INTRODUCTION:

Amla (syn: Emblica officinal’s, Emblica Myrobalan, Phyllanthus Emblica) also known as Indian gooseberry. It is a deciduous tree of the family belong to family Euphorbiaceae¹ and this species is native to India and also grows in tropical and subtropical regions including Pakistan, Srilanka, South East Asia, China and Malaysia.

The fruits of Emblica officinals are widely used in the Ayurveda² and are believed to defence against diseases. Different types of gooseberry species are present world widely, those are Pereskia aculeate (syn: Barbados gooseberry) belongs to family Cactaceae and it is native to tropical America, Physalis peruviana (syn: cape goose berry ) belongs to family Solanaceae and it is native to South Africa, Dovyalis hebecarpa (syn: Ceylon gooseberry) belongs to family Flacourtiaceae and it is native to Sri Lanka and southern India, Kiwifruit (syn: Chinese goose berry) belongs to family Actinidiaceace and it is native to New Zealand, Italy, Chile, Greece and france^’, Phyllanthus acidus (syn: star gooseberry) belongs to family Phyllanthaceae and it is native to South East Asian countries, Ribes grossularia (syn: European berry) belongs to family Grossulariaceae and it is native to Europe east to the Caucasus and south to North Africa, Ribes hirtellum(syn: American goose berry) belongs to family Melastomaceae and it is native to Northern America.

Emblica officinal’s primarily contains tannins (gallic acid, ellagic acid)³, 1-O-galloyl-beta-D-glucose, 3,6-di-O-galloyl-Dglucose, chebulinic acid, quercetin, alkaloids, phenolic compounds, amino acids and carbohydrates⁴, chebulagic acid. Its fruit juice contains the highest vitamin C. The principal constituents of ascorbic acid and gallic acid are shown in Fig. 1, 2.

Figure1: Structure of Ascorbic acid

Mol Formula - C₆H₈O₆

Mol Wt - 176.12 g mol^-

Figure2: Structure of Gallic acid

Mol Formula – C₇H₆O₅

Mol Wt - 170.12 g mol^-

Vitamin C or ascorbic acid is a water-soluble nutrient and it can be extracted from various fruits and vegetables ⁵i.e. Amla, parsley, broccoli, bell peppers, strawberries, oranges, lemon juice, papaya, cauliflower, kale, mustard greens and Brussels sprouts. The following pharmacological actions has been reported for vitamin c which includes anti-diabetic, anti-oxidant⁶, anti-tumour, anti-plasmodial, anti-inflammatory, anti-microbial⁷anti-rheumatic and also possess hepatoprotectivity .Vitamin C was also used for preventing and treating scurvy, common cold, it is applied to the skin to help with damage from radiation therapy⁸.

Gallic acid is a phenolic compound exists in plant material in the form of free acids, esters, catechin derivatives and hydrolysable tannins^9,
10. The interest in this compound is due to its pharmacological activity as radical scavengers. It has been proved to have potential preventive and therapeutic effects in many diseases, where the oxidative stress has been implicated, including cardiovascular diseases, antioxidant¹⁰, antimicrobial, antiviral, anticarcinogenic¹¹, radio protective neurodegenerative disorders and anti purgative.

The objective of the present investigation was to establish and validate the fast and sensitive ultra fast liquid chromatography (UFLC) method for determination of ascorbic acid and gallic acid in ayurvedic formulation.

EXPERIMENTAL WORK:

Apparatus:

The UFLC system Shimadzu SP 20 AD equipped with 20μl loop and PDA Detector. Integration was achieved by using the software LC solutions which was utilized for instrument control, data collection and data processing. Separation was carried out on a prepacked symmetry, 350µm, 4.6 x 150mm C8 column with a vacuum degasser.

Reagents and materials:

The drug samples such as Ascorbic acid from SDFCL (SD Fine Chem Limited) and Gallic acid from (Sigma life sciences) were purchased. Potassium dihydrogen phosphate (SDFCL (SD Fine Chem Limited), Triethylamine (SDFCL (SD Fine Chem Limited), MilliQ water (Merck), Methanol (Merck). All chemicals and solvents used were of GR/HPLC grade, chemicals such as Hydrogen peroxide (SDFCL (SD Fine Chem Limited), Sodium Hydroxide (Mio Chem Pvt. Ltd), and Hydrochloric acid (Merck Chemicals) were of analytical grade dissolved in MilliQ water used for stress degradation studies. Amla capsule (The Rising Pharmaceuticals) with 500 mg of label claim obtained from local drug store.

Chromatographic Conditions:

The mobile phase is a mixture of methanol and Phosphate buffer. The pH of the buffer is maintained at 3.0 with 0.1N hydrochloric acid. The prepared buffer was filtered through a Millipore 0.45μm membrane filter and ultrasonically degassed prior to use. Methanol and Phosphate buffer used in the ratio of 1:5 (v/v) throughout the experiment. The detection wavelength was set at 264 nm. The elution was done at a flow rate of 1.0 ml/min under ambient condition.

Preparation of standard solutions:

Accurately weighed quantity of 10mg of ascorbic acid and 10mg of gallic acid were added into a 10 ml separate volumetric flasks and dissolved under sonication by 5ml of diluent, the volume was made by diluent and mixed well. Then the solutions were filtered through 0.45µ membrane filter and the final concentration of the resulting solutions was 1mg/ml. Further dilutions were made from the std stock solutions.

Preparation of sample:

20 capsules were taken and made into fine powder. Powder equivalent to about 500mg was taken into a 100ml volumetric flask and about 70ml of diluent was added and dissolved by sonication, and the volume was made up with mobile phase and mixed well. The solution was filtered through 0.45µ membrane filter. Final concentration of the resulting solution was 1mg/ml.

Procedure:

Separate and filtered portions of equal volume of (about 20µl) of standard preparations and assay preparation were injected into the system and the chromatogram was recorded shown in fig 3, 4, 5 and the peak responses of the major peak were measured.

Fig 3: chromatogram of Ascorbic acid

Fig 4: Chromatogram of Gallic acid

Fig 5: Chromatogram of sample (Amla Ayurvedic capsule)

Method Validation:

The UFLC method was validated for specificity, linearity, accuracy, precision, limit of detection, limit of quantization and robustness for the analysis of ascorbic acid and gallic acid. All validation studies were performed as per guidelines of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH)^12-14.

Specificity:

Specificity was assessed by comparing the chromatograms obtained from sample and from the standards shown in Figure. The method was found to be specific from the peak purity of analytes and no interference of other constituents with the eluting peaks and also by comparing the retention times of both standard solutions and sample which were identical, hence method was specific for quantitative estimation of both drugs in the ayurvedic formulation.

Linearity:

The linearity was determined separately for ascorbic and gallic acid. Solutions of the standards at different concentrations were analyzed and calibration curves were constructed by plotting mean areas against the respective concentrations. The method was evaluated by determination of the correlation coefficient and intercept values (Table 1, 2). From the calibration plots it was clear that the linear range for ascorbic acid and gallic acid was between 1 - 9μg/ml.

Accuracy:

The accuracy of the method is the closeness of the measured value to the true value; Accuracy was assessed by determination of the recovery of the method by addition of standard drug to pre-analyzed test sample preparation at 3 different concentration levels 80, 100 and 120 %, taking into consideration percentage purity of added bulk drug samples. Each concentration was chromatographed 3 times and average recoveries were measured. The recovery of all these related substances were found to be in between the predefined acceptance criterion of ascorbic acid and gallic acid were found to be 98.60–100.28% and 100.26– 101.35% respectively, and the data is given in Table 3, 4.

Precision:

The closeness of agreement between a series of measurements obtained from multiple samplings of the same homogeneous sample.

Here the precision was established by using two methods:

a) Method Precision:

The repeatability of the method was established by estimating the assay for six different sample preparations of the same batch and the data is given in Table 5.

b) Intermediate Precision (Ruggedness):

Intraday Precision:

The intraday precision of the method was established by estimating the assay of ascorbic acid and gallic acid for six different standard preparations of the same batch by different analysts on a same day and the data is given in Table 6.

Interday Precision:

The interday precision of the method was established by estimating the assay of ascorbic acid and gallic acid for six different standard preparations of the same batch by different analysts on a different day and the data is given in Table 6.

c) System Precision:

The system precision of the method was established by estimating the assay for six different standard preparations of the same batch and the data is given in Table 7.

LOD and LOQ:

In the present study, the LOD and LOQ were calculated according to the 3.3σ/S and 10σ/S criterions, respectively; where σ is the standard deviation of y-intercepts of regression lines and s is the slope of the calibration curve.

The limits of detection and quantification of the method were studied to detect the lowest amount of analyte and quantitative determination of analyte in a sample respectively. The results are reported in Table 8.

Robustness:

The robustness of the method was evaluated by assaying test solutions after slight but deliberate changes in the analytical conditions. For the proposed method it was done by injecting system suitability preparation into UFLC system with 0.9 ml/min and 1.1 ml/min and also by changing the wavelength i.e. (262nm and 266nm) to get the robustness of the assay method. The results are reported in Table 9.

System suitability:

System suitability was defined as the evaluation of system, analytical operations and samples. A system suitability experiment was performed before determination of ascorbic and Gallic acid in sample. Six replicate injections of standard preparation were injected and resolution, repeatability, number of theoretical plates and tailing factor were determined. The results are reported in Table 10.

…

STRESS DEGRADATION STUDIES:

The stress degradation studies such as hydrolytic (in acidic and alkali medium), photolytic, oxidative and dry heat induced degradation studies were performed for API and marketed Ayurvedic formulation as per ICH guidelines ¹⁵Q1A (R2)_.The results are reported in Table 11.

1. Hydrolytic degradation under acidic conditions:

Hydrolytic degradation studies were performed by taking 0.05g of drug in 50ml of 0.1N methanolic hydrochloric acid and it was refluxed in round bottomed flask on boiling water bath for 8hrs. The remaining s was kept at room temperature.

2. Hydrolytic degradation under alkaline condition:

Hydrolytic degradation studies were performed by taking 0.05g of drug in 50ml of 0.1N methanolic sodium hydroxide and it was refluxed in round bottomed flask on boiling water bath for 8hrs. The remaining was kept at room temperature.

3. Oxidative degradation:

Oxidative degradation studies were performed by taking 0.05g of drug in 50ml of 3%hydrogen peroxide and it was refluxed in round bottomed flask on boiling water bath for 8hrs. The remaining was kept at room temperature.

4. Dry heat induced degradation:

Dry heat induced degradation study was performed by taking 0.01g of drug in different weighing bottles were kept at 70^oC and 25^oC for different time intervals i.e.,7,14,30 days.

5. Photolytic degradation:

A photolytic degradation study was performed by exposing 0.01g of drug is evenly spread in a Petri dish and kept in sun light and also blank in dark condition for different time intervals i.e.,7,14,30 days.

RESULTS AND DISCUSSION:

Fig 6: Linearity graph of Ascorbic acid

Fig 7: Linearity graph of Gallic acid

Table 1: Linearity results of ascorbic acid:

S.No	Concentration (µg/ml)	Area
1	1	905174
2	2	1542690
3	3	2185476
4	4	2854186
5	5	3506874
6	6	4125862
7	7	4752811
8	8	5377934
9	9	5987532

Table 2: Linearity results of gallic acid

S.no	Concentration (µg/ml)	Area
1	1	365895
2	2	665892
3	3	964858
4	4	1264897
5	5	1568120
6	6	1836858
7	7	2136845
8	8	2435891
9	9	2698473

Table 3: Accuracy results of Ascorbic acid:

Spike level	Area	Amount added (mg)	Amount recovered (mg)	% Recovered	Mean Recovery	%RSD
80%	571684	792	781.54		98.60	0.24
	569689	792	778.84	98.33
	572416	792	782.57	98.80
100%	911938	990	996.80	100.68	100.28	0.48
	917524	990	1003.50	101.36
	908825	990	993.99	100.40
120%	1286581	1188	1172.6	98.70	99.78	1.41
	1321568	1188	1204.51	101.38
	1293829	1188	1179.23	99.26

Table 4: Accuracy results of Gallic acid:

Spike level	Area	Amount added(mg)	Amount recovered (mg)	% Recovered	Mean Recovery	%RSD
80%	239689	792	810.65	102.35	101.35	0.85
	236581	792	800.14	101.02
	235863	792	797.71	100.70
100%	372953	990	1009.09	101.92	101.37	0.46
	371524	990	1005.2	101.53
	369485	990	999.71	100.98
120%	528369	1188	1191.33	100.28	100.26	0.24
	526943	1188	1188.12	100.01
	529475	1188	1193.83	100.49

Table 5: Method precision results of Ascorbic acid and Gallic acid:

SS.no

Retention time (min)

Area

Gallic acid

Ascorbic acid

Gallic acid

Ascorbic acid

2.612

1.527

382769

915862

2.610

1.527

388571

913587

2.608

1.522

386586

916824

2.620

1.533

385821

904587

2.594

1.522

388746

918751

2.581

1.522

381268

917536

Mean = 385626.8

Mean = 914524.5

S.D =3050.3

S.D = 5169.949

%RSD =0.79

%RSD = 0.56

Table 6: Ruggedness results of Ascorbic acid and Gallic acid:

	Compound	Rt(min)	Mean area	%RSD
Intraday precision	Ascorbic acid	6.826	909623	0.61
Intraday precision	Gallic acid	2.413	377029	1.63
Interday precision	Ascorbic acid	1.540	944174.5	0.23
Interday precision	Gallic acid	2.5857	384715.5	0.78

Table 7: System precision results of Ascorbic acid and Gallic acid:

S. No	Retention time (min)		Area
S. No	Gallic acid Ascorbic acid		Gallic acid Ascorbic acid
1	2.582	1.574	368217	927584
	2.591	1.549	371472	925874
	2.595	1.542	368726	908726
	2.598	1.541	368293	917452
	2.582	1.538	378242	915863
	2.591	0.536	368934	924767
			Mean = 370647.3	Mean =920044.3
			S.D =3909.871	S.D =7286.2
			%RSD = 1.05	%RSD=0.79

Table 8: LOD and LOQ of Ascorbic acid and Gallic acid:

Compound	LOD(µg/ml)	LOQ(µg/ml)
Ascorbic acid	0.03820	0.1159
Gallic acid	0.14811	0.4488

Table 9: Robustness results of Ascorbic acid and Gallic acid:

ASCORBIC ACID					GALLIC ACID
Parameter Modifications		Plate count	Tailing	Rt (min)	Plate count	Tailing	Rt (min)
Flow Rate (ml/min)	0.9	16360	1.475	1.602	33176	1.322	2.742
Flow Rate (ml/min)	1.1	15171	1.506	1.516	30624	1.329	2.589
Detection wavelength(nm)	262	15860	1.554	1.575	31802	1.316	2.715
Detection wavelength(nm)	266	15282	1.601	1.568	31347	1.311	2.699

Table 10: System suitability parameters of Ascorbic acid

S. no	Retention time(min)	Peak area	Theoretical plates	Tailing
1	1.527	915862	20405.346	1.893
2	1.527	913587	17774.411	1.632
3	1.522	916824	17306.976	1.624
4	1.533	904587	18622.976	1.618
5	1.522	918751	18609.311	1.634
6	1.522	917536	18732.719	1.710

Table10: System suitability parameters of Gallic acid

S.no	Retention time(min)	Peak area	Theoretical plates	Tailing	Resolution
1	2.612	382769	33140.521	1.386	7.636
2	2.610	388571	28985.323	1.336	7.873
3	2.608	386586	28278.573	1.354	7.773
4	2.620	385821	29195.173	1.375	7.932
5	2.594	388746	29482.749	1.366	7.945
6	2.581	381268	29085.517	1.399	7.968

Table 11: Stress degradation studies of ascorbic acid and gallic acid

S.NO	Stress degrdation studies	Time period	Ascorbic acid	Gallic acid
1	Hydrolytic degradation under acidic conditions	1hr	Degradation was Observed	Degradation was Observed
2	Hydrolytic degradation under alkali conditions	1hr	Degradation was Observed	Degradation was Observed
3	Photolytic degradation	1hr	Degradation was Observed	Degradation was Observed
4	Oxidative degradation	1 day	Degradation was Observed	Degradation was Observed
5	Dry heat induced degradation	1 day	Degradation was Observed	Degradation was Observed