Development and Validation of a Simple HPLC PDA Method for the Simultaneous Analysis of 13- Docosenamide, Squalene and n-Tetracosanol-1 from the Leaf Extracts of Wagatea spicata

Traditional knowledge of India is gaining increasing acceptance all over the world for its therapeutic efficacy and safety (Chaudhary and Singh, 2011). However, only a small percentage of the entire treasure has been explored and there is a lot more to be put forth. To meet this objective, it is essential that the ethnomedicinal plants of Indian origin are scientifically studied for their known medicinal benefits and documented. Development of a formulation from such plants will also require extensive standardization for establishing the quality and/or efficacy of individual plant components as well as raw material or as a final product component. (Nandini et al. , 2017)

With the above objective in mind, the isolation of the compounds 13docosenamide, squalene and n-Tetracosanol-1 has been previously performed using HPTLC and the structural elucidation was performed with the help of FTIR, NMR and GCMS techniques (Surange and Deokule, 1986; Nandini and Vikas, 2019). Further, it was felt worthwhile to develop simultaneous methods for the quantitative analysis of the isolated components.

Thus, the present work explains the development of a simple, sensitive and accurate high-performance liquid chromatographic method for the simultaneous determination of 13docosenamide, squalene and n-Tetracosanol-1 from the leaf extracts of the plant Wagatea spicata .

MATERIALS AND METHODS

Chemicals and materials

HPLC grade Methanol, Ethanol was procured from E. Merck, Mumbai, India. Reference standards of 13 Docosenamide (Purity>95%), Squalene (Purity>95%) and n-Tetracosanol-1 also called as Lignoceryl alcohol (Purity>95%) were purchased from Sigma-Aldrich (Aldrich Division; Steinheim, Germany).

Plant Material

Wagatea spicata fresh plant was collected from the field area of Kankeshwar, Alibaug, District- Raigad, and Maharashtra, India in the month of November 2014; and the herbarium specimens were identified and authenticated by Botanical Survey of India, Pune.

Sample preparation

1 g of fine leaf powder in 10ml of ethanol was subjected to accelerated maceration by ultrasonication for 30 minutes, followed by overnight steady state extraction. After filtration using syringe filter (pore size 0.2 microns), the clear extract was used for HPLC analysis.

Preparation of standard solution(s)

Individual stock solutions of n-Tetracosanol-1, 13 docosenamide and Squalene (1000 µg/ mL) were prepared in ethanol. 1000 µg/mL stock solution of standard mixture was also prepared in ethanol.

RESULTS AND DISCUSSION

Optimization of the Chromatography

Initial trial experiments were conducted to select a suitable mobile phase for accurate analysis of the standards and HPLC grade acetonitrile containing formic acid (0.1%) in isocratic mode was finalized as the best for the separation of the three analytes 13 docosenamide, squalene and n-tetracosanol-1.

The separation of analytes of interest was also evaluated at different flow rates (0.6-1mL/min). Finally the flow rate was optimized to 1 mL/min to avoid the interference of solvent peaks. Considering the complexity of herbal samples, the total run time for the method was determined to be 35 mins.

The wavelength of 202 nm was selected for the detection of analytes eluting out from the column as phytoconstituents under study demonstrated the maximum absorption at the specified wavelength.

The optimized chromatographic conditions chromatograms and spectra of individual standards standard mixture and sample extract as obtained under the same chromatographic conditions are depicted in Table 1 and Figures 1-7 respectively.

Method Validation

The developed method was validated as per tripartite ICH guidelines (ICH Harmonised Tripartite Guideline, 2005).

Selectivity: As shown in figures 7A and 7B, there was no interference observed from diluent blank (ethanol) and mobile phase overlapping with the retention times of 13 docosenamide, squalene and n-tetracosanol-1. Hence the method is selective.

System suitability : The RSD values for area and retention time of 13 docosenamide, squalene and n-tetracosanol-1 and were found to be <2% indicating that the system was suitable to carry out further analysis. Squalene, 13 docosenamide and n-Tetracosanol-1 were detected at 15.298, 8.963 and 32.054 minutes respectively.

Linearity : The method was found to be linear from 50-300 μg/mL for 13 docosenamide, 100-300 μg/mL for squalene, 5-300 μg/ml for n-Tetracosanol-1. The correlation coefficient was found to be ≥0.99 for all the three components.

Sensitivity: Sensitivity of the method was affirmed in terms of LOD and LOQ for 13 docosenamide squalene and n-Tetracosanol-1.

The value of limit of detection was found to be 50 µg/mL for squalene and 13 docosenamide and while it was 5 µg/mL for n-tetracosanol-1, whereas the limit of quantification was found to be 100 µg/mL for squalene 50 µg/mL for 13 docosenamide and 5 µg/mL for n-Tetracosanol-1 (Griffiths, 2003; Vuppugalla et al ., 2003 Lu et al. , 2004; Wichitnithad et al , 2009; Le et al. , 2019).

Precision : In the repeatability study, intra-day and inter-day precision of the HPLC method were investigated using replicate injection ( n =3) of quality control samples of all the three standards. The developed method was found to be precise with RSD<2%.

Robustness: As inferred from the %RSD values of robustness testing results, it can be said that the proposed method was not influenced by slight change in the wavelength of analysis. Minor change in flow rate and mobile phase composition affected the separation of the three analytes. However, the simplicity of the method increases the robustness by minimizing mobile phase alterations.

Stability: Stability studies showed that the components were found stable in the mixture for at least 24.0 h at room temperature and 48 hours at 2-8°C of storage condition. The individual stock solutions however, were stable for up to 72 hours under both the conditions.

Assay: The percentage of n-tetracosanol-1, 13 docosenamide and Squalene in the leaf extracts of Wagatea spicata extract was found to be 0.009% 2.54% and 1.07% respectively. The method is sensitive and selective for the aforementioned phytoconstituents in presence of other phytochemicals present in the extract.

Recovery: The recovery values for all the three components were within acceptable limits (80.0 to 120.0%). This indicated that the method was reliable and accurate.

Thus, the proposed HPLC method was found to be suitable for qualitative and simultaneous quantitative analysis of 13 docosenamide, squalene and n-Tetracosanol-1 in the ethanolic extract of Wagatea spicata . The summary of validation is charted in Table 2.

Table 1: Optimised Chromatographic Conditions.

Parameter	Description
Instrument	HPLC- Prominence-i, LC-2030C 3D Plus Liquid chromatograph
Pump	LC 2030 pump
Injector	LC 2030, Autosampler
Injection volume	50ul
Column oven	LC 2030 oven,40oC
Column	Shimadzu, C18 250mm X 4.6 mm, 5µm
Mobile Phase	HPLC grade acetonitrile containing formic acid (0.1%)
Flow Rate	1mL/min
Detector	LC 2030/2040 PDA
Detection Wavelength	202nm

3eakS	Ret. Time	Area	Heiqht	Name
1	3.693	7896636	270958
2	8.935	6632864	316005	13 docosenamide

A

Figure 1: A - HPLC chromatogram of standard 13 docosenamide; B - HPLC chromatogram of standard squalene;

<Реак Table>

РПАСМ ?П?пт

=>63^	Ret. Time	Area	Heiaht	Name
1	2.837	2954943	372324
2	3.004	6410179	346734
3	3.696	4516941	179659
4	15.296	6679980	215758	Squalene

A

' ^ПАПМ 909nm

эеакй	Ret. Time	Area	Heiaht	Name
1	8.974	1684717	72330	13 docosenamide
2	11.681	438963	11816
3	15.291	394912	12731	saualene
4	32.084	47666422	844539	n-tetracosanol-1
Total		50185014	941416

Figure 2: A - HPLC chromatogram of standard n-tetracosanol-1 ; B - HPLC chromatogram of standard mixture

B

рпд гы	Fedc^ble Я№™
РУ-	Ret. Г'"?	Am	НчеЬл	__ Name __
1	1.079	3333	352
2	2.457	31-047203	21305 56
3	2.324	1154515"	17-525-
4	2.933 3.717	39405051 423043"9	____________ 1 "25593 13-04431	—
5	4.539	35499	10424
	4.373	140736	14524
в	5 200	359633	53472
g	5.-93	139010	13590
10	5.244	85337	7453
11	5.335	3303191	317631
12	7908	55153	5545
13	3.753	114570	7643
14	9 072	2-9125	12353	13 Дocc«ramida
._______Li.	9.605	-10607	17034
1      16	10.666	4353 36	22043
17	11620	215341	5937
IB	12.321	46645	1423
19	13.022	63122	2236
20	15.040	6917	333	mraffw__________________
21	16.050	632275	24801
22	15.772	615166	14355
23	19 30В	27150	"45
24	20.342	49-251	10234
25	_____________ 22.51-	128474	4120
26	_____________ 23.553	298015	341
27	24.373	54335	1532
23	25.633	54404	1441
29	29.096	______________ 295313.	5953
30	32.453	47626	1039	ibtetracosmol-l
31	34.079	171375	3305
Totil		139096971	755-263

Figure 3: HPLC chromatogram of leaf extract of Wagatea spicata

A

Figure 4: A - Spectrum of 13 docosenamide standard; B - Spectrum of 13 docosenamide from sample.

Figure 5: A - Spectrum of squalene standard; B - Spectrum of squalene from sample.

B

Figure 6: A - Spectrum of n-tetracosanol-1 standard; B - Spectrum of n-tetracosanol-1 from sample.

1243806 223511 184211 155756 110875

Peak#

Ret. Time

B

Figure 7: A - HPLC chromatogram of mobile phase; B - HPLC chromatogram of ethanol blank .

PDA Ch2 202nm

1.601

2.413

2.829

3.699

3.916

4.192

5.291

5.804

6.332

6.875

9.246

9.546

12 "

Total

Table 2: Summary of Validation

Parameter	13 docosenamide	Squalene	n-Tetracosanol-1
System Suitability(%RSD)	Area = 0.696 Rt = 0.52	Area=1.476 Rt = 0.589	Area = 0.393 Rt = 0.585
Specificity and Robustness	Specific and Robust	Specific and Robust	Specific and Robust
Precision(%RSD) Intraday,200(μg/ml)	0.779	0.255	1.558
Precision(%RSD) Interday, 200(μg/ml)	0.094	0.709	0.914
LOD	50 μg/ml	50 μg/ml	5 μg/ml
LOQ	50 μg/ml	100 μg/ml	5 μg/ml
Linearity	50-300 μg/ml	100-300 μg/ml	5-300 μg/ml
Assay	2.54%	1.075%	0.009%
Recovery	100.894%	97.550%	100.787%

CONCLUSION

The current work provides a simple, precise, accurate and reproducible method for the qualitative and quantitative analysis of 13 docosenamide, squalene and n-Tetracosanol-1 from Wagatea spicata . The developed method can be used as a tool to asses phytochemical variation caused due to diverse geographical, climatic genotypic factors. It can also be used as a quality control method for the plant and formulations containing Wagatea spicata .

ACKNOWLEDGEMENTS

The authors acknowledge the support received from Rashtriya Uchchatar Shiksha Abhiyan (RUSA) for the HPLC facility at the Department of Bioanalytical Sciences, Ramnarain Ruia Autonomous College.