Simultaneous determination of chlorogenic acids in green coffee bean extracts with effective relative response factors

Figures & data

Table 1. Chemical structures of seven chlorogenic acids.

Component	R3	R4	R5
3-CQA	C	H	H
5-CQA	H	H	C
4-CQA	H	C	H
5-FQA	H	H	F
3,4-diCQA	C	C	H
3,5-diCQA	C	H	C
4,5-diCQA	H	C	C

Table 2. The relative response factors of seven chlorogenic acids.

Order	LC Column	LC Instrument	F5-CQA	F3-CQA	F4-CQA	F5-FQA	F3,4-diCQA	F3,5-diCQA	F4,5-diCQA
1	Phenomenex	WUFENG	1.00	0.99	0.94	1.20	0.84	0.88	0.69
2	Phenomenex	SHIMADZU	1.00	0.96	0.91	1.11	0.87	0.89	0.70
3	GraceSmart	WUFENG	1.00	0.95	0.95	1.20	0.83	0.86	0.67
4	GraceSmart	SHIMADZU	1.00	0.95	0.94	1.19	0.83	0.91	0.69
5	Promasil	WUFENG	1.00	0.96	0.91	1.15	0.84	0.87	0.67
6	Promasil	SHIMADZU	1.00	0.97	0.93	1.21	0.81	0.87	0.64
7	Kromasil	WUFENG	1.00	0.96	0.91	1.13	0.83	0.86	0.67
8	Kromasil	SHIMADZU	1.00	0.92	0.89	1.13	0.82	0.85	0.66
9	Agilent	WUFENG	1.00	0.97	0.88	1.14	0.84	0.87	0.67
10	Agilent	SHIMADZU	1.00	0.96	0.90	1.17	0.86	0.87	0.66
11	Hedera ODS-2	WUFENG	1.00	0.96	0.87	1.17	0.83	0.87	0.67
12	Hedera ODS-2	SHIMADZU	1.00	0.94	0.90	1.19	0.85	0.86	0.67
13	Venusil MP	WUFENG	1.00	0.94	0.86	1.19	0.83	0.90	0.69
14	Venusil MP	SHIMADZU	1.00	0.94	0.90	1.19	0.83	0.88	0.68
15	Ultimate LP	WUFENG	1.00	0.96	0.88	1.21	0.83	0.88	0.68
16	Ultimate LP	SHIMADZU	1.00	0.95	0.89	1.18	0.82	0.89	0.68
	Means		1.00	0.96	0.90	1.17	0.84	0.88	0.67
	STDEV		0.00	0.02	0.03	0.03	0.01	0.02	0.01
	RSD (%)		0.00	1.61	2.79	2.59	1.75	1.76	2.10

Obtained by minitab v16, Taguchi design, L16 (8**1 2**8) Array.

Table 3. Contents (%) of seven chlorogenic acids in samples of green coffee bean extracts^c.

		Samples
Chemical		1	2	3	4	5	6	7	8	9	10	11	12	13	14
3-CQA	a^1	5.41	6.35	3.53	3.83	6.35	2.66	5.44	5.26	5.94	3.22	3.78	7.00	3.74	2.88
	b^2	5.25	6.17	3.40	3.66	6.07	2.56	5.23	5.12	5.78	3.13	3.68	6.81	3.64	2.80
5-CQA	a^1	18.56	24.24	22.64	26.34	28.02	20.95	20.60	21.12	28.91	25.44	24.97	28.49	24.75	33.07
	b^2	18.56	24.24	22.64	25.34	27.02	20.95	20.60	21.12	28.91	25.44	24.97	28.49	24.75	33.07
4-CQA	a^1	6.98	7.24	6.59	6.76	8.45	4.51	10.41	10.45	8.14	6.82	6.22	8.62	6.17	4.26
	b^2	6.67	6.92	6.25	6.38	7.96	4.26	9.83	10.01	7.80	6.53	5.96	8.25	5.90	4.08
5-FQA	a^1	3.91	4.29	5.15	5.81	3.14	5.41	2.66	3.20	3.48	3.87	5.77	2.82	5.56	2.83
	b^2	3.95	4.33	5.20	5.87	3.17	5.46	2.69	3.29	3.58	3.99	5.93	2.90	5.71	2.91
3,4-diCQA	a^1	2.58	1.94	2.73	2.96	1.35	3.60	2.44	2.38	1.46	2.23	2.86	0.73	2.84	1.07
	b^2	2.56	1.92	2.67	2.87	1.31	3.52	2.38	2.36	1.45	2.21	2.84	0.72	2.81	1.06
3,5-diCQA	a^1	1.93	1.19	2.10	2.15	0.83	3.10	1.80	1.82	1.10	2.30	2.27	0.70	2.25	2.10
	b^2	1.92	1.19	2.09	2.11	0.82	3.02	1.76	1.80	1.09	2.28	2.24	0.70	2.22	2.08
4,5-diCQA	a^1	2.01	1.29	2.18	2.39	0.87	2.98	1.86	1.86	1.11	2.63	3.09	0.64	3.07	1.17
	b^2	2.11	1.36	2.28	2.47	0.90	3.07	1.91	1.94	1.16	2.74	3.22	0.67	3.19	1.22
Total	a^1	41.38	46.54	44.92	50.24	49.01	43.21	45.21	46.09	50.14	46.51	48.96	49.00	48.38	47.38
	b^2	41.02	46.13	44.53	48.70	47.25	42.84	44.40	45.64	49.77	46.32	48.84	48.54	48.22	47.22

^c the content of seven chlorogenic acids is in the dry matter of the green coffee bean extract (with water content lower than 0.5%).

¹ a represents the quantitative analysis of multi-components by multiple standards (QAMM).

² b stands for the quantitative analysis of multi-components by single-marker (QAMS).

Figure 1. Representative HPLC chromatograms. a: Working reference standards solution; b: Sample solution of GCBE (1) 3-CQA, (2) 5-CQA, (3) 4-CQA, (4) 5-FQA, (5) 3,4-diCQA, (6) 3,5-diCQA, (7) 4,5-diCQA.

Table 4. Liner range, liner equation, R², limit of detection (LOD) and limit of quantification (LOQ).

compound	liner range (µg)	liner equation^a	R^2	LOD^b (µg)	LOQ^c (µg)
3-CQA	10.23-102.3	y=78.73x+183.6	0.980	0.0005	0.0012
5-CQA	40.00-400.0	y=64.65x+397.9	0.999	0.0020	0.0050
4-CQA	20.90-209.0	y=74.05x+9.0	0.999	0.0010	0.0026
5-FQA	9.720-97.20	y=35.04x-1.4	0.999	0.0060	0.0016
3,4-diCQA	8.720-87.20	y=74.04x-262.7	0.988	0.0040	0.0130
3,5-diCQA	8.610-86.10	y=72.18x-265.4	0.990	0.0040	0.0128
4,5-diCQA	8.450-84.45	y=70.66x-146.4	0.993	0.0040	0.0126

^a y represents peak area, x represents the content of standard product.

^b signal to noise ratio, S/N is 3.

^c signal to noise ratio, S/N is 10.

Table 5. The relative standard deviation of the precision and repeatability of each standard.

Compound	Inter-day Precision (%)	Intra-day Precision (%)	Repeatability (%)	Stability (%)
3-CQA	0.18	0.68	0.74	0.90
5-CQA	0.12	0.76	0.68	1.09
4-CQA	0.10	0.61	0.77	1.03
5-FQA	0.19	3.49	2.60	1.87
3,4-diCQA	0.18	1.03	1.95	1.9
3,5-diCQA	0.24	0.91	3.80	1.37
4,5-diCQA	0.50	1.15	2.38	1.35

Table 6. Recoveries and relative standard deviations (RSD) of standard compounds (n=3).

Compound	Spiked (µg /mg)	Recovery (%)	RSD (%)
3-CQA	25	97.8	2.58
	50	96.5	0.88
	100	103.6	0.10
5-CQA	100	102.0	0.78
	200	96.6	0.41
	400	96.0	0.36
4-CQA	50	104.9	1.14
	100	103.3	1.16
	200	99.8	3.26
5-FQA	25	101.9	0.98
	50	97.5	1.26
	100	105.9	2.17
3,4-diCQA	45	103.1	1.80
	90	99.0	3.43
	250	105.9	0.76
3,5-diCQA	45	103.2	1.79
	90	102.5	2.56
	250	100.8	1.09
4,5-diCQA	45	99.8	1.87
	90	97.7	0.55
	250	102.8	2.00

Table 7. The results of two-way ANOVA to relative response factors.

Two-way ANOVA: F3-CQA versus LC Column, LC Instrument
Source	DF	SS	MS	F	P
LC Column	7	0.002200	0.0003143	2.26	0.153
LC Instrument	1	0.000625	0.0006250	4.49	0.072
Error	7	0.000975	0.0001393
Total	15	0.003800
S = 0.01180 R-Sq = 74.34% R-Sq (adj) = 45.02%
Two-way ANOVA: F4-CQA versus LC Column, LC Instrument
Source	DF	SS	MS	F	P
LC Column	7	0.007775	0.0011107	3.57	0.057
LC Instrument	1	0.000225	0.000225	0.72	0.423
Error	7	0.002175	0.0003107
Total	15	0.010175
S = 0.01763 R-Sq = 78.62% R-Sq (adj) = 54.19%
Two-way ANOVA: F5-FQA versus LC Column, LC Instrument
Source	DF	SS	MS	F	P
LC Column	7	0.007700	0.0011000	1.1	0.45
LC Instrument	1	0.000025	0.0000250	0.03	0.879
Error	7	0.006975	0.0009964
Total	15	0.014700	0.0147000
S = 0.03157 R-Sq = 52.55% R-Sq (adj) = 0.00%
Two-way ANOVA: F3,4-diCQA versus LC Column, LC Instrument
Source	DF	SS	MS	F	P
LC Column	7	0.002000	0.0002857	1.45	0.317
LC Instrument	1	0.000025	0.000025	0.13	0.732
Error	7	0.001375	0.0001964
Total	15	0.003400
S = 0.01402 R-Sq = 59.56% R-Sq (adj) = 13.34%
Two-way ANOVA: F3,5-diCQA versus LC Column, LC Instrument
Source	DF	SS	MS	F	P
LC Column	7	0.0021438	0.0003063	1.35	0.353
LC Instrument	1	0.0000562	0.0000562	0.25	0.634
Error	7	0.0015938	0.0002277
Total	15	0.0037938
S = 0.01509 R-Sq = 57.99% R-Sq (adj) = 9.98%
Two-way ANOVA: F4,5-diCQA versus LC Column, LC Instrument
Source	DF	SS	MS	F	P
LC Column	7	0.0023437	0.0003348	2.95	0.088
LC Instrument	1	0.0000563	0.0000563	0.50	0.504
Error	7	0.0007937	0.0001134
Total	15	0.0031937
S = 0.01065 R-Sq = 75.15% R-Sq (adj) = 46.74%

DF: degree of freedom; SS: sum of square; MS: mean sum of squares.

Figure 2. Residual plots for F_3–CQA.

Table 8. The response tables for signal to noise ratios, means and standard deviations. Nominal is best (10*Log10(Ybar**2/s**2)).

Level	LC Column	LC Instrument
1	16.34	15.47
2	15.34	15.55
3	15.07
4	15.83
5	15.63
6	15.28
7	15.38
8	15.21
Delta	1.27	0.07
Rank	1	2
Response Table for Means
Level	LC Column	LC Instrument
1	0.9271	0.9170
2	0.9264	0.9163
3	0.9164
4	0.9021
5	0.9136
6	0.9129
7	0.9164
8	0.9179
Delta	0.0250	0.0007
Rank	1	2
Response Table for Standard Deviations
Level	LC Column	LC Instrument
1	0.1422	0.1545
2	0.1585	0.1536
3	0.1623
4	0.1458
5	0.1511
6	0.1571
7	0.156
8	0.1594
Delta	0.0201	0.001
Rank	1	2

Figure 3. Main effects plots for signal-to-noise ratios (SN ratios), means, and standard deviations (SDs).

Figure 4. Interaction plots for signal-to-noise ratios (SN ratios), means, and standard deviations (SDs).