A new fluorescent derivatization reagent and its application to free fatty acid
analysis in pomegranate samples using HPLC with fluorescence detection
Aihong Wang a,b, Guoliang Li b*, Jinmao You a,b*, Zhongyin Ji a,c
a
Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau
Biology, Chinese Academy of Sciences, Xining, PR China;
b
Key Laboratory of Life-Organic Analysis of Shandong Province, Qufu Normal
University, Qufu, PR China;
c
University of Chinese Academy of Sciences, Beijing 100049, PR China;
* To whom correspondence should be addressed; Tel: 86-537-4456305; fax:
86-537-4458301 E-mail: jmyou6304@163.com (J.M.You); 61254368@163.com
(G.L.Li)
Synthesis
of
2-(7-methyl-1H-pyrazolo-[3,4-b]quinoline-1-yl)ethyl-4-methyl
benzenesulfonate (MBP)
1) Synthesis of 7-methyl-1H-pyrazolo[3,4-b]quinoline
2.33 g 2-chloro-7-methylquinoline-3-carbaldehyde, 85% hydrazine hydrate (2.3
mL) and 1 g p-toluenesulfonic acid were fully mixed in a 100 mL round- bottom flask.
The mixture of the flask reacted for 40 min at 130°C, and the solid was washed with
water after reaction. The resume was extracted two times with diluted hydrochloric
acid and then combined filtrates. PH of solution was adjusted to 7-8 with sodium
hydroxide and filtrated to get precipitated solid. The pure product was recovered by
drying and recrystallization from methanol solution. The yield was 92%, mp.
229.5°C-230.2°C. Found, C 72.11, H 4.95; Calculated, C 72.08, H 4.92; IR (KBr),
1625.27 (C=N) 1439.69 (-CH3); 930.73, 905.21, 853.49, 773.78; MS: m/z [M+H]+,
184.1
2) Synthesis of 2-(7-methyl-1H-pyrazolo-[3,4-b]quinoline-1-yl)ethanol
5 g 7-methyl-1H-pyrazolo[3,4-b]quinoline, 4.3 g ethylene carbonate and a trace
of KOH were added to a 500 mL round-bottom flask containing 100 mL DMF and
then rapidly heated to reflux with stirring for 1 h. when the reaction was accomplished,
the solution was concentrated using a rotary evaporator under the condition of oil bath
heating. The resume was washed three times with water, then dried and recrystallized
from methanol solution to produce a yellow needle crystal (yield: 80%, mp:
153.3°C-153.7°C). Found，C 68.70, H 5.77, N 18.49; Calculated, C 68.74, H 5.75, N
18.42; IR (KBr), 3264.60 (-OH); 2959.77, 2902.47 (C-H); 1616.82 (C=N); 1446.66
(-CH3); 1067.95 (-CH2-OH); 906.9, 824.9, 781.19; MS: m/z [M+H]+, 228.2.
3)
Synthesis
of
2-(7-methyl-1H-pyrazolo-[3,4-b]quinoline-1-yl)ethyl-4-methyl
benzenesulfonate
To a solution containing 3.7 g paratoluensulfonyl chloride and 30 mL anhydrous
pyridine (0°C) in a 100 mL round-bottom flask equipped with magnetic stirring and a
constant
pressure
drop
funnel,
5
g
2-(7-methyl-1H-pyrazolo-[3,4-b]quinoline-1-yl)ethanol dissolved in 50 mL pyridine
solution was then added dropwise within 30 min. After stirring at 0 ºC for 4 h, the
contents were kept at ambient temperature for another 4 h period with vigorous
stirring. After reaction, the solution was concentrated using a rotary evaporator. The
residue was washed with water and then dried to produce the yellow solid. The crude
products were recrystallized two times from methanol solution to give the yellow
crystal (yield: 70%, mp: 117-118 ºC). Found, C 62.97, H 5.02, N 11.02, S 8.41;
Calculated, C 62.94, H 5.09, N 11.08, S 8.35; IR (KBr), 1619.2 (C=N); 1448.3 (-CH3),
1361.81 (C-SO2-); 1189.03, 987.93 (ph-S-); 766.79, 666.17, 554.01; MS: m/z [M+H]+,
382.1
Table S1 Comparison of the proposed method with other methods
Analytes
Derivatization
reagent
Derivatization
conditions
Method
LOD
Ref.
Pheromone gland
extracts
DMDS
Methanol containing 5% HCL,
65 °C, 4 h
Capillary
GC-MS-EI
(oven temperature up to
250 °C)
20 - 40 ng/mL
[18]
Conjugated fatty
acid
methyl
esters
MTAD
Dichloromethane, 0°C, 10 s
GC–MS
(oven
temperature up to 350 ºC)
Human serum
AMMP
C3–C20 saturated
fatty acids
BrDMEQ
Human serum
9-AP
Benzene solution
45 min at 70 ºC
CLA-rich soy oil
PTAD
Room temperature for 15 min,
the solvent was evaporated.
Nitraria
tangutorum seed
oil
BDETS
K2CO3, DMF, 90 ºC,
30 min
Notopterygiumfo
rbesii Boiss
CPMS
K2CO3, DMF, 90 ºC,
30 min
Pomegranate
MPB
K2CO3, DMF, 90 ºC,
30 min
BMP and CMP in
50 °C ,30 min
Acetone or toluene,
K2CO3, 18-crown-6,
60 ºC, 30–60 min
CAN,
LC/ESI-MS
HPLC-FLD,
λex = 370 nm,
λem = 450 nm
HPLC-FLD,
λex = 303nm,
λem = 376 nm
LC/ESI-MS/MS
HPLC-FLD,
λex = 333 nm,
λem = 390 nm
HPLC-FLD,
λex = 293 nm,
λem = 360 nm
HPLC-FLD,
λex = 245 nm,
λem = 410 nm
λex : excitation wavelength ; λem : emission wavelength
DMDS : dimethyl disulfide
PTAD : 4-Phenyl-1,2,4-triazoline-3,5-dione
MTAD: 4-methyl- 1,2,4-triazoline-3,5-dione
AMMP : 3-acyloxymethyl-1-methylpyridinium iodide
BDETS: 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-p-toluenesulfonate
BrDMEQ: 3-Bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone
9-AP : 9-aminophenanthrene
CPMS: 1-(9H-carbazol-9-yl) propan-2-yl-methanesulfonate
MPB: 2-(7-methyl-1H-pyrazolo-[3,4-b]quinoline-1-yl)ethyl-4-methyl benzenesulfonate
[19]
1.0 - 4.0 nM
(14.32-27.52 ng/mL)
[20]
10 - 15 fmol/10 µL
(0.12 - 0.46 ng/mL)
[21]
10 - 15 pmol/10µL
(17.2 - 45.6 ng/mL)
[22]
18 - 930 fmol/15µL
(0.53 - 17.30 ng/mL)
[23]
3.39 - 6.57 nmol/L
(8.58 - 18.53 ng/mL)
[24]
1.6 - 15.6 ng/mL
[25]
0.41 - 2.5 ng/mL
this
work