INTRODUCTION:

Chromatograph:

A chromatograph is an instrument or apparatus used in chromatography to separate the components of a mixture based on their different interactions with a stationary phase and a mobile phase. The term can also refer to the output data produced by the instrument, typically in the form of a chromatogram, which displays the separated components as distinct peaks.³

Thin-Layer Chromatography (TLC):

Thin-layer chromatography (TLC) was introduced as a more refined form of paper chromatography. In TLC, a thin layer of an adsorbent material (such as silica gel or alumina) is coated onto a glass or plastic plate⁸. This method provided better resolution and faster separation times compared to paper chromatography.⁹

Gas Chromatography (1950s):

Gas chromatography (GC) was developed in the early 1950s. This technique involves a gaseous mobile phase and a liquid or solid stationary phase. GC revolutionized analytical chemistry by allowing the separation and analysis of volatile compounds with high sensitivity and efficiency. A.J.P. Martin and Anthony T. James significantly contributed to the development of GC.¹

High-Performance Liquid Chromatography (1960s-1970s):

High-Performance Liquid Chromatography (HPLC) emerged in the late 1960s and early 1970s. HPLC uses high-pressure pumps to pass the liquid mobile phase through a column packed with fine particles, resulting in improved separation efficiency and speed⁵. The introduction of HPLC brought about significant advancements in pharmaceutical analysis, environmental testing, and biochemical research.⁷

Advantages and Disadvantages of High-Performance Liquid Chromatography (HPLC) for Pharmaceutical Analysis:

1. High Resolution and Efficiency

2. Versatility

3. Quantitative Precision and Accuracy

4. Reproducibility

5. Sensitivity

6. utomation and High Throughput

7. Diverse Detection Options

Disadvantages of HPLC:

1. High Cost:

2. Complexity and Technical Expertise

3. Time-Consuming Method Development

4. Environmental and Safety Concerns

5. Limitations with Non-Volatile Compounds

6. Potential for Column Degradation

7. Sample Preparation Requirements⁹

RP-HPLC (Reverse Phase HPLC):

1. RP-HPLC is a specific type of HPLC where the stationary phase is non-polar and the mobile phase is polar, allowing for the separation of non-polar compounds⁴.

2. Stationary Phase: Non-polar, usually made of silica derivatized with alkyl chains.

3. Mobile Phase: Polar, often water-based with organic solvents like methanol or acetonitrile. Separation Mechanism: Non-polar compounds interact more strongly with the non-polar stationary phase, while polar compounds are eluted more readily by the polar mobile phase.

4. Benefits: RP-HPLC is highly versatile and widely used for separating a broad range of compounds, including proteins and peptides.¹⁰

MATERIAL AND METHODS:

Material:

The list of material and instrument with their manufacturer which is used in experiment.

Collection of Standard Eleutherosides and Herbal Formulation:

The standard Eleutherosides was obtained/procure from Sigma Aldrich Solution. The herbal formulation was purchase from Amazon (Online Shopping).¹⁶

Reference substances (Marker compound):

Eleutherosides was obtained from Sigma Aldrich Solution.

Instruments:

RP-HPLC- Thermo fishers Scientifics.

Method:

Development and validation of analytical method for determination of Eleutherosides in herbal formulation by RP- HPLC method.

Selection of chromatographic Mode:

For development, reverse phase high performance liquid chromatography was chosen.¹⁷

Optimization of Detection Wavelength:

Because it was dependable and simple to set at the right wavelength, a UV detector was chosen. Analyte concentrations were tested at various wavelengths². Eleutherosides spectrum was measured at 220nm.

Selection of mobile Phase:

The literature review served as the basis for the selection. Acetonitrile: Phosphate Buffer pH 8.5, following an evaluation of the drug's solubility in several solvents and a review of the literature.

Optimization of Chromatographic Parameters:

Finding a collection of parameters that efficiently separate and enable the measurement of the analysts from the endogenous material with acceptable accuracy, precision, sensitivity, specificity, cost, ease, and speed is known as optimization in HPLC. Eleutherosides were separated using the Zodic C18 RP-HPLC column (4.6* 250, 5µm), which provides good resolution and run time. Eleutherosides were obtained by optimizing the mobile phase. At first, different ratios of acetonitrile, methanol, and water were attempted as the mobile phase; however, the tailing of the peak was seen. The drug was resolved by adjusting the pH of the aqueous phase mixture of acetonitrile and water, but the issue remained unresolved. Ultimately, Eleutherosides showed a symmetric peak and acceptable resolution. when the pH is adjusted using mobile phase modifiers in conjunction with phosphate buffer pH 8.5. The mobile phase had a flow rate of 1.0milliliters per minute. Eleutherosides had a retention duration of 6.1minutes under ideal chromatographic conditions, and detection was done at 220nm.¹¹

Table 1: Final chromatographic conditions for HPLC method

Chromatographic Mode	Chromatographic Condition
HPLC System	Thermo Scientific, Vanquish HPLC system
Pump	Quaternary pump
Detector	UV Visible
Data processor	Chromeleon 7.2
Stationary phase	Zodic C₁₈ RP-HPLC column (4.6 * 250, 5 µm)
Mobile phase	Acetonitrile: Phosphate Buffer pH 8.5 (65:35, v/v)
Detection wavelength	220 nm
Flow rate	1.0 mL/min
Sample size	10 µL

Preparation of standard stock solutions:

After adding 7mL of the solvent combination and transferring 10mg of eleutherosides to a 10mL volumetric flask, sonicate for 15minutes. Using the solvent combination, make up the volume and thoroughly mix. Eleutheroside concentration was 1000 µg/mL. 6.6.7 Preparing the sample.¹²

Analysis of Pharmaceutical/Herbal Formulation:

The equivalent of Eleutherosides, 10 450mg capsules of the Eleutherosides formulation (Eleuthero Extract (0.8%) Eleutherosides; manufactured by Terravita) were ingested. After transferring 50mg of the formulation powder into a 50mL volumetric flask, 25mL of the solvent combination was added, sonicated for 25 to 30 minutes while being shaken, and filtered through a 0.2µ membrane. The 1 mL filtered sample was placed in a 10 mL volumetric flask, and the volume was adjusted with diluent and thoroughly mixed.⁶ (Conc. 100µg/mL)

Optimization of mobile phase pH:

Eleutherosides were shown to be compatible with Phosphate Buffer based on the chromatographic conditions.

Linearity Study of Eleutherosides:

Eleutherosides were administered in various amounts on the TLC plate (500–3000ng/spot) using a Linomat 5 sample applicator and a microliter syringe. The chromatographic conditions mentioned above were used to develop and scan the plate. For every drug concentration, peak area was measured; the results are shown in Table 7.3, and a calibration curve was displayed as concentration versus peak area.

Table 2: Linearity study of Eleutherosides

Sr. No	Concentration of Eleutherosides in [ng/spot]	Peak area means± S.D. [n = 3]	%R.S.D.
1	500	528 ± 2.4494	0.4639
2	1000	748 ± 8.5764	1.1465
3	1500	1024.67 ± 2.4944	0.2434
4	2000	1271.67 ± 2.0548	0.1615
5	2500	1495.33 ± 4.4969	0.3007
6	3000	1768.67 ± 4.4969	0.2542

Figure 1. Eleutherosides Calibration Curve

Y = 0.4967x + 270.16; Correlation Coefficient = 0.9991; Intercept = 270.16, Slope = 0.4967

Acceptance Criteria: A correlation coefficient of at least 0.995 is required.

In conclusion, 0.9991 is the correlation coefficient. Eleutherosides can therefore be measured using the linear HPTLC method.

Analysis of Herbal Formulation:

Twenty capsules containing 450 mg of extract (as stated on the label) were precisely weighed and ground into powder in order to determine the amount of eleutherosides in the formulation.¹⁸ After being weighed, a quantity of powder equal to 100 mg of formulation was added to a 100 mL volumetric flask that held around 100 mL of solvent mixture.¹⁵A 0.45 μ membrane filter paper was used to filter the mixture. A 10 mL volumetric flask was filled with 1 mL of the filtered material, and the volume was well mixed and adjusted using diluent. Six injections of the sample solutions were made into the column. ¹³

Validation:

The proposed method was validated as per ICH guideline¹⁴(Q2A).

1. Accuracy

2. Precision

3. Ruggedness

4. Robustness

5. Sensitivity

6. Specificity and Selectivity study for Eleutherosides

RESULTS:

Validation:

The proposed method was validated as per ICH guideline (Q2A).

Accuracy:

The recovery investigation was carried out at 50%, 100%, and 150% levels utilizing the standard addition method. A known quantity of standard Eleutherosides (20.0g/mL of Eleutherosides) was added to the previously examined sample, and they were then exposed to the suggested HPLC procedure. The table displays the percentage recovery, which should be between 98% and 102%.

Precision:

Repeatability and intermediate precision investigations confirmed the method's accuracy. Eleutherosides at 40, 60, and 80µg/mL were analyzed three times on the same day in order to examine intra-day precision. By examining the same concentration on three separate days over the course of a week, inter-day accuracy was examined. Eleutherosides at 60g/mL were analyzed six times to determine repeatability. The RSD percentage shouldn't exceed 3%. The outcomes and repeatability are displayed in the table 4.

Ruggedness:

From stock solutions, sample solution of 60µg/mL of Eleutherosides was produced and evaluated by two different analysts utilizing similar operational and environmental circumstances. Peak area was measured six times for solutions with the same concentration; the percentage amount should fall between 98% and 102%, and the relative standard deviation should not exceed 2.0%. The outcomes are displayed in the table 6.

Table 3: Results of Recovery studies for Eleutherosides

Drug	Initial amount [mg/mL]	Excess drug added to the analyte [%]	Amount recovered ± S.D [mg/mL]	Recovery [%]	%RSD [n = 3]
Eleutherosides	20	50	30.02 ±0.0018	100.06	0.0061
	20	100	40.37 ±0.0048	100.93	0.012
	20	150	49.73 ±0.0045	99.46	0.009

Table 4: Results of Precision study for Eleutherosides

Drug	Conc. [mg/mL]	Intra –day Amount Found [mg/mL] [n = 3]		Inter- day Amount Found [mg/mL] [n = 3]
Drug	Conc. [mg/mL]	Mean	% RSD	Mean	% RSD
Eleutherosides	40	39.96 ±0.25	0.6256	39.89 ±0.1166	0.2924
	60	59.71 ±0.6833	1.1444	60.13 ±0.1564	0.2601
	80	79.98 ±0.3626	0.4534	80.29 ±0.4432	0.552

Table 5: Results of Repeatability study for Eleutherosides

Sr. No.	Conc. (mg/mL)	Amount Found
1	60	59.12
2	60	60.14
3	60	60.09
4	60	59.97
5	60	59.96
6	60	60.09
Mean		59.9
S.D.		0.3508
%R.S.D.		0.0701

Table 6: Results of Ruggedness study Eleutherosides

Drug	Amount in µg/ml	Amount Found [n = 6] Mean ± SD		% Amount Found [n = 6]		% RSD
Drug	Amount in µg/ml	Analyst I	Analyst II	Analyst I	Analyst II	Analyst I	Analyst II
Eleutherosides	60	60.06 ± 0.0644	60.02 ± 0.05	100.1 ± 0.1074	100.04 ± 0.0834	0.1073	0.0833

Robustness:

The method's robustness was examined by purposefully altering a few parameters, specifically the flow rate and the acid concentration (pH modifiers). In order to estimate the effect, one element was adjusted at a time while 10 µg/mL of eluterosides was injected. The results are displayed in a table.

Table 7: Results of Robustness Studies Eleutherosides

Parameters	Eleutherosides R_t
Change in pH
5.5	6.4
6.0	5.4
7.0	4.5
Change in Flow Rate
0.8 ml/min	3.9
1.2 ml/min	2.2
1.8 ml/min	1.8

Sensitivity:

For low concentrations of chemicals in sample matrices, the quantitation limit is a quantitative assay parameter that is specifically used to identify contaminants and/or degradation products.The following formulas were used to calculate the limit of quantitation (LOQ) and the limit of detection (LOD).LOD = 3.3 (SD)/S; LOQ = 10 (SD)/S; where S is the calibration curve's slope and SD is the response's standard deviation. Eleutherosides' LOD and LOQ were determined to be 9.89 µg and 29.98µg, respectively.

Fig 2: RP-HPLC graph of Standard Eleutherosides

Fig 3: RP-HPLC graph of Formulation

Specificity and Selectivity:

The analytes should be well-resolved from other unrelated components and free from interference. The process of quantitatively detecting analytes in the presence of potentially expected components in the sample matrix is known as specificity, whereas the process of qualitatively detecting analytes in the presence of potentially expected components in the sample matrix is known as selectivity. It's a very selective process. Around the retention time of both Eleutherosides, there was no other interfering peak, and there was no discernible noise in the baseline.

CONCLUSION:

The quantitative quantification of Eleutherosides in a herbal formulation comprising Eleuthero extract was accomplished through the successful development and validation of a robust and dependable RP-HPLC technique. With UV detection at 220 nm, the technique used a Zodic C18 column with a mobile phase consisting of acetonitrile and phosphate buffer (pH 8.5) in a 65:35 ratio. Eleutherosides had a retention period of roughly 6.1 minutes, yielding well-resolved, symmetrical peaks.

REFERENCE:

1. Bhardwaj, S.K., Dwivedi, K., Agarwal, D.D. A review: GC method development and validation. Int. J. Anal. Bioanal. Chem. 2016; 6: 1–7.

2. Moein, M.M., El Beqqali, A., Abdel-Rehim, M. Bioanalytical method development and validation: Critical concepts and strategies. J. Chromatogr. B 2017; 1043: 3–11.

3. Allegrone, G., Pecci, Y., Mauro, M., Orsetti, M. Determination of eleutherosides in dietary supplements by LC-MS/MS. Chromatographic. 2013; 76: 41–48. https://doi.org/10.1007/s10337-012-2355-9

4. Kirthi, A., Shanmugam, R., Prathyusha, M.S., Basha, D D.J. A review on bioanalytical method development and validation by RP-HPLC. J. Glob. trends Pharm. Sci. 2014; 5: 2265–2271.

5. Apers, S., Naessens, T., Van Miert, S., Pieters, L., Vlietinck, A. Quality control of roots of Eleutherococcus senticosus by HPLC. Phytochem. Anal.2005; 16: 55–60. https://doi.org/10.1002/pca.811

6. Bhagat, R., Saudagar, R.B. A Review on Analytical method Development and Validation. J. Drug Deliv. Ther. 2019; 9: 1064–1067.

7. Bhardwaj, S.K., Dwivedia, K., Agarwala, D.D. A review: HPLC method development and validation. Int. J. Anal. Bioanal. Chem. 2015; 5: 76–81.

8. Isane, S.P., Waghmare, S.A., Kamble, H. V. A Review on Method Development, Validation, Optimization and Applications of TLC. 2022.

9. Ganjiwale, S. V, Dewani, A.P., Chandewar, A. V. A Comprehensive Overview of HPLC Method Development and Validation. Int. J. Pharm. Sci. 2024; 2: 1.

10. Gupta, S., Verma, P., Mishra, A.P., Omar, N., Mathur, R. A Review on Novel Analytical Method Development and Validation by RP-HPLC Method. Indian J. Forensic Med. and Toxicol. 2021; 15: 3479–3486.

11. Kang, J.S., Linh, P.T., Cai, X.F., Kim, H.S., Lee, J.J., Kim, Y.H. Quantitative Determination of Eleutheroside B and E from Acanthopanax Species by High Performance Liquid Chromatography. Arch. Pharm. Res. 2001; 24: 407–411. https://doi.org/10.1007/BF02975184

12. Kim, Y.I., Jung, Y.J., Yoon, H.J., Kwon, H.J., Hong, S.P. Simultaneous quantification method for eleutheroside B, eleutheroside E, chiisanoside, and sesamin using reverse-phase high-performance liquid chromatography coupled with ultraviolet detection and integrated pulsed amperometric detection. Heliyon. 2023; 9: e12684. https://doi.org/10.1016/j.heliyon.2022.e12684

13. Kumar, A., Kishore, L., Kaur, N., Nair, A. Method development and validation: Skills and tricks. Chronicles young Sci. 2012; 3: 3.

14. Lal, B., Kapoor, D., Jaimini, M. A review on analytical method validation and its regulatory perspectives. J. Drug Deliv. Ther. 2019; 9: 501–506.

15. N. Analytical method validation: an updated review. Int. J. Pharm. Sci. Res. 2013; 4: 1280.

16. Kim, Y.I., Jung, Y.J., Yoon, H.J., Kwon, H.J., Hong, S.P. Simultaneous quantification method for eleutheroside B, eleutheroside E, chiisanoside, and sesamin using reverse-phase high-performance liquid chromatography coupled with ultraviolet detection and integrated pulsed amperometric detection. Heliyon. 2023; 9: e12684. https://doi.org/10.1016/j.heliyon.2022.e12684

17. Gupta, S., Verma, P., Mishra, A.P., Omar, N., Mathur, R. A Review on Novel Analytical Method Development and Validation by RP-HPLC Method. Indian J. Forensic Med. and Toxicol. 2021; 15: 3479–3486.

18. Pathuri, R., Muthukumaran, M., Krishnamoorthy, B., Nishat, A. A review on analytical method development and validation of pharmaceutical technology. J. Curr. Pharma Res. 2013; 3: 855.

Received on 11.06.2025 Revised on 19.07.2025

Accepted on 18.08.2025 Published on 08.10.2025

Available online from October 15, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(4):295-299.

DOI: 10.52711/2231-5675.2025.00046

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.