All the reference standard/reference standards are purchased from commercial sources such as catechin (98%, BLD Pharma), epicatechin (97%, TCI), epigallocatechin (> 97%, TCI) and ellagic acid (98% TCI), and HPLC grade solvents such as orthophosphoric acid (OPA) buffer, acetonitrile, water and isopropyl alcohol are purchased from Qualigens chemicals. Acacia catechu hardwood is collected from Tapovan nursery section in Gwalior, MP, India. Three Ayurveda medications, Khadiradivati, Khadirarishta and Lavangadivati, are obtained from the pharmacy at RARI, Gwalior (MP).

The Agilent Infinity 1260 series HPLC instrument is equipped with quat pump, auto sampler, C18 column (Agilent Eclipse XBD-18, 5 µ, 4.6 mm × 250 mm) and DAD WR detector, and Open Lab CDS software is used to conduct the chromatographic analysis. Gradient elution gradient flow program (Table 1) was applied for each type of chromatography method with Peak width >0.1 min (2 s response time) (2.5 Hz), Slit: 2 nm, Injection volume: 10 µL and run time of 35 min. For different methods, different detection channels/wavelengths are applied as required which are provided in result and discussion section (Table 2).

Preparation of individual standards:

All reference standards, i.e., catechin, epicatechin, epigallocatechin, ellagic acid, are weighed 10 mg in 10 ml volumetric flask separately and dissolved in 10 ml methanol using ultra-sonicator to obtain 1 mg/ml (1000 ppm or 1000 mcg/ml) solutions and diluted this stock solution to 200 ppm. Diluted solution was filtered through 0.22 μ membrane filter before using for HPLC analysis.

Preparation of mix standard:

Each reference standards, i.e., catechin, epicatechin, epigallocatechin, ellagic acid, are weighed 10 mg in a single 10 ml volumetric flask and dissolved in 10 ml methanol using ultra-sonicator to obtain 1 mg/ml (1000 ppm or 1000 mcg/ml) solutions and diluted this stock solution to 200 ppm. Diluted solution was filtered through 0.22 μ membrane filter before using for HPLC analysis.

Acacia catechu (heartwood), Khadiradivati and Lavangadivati extraction:

The dried powdered 10 g of Acacia catechu (heartwood), Khadiradivati and Lavangadivati powder was extracted with 200 ml of 70% hydromethanolic solvent by using Soxhlet extraction for 3 h. The extract was evaporated to dryness under reduced pressure. The obtained extract was collected, dried and weighed; 1.2565 g of residue was obtained from extract which is stored separately at 4 °C for further studies.

Preparation of Acacia catechu (heartwood) extract sample:

Acacia catechu heartwood extract was weighed 100 mg in 10 ml volumetric flask and dissolved in 10ml methanol using ultra-sonicator to obtain 1mg/ml (10000 ppm or 1000 mcg/ml) solutions. This solution was filtered through 0.22μ membrane filter before using for HPLC analysis.

Preparation of formulation sample:

Khadiradivati (13.2) and Lavangadivati (16.0 mg) was weighted in 10 ml volumetric flask separately and dissolved in 10 ml methanol using ultra-sonicator to obtain 1.32 mg/ml (1320 ppm or 1320 mcg/ml) solution and 1.6 mg/ml (1600 ppm or 1600 mcg/ml) solutions. This solution was filtered through 0.22 μ membrane filter before using for HPLC analysis.

MSMS method standardized by using three modes of analytical strategies. Single Signal Single Standard (SSSS) individual detection, 4 individual phytochemical reference standards given 4 single injections separately and detected under single wavelength of 254 nm. Single-Signal-Mixed-Standard (SSMS) simultaneous detection, 4 reference standards mixed together and analyzed under single wavelength 254nm. SSSS, SSMS purpose of doing is to compare the peak area response of standards is same even in mixed condition. Multi-Signal Single Standard (MSSS) individual detection, 4 individual standards injected separately but the detection wavelengths are according to its λmax. These λmax were obtained after spectral extraction for each standard (Fig. 1). The acquisition sequence setup is mentioned in Additional file 1: Table S2. The linearity response and quantification parameters of all individual reference standards are catechin, epicatechin, ellagic acid, epigallocatechin mixed reference standards and Acacia catechu (heartwood) extract Khadiradivati and Lavangadivati (Additional file 1: Table S3). Comparative study of all methods is provided for better understanding of the advancements of new developed analysis method mentioned below

Single Signal Single Standard (SSSS) individual detection:

In this method, for a particular concentration (200 ppm) of each reference standards, HPLC was performed at 254 nm wavelength separately and area under the curve for each marker

Single Signal Mix Standard (SSMS) simultaneous detection:

In this method, for a mix solution of Reference standard, where each marker has particular concentration (200 ppm), HPLC was performed at 254 nm wavelength

Multi-Signal Single Standard (MSSS) individual detection:

In this method, for a particular concentration (200 ppm) of each reference standards, HPLC were performed at different wavelengths, i.e., 254 nm, 274 nm, 280 nm and 280 nm, separately.

Multi-Signal Mix Standard (MSMS) simultaneous detection:

In this method, for a mix solution of reference standard, each marker concentration (200 ppm) was observed under detection wavelength as mentioned for MSSS method.

UV absorption spectrum overlay stack of individual reference standards extracted from HPLC–DAD system. Catechin: 280 nm, epi catechin: 280 nm, epigallocatechin: 274 nm, ellagic acid: 254 nm

MSMS method are validated for specificity, linearity, precision the calibration curve of all 4 standards in mixed condition. Four standards are individually prepared on 5-level (L-1 to L-5) calibration with 100–500 ppm. When 4 different standards are mixed together, the diluted final concentration actually goes to 8–40 ppm. Then, these individual standards are mixed based on their linearity concentration 8, 16, 24, 32, 40 ppm level into one vial, and sequence set has created for calibration including plant material and three Ayurveda formulations. Different levels of calibration for different compounds in mixed condition processed with different wavelengths are provided in Table 3 with particular detection signal. Quantification was processed by Open lab CDS 3.5.0, and 3D UV quantitative default method with linear curve fitting mode of processing was employed.