Development And Validation of RP-HPLC Method for Simultaneous Estimation of Folic Acid and Lysine Hydrochloride in Their Combined Oral Liquid Formulation

The chemical name of Dapagliflozin Folic Acid isN-(4-((2-amino-4-oxo-1,4-dihydropteridin-6-yl) methylamino) benzoyl)-L-glutamic acid and Lysine Hydrochloride chemical name is(S)-2,6-diaminohexanoic acid monohydrochloride. Folic acid (Vitamin B9) and lysine hydrochloride are commonly combined in oral liquid formulations used as nutritional supplements. While folic acid plays a key role in DNA synthesis, cell division, and hematopoiesis, lysine hydrochloride, an essential amino acid, supports protein synthesis, immune function, and overall growth. Given their widespread use, especially in pediatric and nutritional applications, it is essential to establish a robust method for their simultaneous estimation in combined formulations. Simultaneous estimation of compounds with distinct physicochemical properties, such as folic acid (a vitamin) and lysine hydrochloride (an amino acid salt), poses unique analytical challenges. These include differences in polarity, solubility, and UV absorbance, which require careful selection of chromatographic conditions to achieve proper resolution and detection. Reversed-phase high-performance liquid chromatography (RP-HPLC) is a preferred analytical technique due to its high sensitivity, specificity, and suitability for a wide range of pharmaceutical compounds. This study focuses on the development and validation of a simple, reliable, and reproducible RP-HPLC method for the simultaneous estimation of folic acid and lysine hydrochloride in their combined oral liquid formulation. The method was optimized for key parameters such as mobile phase composition, pH, flow rate, detection wavelength, and retention time to ensure adequate separation and quantification of both analytes. Validation of the developed method was performed in accordance with ICH Q2(R1) guidelines, evaluating parameters such as linearity, accuracy, precision, specificity, and robustness.

Folic Acid

Lysine Hydrochloride

MATERIALS AND METHOD:

Materials

Table 1.0 Materials & Sources

Instrumentation

• HPLC instrument with UV-visible detector
• Software: LC Solution
• Column - HYPERSIL ODS C18, 250 mm*4.6 mm
• UV-visible Spectrophotometer - SHIMADZU 1800 Software - UV probe, version- 2.42
• Digital Analytical Balance - Mettler Toledo
• Ultra Sonicator -PS 21
• Controlled temperature water bath
• Hot air Oven – Kesar
• FTIR – SHIMADZU
• Melting Point Apparatus – Gallenkamp Model: 889339

Chemicals and Reagents: Folic Acid API, Lysine Hydrochloride API, Acetonitrile HPLC Grade, Methanol HPLC Grade, Phosphate Buffer HPLC Grade and HPLC Grade water.

Preparation of Phosphate Buffer 0.05M: Weight accurately 9.8g of Potassium Dihydrogen Phosphate, Transfer in 1000mL of volumetric flask add 700mL of distilled water sonicate properly and markup to volume.

Preparation of Mobile Phase:  The mobile phase was prepared by adding the ratio of (50:20:30 v/v/v) Methanol: Acetonitrile: Buffer 0.05M (Potassium Dihydrogen Phosphate) and then filtered through 0.45 μm membrane filter; sonicated for 15 min.

Preparation of Standard Stock Solution

Standard Stock Solution for Folic acid Propionate: Accurately weighed quantity of Folic acid Propionate 10 mg was transferred into 100 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 100 μg/mL. Withdraw 1 ml from Stock Solution and make up to 0.1 ml with to get 1 μg/mL.

Standard Stock Solution for Lysine Hydrochloride: Accurately weighed quantity of Lysine Hydrochloride 10 mg was transferred into 100 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 100 μg/mL. Withdraw 7 ml from Stock Solution and make up to 10 ml with to get 70 μg/mL

Chromatographic Conditions:

Table 2.0 Chromatographic Conditions

Identification And Characterization

The identification of taken standard API for experimental work had done for confirmation of its identity, standard quality and purity. The identification had done by taking IR and UV spectra, solubility study and melting point determination.

Solubility Study:

The solubility of Folic Acid & Lysine Hydrochloride practically determined separately by taking 100 mg of both the drugs in 100 ml volumetric flasks, adding required quantity of solvent at room temperature and shaken for few minutes. Solubility data for each study was observed and recorded in Table 4.0.

Table 4.0 Solubility Data for Dapagliflozin & Bisoprolol

Identification by Melting Point Determination:

Melting point of Dapagliflozin & Bisoprolol hydrochloride has been determined. The melting points of the compounds were taken by open capillary method.

IR Spectra:

The IR Spectra of Lysine Hydrochloride with its functional group identification, were shown in the following graph. IR Spectra scanning of sample: Lysine Hydrochloride.

Fig 1.0 IR Spectra of Standard Lysine Hydrochloride

The IR Spectra of Folic Acid with its functional group identification, were shown in the following graph. IR Spectra scanning of sample: Folic Acid.

Fig 2.0 IR Spectra of Standard Bisoprolol

Table 7.0 IR Spectra Interpretation for Folic Acid

Method Development

Selection of Wavelength:

To determine wavelength for measurement, standard spectra of Folic acid & Lysine Hydrochloride were scanned between 200-400 nm against diluents. Absorbance maxima of Folic acid & Lysine Hydrochloride have detected at 300. Chromatogram was taken at 300 nm, both drugs give good peak height and shape. So, 300 nm was selected for Simultaneous estimation of Folic acid & Lysine Hydrochloride in their formulation.

Fig 3.0 Overlay UV Spectra of Lysine Hydrochloride and Folic Acid

Selection Of Column:

For RP-HPLC Method, various columns are available but based on literature survey C-18 (id 4.6 x 250 mm, 5 µm) was selected over the other columns.

Selection of Mobile phase:

Table 8.0 Trial 1: Selection of Mobile Phase

Fig 4. Trial 1: Chromatogram of Folic acid & Lysine Hydrochloride Acetonitrile: Phosphate Buffer(30:90v/v)

Fig 5.0 Trial 2: Chromatogram of Folic acid & Lysine Hydrochloride Acetonitrile: Phosphate Buffer(50:50v/v)

Fig. 6.0 Trial 3: Chromatogram of Folic acid & Lysine Hydrochloride Acetonitrile: Phosphate Buffer(80:20v/v)

Fig 7.0 Trial 4: Chromatogram of Folic acid & Lysine Hydrochloride Methanol: Phosphate Buffer(60:40v/v)

Fig 8.0 Trial 5: Chromatogram of Folic acid & Lysine Hydrochloride Methanol: Phosphate Buffer(70:30v/v)

Fig 9.0 Trial 6: Chromatogram of Folic acid & Lysine Hydrochloride Methanol: Phosphate Buffer(80:20v/v)

Fig 11.0 Trial 8: Chromatogram of Folic acid & Lysine Hydrochloride Chloroform: Acetonitrile (60:40 v/v)

Table 16.0 Trial 9: selection of mobile phase

Fig 12.0 Trial 9: Chromatogram of Folic acid & Lysine Hydrochloride Chloroform: Acetonitrile(50:50v/v)

Fig 13.0 Trial 10: Chromatogram of Folic acid & Lysine Hydrochloride Acetonitrile: Water(30:70v/v)

Fig 14.0 Trial 11: Chromatogram of Folic acid & Lysine Hydrochloride Methanol: Acetonitrile: Phosphate buffer (55:25:25 v/v/v)

Fig 15.0 Trial 12: Chromatogram of Dapagliflozin & Bisoprolol Acetonitrile: Water (80:20v/v)

Table 20.0 Trial 13: selection of mobile phase

Fig 16.0 Trial 13 Optimized mobile phase trial for optimized chromatogram of Std. Folic acid:3.215 min, Lysine Hydrochloride: 5.115 min

Optimized mobile phase trial for optimized chromatogram of Std. Folic acid:3.215 min, Lysine Hydrochloride: 5.115 min

Fig 17.0: Chromatogram of blank Methanol: Acetonitrile: Phosphate buffer (50:20:30 v/v/v)

Table 23.0: optimized mobile phase trial of Folic Acid &Lysine Hydrochloride

Fig 19.0: Optimized mobile phase trial for optimized chromatogram of Std Folic Acid :3.215 min Lysine Hydrochloride: 5.115 min

Method validation is the process of documenting or proving that an analytical method provides analytical data acceptable for the intended use. The need to validate a method and the procedure to be followed are matters of professional judgement, although well-prescribed procedures and guidelines are now available that aid in decision making. According to that the various validation parameters to validate each and every above stated method are:

• Accuracy and Recovery
• Precision (Repeatability and Reproducibility)
• Linearity
• Range
• Robustness/ Ruggedness
• Limit of Detection (LOD)/ Limit of Quantification (LOQ)

Accuracy and Recovery:

The capability of a procedure to generate outcomes close to the actual or standard value (Standard value may be reference value given in official compendia).

The chosen concentration for precision investigations must encompass the complete concentration range (i.e. one may the lowest concentration, one may the middle and one may be the last of range).

Accuracy is performed by performing recovery studies by spiking in 2 ways:

1. Spiking of sample with standard (in case placebo are not available).
2. Spiking of placebo with standard.

It can be performed at different level like 50, 100 and 150 % of test concentration or 80, 100, 120% of test concentration.

Reproducibility and Precision:

It is defined as the degree of similarity between test findings obtained from many samplings of the same sample. C.V. or relative standard deviation is capable of expressing it is further classified as reproducibility, repeatability, and intermediate precision.  Repeatability is the capability of an analytical procedure to produce identical test findings when performed in a similar setting by the same operator over a brief period of time. It is stated as R.S.D. when a minimum of three concentrations is analysed at least six times each, and R.S.D. is determined.   At least 6 repetitions encompassing 100% of the target concentration or 9 repetitions covering the complete linear range must be assessed. (i.e., three replications for every concentration)  Reproducibility is the capacity of an analytical procedure to yield same test findings when applied to identical samples under various conditions and by different analysts. Conditions of operation vary, but the variance is still within acceptable parameters. It is a crucial validation parameter if the procedure must be executed under diverse situations.

Range and Linearity:

It is the capability of the method to generate findings that are directly proportional to the concentration of a specific component in the samples. It is shown via certain mathematical modifications.

1. It may be conducted directly on the material being examined or using a suitable standard and dilution using the suggested approach.
2. It can be accomplished by measuring at least five injections in a series of standards. Eighty to one hundred twenty percent of the recommended concentration range is permissible.
3. A mathematical equation must illustrate the reaction of the sample to the concentration.
4. The linear regression approach requires a substantial zero intercept, and if it is not reached, one must demonstrate that there is no significant influence on accuracy. 
5.  Another method that may be applied is the response factor, which is generated by dividing the response by its corresponding concentration to obtain the relative response. On a logarithmic scale, a graph showing relative reaction against concentration is presented. Therefore, the resultant horizontal line must be linear over the full range. Negative deviation is often observed at greater doses. On the graph, a horizontal line is drawn parallel to the x axis to represent 95 to 105 percent of the horizontal line. The reaction is linear with respect to concentration up until the intersection of the horizontal line and the 95 percent line.
6. It is the concentration interval between which quantitative analysis may be done with appropriate precision and linearity. The analytical method's range is stated in the same units (such as PPM or %).

Robustness:

A technique has the ability to be unaffected by tiny, purposeful changes to operating settings, but these changes are still within the method's range. The effects of these adjustments on the method's output are evaluated. By conducting robustness, one may assess if revalidation is necessary or not. Variable technique parameters include flow rate, mobile phase composition, mobile phase pH, and detection wavelength. This modification can be within the permissible range (i.e., 2% to 5% of the original value). The essential method parameter that identifies the susceptibility of the technique to change must be reported following ICH rules. However, it is not a registration requirement.

Robustness:

A technique has the ability to be unaffected by tiny, purposeful changes to operating settings, but these changes are still within the method's range. The effects of these adjustments on the method's output are evaluated. By conducting robustness, one may assess if revalidation is necessary or not. Variable technique parameters include flow rate, mobile phase composition, mobile phase pH, and detection wavelength. This modification can be within the permissible range (i.e., 2% to 5% of the original value). The essential method parameter that identifies the susceptibility of the technique to change must be reported following ICH rules. However, it is not a registration requirement.

Limit of Quantification and Limit of Detection:

It refers to the analytical method's capacity to identify analyte in the presence of a matrix with sufficient accuracy and precision. The method may be able to detect the analyte but not quantify it. There may be confusion between technique sensitivity and LOD.  Sensitivity can distinguish between minute differences in concentration. It may be described as the slope of the regression line.

Following methods are available other than signal to noise ratio that are as follows:

1. Visual determination: it is possible to determine by personally injecting the lowest known concentration that is detectable but cannot be measured by mathematical manipulations.
2. Standard deviation of response based on standard deviation of blank: it may be determined by examining the number of blank samples and determining the standard deviation of the blank answers. This approach suggests the capability of a method to provide a response other than analyte concentration.
3. Using slope of regression line to estimate standard deviation of response: one may measure the S.D. of regression line of a given calibration curve or use S.D. of intercept.
4. It is also calculable using the mathematical formulae shown below.

RESULT AND DISCUSSION:

Analytical method Validation

Linearity

For the purpose of linearity, accurately weighed amount of Folic acid (10 mg), and Lysine Hydrochloride (10 mg) was taken into the volumetric flask (10 ml) and volume of the flask was raised to 10 ml with methyl alcohol to give stock solution containing 100 µg/ml of Folic acid, and 100 µg/ml of Lysine Hydrochloride. Various aliquots from this stock solution were transferred to another 10 ml volumetric flask and volume was raised to the mark with mobile phase to give final solutions containing 0.5+35, 0.8+56, 1.0+70, 1.2+84 µg/ml and 1.5+105 µg/ml of Folic acid and Lysine Hydrochloride respectively.

Table 25.0 Linearity data for Lysine Hydrochloride

Fig 20.0: Overlain Linearity Spectra of Folic Acid and Lysine Hydrochloride

Fig 21.0: Calibration curve of Folic Acid

Fig 22.0: Calibration curve of Lysine Hydrochloride

Repeatability

The data for repeatability for Folic acid and Lysine Hydrochloride is shown in table 27.0. The % R.S.D For Repeatability data was found to be 0.64 % for Folic acid and 0.45 % for Lysine Hydrochloride.

The data for interday precision for Folic acid and Lysine Hydrochloride is shown in table 28.0 & 29.0 The % R.S.D for intraday precision was found to be 0.36-0.79% for Folic acid and 0.13 -0.41 % for Lysine Hydrochloride.

Inter-Day Precision for Folic Acid:

The data for intra-day precision for Folic acid and Lysine Hydrochloride is shown in table 9.5. The % R.S.D for intraday precision was found to be 0.47-1.01 % for Folic acid and 0.11 – 0.24 % for Lysine Hydrochloride.

Accuracy of the method was confirmed by recovery study from synthetic mixture at three level standard additions. Percentage recovery for Folic acid & Lysine Hydrochloride was found to be 99.48- 99.78% and 99.33-100.59 % respectively. The results are shown in table31.0-32.0.

Recovery For Dapagliflozin:

The limit of detection (LOD) and Limit of Quantification (LOQ) was found to be as per below:

The method is found to be robust as the results were not significantly affected by slight variation in Mobile Phase Composition and flow rate of mobile phase. The results are shown in table 35.0. Variation seen was within the acceptable range respect to peak asymmetry and theoretical plates, so the method was found to be robust.

The proposed method was successfully applied to analysis of the commercially available tablet formulation. The % drugs were found satisfactory, which is comparable with the corresponding label claim.

Table 36.0 Analysis of marketed formulations

Summary Of Method Validation:

Summary of validation parameter are shown in below table. All the parameters for substance met the criteria of ICH guideline for the method validation and found to be suitable for routine quantitative analysis in pharmaceutical dosage forms. The result of linearity, accuracy, precision proved to be within limits with lower limits of detection and quantification. Robustness of method was confirmed as no significant in the were observed on analysis by subjecting the method to slight change in the method condition. Assay results obtained by proposed method are fair agreement.

Table 38.0 Validation Parameters