Fast Separation of Capsaicinoids from Peppers by Reversed Phase Ultra-Performance Liquid Chromatography: Comparation with Traditional High-Performance Liquid Chromatography Methods

Figures & data

FIGURE 1 UHPLC Chromatogram of hot pepper extract [1-nordihydrocapsaicin (n-DHC); 2-capsaicin (C); 3-dihydrocapsaicin (DHC); 4-homocapsaicin (h-C); 5-homodihydrocapsaicin (h-DHC)]. Absorbance detection at 280 nm.

TABLE 1 Retention times in minutes (n = 3) of the chromatographic peaks corresponding to the capsaicinoids studied with each chromatographic separation method [reversed-phase HPLC using a C-18 convenctional column (rp-HPLC cc), reversed-phase HPLC using a C-18 monolithic column (rp-HPLC-mc) and reversed-phase UHPLC (rp-UHPLC)]

	n-DHC	C	DHC	h-C	h-DHC
rp-HPLC cc	14.03 ± 0.01	14.84 ± 0.01	19.42 ± 0.01	20.35 ± 0.01	24.67 ± 0.01
rp-HPLC mc	4.29 ± 0.05	4.61 ± 0,05	5.78 ± 0.04	6.12 ± 0.03	7.21 ± 0.01
rp-UHPLC	1.97 ± 0.01	2.06 ± 0.01	2.48 ± 0.01	2.58 ± 0.01	2.97 ± 0.01

TABLE 2 Intraday (%) and interday (%) relative standard deviations in respect of the retention time and peak area

		n-DHC	C	DHC	h-C	h-DHC
Retention time	RSD Intraday (%)	0.05	0.05	0.05	0.06	0.03
	RSD Interday (%)	0.46	0.43	0.30	0.46	0.32
Peak area	RSD Intraday (%)	2.07	1.66	1.69	2.46	3.60
	RSD Interday (%)	2.22	1.97	2.23	3.20	4.33

TABLE 3 Analysis of the variance of a factor for the peak time and peak area, with different percentages of methanol (25–100%) in the extracts

	% MeOH	n-DHC	C	DHC	h-C	H-DHC
Retention time	25	2.059^a,b	2.145^a,b	2.558^a,b	2.659^a,c	3.021^a
	50	2.046^a	2.132^a	2.543^a	2.644^a	3.010^a
	75	2.038^a,b	2.124^a,b	2.534^a,b	2.634^b,c	3.004^a,b
	100	2.034^b	2.119^b	2.529^b	2.629^c	3.000^b
Peak area	25	14783^a	53259^a,b	43488^a,b	4816^a	3753^a
	50	14655^a	54814^a	45027^a	5081^a	3929^a
	75	14980^a	55146^a	45237^a	4894^a	3680^a
	100	14258^a	52719^b	43279^b	4817^a	3561^a

Means with the same superscripts (a, b, c) in the same column are not statistically different (p < 0.05).

TABLE 4 Resolution of the chromatographic peaks corresponding to the capsaicinoids studied with each chromatographic separation method [reversed-phase HPLC using a C-18 conventional column (rp-HPLC cc), reversed-phase HPLC using a C-18 monolithic column (rp-HPLC-mc) and reversed-phase UHPLC (rp-UHPLC)]. n.a. = not available, peaks partially overlapping

	n-DHC/C	C/DHC	DHC/h-C	h-C/h-DHC
rp-HPLC cc	1.56 ± 0.02	8.47 ± 0.08	n.a.	n.a.
rp-HPLC mc	1.77 ± 0.04	6.62 ± 0.09	1.86 ± 0.03	5.90 ± 0.10
rp-UHPLC	0.71 ± 0.05	3.02 ± 0.25	0.86 ± 0.06	4.18 ± 0.16

TABLE 5 Quantity (µmol) of capsaicinoid per kilogram of fresh pepper in the samples analysed (n = 3)

Species	Sample	n-DHC	C	DHC	h-C	h-DHC	Total
C. annuum	1	141.19 ± 3.13^a	579.03 ± 11.41^a	528.65 ± 11.79^a	22.47 ± 0.72^a	43.43 ± 1.88^a	1314.77 ± 28.93^a
	2	150.61 ± 3.34^a	593.23 ± 11.69^a	56.17 ± 1.25^b	15.99 ± 0.51^a	37.80 ± 1.64^a	853.8 ± 18.43^b
	3	34.91 ± 0.78^b	190.30 ± 3.75^b	140.75 ± 3.14^c	7.65 ± 0.24^b	14.98 ± 0.65^b	388.59 ± 8.56^c
	4	114.10 ± 2.53^c	554.38 ± 10.92^a	440.74 ± 9.83^a	9.67 ± 0.31^b	37.39 ± 1.62^a	1156.28 ± 25.21^a
	5	53.56 ± 1.19^d	278.34 ± 5.48^c	262.87 ± 5.86	15.43 ± 0.49^a	20.28 ± 0.88^b	630.48 ± 13.91^d
	6	13.34 ± 0.30 ^e	60.08 ± 1.18^d	52.64 ± 1.17^b	n.d.	3.52 ± 0.15^c	129.58 ± 2.81^e
	7	30.57 ± 0.68^b	130.20 ± 2.56^e	88.71 ± 1.98^d	5.36 ± 0.17^b	13.22 ± 0.57^b	268.06 ± 5.97^f
	8	238.49 ± 5.29	1539.10 ± 30.32^f	1048.8 ± 23.39^e	66.67 ± 2.13	82.76 ± 3.58^d	2975.82 ± 64.72^g
	9	55.15 ± 1.22^d	282.54 ± 5.57^c	195.41 ± 4.36^f	9.87 ± 0.32^b	19.77 ± 0.86^b	562.74 ± 12.32^h
C. frutescens	10	329.98 ± 7.33^f	974.61 ± 19.20^g	1070.74 ± 23.88^e	34.45 ± 1.10^c	52.37 ± 2.27^a	2462.15 ± 53.77^i
	11	271.74 ± 6.03^f	800.93 ± 15.78^g,h	857.83 ± 19.13^g	78.20 ± 2.50^d	95.60 ± 4.14^d	2104.30 ± 47.58^i
	12	143.10 ± 3.18^a	1956.81 ± 38.55^i	1394.18 ± 31.09^h	64.48 ± 2.06^d	50.99 ± 2.21^a	3609.56 ± 77.09^j
	13	198.16 ± 4.40^g	706.09 ± 13.91^h	779.58 ± 17.38^g	47.96 ± 1.53^e	56.93 ± 2.47^a	1788.72 ± 39.69^i
	14	451.46 ± 10.02^h	1151.76 ± 22.69	1404.00 ± 31.31^h	71.28 ± 2.28^d	156.06 ± 6.76	3234.56 ± 73.06^j
C. baccatum	15	24.36 ± 0.54^e	158.88 ± 3.13^b,e	95.39 ± 2.13^d	10.80 ± 0.35^b	10.94 ± 0.47^e	300.37 ± 6.62^f
	16	54.00 ± 1.20^d	198.64 ± 3.91^b,c	196.23 ± 4.38^f	12.50 ± 0.40^b	20.61 ± 0.89^b	481.98 ± 10.78^c

Means with the same superscripts (a–j) in the same column are not statistically different (p < 0.05). Samples 1–2: Bola Pepper varieties; Samples 3–5: Jeromin Pepper varieties; Samples 6–9: Jariza Pepper varieties; Samples 10–14: Malagueta Pepper varieties; Samples 15–16: Bishop Crown varieties.