Enicostemma littorale (whole plant) was collected from three different geographical regions viz- Shivapuri (M.P), Ajmer (RAJ) in the month of February whereas sample from Haridwar (UA) was collected in the month of March. These plants were assigned batches like EL/SGTC/MP, EL/SSH/RAJ and EL/SSH/UA respectively in the samples obtained from M.P, Rajasthan and Uttaranchal (India). The identity of these batches was confirmed by taxonomist of Ranbaxy Research Laboratories, Gurgaon, India. The voucher specimen was deposited in Phytochemistry research laboratory, Jamia Hamdard (New Delhi), India. All the solvents were of HPLC grade and other chemicals used were of analytical reagent (AR) grade and obtained from E-Merck, Mumbai, India.

Two traditional ayurvedic marketed formulations A (tablet) and B (capsule) containing E. littorale extract was purchased from local market and used for quantification of swertiamarin.

About 5 g of the crude drugs weighed and refluxed with 100 ml of methanol for 1 h in water bath and filtered through whatmann filter paper (No. 41). The marc left out was refluxed again with 50 ml of methanol for 1 h and filtered. The filtrates were combined and concentrated to 25 ml in rotary vacuum evaporator (Medica Instrument Mfg. Co. Mumbai, India) to a final volume of 25 ml. This solution was used as test solution in the HPLC analysis.

6 tablets (formulation A) or 6 capsules (formulation B) were crushed and dissolved in 50 ml of distilled water, transferred to a separating funnel. It was then extracted thrice with 140 ml each of extraction media (chloroform: methanol: butanol-25:2:3). The organic layer was passed over anhydrous sodium sulphate and evaporated to dryness on water bath under vacuum. The residue left was reconstituted in 20 ml of methanol, which was used as test solution in HPLC analysis.

Accurately weighed 10.2 mg of swertiamarin (purity 98 %) reference standard was transferred to 25 ml volumetric flask. Methanol was added and sonicated in ultrasonic water bath to dissolve. Volume was made up with methanol to 25 ml. This gives concentration of 399.84 μg/ml. 10 ml of this solution was further diluted to 13.32 ml with methanol to get concentration of 300 μg/ml This solution was used as a reference solution (stock solution) for swertiamarin.

A Shimadzu model HPLC equipped with quaternary LC-10A VP pumps, variable wavelength programmable UV/VIS detector SPD-10AVP column oven (Shimadzu), SCL 10AVP system controller (Shimadzu), Rheodyne injector fitted with a 20 μl loop and Class-VP 5.032 software was used. The chromatographic column used was a reverse phase C18, 250 X 4.6mm, 5mic Zorax RP-HPLC. The column and HPLC system were kept at ambient conditions. The mobile phase was Methanol: water (9:1) with the flow rate of 1.0 ml/min. The injection volume was 20 μl and the elutes were analyzed at a wavelength of 338 nm.

Various solvent systems were tried for the development of a suitable HPLC method for estimation of swertiamarin in 60% methanolic extract and marketed formulations. Mobile phases tried for these purposes were methanol: water (10:90 %), methanol: water (40:60 %), methanol: water (90:10 %), acetonitrile: water (10:90 %) acetonitrile: water (40:60 %) and acetonitrile: water (90:10 %). The suitability of the solvent system was decided by cost, sensitivity of the assay and time required for the analysis.

Various concentrations (50-300 μg/ml) were made for the preparation of calibration curve from the prepared standard stock solution. The mobile phase after filtration through 0.45 μm membrane filter was delivered at 1.0 ml/min for column standardization. The baseline was continuously monitored during the column standardization. The wavelength of detection was selected at 338 nm. The prepared dilutions (concentrations) were injected serially and areas under the peaks were recorded for each concentration. The stability of drug in solution during analysis was also determined by repeated analysis of samples during the course of experimentation on the same day and also after 48 h storage of drug solution at laboratory conditions and in the refrigerator.

The linearity of swertiamarin was checked between 50-300 μg/ml concentration range. Graph was plotted between concentration and area for linearity.

Accuracy was determined as % recovery by standard addition method. The preanalyzed sample of swertiamarin (100 μg/ml) was spiked with the extra 0, 50, 100 and 150 % of the standard swertiamarin and the mixtures were reanalyzed by the proposed method. The % recovery and percent relative standard deviation (% RSD) were calculated at each concentration level.

Precision was determined at two levels according to ICH guidelines (repeatability and intermediate precision). Repeatability was determined as intraday precision whereas intermediate precision was determined by carrying out inter-day variation for the determination of swertiamarin at four different concentration levels of 100, 150, 200 and 250 μg/ml in triplicates.

Reproducibility of the proposed method was checked by analyzing precision on a different instrument, which was analyzed by another person in different laboratory. Both intraday and intermediate precision was calculated at four different concentration levels of 100, 150, 200 and 250 μg/ml in triplicates.

LOD and LOQ were determined by standard deviation (SD) method. LOD and LOQ were determined by injecting blank sample to the chromatograph in triplicates, peak area of this blank was recorded. LOD and LOQ were determined using the slope of the calibration curve and SD of the blank sample using following formulae:

LOD=3.3×SD/S

LOQ=10×SD/S

Where SD is the standard deviation of the blank response and S is the slope of the calibration curve.

Robustness of the method was determined by changing the flow rate (1.1 and 0.9 ml/min) of mobile phase. The change in retention time (R_t) was recorded and % RSD was calculated.

The test samples were injected and chromatograms were obtained in same conditions as that of standard swertiamarin. The peak area of the peak corresponding to the R_t of standard swertiamarin was recorded and content of the same was calculated from the regression equation obtained from calibration curve.

The mobile phase composition was optimized to develop a suitable and accurate HPLC method for quantification of swertiamarin. Mobile phases tried for this purposes were methanol: water (90:10 %), acetonitrile: water (25:75 %), methanol: water (50:50 %), acetonitrile: water (50:50 %) and acetonitrile: water (75:25 %). The chromatogram obtained with methanol: water (90:10 %) solvent system was found to have very good symmetry (1.15), with lowest Rt (10.15 min) and sharp well defined peak (Figure 1). Therefore the mixture of methanol: water (90:10 %) was optimized as the mobile phase. The drug was stable for a period of 48 h storage at laboratory temperature and under refrigerator temperature in methanol: water (90:10 %) mixture.

The calibration curve area versus concentration (μg/ml) was found linear in the range of 50-300 μg/ml. Values of mean area with corresponding concentration, their standard deviation, % RSD and standard error are shown in Table 1. Statistical calculations were done at 5% level of significance. One-way analysis of variance (ANOVA) statistical test was performed to compare these results. The linear regression data for the calibration curve showed a good linear relationship over the concentration ranges of 50-300 μg/ml with respect to peak area as shown in Table 2. The correlation coefficient value (R2) was highly significant (Table 2) (p<0.05). No significant differences were observed in the slope of standard curves (p'0.05). The retention time and asymmetry factor were found to be 10.15±1.52 min and 1.15 ±0.16 respectively.

The linearity range of swertiamarin was obtained as 50- 300 μg/ml as shown in Table 1. The regression equation was Y= 25653X+490912 with correlation coefficient (R²) of 0.998 (Table 2).

The accuracy of the proposed method was calculated by recovery analysis which afforded recovery of 99.16-99.89 % after spiking the additional standard drug solution to the previously analyzed test solution. The values of % recovery, % RSD and standard error are shown in Table 3, which indicated the good accuracy of the proposed method.

Precision was considered repeatability (intraday precision) and intermediate precision. Results of repeatability and intermediate precision were expressed in terms of % RSD and are shown in Table 4. The low values of % RSD indicated the repeatability and intermediate precision of the proposed method.

Reproducibility of the proposed method was checked by obtaining precision of the method in another laboratory using different instrument and analyzed by another person but in the same condition. Both intraday and interday precision was examined in labs. There were no significant differences observed in the % RSD values of intraday and interday precision, which indicates the reproducibility of the method Table 5.

LOD and LOQ of the proposed method were determined by the standard deviation method and were found to be 17.25 and 56.92 μg/ml respectively, which indicated that the proposed method can be used in wide range for detection and quantification of swertiamarin effectively.

Robustness was determined to evaluate the influence of small but deliberate variation in the chromatographic conditions for the determination of swertiamarin. There was no significant change in the R_t of swertiamarin by changing the flow rate (Table 6). Low value of the % RSD indicated the robustness of the method as shown in Table 6.

The peaks of swertiamarin from sample solution were identified by comparing their Rt obtained from the peaks with those of standard. HPLC profile of the 60 % methanolic extract of the E. littorale was developed through the same condition as estimation of standard swertiamarin (R_t value 10.15 min). The R_t value of swertiamarin was 10.17 min in extract as shown in Figure 2. Some other peaks were also obtained in extract at different values of R_t because of presence of some impurities in the extract as shown in Figure 2. The peak purity of extract was confirmed by superimposing the peaks of standard and extract. It was observed that extract of E. littorale collected from UA zone showedgood content of swertiamarin followed by samples from MP and RAJ regions. The swertiamarin was quantified in extract of E. littorale using regression equation and value was found to be 11.70 %, 9.20 % and 7.10 % w/w in UA, MP and RAJ samples respectively.

A single HPLC peak was observed at the same R_t in the samples of marketed formulations A and B. There was no interaction between the swertiamarin and other excipients present in the marketed formulations. The swertiamarin content was found to be 7.50 % and 8.11 % w/w in formulation A and B respectively. The value of % RSD was also too low which indicated the suitability of this method for the routine analysis of swertiamarin in marketed formulations.

The method was used to determine the presence swertiamarin in E. littorale from three different parts of the India. The swertiamarin was present in all the three extract and in marketed formulations, but there was considerable variation in the amounts present. It may be because of genetic variability, source of plant as well as external factors like seasonal and environmental variations, drying processes and storage conditions cannot be ruled out.