Development and validation of a quantification method for α-humulene and trans-caryophyllene in <i>Cordia verbenacea</i> by high performance liquid chromatography

Gomes, Márcio Vinícius da Silva; Silva, J.D. da; Ribeiro, Ana Ferreira; Cabral, Lúcio M.; de Sousa, V.P.

doi:10.1016/j.bjp.2019.01.009

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Revista Brasileira de Farmacognosia

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Original articles • Rev. bras. farmacogn. 29 (2) • Mar-Apr 2019 • https://doi.org/10.1016/j.bjp.2019.01.009 copy

Development and validation of a quantification method for α-humulene and trans-caryophyllene in Cordia verbenacea by high performance liquid chromatography

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

Acheflan^® herbal medicine is the first medicine developed and produced in Brazil using Cordia verbenacea DC., Boraginaceae, essential oil as a constituent. C. verbenacea has anti-inflammatory properties, which have been directly related to α-humulene and trans-caryophyllene. Currently, the quantification of α-humulene and trans-caryophyllene in C. verbenacea has only been described by GC-MS. Although this technique is widely used it cannot be directly applied to finished medicinal products since they contain aqueous constituents. In this context, the objective of this work was to develop a methodology for quantifying C. verbenacea in raw materials and in pharmaceutical formulations using liquid chromatography. The Box–Behnken experimental design was used successfully to optimize the analytical method. The method developed in this study was validated and proven to be effective in the proper separation of α-humulene and trans-caryophyllene. In addition, the developed method was applied to commercial formulations (cream and aerosol) containing C. verbenacea essential oil, which resulted in very satisfactory separation without interference from excipients. C. verbenacea oil contains four times higher concentrations of trans-caryophyllene than α-humulene. Therefore, we propose that trans-caryophyllene can be used as a marker of C. verbenacea essential oil. Evaluation of trans-caryophyllene content would be especially valuable for applications where the concentrations are very low such as in permeation and release studies of dermatological formulations.

Keywords:
Box–Behnken; Experimental design; α-humulene; Trans-caryophyllene; Acheflan®; HPLC

Column	Method	Cromatographic conditions					Cromatographic Parameters
		t (min)	OP%	AP%	F	T	HUM	CAR	R HUM	R CAR
							t _R	t _R
CN	1	0	43.6	56.4	1.2	25	21	22.12		0.65
	14	50.3	49.7
	22	50.3	49.7
	22.1	43.6	56.4
	30	43.6	56.4
	2	0	43.6	56.4	1.2	25	10.39	10.97		0.611
	4	50.3	49.7
	22	50.3	49.7
	23	43.6	56.4
	31	43.6	56.4
	3	0	43.6	56.4	1.2	25	13.66	14.68		1.074
	15	43.6	56.4
	5	50.3	49.7
	5	50.3	49.7
	1	43.6	56.4
	5	43.6	56.4
	4	0	43.6	56.4	1.2	25	16.01	17.12		0.925
	20	50.3	49.7
	40	50.3	49.7
	41	43.6	56.4
	50	43.6	56.4
C₂	5	0	10	90	1.2	25	-	-	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.
		30	50	50
	6	0-40^a a Isocratic elution.	25	75	1.2	25	3.51	3.62	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.
	7	0-40^a a Isocratic elution.	15	85	1.2	25	3.82	3.9	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.
	8	0-40^a a Isocratic elution.	25	75	1.0	25	4.01	4.22	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.
	9	0-40^a a Isocratic elution.	15	85	1.0	25	4.12	4.18	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.
C₃	10	0	50	50	1.0	25	9.8	10.2	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.
		30	90	10
	11	0-30^a a Isocratic elution.	60	40	1.0	25	6.22	6.56	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.
	12	0-40^a a Isocratic elution.	50	50	1.0	25	16.92	18.33	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.
C₄	13	0	10	90	1.2	25	44.01	44.79	0.71	0.32
		60	90	10
	14	0	50	50	1.2	25	26.08	26.73	0.92	0.88
		30	90	10
	15	0-30^a a Isocratic elution.	57	43	1.2	25	16.89	18.97	2.8	0.88
	16	0-45^a a Isocratic elution.	52	48	1.4	40	18.16	20.64	1.54	0.9
	17	0-45^a a Isocratic elution.	52	48	1.4	25	21.81	24.81	0.92	1.23
	18	0-45^a a Isocratic elution.	45	55	2.0	40	26.55	31.07	1.63	0.63
	19	0-45^a a Isocratic elution.	45	55	2.0	25	34.03	40.06	1.79	0.72
	20	0-45^a a Isocratic elution.	43	57	2.2	40	31.26	36.97	1.81	0.98
	21	0	44	56	2.0	40	19.45	23.72	1.46	0.96
		10	44	56
		10.1	53	47
		14	53	47
		14.1	45	55
		50	45	55
C₆-Phenyl	22	0	10	90	1.0	25	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.
		60	90	10
	23	0	5	95	1.0	25	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.
		60	25	75
	24	0-45^a a Isocratic elution.	62	38	0.8	25	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.	^b b Resolution not calculated due to high quantity of components coeluted with HUM and CAR. t, run time (min); OP, organic phase (acetonitrile in all methods); AP, Aqueous phase (9 mM Phosphate buffer pH 5.0 + 1.1% TEA for 1-5 method and MilliQ water for 5-30 method); F, flow rate (ml/min); T, temperature (°C); R, resolution; tR (min), retention time.
C₈	25	0	50	50	1.0	25	25.76	26.73	1.01	0.81
		30	90	10
	26	0	65	35	1.2	25	17.37	18.76	0.48	0.4
		30	95	5
	27	0	62	38	1.5	25	17.07	18.57	1.22	1.27
		30	90	10
C₁₈	28	0	50	50	1.0	25	22.79	23.33	1.29	0.65
		30	90	10
	29	0-30^a a Isocratic elution.	70	30	1.0	25	10.21	12.05	0.33	1.49
	30	0-30^a a Isocratic elution.	60	40	1.0	25	25.13	29.69	0.6	0.95

Method	Chromatographic conditions						Chromatographic parameters
	t (min)	ACN%	HP-β-CD%	MeOH	F	T	HUM	CAR	R HUM	R CAR
							t _R	t _R
31	0-45^* * means isocratic elution of the mobile phase	40	50	10	1.0	50	24.64	28.84	1.13	1.7
32	0-45^* * means isocratic elution of the mobile phase	38	52	10	1.0	50	26.26	31.04	1.72	1.85
33	0-45^* * means isocratic elution of the mobile phase	34	50	16	1.0	50	24.97	29.48	1.08	^ means resolution not calculated due to high amount of component coeluted with HUM and CAR
34	0-45^* * means isocratic elution of the mobile phase	41	51	8	1.0	50	26.65	29.61	1.16	1.64
35	0-45^* * means isocratic elution of the mobile phase	42	51	7	1.0	50	25.01	29.33	1.21	1.09

Factors (Independent variables)	Level (-)	Level (0)	Level (+)
X₁: Flow rate (ml/min)	0.8	1.0	1.2
X₂: Column temperature (°C)	30	40	50
X₃: Methanol proportion in the mobile phase (%)	6	8	10
X₄: HP-β-CD concentration (mM)	15	20	25
Responses (Dependent variables)	Abbreviation
Y₁: Resolution between component 1 and caryophyllene	R_CMP1-CAR
Y₂: Resolution between caryophyllene and component 2	R_CAR-CMP2
Y₃: Retention time of caryophyllene	RT_CAR
Y₄: Resolution between component 3 and humulene	R_CMP3-HUM
Y₅: Resolution between humulene and component 4	R_HUM-CMP4
Y₆: Retention time of humulene	RT_HUM

Trial	Factors				Responses
	X₁	X₂	X₃	X₄	Y₁	Y₂	Y₃	Y₄	Y₅	Y₆
1	0.8	30	8	20	0.947	1.332	45.32	1.230	1.124	37.87
2	0.8	50	8	20	1.593	2.286	36.26	1.336	0.000	31.15
3	1.0	40	6	15	1.261	1.079	31.93	1.380	0.460	27.31
4	1.0	40	10	15	1.840	2.25	34.53	1.282	0.000	29.29
5	0.8	40	8	15	1.562	1.274	41.24	1.210	0.000	35.19
6	1.2	40	8	15	1.694	1.343	28.16	1.311	0.000	24.00
7	1.0	30	6	20	0.268	1.414	35.37	0.000	1.607	29.66
8	1.0	30	10	20	1.213	0.741	37.44	1.840	0.000	31.10
9	0.8	40	6	20	2.288	1.721	39.46	1.138	0.819	33.53
10	0.8	40	10	20	1.935	1.598	41.78	1.891	1.574	35.26
11	1.0	30	8	15	1.716	1.717	38.43	1.148	1.035	32.26
12	1.0	50	8	15	0.000	1.163	29.93	1.334	0.633	25.79
13^c	1.0	40	8	20	2.227	1.852	30.02	1.249	0.663	25.27
14^c	1.0	40	8	20	1.989	1.634	32.71	1.163	1.088	27.70
15^c	1.0	40	8	20	1.852	1.566	32.41	1.117	1.820	27.44
16	1.2	30	8	20	0.951	1.113	30.05	0.000	0.407	25.03
17	1.2	50	8	20	1.561	2.334	23.95	1.254	0.400	20.54
18	1.0	40	6	25	1.534	1.465	28.79	1.054	1.391	24.43
19	1.0	40	10	25	0.617	1.097	30.30	0.925	0.509	25.55
20	0.8	40	8	25	0.000	1.493	37.58	1.669	1.055	31.79
21	1.2	40	8	25	0.000	1.396	24.72	0.996	0.908	20.94
22	1.0	50	6	20	1.549	3.105	28.25	1.319	0.000	24.29
23	1.0	50	10	20	1.715	2.115	29.63	1.221	0.623	25.35
24	1.2	40	6	20	2.331	1.768	26.49	1.192	0.904	22.50
25	1.2	40	10	20	1.940	1.548	27.92	1.795	1.512	23.55
26	1.0	30	8	25	1.297	0.000	32.97	1.033	2.277	27.48
27	1.0	50	8	25	1.949	1.773	26.99	1.172	0.845	23.21
28^c	1.0	40	8	20	1.940	1.548	32.67	1.795	1.512	27.67
29^c	1.0	40	8	20	2.269	1.632	32.63	1.107	1.238	27.61
30^c	1.0	40	8	20	2.054	1.683	32.83	1.178	1.158	27.79

Parameters			HUM		CAR
Linearity	Range		5-25 µg/ml		64-96 µg/ml
	Coefficient of determination (R²)		0.9987		0.9932
	F regression slope non-zero (F_critic = 7.71)		2963.0		591.7
	Slope ¹/F_calc (F_critic = 4.26)		[153255.6 ± 1982/0.398]		[180,008.8 ± 1033.7/0.014]
	Intercept^1/ F_calc (F_critic = 3.98)		[-87,666.4 ± 3760.3/2.513]		[-1,755,483 ± 47,318/0.214]
	Concentration (%)	Day	Intraday (DPR%)	Interday (DPR%)	Intraday (DPR%)	Interday (DPR%)
Precision	80	1	0.69	0.57	0.79	1.04
	80	2	0.59	0.57	1.44	1.04
	100	1	0.23	0.17	1.53	1.76
	100	2	0.05	0.17	1.04	1.76
	120	1	0.92	0.59	0.69	0.79
	120	2	0.07	0.59	1.04	0.79
Accuracy	Low		101.83 ± 0.69		98.72 ± 0.79
	Medium		100.56 ± 0.22		100.74 ± 1.53
	High		99.33 ± 0.92		98.37 ± 0.69
Limits	LD (µg/ml)		0.074		0.789
Limits	LQ (µg/ml)		0.245		2.629

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[1] * Corresponding author. E-mail:valeria@pharma.ufrj.br (V. de Sousa).