A method for determination of
There are few methods for determination of BCA and GCA in
Seeds were collected from eight
DEEMM, BCA,
The HPLC system (Beckman-Coulter) consisted of a 126 solvent module, 166 detector, and IBM personal computer. Data acquisition and processing were carried out using 32 Karat 7.0 version software (Beckman-Coulter). Samples (20
Chromatographic gradient conditions for the analysis of GCA, BCA, and free amino acids.
Time (min) | Eluent Aa | Eluent Bb |
---|---|---|
0 | 96 | 4 |
3 | 88 | 12 |
13 | 88 | 12 |
30 | 69 | 31 |
35 | 69 | 31 |
40 | 96 | 4 |
bAcetonitrile.
Samples (2.0 mg) were stirred in ethanol : water (3 : 7 v/v, 1 mL) for 30 min at room temperature and centrifuged at 12000 rpm for 10 min. Pellets were reextracted twice more, and the resulting supernatants were pooled and taken to dryness under nitrogen.
Internal standard (24
Evaluation was carried out by determination of linearity, limit of detection (LOD), limit of quantification (LOQ), repeatability, and accuracy (recovery) [
The RSD was calculated according to the formula
Like other
Precolumn derivatization of BCA, GCA, and standard amino acids by reaction with DEEMM resulted in stable derivatives with a very good chromatographic behavior in reversed-phase HPLC. These derivatives were readily detected at 280 nm with low detection limits and with no interference from the reagent. Most of these components were identified in the
HPLC analysis of the DEEMM derivatives of GCA, BCA, and amino acid standards (a) and seed extracts (b). 1 = Asp; 2 = Glu; 3 = GCA; 4 = Asn; 5 = Ser; 6 = Gln; 7 = His; 8 = Gly; 9 = Thr; 10 = Arg; 11 = Ala; 12 = BCA; 13 = Pro; 14 = I.S.; 15 = Tyr; 16 = ammonium ion; 17 = Val; 18 = Met; 19 = Cys-Cys; 20 = Ile; 21 = Trp; 22 = Leu; 23 = Phe; 24 = Cys; 25 = Lys.
Analysis of 0.50 to 200
Precision, calibration parameters, and sensitivity of the determination of GCA and BCA by HPLC.
Compound | Repeatability (%) | Calibration | LOQd ( |
LODe ( | |||
---|---|---|---|---|---|---|---|
Intradaya | Interdayb | Regression equation |
|
Linear rangec ( | |||
GCA | 0.28 | 2.76 |
|
0.9998 | 0.50–200 | 0.50 | 0.15 |
BCA | 0.31 | 3.08 |
|
0.9997 | 0.50–200 | 0.50 | 0.15 |
bRSD of peak area based on 8 runs of a solution of the standard on independent days.
cConcentration range between the limit of quantification and the upper linear limit.
dLimit of quantification: signal/noise ratio = 10.
eLimit of detection: signal/noise ratio = 3.
Recovery (mean and RSD) of the HPLC method for determination of GCA and BCA in
Compound |
Initial content ( |
First concentration added (10 |
Second concentration added (30 |
Third concentration added (90 | |||
---|---|---|---|---|---|---|---|
Content I ( |
Recovery | Content II ( |
Recovery | Content III ( |
Recovery | ||
GCA | 37.83 | 47.81 | 99.83 (0.76) | 67.82 | 99.97 (0.16) | 127.67 | 99.82 (0.33) |
BCA | 0.61 | 10.58 | 99.67 (0.55) | 30.56 | 99.82 (0.20) | 90.53 | 99.91 (0.09) |
This method was applied to the determination of BCA and GCA in the seeds of eight
Contents (% w/w of dry weight) of GCA, BCA, and free amino acids in
Compound/sample | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
---|---|---|---|---|---|---|---|---|
GCA | 0.887 ± 0.008 | 1.071 ± 0.021 | 0.572 ± 0.015 | 1.029 ± 0.031 | 1.134 ± 0.020 | 0.946 ± 0.027 | 1.252 ± 0.018 | 1.010 ± 0.075 |
BCA | 0.003 ± 0.000 | 0.005 ± 0.000 | 0.006 ± 0.000 | 0.017 ± 0.001 | 0.022 ± 0.001 | 0.007 ± 0.001 | 0.009 ± 0.000 | 0.003 ± 0.000 |
Asp | 0.094 ± 0.002 | 0.029 ± 0.002 | 0.016 ± 0.001 | 0.013 ± 0.001 | 0.029 ± 0.000 | 0.013 ± 0.001 | 0.031 ± 0.002 | 0.027 ± 0.001 |
Glu | 0.036 ± 0.000 | 0.058 ± 0.002 | 0.081 ± 0.002 | 0.083 ± 0.003 | 0.082 ± 0.005 | 0.040 ± 0.001 | 0.099 ± 0.003 | 0.066 ± 0.000 |
Asn | 0.010 ± 0.001 | 0.031 ± 0.003 | 0.189 ± 0.003 | 0.045 ± 0.002 | 0.045 ± 0.000 | 0.021 ± 0.002 | 0.048 ± 0.004 | 0.046 ± 0.002 |
Ser | 0.010 ± 0.001 | 0.007 ± 0.000 | 0.006 ± 0.000 | 0.005 ± 0.000 | 0.006 ± 0.000 | 0.005 ± 0.000 | 0.007 ± 0.001 | 0.006 ± 0.000 |
Gln | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 |
His | 0.009 ± 0.001 | 0.006 ± 0.000 | 0.037 ± 0.001 | 0.003 ± 0.000 | 0.002 ± 0.000 | 0.014 ± 0.001 | 0.013 ± 0.002 | 0.013 ± 0.001 |
Gly | 0.009 ± 0.000 | 0.007 ± 0.000 | 0.007 ± 0.000 | 0.006 ± 0.001 | 0.006 ± 0.000 | 0.001 ± 0.000 | 0.005 ± 0.001 | 0.002 ± 0.000 |
Thr | 0.065 ± 0.005 | 0.140 ± 0.003 | 0.014 ± 0.001 | 0.071 ± 0.003 | 0.028 ± 0.002 | 0.005 ± 0.001 | 0.007 ± 0.001 | 0.012 ± 0.001 |
Arg | 0.072 ± 0.001 | 0.098 ± 0.009 | 0.301 ± 0.012 | 0.074 ± 0.012 | 0.157 ± 0.017 | 0.059 ± 0.002 | 0.124 ± 0.006 | 0.026 ± 0.003 |
Ala | 0.007 ± 0.001 | 0.008 ± 0.001 | 0.014 ± 0.001 | 0.009 ± 0.001 | 0.014 ± 0.001 | 0.010 ± 0.001 | 0.012 ± 0.002 | 0.010 ± 0.001 |
Pro | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 |
Try | 0.003 ± 0.000 | 0.002 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.002 ± 0.000 | 0.002 ± 0.000 | 0.003 ± 0.001 | 0.002 ± 0.000 |
Val | 0.009 ± 0.001 | 0.007 ± 0.001 | 0.007 ± 0.000 | 0.013 ± 0.001 | 0.004 ± 0.000 | 0.003 ± 0.000 | 0.003 ± 0.001 | 0.004 ± 0.001 |
Met | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 |
Cys-Cys | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 |
Ile | 0.047 ± 0.001 | 0.002 ± 0.000 | 0.005 ± 0.000 | 0.005 ± 0.000 | 0.000 ± 0.000 | 0.001 ± 0.000 | 0.009 ± 0.000 | 0.009 ± 0.001 |
Trp | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 |
Leu | 0.011 ± 0.001 | 0.014 ± 0.000 | 0.020 ± 0.001 | 0.007 ± 0.000 | 0.011 ± 0.000 | 0.002 ± 0.000 | 0.007 ± 0.000 | 0.019 ± 0.001 |
Phe | 0.003 ± 0.000 | 0.003 ± 0.000 | 0.007 ± 0.001 | 0.005 ± 0.000 | 0.007 ± 0.000 | 0.008 ± 0.000 | 0.004 ± 0.000 | 0.008 ± 0.000 |
Cys | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 | 0.000 ± 0.000 |
Lys | 0.003 ± 0.000 | 0.003 ± 0.000 | 0.004 ± 0.001 | 0.003 ± 0.000 | 0.004 ± 0.000 | 0.004 ± 0.001 | 0.004 ± 0.000 | 0.002 ± 0.000 |
As compared to other HPLC methods previously reported for determination of BCA and GCA [
Reverse phase HPLC of DEEMM derivatives allows for determination of BCA, GCA, and other free amino acids in the seeds of
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors have contributed equally to this work.
This work was carried out with the financial support of Junta de Andalucía (Spain) to the Laboratory of Bioactive and Functional Components of Plant Products (Instituto de la Grasa, C.S.I.C.). Cristina Megías and Isabel Cortés-Giraldo are recipients of a JAE-Doc (C.S.I.C.) contract and a JAE-Pre (C.S.I.C.) fellowship from the “Junta para la Ampliación de Estudios” program (cofinanced by the European Social Fund), respectively. Thanks are due to María Dolores García Contreras for technical assistance.