TITLE: Binary HPLC method fot determination of 4-hydroxybenzoic acid (4-HBA), chlorogenic acid (CGA), quercetin (QUE), and salicilic acid (SAL)
AUTHORS: Mariusz Krzysztof Janczur, Emilio González Camarena

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>We cut 1 g of each sample in such way that it contained the cuticle from one side of the sample. We homogenized it with 35 ml of the methanol 100%, and left them in ultrasonic 6 l bath during 30 min at room temperature. Methanol extracts obtained were filtered, placed in amber bottles to avoid degradation by light, and stored at –20°C until processed. We used Waters 717 liquid chromatograph with autosampler, Waters 2487 HPLC Absorbance UV-Vis Detector, Waters 1525 Binary HPLC Pump, Waters control module with SAT/IN Bus (Waters, Milford, MA, USA), Symmetry HPLC C18 column (particle size 5 µm, length 250 mm, internal Ø = 4.6 cm; Waters, Milford, MA, USA). We filtered the extracts with 0.45 µm pore size nylon-membrane filter. To obtain the calibration curves we used standards for salicylic acid (SAL), 4-hydroxybenzoic acid (4-HBA), chlorogenic acid (CGA), and quercetin (QUE), all supplied by Sigma-Aldrich. The mobile phase consisted of 0.1% v/v acetic acid (A) together with 100% acetonitrile (B). For the mobile phase A, we dissolved 1ml of glacial acetic acid with HPLC water, until 1 L. For the mobile phase B we used 100% acetonitrile. We filtered both mobile phases with 0.45 µm nylon membrane. We degasified them with ultrasonic bath during 30 min. We put the column temperature at 25°C, used the 254 nm UV detector, established the flow of the mobile phase at 0.2–0.8 mL/min, injection volume at 8 µL, and run time at 35 min. To wash the piston seals we used MeOH : H</span><span style = "vertical-align:sub;">2</span><span>O (60 : 50). To quantify the four compounds, we used the calibration curves prepared out of 10 mg stock solution in 5 mL of MeOH. We took known concentrations from these solutions to obtain the relationship between the area corresponding to absorbance and concentration of each metabolite.</span></div></div>

[STEPS]
?. [Extraction of secondary metabolites ]
We cut 1 g of each sample in such way that it contained the cuticle from one side of the sample. We homogenized it with 35 ml of the methanol 100%, and left them in ultrasonic 6 l bath during 30 min at room temperature.
[methanol]
[methanol]
?. [Filtration of mobile phases]
We used 0.45 µm nylon membrane.
?. [Degasification of mobile phases]
We degasified them with ultrasonic bath during 30 min.
?. [Column temperature]
We put the column temperature at 25°C
?. [UV detection light frequency]
We used the 254 nm UV detector
?. [Mobile phaase flow rate]
ABCD1Time
Flow rate (ml/min)
%A (acetic acid)
%B (acetonitrile)
20.00
0.30
80.0
20.0
35.30
0.30
70.0
30.0
411.00
0.20
65.0
35.0
513.00
0.20
50.0
50.0
613.30
0.20
40.0
60.0
715.00
0.20
30.0
70.0
816.00
0.30
40.0
60.0
917.00
0.70
50.0
50.0
1019.00
0.70
65.0
35.0
1122.0
0.80
70.0
30.0
1230.0
0.80
80.0
20.0
1335.0
0.80
80.0
20.0
ABCD1Time
Flow rate (ml/min)
%A (acetic acid)
%B (acetonitrile)
20.00
0.30
80.0
20.0
35.30
0.30
70.0
30.0
411.00
0.20
65.0
35.0
513.00
0.20
50.0
50.0
613.30
0.20
40.0
60.0
715.00
0.20
30.0
70.0
816.00
0.30
40.0
60.0
917.00
0.70
50.0
50.0
1019.00
0.70
65.0
35.0
1122.0
0.80
70.0
30.0
1230.0
0.80
80.0
20.0
1335.0
0.80
80.0
20.0
?. [Mobile phase composition]
The mobile phase consisted of 0.1% v/v acetic acid (A) together with 100% acetonitrile (B).For the mobile phase A, we dissolved 1ml of glacial acetic acid with HPLC water, until 1 L. For the mobile phase B we used 100% acetonitrile.
?. [Calibration curves]
To quantify the four compounds, we used the calibration curves prepared out of 10 mg stock solution in 5 mL of MeOH. We took known concentrations from these solutions to obtain the relationship between the area corresponding to absorbance and concentration of each metabolite. We obtained the fallowing calibration curves:yi = 1109.4xi + 481.67,yi = 296.01xi + 133.74,yi = 551.41xi + 263.64, andyi = 919.96xi + 201.64,where yi – area below the absorbance curve, xi – concentration of the secondary metabolite, and i = 1, 2, 3 or 4 for 4-hydroxybenzoic acid (4-HBA), chlorogenic acid (CGA), quercetine (QUE), or salicilic acid (SAL), respectively.
?. [Calibration curves]