INTRODUCTION:

Mostly found in citrus fruits, including grapes, apples, papayas, strawberries, and mangoes, tartaric acid is a white, crystalline organic acid. Citrus fruits naturally contain tartaric acids, which are weak organic acids. It is a fantastic source of antioxidants and is often employed as a buffer¹. For individuals who do their homework, tartaric acid may also be utilized in cosmetic products like serums and masks to lessen fine lines, acne scars, and a dull skin tone. These organic acids are being quantified in order to confirm the RP-HPLC analytical technique used to separate the tartaric acid medication.

The most popular HPLC methods for determining organic acids A typical method for determining the contaminants or components in drugs is high-performance liquid chromatography². For this determination, an isocratic technique with a mobile phase of 0.01M potassium dihydrogen phosphate (KH₂PO₄) and a pH of 2.6 is developed. Shimadzu GIST RP-column C18 chromatographic conditions were used. Shim pack (4.6mm x 250mm, 5um) with a flow rate of 1ml, wavelength of 210nm, and injection volume of 20 l. The oven is set to a temperature of around 30°C, and the RP-HPLC method takes about 15 minutes to complete³. Accurately presenting the findings with their acceptance requirements are the validation parameters' accuracy, system appropriateness, linearity, precision, and robustness. With the use of novel chromatographic conditions, we performed the analysis in this study and compared various circumstances⁴. The results of the supplied technique were accurate and exact, and they fulfilled all the requirements.

Fig No 01: Tartaric Acid

MATERIALS AND METHODS:

1.1) Chemicals:

Orthophosphoric acid (H₃PO₄) and potassium dihydrogen phosphate (KH₂PO₄) were produced by Rankem Pvt. Ltd. and Loba Cheme Pvt. Ltd., respectively. Sci Tech Specialties Pvt. Ltd. used HPLC-grade water as the solvent and for sample preparation in the mobile phase.

1.2) Equipments:

To validate the analytical procedure, an Agilent Technologies 1260 Infinity instrument with EZ Chrome software and a UV/Vis detector, Column C18 Shimadzu GIST, was used. (4.6mm, 250mm, 5um) Shim pack. The ultrasonic cleaner sonicator, PH meter, and analytical balance were all Shimadzu AP225WD models made by the Shimadzu Corporation. The PH meter was a picometer made by Dakshin.

1.3) Chromatographic Conditions:

The technique was validated using Column C18 Shimadzu GIST Shim Pack (4.6mm, 250mm, and 5um), with a flow rate of 1ml and an injection volume of 20 l. The HPLC-RP method runs for 15minutes at a temperature of roughly 30°C in the oven. There is a 4-minute retention period.

1.4) Solubility studies:

In numerous solvents, including water and ethanol, tartaric acid's solubility was studied. The substance was thoroughly dissolved using a sonicator for 15minutes after being correctly weighed out to be 2.0grams of tartaric acid in water and 95% ethanol. Tartaric acid was discovered to be soluble in water and 95% ethanol after the tests were conducted⁵.

Table No 01: Table of Solubility studies

Sr. No	Solvent	Solubility
1	Water	Soluble
2	95 %Ethanol	Soluble
3	Potassium Dihydrogen Phosphate	Soluble

1.5) Preparation of solution Mobile Phase (Diluent):

In 1000ml of water, potassium dihydrogen phosphate was dissolved at a concentration of 1.36089. Sonicate it for 5minutes. Utilizing orthophosphoric acid, precisely adjust the pH to 2.6. The diluent is potassium dihydrogen phosphate, 0.01M.

1.6) Blank Solution:

The blank solution is diluent.

1.7) Preparation of Standard Solution:

Transfer the 50mg of tartaric acid working standard, which has been weighed, into a volumetric flask with 100ml of diluent, enough to thoroughly dissolve it, and enough makeup to fill the flask up to 100ml. Take 5ml of the aforementioned solution, then dilute it to the proper strength—25ml—with diluent.

1.8) Preparation of Sample Solution:

Take the mixture from the 10sachets and grind it into a fine powder. 1.5g of crushed powder should be weighed and transferred into a 100ml volumetric flask. To thoroughly dissolve it, add 50ml of diluent. Then, dilute the solution to the proper strength—100ml. Take 2ml of the aforementioned solution, add it to a 50ml volumetric flask with the aid of diluent, and fill the flask to the required level. 0.2micron filtering is required.

RESULTS AND DISCUSSION:

Citric acid and tartaric acid are widely used in food and arious pharmaceutical industries as well as in wine production they play very important role in taaste. Their quanititation and estimation of organic acids helps to determine the quality of pharmaceutical products. In this reverse phase HPLC with UV- Vis detection is commonly technique used for this analysis. In the various review literature, the wavlength was set at 214 nm and the mobile phase was phosphate buffer solution pH = 2.8 is adjusted. Although the chromatogram conditions were different in literature survey like column were used HPLC LiChrosorb RP-18 (10um, 25cm × 4.0 mm.

The further experiment was conducted by using the RP- HPLC Method by using Column C18 Shimadzu GIST Shim pack (4.6mm × 250mm, 5um) with flow rate of 1 ml, having wavelength 210nm with injection volume 20 µl. oven temp is about 30^oC and run time of RP- HPLC Method is about 15min. The mobile phase is used as common diluent for preparing the samples and standards. In this we have disscussed the various analytical method validation parameters. Additionally, we have noted the findings of the accuracy, specificity, precision, repeatability between and within days, robustness, and stability conditions.

1.1) Validation of the Method:

The tartaric acid working standard concentration range of 80 to 120ug/ml was used to assess the method's linearity. All concentration ranges were examined using three distinct samples, and the correlation coefficient and intercept were reported. They should have a correlation coefficient of at least 0.999. In terms of system appropriateness, the standard peak's relative standard deviation for five replicates was 0.044, with a standard deviation of 1485.68. In order to verify the system's appropriateness requirements, a solution was created and injected using the test technique. The acceptance requirements were determined to be well satisfied by all factors. RSD should not exceed 2.0% for any absorbance. We have observed that the findings of system appropriateness and linearity using this approach were exact, accurate, and fulfilled acceptance requirements⁶.

1.2) System Suitability:

The relative standard deviation of the standard peak for five replicates was 0.044 in terms of system appropriateness, with a standard deviation of 1485.68. A solution was made and injected as directed by the test procedure to evaluate the system's appropriateness requirements. All system suitability replicates were determined to be well within the acceptance standards ⁷. RSD should not exceed 2.0% for everyone. All samples of specificity were determined to be well within the parameters for approval. The RSD of specificity was discovered to be 0.044%.

Table No 02: Result Of System Suitability

Sr. No	Retention times	Number of theoretical plates	Observed asymmetry factor	Area of tartaric acid
1	3.393	9435	1.20857	3394296
2	3.393	9436	1.21165	3390738
3	3.397	9469	1.12959	3391735
4	3.393	9415	1.19628	3393257
5	3.397	9497	1.14151	3393801
Avg				3392765.4
Std.Dev				1485.68
%RSD				0.044 %

1.3) Linearity:

The working standard tartaric acid concentration range of 80 to 120ug/ml was used to assess the method's linearity. The correlation coefficient and intercept were collected for each of the concentration ranges that were evaluated using three distinct samples. The correlation between them should have a coefficient of 0.999 or more. In terms of the acceptance criteria, all linearity samples were judged to be in good shape. The co-relation found to be 0.99998 was the linearity coefficient ⁸.

Table No 03: Result Of Linearity

Conc. Level	Weight Taken	Area
80%	40	2699267
90%	45	3065706
100%	50	3398740
110%	55	3745693
120%	60	4074739
Slope		34004.83
Intercept		3044.82
Co-relation Coefficient		0.99998

Fig No 02: Linearity graph

1.4) Accuracy:

The agreement between the measured and real values served as the benchmark for the method's accuracy. Three samples were gathered for each concentration to determine the accuracy parameter. Six concentrations—100%, 110%, 120%, 130%, 140%, and 150%—have been created. There are three replicates for each concentration, where all five are operating at the required standard and 100% are spike-free. Each sample's total recovery should range between 95 and 105%, and each stage's relative standard deviation should not be more than 2.0%. All accuracy samples were determined to be well within the acceptable range. The RSD for accuracy was discovered to be 1.22%⁹.

Table No 04: Result of Accuracy

Sample Recovery	Sample without spike	Sample spike with 10 % spike	Sample spike with 20 % spike	Sample spike with 30 % spike	Sample spike with 40 % spike	Sample spike with 50 % spike
Sample 1	99.29 %	110.80 %	119.42%	130.60%	141.32%	151.27%
Sample 2	101.35%	110.69%	120.96%	131.0%2	140.85%	151.39%
Sample 3	101.49 %	110.80%	117.91%	131.26%	140.83%	151.05%
Average Assay	100.71%	110.76%	119.43%	131.96%	141.00%	151.24%
STD DEV	1.23	0.06	1.53	0.33	0.28	0.17
RSD	1.22%	0.06%	1.28%	0.26%	0.20%	0.11%
% Recovery of SPL 1	99.29%	100.73%	99.52%	100.46%	100.94%	100.85%
% Recovery of SPL 1	101.35%	100.63%	100.80%	100.78%	100.61%	100.93%
% Recovery of SPL 1	101.49%	100.73%	98.26%	100.97%	100.59%	100.70%
Average % Recovery	100.71%	100.69%	99.53%	100.74%	100.71%	100.82%
STD DEV	1.23	0.06	1.27	0.26%	0.20	0.11
RSD	1.22%	0.06%	1.28%	0.26%	0.20%	0.11%

Table No 05: Result Of Specificity

Sample Results	Sample without spike	Sample spike with 10% placebo	Sample spike with 20% placebo	Sample spike with 30% placebo	Sample spike with 40% placebo	Sample spike with 50% placebo
Assay of Sample-1	102.43%	98.89%	100.74%	102.50%	99.22%	101.61%
Assay of Sample-2	102.52%	98.95%	100.91%	102.53%	99.12%	102.21%
Assay of Sample-3	101.81%	98.87%	100.85%	102.40%	99.30%	102.16%
Average assay	102.25%	98.90%	100.83%	102.48%	99.21%	101.99%
STDEV	0.39	0.04	0.09	0.07	0.09	0.33
RSD	0.38%	0.04%	0.09%	0.07%	0.09%	0.33%

1.5) Specificity:

The ability to clearly identify components that would be expected to be present in the analyte in the present is referred to as specificity. Six identical samples were specifically created by spiking them with various dosages of placebos. The first sample consisted of 10%, 20%, 30%, 40%, and 50% placebo. We have three duplicates of each concentration. All sample solutions should have an RSD of no more than 2.0%. All accuracy test samples were determined to be within acceptable limits. For specificity, RSD was calculated to be 0.38 percent ¹⁰.

1.6) Precision:

Repeatability:

Six samples, including contaminated wine samples and standard successive samples, were used to assess the accuracy of the procedure¹¹. The repeatability is the assumption that the results fall within the predetermined ranges and that the technique supplied for making tartaric acid from sodium bicarbonate, sodium citrate, citric acid, and tartaric acid granules satisfies all requirements for acceptance under the repeatability study. The total sample RSD should not be more than 2.0%. The linked sample's II% RSD was discovered to be 0.18%¹².

Table No 06: Result Of Linearity

Sr. No	Standard	Weight Of Tartaric Acid	% Assay Of Tartaric Acid
1	Sample 1	1.5 gm	100.21%
2	Sample 2	1.5 gm	100.09%
3	Sample 3	1.5 gm	100.48%
4	Sample 4	1.5 gm	100.45%
5	Sample 5	1.5 gm	100.57%
6	Sample 6	1.5 gm	100.31%
Average			100.35%
STD DEV			0.18
% RSD			0.18%

Intermediate Precision :

Lab variations, such as those caused by various analysts and instruments, include intermediate precision. Three samples are produced for moderate precision, each with analysts 1 and 2. The combined RSD of analysts 1 and 2 should not be more than 2.0%. The RSD of both findings was determined to be 0.67% ¹³.

1.7) Robustness:

· Change In Temperature:

· Change In Flow Rate

When the analytical method's parameters are intentionally changed, robustness indicates the analytical method's dependability. Three samples, each made from a homogeneous mixture of the identical components, were prepared and examined at three different temperatures in the column compartment: 270C, 30C, and 330C. All samples' RSD shouldn't be higher than 2.0%. These three samples all revealed that they met the requirements for admission. The temperature-related RSD of robustness change was 0.03%¹⁴.

Three samples, each with a flow rate of 0.8, 1.0, and 1.2 ml/min, were prepared from a homogeneous mix of the same samples and evaluated. All samples' RSD shouldn't be higher than 2.0%¹⁵.

Table No 07: Result of Change in Temperature

Sample Temperature	Sample 1 Assay	Sample 2 Assay	Sample 3 Assay
27.0⁰C	100.13%	99.96%	100.29%
30.0⁰C	100.17%	99.94%	100.33%
33.0⁰C	100.18%	100.04%	100.45%
Average Assay	100.16%	99.98%	100.36%
STDEV	0.03	0.05	0.08
RSD	0.03%	0.05%	0.08%

Table No 08: Result of Change in flow rate

Sample Flow rate	Sample 1 Assay	Sample 2 Assay	Sample 3 Assay
0.8 ml/min	101.15%	100.21%	101.40%
1.0 ml/min	100.20%	100.07%	100.32%
1.2 ml/min	100.27%	100.06%	100.44%
Average Assay	100.54%	100.45%	100.72%
STDEV	0.53	0.66	0.59
RSD	0.53%	0.66%	0.59%

1.7) Stability Conditions:

The shift in time where the samples reveal correct results or incorrect ones corresponds to the stability condition¹⁶. Three samples were made from a homogeneous mix of the same components, and each sample was then tested for tartaric acid at various intervals: immediately after production, two hours later, four hours later, and six hours later. The stability samples' RSD shouldn't be more than 2.0%. All stability solution samples met the requirements for approval. The overall stability condition change's RSD was 0.10%¹⁷.

CONCLUSION:

Assaying tartaric acid from granules of tartaric acid, citric acid, sodium citrate, and sodium bicarbonate was done as part of the analytical method vaporation investigation. It has been shown that the validation of the supplied analytical technique meets every acceptance criterion for the various validation parameters, including system appropriateness, linearity, accuracy, specificity, precision, and robustness. As a result, it can be said that the tartaric acid test analytical technique provided is reliable and suitable for routine analysis.

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Received on 15.01.2023 Modified on 23.09.2023

Asian J. Pharm. Ana. 2024; 14(2):81-85.

DOI: 10.52711/2231-5675.2024.00015

Sample % Assay	Assay of Tartaric acid Immediately after preparation	Assay of Tartaric acid After 2 hours	Assay of Tartaric acid After 4 hours	Assay of Tartaric acid After 6 hours
Assay of sample 1	101.75%	101.82%	102.09%	101.90%
Assay of sample 2	101.58%	101.65%	101.68%	101.67%
Assay of sample 3	101.75%	101.74%	101.78%	101.77%
Average Assay	101.69%	101.74%	101.85%	101.78%
STDEV	0.10	0.09	0.21	0.12
RSD	0.10%	0.08%	0.21%	0.11%