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Journal of Stress Physiology & Biochemistry, Vol. 16, No. 3, 2020, pp. 5-13 ISSN 1997-0838 Original Text Copyright © 2020 by Nandini and Vikas
ORIGINAL ARTICLE
OPEN /71 ACCESS
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
Girish Nandini and Vaidya Vikas
Ramnarain Ruia Autonomous College, L. Nappo Road, Dadar East, Matunga, Mumbai, Maharashtra 400019, (India)
*E-Mail: [email protected]
Received February 29, 2020
Wagatea spicata (Dalzell) Wight [Moullava spicata (Dalzell) Nicolson] exhibits a diverse concentration of biologically active constituents such as Lupeol, Bergenin, Stigmasterol, Friedelin, n-Hexadecanoic acid, Palmitic acid, Gamma sitosterol,13docosenamide, squalene and n-Tetracosanol-1.
Present work focuses on development and validation of a precise, accurate and reproducible HPLC method for simultaneous quantification of three pharmacologically active phytochemical markers 13docosenamide, squalene and n-Tetracosanol-1 from the aerial parts of Wagatea spicata.
Key words: HPLC, 13-docosenamide, squalene, n-Tetracosanol-1, simultaneous, method development, method validation
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 |g/ mL) were prepared in ethanol. 1000 |g/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 Mg/mL for 13 docosenamide, 100-300 Mg/mL for squalene, 5-300 Mg/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 |ig/mL for squalene and 13 docosenamide and while it was 5 |g/mL for n-tetracosanol-1, whereas the limit of quantification was found to be 100 |g/mL for squalene, 50 |g/mL for 13 docosenamide and 5 |g/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, 5^m
Mobile Phase HPLC grade acetonitrile containing formic acid (0.1%)
Flow Rate 1mL/min
Detector LC 2030/2040 PDA
Detection Wavelength 202nm
A
B
Figure 1: A - HPLC chromatogram of standard 13 docosenamide; B - HPLC chromatogram of standard squalene;
A
Figure 2: A - HPLC chromatogram of standard n-tetracosanol-1 ; B - HPLC chromatogram of standard mixture
Figure 3: HPLC chromatogram of leaf extract of Wagatea spicata
Figure 4: A - Spectrum of 13 docosenamide standard; B - Spectrum of 13 docosenamide from sample. mAU
250-
a-
5.296/1.00 Wavelength 270.232 mAU 0.0S&
-274 Lfi LD T I I T-CM LM'JJ i i i i oriLii m co into r-- CH W V IV
1 ■ ii i i i i ■ i II i i i i .........................
200 mAU 75-j
0-
300
400
500
600
700
nm
1 M 5.326/1.00 (N A 1 vTv __j____ Wavelength 201.096 _______j______________i__ mAU 16.421
1 / 1 \ Id) / \ — — h — — — - ---— — — — h — — — — —
i iD n ---1---- CD Lj'i T --------i----- CO iJ? C3 CO l> -765
----------- ---1---- — 1 ---- — - - — 1 1 i
200
3,00
400
500
600
700
A
B
A
nm
B
Figure 5: A - Spectrum of squalene standard; B - Spectrum of squalene from sample.
im a
B
Figure 6: A - Spectrum of n-tetracosanol-1 standard; B - Spectrum of n-tetracosanol-1 from sample.
A
<Peak Table>
PDA C h2 202nm
Peaktf Ret Time Area Height Cone Unit Mark Name
1 1.237 1284 46 0.003
2 1601 1214 198 0.003 V
3 2 413 3132303 109685 6.697
4 2.829 18676098 1243806 35.656
5 3,699 3882022 223511 8.300 V
6 3 916 32S38B4 184211 7.043 V
7 4 192 8065380 155756 17.245 V
8 5.291 1575381 110875 3.368 V
9 5,604 4536933 99339 9.701 V
10 6 332 1867379 74910 3.993 V
11 6 875 3732673 84721 7 931 V
12 9.246 2603 237 0.006
13 9,546 1987 224 0.004
Tola 46769121 2287520
B
Figure 7: A - HPLC chromatogram of mobile phase; B - HPLC chromatogram of ethanol blank
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(^g/ml) 0.779 0.255 1.558
Precision(%RSD) Interday, 200(^g/ml) 0.094 0.709 0.914
LOD 50 Mg/ml 50 Mg/ml 5 Mg/ml
LOQ 50 Mg/ml 100 Mg/ml 5 Mg/ml
Linearity 50-300 Mg/ml 100-300 Mg/ml 5-300 Mg/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.
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