key: cord-0052335-zxhve7tg
authors: Girme, Aboli; Saste, Ganesh; Pawar, Sandeep; Balasubramaniam, Arun Kumar; Musande, Kalpesh; Darji, Bhaumik; Satti, Naresh Kumar; Verma, Mahendra Kumar; Anand, Rajneesh; Singh, Ruchi; Vishwakarma, Ram A.; Hingorani, Lal
title: Investigating 11 Withanosides and Withanolides by UHPLC–PDA and Mass Fragmentation Studies from Ashwagandha (Withania somnifera)
date: 2020-10-21
journal: ACS Omega
DOI: 10.1021/acsomega.0c03266
sha: 7f5137882ecccf52581790f48430c33a898a9b54
doc_id: 52335
cord_uid: zxhve7tg

[Image: see text] Withania somnifera (WS), also known as ashwagandha or Indian ginseng, is known for its pharmacological significance in neurodegenerative diseases, stress, cancer, immunomodulatory, and antiviral activity. In this study, the WS extract (WSE) from the root was subjected to ultrahigh-performance liquid chromatography with photodiode array detection (UHPLC–PDA) analysis to separate 11 withanoside and withanolide compounds. The quantification validation was carried out as per ICHQ2R1 guidelines in a single methodology. The calibration curves were linear (r(2) > 0.99) for all 11 compounds within the tested concentration ranges. The limits of detection and quantification were in the range of 0.213–0.362 and 0.646–1.098 μg/mL, respectively. The results were precise (relative standard deviation, <5.0%) and accurate (relative error, 0.01–0.76). All compounds showed good recoveries of 84.77–100.11%. For the first time, withanoside VII, 27-hydroxywithanone, dihydrowithaferin A, and viscosalactone B were quantified and validated along with bioactive compounds withanoside IV, withanoside V, withaferin A, 12-deoxywithastramonolide, withanolide A, withanone, and withanolide B simultaneously in WS. This UHPLC–PDA method has practical adaptability for ashwagandha raw material, extract, and product manufacturers, along with basic and applied science researchers. The method has been developed on UHPLC for routine analysis. The 11 withanosides and withanolides were confirmed using the fragmentation pattern obtained by the combined use of electrospray ionization and collision-induced dissociation in triple-quadrupole tandem mass spectrometry (TQ–MS/MS) in the WSE.

Withania somnifera (WS) (L.) Dunal is commonly known as ashwagandha, winter cherry, and "Indian ginseng". 1 WS is one of the essential herbs in Ayurveda known as Medhya Rasayana (promotes learning and a good memory) and Rasayana (increases longevity, prevents aging, imparts resistance, and improves immunity).

The traditional use of "ashwagandha" is to increase energy and endurance, improve health, and nurture the elements of the body. 2 Because of its medicinal potential, WS roots are a part of over 200 formulations in Ayurveda, Siddha, and Unani medicines used in treatment. 3 Traditionally, it has multiple pharmacological actions that have been validated by numerous clinical studies for stress, neuroprotective, anti-inflammatory, and aphrodisiac properties. 4−6 The compounds of WS have acquired active research interest globally because of their multidimensional significance.

The primary chemical constituents of WS have been identified as bioactive steroidal lactones called withanolides. It is a group of naturally occurring C-28 steroidal lactones which are formed on an intact or rearranged ergostane framework, where C-22 and C-26 are oxidized and make a six-membered lactone ring. There are many novel structural variants of withanolides that have been reported and studied with modifications such as withanone, withaferin A, and withanolide A−D. The characteristic feature of withanolides and ergostane-type steroids is a C8 or C9 side chain with a lactone or lactol ring, where the lactone ring may be either six-membered or five-membered. It may be fused with the carbocyclic part of the molecule through a carbon−carbon bond or an oxygen bridge. 7−10 WS also contains withanolide glycosides or glycowithanolides known as withanosides with mostly a 6-O-β-D-glucopyranosyl-β-Dglucopyranosyl type of glycosidic linkage. Withanosides I− XI 4, 11 have been reported so far from WS and have anti-Alzheimer, antistress, and neuroprotective activity. 12 The isolated compounds of WS have a range of medicinal properties such as anticancer, anti-inflammatory, and antiangiogenic effects. 13 −15 These WS compounds have also shown antiviral activity 16, 17 with distinct effects on the viral receptor, which might be potent against COVID-19. 18, 19 The current reports call for the need to quantify these compounds and validating in the extract and dietary supplements.

The literature on standardization of WS shows reports of high-performance liquid chromatography (HPLC), 20−24 highperformance thin-layer chromatography, 25−27 and liquid chromatography−mass spectrometry (LC−MS)-based 28−31 methodologies with the single or multiple bioactive or chemical markers. In these, withanolides and withanosides have been studied separately or together. However, there is no report on the simultaneous validated determination of 11 markers, namely, withanoside IV, withanoside V, withaferin A, 12-deoxywithastramonolide, withanolide A, withanone, withanolide B, viscosalactone B, 27-hydroxywithanone, withanoside VII, and dihydrowithaferin A (Figure 1) , with a confirmative mass fragmentation from WS.

The challenge in analyzing this botanical is the separation of these isobaric and isomeric compounds by a reverse-phase (RP) approach with the confirmation. The analytical investigations for WS require a simple methodology with optimum extraction for withanosides and withanolides by LCbased photodiode array detection (PDA)/UV or MS detection methods. Currently, most of the quality control (QC) and research labs are equipped with LC-based systems, which are economical for routine analysis. Thus, this research study has validated an ultrahigh-performance liquid chromatography with photodiode array detection (UHPLC−PDA) methodology with the novelty report of quantification of viscosalactone B, 27-hydroxywithanone, withanoside VII, and dihydrowithaferin A for the first time in WS along with seven compounds, which may increase valuation as a standardized botanical. Also, this developed fingerprinting aimed for the better separation of a major withanolide, that is, withaferin A, along with other withanolides and withanosides by rapid and simple sample processing.

Hence, in the present study, the sensitive UHPLC−PDA method developed and validated the simultaneous determination of the 11 markers in the WS extract (WSE) from the roots by establishing the linearity and range with the limit of detection (LOD), the limit of quantitation (LOQ), precision, accuracy, and recovery with stability. The UHPLC−PDA method has practical adaptability to raw material, extract, and product manufacturers and researchers of ashwagandha as the availability of UHPLC instruments are common in QC, analytical, and research laboratories. Further, these compounds were identified and confirmed by triple-quadrupole tandem mass spectrometry (TQ-MS/MS) with this optimized RP-UHPLC method in the WSE. The mass fragmentation patterns and the distinctive data of 11 markers are being reported for the first time based on the precursor ion and possible fragmented ions from MS/MS.

2.1. UHPLC−PDA Method Optimization. The RP method was optimized using a simple matrix extraction process from WSE samples. The aim of the optimization was the separation of minor and major withanolides and withanosides with MS-compatible solvents for confirmation in the WSE. The four wavelengths (210, 227, 254, and 350 nm) were tested for the largest number of peak responses and relatively high 

http://pubs.acs.org/journal/acsodf Article sensitivity. The optimal wavelength for WS was found to be 227 nm. After several trials, the polar solvent system of water and acetonitrile was found effective in the separation of nonpolar and midpolar compounds. Compared with isocratic elution, programmed gradient elution was more suitable for better profiling a complex multicomponent from WS. The MS Figure 2 ).

The system suitability was performed on different parameters such as retention time (rT), response, number of theoretical plates (n), tailing factor (Tf), resolution factor (Rs), and capacity factor (K′) (Table S1).

2.2.1. Selectivity, Linearity, LOD, and LOQ. After establishing the suitability of the system, the comparison of the representative chromatogram for standards and spiked samples and negatively proved that the optimized method was able to differentiate the analytes from the matrix interferents. Further, the recovery, precision, and linearity were validated according to guidelines. The LOD and LOQ were determined for each analyte under the acquired chromatographic conditions as per ICHQ2R1 by linearity and the calibration curve slope (CCS) method. The LOD and LOQ values for these specific analytes were in the range of 0.213−0.362 and 0.646−1.098 μg/mL, respectively. The calibration curve found excellent linearity for all compounds with r 2 > 0.99 ( Figure S1 ). Linear regression analyses of the calibration curves of these compounds are provided in Table 1. 2.2.2. Accuracy, Precision, and Recovery. The precision of the developed UHPLC−PDA method was verified by repeated injections of the WSE for quantification (1−11) at six concentrations. The intraday and interday variations by the developed UHPLC method were evaluated by the relative standard deviation (RSD) values of active compounds in the WSE. The analytical recovery was performed by analyzing the analytes by spiking with the 11 reference standards in the WSE. The overall recovery percentages ranged between 84.77 ± 0.547 and 100.11 ± 0.631% for 3 different concentrations in 3 replicates for 11 compounds. The developed method was specific for determining compounds 1−11 as their peak purity values were established with the absence of any coeluting peaks (Table 1) (6), withanoside V (7), 12-deoxywithastramonolide (8), withanolide A (9), withanone (10) , and withanolide B (11) in the WSE ( Figure 3C ). There was no significant difference in rTs of UHPLC−PDA and ESI-MS/MS combined as per the t-test (Table 2 ).

The WS compounds such as withanolide A, withanolide B, withanone, 12-deoxywithastramonolide, and withaferin A have been discussed in a few reports for their mass ions and possible fragmentation characteristics. 31, 33 We have studied viscosalactone B; withanoside IV, V, and VII; 27-hydroxywithanone; and dihydrowithaferin A with these compounds under the same set of experiments for more clarity. Therefore, the precursor ions, mass ions, and fragmentation patterns for compounds 1−11 from WS are discussed below. 

Calculations of the WSE. The fingerprinting and quantitative analysis of the standardized extract is a robust QC tool. Many QC laboratories are equipped with UHPLC or HPLC instruments, which facilitate routine analysis. This method has the advantage of using protic solvents such as water with acetonitrile, with improved separation of target analytes. It could also be adopted for LC-based MS/MS identification of these compounds in WS if coupled with hyphenated techniques. The profile pattern has been generated for 12 batches of the WSE (n = 3) by UHPLC−PDA for studying the robustness of this methodology (Figure 4 ). The quantitative results indicated that the analysis is robust (% RSD, 1.64) for the total content of 11 compounds with good reproducibility (Table 3) .

With the multicomponent presence, the precise quantification of these bioactive compounds is key to the assessment of WS quality. The current analytical studies use multiple reference standards for the quantitative determination of these constituents in the routine analysis by an external standard method. 20, 21, 35 However, the cost, stability, and shortage of these reference standards are the issues. Therefore, after generating the UHPLC profile for 12 samples of the WSE (n = 3), the mean content was calculated. The mean quantitative results of these 12 batches confirmed that by adopting this methodology, as set A (4.14 ± 0.07%) in the WSE. The second quantitation set B (3.92 ± 0.06%) was generated using three representative external standards, that is, withanoside IV for withanosides (1, 2, and 7), withaferin A for withaferin A (6), and withanolide A for withanolides (3−5, 8− 11) in the calculations (Table S2 ). These quantitative data sets (A and B) were analyzed using a percent relative error (% RE), representing a percent relative difference between both sets, which was found to be 5.20 ± 0.38%. To find the similarity of data sets A and B, the Pearson correlation coefficient (R) was calculated and found to be 0.9704 (a value closer to 1.0 indicates similarity in data sets). These preliminary investigations indicate that the use of withanoside IV, withaferin A, and withanolide A can represent precise results as a validated method (A). 

http://pubs.acs.org/journal/acsodf Article Standardization is a pivotal link between the preformulation, formulation, and biological application of any botanical medicine. The current research and applications of WS are dominant by only withanolide A and withaferin A. 10, 13, 14, 22, 30 Analytical research focused only on these compounds is not sufficient in the evolved research of this botanical. 33−35 Thus, with the present methodology, the evaluation of the WSE increases in terms of 11 bioactive molecules for any further research study with a simple and validated UHPLC technique.

The selected compounds in this research, namely, withanoside IV, withanoside V, withaferin A, 12-deoxywithastramonolide, withanolide A, withanone, and withanolide B, are prominent in therapeutic effects from the withanolide class and withanoside class. 36−39 All these compounds have proven their role in different biological applications such as antitumor activity, inhibition of pancreatic cancer, antiviral activity, immunomodulatory activity, stress, and treatment for Alzheimer's disease. 40−45 In this research, we have combined the UHPLC confirmation and quantitation of these compounds with additional bioactive markers, that is, withanoside VII, 27-hydroxywithanone, dihydrowithaferin A, and viscosalactone B in a validated way. 29, 33 This analytical method will be an advantage in addressing biological and pharmacokinetic studies, which are currently only focused on withaferin A and withanolide A. 46, 47 More studies targeting one molecule or a combination of molecules will be possible with the help of this method. This method is also advantageous for studying more molecules in formulations and mixtures. The availability of standards and methods of analysis of new withanolides and withanosides will open the chances of finding the activity of these compounds, which are present in the right concentration in the WSE (ashwagandha extract).

A novel UHPLC−PDA method for simultaneous identification and quantification of 11 compounds, that is, withanoside IV, withanoside VII, withanoside V, withaferin A, 12-deoxywithastramonolide, withanolide A, withanone, withanolide B, viscosalactone B, 27-hydroxywithanone, and dihydrowithaferin A, has been reported the first time. The developed method was successfully validated and applied for ESI-MS/MS confirmation and the mass fragmentation study of these compounds in WS (L.) Dunal roots. The developed method showed the selectivity, reliable sensitivity (an LOQ from 0.646 to 1.098 μg/mL), an acceptable range of precision (lower than 5.0%), and recoveries (from 84.77 to 100.11%). This method has an application in routine laboratory analysis of WS and its raw material, extracts, and products considering its sensitivity and satisfactory performance. (2), viscosalactone B (3), 27-hydroxywithanone (4), dihydrowithaferin A (5), withaferin A (6), withanoside V (7), 12-deoxywithastramonolide (8), withanolide A (9), withanone (10) , and withanolide B (11) were the compounds used in this study. Compounds 1 and 9 were procured from USP, USA, and compounds 7, 10, and 11 were procured from Phytocompounds (Bangalore, IN); compounds 6 and 8 were procured from Chromadex (CA, USA). Compounds 2−5 were isolated from the roots of WS in our in-house laboratory by previously reported methods. 48 with product ion confirmation by a precursor scan. The interface for ESI was set at 300°C. The desolvation line and heat block temperatures were set to 250 and 400°C, respectively. The nebulizing gas flow was 2.5 L/ min, the heating gas flow was 10.00 L/min, and the drying gas flow was 10.00 L/min. The fragmentation was performed at CE 20 eV. The WSE sample was analyzed in the Q1 scan mode, where the scan range was 100−2000 m/z in the positive and negative modes, which was later confirmed in the product ion scan mode for m/z 781, 489, 487, 473, 783, 767, 471, 469, and 455 for the compounds as per their mass ion. All data were analyzed using the Lab Solution software (Version 6.80).

The stock solution of each of the standard compounds (1−11) was prepared with a concentration of 1 mg/mL in methanol. Five different concentration levels of each compound were injected in triplicate to prepare the calibration curves. The range of calibration curves was between 1-150 μg/mL for compounds 1−4 and 6−11 and 1−100 μg/mL for compound 5. All the working solutions were stored at 4°C. 4.5. Sample Solutions. The simple and rapid method was employed to extract the target analytes from the WSE. The samples from Section 4.1 were accurately weighed, and ACS Omega http://pubs.acs.org/journal/acsodf Article methanol (100 mg/10 mL) was added, followed by sonication for 30 min. This sample solution was passed through a 0.22 μm filter and injected into the UHPLC system for the analysis. 4.6. UHPLC−PDA Method Validation. A calibration curve was plotted by preparing five different concentrations of compounds (1−11) and injected separately in the UHPLC system, followed by recording their peak area and plotting a curve between peak area and concentration. The regression equations were obtained after plotting peak areas versus the corresponding concentration of five standards individually. The CCS method was used to determine the LOD and LOQ ( Table 1) .

The intraday and interday precision and accuracy were determined by assaying six same concentrations of the WSE. The experiment was repeated (n = 6) on the same day and the consecutive day (n = 6). The measurement precision in the WSE for the content of marker compounds 1−11 separately was expressed in the RSD. The recovery was determined based on the recovery of known amounts of the analyte added to the extracts. Each level was prepared by three replicate samples (n = 3), as shown in Table 1 . The analyte concentrations were determined by the external standard method against the individual standard area under the curve (AUC) method under identical conditions for the experiments. The recoveries were obtained (between 80.0 and 120.0%), and the precisions were lower than 5.0% as per the guidelines of ICH. Table 2 .

The combined use of ESI and collision-induced dissociation in TQ-MS/MS was applied to investigate the structural characterization of 11 compounds of WS in the protonated and deprotonated forms. The data obtained by this experiment performed on the reference compounds (1−11) generated proposed fragmentation mechanisms with ion structures.

4.8. Application in the Quantification of WSE Samples by External Standard Calibration. The 12 WSE batches were prepared from the same WSR with the extraction methodology as per Section 4.1 and analyzed (n = 3) by the validated UHPLC−PDA method as per Section 4.3. The quantification was done by an external standard calibration method. The percent relative error (% RE) was calculated to determine the arithmetic difference of two sets of values in content. Experimental data analysis and parameter calculation were achieved using Office Excel 2010 (Microsoft, Redmon, WA, USA)..

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c03266.

System suitability and specificity parameters for the UHPLC−PDA methodology; quantification results in the total content of compounds (1−11) in the WSE by an external standard with 11 standards and with three standards, withanoside IV for withanosides (1, 2, and 7), withaferin A for withaferin A (6), and withanolide A for withanolides (3−5, 8−11) ; linearity and residual plots of validation by the UHPLC−PDA method for compounds (1−11) ; and ESI-MS/MS data for 11 withanosides and withanolides in the ESI (+ve and −ve) mode (PDF)

μg, microgram; μm, micrometer; C, celsius; CV, coefficient of variation; ESI, electrospray ionization; eV, electronvolt; g, gram; HPLC, high-performance liquid chromatography

L, liter; LC, liquid chromatography; m/z, mass-to-charge ratio

min, minutes; mL, milliliter; mm, millimeter; MS, mass spectrometry

tandem mass spectrometry; nm, nanometer; PDA, photodiode array detection; r 2 , regression coefficient; RE, relative error

RSD, relative standard deviation

SD, standard deviation; SEM, standard error of mean; TIC, total ion chromatogram; TQ-MS/MS, triple-quadrupole tandem mass spectrometry; UHPLC, ultrahigh-performance liquid chromatography

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The authors declare no competing financial interest.