Synthesis of toluene-4-sulfonic acid 2-(2-thiophen-2-yl-phenanthro[9,10-: D] imidazol-1-yl)-ethyl ester and its application for sensitive determination of free fatty acids in ginkgo nut and ginkgo leaf by high performance liquid chromatography with fluorescence detection

Article abstract of DOI:10.1039/c8ra01875h

A novel fluorescent labeling reagent toluene-4-sulfonic acid 2-(2-thiophen-2-yl-phenanthro[9,10-d]imidazol-1-yl)-ethyl ester has been designed and synthesized. It was used to label twenty-six fatty acids (C5-C30) successfully in the presence of K₂CO₃ catalyst in N,N-dimethylformamide solvent. The reaction conditions were optimized by employing a three-factor, three-level Box-Behnken design. Derivatives were sufficiently stable to be efficiently analyzed by high-performance liquid chromatography with fluorescence detection. All fatty acid derivatives were separated on a hypersil BDS-C8 column in conjunction with a gradient elution with a good baseline resolution. Good linear correlations were observed for all fatty acids with correlation coefficients > 0.993. The established method exhibited high sensitivity and excellent repeatability. The limit of detection (at a signal-to-noise ratio of 3:1) was 8.8-45.5 fmol. The method was used to quantify free fatty acids in ginkgo nut and ginkgo leaf samples with satisfactory results.

Full text of DOI:10.1039/c8ra01875h

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PAPER
Synthesis of toluene-4-sulfonic acid 2-(2-
thiophen-2-yl-phenanthro[9,10-d]imidazol-1-yl)-
ethyl ester and its application for sensitive
determination of free fatty acids in ginkgo nut and
ginkgo leaf by high performance liquid
Cite this: RSC Adv., 2018, 8, 18549
chromatography with fluorescence detection
*
Xiuli Dong, Xueying Liu and Xiangming Chen
A novel fluorescent labeling reagent toluene-4-sulfonic acid 2-(2-thiophen-2-yl-phenanthro[9,10-d]
imidazol-1-yl)-ethyl ester has been designed and synthesized. It was used to label twenty-six fatty acids
(C5–C30) successfully in the presence of K2CO3 catalyst in N,N-dimethylformamide solvent. The
reaction conditions were optimized by employing a three-factor, three-level Box–Behnken design.
Derivatives were sufficiently stable to be efficiently analyzed by high-performance liquid
chromatography with fluorescence detection. All fatty acid derivatives were separated on a hypersil BDS-
C8 column in conjunction with a gradient elution with a good baseline resolution. Good linear
correlations were observed for all fatty acids with correlation coefficients > 0.993. The established
method exhibited high sensitivity and excellent repeatability. The limit of detection (at a signal-to-noise
ratio of 3 : 1) was 8.8–45.5 fmol. The method was used to quantify free fatty acids in ginkgo nut and
ginkgo leaf samples with satisfactory results.
Received 3rd March 2018
Accepted 9th May 2018
DOI: 10.1039/c8ra01875h
rsc.li/rsc-advances
fatty acids directly by GC because of their high polarity and low
volatility. One method of overcoming this difficulty is to prepare
1. Introduction
volatile alkyl derivatives or silylation derivatives which can
decrease the boiling point of fatty acids,11 but this procedure is
usually very time-consuming, and the limited stability of the
derivatives is also an obstacle for accurate quantitative analysis.
There is a growing interest in the use of high performance
liquid chromatography (HPLC) for analyzing fatty acids.12–15
HPLC analysis is usually performed at room temperature,
a lower temperature compared with GC would reduce the risk of
damage of heat-liable compounds. HPLC methods for the
determination of fatty acids involving detectors such as mass
spectrometer,15,16 chemiluminescence,17 evaporative light scat-
tering detector,14,18 and contactless conductivity detector13 have
been reported. The frequently-used detector for HPLC is an UV-
vis and uorescence detector, but most fatty acids show weak
natural absorption in the visible or UV regions and no uores-
cence, so it is of low sensitivity for direct analysis of fatty acids
by HPLC.19 For these reasons, derivatization procedures allow-
ing a chromophore or uorophore to label fatty acids have been
widely adopted, which allows fatty acids to be detected by an
UV-vis or uorescence detector and increases the sensitivity of
the methods.11,20–23 Many pre-column ultraviolet and uores-
cence labeling reagents have been developed for analysis of the
compounds with carboxy groups in the last decades. These
Fatty acids are widely distributed in all kinds of samples, such
as living organisms, vegetable oils, and food products. They are
essential structural elements of biological membranes, and play
important roles at trace levels in the regulation of a variety of
physiological and biological functions. Saturated fatty acids
have been considered to be bad fatty acids that could cause
hypercholesterol and cardiovascular disease if people eat too
much every day. However, many research studies revealed that
saturated fatty acids have a therapeutic action for some diseases
such as alcoholic liver injury,1,2 intraparenchymal hemorrhage,3
metabolic syndrome4 and insulin resistance syndrome.5
Therefore, sensitive and accurate isolation and quantization of
trace fatty acids is of great importance both in biology and
medicine.
The most commonly used method for the determination of
free fatty acids is capillary gas chromatography (GC) with
a ame ionization detector6,7 or GC coupled with mass spec-
trometry.8–10 GC provides excellent separation and quantica-
tion together with high sensitivity and selectivity for the analysis
of multi-component samples. However, it is difficult to analyze
School of Pharmacy, Binzhou Medical University, Yantai, 264003, P. R. China. E-mail:
xmch913@163.com; Tel: +86(535)6913406
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regents are classied into diazomethane-type reagents,24,25 synthesized in our lab as described in the synthesis section and
coumarin-type derivatives,26,27 benzofurazan-type reagents,28,29 Fig. 1. Water was puried on a Milli-Q system (Millipore, Bed-
and alcohol reagents.30,31 Typical labeling reagents for carboxyl ford, MA). All other reagents used in this study were also of
include 9-anthryldiazomethane (ADAM),25 4-aminomethyl-6,7- analytical grade unless otherwise stated. Ginkgo nut and ginkgo
dimethoxycoumarin (ADC),27 7-acetylamino-4-mercapto-2,1,3- leaf samples were collected from Shandong province, Jiangsu
benzoxadiazole (AABD),29 1,2-benzo-3,4-dihydrocarbazole-9- province and Henan province of China.
ethanol (BCE),31 and so on. ADAM is not stable enough and
thus cannot be stored in solution for a long time, although it
can react with carboxylic acids under mild conditions without
a catalyst. ADC is easily be decomposed when was exposed to
moist environment and benzene or toluene which is highly toxic
is used in derivatization procedure. Derivatization procedure is
complicated and time-consuming. Other labeling reagents have
many disadvantages in their applications such as low detection
sensitivity, short detection wave-lengths, poor stability, tedious
analytical procedures, and so on.32–34
Phenanthroimidazole is an excellent luminophore which
could emit strong blue light with high efficiency when excited by
light or electric. A serious of phenanthroimidazole-derivatives
were designed and synthesized to study the possibility of use
as photoelectric material. Inspired by this, we think that phe-
nanthroimidazole could be used as a good pre-column deriva-
tive reagent aer structure modied. Therefore, we designed
and synthesized a novel uorescence labeling reagent, toluene-
4-sulfonic acid 2-(2-thiophen-2-yl-phenanthro[9,10-d]imidazol-
2.2 Instruments and conditions
Agilent 1100 series HPLC (Agilent, USA) was used to perform the
experiment. The HPLC system was equipped with a quaternary
pump (model G1311A), an online vacuum degasser (model
G1322A), an auto-sampler (model G1329A), a thermostated
column compartment (model G1316A) and a uorescence
detector (FLD) (model G1321A). The HPLC system was
controlled by Agilent Chemstation soware (version B.01.01).
Chromatographic conditions: derivatives were separated on
a reversed-phase Hypersil BDS-C8 column (150 ꢁ 4.6 mm, 5 mm,
Dalian Elite Analytical Instruments Co., Ltd., China) by
a gradient elution. Eluent A was 5% acetonitrile in water, and B
was acetonitrile. Elution conditions were as follows: initial ¼
60% B; 15 min ¼ 80% B; 40 min ¼ 100% B (kept for 15 min). The
ow rate was constant at 1.0 mL minꢂ1 and the column
temperature was set at 30 ꢀC. Before the injection of next
sample, the column was equilibrated with the initial mobile
phase for 5 min. The uorescence excitation and emission
wavelengths were set at 314 nm and 398 nm, respectively.
1-yl)-ethyl ester (TSTPE) that emits strong uorescence (lem
¼
398 nm) when exposed to excitation light (lex ¼ 314 nm). TSTPE
can react with carboxylic acid compounds in the presence of
a basic catalyst and a highly sensitive method for the analysis of
fatty acids using TSTPE as labeling reagent was developed. The
2.3 Synthesis of TSTPE
2.3.1 Synthesis of 2-(thiophen-2-yl)-1H-phenanthro[9,10-d]
optimꢀum derivatization of fatty acids with TSTPE was obtained imidazole. 9,10-Phenanthraquinone (16 g), 2-thiophenecar-
at 94 C for 32.6 min in the presence of potassium carbonate boxaldehyde (12 mL), ammonium acetate (120 g) and glacial
(K2CO3) in N,N-dimethylformamide (DMF) solvent. A total of 26 acetic acid (300 mL) were fully mixed in a 1000 mL of round-
saturated fatty acids were separated in 50 min on a Hypersil bottom ask. Then, the contents of the ask were rapidly
ꢀ
BDS-C8 column with gradient elution. The established method heated to 80–90 C with stirring for 3 h. Aer cooling to room
has been used to determine the saturated fatty acid composi- temperature, the mixture was poured into a 1000 mL beaker and
tions in ginkgo nut and ginkgo leaf with satisfactory results. ammonium hydroxide was added to adjust pH of the solution to
Ginkgo nut, the seed of Ginkgo biloba, is an important nut for 7–8. The precipitated solid was recovered by ltration and
most Chinese. It's also a traditional Chinese medicine for washed with water for three times. Aer dried at room
treating cough, asthma, enuresis, alcohol misuse, pyogenic skin temperature in dark for 3 days, the crude product was recrys-
infections, and worm infections in the intestinal tract.35 Ginkgo tallized twice from acetonitrile/DMF mixed solvent (5 : 1, v/v) to
leaf is also a traditional Chinese medicine used to lower lipid afford a brown crystal, yield 88%.
levels and prevent cardiovascular and cerebrovascular disease.
2.3.2 Synthesis of 2-(2-thiophen-2-yl-phenanthro[9,10-d]
In this study, the content of free fatty acids in ginkgo leaf and imidazol-1-yl)-ethanol. 2-(Thiophen-2-yl)-1H-phenanthro[9,10-
ginkgo nut was determined for the rst time.
d]imidazole (9 g), KOH (0.1 g) and DMF (100 mL) were mixed in
a 500 mL of round-bottom ask and rapidly heated to reux
with vigorous stirring, Then, ethylene carbonate (3.2 g) was
added to the mixture in 3 portions in 1 h. Aer kept reux for
6 h, the contents were transferred into 300 mL of water. The
2. Experimental
2.1 Chemicals and materials
All fatty acids (C5–C30) standards were of analytical grade and precipitated solid was recovered by ltration, and washed
purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC successively with water, 60% ethanol solution (ethanol/water
grade acetonitrile (ACN) was purchased from Yucheng (Shan- 3 : 2, v/v). The crude product was dried at room temperature
dong Province, China). N,N-Dimethylformamide (DMF), potas- for 2 days and recrystallized from acetonitrile/DMF mixed
sium carbonate (K2CO3), pyridine, and chloroform were of solvent (acetonitrile/DMF, 4 : 1, v/v) to afford a yellow acicular
analytical grade obtained from Shanghai Chemical Reagent Co. type crystal, yield 51%.
(Shanghai, China). Toluene-4-sulfonic acid 2-(2-thiophen-2-yl-
2.3.3 Synthesis of toluene-4-sulfonic acid 2-(2-thiophen-2-
phenanthro[9,10-d]imidazol-1-yl)-ethyl ester (TSTPE) was yl-phenanthro[9,10-d]imidazol-1-yl)-ethyl ester (TSTPE). To
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Fig. 1 Synthesis route of TSTPE and derivatization scheme of TSTPE with fatty acids.
a solution of 3 g toluenesulfonyl chloride in 20 mL pyridine (0 4.45, N 5.62, S 12.86, O 9.62. 1H NMR (500 MHz, DMSO) d 8.98–
ꢀC) in a 100 mL round-bottom ask, a solution of 2-(2-thiophen- 8.93 (m, 1H), 8.89 (d, J ¼ 8.3 Hz, 1H), 8.60 (d, J ¼ 7.8 Hz, 1H),
2-yl-phenanthro[9,10-d]imidazol-1-yl)-ethanol (6.0 g) in 10 mL 8.26–8.21 (m, 1H), 7.90 (d, J ¼ 5.0 Hz, 1H), 7.79 (t, J ¼ 7.4 Hz,
of pyridine was added dropwise within 30 min under vigorous 1H), 7.70 (ddd, J ¼ 17.8, 11.5, 6.2 Hz, 4H), 7.37–7.32 (m, 1H),
stirring. Aer stirring at 0 ꢀC for 4 h, the contents were kept at 7.04 (d, J ¼ 8.1 Hz, 2H), 6.58 (d, J ¼ 8.0 Hz, 2H), 5.17 (t, J ¼
ambient temperature for 10 h with vigorous stirring. Then the 4.4 Hz, 2H), 4.54 (t, J ¼ 4.4 Hz, 2H), 1.78 (s, 3H). m/z [M + H]:
contents was transferred into a 100 mL of ice water with 499.7.
vigorous stirring for 0.5 h, the precipitated solid was recovered
by ltration and washed with distilled water for three times.
Aer dried at ambient temperature for 48 h, the crude products
were recrystallized twice from acetonitrile to give pale yellow
crystals 7.93 g, yield 91%, mp 175.1–177.2 ꢀC. Found (%): C
67.43, H 4.46, N 5.60, S 12.89, O 9.62; calculated (%): C 67.45, H
2.4 Preparation of solutions
Individual stock solutions of fatty acids (1.0 ꢁ 10ꢂ2 mol Lꢂ1
)
were prepared in ACN/DMF (1 : 1, v/v), the standard fatty acids
for HPLC analysis at individual concentrations of 1.0 ꢁ
10ꢂ4 mol Lꢂ1 were prepared by diluting the corresponding stock
Table 1 The Box–Behnken design matrix of three test variables in coded and natural units along with observed responses
Independent variable
V (molar
ratio)
Response
(total peak area)
Run number
T (temperature, ꢀC)
t (time, min)
1
2
3
4
5
6
7
8
10(0)
10(0)
10(0)
10(0)
10(0)
8(ꢂ1)
10(0)
8(ꢂ1)
12(+1)
10(0)
12(+1)
12(+1)
10(0)
12(+1)
8(ꢂ1)
10(0)
8(ꢂ1)
85(0)
30(0)
15 591
6905
12 371
14 560
9448
4184
14 393
9576
100(+1)
100(+1)
85(0)
20(ꢂ1)
40(+1)
30(0)
40(+1)
20(ꢂ1)
30(0)
40(+1)
20(ꢂ1)
30(0)
30(0)
40(+1)
30(0)
30(0)
30(0)
70(ꢂ1)
85(0)
85(0)
85(0)
85(0)
85(0)
100(+1)
85(0)
85(0)
70(ꢂ1)
70(ꢂ1)
70(ꢂ1)
100(+1)
9
5365
10
11
12
13
14
15
16
17
14 713
13 901
10 605
15 350
11 858
8616
20(ꢂ1)
5963
11 707
30(0)
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Fig. 2 Excitation and emission spectra of TSTPE in DMF (A) and TSTPE-C18 in acetonitrile (B). 1: 100% acetonitrile, 2: 90% acetonitrile, 3: 80%
acetonitrile, 4: 70% acetonitrile, 5: 60% acetonitrile, 6: 50% acetonitrile.
solutions with ACN/DMF (1 : 1, v/v). The derivatization reagent
solution (1.0 ꢁ 10ꢂ2 mol Lꢂ1) was prepared by dissolving 0.5 mg
TSTPE in 10 mL DMF. When not in use, all solutions were
2.5 Extraction of fatty acids from samples
All ginkgo nut and ginkgo leaf samples were baked at 80 ꢀC for
4 h and grinded by a Chinese medicine pulverizer. 1.0 g
ꢀ
stored at 4 C in a refrigerator.
Fig. 3 The 3D response surface plots showing effects of the examined factors on the response. (A) The effect of reaction time and TSTPE
amounts on the peak area; (B) the effect of derivatization temperature and TSTPE amounts on the peak area; (C) the effect of derivatization
temperature and reaction time on the peak area.
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Fig. 4 Chromatogram of standard fatty acids derivatives. Chromatographic conditions: Hypersil BDS-C8 column (4.6 ꢁ 150 mm, 5 mm), column
temperature 30 ꢀC; flow rate 1.0 mL minꢂ1; excitation and emission: lex/lem ¼ 314/398 nm. Peaks are labeled with abbreviations for all fatty acids:
C5 (pentanoic acid); C6 (hexanoic acid); C7 (heptanoic acid); C8 (caprylic acid); C9 (pelargonic acid); C10 (decanoic acid); C11 (undecanoic acid);
C12 (dodecanoic acid); C13 (tridecanoic acid); C14 (myristic acid); C15 (pentadecanoic acid); C16 (hexadecanoic acid); C17 (heptadecanoic acid);
C18 (octadecanoic acid); C19 (nonadecanoic acid); C20 (arachidic acid); C21 (heneicosoic acid); C22 (docosanoic acid); C23 (tricosanoic acid);
C24 (tetracosanoic acid); C25 (pentacosanoic acid); C26 (hexacosanoic acid); C27 (carboceric acid); C28 (montanic acid); C29 (motanic acid);
C30 (triacontanoic acid).
powdered ginkgo nut sample or ginkgo leaf sample and 10 mL the sample was sonicated for 60 min. The mixture was ltered
chloroform were added in a round-bottom ask (50 mL) in turn. and the ltrate was collected. 10 mL chloroform was added into
The ask was then immersed in an ultrasonic water-bath and the residue for further extraction. The twice ltrate was united
Table 2 Linearity, correlation coefficient, LOD, LOQ and recovery for the method
Correlation
Fatty acids
Y ¼ AX + B
coefficient
LOD (fmol)
LOQ (fmol)
Recovery (%)
RSD (%)
C5
C6
C7
C8
Y ¼ 32.46X ꢂ 17.60
Y ¼ 31.25X ꢂ 28.20
Y ¼ 32.21X ꢂ 28.34
Y ¼ 26.27X ꢂ 22.43
Y ¼ 23.71X ꢂ 24.20
Y ¼ 26.82X ꢂ 27.24
Y ¼ 22.02X ꢂ 22.25
Y ¼ 24.41X ꢂ 21.55
Y ¼ 24.52X ꢂ 20.60
Y ¼ 27.39X ꢂ 21.33
Y ¼ 25.31X ꢂ 20.35
Y ¼ 31.60X ꢂ 24.53
Y ¼ 24.04X ꢂ 14.70
Y ¼ 25.09X ꢂ 16.94
Y ¼ 23.55X ꢂ 15.91
Y ¼ 24.96X ꢂ 17.23
Y ¼ 24.05X ꢂ 17.01
Y ¼ 24.47X ꢂ 17.88
Y ¼ 23.12X ꢂ 17.81
Y ¼ 24.63X ꢂ 18.82
Y ¼ 21.95X ꢂ 18.38
Y ¼ 21.40X ꢂ 19.05
Y ¼ 20.83X ꢂ 21.39
Y ¼ 23.42X ꢂ 22.61
Y ¼ 20.56X ꢂ 20.36
Y ¼ 16.38X ꢂ 18.60
0.9957
0.9932
0.9971
0.9983
0.9983
0.9985
0.9985
0.9991
0.9992
0.9993
0.9992
0.9993
0.9998
0.9994
0.9994
0.9994
0.9994
0.9994
0.9993
0.9993
0.9993
0.9992
0.9990
0.9991
0.9990
0.9990
8.82
11.06
10.82
13.44
15.76
14.79
18.52
17.28
17.61
16.20
18.52
15.69
21.74
20.78
22.94
21.93
22.59
22.52
23.29
21.80
24.43
24.83
26.79
23.89
30.61
45.45
29.41
36.87
36.08
44.80
52.52
49.31
61.73
57.60
58.69
54.00
61.73
52.30
72.46
69.25
76.45
73.10
75.30
75.08
77.64
72.67
81.43
82.78
89.29
79.62
102.04
151.52
92.24
91.54
89.42
94.49
92.87
87.29
94.60
96.27
92.28
95.40
89.74
92.78
94.00
91.25
90.75
92.54
90.22
87.98
90.42
92.22
91.29
96.42
92.87
92.04
87.49
90.98
4.5
3.9
5.1
4.2
3.1
4.7
2.9
6.5
4.6
5.2
4.9
3.8
3.7
4.1
3.1
3.9
5.3
3.5
6.2
3.6
5.9
6.1
6.3
5.8
6.7
6.2
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
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Fig. 5 Chromatogram of fatty acid derivatives from the extracted ginkgo leaf sample (A) and ginkgo nut sample (B). Chromatographic conditions
and peaks labels are the same with Fig. 4.
and evaporated to dryness under a gentle stream of nitrogen, experiments. Compared to the classical experimental design
the residue was re-dissolved in 1.0 mL of DMF, and ltered method, RSM is more efficient and provides interaction effects
through a 0.2 mm nylon membrane lter. The nal solution was on the response. The optimum conditions could be found by
ꢀ
stored at 4 C until derivatization and HPLC analysis.
a small amount of tests. In this study, three factors including
the amount of TSTPE (V: molar ratio of TSTPE to total free fatty
acids), reaction temperature (T) and reaction time (t) were
selected and optimized by employing a three-level, three-
variable Box–Behnken design (BBD) from RSM. The coded
and uncoded independent variables used in the RSM design
and their respective levels were listed in Table 1. A total of 17
experiments were designed. The total peak area of all fatty
acids was taken as the response. The surface response models
were obtained by tting the data to a polynomial model as
shown below in the following equation.
2.6 Derivatization procedure
20 mg K2CO3 and 400 mL DMF were added into a 2.0 mL vial,
then, 100 mL fatty acids solution and 270 mL TSTPE were added
successively. The vial was sealed and shaken for 1 min, and
then heated at 94 ꢀC for 32.6 min. Aer derivatization, 230 mL
acetonitrile was added into the mixture. The nal solution was
directly injected into the HPLC system for analysis. The
derivatization scheme of TSTPE with fatty acids is shown in
Fig. 1.
Y ¼ b0 + b1V + b2t + b3T ꢂ b12Vt ꢂ b13VT + b23tT + b11V2
+ b22t2 + b33T2
2.7 Experimental design for optimization of derivatization
Based on previous studies,21,40 the labeling reagent with active
sulfonic ester group could easily react with fatty acids in the
presence of basic catalyst and DMF. In this study, K2CO3 was
used as catalyst and DMF was used as reaction medium,
because TSTPE has the same sulfonic ester group as APIETS21
and TSPP.40 In order to ensure the sufficient labeling of the
analyzed components by TSTPE, derivatization parameters
were optimized by response surface methodology (RSM) which
was based on the results obtained by single factor
where Y represents the response variable, b0 is a constant, bi, bii
and bij are the linear, quadratic and interactive coefficients,
respectively.
A soware Design-Expert 8.0.6 (State-Ease, Inc., Minneapolis
MN, USA) was used to obtain the coefficients of the quadratic
polynomial model. The quality of the tted model was
expressed by the determined coefficient (R2), and its statistical
signicance was checked by an F-test.
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Table 3 Analytical results of gingkgo nut sample and gingkgo leaf sample from different areas
Shandong (mg gꢂ1
)
Jiangsu (mg gꢂ1
)
Henan (mg gꢂ1
)
Fatty acids
Gingko nut
Gingko leaf
Gingko nut
Gingko leaf
Gingko nut
Gingko leaf
C5
C6
C7
C8
7.88
12.97
6.17
7.52
11.15
6.47
2.53
5.12
2.57
8.58
6.07
11.02
2.55
5.17
6.21
2.87
1.89
2.91
1.93
5.04
2.82
70.86
2.11
48.68
0.73
2.90
1.26
3.65
1.61
3.24
1.17
2.53
0.97
4.62
1.20
4.59
9.28
17.88
3.78
10.99
14.14
37.63
3.48
4.46
9.12
1.41
4.14
7.96
3.20
1.37
3.05
1.75
5.05
3.9
82.96
6.21
43.37
4.09
2.75
5.93
1.91
2.78
2.91
2.92
6.74
2.11
4.09
1.82
6.98
12.43
18.18
4.48
12.64
12.05
5.40
3.45
5.78
4.06
16.68
14.45
181.59
12.69
108.14
4.59
44.14
14.89
46.67
14.76
50.42
8.03
5.14
9.05
1.82
5.40
6.88
3.61
1.28
3.01
1.61
3.90
3.91
102.8
4.06
50.21
1.86
4.12
2.63
1.76
2.45
2.47
2.65
4.15
1.74
2.67
1.58
4.19
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
4.80
3.65
12.22
10.91
228.92
16.12
112.38
10.94
34.00
15.14
36.56
13.35
24.86
6.61
8.97
176.14
12.15
90.05
5.66
26.11
8.51
34.25
11.72
25.59
5.72
16.23
4.17
12.67
6.39
15.96
3.03
49.35
5.62
17.53
2.67
16.04
3.09
10.50
13.15
5.39
potassium citrate, pyridine, triethylamine, 4-dimethylamino-
pyridine (DMAP) were tested and evaluated for their derivati-
zation efficiency. The strongest uorescence intensity was
obtained by using K2CO3 as catalyst. The effect of the K2CO3
amount on derivatization yield was also tested and maximum
response was obtained when the amount of K2CO3 was 20 mg.
When the amount of K2CO3 was increased to 50 mg, no obvious
increase in response was observed. Therefore, 20 mg of K2CO3
was applied in all subsequent derivatization reactions.
The reaction temperature and time are always the important
factors on the derivative yield. The effect of the reaction
temperature was examined over the range of 60–100 ꢀC. The
uorescence intensity increased with the rise of the tempera-
ture, and the maximum uorescence intensity was obtained at
85 ꢀC. When exceeding this temperature, the decreased
responses and more by-product peaks were observed, while
below 85 ꢀC, the rate of reaction was decreased which led to long
derivatization time. Different reaction time was tested over the
range of 10–60 min to investigate the effect of time on the
derivatization reaction. The maximum uorescence intensity
was obtained at 30 min, and there was little decrease for uo-
rescence intensity when prolonging reaction time.
3. Results and discussion
3.1 Fluorescence spectra of TSTPE and fatty acid derivatives
In order to nd the uorometric detector wavelengths, the
excitation and emission spectra of TSTPE (Fig. 2A) and fatty acid
derivatives (Fig. 2B) were determined at the wavelength range of
200 to 550 nm. Octadecanoic acid (C18) was selected as repre-
sentative fatty acid that was used to study the excitation and
emission spectra of derivatives. As was shown in Fig. 2, the
uorescence spectra of TSTPE-C18 and TSTPE were very similar,
but the excitation spectra of the two compounds changed
a little. There was no obviously different in maximum excitation
and emission wavelength for the two compounds. The uores-
cence spectra of TSTPE-C18 in acetonitrile solution with
different concentrations (50–100%) were also studied to eval-
uate the effect of mobile phase gradient on uorescence prop-
erties. The result (Fig. 2B) showed that the maximum excitation
and emission wavelengths are 314 and 398 nm, respectively.
Therefore, these wavelengths were chosen as detecting wave-
lengths for further experiments. The uorescence intensity of
TSTPE-C18 decreased with the decrease of acetonitrile concen-
tration, and a 40% decrease was found when acetonitrile
concentration varied from 100% to 50%.
The effect of TSTPE concentrations on derivatization was
investigated in detail to ensure sufficient reaction of fatty acids.
The result indicated that constant uorescence intensity was
obtained when ten-fold molar excess to total molar fatty acids,
further increasing TSTPE concentration beyond this level had
no signicant effect on uorescence intensity.
3.2 Single factor experiments
TSTPE can react with fatty acid in the existence of basic catalyst.
Several different basic catalysts including K2CO3, Na2CO3,
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experiments. It is an efficient tool that can decrease labor time
and reagent consumption and includes interaction effects of
derivatization conditions in addition to single-factor effects.
3.3 Response surface methodology
The optimum derivatization conditions including the concen-
tration of TSTPE, derivatization temperature, and derivatization
time were optimized through Box–Behnken design and
response surface analysis. A total of 17 runs were tested, and the
experimental results are shown in Table 1. The analysis of
variance (ANOVA) for the response surface quadratic model
showed that the model was signicant at the level of p < 0.0001;
none of the linear parameters and interaction parameters was
signicant; quadratic parameters were insignicant except
derivatization time. The R2 value was calculated to be 0.9866,
revealing that the experimental data were in good agreement
with the predicted peak area values. The F-value of 2.28 for the
lack of t was insignicant (p > 0.05), indicating that the model
was sufficiently accurate for predicting the relevant responses.
The coefficient of variation (CV%) of less than 5.95 indicates
that the model was reproducible. The nal estimative response
model equation (based on the actual value) is as follows:
3.4 Stability of TSTPE and derivatives
There was no obvious difference for the derivatization yields
when solution of TSTPE in DMF was stored at 4 ꢀC for at least 3
days. The corresponding neutral derivatives solution was stored
at 4 ꢀC for a period of 24 hours, during which time the solutions
were analyzed six times. Results showed that derivatives were
stable enough for accurate HPLC with normalized peak areas
varying <4.5%.
3.5 Solubility of TSTPE
In order to evaluate the solubility of TSTPE in DMF, 10 mg of
TSTPE was added into a 2.0 mL vial, then, a denite proportion
of DMF was added. The vial was treated by ultrasonic for 5 min,
and no solid particle was observed when 200 mL DMF was
added. Therefore, we inferred that TSTPE dissolve in DMF
easily, and the solubility of TSTPE in DMF is >0.1 mol Lꢂ1 which
could well meet the requirement of derivatization.
Y ¼ 14921 + 956V + 2448t + 1125T ꢂ 38Vt ꢂ 262VT + 495tT
ꢂ 2320V2 ꢂ 5169t2 ꢂ 1080T2
Three-dimensional response surfaces (Fig. 3) were plotted on
the basis of this model equation to investigate the interaction
among the variables and to determine the optimum values of
each factor for the maximum derivatization yield. Fig. 3A intu-
itively presents the variations of peak areas with derivatization
time and the molar ratio of TSTPE to fatty acids at a constant
derivatization temperature of 85 ꢀC. Fig. 3B is the response
surface showing the effect of the derivatization temperature and
molar ratio of TSTPE to fatty acids on the response (peak area)
at a xed derivatization time of 30 min. Fig. 3C describes the
effects of different derivatization time and temperature at
a xed TSTPE-to-fatty acid molar ratio of 10 : 1.
3.6 Chromatographic separation
The polarity of fatty acid derivatives decreases when the length
of carbon chain increases, which would increase the retention
time of analytes. In order to shorten analysis time, the separa-
tion of fatty acid derivatives was carried out on a hypersil BDS-
C8 column, rather than C18 column. In order to obtain complete
baseline resolution and good peak shape within shorter time,
gradient elution was utilized with water–acetonitrile as mobile
phase. The chromatogram of twenty-six fatty acid derivatives
was obtained within 50 min (Fig. 4).
The optimal values of the selected variables were obtained by
solving the regression equation using Design-Expert soware.
The optimal derivatization conditions estimated by the model
equation were molar ratio of TSTPE to fatty acid ¼ 10.4, deriva-
tization temperature ¼ 94 ꢀC, and derivatization time ¼ 32.6 min.
According to the results of response surface methodology, the
3.7 Validation parameters
The developed method was validated in terms of linearity, limit
of detection (LOD), limit of quantitation (LOQ) and precision
calculated as relative standard deviation (RSD).
The calibration graph was established with the peak area (Y)
versus fatty acid concentration (X, pmol, injected amount), and
injection amount varied from 100 pmol to 40 fmol. The linear
regression equations were shown in Table 2. All fatty acids were
found to give excellent linear responses over this range, with
correlation coefficients of >0.993. LODs (at signal-to-noise ratio of
3 : 1) were 8.8–45.5 fmol for the labeled fatty acids and LOQs (at
signal-to-noise ratio of 10 : 1) were 29.5–152 fmol. A standard
solution consisting of 25 pmol fatty acid derivatives was prepared
and analyzed to examine the method repeatability. The relative
standard deviations (RSDs; n ¼ 6) for the peak areas was <3.1%. A
high repeatability in the retention time was obtained with RSD
values lower than 0.18%. The recoveries of fatty acids were eval-
uated by the addition of a known amount of standard into the
sample. The sample was treated according to the method as
described above and derivatized with TSTPE. The analysis was
carried out in triplicate. The experimental recoveries were in the
ꢀ
peak area changed little in the range of 90–100 C with RSD ¼
0.37% which means that there is little change for derivatization
yield in this temperature range. The peak area also changed little
in the range of 30–35 min with RSD ¼ 1.4%. When reaction time
was more than 35 min, the peak area dramatically decreased
which maybe caused by the decomposition of derivatives.
Therefore the optimal derivatization procedure is as follows:
20 mg of K2CO3 and 400 mL of DMF are added into a 2.0 mL vial,
and then 100 mL of fatty acid solution and 270 mL of TSTPE are
added successively. The vial is sealed and shaken for 1 min and
ꢀ
then heated at 94 C for 32.6 min. Aer derivatization, 230 mL
acetonitrile is added into the mixture. The nal solution is
directly injected into the HPLC system for analysis.
There are several advantages of RSM over traditional
methods for derivatization optimization based on single-factor
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range of 87.29–96.27% (see Table 2) and the method was suffi- reaction times, simplied sample preparation and higher
ciently accurate for the determination of free fatty acids.
sensitivity.
3.8 Analysis of free fatty acids
Conflicts of interest
The proposed method was applied to determine free fatty acids
extracted from ginkgo nut and ginkgo leaf samples. The ob-
tained chromatogram is shown in Fig. 5, and the analytical
results are listed in Table 3. It was found that the content of fatty
acids with even number carbon atom was much higher than
that of fatty acids with odd number carbon atom, and C16 and
C18 are the main saturated fatty acids in both samples. These
two kinds of fatty acids are widely used in chemical enterprises.
It is clear that the established method is suitable for the
determination of these components from ginkgo nut and
ginkgo leaf with satisfactory results. It can also be used to
determinate free fatty acids in other plant samples.
There are no conicts to declare.
References
1 A. A. Nanji, K. Jokelainen, G. L. Tipoe, A. Rahemtulla and
A. J. Dannenberg, J. Pharmacol. Exp. Ther., 2001, 299, 638–
644.
2 M. J. J. Ronis, S. Korourian, M. Zipperman, R. Hakkak and
T. M. Badger, J. Nutr., 2004, 134, 904–912.
3 H. Iso, M. J. Stampfer, J. E. Manson, K. Rexrode, F. B. Hu,
C. H. Hennekens, G. A. Colditz, F. E. Speizer and
W. C. Willett, Circulation, 2001, 103, 856–863.
´
4 L. I. Mennen, L. Lafay, E. J. M. Feskens, M. Novak, P. Lepinay
3.9 Comparisons of the proposed method with the reported
methods
and B. Balkau, Nutr. Res., 2000, 20, 335–347.
5 M. A. Pereira, D. R. Jacobs, L. Van Horn, M. L. Slattery,
A. I. Kartashov and D. S. Ludwig, JAMA, J. Am. Med. Assoc.,
2002, 287, 2081–2089.
6 C. O. Kangani, D. E. Kelley and J. P. Delany, J. Chromatogr. B:
Anal. Technol. Biomed. Life Sci., 2008, 873, 95–101.
7 S. G. Woo, K. Yoo, J. Lee, S. Bang, M. Lee, K. On and J. Park,
Talanta, 2012, 97, 103–110.
As mentioned above, there were several GC and HPLC
methods reported in other papers for the determination of free
fatty acids. The comparison of those methods with this work is
listed in Table 4. The main advantage of this study is that the
LOD of the proposed method was lower than those of GC,36
GC-MS6,37 and HPLC,12,13,38 which means a higher sensitivity.
The commonly-used detector for HPLC is UV-vis detector.
However, fatty acids show weak UV absorption at the wave-
length of 210 nm nearby, so the LOD of the method for direct
determination of fatty acids without derivatization was only
6.4–9.4 nmol.19 To increase the sensitivity, fatty acids were
usually derivatived by UV39 or uorescence reagent.21,31,40–47
TSTPE was evaluated by comparing with those labeling
reagents used for the derivatization of free fatty acids in other
reports in term of derivatization condition and detection limit
(see Table 4). The LOD for free fatty acids in this study is 8.8–
45.5 fmol which is lower than most other methods. Therefore,
TSTPE can act as a new derivatization reagent with a high
sensitivity for free fatty acids.
8 J. Ecker, M. Scherer, G. Schmitz and G. Liebisch, J.
Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2012, 897,
98–104.
9 M. Vosough and A. Salemi, Talanta, 2007, 73, 30–36.
´
´
10 A. G. Casado, E. J. A. Hernandez and J. L. Vılchez, Water Res.,
1998, 32, 3168–3172.
11 T. Toyo'oka, Anal. Chim. Acta, 2002, 465, 111–130.
12 J. Zhao, S. P. Li, F. Q. Yang, P. Li and Y. T. Wang, J.
Chromatogr. A, 2006, 1108, 188–194.
13 A. Makahleh, B. Saad, G. H. Siang, M. I. Saleh, H. Osman and
B. Salleh, Talanta, 2010, 81, 20–24.
14 C. Orellana-Coca, D. Adlercreutz, M. M. Andersson,
B. Mattiasson and R. Hatti-Kaul, Chem. Phys. Lipids, 2005,
135, 189–199.
ˇ
15 M. Spitsmeister, K. Adamberg and R. Vilu, J. Microbiol.
4. Conclusion
Methods, 2010, 82, 288–295.
In this paper, a new uorescence labeling reagent, toluene-4- 16 J. P. C. L. Lacaze, L. A. Stobo, E. A. Turrell and M. A. Quilliam,
sulfonic acid 2-(2-thiophen-2-yl-phenanthro[9,10-d]imidazol- J. Chromatogr. A, 2007, 1145, 51–57.
1-yl)-ethyl ester (TSTPE), was designed and synthesized. A 17 Y. Ohba, N. Kuroda and K. Nakashima, Anal. Chim. Acta,
simple and highly sensitive method to determine twenty-six 2002, 465, 101–109.
fatty acids was developed utilizing TSTPE as the novel pre- 18 E. Bravi, G. Perretti and L. Montanari, J. Chromatogr. A, 2006,
column derivatization reagent followed by high-performance 1134, 210–214.
liquid chromatography with uorescence detection. TSTPE 19 L. R. V. de Sa, M. A. L. de Oliveira, M. C. Cammarota,
´
˜
was stable enough for the derivatization procedure. The
derivatization reaction was carried out under mild conditions
A. Matos and V. S. Ferreira-Leitao, Int. J. Hydrogen Energy,
2011, 36, 15177–15186.
and the derivative was stable enough for HPLC analysis. The 20 B. Bravo, G. Chavez, N. Pina, F. Ysambertt, N. Marquez and
described method shows good correlation, precision and A. Caceres, Talanta, 2004, 64, 1329–1334.
accuracy which were useful in the analysis of free fatty acids in 21 Z. Sun, J. You, C. Song and L. Xia, Talanta, 2011, 85, 1088–
ginkgo nut and ginkgo leaf. In contrast to the existing methods 1099.
using other derivatization reagents, the method established in 22 G. Li, J. You, Y. Suo, C. Song, Z. Sun, L. Xia, X. Zhao and
this paper has advantage such as shorter derivatization
J. Shi, Food Chem., 2011, 125, 1365–1372.
18558 | RSC Adv., 2018, 8, 18549–18559
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
23 N. Jing, J. Shi, G. Li, Z. Sun and J. You, Food Res. Int., 2012, 37 O. Quehenberger, A. M. Armando and E. A. Dennis, BBA,
48, 155–163.
Biochim. Biophys. Acta, Mol. Cell Biol. Lipids, 2011, 1811,
648–656.
38 H. Yoshida, K. Ureshino, J. Ishida, H. Nohta and
M. Yamaguchi, Anal. Sci., 1999, 15, 937–941.
39 H. Miwa and M. Yamamoto, J. Chromatogr., 1987, 416, 237–
245.
40 J. You, X. Zhao, Y. Suo, Y. Li, H. Wang and G. Chen, J.
Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2007, 848,
283–291.
24 T. Yoshida, A. Uetake, H. Yamaguchi, N. Nimura and
T. Kinoshita, Anal. Biochem., 1988, 173, 70–74.
25 M. A. Quiliam, A. Gago-martınez and J. A. Rodrıguez-
´
´
´
Vazquez, J. Chromatogr. A, 1998, 807, 229–239.
26 Y. Amet, F. Berthou and J. F. Menez, J. Chromatogr. B:
Biomed. Sci. Appl., 1996, 681, 233–239.
27 K. Sasamoto, T. Ushijima, M. Saito and Y. Ohkura, Anal. Sci.,
1996, 12, 189–193.
28 P. Pradosa, T. Fukushima, T. Santa, H. Homma, M. Tsunoda, 41 Y. Tsukamoto, T. Santa, H. Saimaru, K. Imai and T. Funatsu,
S. Al-Kindy, S. Mori, H. Yokosu and K. Imai, Anal. Chim. Acta,
1997, 344, 227–232.
29 S. Uchiyama, T. Santa and K. Imai, Anal. Chem., 2001, 73,
Biomed. Chromatogr., 2005, 19, 802–808.
42 X. Du, H. Zhang, X. Guo, Y. Deng and H. Wang, J.
Chromatogr. A, 2007, 1169, 77–85.
2165–2170.
43 Z. Zeng, Z. Ji, N. Hu, B. Bai, H. Wang and Y. Suo, J.
Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2017, 1064,
151–159.
30 J. You, W. Zhang and Y. Zhang, Anal. Chim. Acta, 2001, 436,
163–172.
31 X. Chen, X. Dong and J. You, Chromatographia, 2010, 72, 44 M. Jonnada, G. D. E. Rassi and Z. E. Rassi, Electrophoresis,
171–175. 2017, 38, 1592–1601.
32 Y. Cao and Y. Suo, J. Food Compos. Anal., 2010, 23, 100–106. 45 Y. Sun, X. Zhang, Z. Ji, C. Song, Z. Sun and J. You,
33 J. H. Wolf and J. Korf, J. Pharm. Biomed. Anal., 1992, 10, 99–
Chromatographia, 2016, 79, 547–559.
46 M. B. Majnooni, B. Mohammadi, R. Jalili, A. Babaei and
G. Bahrami, J. Liq. Chromatogr. Relat. Technol., 2016, 39,
877–881.
107.
34 T. Rezanka and J. Votruba, Anal. Chim. Acta, 2002, 465, 273–
297.
35 T. A. van Beek and P. Montoro, J. Chromatogr. A, 2009, 1216, 47 M. Nishikiori, H. Iizuka, H. Ichiba, K. Sadamoto and
2002–2032.
T. Fukushima, J. Chromatogr. Sci., 2015, 53, 537–541.
36 K. Takahashi and N. Goto-Yamamoto, J. Chromatogr. A, 2011,
1218, 7850–7856.
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