Analytical Method Development for Determination of Salasia and Guduchi in Pharmaceutical Formulation Using Advanced Chromatographic and Spectroscopic Method

The rich heritage of Indian traditional medicine, particularly Ayurveda, has provided a vast array of medicinal plants that have been used for centuries to treat various ailments. Among these, Salacia and Guduchi (Tinospora cordifolia) hold a significant position due to their diverse pharmacological properties and potential therapeutic benefits. Both plants have gained widespread recognition in recent years for their applications in managing metabolic disorders, particularly diabetes mellitus, and other chronic conditions. Their extensive use in traditional practices underscores their importance as natural remedies in contemporary medicine. ^1-2

The Historical Significance of Medicinal Plants:

Medicinal plants have been integral to human health since ancient times, serving as a foundation for the development of modern pharmaceuticals. In Ayurveda, one of the oldest systems of medicine, plants like Salacia and Guduchi have been revered for their ability to restore balance within the body. These plants are often described as "Rasayana" herbs, known for their Adaptogenic, rejuvenating, and therapeutic effects. The increasing interest in herbal medicine globally is driven by the quest for safer, more sustainable, and cost-effective healthcare solutions. 

Diabetes Mellitus: A Global Health Challenge:

Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The prevalence of diabetes has risen to pandemic levels, with the International Diabetes Federation (IDF) reporting over 500 million cases worldwide as of 2023. The disease poses significant challenges to global healthcare systems due to its associated complications, including cardiovascular disease, neuropathy, nephropathy, and retinopathy.   The current therapeutic options for diabetes, including insulin therapy and oral hypoglycaemic agents, are often associated with limitations such as adverse effects, cost, and incomplete glycemic control. This has driven the search for alternative and complementary therapies, particularly those derived from natural sources. Plants like Salacia and Guduchi have emerged as promising candidates due to their multifaceted mechanisms of action and minimal side effects.  The pharmaceutical industry relies on accurate and reliable analytical methods for the quality control of herbal formulations. Salasia and Guduchi, two medicinally important plants, have been widely used for their therapeutic benefits, particularly in the treatment of metabolic disorders. However, the standardization and quality assessment of herbal formulations remain a challenge due to their complex phytochemical composition.  Advanced chromatographic and spectroscopic techniques, such as High-Performance Liquid Chromatography (HPLC), UV-visible spectroscopy, and FT-IR analysis, provide robust and precise methods for the identification and quantification of bioactive constituents in pharmaceutical preparations. These techniques ensure the efficacy, stability, and safety of herbal formulations by providing accurate analytical data. ^3-8

Fig. No. 01: Image of Salasia     Image of Guduchi

(Salacia reticulata)           (Tinospora cordifolia)

Instruments: Various instruments were employed for RP-HPLC analysis, including a Shimadzu analytical balance, a Cadmach tablet compression machine, an Agilent 1200 RP-HPLC system, a Shimadzu UV-1800 spectrophotometer, and a Bruker FTIR spectrophotometer.

Experimental Work: Analytical Method Development for Determination of Salacia and Guduchi in Pharmaceutical Formulation Using Advanced Chromatographic and Spectroscopic Methods

Procurement and Identity Confirmation:  Marketed Salacia reticulata and Guduchi tablets were procured from: Salacia reticulata Tablet (Batch 61-66):  Genius Nature Herbs Pvt. Ltd., Coimbatore, Tamil Nadu. Guduchi Tablet (Batch 67-70): Pasari Medical, Dhule, Maharashtra. Packaging details, including batch number, manufacturing, and expiry dates, were recorded. Storage was maintained in a controlled environment. 

Confirmation of Identity Using UV-Visible Spectroscopy:  Sample Preparation: Tablets were finely powdered (100 mg) and extracted using methanol (10 mL) with sonication for 15 minutes, followed by filtration and dilution for analysis.

Method Development:  HPLC Optimization: Chromatographic conditions were optimized using a C18 column (5μm, 250 x 4.6 mm) with a mobile phase of Acetonitrile, methanol, and glacial acetic acid (50:20:30 v/v/v) at a 1 mL/min flow rate. Detection wavelengths: 312 nm and 270 nm. The mobile phase was filtered (0.45μm) and degassed. UV Analysis: Standard solutions (1.0-40.0 μg/mL) of Salacia reticulata and Guduchi were prepared in mobile phase for calibration.

Preparation of Standard Solutions: Stock Solutions: Prepared by dissolving 100 mg of Salacia reticulata and Guduchi in 100 mL volumetric flask with water, yielding 1000 μg/mL concentration. Dilutions were made in water for analysis.^9-15

System Suitability Testing:

1. Chromatographic Performance Parameters
2. Retention Time: Consistent and reproducible.
3. Peak Symmetry: Ensured uniform elution.
4. Resolution & Selectivity: Verified clear separation from impurities.
5. Peak Area: Proportional to analyte concentration. ¹⁶

Method Validation: Validation studies were conducted per regulatory guidelines:

1. Linearity: Calibration curve (1.0-40.0 μg/mL) showed a high correlation coefficient.
2. LOD & LOQ: Determined based on regression line standard deviation.
3. Precision: System suitability, method precision, and intermediate precision studies showed RSD < 2%.
4. Accuracy & Recovery: Performed using recovery experiments at 50% and 100% concentrations with satisfactory results.
5. Robustness: Small variations in mobile phase composition and flow rate had minimal impact (<2%) on retention time and peak area.

Assay Method: Twenty tablets were weighed, crushed, and an equivalent amount of Salacia reticulata, and Guduchi (100 mg) was dissolved in 100 mL water, sonicated for 20 minutes, filtered (0.45μm), and diluted. HPLC Analysis: 20μL sample injection, optimized conditions, and calibration curve were used to quantify active components. Retention time: 3.56 min.

Forced Degradation Studies: Samples were subjected to stress conditions: heat, light, acid/base hydrolysis, oxidation, and photolysis. HPLC monitored degradation products to ensure accurate quantification in presence of degradation.^17-20

RESULTS AND DISCUSSION:

Result of Procurement and Confirmation of Identity of Salasia and Guduchi: HPLC HPLC-grade Acetonitrile, methanol, and glacial acetic acid were sourced from Rankem (Mumbai, India), while pure Salacia reticulata and Guduchi samples were obtained from Genius Nature Herbs Pvt. Ltd. (Coimbatore) and Pasari Medical (Dhule). Milli-Q ultra-pure water (Millipore, Bangalore) was used for all solution preparations.  FT-IR Analysis: FT-IR spectra were recorded using KBr pellet method, confirming characteristic absorption bands for ester carbonyl, aromatic rings, hydroxyl, and C-H stretching in both Salacia reticulata and Guduchi. UV-Visible Spectroscopy: Salacia reticulata exhibited a λmax at 275 nm, with a calibration curve showing strong linearity (R² = 0.999). Guduchi showed absorption peaks at 216 nm, 266 nm, and 355 nm using methanol as solvent. These results confirm the identity of the procured Salacia reticulata and Guduchi samples for further analytical studies.

Result of Method Development: A stock standard solution (1000 μg/mL) of Salacia reticulata and Guduchi was prepared by dissolving 100 mg of each in 100 mL water. Standard solutions (1.0–40.0 μg/mL) were obtained by serial dilution using mobile phases (Acetonitrile: Methanol: Glacial Acetic Acid, 50:06:10 v/v/v and 50:10:10 v/v/v).

Mobile phase: Acetonitrile, methanol, and glacial acetic acid in the ratio 50:06:10 v/v/v for Salacia reticulata determination peak shows HPLC 275 nm with RT 2.5 min, and 50:10:10 v/v/v for Salacia reticulata determination peak shows for Guduchi HPLC 355 nm with RT 3.52 min shown in figure no. 05, and 06.

Result of Preparation of Standard Solutions: A stock standard solution (1000 μg/mL) of Salacia reticulata and Guduchi was prepared by dissolving 100 mg of each in 100 mL water. Standard solutions (1.0–40.0 μg/mL) were obtained by serial dilution using water, as shown in Figure 02 and 03.

Result of Method Validation:

Linearity: Calibration curves were constructed using three series of standard Salacia reticulata, and Guduchi solutions in the range of 0.0 - 40.0 μg/ml. The equation of linear regression and statistical data are presented in Table no. 02 and figure no 07. The linearity of the calibration curve was validated by the high value of the correlation coefficient.

Table no.02: Linearity of Salacia reticulata, and Guduchi

Figure no. 07: Linearity of Salacia reticulata, and Guduchi.

HPLC for Salacia = LOD =3.3 σ/s = 0.310 and HPLC for Guduchi = LOD =3.3 σ/s = 0.290

HPLC for Salacia = LOQ=10 σ/s = 1.022 and HPLC for Guduchi = LOQ =10 σ/s = 1.322

Precision: The assay was evaluated for system suitability, method precision, and intermediate precision. Repeatability was assessed using five consecutive injections, analyzing peak area values of Salacia reticulata and Guduchi. Precision (R.S.D. < 2%) results are shown in Table no. 03. Intra-day (same day, three concentrations) and inter-day (three days, six injections per sample) precision studies showed R.S.D. between 0.16–0.50 percent, confirming the method's reliability.

*Average of six determinations. R.S.D. (%): relative standard deviation; bias (%):

[(found – taken)/taken] x 100.

Accuracy and recovery studies: The accuracy of the method was assessed by comparing measured and reference values, with results detailed in Tables no. 04 to 07. Recovery experiments at 50% and 100% concentrations of Salacia reticulata and Guduchi were conducted using six samples per level. The results confirm high accuracy and repeatability of the method.

Table no. 04: Precision: Intra- and inter-day precision of Salacia reticulata for HPLC:

Average of six determinations.

Average of six determinations.

[(found – taken)/taken] x 100.

Robustness: The robustness of the method was evaluated by introducing ±10% variations in mobile phase composition and flow rate. Peak area and retention time variations remained below 2%, with no significant impact on quantification. RSD values (Table 20 and 21) were consistently below 2%, confirming the method's reliability under minor parameter changes.

[(found – taken)/taken] x 100.

Table no. 09: Guduchi content and their respective RSD values following each HPLC

 (Chromatographic condition variation):

[(found – taken)/taken] x 100.

Result of System Suitability Testing: Evaluated parameters such as retention time, peak symmetry, resolution, and column efficiency to ensure the reliability of the method shown in table no. 10 and 11.

Table no.11: System Suitability Test for HPLC Parameters for Guduchi:

Result of Assay method: Pharmaceutical Formulation: Twenty tablets were crushed, and 100 mg of Salacia reticulata and Guduchi powder was dissolved in 100 mL water, sonicated for 20 min, and filtered through a 0.45μm membrane. The solution was diluted with the mobile phase, and 20μL was injected into the HPLC system. Retention times were 2.5 min (Salacia reticulata) and 3.1 min (Guduchi) (Figure 07 & 08). The drug content was calculated using the calibration curve.

Table no. 12: HPLC Analysis of Salacia reticulata and Guduchi from pharmaceutical formulations by proposed method on Third day:

Result of Forced Degradation Studies:

Figure no. 09: Result of Forced Degradation Studies Salacia reticulata and Guduchi.