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Food Chemistry 81 (2003) 631–638 www.elsevier.com/locate/foodchem
Analytical, Nutritional and Clinical Methods Section
Analysis of green tea catechins: comparative study between HPLC and HPCE Matteo Bonoli*, Marco Pelillo, Tullia Gallina Toschi, Giovanni Lercker Dipartimento di Scienze degli Alimenti, Universita` di Bologna, Via Ravennate 1020, Cesena (FC), 47023, Italy Received 11 July 2002; received in revised form 13 November 2002; accepted 13 November 2002
Abstract A comparison between a borate–phosphate–SDS based MEKC and an RP-HPLC method for the separation of seven tea catechins and gallic acid in a green tea extract is here proposed. Under optimised conditions, HPCE offered several advantages respect to time of analysis (compounds were separated within 4.5 min), sensitivity (HPCE LODs were about 20–100 times lower than HPLC ones) and solvent consumption. HPCE displayed excellent migration time repeatability (RSD% on MT < 2%, and RSD% on RMT < 1%), whereas HPLC showed slightly more quantification ruggedness (total amount catechins RSD% was < 2% for HPLC and 30%), the fact that it was possible to detect this compound by this technique is quite important from the qualitative standpoint. The slight difference between migration times of the components of the standard mixture and those of the GTE samples (see Fig. 2A and B) could be due to the different analytes concentration employed. The elution order is listed as follows: GC, C, EGC, EGCG, GCG, ECG, EC and GA. These eight compounds were completely separated within 4.5 min; a whole analysis required about 12 min (rinse steps included) and no sample clean-up pre-treatment was necessary. 3.2. HPLC and MEKC performances Table 1 shows performance parameters (calibration parameters, intra- and inter-assay precision), of GTE samples of both HPLC and MEKC methods. The
Table 1 HPLC and HPCE calibration curves parameters and repeatability study on quantification (values are expressed in mg of catechins for 100 mg of extract) Analytes
r Areasa
LOD (mg/ml)b
First quantification (n=5)
After 1 week quantification (n=5)
INTRA-assay
INTRA-assay
INTER-assay
INTER-assay
MeanS.D.
%RSD
MeanS.D.
%RSD
MeanS.D.
%RSD
Mean S.D.
%RSD
GC-HPLC GC-HPCE
0.995 0.997
0.166 0.0013
4.23 0.15 1.21 0.05
3.58 4.38
4.400.13 1.160.11
2.95 9.62
4.550.11 1.160.08
2.50 7.26
4.580.10 1.190.04
2.10 3.07
C-HPLC C-HPCE
0.999 0.992
0.224 0.0012
0.83 0.02 0.75 0.02
2.59 2.38
0.800.06 0.720.04
7.06 5.17
0.890.08 0.770.03
8.70 3.32
0.860.03 0.760.01
3.53 1.39
EGC-HPLC EGC-HPCE
0.999 0.999
0.385 0.0017
11.82 0.15 10.34 0.21
1.27 2.06
11.920.16 10.740.77
1.38 7.13
12.070.12 10.080.89
0.96 8.79
12.130.26 10.680.35
2.17 3.23
EGCG-HPLC EGCG-HPCE
0.999 0.999
0.192 0.0018
38.78 0.40 34.46 0.51
1.02 1.47
39.070.30 34.521.95
0.77 5.66
40.300.50 36.651.91
1.24 5.20
40.290.67 37.161.12
1.67 3.03
GCG-HPLC GCG-HPCE
0.997 0.994
0.084 0.0033
0.69 0.03 1.10 0.11
4.35 10.12
0.720.03 1.190.11
3.94 9.36
0.850.04 1.260.15
4.56 11.69
0.790.05 1.240.08
6.60 6.44
ECG-HPLC ECG-HPCE
0.998 0.999
0.040 0.0023
10.10 0.16 8.77 0.24
1.56 2.77
10.040.09 8.810.30
0.87 3.37
10.550.30 9.520.49
2.80 5.13
10.310.15 9.740.51
1.47 5.28
EC-HPLC EC-HPCE
0.999 0.999
0.321 0.0023
5.55 0.14 5.07 0.14
2.57 2.70
5.570.12 4.920.29
2.22 5.82
6.210.33 5.330.12
5.37 2.27
5.910.24 5.470.13
4.11 2.29
GA-HPLC GA-HPCE
0.999 0.999
0.0250 0.0051
NDc 0.06 0.02
– 31.49
NDc 0.090.03
– 31.07
NDc 0.100.04
– 37.14
NDc 0.110.04
– 35.92
71.98 0.56 61.77 0.62
0.77 1.01
72.520.64 62.153.45
0.88 5.54
75.400.73 64.382.72
0.96 4.22
74.881.29 66.341.91
1.72 2.88
TOT-HPLC TOT-HPCE a b c
Correlation coefficients of the calibration curves using peak area. For S/N=3 Not detectable.
M. Bonoli et al. / Food Chemistry 81 (2003) 631–638 Table 2 Differences in total amount of catechins and gallic acid (TACs) found by HPLC and HPCE a d S:D:b t0 Significativityc Total amount of catechins a b c
10.04
1.06
18.94
S
Pn
ðTACsHPLC;i TACsMEKC;i Þ d ¼ i¼1 ; n ¼ 4: n Standard deviation (n =4). S=significant; NS=not significant.
bility in HPCE is highly acceptable for a hyphenated analytical technique. As you can see in Table 1, no GA data have been reported in HPLC quantification. Only GC quantification data have been tabulated because, from further mass spectrometry analyses, GA was not detected (Pelillo et al., 2002). This evidence confirmed that the sensitivity of capillary electrophoresis permitted a better qualitative investigation of this extract than liquid chromatography. Regarding elution time, HPCE is much faster than HPLC, since total analysis time for the eight components is 12 min for this MEKC method whereas HPLC separation is achieved in 40 min. Table 2 shows the statistical interpretation of results. The following formula was applied: d pffiffiffi t0 ¼ SD= n where d is defined as the average difference between total amount of catechins found by HPLC and that found by MEKC, SD is the standard deviation and n is the number of replications. Finally, value was compared with t-Student value (t ¼0:05 n1¼3 ¼ 2:35); the repeatability hypothesis was rejected if t0 > t ¼0:05 n1¼3 . In fact, the significant difference between the total amount of catechins found with the two analytical methods can be attributed to the different peak area integration software used (Faller & Engelhardt, 1999; Reijenga & Lee, 2001), and to the higher efficiency and sensitivity of HPCE than HPLC. Nevertheless, another factor that could have influenced these results could be the response variation related to the different wavelength used for the component detection (200 and 270 nm for CE and HPLC, respectively). Indeed, significant difference between the quantification of some compounds by the HPCE and HPLC methods could be due to interferences in the sample which could have a different weight at the two selected wavelength. As earlier mentioned, it was not possible to use 200 nm as detection wavelength for both methods because the HPLC underwent interference by the mobile phase.
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4. Conclusions Two different analytical techniques (MEKC and HPLC) for the analysis of green tea catechins, were compared. This study highlights the effective possibility of application of HPCE in the food chemistry field. The MEKC method here suggested show higher sensitivity, resolution, efficiency and migration times repeatability than the HPLC method, even if the latter displayed a slightly better repeatability in the quantification of total amount of catechins. It should be pointed out that HPCE LODs were about 20–100 times lower than HPLC ones, which is extremely advantageous when analyzing real systems, such as food products. Moreover, HPCE is very convenient in terms of analysis time (12 min for total analysis against 40 min for the HPLC one) and solvents consumption for routine analysis of tea extracts.
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