1. Chemicals and reagents
The solvents and chemicals were procured from E. Merck India. Reserpine (Lot No. 3819921) was purchased from Sigma-Aldrich.
2. Collection and processing of plant materials
Roots of Sarpagandha (Rauvolfia serpentine) were collected from the medicinal plant garden of the Institute, and the sample was stored for future reference with specimen no. 569874. Roots were appropriately washed with water to remove adhered foreign matter and dried under shade. Dried materials were examined carefully and handpicked to remove any foreign matter and stored in an airtight glass container.
3. Brief outline for the preparation of Sarpagandha Ghana
Sarpagandha Ghana (a dried and powdered decoction) was prepared as per classical text8. The root was crushed into pieces of 1–3 cm to obtain kwatha churna (coarsely grounded). This kwacha churn was boiled with 16 times potable water to reduce it to 1/8th of the original volume and filtered through a muslin cloth. Filtrate was further heated to get concentrated and subsequently solidified under vacuum to get a powdered mass called ghana. Ghana was carefully preserved in an airtight container to avoid any moisture or humidity exposure.
4. Preparation of Stock and standard solution of reserpine
A stock solution of reserpine (1000 ppm) was prepared by dissolving 100 mg accurately weighed quantity in 100 mL methanol. Variable aliquots of this stock solution were transferred to a 10 mL volumetric flask and diluted up to the mark with methanol to get a working standard of 20–100 ppm for chromatographic experiments.
5. Preparation of working extracts of plant, ghana
Root and ghana were extracted separately in methanol by continuous extraction method to obtain the extracts of 10mg/mL.
6. High Performance Thin Layer Chromatographic (HPTLC) analysis
6.1. Instrumentation
For the HPTLC study, sample (ghana and root) and standard were applied as a band by ATS-4, CAMAG, on aluminium supported precoated 60F254 silica gel plate, developed in CAMAG make twin trough chamber. The developed plate was scanned densitometrically by TLC Scanner 4 and photo documented by using TLC visualiser-3 of CAMAG make. For all the instrumental steps, winCATS software of version 1.4.10 was taken into account.
6.2. Chromatographic condition and method development
On few trial in optimizing the mobile phase, the most suitable one was found to be of hexane: ethyl acetate: methanol: formic acid (3:5:1:0.5, v/v), which enables to the well resolute, sharp and compact band of reserpine at Rf 0.46. Samples and standard were applied as 8 mm band at 10 mm height from bottom edge of the plate by keeping 11 mm distance between the track. Plate was developed up to 80 mm height at an average relative humidity of 53% and tank saturation was allowed for 15 min. The developed plate was dried in ambient air and densitometrically scanned at absorption maxima that is 336 nm and photo documented at 366 nm to visualise the track pattern (Fig. 1).
Different volumes of working standard solutions (3, 4, 5, 6, 7, and 8 µL) were applied as band of 8 mm, in triplicate by keeping the distance between the track 10.2 mm with constant application rate of 100 nL s–1, on 20 cm x 10 cm precoated aluminium supported silica gel 60F254 TLC plates with 200 mm thickness (E. Merck, Germany). Linear ascending development was carried out in a twin trough chamber up to a distance of 80 mm with the optimised mobile phase as stated earlier. Subsequent to the development, plate was scanned densitometrically at absorption maxima 360 nm, with slit dimension of 6 mm × 0.45 mm at scanning speed of 10 mm s–1. The peak areas were recorded. Calibration curves (Fig. 2) were obtained by plotting peak areas vs. applied concentrations. Variable volumes (2µL, 4µL and 6µL) of the prepared sample solutions were applied in triplicate on TLC plate. Plate was developed and scanned as mentioned above. Peak areas and absorption spectra were recorded. Amount of reserpine in root and ghana were calculated from quantification curve (Fig. 3) by considering the area under the curve using the corresponding linear calibration graph.
7. High Performance Liquid Chromatographic (HPLC) analysis
7.1 Instrumentation
HPLC measurement was performed in Agilent LC 1260 Infinity series II chromatographic system consisting of Quaternary pump 03076, auto sampler 41150, diode array detector 14541 and a sampling thermostat 28636.
7.2 Chromatographic condition and method development
The method was developed in octadecylsilane end-capped Poroshell (Agilent) column of 4µ, 150 x 4.6 mm, as stationary phase. The system was operated at pressure 430–432 psi and UV detection at 218 nm. The mobile phase plays a crucial role during the HPLC analysis for the exact measurement of analytes. A solvent system that would give well resolute peak with appropriate and significantly separated in Rt values was highly desired. In view of this, a number of mobile phases have been tried and found that gradient elution with acetonitrile: water (1% NH4Cl) (60: 40, v/v) for 0–4 min and vice versa for 4–8 min, with a flow rate of 1.0 mL min− 1 for total run time of 8 min. gave well separated peak (Fig. 4) of reserpine at Rt 4.587. The injection volume was 10µL. Standard solution of variable concentration (20, 40, 60, 80 and 100 ppm) were injected and responses were recorded (Fig. 5) to get calibration curve (Fig. 6). Both the sample extract (root and ghana) were individually injected in equivolume (10 µL) quantity to get the response in terms of absorbance. Absorbances noted were evaluated by calibration curve, to get the concentration of reserpine present therein (Fig. 7).
8. Chromatographic method validation
Any newly developed analytical method must be validated. The authors of this work have followed the guidelines laid down in ICH9–11. Limit of detection (LOD) and limit of quantification (LOQ) were estimated by applying or injecting the blank (methanol) followed by signal to noise determination. LOD was considered as 3σ (σ is signal to noise ratio) and LOQ as 10σ. LOD and LOQ were experimentally determined by applying different dilution of the known concentration of standards until the average responses were approximately 3 and 10 times of the responses for the six replicate determinations.
The precision is the parameter that expresses the closeness of agreement between a series of measurements obtained from multiple analyses under certain conditions. To determine precision, the repeatability of sample application and of measurement of reserpine peak area was measured by performing six replicate analyses of the same concentration. Method precision was assessed by measuring intra-day and inter-day variation of results of standard solution at three different concentrations. In HPTLC experiment 60, 80, and 100 ng per band was applied and in HPLC 60, 80 and 120 ppm was injected, area under the curve was taken in account for calculation of precision. This was repeated thrice in a day for consecutive three days to calculate intraday precision and interday precision.
For both HPTLC and HPLC methods, accuracy of the method was tested by performing external standard addition method at three different levels. Preanalyzed samples were spiked with 80%, 100%, and 120% of standard solutions and quantified by the proposed method. This experiment was repeated thrice.
Stability of reserpine in solution (methanol) and on plate was checked. The stability of reserpine in methanol was performed by applying 60 ng per band on TLC plate and scanning the plate by proposed method, this has been performed for consecutive six days. The stability of reserpine on TLC plate was performed by rescanning the developed TLC plate after a regular interval of 24 h for consecutive six days. Stability in both cases was determined by calculating the %RSD of peak area obtained by each densitometric scanning.
Ruggedness or robustness of any analytical or instrumentation method is generally performed to test the method’s sturdiness and to check how much the method can withstand against little variations in method parameters. Robustness of the proposed methods was noted by calculating the %RSD of peak areas for every small changes in different parameters like, mobile phase volume, tank saturation, development distance, run time, flow rate, minor alteration in mobile phase composition, changing time from application to chromatogram, time from plate development to scanning etc.
9. Qualitative identification of Phytocompounds by LC-MS/MS (QTOF)
Previously prepared extracts of root and Ghana were subjected to Liquid Chromatography tandem Mass Spectrometry LC-MS/MS Quadrupole Time of Flight (QTOF) to reveal the presence of phytocompounds present therein.
9.1 Instrumentation
Q-Exactive Plus Biopharma, Thermo Scientific make equipment was used for analysis and for data acquisition and processing Xcalibur-Thermo Scientific, Version 4.2.28.14 and Compound Discoverer 3.2 SP1 were used respectively. In the whole experiment SB-C18 RRHD 100 x 2.1 MM, 1.8 microns (make Agilent Technologies) column was used.
Mass spectrometry was performed using a QSTAR Elite LC = MS = MS system from Applied Biosystems = MDS Sciex (Concord, ON, Canada) equipped with an ESI ion source.
9.2 Chromatographic condition
The aim of this analysis was to identify the secondary metabolites present in plant extract and Ghana extract using QTOF. An Agilent make SB-C18 RRHD 100 x 2.1 MM, 1.8 microns was found to be suitable for analysis after tried different columns like BEHC18, CSH Fluro-phenyl, and BEH amide. Acetonitrile and methanol were tried, and acetonitrile was chosen, as it was giving good separation with sensitivity. Different pHs were tried acidic, neutral, and basic, acidic pH gave good separation and sensitivity, so acidic pH was chosen, as all peaks were well resolved. pH of the mobile phase was important as little change in pH resulted in the loss of resolution between peaks. Different column temperatures 30°C, 40°C, and 50°C were tried, but keeping the column temperature constant at 35°C found most suitable in generating a resolute and chromatogram. An isocratic program was adopted with mobile phase consisting of (A) 0.1% formic acid in milliq water (30%) and (D) acetonitrile (70%) with flow rate of 0.4 mL min− 1 keeping the runtime 40 min. The representative chromatograms are shown in Fig. 8 to 11.
9.3 Mass Spectrometry condition
The source parameters were optimized as follows: ESI voltage, 5500 V (Positive), 4500 V (Negative); nebulizer gas, 60; auxiliary gas, 50; curtain gas, 35; Turbo gas temperature, 450 ⸰C ; declustering potential, 60 V (Positive), 60 V (Negative); focusing potential, 350 V (Positive), 350 V (Negative); declustering potential 2, 10 V (Positive), 10 V (Negative). Nitrogen was used in all cases. The samples were analyzed with IDA (Information Dependent Acquisition) method which can automatically select candidate ions for MS = MS study. The TOF mass range was set from m/z 50 to 1000 and the mass range for product ion scan was m/z 100–900. The collision energy (CE) was set from 20 eV to 70 eV to optimize signals to obtain maximal structure information from the ions of interest. Accurate mass measurements of each peak from the total ion chromatograms (TICs) were obtained by the dynamic auto calibration method that allows for real-time internal calibration during data acquisition.