Development and Validation of RP-UHPLC and HPTLC Methods for a Polyherbal Active Ingredient Boswellia serrata

 

Sufiyan Ahmad¹*, Amle Poonam¹, Mukesh Patil², Tushar Salunke¹, Rakhi Khabiya³

¹Department of Quality Assurance, Gangamai College of Pharmacy, Nagaon, Dist. Dhule (M.S.), India.

²Department of Quality Assurance, Shri D. D. Vispute College of Pharmacy and Research Center,

New Panvel, Dist. Raigad, 410206, India.

³Acropolis Institute of Pharmaceutical Education and Research, Indore, Madhya Pradesh, India.

 

ABSTRACT:

The aim of present research work was to create a quick, accurate, and reliable RP-UHPLC and HPTLC method for estimating Boswellic acid from Boswellia Serrata extracts and formulations. RP-UHPLC method has been developed and validated for the determination of Boswellic acidin extract and in formulation. The RP-UHPLC analysis was performed on the Cosmosil C₁₈, (250mm x 4.6mm i.d., 5 µm) in isocratic mode, at ambient temperature using Methanol : Water (0.1 % OPA) (80:20, v/v) as mobile phase; flow rate was set at 1.0mL/min. The detection was carried out at 250nm. The retention time for Boswellic acid was found to be 12.5 min. Linearity of Boswellic acid followed in the concentration range of 10-60µg/mLand correlation coefficient was found 0.9997. The HPTLC method was performed on 10cm x 10cm aluminium plate coated with 0.2 mm layer of silica gel 60 F₂₅₄ (E. Merck, Germany). Samples were applied to the plate as bands width 6 mm by using Camag (Muttenz, Switzerland) Linomat-5 applicator fitted with 100µl syringe (Hamilton, Switzerland). The rate of application was constant at 150nl sec^-1 and space between two bands was 14mm. The mobile phase consisted of toluene: ethyl acetate: methanol (4:1:0.5). Densitometric scanning was performed at 254nm using Camag TLC 3-Scanner. The average R_f is 0.54. The calibration curve was linear in concentration range of 500-3000ng/spot and correlation coefficient is 0.9996. The Limit of detection was calculated and it was found to be 5.42ng and Limit of quantification was found to be 16.45ng. The developed RP-UHPCL and HPTLC method was validated as per ICH Q2 (R1) analytical method validation guidelines. All developed methods could be used for routine analysis of Boswellic Acidin the dosage form.

 

 

 

INTRODUCTION: 

Boswellia serrata is found in India and East and North region of Africa. The gummy extract or resin obtained by pealing the bark is generally known as Frankincense or Olibanum used to cure joint pain, Colitis Gravis., sneezes, ulcers, reduce inflammation, and breathing problems. Literature survey revealed nonaqueous titration¹, RP-HPLC^2-3_, HPTLC^4-7 and capillary electrochromatographic⁸ methods for estimation of boswellic acids; whereas HPTLC⁹, HPLC^10-12,

 

GC-MS¹³ and LC-MS¹⁴ methods arequantified in human plasma.Few methods are available for the analysis of boswellic acids including HPLC² and HPTLC^15-16 in the marketed formulation, estimation as per Indian Pharmacopoeial¹² and Ganzeraet al¹¹ reported an HPLC method in thecrude gum resin of Boswellia serrate with an isocratic mobile phase at acidic pH 2.8 which may cause column damage.Further gradient elution¹⁷ at 60ºC for betterseparation withhigher retention timefor 11-KBA and A-11-KBA.

 

MATERIALS AND METHODS:

Instrument:

The RP-UHPLCsystem (Thermo fishers Scientific Vanquish UHPLC system) consists of Cosmosil C₁₈, (250mm x 4.6mm i.d., 5µm) column with UV detector. Whereas HPTLC system from CAMAG with Linomat-5 applicator and CAMAG-3 Scanner.

 

Chemicals:

Boswellia serrata extract procured from Sunpure herbal Pvt. Ltd, Delhi and herbal formulation was procured from local market. Boswellic acid were procured from Sigma Aldrich Ltd., USA which was used as working standard. All the chemicals used were of HPLC grade used without further purification.

 

Chromatographic Condition:

Selection of chromatographic conditions for RP-UHPLC was done on the basis of literature survey. After assessing the solubility of drug in different solvents, Methanol: Water (0.1 % OPA) (80:20, v/v) was used as mobile phase at detection wavelength 250nm. HPTLC was performed on 10cm x 10cm aluminium plate precoated with 0.2mm layer of silica gel 60 F₂₅₄ (E. Merck, Germany). The mobile phase consisted of toluene: ethyl acetate: methanol (4:1:0.5). Densitometric scanning was done at 254nm using Camag TLC scanner 3.

 

Preparation of standard stock solution:

10mg standardBoswellic acid was dissolved in sufficient mobile phase to get the concentration of 1000µg/mL after 15min sonication. While for HPLTC 1mg of Boswellic acid was transferred to 10ml volumetric flask, dissolved in 10ml methanol to obtain concentration i.e. 100μg/ml.

 

Preparation of Sample solution:

10mg of Boswellia serrata extract was transfer in 10mL volumetric flask then add 10mL solvent mixture sonicate for 25-30 min. with shaking then filtered using 0.2µ membrane. Further 1mL filtered sample taken in 10 mL volumetric flask and made up volume with diluent and mixed well (100µg/mL). 20 capsules of Boswellia serrata extract (Equivalent to 50mg of Boswellic acid) was transfer in 50mL volumetric flask then 25mL solvent mixture was addedand sample solution was sonicated for 30min. with shaking then filter using 0.2µ membrane. The 1mL filtered sample takes in 10 mL volumetric flask and made up volume with diluent and mixed well (100µg/mL).

 

RESULTS AND DISCUSSION:

Method development^18-30

After taking number of trials for mobile phase based on trial and error basis and eluotropic series, Methanol: Water (0.1% OPA) (80:20, v/v) with isocratic elution, shows good chromatographic conditions and resolution (Fig: 1).

 

 

Figure 1.RP-UHPLC curve of Boswellic Acid

 

For HPTLC method development, samples were applied as bands width 6mm using Camag (Muttenz, Switzerland) Linomat-5 applicator fitted with 100µl syringe (Hamilton, Switzerland). The rate of application was constant at 150nl sec^-1 and space between two bands was 14mm. After plate development and air drying densitometric scanning was performed at 254nm using Camag TLC scanner-3 which shows typical chromatogram as shown in (Fig: 2).

 

 

Figure 2: HPTLC chromatogram of Boswellic acid

 

Validation:

As per ICH Q2(R1) guidelines method was validated including linearity, precision, accuracy, limit of detection, limits of quantitation and robustness as discussed under.

 

Linearity:

From the prepared solution 10-60µg/mL was calculated by calibration curve (Fig: 3) using Peak area vs. concentration. Equation and correlation coefficient was calculated for statistical analysis.The linear regression equationsobtainedarey=0.2462 X+0.2782 and Correlation coefficient was 0.9997. The retention time of standard Boswellic acidwere found to be12.5 min.

 

 

Different aliquots for HPTLC ranged 500– 3000ng/spot were applied on TLC plate. After development TLC plate scanned at 254nm. Calibration curve (Fig: 4) Correlation coefficient and y-interceptof the linearity curve was calculated. The linear regression equations obtained are y=1.3485X+92.24 and Correlation coefficient was 0.9996.

 

Figure 3: Calibration Curve of Boswellic acid by RP-UHPLC

 

Figure 3: Calibration Curve of Boswellic acid by HPTLC

 

Precision:

By RP-UHPLC method, Precision of the method was verified by repeatability and intermediate precision studies. Intra-day precision was studied by analyzing 10, 20, 30µg/mL of Boswellic acid for three times on the same day. Inter-day precision was checked analyzing the same concentration for three different days over a period of week. The %RSD should not be more than 2% shown in (Table 1).

 

By HPTLC method, Precision of the method was determined as intra-day and inter-day variations. Intra-day variations were determined by analyzing 1500, 2000, 2500/spot of standard solution of Boswellic acid for three times on the same day. Inter-day precision was determined by analyzing 1500, 2000, 2500ng/spot of standard solution of Boswellic acid for three consecutive days over a period of a week. The %RSD should not be more than 2% shown in (Table 2).

 

 

 

 

Table 1: Precision Study by RP-UHPLC (n = 6) (acceptance limit % RSD < 2)

 

Table 2. Precision Study by HPTLC (n = 6) (acceptance limit % RSD < 2)

Day 1	Day 2	Day 3
2797.66	2796.33	2120.98
1.95	1.54	1.18
1.62%	1.26%	1.07%

 

Accuracy:

For RP-UHPLC, It was done by recovery study using standard addition method at 80%, 100% and 120% level; known amount of standard Boswellic acid was added to pre-analyzed sample (10.0mg/mL of Boswellic acid) and subjected them to the proposed UHPLC method % recovery should be between 98% to 102% results are shown in (Table 3).

 

For HPTLC, Recovery experiments were performed at three different levels i.e. 80, 100 and 120%. To the pre-analysed sample solutions; a known amount of standard drug solution of boswellic acid was over spotted at three different levels. The chromatogram was developed and scanned. The results shown in (Table 4).

 

Table 3. Results for RP-UHPLC method for % recovery

Drug	Initial amount [mg/mL]	Excess drug added to the analyte [%]	Amount recovered ± S.D. [mg/mL]	Recovery [%]	%RSD [n = 3]
Boswellic acid	10	80	17.96 ± 0.052	99.77778	0.0432
	10	100	19.98 ± 0.025	99.91667	0.0205
	10	120	21.96 ± 0.015	99.83333	0.0124

 

Table 4. Results for HPTLC method for % recovery

Initial amount [mg/mL]	Excess drug added to the analyte [%]	Amount recovered ± S.D. [mg/mL]	Recovery [%]	% RSD [n = 3]
1500	1200	2696.03 ± 7.34	99.8530864	5.99
1500	1500	3001.3 ± 2.56	100.043333	2.09
1500	1800	3299.36 ± 0.41	99.9808081	0.33

 

Sensitivity:

For RP-HPLC method, limit of detection (LOD) and limit of quantitation (LOQ) were determined and LOD and LOQ were found to be 0.9076mg and 2.7503mg for Boswellic acid.

 

 

Whereas HPTLC method sensitivity of measurements of Boswellic acid by the use of the proposed method was estimated in terms of the Limit of Detection (LOD) and Limit of Quantitation (LOQ). The LOD and LOQ for Boswellic acid for were found to be 5.429ng and 16.453 ng, respectively.

 

Ruggedness:

For RP-UHPLC and HPTLC method, solution of 10 µg/mL of Boswellic acid was prepared and analyzed by two different analysts using similar operational and environmental conditions.Percentage relative standard deviation should not be more than 2.0% given in (Table 5).

 

Robustness:

For RP-UHPLC, Robustness of the method was studied by making deliberate changes in few parameters viz; change in mobile phase composition, acid concentration (pH modifiers), and flow rate. The effects on the results were studied by injecting 10µg/mL of Boswellic acid; one factor was changed at one time to estimate the effect; the results are shown in (Table 6).

 

 

Table 5: Ruggedness results for RP-UHPLC and HPTLC

Method	Amount in µg/mL	Amount Found [n = 6] Mean ± SD		% Amount [n = 6]		% RSD
		Analyst I	Analyst II	Analyst I	Analyst II	Analyst I	Analyst II
RP-UHPLC	10	9.96 ± 0.031	9.93 ± 0.049	99.6 ± 0.316	99.31 ± 0.491	0.0288	0.0448
HPTLC	10	9.94 ± 0.035	9.95±0.037	99.18 ± 0.389	99.45 ± 0.478	0.1961	0.2857

 

Table 6: Robustness study by RP-UHPLC

Parameters	Boswellic acid Rt
Change in Acid Concentration for pH adjustment
0.15 %	13.01
0.2 %	12.23
0.3 %	12.49
Change in Flow Rate
0.8 ml/min	15.36
1.2 ml/min	10.69
1.5 ml/min	08.64
Change in mobile phase composition
(60 : 40 v/v)	22.36
(65 : 35 v/v)	18.65
(70 : 30 v/v)	16.13

 

By HPTLC, Robustness of the method was studied by making deliberate changes in few parameters viz; change in mobile phase composition, stability of the stock solution. The effects on the results were studied by applying 1000.0 ng/band of Boswellic acid and one factor was changed at one time to estimate the effect; the results are shown in (Table 7).

 

Table 7: Robustness study by HPTLC

Parameters	Boswellic acid
	SD of peak area		% RSD
Mobile phase composition
aToluene: ethyl acetate: methanol (4:1:1)	23.89		0.22
bToluene: ethyl acetate: methanol (4:1.5:0.5)	15.41		0.14
Mobile phase volume (mL)
4.7	15.38		0.14
9.4	11.37		0.10
Development distance (mm)
70	18.84		0.39
75	08.05		0.16
80	13.85		0.29
Relative humidity (%)
55	16.73		0.15
65	13.80		0.12
Duration of saturation (min)
20	19.31	0.18
25	17.10	0.16
30	12.22	0.11
Activation of prewashed TLCPlates (min)
08	12.66	0.11
10	5.22	0.04
12	7.19	0.06
Time from spotting tochromatography	7.22	0.06
Time from chromatography to scanning	11.18	0.10

 

Specificity and Selectivity:

The analytes should have no interference from other extraneous components and be well resolved from them. Specificity is a procedure to detect quantitatively the analyte in presence of component that may be expected to be present in the sample matrix, while selectivity is the procedure to detect qualitatively the analyte in presence of components that may be expected to be present in the sample matrix.

 

CONCLUSION:

The method is quite selective. There was no other interfering peak around the retention time of both the Boswellic acid; also the base line did not show any significant noise.The mobile phase was developed to get well separated peaks of sample of Boswellic acid. Results of estimation of Boswellic acid in extract and formulation were accurate, precise and rugged. The developed method was validated by means of accuracy, precision and linearity range as per ICH guidelines. The developed method was specific, linear, precise, accurate and rugged, for assay of Boswellic acidin dosage form. A validated RP-UHPLC method for the determination of Boswellic acid in extract and in formulation was found to be simple and precise. Further, developed method was rugged, robust and sensitive. Avalidated HPTLC method for the determination of Boswellic acid in extract and in formulation was found to be simple and precise. Further, developed method was rugged, robust and sensitive. All developed methods could be used for routine analysis of Boswellic acidin the dosage form.

 

ACKNOWLEDGEMENTS:

The authors are thankful to the Principal, Gangamai College of Pharmacy, Nagaon, Dist. Dhule for providing necessary facilities for research work. They are also grateful to Sigma Aldrich Ltd., USA. for giving gift samples of pure drugs.

 

ABBREVIATION USED:

HPLC: High performance liquid chromatography; ICH: International Conference on Harmonization; LOQ: Limit of quantitation; LOD: Limit of detection; RSD: Relative standard deviation; RT: Retention time;FDA: Food and Drug Administration; SD: Standard deviation.

 

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Accepted on 06.02.2023          © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(10):4658-4662.