New Method for Carotenoid Extraction and Analysis by HPLC-DAD-MS/MS in Freeze-Dried Citrus and Mango Pulps

Petry, Fabiane C.; Mercadante, Adriana Z.

doi:10.21577/0103-5053.20170127

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Short Reports • J. Braz. Chem. Soc. 29 (1) • Jan 2018 • https://doi.org/10.21577/0103-5053.20170127 copy

New Method for Carotenoid Extraction and Analysis by HPLC-DAD-MS/MS in Freeze-Dried Citrus and Mango Pulps

Authorship SCIMAGO INSTITUTIONS RANKINGS

Carotenoid analysis in citrus is still a challenge due to their complex carotenoid profile and matrix acidity, which can promote isomerization and epoxide-furanoid rearrangements of these compounds. A new method for carotenoid extraction from freeze-dried citrus was developed. Extraction was carried out by magnetic stirring using ethyl acetate and methanol as solvents and Na₂CO₃ to neutralize organic acids. Liquid-liquid partition was achieved by centrifugation. Carotenoid quantification was accomplished by both UV-Vis and high-performance liquid chromatography-diode array detection (HPLC-DAD). Method repeatability (2.9 to 8.5%) and recovery (82 to 88%) were similar to reported values for procedures including all the carotenoid analysis steps. In 'Valencia' orange pulp, xanthophylls accounted for 98% of the total carotenoid content and the predominant carotenoids were (9Z)-violaxanthin, (all-E)-lutein and (9Z)-antheraxanthin. This method was further applied for carotenoid analysis in freeze-dried 'Tommy Atkins' mango pulp. The developed method is precise, accurate, fast and able to maintain epoxy-carotenoids stability during analysis.

Keywords:
chromatography; epoxy-carotenoids; epoxy-furanoid rearrangement; identification; quantification

Procedure	Extraction	Solvent	Number of extraction	Solvent volume / mL
A	maceration with mortar and pestle (1 min)	EtOAc:MeOH (1:1, v/v)	7	20 mL (first extraction), 15 mL (further extractions) total: 110 mL
B	maceration with mortar and pestle (1 min)	EtOAc MeOH	5 2	20 mL (first extraction), 15 mL (further extractions) total: 110 mL
C	magnetic stirring (5 min)	EtOAc:MeOH (1:1, v/v)	4	50 mL (first extraction), 30 mL (further extractions) total: 140 mL
D	magnetic stirring (5 min)	EtOAc:MeOH (1:1, v/v) MeOH	3 1	50 mL (first extraction), 30 mL (further extractions) total: 140 mL
E	magnetic stirring (5 min)	EtOAc MeOH	2 2	50 mL (first extraction), 30 mL (further extractions) total: 140 mL

Procedure	Total carotenoid content^a a Mean and standard deviation of three replicates (n = 3), expressed in dry basis; / (µg g^-1)	RSD^b b relative standard deviation. / %
A	124.68 ± 3.32^B A,B Values with different letters were significantly different (Tukey's test; p < 0.05). A: 7 maceration steps, using a mixture of EtOAc:MeOH 1:1 as solvent; B: 5 maceration steps with EtOAc and 2 maceration steps with MeOH; C: 4 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1; D: 3 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1 and one further extraction with MeOH; E: 2 extraction steps by magnetic stirring with EtOAC and 2 extraction steps with MeOH. Further details of the extraction procedures can be found in Material and Methods section.	2.66
B	137.58 ± 2.09^A A,B Values with different letters were significantly different (Tukey's test; p < 0.05). A: 7 maceration steps, using a mixture of EtOAc:MeOH 1:1 as solvent; B: 5 maceration steps with EtOAc and 2 maceration steps with MeOH; C: 4 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1; D: 3 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1 and one further extraction with MeOH; E: 2 extraction steps by magnetic stirring with EtOAC and 2 extraction steps with MeOH. Further details of the extraction procedures can be found in Material and Methods section. ^B A,B Values with different letters were significantly different (Tukey's test; p < 0.05). A: 7 maceration steps, using a mixture of EtOAc:MeOH 1:1 as solvent; B: 5 maceration steps with EtOAc and 2 maceration steps with MeOH; C: 4 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1; D: 3 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1 and one further extraction with MeOH; E: 2 extraction steps by magnetic stirring with EtOAC and 2 extraction steps with MeOH. Further details of the extraction procedures can be found in Material and Methods section.	1.52
C	132.45 ± 7.32^A A,B Values with different letters were significantly different (Tukey's test; p < 0.05). A: 7 maceration steps, using a mixture of EtOAc:MeOH 1:1 as solvent; B: 5 maceration steps with EtOAc and 2 maceration steps with MeOH; C: 4 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1; D: 3 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1 and one further extraction with MeOH; E: 2 extraction steps by magnetic stirring with EtOAC and 2 extraction steps with MeOH. Further details of the extraction procedures can be found in Material and Methods section. ^B A,B Values with different letters were significantly different (Tukey's test; p < 0.05). A: 7 maceration steps, using a mixture of EtOAc:MeOH 1:1 as solvent; B: 5 maceration steps with EtOAc and 2 maceration steps with MeOH; C: 4 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1; D: 3 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1 and one further extraction with MeOH; E: 2 extraction steps by magnetic stirring with EtOAC and 2 extraction steps with MeOH. Further details of the extraction procedures can be found in Material and Methods section.	5.52
D	133.25 ± 5.50^A A,B Values with different letters were significantly different (Tukey's test; p < 0.05). A: 7 maceration steps, using a mixture of EtOAc:MeOH 1:1 as solvent; B: 5 maceration steps with EtOAc and 2 maceration steps with MeOH; C: 4 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1; D: 3 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1 and one further extraction with MeOH; E: 2 extraction steps by magnetic stirring with EtOAC and 2 extraction steps with MeOH. Further details of the extraction procedures can be found in Material and Methods section. ^B A,B Values with different letters were significantly different (Tukey's test; p < 0.05). A: 7 maceration steps, using a mixture of EtOAc:MeOH 1:1 as solvent; B: 5 maceration steps with EtOAc and 2 maceration steps with MeOH; C: 4 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1; D: 3 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1 and one further extraction with MeOH; E: 2 extraction steps by magnetic stirring with EtOAC and 2 extraction steps with MeOH. Further details of the extraction procedures can be found in Material and Methods section.	4.20
E	140.45 ± 4.96^A A,B Values with different letters were significantly different (Tukey's test; p < 0.05). A: 7 maceration steps, using a mixture of EtOAc:MeOH 1:1 as solvent; B: 5 maceration steps with EtOAc and 2 maceration steps with MeOH; C: 4 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1; D: 3 extraction steps by magnetic stirring with a mixture of EtOAc:MeOH 1:1 and one further extraction with MeOH; E: 2 extraction steps by magnetic stirring with EtOAC and 2 extraction steps with MeOH. Further details of the extraction procedures can be found in Material and Methods section.	3.53

Liquid-liquid partition	Total carotenoid content^a a Values are mean and standard deviation of three replicates (n = 3), expressed in dry basis; / (µg g^-1)	RSD^b b relative standard deviation. / %
Separatory funnel	140.45 ± 4.96^B A,B Values with different letters were significantly different (t test for independent samples, p< 0.05).	3.53
Centrifuge	156.81 ± 1.84^A A,B Values with different letters were significantly different (t test for independent samples, p< 0.05).	1.18

Carotenoid	Linear range / (µg mL^-1)	Linear regression^b b y is the DAD response in area units (mAU) and x is the carotenoid concentration (µg mL-1). r2: determination coefficient; LOD: limit of detection; LOQ: limit of quantification.	r²	LOD / (µg mL^-1)	LOQ / (µg mL^-1)
(all-E)-Lutein	1.53-24.43	y = 373591.5x - 210030.2	0.998	0.51	1.54
(all-E)-Zeaxanthin	1.46-21.64	y = 430716.1x - 83713.1	0.997	0.48	1.45
(all-E)-β-Cryptoxanthin	2.52-26.23	y = 416456.7x - 80162.1	0.995	0.83	2.52
(all-E)-α-Carotene	0.99-8.44	y = 375635.2x + 93477.8	0.995	0.38	1.15
(all-E)-β-Carotene	2.77-25.71	y = 443449.9x + 47112.9	0.993	0.91	2.77

Carotenoid	Concentration in the matrix^a a Means and standard deviations of three replicates; ,^b b recovery assays were carried out using freeze-dried pulp of unripe orange cv. "Valencia". / (µg g^-1)	Spiked amount / (µg g^-1)		Experimental amount^a a Means and standard deviations of three replicates; / (µg g^-1)	Recovery / %	Repeatability (% RSD)
(all-E)-Lutein	5.52 ± 0.58	level 1	5.16	9.91 ± 0.22	85.1 ± 4.2	4.9
		level 2	10.30	14.26 ± 0.31	84.9 ± 3.1	3.6
		level 3	14.72	18.53 ± 0.44	88.4 ± 3.0	3.4
(all-E)-β-Cryptoxanthin	1.38 ± 0.02	level 1	1.88	2.94 ± 0.12	82.9 ± 6.4	7.8
		level 2	3.76	4.61 ± 0.15	85.9 ± 3.9	4.5
		level 3	7.54	7.67 ± 0.31	83.4 ± 4.1	4.9
(all-E)-β-Carotene	0.37 ± 0.02	level 1	0.64	0.90 ± 0.03	83.4 ± 4.7	5.6
		level 2	1.26	1.44 ± 0.04	85.0 ± 3.4	4.0
		level 3	3.08	2.93 ± 0.09	83.1 ± 2.8	3.4

Peak^a a Numbered according to the chromatograms shown in Figure 1;	Carotenoid	Concentration of freeze dried pulp^b b mean and standard deviation of three replicates (n = 3); / (µg g^-1)		t_R^c c retention time on the C30 column; / min	λ_max^d d linear gradient of MeOH/MTBE; / nm	III/II / %	A_B/A_II / %	[M + H]⁺ (m/z)	Fragment ions (m/z)
	Carotenoid	Orange	Mango	t_R^c c retention time on the C30 column; / min	λ_max^d d linear gradient of MeOH/MTBE; / nm	III/II / %	A_B/A_II / %	[M + H]⁺ (m/z)	Fragment ions (m/z)
1	mixture	1.8 ± 0.1	nd^e e not detected;	6.2-6.3	395, 421, 441, 465	nc^f f not calculated;	nd^e e not detected;	nd^e e not detected;	nd^e e not detected;
2	(di-Z)-violaxanthin	3.9 ± 0.2	nd^e e not detected;	6.6	328, 409, 430, 455	15	30	601	nd^e e not detected;
3	(9Z)-neoxanthin	nd^e e not detected;	15.2 ± 1.5	6.7	330, 415, 440, 469	84	24	601	583[M + H - 18]⁺, 565[M + H - 18 - 18]⁺, 509[M + H - 92]⁺, 491[M + H - 92 - 18]⁺, 221
4	(di-Z)-violaxanthin	4.1 ± 0.1	nd^e e not detected;	6.8-6.9	328, 408, 429, 456	35	32	601	nd^e e not detected;
5	(all-E)-violaxanthin	8.8 ± 0.5	66.9 ± 1.1	7.3-7.4	414, 438, 467	78	0	601	583[M + H - 18]⁺, 565[M + H - 18 - 18]⁺, 547[M + H - 18 - 18 - 18]⁺, 509[M + H - 92]⁺, 491[M + H - 92 - 18]⁺, 221
6	(all-E)-luteoxanthin	5.1 ± 0.2	4.7 ± 0.2	8.5	400, 421, 447	93	0	601	583[M + H - 18] ⁺, 565[M + H - 18 - 18] ⁺, 547[M + H - 18 - 18 - 18]⁺, 509[M + H - 92]⁺, 491[M + H - 92 - 18]⁺, 221
7	not identified	2.6 ± 0.01	nd^e e not detected;	9.1	329, 402, 424, 445	19	27	601	583[M + H - 18]⁺, 565[M + H - 18 - 18]⁺, 509[M + H - 92]⁺, 491[M + H - 92 - 18]⁺, 221
8	(9Z)-violaxanthin + (all-E)-antheraxanthin	30.3 ± 1.9	41.5 ± 1.3	10.4	328, 412, 435, 463	88	8	585, 601	[585]: 567[M + H - 18]⁺, 549[M + H - 18 - 18]⁺, 493[M + H - 92]⁺, 221; [601]: 583[M + H - 18]⁺, 565[M + H - 18 - 18]⁺, 509[M + H - 92]⁺, 491[M + H - 92 - 18]⁺, 221
9	(9Z) or (9'Z)-luteoxanthin	2.3 ± 0.2	nd^e e not detected;	10.8	310, 396, 417, 442	98	13	601	nd^e e not detected;
10	(9Z) or (9'Z)-luteoxanthin	5.5 ± 0.3	nd^e e not detected;	11.4	310, 395, 417, 443	94	10	601	583[M + H - 18]⁺, 565[M + H - 18 - 18]⁺, 509[M + H - 92]⁺, 491[M + H - 92 - 18]⁺, 221
11	mutatoxanthin epimer	2.1 ± 0.1	nd^e e not detected;	12.0-12.1	400, 428, 449	70	0	585	567[M + H - 18]⁺, 549[M + H - 18 - 18]⁺, 493[M + H - 92]⁺, 221
12	(all-E)-lutein	11.5 ± 0.7	nd^e e not detected;	12.4	415, 444, 472	50	0	569	551[M + H - 18]⁺,^g g in source fragmentation. Bold numbers indicate cis peak absorption wavelengths. III/II: spectral fine structure; AB/AII: cis peak intensity. 533[M + H - 18 - 18]⁺, 477[M + H - 92]⁺
13	mutatoxanthin epimer	1.3 ± 0.1	nd^e e not detected;	13.0	402, 427, 451	63	0	585	567[M + H - 18]⁺, 549[M + H - 18 - 18]⁺, 493[M + H - 92]⁺, 221
14	(all-E)-zeaxanthin	7.9 ± 0.5	nd^e e not detected;	14.7	413, 450, 476	29	0	569	551[M + H - 18]⁺, 533[M + H - 18 - 18]⁺, 495[M + H - 56 - 18]⁺, 476[M - 92] ⁺
15	(9Z)-antheraxanthin	10.6 ± 0.7	nd^e e not detected;	15.7	330, 410, 440, 468	60	9	585	567[M + H - 18]⁺, 549[M + H - 18 - 18]⁺, 493[M + H - 92]⁺, 475[M + H - 92 - 18]⁺, 221
16	(15Z) or (13Z)-zeinoxanthin + phytoene	1.0 ± 0.02	nd^e e not detected;	17.5	330, 412, 443, 467	16	51	553, 545	[553]: 535[M + H - 18]⁺, 461[M + H - 92]⁺; [545]: 489[M + H - 56]⁺
17	(all-E)-zeinoxanthin	2.4 ± 0.1	nd^e e not detected;	19.0-19.1	414, 445, 472	56	0	553	535[M + H - 18]⁺, 460[M - 92]⁺
18	(all-E)-β-cryptoxanthin	4.2 ± 0.3	0.3 ± 0.04	22.5	415, 450, 477	26	0	553	535[M + H - 18]⁺, 461[M - H - 92]⁺
19	(13Z)-β-carotene	nd^e e not detected;	3.7 ± 0.002	27.8	338, 422, 445, 470	7	44	537	nd^e e not detected;
20	(all-E)-α-carotene	0.8 ± 0.07	nd^e e not detected;	28.1	410, 445, 472	53	0	537	481[M + H - 56]⁺, 444[M - 92]⁺, 413, 401, 299, 281
21	(all-E)-β-carotene	1.3 ± 0.1	43.3 ± 0.2	32.3	412, 451, 478	17	0	537	444[M - 92]⁺, 413
22	(9Z)-β-carotene	nd^e e not detected;	3.3 ± 0.2	34.5	339, 421, 447, 473	17	6	537	nd^e e not detected;
	Total carotenoids of freeze-dried pulp / (µg g^-1)	107 ± 5	180 ± 13
	Total carotenoids of fresh pulp / (µg g^-1)	14.2 ± 0.7	25.2 ± 1.8

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