17
9626.14
0.8121
SP5
5.58
6257.00
0.0892
29.04
13866.45
0.2094
34.93
23518.46
0.1485
0.86
11427.08
0.0075
6.41
4312.95
0.1486
0.96
9753.24
0.0098
16.39
18654.70
0.0879
21.25
11094.76
0.1915
5.53
10174.01
0.0544
SP6
27.42
6245.68
0.4390
273.35
12005.84
2.2768
49.09
18787.83
0.2613
2.55
10284.72
0.0248
45.46
3424.75
1.3274
13.55
8465.53
0.1601
132.84
15752.94
0.8433
139.27
8608.52
1.6178
77.36
7010.22
1.1035
SP7
39.61
5884.11
0.6732
229.20
12123.87
1.8905
71.86
18146.81
0.3960
4.90
8585.01
0.0571
46.17
4088.80
1.1292
11.22
7231.96
0.1551
94.84
14833.85
0.6393
230.52
7636.34
3.0187
65.35
5417.04
1.2064
SP8
5.71
6124.81
0.0932
38.00
9734.57
0.3904
53.14
17640.33
0.3012
1.17
8527.84
0.0137
4.56
3751.52
0.1216
1.40
7266.84
0.0193
42.82
14315.75
0.2991
22.77
7392.06
0.3080
5.63
7832.50
0.0719
SP9
13.90
6054.66
0.2296
120.58
9953.18
1.2115
70.75
16620.79
0.4257
3.68
8384.54
0.0439
18.01
3496.99
0.5150
5.25
6745.54
0.0778
94.10
11606.55
0.8107
73.99
6323.41
1.1701
21.63
7899.86
0.2738
SP10
16.70
6097.17
0.2739
118.61
13272.16
0.8937
74.72
25451.33
0.2936
3.72
11056.99
0.0336
28.19
4110.01
0.6859
4.41
9671.14
0.0456
36.11
17757.11
0.2034
70.81
8546.75
0.8285
25.55
7505.72
0.3404
SP11
23.10
6004.10
0.3847
205.08
11155.38
1.8384
67.31
26088.53
0.2580
10.26
9883.88
0.1038
61.35
3238.60
1.8943
14.23
8891.25
0.1600
56.14
15601.46
0.3598
133.66
8386.70
1.5937
87.43
9088.41
0.9620
SP12
9.38
6127.68
0.1531
93.06
9760.92
0.9534
51.58
26516.14
0.1945
7.60
7997.37
0.0950
31.87
3300.60
0.9656
4.09
9260.26
0.0442
225.20
12413.46
1.8142
74.86
5684.24
1.3170
24.76
6908.86
0.3584
SP13
93.61
6130.06
1.5271
195.69
8956.55
2.1849
52.85
16377.44
0.3227
23.33
7668.17
0.3042
46.78
2889.26
1.6191
14.85
7650.07
0.1941
4.76
10136.24
0.0470
135.91
6635.82
2.0481
73.19
5663.18
1.2924
SP14
19.98
6120.49
0.3264
161.86
13337.15
1.2136
96.09
15114.77
0.6357
5.94
10678.41
0.0556
26.03
4189.64
0.6213
6.56
9933.71
0.0660
29.20
13225.32
0.2208
102.55
11141.92
0.9204
34.50
7778.37
0.4435
SP15
1.13
6057.44
0.0187
19.99
13617.28
0.1468
17.01
21045.25
0.0808
0.62
11056.19
0.0056
9.77
4213.32
0.2319
0.30
8452.34
0.0035
0.95
12630.34
0.0075
17.70
8018.34
0.2207
6.64
6872.17
0.0966
SP16
61.29
5884.22
1.0416
280.92
12551.36
2.2382
46.82
20775.77
0.2254
19.27
10311.88
0.1869
54.60
3881.89
1.4065
20.18
9290.92
0.2172
112.81
13791.37
0.8180
158.24
7293.79
2.1695
52.41
8307.13
0.6309
SP17
0.89
5938.26
0.0150
23.96
10170.30
0.2356
46.90
15656.17
0.2996
0.42
10562.87
0.0040
2.49
5358.17
0.0465
1.11
10201.54
0.0109
2.95
12263.36
0.0241
13.08
8069.24
0.1621
4.44
5669.36
0.0783
SP18
12.28
5985.32
0.2052
157.75
13673.70
1.1537
81.27
15272.53
0.5321
15.58
10449.06
0.1491
28.93
4949.42
0.5845
10.04
9948.89
0.1009
85.38
20641.51
0.4136
79.39
11463.15
0.6926
29.19
6848.95
0.4262
SP19
45.66
5897.55
0.7742
182.98
12217.80
1.4977
87.43
20888.93
0.4185
28.84
10128.19
0.2847
48.02
4231.77
1.1347
20.85
9161.44
0.2276
10.22
14574.99
0.0701
83.12
9060.52
0.9174
34.17
6664.53
0.5127
SP20
28.03
5860.01
0.4783
241.91
9302.34
2.6005
46.17
14837.19
0.3112
23.18
7603.97
0.3048
64.10
3206.91
1.9988
14.80
6774.94
0.2185
109.03
11544.71
0.9444
164.87
6737.28
2.4471
73.15
6715.81
1.0892
SP21
2.75
6264.80
0.0439
55.55
10989.25
0.5055
91.79
22493.65
0.4081
0.84
9615.10
0.0087
23.80
4257.07
0.5591
1.00
8364.75
0.0120
—
11442.81
—
31.30
6932.76
0.4515
9.46
6910.06
0.1369
SP22
7.95
5839.15
0.1361
29.20
10343.17
0.2823
22.86
16692.99
0.1369
1.07
8957.29
0.0119
5.17
3585.28
0.1442
1.06
8011.79
0.0132
0.91
13590.39
0.0067
15.15
6361.56
0.2381
11.21
7059.65
0.1588
SP23
6.86
6265.35
0.1095
68.62
11510.74
0.5961
100.28
20064.52
0.4998
2.53
9584.25
0.0264
32.22
3573.51
0.9016
1.84
7753.50
0.0237
33.31
15126.56
0.2202
42.69
7622.22
0.5601
11.21
8763.05
0.1279
SP24
2.86
6318.22
0.0453
121.06
11508.52
1.0519
133.69
16946.16
0.7889
1.99
9477.13
0.0210
25.81
4474.44
0.5768
2.27
7926.91
0.0286
47.91
15776.45
0.3037
30.02
7939.55
0.3781
10.04
8867.42
0.1132
SP25
43.24
5967.19
0.7246
261.74
11024.75
2.3741
53.23
21609.69
0.2463
16.89
9293.80
0.1817
64.22
4222.87
1.5208
20.92
9560.51
0.2188
162.59
18624.39
0.8730
209.44
7299.84
2.8691
63.50
7917.96
0.8020
LP1
15.93
5971.26
0.2668
66.50
12151.90
0.5472
30.75
24958.79
0.1232
11.80
10826.10
0.1090
35.08
4061.19
0.8638
4.88
9319.60
0.0524
108.30
14896.35
0.7270
47.16
8613.79
0.5475
14.76
9583.97
0.1540
LP2
38.97
5921.55
0.6581
140.81
11584.07
1.2155
63.83
21969.13
0.2905
15.15
9814.43
0.1544
30.41
4647.49
0.6543
2.61
8750.97
0.0298
59.96
16728.80
0.3584
134.72
7842.84
1.7177
30.64
6109.14
0.5015
LP3
10.25
6287.19
0.1630
65.63
11013.29
0.5959
55.16
18366.66
0.3003
2.58
10992.07
0.0235
16.14
3302.54
0.4887
0.65
8567.75
0.0076
28.45
14101.74
0.2017
40.15
6593.31
0.6090
7.10
4632.59
0.1533
LP4
47.76
6004.07
0.7955
180.48
11148.05
1.6189
83.06
18093.89
0.4590
7.65
9312.27
0.0821
48.49
3515.42
1.3794
9.18
8187.52
0.1121
103.28
15513.40
0.6657
94.68
8007.43
1.1824
74.60
8640.35
0.8634
LP5
74.45
6011.02
1.2386
318.14
9785.11
3.2513
50.05
16830.48
0.2974
17.43
8490.88
0.2053
38.14
3571.09
1.0680
11.66
7900.11
0.1476
218.70
13106.01
1.6687
129.60
8308.91
1.5598
44.93
5574.15
0.8060
LP6
22.44
6404.35
0.3504
187.93
9320.51
2.0163
51.11
19093.97
0.2677
10.76
10746.47
0.1001
17.66
4145.39
0.4260
4.29
8189.38
0.0524
137.49
13879.01
0.9906
57.61
6486.39
0.8882
13.94
6180.53
0.2255
LP7
17.40
6413.54
0.2713
167.19
10950.61
1.5268
73.42
20616.00
0.3561
10.75
9606.73
0.1119
20.94
3464.43
0.6044
2.23
8717.93
0.0256
207.21
15567.27
1.3311
51.72
8761.56
0.5903
14.60
6213.59
0.2350
LP8
35.02
6285.44
0.5572
139.27
11569.10
1.2038
72.38
18381.09
0.3938
18.51
10138.80
0.1826
28.90
4434.85
0.6517
7.46
9558.72
0.0780
73.30
15357.08
0.4773
78.65
7886.32
0.9973
24.80
8792.99
0.2820
LP9
41.17
5951.60
0.6917
149.55
11075.37
1.3503
48.55
22021.01
0.2205
9.10
11619.61
0.0783
52.62
3058.17
1.7206
12.36
8587.29
0.1439
182.44
15444.00
1.1813
202.10
10583.78
1.9095
176.76
6207.16
2.8477
LP10
23.74
6012.91
0.3948
187.15
10077.16
1.8572
57.20
22847.31
0.2504
3.20
10674.83
0.0300
11.62
3881.87
0.2993
6.47
10514.94
0.0615
48.04
19845.70
0.2421
111.09
8283.89
1.3410
107.73
7557.95
1.4254
LP11
14.86
6110.90
0.2432
94.14
9256.17
1.0171
16.70
26967.52
0.0619
5.60
7780.40
0.0720
7.52
3541.02
0.2124
3.81
7300.09
0.0522
6.45
13678.56
0.0472
75.66
7596.11
0.9960
46.74
6276.56
0.7447
LP12
8.06
6259.52
0.1288
25.80
10729.35
0.2405
11.61
28848.73
0.0402
1.45
9609.21
0.0151
4.31
4095.51
0.1052
0.93
8635.84
0.0108
2.44
11847.35
0.0206
29.23
8736.23
0.3346
7.00
7434.59
0.0942
LP13
33.36
6270.49
0.5320
274.21
10059.65
2.7258
85.36
26195.21
0.3259
5.22
11114.42
0.0470
44.10
3500.71
1.2597
8.74
9771.06
0.0894
78.86
17064.92
0.4621
148.06
8026.46
1.8446
92.39
7807.71
1.1833
LP14
29.35
6070.40
0.4835
160.58
13818.32
1.1621
57.00
27399.97
0.2080
5.18
12682.81
0.0408
38.41
5535.27
0.6939
9.59
11826.29
0.0811
139.52
14718.19
0.9479
158.90
7779.67
2.0425
85.79
7101.48
1.2081
LP15
14.08
6310.67
0.2231
108.54
12226.01
0.8878
11.43
20415.24
0.0560
4.29
10235.21
0.0419
6.83
4786.65
0.1427
3.49
10163.51
0.0343
4.42
16813.25
0.0263
70.82
8454.76
0.8376
57.87
8826.59
0.6556
LP16
38.10
6308.99
0.6039
224.42
10920.13
2.0551
58.19
18915.27
0.3076
12.98
9252.57
0.1403
25.58
3501.54
0.7305
6.06
8361.97
0.0725
187.80
14243.86
1.3185
79.64
8381.28
0.9502
24.49
7349.44
0.3332
LA1
39.53
5844.15
0.6764
153.40
11080.16
1.3845
53.94
17900.19
0.3013
5.26
8940.22
0.0588
38.98
3436.65
1.1342
10.44
7739.17
0.1349
92.27
11396.06
0.8097
116.20
8514.88
1.3647
16.63
8366.72
0.1988
LA2
7.06
6163.58
0.1145
110.58
11359.90
0.9734
38.25
21707.93
0.1762
8.65
9105.55
0.0950
45.78
3419.43
1.3388
10.95
9140.90
0.1198
84.67
16077.78
0.5266
87.09
8669.05
1.0046
14.22
7375.81
0.1928
LA3
49.51
6127.32
0.8080
189.37
10480.08
1.8070
47.31
23969.23
0.1974
23.46
8251.42
0.2843
73.17
3213.54
2.2769
30.13
8053.11
0.3741
190.71
18067.61
1.0555
206.67
10157.90
2.0346
118.69
11012.41
1.0778
LA4
9.45
6231.43
0.1517
101.15
10764.28
0.9397
43.93
18445.78
0.2382
6.83
9106.47
0.0750
5.48
3968.13
0.1381
2.07
7183.48
0.0288
14.98
11095.78
0.1350
44.31
8400.33
0.5275
18.02
5093.33
0.3538
LA5
8.21
6468.09
0.1269
101.70
10782.93
0.9432
49.41
17857.46
0.2767
4.67
9378.50
0.0498
27.88
4320.50
0.6453
7.67
8732.91
0.0878
245.22
11348.94
2.1607
101.42
9642.82
1.0518
59.10
7589.10
0.7787
LA6
95.03
6441.94
1.4752
53.56
10969.89
0.4882
45.63
19113.36
0.2387
34.05
8826.04
0.3858
22.67
3432.16
0.6605
15.73
7873.91
0.1998
194.49
13395.53
1.4519
203.37
9453.82
2.1512
165.17
5220.68
3.1638
LA7
43.57
6390.61
0.6818
136.48
9697.57
1.4074
44.59
17227.16
0.2588
26.85
10617.54
0.2529
53.25
3342.84
1.5930
8.20
8078.65
0.1015
99.41
13308.10
0.7470
156.91
8444.81
1.8581
124.79
6172.25
2.0218
LA8
25.51
5969.99
0.4273
159.29
9551.72
1.6677
21.69
22493.72
0.0964
7.29
12500.69
0.0583
28.21
5068.31
0.5566
12.52
10500.36
0.1192
2.23
19936.15
0.0112
66.00
11282.00
0.5850
75.19
11732.39
0.6409
LA9
3.26
6188.23
0.0527
19.10
14974.86
0.1275
71.65
23929.16
0.2994
0.84
12303.21
0.0068
3.65
4847.99
0.0753
0.36
10895.91
0.0033
2.47
21371.14
0.0116
18.43
10548.00
0.1747
7.33
7698.82
0.0952
LA10
29.15
5884.38
0.4954
95.43
11229.43
0.8498
89.66
20489.06
0.4376
16.96
9051.65
0.1874
21.61
3412.09
0.6333
16.06
8721.17
0.1841
2.82
17303.92
0.0163
74.14
8057.43
0.9201
27.50
5058.68
0.5436
LA11
10.88
6259.53
0.1738
40.56
13131.47
0.3089
121.99
22020.10
0.5540
2.48
10555.63
0.0235
5.92
4847.60
0.1221
0.48
9784.56
0.0049
7.09
16593.97
0.0427
26.86
7655.07
0.3509
7.73
7284.80
0.1061
LA12
48.02
6403.75
0.7499
149.63
9735.88
1.5369
62.74
14671.39
0.4276
20.20
8380.51
0.2410
56.05
3384.94
1.6559
5.41
6896.77
0.0784
91.38
11321.11
0.8072
109.18
5802.35
1.8817
46.13
7209.85
0.6398
LA13
22.08
5983.25
0.3690
77.34
12359.55
0.6258
49.89
24426.48
0.2042
13.64
10650.97
0.1281
31.43
4286.46
0.7332
3.68
8877.22
0.0415
184.36
14885.17
1.2385
77.19
10677.05
0.7230
23.24
10012.96
0.2321
LA14
29.06
6197.60
0.4689
219.41
12097.18
1.8137
68.78
22967.64
0.2995
7.41
10651.10
0.0696
33.00
4079.19
0.8090
10.50
8376.22
0.1254
21.77
16404.09
0.1327
122.70
8502.69
1.4431
92.02
9152.03
1.0055
LA15
28.90
6409.17
0.4509
11.61
12035.67
0.0965
43.15
25371.86
0.1701
5.48
11118.01
0.0493
35.81
3597.27
0.9955
6.72
6464.97
0.1039
159.51
12763.92
1.2497
120.12
7008.78
1.7139
55.67
5676.13
0.9808
LA16
20.91
6243.38
0.3349
95.37
11719.51
0.8138
41.47
21759.15
0.1906
2.48
11669.90
0.0213
27.93
5249.47
0.5321
5.48
10752.39
0.0510
113.55
14828.06
0.7658
123.52
9586.25
1.2885
34.37
10915.62
0.3149
LA17
33.89
6029.51
0.5621
12.57
12627.27
0.0995
65.10
23746.45
0.2741
13.69
10538.29
0.1299
34.97
4468.75
0.7825
7.61
9975.36
0.0763
117.69
13313.53
0.8840
122.84
9248.08
1.3283
36.44
8620.93
0.4227
LA18
29.56
6046.27
0.4889
137.19
10245.05
1.3391
54.86
18325.38
0.2994
10.74
8928.25
0.1203
48.27
3846.06
1.2551
6.44
7467.00
0.0862
91.91
12882.36
0.7135
125.43
7366.73
1.7027
36.87
4820.33
0.7649
Sample
Val
Cit
GABA
Cys2
Pro
Trp
Leu
Arg
Cys
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
SP1
4.42
6012.65
0.0735
2.49
4673.88
0.0533
3.46
3353.11
0.1032
—
11525.08
—
4.15
6607.74
0.0628
0.24
3637.74
0.0066
1.37
12211.14
0.0112
34.88
9489.73
0.3676
—
5538.05
—
SP2
15.20
10648.71
0.1427
6.07
3955.21
0.1535
21.27
4028.12
0.5280
—
13123.45
—
12.90
6341.28
0.2034
0.51
6868.79
0.0074
4.08
11230.44
0.0363
87.38
8979.88
0.9731
—
8675.28
—
SP3
15.36
6327.29
0.2428
24.59
4218.39
0.5829
83.54
2906.04
2.8747
0.67
9520.68
0.0070
27.55
7911.24
0.3482
2.67
6256.52
0.0427
33.86
9531.17
0.3553
68.39
8667.37
0.7891
—
3752.42
—
SP4
16.09
10136.97
0.1587
9.45
4525.17
0.2088
112.07
1860.38
6.0240
0.71
8713.26
0.0081
22.64
9816.87
0.2306
3.14
6886.81
0.0456
25.37
11900.95
0.2132
41.42
7830.13
0.5290
—
5020.00
—
SP5
12.81
12270.45
0.1044
3.68
5960.38
0.0617
9.31
2163.00
0.4304
—
11194.54
—
5.92
8973.82
0.0660
0.39
6741.47
0.0058
3.03
17710.97
0.0171
75.96
11624.78
0.6534
—
8152.58
—
SP6
18.63
6921.90
0.2691
54.08
6475.87
0.8351
110.08
2010.87
5.4742
0.26
9803.15
0.0027
28.19
7126.12
0.3956
4.73
3570.11
0.1325
26.75
15586.82
0.1716
43.86
5689.69
0.7709
—
3789.97
—
SP7
17.07
9259.39
0.1844
29.75
4140.35
0.7185
103.95
2825.91
3.6785
2.41
9778.44
0.0246
24.62
8226.72
0.2993
3.95
4856.66
0.0813
27.66
10861.08
0.2547
34.40
4988.56
0.6896
—
3471.13
—
SP8
15.86
7296.00
0.2174
3.13
4702.29
0.0666
20.48
1682.03
1.2176
—
6139.57
—
10.52
6680.26
0.1575
0.56
4883.67
0.0115
4.38
9640.58
0.0454
64.28
4886.46
1.3155
—
3120.50
—
SP9
37.20
6514.46
0.5710
10.18
5254.55
0.1937
57.02
2501.97
2.2790
0.14
10056.41
0.0014
15.77
5340.61
0.2953
1.20
4253.18
0.0282
14.42
11713.99
0.1231
124.64
5090.33
2.4486
—
3897.56
—
SP10
38.29
9908.44
0.3864
13.50
7105.23
0.1900
57.85
1953.05
2.9620
0.26
10687.68
0.0024
19.03
7381.05
0.2578
1.17
7036.56
0.0166
13.77
14235.69
0.0967
108.51
4268.65
2.5420
—
8653.16
—
SP11
21.90
10089.65
0.2171
8.30
5747.51
0.1444
108.20
3155.71
3.4287
0.79
9550.72
0.0083
24.66
5804.81
0.4248
1.94
7077.68
0.0274
26.89
14118.06
0.1905
67.72
5222.67
1.2967
—
6165.20
—
SP12
36.91
5104.20
0.7231
31.74
3267.50
0.9714
56.19
3092.80
1.8168
0.58
8821.94
0.0066
11.01
6511.44
0.1691
2.35
4185.92
0.0561
10.46
8633.77
0.1212
99.42
6433.17
1.5454
—
2940.84
—
SP13
27.76
5423.78
0.5118
27.14
3816.49
0.7111
82.12
2484.15
3.3058
0.99
6828.14
0.0145
22.06
7524.55
0.2932
11.42
3149.92
0.3625
31.25
8945.92
0.3493
73.82
5010.51
1.4733
—
6503.52
—
SP14
44.12
10896.32
0.4049
11.57
6328.11
0.1828
70.23
4220.23
1.6641
0.28
9165.62
0.0031
21.63
8005.88
0.2702
2.44
4732.57
0.0516
18.29
13035.53
0.1403
65.62
6826.41
0.9613
—
8677.59
—
SP15
6.17
7444.35
0.0829
5.45
3756.96
0.1451
4.23
3324.82
0.1272
—
10198.26
—
14.42
5416.47
0.2662
—
5143.26
—
1.80
14814.40
0.0122
30.19
5059.02
0.5968
—
6694.08
—
SP16
26.04
6396.60
0.4071
36.82
3762.26
0.9787
134.46
3870.97
3.4735
0.72
8704.16
0.0083
27.10
7353.81
0.3685
15.39
7545.48
0.2040
30.79
12588.03
0.2446
43.32
6483.46
0.6682
—
4369.57
—
SP17
9.24
8969.61
0.1030
4.12
3703.39
0.1112
2.34
3148.92
0.0743
—
8001.88
—
14.78
8403.61
0.1759
—
4014.56
—
5.35
10274.36
0.0521
9.96
4876.50
0.2042
—
6360.07
—
SP18
26.19
12386.71
0.2114
26.35
5209.87
0.5058
98.88
3534.87
2.7973
0.12
12402.18
0.0010
17.21
9211.57
0.1868
2.08
7798.78
0.0267
22.77
17831.20
0.1277
108.35
4662.91
2.3237
—
7665.04
—
SP19
25.16
8312.52
0.3027
8.51
4849.89
0.1755
73.42
1868.93
3.9285
0.28
8968.53
0.0031
17.64
8754.89
0.2015
3.68
5290.70
0.0696
16.70
11607.35
0.1439
80.47
7406.94
1.0864
—
7063.71
—
SP20
25.85
8395.03
0.3079
17.22
4586.93
0.3754
131.28
2873.62
4.5685
0.40
8894.24
0.0045
18.50
4472.47
0.4136
9.86
3888.62
0.2536
24.40
8829.37
0.2764
42.54
4662.51
0.9124
—
4657.46
—
SP21
11.27
5982.04
0.1884
24.90
4366.50
0.5703
10.82
3551.90
0.3046
0.24
8933.72
0.0027
28.86
6002.68
0.4808
0.15
6460.55
0.0023
3.89
12300.36
0.0316
55.98
5963.67
0.9387
—
5955.22
—
SP22
10.06
9469.47
0.1062
5.09
5545.62
0.0918
1.90
1902.37
0.0999
—
10023.81
—
18.22
5611.46
0.3247
0.14
3261.78
0.0043
9.76
12591.58
0.0775
18.52
8217.10
0.2254
—
6884.34
—
SP23
19.80
6941.68
0.2852
35.46
4560.09
0.7776
10.96
2519.87
0.4349
0.31
11078.52
0.0028
25.31
7474.12
0.3386
0.55
4776.94
0.0115
5.34
9323.61
0.0573
75.70
8379.33
0.9034
—
4296.33
—
SP24
24.68
7991.34
0.3088
29.19
6319.28
0.4619
4.94
3668.10
0.1347
0.09
10178.72
0.0009
23.29
5293.61
0.4400
0.60
4551.39
0.0132
3.83
14205.93
0.0270
58.04
6170.55
0.9406
—
5644.77
—
SP25
28.45
7459.65
0.3814
46.15
5088.78
0.9069
163.48
3897.18
4.1948
1.16
11215.85
0.0103
24.67
7584.57
0.3253
7.63
7080.31
0.1078
35.40
14162.74
0.2500
74.34
9656.72
0.7698
—
6166.09
—
LP1
21.36
7244.74
0.2948
5.16
4405.39
0.1171
58.37
2407.83
2.4242
0.13
13065.90
0.0010
11.76
8651.28
0.1359
3.62
5123.83
0.0707
13.93
10764.87
0.1294
30.24
8884.37
0.3404
—
4324.94
—
LP2
14.46
7373.76
0.1961
16.36
4874.65
0.3356
40.10
2138.03
1.8756
0.77
7988.91
0.0096
14.86
8851.40
0.1679
14.60
3817.53
0.3824
15.17
13357.73
0.1136
26.71
9701.11
0.2753
—
7977.29
—
LP3
9.83
8734.76
0.1125
60.38
3744.80
1.6124
14.14
2099.63
0.6735
—
9725.45
—
18.43
4540.45
0.4059
0.45
4728.65
0.0095
3.52
9822.04
0.0358
74.16
5810.06
1.2764
—
3530.69
—
LP4
35.80
9919.45
0.3609
3.79
3526.80
0.1075
122.04
2438.49
5.0047
0.37
9387.49
0.0039
18.34
8296.58
0.2211
1.14
5349.26
0.0213
22.21
9157.07
0.2425
48.84
8460.67
0.5773
—
6861.56
—
LP5
35.99
8871.65
0.4057
11.90
4587.62
0.2594
133.05
3159.85
4.2106
4.21
7298.47
0.0577
25.23
5061.04
0.4985
4.34
5510.02
0.0788
31.89
11224.14
0.2841
45.88
6939.12
0.6612
—
3854.06
—
LP6
24.78
7215.09
0.3434
9.73
4416.05
0.2203
91.48
2092.89
4.3710
2.32
7641.78
0.0304
14.71
6655.83
0.2210
5.76
3078.31
0.1871
13.10
10225.91
0.1281
77.03
6842.51
1.1258
—
3272.62
—
LP7
18.41
6396.21
0.2878
10.84
4275.51
0.2535
64.21
2197.40
2.9221
4.25
11232.05
0.0378
9.15
8097.10
0.1130
4.71
3289.73
0.1432
11.93
9991.64
0.1194
30.66
7921.66
0.3870
0.14
6607.63
0.0021
LP8
23.88
7401.70
0.3226
16.38
4758.26
0.3442
69.10
3565.33
1.9381
0.55
8739.92
0.0063
14.73
8982.82
0.1640
2.82
4541.13
0.0621
24.29
16149.82
0.1504
31.42
6643.33
0.4730
—
5673.14
—
LP9
22.74
8090.74
0.2811
32.17
6784.16
0.4742
95.67
3874.88
2.4690
0.34
11756.40
0.0029
25.48
9895.35
0.2575
3.59
4755.18
0.0755
30.55
11213.15
0.2724
118.53
7876.17
1.5049
0.22
4743.85
0.0046
LP10
44.70
10435.87
0.4283
15.47
3862.27
0.4005
82.77
2085.51
3.9688
0.95
13573.29
0.0070
21.56
8888.77
0.2426
0.77
7378.31
0.0104
16.31
14411.77
0.1132
63.57
9539.07
0.6664
0.14
8866.38
0.0016
LP11
30.18
7118.23
0.4240
10.12
3840.01
0.2635
38.62
2188.18
1.7649
0.39
9579.85
0.0041
14.31
6477.73
0.2209
0.56
4389.22
0.0128
9.34
10905.75
0.0856
74.49
5696.30
1.3077
—
5334.87
—
LP12
10.67
9073.21
0.1176
9.45
4795.94
0.1970
6.74
2798.07
0.2409
—
9754.10
—
10.14
5687.18
0.1783
0.22
6220.07
0.0035
2.99
11120.12
0.0269
89.48
9822.46
0.9110
—
7787.85
—
LP13
24.43
7986.97
0.3059
10.20
5208.39
0.1958
105.99
3587.82
2.9542
1.19
10899.43
0.0109
25.32
9578.40
0.2643
2.01
6255.14
0.0321
24.85
10572.72
0.2350
76.46
7230.87
1.0574
—
8350.79
—
LP14
21.85
12895.61
0.1694
10.61
6247.35
0.1698
114.88
3385.86
3.3929
0.65
12941.50
0.0050
24.21
10165.28
0.2382
0.42
5801.66
0.0072
24.35
12767.46
0.1907
38.76
9267.96
0.4182
0.10
4749.52
0.0021
LP15
30.04
10600.31
0.2834
8.02
6112.10
0.1312
39.61
2063.22
1.9198
0.26
10874.29
0.0024
14.42
6109.32
0.2360
0.63
7137.55
0.0088
7.68
12394.41
0.0620
71.29
6514.00
1.0944
—
4351.01
—
LP16
26.39
5728.98
0.4606
10.83
4203.58
0.2576
96.25
3176.63
3.0299
3.59
6782.15
0.0529
16.36
8733.17
0.1873
4.94
6147.21
0.0804
18.97
9376.15
0.2023
51.19
8498.90
0.6023
0.14
5213.83
0.0027
LA1
45.00
5702.67
0.7891
13.87
3437.80
0.4035
97.36
3259.71
2.9868
0.34
9934.14
0.0034
16.88
5375.18
0.3140
5.39
5649.12
0.0954
25.80
11813.05
0.2184
46.47
7148.37
0.6501
—
6609.48
—
LA2
19.62
10334.80
0.1898
28.58
3747.09
0.7627
81.64
2390.45
3.4153
0.86
7845.80
0.0110
12.38
8095.98
0.1529
2.57
5265.52
0.0488
23.74
15177.45
0.1564
33.03
7150.11
0.4620
—
5064.86
—
LA3
39.08
11531.59
0.3389
11.17
7658.73
0.1458
96.78
3420.33
2.8296
0.34
9648.03
0.0035
25.22
6740.71
0.3741
14.39
6990.99
0.2058
28.29
16947.54
0.1669
35.87
11157.91
0.3215
0.12
5317.49
0.0023
LA4
30.49
9094.55
0.3353
5.75
3337.54
0.1723
59.10
1960.88
3.0140
—
7030.26
—
21.76
7834.00
0.2778
1.38
6225.95
0.0222
12.30
8279.16
0.1486
67.99
6933.19
0.9806
—
5458.32
—
LA5
41.41
10636.04
0.3893
53.62
5018.49
1.0684
47.11
3686.56
1.2779
1.53
7468.47
0.0205
28.74
6068.81
0.4736
1.29
4893.15
0.0264
22.91
11517.82
0.1989
104.70
5679.74
1.8434
—
6326.99
—
LA6
26.86
10152.01
0.2646
10.02
5879.06
0.1704
109.90
3477.43
3.1604
0.66
10000.06
0.0066
23.06
5135.21
0.4491
7.12
3736.03
0.1906
33.26
15229.07
0.2184
38.54
6481.81
0.5946
0.24
5289.57
0.0045
LA7
37.39
5299.21
0.7056
7.21
4582.47
0.1573
137.64
3164.85
4.3490
—
6820.33
—
25.78
4508.55
0.5718
2.81
5062.03
0.0555
30.16
14035.50
0.2149
37.11
8557.89
0.4336
—
6719.24
—
LA8
33.07
10470.63
0.3158
14.37
6157.76
0.2334
26.46
2514.35
1.0524
—
12359.55
—
27.27
8147.42
0.3347
3.27
7846.61
0.0417
18.76
12842.68
0.1461
88.93
8060.89
1.1032
—
4516.77
—
LA9
3.40
12345.60
0.0275
9.98
5675.92
0.1758
2.04
4742.21
0.0430
—
15127.20
—
6.76
10585.00
0.0639
0.29
8961.94
0.0032
1.34
12900.12
0.0104
13.42
10716.38
0.1252
—
5820.67
—
LA10
32.37
8506.23
0.3805
15.12
4869.46
0.3105
40.05
3515.47
1.1393
0.11
11225.08
0.0010
15.86
4962.73
0.3196
1.40
6261.69
0.0224
8.88
9143.75
0.0971
86.86
5400.65
1.6083
—
7815.34
—
LA11
5.06
11187.35
0.0452
16.69
6130.94
0.2722
2.35
2533.92
0.0927
0.36
13012.18
0.0028
5.43
10083.36
0.0539
0.97
5607.42
0.0173
1.87
15543.78
0.0120
7.40
7751.63
0.0955
—
4413.68
—
LA12
38.36
6567.71
0.5841
37.15
4331.21
0.8577
73.93
1638.83
4.5111
0.13
10001.78
0.0013
19.64
5219.17
0.3763
1.98
3334.61
0.0594
16.66
13924.60
0.1196
43.90
4477.46
0.9805
—
3708.31
—
LA13
33.49
9334.49
0.3588
21.25
5427.96
0.3915
57.89
3900.56
1.4841
0.15
11927.09
0.0013
18.94
7570.98
0.2502
1.52
3832.52
0.0397
14.66
14801.24
0.0990
130.27
9104.06
1.4309
—
4437.40
—
LA14
48.22
9862.78
0.4889
10.25
5078.48
0.2018
68.84
1820.89
3.7806
0.18
12167.89
0.0015
20.49
9331.57
0.2196
3.12
6487.16
0.0481
27.88
15343.15
0.1817
73.66
5754.34
1.2801
—
4845.05
—
LA15
33.62
4870.39
0.6903
38.52
4318.57
0.8920
46.08
2538.52
1.8152
—
8893.47
—
18.06
5895.62
0.3063
0.58
3366.22
0.0172
18.67
7744.33
0.2411
97.99
6807.78
1.4394
0.19
6361.27
0.0030
LA16
28.73
10676.60
0.2691
17.16
5956.75
0.2881
30.34
4234.41
0.7165
0.10
10936.85
0.0009
20.90
7579.89
0.2757
1.06
6621.76
0.0160
17.03
15351.32
0.1109
39.65
7199.84
0.5507
0.16
4666.63
0.0034
LA17
25.33
11534.16
0.2196
8.43
5093.69
0.1655
57.74
3692.35
1.5638
0.41
10297.60
0.0040
12.50
7491.18
0.1669
4.43
3719.53
0.1191
16.02
17696.48
0.0905
32.56
9782.67
0.3328
—
8946.44
—
LA18
24.44
9269.41
0.2637
7.67
3801.25
0.2018
56.82
3179.11
1.7873
0.60
9490.75
0.0063
15.74
6342.64
0.2482
2.92
3811.54
0.0766
15.43
11215.99
0.1376
37.22
8358.76
0.4453
—
3946.18
—
Sample
Hpro
Met
Thr
Asn
Gln
Ser
Total free amino acids
Total proteins (%)
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
Free
Total
Ratio
SP1
0.34
12275.50
0.0028
—
2102.26
—
12.78
7963.72
0.1605
39.82
3834.80
1.0384
1.04
1971.45
0.0528
4.76
6466.09
0.0736
172.07
16.19
SP2
0.84
11585.53
0.0073
—
1927.32
—
38.01
7239.98
0.5250
215.37
4986.79
4.3188
3.20
2096.01
0.1527
22.43
5929.40
0.3783
646.85
18.88
SP3
10.37
9975.53
0.1040
4.20
1384.33
0.3034
157.92
4945.98
3.1929
133.70
3711.29
3.6025
68.98
1951.23
3.5352
144.02
5954.58
2.4186
1437.51
15.19
SP4
3.50
12741.33
0.0275
—
1958.62
—
168.24
8709.13
1.9318
139.13
4253.91
3.2706
143.70
2036.62
7.0558
163.42
5609.49
2.9133
1520.16
17.82
SP5
0.48
15829.31
0.0030
—
2151.19
—
34.91
8355.57
0.4178
258.84
4912.45
5.2691
3.06
2256.83
0.1356
16.69
10234.90
0.1631
546.01
20.46
SP6
6.53
12371.35
0.0528
—
1608.64
—
185.93
5726.31
3.2469
131.14
4602.59
2.8493
53.20
2164.53
2.4578
144.62
8041.81
1.7984
1568.88
17.51
SP7
3.37
12423.61
0.0271
0.10
1470.47
0.0068
168.48
4848.42
3.4749
137.82
3978.83
3.4638
115.64
2145.57
5.3897
153.92
8601.82
1.7894
1616.80
15.59
SP8
0.83
9760.19
0.0085
—
1320.98
—
36.00
5251.98
0.6855
243.18
3721.89
6.5338
4.33
2074.31
0.2087
23.02
7285.14
0.3160
601.76
14.41
SP9
2.33
10641.34
0.0219
0.10
1927.64
0.0052
101.08
6224.18
1.6240
287.73
3643.59
7.8969
22.11
2123.14
1.0414
80.72
7300.64
1.1057
1176.53
14.43
SP10
5.44
15070.02
0.0361
0.26
2233.47
0.0116
111.26
9030.91
1.2320
295.67
4158.01
7.1109
19.66
2820.23
0.6971
98.55
9877.31
0.9977
1162.05
19.14
SP11
11.23
13641.99
0.0823
0.28
1744.92
0.0160
176.59
7985.90
2.2113
140.18
4655.28
3.0112
144.55
2112.77
6.8417
179.12
6088.37
2.9420
1570.89
17.01
SP12
12.01
11162.90
0.1076
0.24
1881.32
0.0128
125.79
4403.32
2.8567
137.25
3369.96
4.0727
41.76
1600.68
2.6089
57.70
6605.01
0.8736
1145.82
14.13
SP13
2.39
10615.66
0.0225
3.51
1382.26
0.2539
228.15
6391.62
3.5695
165.25
4095.51
4.0349
269.51
1696.44
15.8868
168.80
6708.25
2.5163
1755.14
13.75
SP14
1.44
13205.06
0.0109
—
2393.23
—
132.66
8976.95
1.4778
149.56
4798.05
3.1171
125.79
2465.90
5.1012
129.14
7298.41
1.7694
1255.50
19.06
SP15
6.10
10366.51
0.0588
—
1814.22
—
15.94
7685.12
0.2074
22.02
4676.65
0.4708
6.45
2382.45
0.2707
7.22
5548.49
0.1301
194.11
15.85
SP16
7.67
12903.86
0.0594
0.23
2509.21
0.0092
85.06
5550.87
1.5324
142.06
4329.68
3.2811
176.62
3056.34
5.7788
145.74
5866.53
2.4843
1678.57
18.29
SP17
2.03
10626.57
0.0191
—
1794.68
—
10.28
5329.55
0.1929
3.24
3731.24
0.0868
2.10
2828.19
0.0743
6.37
4657.92
0.1368
166.05
15.26
SP18
3.88
12874.45
0.0301
0.25
2623.84
0.0095
124.83
7649.77
1.6318
148.44
4745.86
3.1278
123.11
3038.81
4.0513
102.24
8464.54
1.2079
1304.51
19.91
SP19
6.64
13923.42
0.0477
0.39
1611.21
0.0242
116.76
6748.38
1.7302
49.92
4539.22
1.0997
45.19
3232.29
1.3981
77.95
5525.06
1.4108
1063.99
17.50
SP20
1.26
8623.89
0.0146
0.17
1655.87
0.0103
184.54
4751.82
3.8836
152.52
3938.78
3.8723
120.92
2150.51
5.6229
176.26
7450.83
2.3656
1670.95
13.61
SP21
10.27
10862.57
0.0945
—
1789.85
—
19.55
7093.47
0.2756
38.03
4126.30
0.9216
7.72
2908.79
0.2654
11.94
6041.00
0.1976
460.11
16.55
SP22
3.58
11476.96
0.0312
0.36
1441.11
0.0250
11.15
5896.68
0.1891
3.63
3801.35
0.0955
8.88
2075.29
0.4279
6.66
7009.63
0.0950
192.55
15.30
SP23
6.80
11931.55
0.0570
—
1557.75
—
48.22
5634.77
0.8558
90.90
3869.18
2.3493
14.04
1927.16
0.7285
18.66
8290.65
0.2251
651.61
16.73
SP24
6.27
10593.16
0.0592
0.12
2127.69
0.0056
64.57
5101.21
1.2658
104.24
3837.96
2.7160
14.64
2823.06
0.5186
24.13
9012.82
0.2677
734.27
16.87
SP25
1.86
13824.44
0.0135
0.44
2716.54
0.0162
186.66
4842.06
3.8550
146.65
4187.28
3.5023
97.55
2771.87
3.5193
189.30
8595.86
2.2022
1899.50
19.76
LP1
0.47
14631.38
0.0032
1.18
1948.75
0.0606
65.83
6231.55
1.0564
95.95
4013.49
2.3907
86.11
3138.12
2.7440
85.73
5753.40
1.4901
814.99
18.47
LP2
0.64
12206.18
0.0052
1.85
2297.38
0.0805
78.50
5838.37
1.3446
145.43
4899.76
2.9681
52.18
2104.26
2.4797
73.92
6212.65
1.1898
1012.65
17.73
LP3
4.52
8885.71
0.0509
0.13
1858.23
0.0070
41.09
5789.60
0.7097
124.04
2882.55
4.3031
10.24
2456.95
0.4168
10.18
6286.97
0.1619
597.23
13.95
LP4
5.45
10112.04
0.0539
—
1630.58
—
163.52
7525.00
2.1730
143.69
3758.46
3.8231
51.80
1875.09
2.7625
160.80
7731.94
2.0797
1426.96
16.05
LP5
1.71
9028.04
0.0189
—
1621.37
—
152.81
4615.56
3.3108
134.30
3260.74
4.1187
112.30
1704.64
6.5879
122.16
4651.12
2.6265
1718.87
15.03
LP6
0.71
11053.96
0.0064
—
1379.86
—
85.21
6409.94
1.3293
270.66
3875.46
6.9839
136.75
1528.04
8.9494
72.78
6453.61
1.1277
1308.25
13.94
LP7
0.41
10437.44
0.0039
0.99
1616.03
0.0613
48.37
6713.60
0.7205
199.42
3616.48
5.5142
71.18
2426.57
2.9334
73.61
5273.31
1.3959
1113.74
16.11
LP8
0.60
12028.82
0.0050
2.77
2130.37
0.1300
64.52
8264.66
0.7807
93.71
4102.87
2.2840
128.62
3030.50
4.2442
77.21
8764.09
0.8810
1028.91
17.52
LP9
2.85
15395.17
0.0185
1.09
2611.93
0.0417
95.31
6079.19
1.5678
135.68
5800.10
2.3393
21.02
2847.50
0.7382
160.76
6714.80
2.3941
1620.64
20.09
LP10
3.15
12510.19
0.0252
0.19
2294.16
0.0083
139.99
5986.95
2.3383
316.13
4149.54
7.6184
20.34
2755.14
0.7383
126.41
9249.73
1.3666
1408.67
18.88
LP11
2.02
11304.83
0.0179
0.44
1616.02
0.0272
92.06
7087.95
1.2988
100.62
3134.85
3.2097
9.67
2255.64
0.4287
81.26
7479.28
1.0865
735.57
14.11
LP12
1.26
11500.95
0.0110
0.34
1721.63
0.0197
24.77
6419.44
0.3859
25.52
4463.29
0.5718
3.93
2645.29
0.1486
15.06
7598.99
0.1982
291.40
16.46
LP13
6.41
13163.18
0.0487
0.30
2716.29
0.0110
172.44
9432.07
1.8282
143.41
4225.29
3.3941
134.72
2932.22
4.5945
169.75
8497.59
1.9976
1667.79
19.79
LP14
3.64
16225.57
0.0224
0.17
2793.92
0.0061
175.47
6457.65
2.7172
140.65
5052.79
2.7836
118.28
3874.56
3.0527
171.21
9352.55
1.8306
1529.56
21.15
LP15
1.70
11614.17
0.0146
0.09
2501.01
0.0036
89.92
6615.18
1.3593
269.56
3725.94
7.2347
3.34
2877.91
0.1161
82.11
8616.92
0.9529
900.44
18.46
LP16
0.94
11631.20
0.0081
0.99
1651.61
0.0599
95.46
5189.08
1.8396
201.46
3964.71
5.0813
106.74
2083.97
5.1220
89.52
5649.83
1.5845
1381.04
15.81
LA1
0.75
10525.37
0.0071
0.33
1722.41
0.0192
119.80
5747.08
2.0845
146.08
4354.39
3.3548
117.79
2828.86
4.1639
99.93
8354.17
1.1962
1262.44
15.27
LA2
0.71
11050.46
0.0064
2.14
2075.76
0.1031
88.95
5755.14
1.5456
114.22
4714.20
2.4229
113.30
2590.48
4.3737
128.49
8446.54
1.5212
1057.50
16.42
LA3
1.58
14292.88
0.0111
—
2387.79
—
85.96
9803.09
0.8769
129.12
5651.48
2.2847
172.44
2107.17
8.1835
147.16
11408.14
1.2900
1716.54
20.72
LA4
0.78
11457.33
0.0068
—
2109.40
—
75.39
7598.12
0.9922
177.20
4225.71
4.1934
34.18
2341.13
1.4600
45.77
7073.90
0.6470
778.32
15.58
LA5
6.18
11098.92
0.0557
0.60
1527.50
0.0393
127.09
6426.35
1.9776
196.17
4798.09
4.0885
29.66
2500.29
1.1863
66.00
7750.15
0.8516
1332.28
16.58
LA6
1.69
11256.40
0.0150
0.14
1485.02
0.0094
92.57
6651.83
1.3916
129.99
4328.83
3.0029
86.79
1722.57
5.0384
157.13
8406.98
1.8690
1547.66
16.04
LA7
2.07
11307.16
0.0183
—
1577.93
—
92.43
4656.66
1.9849
145.34
4362.67
3.3314
135.71
1877.25
7.2292
170.21
8033.47
2.1188
1517.92
14.71
LA8
10.79
15918.39
0.0678
0.19
2259.56
0.0084
117.78
7883.83
1.4939
154.15
5792.38
2.6613
216.67
2621.75
8.2643
61.37
7941.33
0.7728
1171.00
21.30
LA9
0.76
14178.74
0.0054
0.14
2618.77
0.0053
6.51
6538.81
0.0996
7.87
4624.51
0.1702
2.68
2321.32
0.1155
6.76
10561.97
0.0640
189.02
21.46
LA10
1.44
11088.93
0.0130
0.26
1806.72
0.0144
62.25
6942.43
0.8967
53.97
4802.60
1.1238
47.46
2041.76
2.3245
36.17
5403.37
0.6694
775.54
16.41
LA11
0.84
15220.71
0.0055
1.65
2528.91
0.0652
8.60
7038.75
0.1222
1.31
5236.37
0.0250
4.91
2429.40
0.2021
7.80
6092.24
0.1280
289.21
19.05
LA12
2.55
9164.65
0.0278
0.18
1894.44
0.0095
174.89
4404.00
3.9712
138.27
3675.47
3.7620
72.48
1770.28
4.0943
85.28
5128.63
1.6628
1294.14
14.54
LA13
2.16
13906.13
0.0155
—
2514.70
—
116.52
7070.17
1.6481
294.15
3964.62
7.4194
42.77
2727.93
1.5679
65.75
7050.95
0.9325
1282.38
18.31
LA14
9.08
13760.74
0.0660
0.60
2297.56
0.0261
164.90
9014.10
1.8294
131.85
5045.39
2.6133
63.90
2704.59
2.3627
144.27
5601.90
2.5754
1371.90
17.81
LA15
3.69
8467.71
0.0436
0.30
1826.09
0.0164
135.18
5450.03
2.4804
62.94
3497.92
1.7994
46.02
1747.54
2.6334
51.56
4656.58
1.1073
1020.37
13.89
LA16
2.78
16217.53
0.0171
0.29
2603.75
0.0111
97.73
8455.20
1.1559
22.61
5055.17
0.4473
18.70
2206.25
0.8476
48.71
9026.29
0.5396
811.03
20.42
LA17
0.68
12202.21
0.0056
2.23
2011.22
0.1109
81.41
5966.49
1.3645
207.86
3770.32
5.5131
55.84
3327.23
1.6783
76.89
7660.33
1.0037
1027.14
18.44
LA18
0.55
9875.86
0.0056
2.42
1754.15
0.1380
76.82
5468.75
1.4047
143.75
3704.63
3.8803
52.67
1760.52
2.9917
90.61
6192.83
1.4631
1068.96
14.93
—, analytes were not detected.Twenty-six minerals, total proteins, and the content of amino acids after hydrolysis are shown in Tables5 and 6. From the tables, it could be found that the total protein contents in 59 samples ranged from 13.61% to 21.46%. The relative contents of amino acids such as Lys, Ala, Ser, Thr, GABA, Gln, and Asn were all a little higher than other components based on the average value of each species among three different varieties of duckweed. The contents of Cys, Cys2, and Met were higher after hydrolysis. Obviously, it could be seen that the contents of free amino acids in duckweed were much lower than that of protein-bound amino acids. Moreover, the data showed that the contents of free amino acids in duckweed were negatively correlated with binding amino acids.Table 6
Contents of 26 minerals in living water of three species of duckweed (μg/L).
Sample
Ti
V
Cr
Mn
Co
Ni
Cu
Zn
As
Se
Sr
Mo
Cd
Sn
Sb
Ba
Tl
Pb
SP1
22.831
2.900
2.178
47.015
0.181
1.860
1.061
90.405
1.530
1.578
261.614
2.840
0.007
0.087
0.440
96.840
0.003
0.058
SP2
27.277
1.017
2.922
36.990
0.177
1.648
0.806
80.395
2.091
1.288
199.006
3.275
0.006
0.007
0.516
60.613
0.003
0.001
SP3
35.621
1.915
1.838
41.295
0.162
2.899
1.500
56.437
1.127
1.983
172.960
3.017
0.016
0.168
0.386
70.196
0.002
0.024
SP4
22.183
3.099
1.687
55.683
0.214
1.767
1.690
63.717
3.164
1.935
178.985
0.407
0.014
0.168
0.487
77.659
0.001
0.041
SP5
45.839
3.162
1.413
24.431
0.189
2.829
0.615
71.741
0.920
1.861
275.418
2.211
0.011
0.098
0.542
63.126
0.004
0.074
SP6
41.446
1.783
2.371
2.110
0.240
2.157
2.805
87.665
2.146
1.464
193.120
0.577
0.002
0.121
0.536
39.704
0.004
0.046
SP7
37.099
2.423
2.068
13.993
0.219
2.583
1.424
88.936
2.601
1.747
165.967
1.562
0.010
0.095
0.316
69.916
0.003
0.050
SP8
28.377
2.251
2.276
54.028
0.197
1.709
2.941
46.581
2.262
2.139
249.180
2.345
0.015
0.125
0.359
110.809
0.004
0.047
SP9
17.084
1.069
2.452
40.559
0.192
3.140
1.247
29.974
2.770
1.664
163.694
2.659
0.015
0.139
0.637
60.895
0.004
0.018
SP10
37.166
3.316
1.705
35.254
0.184
1.793
2.040
78.451
1.247
1.407
174.872
0.632
0.002
0.137
0.720
86.360
—
0.003
SP11
24.163
1.033
1.368
60.738
0.190
3.261
2.894
66.981
1.135
2.291
207.084
1.840
0.003
0.160
0.488
117.603
0.004
0.046
SP12
38.630
2.597
1.146
53.768
0.252
2.550
1.505
76.263
1.772
2.126
180.679
0.626
0.003
0.124
0.628
142.280
0.001
0.062
SP13
39.728
2.347
1.577
26.733
0.173
3.173
2.882
75.098
1.628
1.112
225.261
0.323
0.012
0.106
0.550
95.230
0.001
0.001
SP14
39.483
2.488
2.539
3.742
0.249
2.137
1.427
55.271
1.670
1.581
154.375
3.214
0.019
0.135
0.338
119.138
0.003
0.043
SP15
19.629
2.734
2.326
22.405
0.198
3.153
1.427
55.631
1.175
2.065
247.855
2.928
0.011
0.011
0.633
136.536
0.004
0.030
SP16
34.619
1.068
2.064
52.250
0.163
2.169
3.017
54.488
2.321
1.985
253.553
2.155
0.002
0.120
0.377
68.487
0.001
0.022
SP17
28.672
3.244
2.192
31.414
0.159
1.880
2.871
91.946
1.444
1.287
175.165
1.721
0.018
0.138
0.669
91.883
0.004
0.030
SP18
25.018
1.789
2.378
45.984
0.183
2.570
2.365
24.786
1.877
2.146
194.164
3.450
0.007
0.049
0.318
95.469
0.002
0.057
SP19
22.802
2.136
2.199
53.284
0.212
2.704
1.054
85.613
0.913
2.073
269.684
3.174
0.014
0.043
0.423
111.149
0.003
0.004
SP20
40.638
1.682
1.746
14.621
0.180
1.611
2.705
73.740
3.221
2.055
225.646
3.023
0.013
0.159
0.450
137.841
0.004
0.019
SP21
23.513
1.413
2.811
12.908
0.236
1.959
0.607
22.616
2.711
1.170
227.681
3.187
0.005
0.037
0.205
62.615
0.003
0.016
SP22
32.010
1.755
2.461
56.750
0.202
2.515
1.114
80.116
1.210
2.322
223.754
1.075
0.015
0.155
0.573
96.469
0.002
0.012
SP23
17.817
2.259
2.391
43.452
0.219
1.903
0.700
88.007
3.277
1.855
155.450
2.793
0.014
0.164
0.311
82.020
0.002
0.010
SP24
24.306
2.384
1.841
32.935
0.253
1.896
3.027
63.243
1.975
1.109
214.325
2.979
0.008
0.044
0.642
47.431
0.003
0.061
SP25
30.842
2.878
1.693
29.377
0.254
2.786
1.811
69.276
1.082
1.349
218.438
0.896
0.011
0.125
0.474
126.036
0.001
0.032
LP1
37.974
1.441
2.756
42.898
0.251
1.828
2.325
46.010
2.655
2.264
162.610
0.298
0.004
0.049
0.368
106.287
0.002
0.055
LP2
25.601
3.144
1.914
10.035
0.173
2.425
2.422
53.173
1.031
1.740
211.860
0.256
0.017
0.153
0.596
73.995
0.002
0.001
LP3
40.576
2.424
1.579
36.158
0.203
2.635
1.373
32.781
1.839
1.363
234.793
1.450
0.001
0.145
0.697
107.956
0.001
0.032
LP4
29.919
1.451
2.468
31.060
0.239
3.221
2.253
43.918
2.873
1.754
193.086
2.154
0.013
0.027
0.249
36.637
0.001
0.015
LP5
43.240
2.466
2.331
46.991
0.209
3.218
1.577
26.153
1.994
2.211
234.265
1.895
0.010
0.017
0.224
84.449
0.004
0.006
LP6
29.925
2.796
2.933
11.764
0.248
2.974
2.174
43.896
2.394
1.784
247.178
3.487
0.012
0.017
0.336
125.785
0.003
0.016
LP7
28.783
1.090
2.067
41.129
0.244
2.388
1.514
76.640
1.923
1.886
157.760
2.486
0.003
0.031
0.241
87.240
0.004
0.053
LP8
45.076
3.030
1.857
7.724
0.247
2.328
2.152
87.415
2.455
2.044
225.691
2.976
0.011
0.009
0.607
111.718
0.003
0.023
LP9
20.651
2.339
2.731
42.403
0.257
1.681
1.012
89.960
1.066
1.855
210.521
2.237
0.007
0.052
0.424
32.520
—
0.033
LP10
23.769
1.646
1.288
52.927
0.243
2.319
2.950
93.752
2.929
1.882
217.655
2.496
0.017
0.165
0.196
135.624
0.003
0.051
LP11
32.364
2.560
3.084
33.335
0.208
3.219
2.180
82.812
1.451
1.918
269.769
3.250
0.019
0.008
0.406
66.278
0.003
0.051
LP12
24.918
1.470
2.999
37.041
0.243
1.893
1.928
95.305
2.415
1.698
224.103
1.906
0.008
0.041
0.570
64.033
0.004
0.008
LP13
33.468
1.704
2.451
45.575
0.176
2.507
1.882
38.837
2.524
1.580
187.087
1.245
0.003
0.133
0.285
44.075
0.003
0.030
LP14
28.614
2.426
1.879
39.614
0.209
3.272
1.963
74.622
2.875
1.962
261.572
2.514
0.013
0.136
0.287
100.138
0.002
0.048
LP15
35.617
2.088
2.116
42.214
0.162
2.284
1.641
39.037
1.412
1.185
244.588
0.489
0.007
0.158
0.512
110.953
0.003
0.032
LP16
32.522
2.845
1.545
41.087
0.186
2.100
1.860
94.105
3.060
1.921
165.053
1.398
0.015
0.008
0.451
69.796
0.002
0.066
LA1
36.451
2.711
1.271
38.156
0.233
2.893
0.909
65.183
3.041
1.926
194.455
3.342
0.005
0.055
0.262
51.502
0.002
0.023
LA2
43.394
2.188
2.067
17.427
0.165
2.206
1.564
58.425
3.159
1.247
254.469
1.789
0.006
0.146
0.280
33.628
0.003
0.052
LA3
24.311
1.120
2.917
35.712
0.247
2.359
2.087
59.228
3.345
2.092
247.432
1.778
0.010
0.108
0.362
105.945
—
0.069
LA4
26.794
1.110
1.710
13.192
0.232
1.909
1.963
25.002
3.351
1.618
198.614
1.801
—
0.011
0.452
33.107
0.003
0.024
LA5
39.248
2.722
1.585
24.608
0.203
2.755
2.186
68.836
1.896
1.634
279.990
3.413
0.016
0.037
0.213
37.247
0.004
0.077
LA6
20.981
2.024
1.392
25.368
0.248
2.878
2.542
45.526
1.863
1.095
215.126
3.019
0.009
0.148
0.651
117.017
0.003
0.010
LA7
30.279
1.148
2.330
62.335
0.231
2.772
2.791
44.135
2.816
1.556
187.777
3.290
0.012
0.151
0.436
36.940
0.003
0.041
LA8
39.053
0.971
3.084
15.630
0.243
3.097
1.738
42.571
2.567
1.411
231.825
2.281
0.010
0.013
0.499
33.670
0.003
0.053
LA9
41.198
2.925
2.254
52.257
0.230
2.490
2.132
95.196
2.872
2.220
170.239
1.302
0.002
0.132
0.395
80.576
0.003
0.015
LA10
16.833
1.384
2.832
34.567
0.188
2.044
1.951
62.570
0.922
1.571
169.715
0.952
0.001
0.110
0.496
81.418
0.002
0.044
LA11
38.564
2.900
3.171
30.344
0.257
2.860
2.083
37.280
1.155
1.073
172.207
1.959
0.008
0.117
0.433
108.524
0.002
0.014
LA12
43.364
1.975
2.442
7.660
0.253
1.986
0.775
85.359
2.247
1.143
237.191
0.673
0.009
0.155
0.201
41.025
—
0.002
LA13
30.181
3.229
1.146
53.644
0.230
2.341
2.091
77.183
0.842
2.028
177.614
2.395
0.009
0.105
0.689
97.023
0.001
0.022
LA14
31.940
3.492
1.508
64.537
0.181
2.947
2.883
24.336
3.049
2.137
243.782
1.989
0.017
0.117
0.272
125.192
0.003
0.021
LA15
22.170
2.327
3.077
51.812
0.175
3.050
0.998
60.176
2.860
1.716
206.597
0.281
0.006
0.058
0.397
108.215
—
0.025
LA16
42.638
1.570
1.978
49.228
0.258
1.959
0.924
88.563
2.498
1.683
201.422
3.242
0.014
0.029
0.295
80.373
0.003
0.075
LA17
39.670
1.249
2.650
27.755
0.259
2.112
2.250
55.219
2.830
1.217
154.053
1.978
0.006
0.099
0.561
116.452
—
0.047
LA18
23.423
2.528
2.932
10.179
0.199
2.743
2.953
66.269
3.199
1.527
209.326
3.075
0.016
0.116
0.306
79.441
0.001
0.040
Sample
Na
Mg
Al
K
Ca
Fe
N
P
Total
SP1
410579.062
209771.718
18661.431
178472.536
591631.101
334974.476
4.303
0.914
1744628.970
SP2
546214.809
232797.639
9209.284
416708.514
386703.829
349989.801
2.389
0.675
1942044.979
SP3
384732.341
302374.639
16864.219
351597.651
645593.344
330485.336
18.804
5.589
2032063.470
SP4
763978.060
217006.139
24174.116
515345.319
525036.011
165789.077
19.687
4.182
2211765.493
SP5
498927.575
304758.187
29743.190
617594.946
663524.773
200323.584
25.750
23.839
2315416.327
SP6
568688.625
219822.814
31062.346
305039.982
486491.766
202085.631
25.562
22.681
1813617.704
SP7
815621.400
203217.299
32660.784
559562.408
566642.730
171755.702
2.565
1.507
2349855.405
SP8
704283.730
188285.004
23602.584
315772.349
624642.806
439955.497
23.372
6.689
2297077.677
SP9
876213.262
151574.474
27629.879
516294.381
409003.501
35506.241
18.458
4.258
2016572.669
SP10
873643.979
294027.085
21164.307
737471.818
383002.078
37324.576
33.550
19.033
2347111.719
SP11
491391.823
256934.016
10147.423
610308.309
636084.667
367347.644
31.694
25.205
2372762.062
SP12
796497.255
269227.175
17999.811
414658.818
512138.780
171064.616
16.646
2.703
2182110.814
SP13
366808.463
156147.673
11576.232
471660.374
756208.109
347515.518
11.647
4.202
2110408.155
SP14
512150.165
243500.636
31493.169
641312.090
499270.353
208734.109
11.077
1.602
2136861.051
SP15
533557.790
280721.134
16614.646
267728.355
378226.451
306162.902
13.593
4.564
1783528.184
SP16
644403.624
276057.049
21110.856
521532.157
583524.045
59176.189
11.942
25.264
2106319.987
SP17
751229.025
173163.933
10495.020
625004.125
472688.229
345116.023
8.458
5.204
2378144.753
SP18
522140.520
176639.957
29371.612
557932.109
690911.336
157124.877
29.906
10.221
2134563.150
SP19
579415.759
238453.094
30677.565
439717.245
686006.551
382672.972
10.823
3.662
2357515.153
SP20
476281.317
182915.790
9070.049
325811.742
373586.252
432553.750
34.616
21.891
1800784.763
SP21
760676.992
188574.381
12126.295
629919.272
484913.883
230880.412
26.927
9.833
2307491.687
SP22
709408.940
239417.173
10783.138
619462.993
550226.213
88795.927
20.170
9.877
2218626.941
SP23
513303.754
213899.504
26441.948
351694.267
732144.084
326876.462
34.159
26.699
2164823.520
SP24
824465.276
238490.190
31283.397
717257.458
679154.325
297001.594
14.031
10.796
2788075.528
SP25
815267.536
215764.623
30718.721
536395.881
508251.297
371174.761
24.650
2.494
2478087.325
LP1
897950.747
158686.979
27871.364
590366.194
696960.175
327232.239
17.263
7.080
2699502.116
LP2
531566.916
294929.433
14640.021
570417.870
648189.841
273059.464
15.536
1.239
2333208.859
LP3
858626.936
266231.149
32272.481
727712.714
480257.660
348345.090
12.319
1.894
2713926.251
LP4
431098.253
221743.162
34467.794
311381.975
785570.359
339960.811
26.584
17.225
2124617.501
LP5
818404.736
237360.411
25820.309
683695.992
566015.946
406722.563
20.219
7.521
2738498.958
LP6
614726.783
203772.151
23626.598
170198.351
715325.262
132978.487
8.402
1.447
1861115.202
LP7
498622.492
200069.361
34249.218
182114.487
450498.128
213156.897
26.337
12.664
1579155.065
LP8
407734.217
308322.609
12414.630
462642.984
726996.054
273473.025
23.952
9.428
2192112.264
LP9
591923.425
196758.141
20795.603
169080.807
379447.395
244832.850
24.638
15.719
1603288.325
LP10
506188.280
278745.249
26183.977
548614.526
619327.368
71487.282
27.250
10.475
2051124.319
LP11
484360.416
252740.828
27267.497
394747.762
420528.173
411590.345
15.427
3.462
1991756.825
LP12
755545.368
290158.455
18713.693
269669.218
653034.467
303936.794
26.120
0.889
2291545.587
LP13
644431.270
171083.444
14922.153
701260.007
799614.153
241821.499
27.383
10.915
2573534.387
LP14
855511.785
226105.300
11812.507
382201.190
657443.790
421778.147
4.108
0.257
2555378.850
LP15
840333.425
261183.216
19461.126
468066.062
568653.040
96502.984
17.469
12.422
2254714.244
LP16
662138.232
247763.574
23217.103
653430.420
481555.835
52632.672
16.299
0.242
2121172.397
LA1
542867.038
291631.774
16101.327
208393.119
534546.718
287585.245
20.487
11.217
1881559.346
LA2
768502.457
174767.313
34293.763
538598.282
767009.154
130620.652
24.093
8.098
2414246.025
LA3
836100.485
232547.859
8383.317
197659.044
475008.913
70420.274
26.668
18.771
1820654.452
LA4
554969.006
231606.445
11451.728
365538.304
505163.079
269818.459
11.932
3.908
1938873.753
LA5
794677.014
293809.893
30832.050
293530.490
510493.154
262663.280
12.498
6.307
2186491.358
LA6
704360.729
287183.610
31019.483
189142.179
384371.287
327789.933
12.239
4.852
1924324.212
LA7
894339.623
204701.453
27985.214
281212.437
595254.900
371594.312
24.934
18.350
2375510.268
LA8
427283.433
184599.086
23700.009
211637.098
596714.072
325251.967
22.097
1.616
1769588.097
LA9
879491.821
180076.383
12682.543
611024.725
416252.729
426535.290
22.887
10.851
2526553.669
LA10
712373.467
297199.560
9966.649
218385.532
678770.388
129164.596
32.940
4.216
2046274.948
LA11
625486.302
275066.946
25810.603
496586.040
470812.328
438093.869
14.927
1.233
2332275.197
LA12
796469.226
199565.531
26532.869
616737.846
538184.180
286481.065
25.413
16.087
2464438.679
LA13
587462.535
234893.095
16533.152
573600.610
441381.833
215218.753
5.375
3.154
2069549.281
LA14
598797.362
200101.308
32285.037
179408.885
737688.278
321911.868
10.937
7.808
2070719.888
LA15
775176.535
182419.544
9875.652
375172.731
686178.073
146549.529
20.767
18.862
2175875.636
LA16
486462.514
269482.486
19514.461
382028.835
385846.710
419515.606
25.566
4.690
1963357.619
LA17
397975.248
220647.104
20225.722
404790.872
406870.509
69049.196
25.782
16.448
1520009.288
LA18
852308.317
145491.528
11612.476
499143.838
570244.949
84828.689
23.929
16.569
2164078.568
—, analytes were not detected.The elements with higher content in the samples were sodium, calcium, potassium, iron, magnesium, and aluminum. Their concentration ranges were 366808.463–897950.747, 373586.252–799614.153, 169080.807–737471.818, 35506.241–439955.497, 145491.528–308322.609, and 8383.317–34467.794μg/L, respectively. The concentration ranges of strontium, barium, zinc, manganese and titanium were 154.053–279.990, 32.520–142.280, 22.616–95.305, 2.110–64.537, 16.833–45.839 μg/L. The concentrations of vanadium, chromium, cobalt, nickel, copper, arsenic, selenium, molybdenum, cadmium, tin, antimony, thallium, and lead are very low. It ranged from 0.0 to 3.492 μg/L. Evidently, the contents of macroelements in the samples were higher while they contained much lower trace elements.


## 3.6. Statistical Analysis

As one of the most important multivariate analysis techniques, PCA was employed to evaluate the variation among three species of duckweed. PCA is an unsupervised pattern recognition method without prior information of the data set and retained maximum variance of multidimensional data while reducing its dimensionality into two- or three-dimension [29–31]. Furthermore, all of the data information about amino acids was subjected to a supervised method, OPLS-DA [32], to get more information about different duckweeds. All the raw data were processed by normalization and Pareto scaling (Par) before modeling. Three dimensions were established in both the PCA and OPLS-DA models based on the content of free amino acids and the ratio of free to total amino acids with R2X (cum) value of 0.628, 0.596 and 0.758, 0.676, respectively. As shown in Figures 2–5, both PCA and OPLS-DA methods demonstrated that Spirodela polyrhiza, Landoltia punctata, and Lemna aequinoctialis were clustered together with each other in the score plot. There was no remarkable difference in different duckweeds.Figure 2
Three-dimensional score plot of principle component analysis (PCA) based on the content of free amino acids.Figure 3
Three-dimensional score plot of orthogonal partial least square-discriminant analysis (OPLS-DA) based on the content of free amino acids.Figure 4
Three-dimensional score plot of principle component analysis (PCA) based on the ratio of free to total amino acids.Figure 5
Three-dimensional score plot of orthogonal partial least squares-discriminant analysis (OPLS-DA) based on the ratio of free to total amino acids.


## 4. Conclusions

In this study, a simple, fast, and convenient method with UPLC-QTRAP-MS/MS was established for the identification and quantification control of duckweeds by HILIC separation without derivatization. The results of method validation suggested that the developed method was sensitive, accurate, and precise for determination of 24 free amino acids. 59 batches of sample in three species were quantified, and then the data were statistically analysed by using PCA and OPLS-DA. The multivariate analysis illustrated that the content of free amino acids was not significantly different amongSpirodela polyrhiza, Landoltia punctata, and Lemna aequinoctialis in pattern recognition. But as a food, more amino acids mean more nutrition it has and more valuable it is. In other words, all of the three species of duckweed could be used as food equally if the absolute amount of AAs was more or less the same. In conclusion, a reliable and feasible method was developed for the nutritional evaluation of duckweeds.



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*Source: 7673652-2018-12-05.xml*