Authentication of Rhodiola rosea , Rhodiola quadrifida and Rhodiola rosea liquid extract from the Ukrainian market using HPTLC chromatographic profiles

Rhodiola rosea and Rhodiola quadrifida are widely distributed and sold in Eastern Europe. The purpose of this paper was to identify R.rosea , R.quadrifida and Rhodiola rosea liquid extract (RRLE) in the Ukrainian market and bring out adulteration cases using chromatographic characterisation by HPTLC. The multiple samples of R.rosea , R.quadrifida and RRLE were compared; the optimal chromatographic conditions for identification of R.rosea and RRLE based on the presence of rosavins and salidroside as well as for identification of R.quadrifida based on the presence of salidroside were proposed; the specific HPTLC fingerprints were obtained; the acceptance criteria for each product were set. The adulteration cases for R.rosea and RRLE samples were established. The dependence on handling R.rosea and presence of rosavins was determined. It was assumed that low-quality raw materials or inefficient technology process were used for RRLE. The consistency of HPTLC fingerprints for R.quadrifida samples was established.

whole-piece, seven samples were longitudinally cut, one sample was powdered material. The collected samples of R.quadrifida were dried pieces of caudex with dead stems and roots. Three samples of RRLE acquired randomly in the drug stores from different manufacturers of Russia and Ukraine were analyzed. Details of the samples are shown in the supplement material (Table S3).
Before the HPTLC analysis was conducted, all the samples of R. rosea and R. quadrifida were morphologically authenticated by the specialists of the State Pharmacopoeia of Ukraine and the National University of Pharmacy, Ukraine (Vovk O.G. and Prokopenko Yu.S., PhD). The sample of R. crenulata, collected in China, was shared by Dr.E.Reich (China) as a botanical reference material was included in the analysis. The voucher specimens of R.rosea (S15144), RRLE (S15129), R.quadrifida (S15132) are deposited at the Herbarium of the National University of Pharmacy, Ukraine. Details and voucher specimens numbers are shown in Table S1. The pictures of HRM samples for R. rosea, R. quadrifida, and R. crenulata are shown in the supplement material (Fig. S8).
Preparation of RRLE (laboratory samples used as a reference). These were prepared from the mixture of properly authenticated samples of R.rosea S15144, S15145, S15147, S15148, S15149 in the same ratio (1:1) and using the same solvent (Ethanol 40 v/v) as the RRLE samples acquired from manufactures. 100 ml of liquid extract was prepared from 100 g of dry roots and rhizomes of R. rosea. Maceration was used as the extraction method (2-day extraction time, room temperature).

HPTLC Method
Methodology A comparative study of multiple samples of R. rosea and R. quadrifida, as well as the commercial preparation of RRLE, was carried out using a HPTLC method. Different chromatographic conditions were compared, the optimal parameters for each product were selected (Table S2), the acceptance criteria were set ( Fig.S4-S6). The voucher specimen of potential adulteration such as R. crenulata was compared.

Source of the method
The HPTLC method for R. rosea from USP (USP HMC 2015) was modified; R. quadrifida and RRLE were included. The improved HPTLC method from the USP included the modified sample preparation (solventethanol absolute for R.rosea, methanol absolute for R.quadrifida, ethanol 40% for RRLE), extraction techniqueshaking), different combination of markers (rosavin, rosarin, salidroside for R.rosea and RRLE; salidroside for R.quadrifida) and documentation (additional detection mode at 254 nm before derivatization for R.rosea and RRLE and in white light and 254 nm before derivatization, white light after derivatization).

Chromatographic Conditions
The sample application, plate layout and developing distance were chosen according to the General Chapter 2.8.25 High-performance thin-layer chromatography (European Pharmacopoeia 9.0). Merck HPTLC silica gel F254 (Darmstadt, Germany). ethyl acetate-methanol-water-formic acid, 77:13:10:2 (v/v/v/v) were used for the analysis. For identification, 3 µL of standard solutions and 5 µL of sample solutions were applied to the plate. Relative humidity was 33%, the chamber was saturated for 20 min, the temperature was 25°C.

Visualisation of Fingerprints
The multiple detection modes used were 254 nm before derivatization and white light after derivatization for R.rosea and R.quadrifida. The white light before derivatization was additionally used for R.quadrifida. The plates were heated at 120°C for 5 min and derivatized by dipping (speed of 3, t=0) while still hot into the aniline-diphenylaminephosphoric acid reagent. The reagent was prepared as follows: 4.0 g of diphenylamine was dissolved in 160 mL of acetone; then 4 mL of aniline and 30 mL of o-phosphoric acid were carefully added. Other derivatization reagents used in the research were prepared in accordance with the books (Jork et al. 1990, Reich andSchibli 2007).

Preparation of Samples and Standards
The sample solution of R. rosea: 1.0 g of powdered sample was mixed with 10 mL of ethanol absolute, shaken for 10 min and centrifuged. The supernatant was used as the sample solution.
The sample solution of R. quadrifida: 1.0 g of powdered sample was mixed with 10 mL of methanol absolute, shaken for 10 min and centrifuged. The supernatant was used as the sample solution.
The sample solution of RRLE. 9 ml of ethanol 40 v/v were added to 1 ml of RRLE. The mixture was centrifuged and the supernatant was used as the sample solution.
Standard solutions: 1.0 mg/mL of rosavin in methanol; 1.0 mg/mL of rosarin in methanol; 1.0 mg/mL of salidroside in methanol.

Instruments and Reagents
Instruments The CAMAG HPTLC system (Muttenz, Switzerland) included a visualizer, an Automatic TLC Sampler 4, an Automatic Developing Chamber 2, and a Scanner 4 controlled by visionCATS software.
*Two other Rhodiola species, such as R.coccinea and R.gelida, in Russian speaking countries have the similar folk namered brush for the caudex with dead stems. As the morphological differentiation of R.quadrifida and R.coccinea is almost impossible, some sources suggest that these are the same species, if chromosomes are not varying (Volkov Igor email to Kateryna Khokhlova, Nov 2, 2016). Anisaldehydesulfuric acid reagent -sulfuric acid reagent; -fast blue salt B reagent; -2,6-dibromoquinone 4-chloroimide reagent (Gibbs' reagent); -antimony (V) chloride reagent; Aniline-diphenylamine-phosphoric acid reagent.
Detection a: 254 nm, before derivatization; detection b: white light, after derivatization. Figure S2. Selection of the mobile phase and detection mode Figure S3. Selection of the solvent for the extraction.
Detection a Detection b Figure S4.