Profiling Analysis of 15 Prominent Naturally Occurring
Profiling Analysis of 15 Prominent Naturally Occurring Phenolic Acids by LC-MS Leo Wang, Stacy Henday, Xiaodong Liu, and Bill Schnute, Dionex Corporation, Sunnyvale, CA, USA
INTRODUCTION
INSTRUMENTATION
Phenolic acids have attracted interests for decades. They serve various roles in plant life, from structural to protective.1 The naturally occurring phenolic acids share the frame structure of hydroxycinnamic acid or hydroxybenzoic acid and are present in many foods and plants. Research interests also arise for food quality since they are associated with color, sensory qualities, and nutritional and antioxidant properties.2 Recent interest has focused on their antioxidant properties and potential health implications.
HPLC:
P680 dual ternary pump ASI-100 autosampler TCC-100 column temperature compartment UVD340U detector
Mass Spectrometer:
MSQ™ Plus single quadruple mass spectrometer with electrospray ionization (ESI) interface
Software:
Chromeleon® (6.8, SP3) Chromatography Management System
Xcalibur™ (1.4)
Methods for quantitative analysis of phenolic acids include gas chromatography (GC) and liquid chromatography (LC) with or without mass spectrometric (MS) detection. Due to their low volatilities, GC methods involve derivatization, which could be challenging for the determination of analytes in complex matrices. LC methods generally use reversedphase (RP) columns with low pH mobile phases to achieve retention, and usually suffer from problems of sensitivity and/or resolution. The use of MS detection for LC enhances the selective capability for traclevel detection and identity confirmation. However, the mobile phase condition (low pH, low organic composition) for general reversed-phase separations is not ideal for MS detection. This study introduces a mixed-mode LC method to retain and resolve 15 prominent naturally-occurring phenolic acids with a mild, acidic (pH 4.6), high organic composition mobile phase to improve MS detection.
CHROMATOGRAPHIC CONDITIONS Column:
Acclaim® Mixed-Mode WAX-1 (150 × 2.1 mm, 5 µm)
Column Temperature:
50 °C
Mobile Phase:
A: CH3CN B: NH4OAc 200 mm pH 4.6 C: H2O
Flow Rate:
0.5 mL/min
Injection Volume:
5 µL
HPLC 2008 Presentation
83% 5% 12%
MASS SPECTROMETRIC CONDITIONS
STANDARD PREPARATION
Ionization Interface:
Electrospray ionization (ESI)
Detection Mode:
Selected ion monitoring (SIM)
Needle Temperature:
500 °C
Needle Voltage:
2000 V
All phenolic acids were obtained from Sigma-Aldrich in either pure organic or sodium salt form. Each standard was weighed and dissolved in methanol/water (50/50, v/v) to the concentration of 1000 ppm (µg/mL). The 15 stock solutions were then mixed in acetonitrile to 50 ppm as a primary standard mixture.
Cone Voltage:
60 V
Span:
0.5 amu
Dwell Time:
0.2 s for each SIM
N2 Nebulizer Gas Pressure:
80 psi
Scan Events:
See Table 1 for details.
A 1 to 10 dilution of the primary standard mixture in mobile phase was used as a working solution for method development. Calibration standards were prepared by series dilution from the primary standard mixture to 9 levels: 5000, 2000, 1000, 500, 200, 100, 50, 20, and 10 ppb.
Table 1. SIM Scan Event, Method Linearity, and Detection Limits Name
F.W.
R.T. min
SIM m/z
SIM Time Window min
Linearity
Cal. MDL pg
Sinapic acid
224.21
5.6
223.1
5.0–7.0
0.25
25.0
0.9990
157
Ferulic acid
194.18
6.2
193.1
5.0–7.0
0.05
25.0
0.9992
49.3
Veratric acid
182.17
6.7
181.1
6.0–7.5
0.25
25.0
0.9998
235
p-coumaric acid
164.16
7.6
163.1
6.5–8.5
0.05
25.0
0.9990
11.3
Vanillic acid
168.15
7.6
167.0
7.0–9.0
0.25
25.0
0.9959
65.1
Syringic acid
198.17
7.6
197.1
6.5–8.5
0.10
25.0
0.9945
31.4
p-hydroxybenzoic acid
138.12
8.3
137.1
7.0–9.0
0.10
25.0
0.9998
9.00
Cinnamic acid
148.16
8.8
147.1
8.0–10.0
0.50
25.0
0.9981
364
Benzoic acid
122.12
10.3
121.0
9.5–11.0
1.00
25.0
0.9991
540
o-coumaric acid
164.16
11.1
163.1
10.0–12.5
0.10
25.0
0.9975
41.3
m-coumaric acid
164.16
11.9
163.1
10.0–12.5
0.05
25.0
0.9998
24.2
Caffeic acid
180.16
11.9
179.0
11.0–13.5
0.25
25.0
0.9986
476
Salicylic acid
138.12
12.5
137.1
11.5–14.0
0.05
5.0
0.9950
34.2
Protocatechuic acid
154.12
14.5
153.0
13.0–18.0
0.25
25.0
0.9992
243
Gentisic acid
154.12
16.8
153.0
13.0–18.0
0.05
5.0
0.9952
26.3
2
Profiling Analysis of 15 Prominent Naturally Occurring Phenolic Acids by LC-MS
RESULT AND DISCUSSION
Chromatography and Buffer pH
Chromatography and Organic Solvent
The pH of buffer solutions in mobile phase is one of the key parameters to adjust the chromatographic behavior of organic acids. Adjusting buffer pH determines the charge of organic acids, thus determining the anion exchange retention. Generally, decreasing buffer pH switches organic acids towards neutral form, suppresses the anionic exchange retention, and reduces the overall retention. Selectivity for particular organic acids could be significantly affected by tuning buffer pH. As shown in Figure 1, a pH change from 4.6 to 4.3 does not affect the retention of salicylic acid (pKa = 2.98). However, with the same pH change, retention was significantly suppressed for sinapic acid (pKa ~ 4.6) . Figure 2 shows the retention pH dependence of three organic acids. The retention of gentisic and sinapic acids showed strong dependence on buffer pH: increased mobile phase pH promoted overall retention. On the other hand, the retention of caffeic acid remained relatively unaffected.
Although mixed-mode chromatography features an additional anionicexchange retention mechanism, the linear solvent strength theory for reversed-phase chromatography still supplies an appropriate approximation. Figure 1 shows Logk-%CH3CN plots of sinapic acid (the first eluted peak) and salicylic acid (a late eluted peak). Increase of mobile phase strength by increasing organic solvent composition reduces the retention of organic acids.
logk and % Organic 1.6
Salicylic Acid pH 4.6 Sinapic Acid pH 4.6 Sinapic Acid pH 4.3 Salicylic Acid pH 4.3
1.4 logk and Buffer pH 1.4 Gentisic Acid 1.2
Sinapic Acid
logk
Caffeic Acid 1.2
1.0
1.0 logk
0.8
0.8 0.6
0.6 0.4
40
50
60
70 % CH3CN
80
90 25349
Figure 1. Effect of organic solvent composition on retention under different pH conditions. Mobile phase: varying %CH3CN with constant ammonium acetate concentration at 20 mM. Column temperature: 40 °C. Flow rate: 0.5 mL/min.
0.4
0.2 3.7
3.9
4.1
4.3
4.5 Buffer pH
4.7
4.9
5.1 25350
Figure 2. Effect of buffer pH on retention. Mobile phase: 80% CH3CN and 20 mM ammonium acetate. Column temperature: 40 °C. Flow rate: 0.5 mL/min.
HPLC 2008 Presentation
3
Chromatography and Buffer Concentration
Mass Spectrometry
Buffer concentration determines the eluent strength for ion exchange chromatography and can be used to adjust total chromatographic run time. A chromatographic selectivity change was not observed when buffer concentration was increased from 10 mM to 20 mM but total run time was reduced to 60%.
Chromatographic separation was carefully controlled to resolve most phenolic acids, especially phenolic acid isomers, such as p-, m- and o-coumaric acids and p-hydroxybenzoic acid and salicylic acid. Phenolic acids predominantly form de-protonated ions [M-H]– in negative ionization mode and were used for quantification. Optimized cone voltage ranged from 40 to 70 volts and 60 volts was selected for every phenolic acid for method simplicity.
Chromatography and Column Operating Temperature Column temperature is often used to reduce HPLC system backpressure, reduce chromatographic run time, and maintain system reproducibility. Column temperature is also an important parameter affecting selectivity in mixed-mode chromatography. Figure 3 shows the temperature dependence of retention for 5 organic acids. As a practical approach, logk was plotted against temperature instead of 1/T. The retention of gentisic and caffeic acid remained relatively constant, while three other acids showed decreasing retention with increasing column temperature. Column temperature was set at 50 °C to aid in resolving critical pairs, such as o-coumaric acid and m-coumaric acid which have the same mass and can not be resolved by mass spectrometry.
Linearity and Method Detection Limits (MDL) Linear response was observed for each analyte over two orders of magnitude with correlation coefficient greater than 0.99. Details are shown in Table 1. Seven replicated injections of calibration standard at 500 ppb or 50 ppb were performed and were used for MDL calculations. MDL was calculated by the equation: MDL = t99% × S(n = 7) Where t is Student’s t at 99% confidence intervals (t99%, n=7 = 3.143) and S is the standard deviation. Calculated MDLs range from 9.0 pg (p-hydroxybenzoic acid) to 540 ng (benzoic acid) per injection. Details are shown in Table 1. The calibration curve for o-coumaric acid is shown in Figure 4; the insert shows the calibration curve at the lower range.
logk and Column Temperature 1.55 Gentisic Acid 3.5E4
p-hydroxybenzoic
m-coumaric acid 0.05 ~ 25 ng (5 µL) R2 = 0.9998
Cinamic Acid
1.45
Caffeic Acid Area (counts*min)
m-coumaric
logk
1.35
2.0E3 1.0E3
1.25
0 0 0
1,000
2,000
1.15
20
25
30
35
40
45
Column Temperature (°C)
50
55
60 25351
Figure 3. Effect of column temperature on retention. Mobile phase: 84% CH3CN and 20 mM ammonium acetate, pH 4.6. Flow rate: 0.5 mL/min.
4
0
100
200
300
3,000 4,000 Amount (ppb = ng/mL)
400 5,000
550 6,000 25352
Figure 4. Calibration curve of m-coumaric acid. Calibration standards range from 10 ppb to 5000 ppb; injected amounts range from 0.05 to 25 ng with 5 µL injection.
1.05
0.95
SIM = 163.1 m/z
3.5E3
Profiling Analysis of 15 Prominent Naturally Occurring Phenolic Acids by LC-MS
SIM Chromatograms
6
2
3
7
8 12
5 4
600
15
13 14
10 9
1
UV, WVL: 225 nm
mAU 11
0 40 1
2
3 7
2 # 1. 2. 3. 4. 5. 6. 7. 8.
4
6
Name Sinapic acid Ferulic acid Veratric acid p-coumaric acid Vanillic acid Syringic acid p-hydroxybenzoic acid Cinnamic acid
8
11,12 9 10 13
8 10 12 Retention Time (min) R.T. 5.7 6.2 6.7 7.3 7.5 7.5 8.1 8.8
4
6
8
10
p-hydroxybenzoic
4,5,6
Absorbance (mAU) –1
2
SIM 223.1 193.1 181.1 163.1 167.0 197.1 137.1 147.1
# 9. 10. 11. 12. 13. 14. 15.
14
16
Name Benzoic acid o-coumaric acid m-coumaric acid Caffeic acid Salicylic acid Protocatechuic acid Gentisic acid
protocatechuic
18
syringic
20
SIM 121.0 163.1 163.1 179.0 137.1 153.0 153.0 25351
Figure 5. SIM and UV chromatograms of 15 predominant naturally occurring phenolic acids. Analytical column: Acclaim Mixed-Mode WAX-1, 150 × 2.1 mm; 83% CH3CN, 5% 200 mM NH4OAc pH 4.6, 12% H2O. Column Temperature: 50 °C. Flow rate: 0.5 mL/min. 5 ng injected for MS SIM detections, 25 ng injected for UV detection. SIM chromatogram is normalized to 100% of the greatest peak in each channel.
Application for Beverage Analysis This method has been evaluated for quantitative analysis of phenolic acids found in beverages. A sample chromatogram of green tea analysis is shown in Figure 6. Green tea solution was injected directly after simple filtration through a 0.45 µm membrane. Eleven phenolic acids were detected with individual content ranging from 2.4 to 109 µg per gram of dry leaves.
16
18
20
MS, SIM*
m-coumaric o-coumaric
ferulic R.T. 10.3 10.9 11.6 11.8 12.5 14.0 16.3
14 gentisic
salicylic
p-coumaric
15
14
12
caffeic sinapic
2
4
6
8
10 12 14 Retention Time (min)
16
18
20 25354
Figure 6. SIM and UV chromatograms of phenolic acids in green tea. Green tea leaves were soaked in hot water for 45 minutes (non-thermostated), the solution was filtered through a 0.45 µm membrane, and 5 µL filtered solution was injection directly for quantification. Calculated amount (µg/g): p-coumaric acid: 11.1; o-coumaric acid: 6.0; m-coumaric acid: 44.1; protocatechuic: 16.9; gentisic: 4.0; caffeic acid: 109; salicylic acid: 11.4; p-hydroxybenzoic acid: 23.3; ferulic acid: 4.5; syringic acid: 8.0; sinapic acid: 2.4. *Each SIM chromatogram is normalized to 100% of the greatest peak in that channel.
CONCLUSION An LC-MS method for quantitative analysis of 15 prominent naturally occurring phenolic acids was developed using mixed-mode liquid chromatography and single-quadrupole mass spectrometry. Parameters affecting chromatographic separation and mass spectrometric detection were explored. Linearity was achieved for each phenolic acid over two orders of magnitude with R2 > 0.99. Method detection limits ranged from 9.0 pg (p-hydroxybenzoic acid) to 540 pg (benzoic acid) per injection.
REFERENCES 1. Robbins, R. J. J. Agric. Food Chem. 2003, 51, 2866–2887. 2. Shahidi, F.; Wanasundara, P. K. Phenolic Antioxidants. Crit. ReV. Food Sci. Nutr. 1992, 32, 67.
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LPN 2062-01 06/08 ©2008 Dionex Corporation
INTRODUCTION
INSTRUMENTATION
Phenolic acids have attracted interests for decades. They serve various roles in plant life, from structural to protective.1 The naturally occurring phenolic acids share the frame structure of hydroxycinnamic acid or hydroxybenzoic acid and are present in many foods and plants. Research interests also arise for food quality since they are associated with color, sensory qualities, and nutritional and antioxidant properties.2 Recent interest has focused on their antioxidant properties and potential health implications.
HPLC:
P680 dual ternary pump ASI-100 autosampler TCC-100 column temperature compartment UVD340U detector
Mass Spectrometer:
MSQ™ Plus single quadruple mass spectrometer with electrospray ionization (ESI) interface
Software:
Chromeleon® (6.8, SP3) Chromatography Management System
Xcalibur™ (1.4)
Methods for quantitative analysis of phenolic acids include gas chromatography (GC) and liquid chromatography (LC) with or without mass spectrometric (MS) detection. Due to their low volatilities, GC methods involve derivatization, which could be challenging for the determination of analytes in complex matrices. LC methods generally use reversedphase (RP) columns with low pH mobile phases to achieve retention, and usually suffer from problems of sensitivity and/or resolution. The use of MS detection for LC enhances the selective capability for traclevel detection and identity confirmation. However, the mobile phase condition (low pH, low organic composition) for general reversed-phase separations is not ideal for MS detection. This study introduces a mixed-mode LC method to retain and resolve 15 prominent naturally-occurring phenolic acids with a mild, acidic (pH 4.6), high organic composition mobile phase to improve MS detection.
CHROMATOGRAPHIC CONDITIONS Column:
Acclaim® Mixed-Mode WAX-1 (150 × 2.1 mm, 5 µm)
Column Temperature:
50 °C
Mobile Phase:
A: CH3CN B: NH4OAc 200 mm pH 4.6 C: H2O
Flow Rate:
0.5 mL/min
Injection Volume:
5 µL
HPLC 2008 Presentation
83% 5% 12%
MASS SPECTROMETRIC CONDITIONS
STANDARD PREPARATION
Ionization Interface:
Electrospray ionization (ESI)
Detection Mode:
Selected ion monitoring (SIM)
Needle Temperature:
500 °C
Needle Voltage:
2000 V
All phenolic acids were obtained from Sigma-Aldrich in either pure organic or sodium salt form. Each standard was weighed and dissolved in methanol/water (50/50, v/v) to the concentration of 1000 ppm (µg/mL). The 15 stock solutions were then mixed in acetonitrile to 50 ppm as a primary standard mixture.
Cone Voltage:
60 V
Span:
0.5 amu
Dwell Time:
0.2 s for each SIM
N2 Nebulizer Gas Pressure:
80 psi
Scan Events:
See Table 1 for details.
A 1 to 10 dilution of the primary standard mixture in mobile phase was used as a working solution for method development. Calibration standards were prepared by series dilution from the primary standard mixture to 9 levels: 5000, 2000, 1000, 500, 200, 100, 50, 20, and 10 ppb.
Table 1. SIM Scan Event, Method Linearity, and Detection Limits Name
F.W.
R.T. min
SIM m/z
SIM Time Window min
Linearity
Cal. MDL pg
Sinapic acid
224.21
5.6
223.1
5.0–7.0
0.25
25.0
0.9990
157
Ferulic acid
194.18
6.2
193.1
5.0–7.0
0.05
25.0
0.9992
49.3
Veratric acid
182.17
6.7
181.1
6.0–7.5
0.25
25.0
0.9998
235
p-coumaric acid
164.16
7.6
163.1
6.5–8.5
0.05
25.0
0.9990
11.3
Vanillic acid
168.15
7.6
167.0
7.0–9.0
0.25
25.0
0.9959
65.1
Syringic acid
198.17
7.6
197.1
6.5–8.5
0.10
25.0
0.9945
31.4
p-hydroxybenzoic acid
138.12
8.3
137.1
7.0–9.0
0.10
25.0
0.9998
9.00
Cinnamic acid
148.16
8.8
147.1
8.0–10.0
0.50
25.0
0.9981
364
Benzoic acid
122.12
10.3
121.0
9.5–11.0
1.00
25.0
0.9991
540
o-coumaric acid
164.16
11.1
163.1
10.0–12.5
0.10
25.0
0.9975
41.3
m-coumaric acid
164.16
11.9
163.1
10.0–12.5
0.05
25.0
0.9998
24.2
Caffeic acid
180.16
11.9
179.0
11.0–13.5
0.25
25.0
0.9986
476
Salicylic acid
138.12
12.5
137.1
11.5–14.0
0.05
5.0
0.9950
34.2
Protocatechuic acid
154.12
14.5
153.0
13.0–18.0
0.25
25.0
0.9992
243
Gentisic acid
154.12
16.8
153.0
13.0–18.0
0.05
5.0
0.9952
26.3
2
Profiling Analysis of 15 Prominent Naturally Occurring Phenolic Acids by LC-MS
RESULT AND DISCUSSION
Chromatography and Buffer pH
Chromatography and Organic Solvent
The pH of buffer solutions in mobile phase is one of the key parameters to adjust the chromatographic behavior of organic acids. Adjusting buffer pH determines the charge of organic acids, thus determining the anion exchange retention. Generally, decreasing buffer pH switches organic acids towards neutral form, suppresses the anionic exchange retention, and reduces the overall retention. Selectivity for particular organic acids could be significantly affected by tuning buffer pH. As shown in Figure 1, a pH change from 4.6 to 4.3 does not affect the retention of salicylic acid (pKa = 2.98). However, with the same pH change, retention was significantly suppressed for sinapic acid (pKa ~ 4.6) . Figure 2 shows the retention pH dependence of three organic acids. The retention of gentisic and sinapic acids showed strong dependence on buffer pH: increased mobile phase pH promoted overall retention. On the other hand, the retention of caffeic acid remained relatively unaffected.
Although mixed-mode chromatography features an additional anionicexchange retention mechanism, the linear solvent strength theory for reversed-phase chromatography still supplies an appropriate approximation. Figure 1 shows Logk-%CH3CN plots of sinapic acid (the first eluted peak) and salicylic acid (a late eluted peak). Increase of mobile phase strength by increasing organic solvent composition reduces the retention of organic acids.
logk and % Organic 1.6
Salicylic Acid pH 4.6 Sinapic Acid pH 4.6 Sinapic Acid pH 4.3 Salicylic Acid pH 4.3
1.4 logk and Buffer pH 1.4 Gentisic Acid 1.2
Sinapic Acid
logk
Caffeic Acid 1.2
1.0
1.0 logk
0.8
0.8 0.6
0.6 0.4
40
50
60
70 % CH3CN
80
90 25349
Figure 1. Effect of organic solvent composition on retention under different pH conditions. Mobile phase: varying %CH3CN with constant ammonium acetate concentration at 20 mM. Column temperature: 40 °C. Flow rate: 0.5 mL/min.
0.4
0.2 3.7
3.9
4.1
4.3
4.5 Buffer pH
4.7
4.9
5.1 25350
Figure 2. Effect of buffer pH on retention. Mobile phase: 80% CH3CN and 20 mM ammonium acetate. Column temperature: 40 °C. Flow rate: 0.5 mL/min.
HPLC 2008 Presentation
3
Chromatography and Buffer Concentration
Mass Spectrometry
Buffer concentration determines the eluent strength for ion exchange chromatography and can be used to adjust total chromatographic run time. A chromatographic selectivity change was not observed when buffer concentration was increased from 10 mM to 20 mM but total run time was reduced to 60%.
Chromatographic separation was carefully controlled to resolve most phenolic acids, especially phenolic acid isomers, such as p-, m- and o-coumaric acids and p-hydroxybenzoic acid and salicylic acid. Phenolic acids predominantly form de-protonated ions [M-H]– in negative ionization mode and were used for quantification. Optimized cone voltage ranged from 40 to 70 volts and 60 volts was selected for every phenolic acid for method simplicity.
Chromatography and Column Operating Temperature Column temperature is often used to reduce HPLC system backpressure, reduce chromatographic run time, and maintain system reproducibility. Column temperature is also an important parameter affecting selectivity in mixed-mode chromatography. Figure 3 shows the temperature dependence of retention for 5 organic acids. As a practical approach, logk was plotted against temperature instead of 1/T. The retention of gentisic and caffeic acid remained relatively constant, while three other acids showed decreasing retention with increasing column temperature. Column temperature was set at 50 °C to aid in resolving critical pairs, such as o-coumaric acid and m-coumaric acid which have the same mass and can not be resolved by mass spectrometry.
Linearity and Method Detection Limits (MDL) Linear response was observed for each analyte over two orders of magnitude with correlation coefficient greater than 0.99. Details are shown in Table 1. Seven replicated injections of calibration standard at 500 ppb or 50 ppb were performed and were used for MDL calculations. MDL was calculated by the equation: MDL = t99% × S(n = 7) Where t is Student’s t at 99% confidence intervals (t99%, n=7 = 3.143) and S is the standard deviation. Calculated MDLs range from 9.0 pg (p-hydroxybenzoic acid) to 540 ng (benzoic acid) per injection. Details are shown in Table 1. The calibration curve for o-coumaric acid is shown in Figure 4; the insert shows the calibration curve at the lower range.
logk and Column Temperature 1.55 Gentisic Acid 3.5E4
p-hydroxybenzoic
m-coumaric acid 0.05 ~ 25 ng (5 µL) R2 = 0.9998
Cinamic Acid
1.45
Caffeic Acid Area (counts*min)
m-coumaric
logk
1.35
2.0E3 1.0E3
1.25
0 0 0
1,000
2,000
1.15
20
25
30
35
40
45
Column Temperature (°C)
50
55
60 25351
Figure 3. Effect of column temperature on retention. Mobile phase: 84% CH3CN and 20 mM ammonium acetate, pH 4.6. Flow rate: 0.5 mL/min.
4
0
100
200
300
3,000 4,000 Amount (ppb = ng/mL)
400 5,000
550 6,000 25352
Figure 4. Calibration curve of m-coumaric acid. Calibration standards range from 10 ppb to 5000 ppb; injected amounts range from 0.05 to 25 ng with 5 µL injection.
1.05
0.95
SIM = 163.1 m/z
3.5E3
Profiling Analysis of 15 Prominent Naturally Occurring Phenolic Acids by LC-MS
SIM Chromatograms
6
2
3
7
8 12
5 4
600
15
13 14
10 9
1
UV, WVL: 225 nm
mAU 11
0 40 1
2
3 7
2 # 1. 2. 3. 4. 5. 6. 7. 8.
4
6
Name Sinapic acid Ferulic acid Veratric acid p-coumaric acid Vanillic acid Syringic acid p-hydroxybenzoic acid Cinnamic acid
8
11,12 9 10 13
8 10 12 Retention Time (min) R.T. 5.7 6.2 6.7 7.3 7.5 7.5 8.1 8.8
4
6
8
10
p-hydroxybenzoic
4,5,6
Absorbance (mAU) –1
2
SIM 223.1 193.1 181.1 163.1 167.0 197.1 137.1 147.1
# 9. 10. 11. 12. 13. 14. 15.
14
16
Name Benzoic acid o-coumaric acid m-coumaric acid Caffeic acid Salicylic acid Protocatechuic acid Gentisic acid
protocatechuic
18
syringic
20
SIM 121.0 163.1 163.1 179.0 137.1 153.0 153.0 25351
Figure 5. SIM and UV chromatograms of 15 predominant naturally occurring phenolic acids. Analytical column: Acclaim Mixed-Mode WAX-1, 150 × 2.1 mm; 83% CH3CN, 5% 200 mM NH4OAc pH 4.6, 12% H2O. Column Temperature: 50 °C. Flow rate: 0.5 mL/min. 5 ng injected for MS SIM detections, 25 ng injected for UV detection. SIM chromatogram is normalized to 100% of the greatest peak in each channel.
Application for Beverage Analysis This method has been evaluated for quantitative analysis of phenolic acids found in beverages. A sample chromatogram of green tea analysis is shown in Figure 6. Green tea solution was injected directly after simple filtration through a 0.45 µm membrane. Eleven phenolic acids were detected with individual content ranging from 2.4 to 109 µg per gram of dry leaves.
16
18
20
MS, SIM*
m-coumaric o-coumaric
ferulic R.T. 10.3 10.9 11.6 11.8 12.5 14.0 16.3
14 gentisic
salicylic
p-coumaric
15
14
12
caffeic sinapic
2
4
6
8
10 12 14 Retention Time (min)
16
18
20 25354
Figure 6. SIM and UV chromatograms of phenolic acids in green tea. Green tea leaves were soaked in hot water for 45 minutes (non-thermostated), the solution was filtered through a 0.45 µm membrane, and 5 µL filtered solution was injection directly for quantification. Calculated amount (µg/g): p-coumaric acid: 11.1; o-coumaric acid: 6.0; m-coumaric acid: 44.1; protocatechuic: 16.9; gentisic: 4.0; caffeic acid: 109; salicylic acid: 11.4; p-hydroxybenzoic acid: 23.3; ferulic acid: 4.5; syringic acid: 8.0; sinapic acid: 2.4. *Each SIM chromatogram is normalized to 100% of the greatest peak in that channel.
CONCLUSION An LC-MS method for quantitative analysis of 15 prominent naturally occurring phenolic acids was developed using mixed-mode liquid chromatography and single-quadrupole mass spectrometry. Parameters affecting chromatographic separation and mass spectrometric detection were explored. Linearity was achieved for each phenolic acid over two orders of magnitude with R2 > 0.99. Method detection limits ranged from 9.0 pg (p-hydroxybenzoic acid) to 540 pg (benzoic acid) per injection.
REFERENCES 1. Robbins, R. J. J. Agric. Food Chem. 2003, 51, 2866–2887. 2. Shahidi, F.; Wanasundara, P. K. Phenolic Antioxidants. Crit. ReV. Food Sci. Nutr. 1992, 32, 67.
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