RP-HPLC Method for Estimation of Ferrous Ascorbate

Development and validation of RP-HPLC method for estimation of ferrous ascorbate and folic acid in bulk and in pharmaceutical formulation

ABSTRACT

A simple, sensitive, precise and reproducible RP-HPLC method has been developed and validated for estimating the contents of ferrous ascorbate and folic acid combination in bulk and pharmaceutical formulation. The analysis was performed with Gemini-NX-C18 (250×4.60 mm, 5µm) column using mobile phase phosphate buffer (pH 7): methanol (98:2, v/v) for ferrous ascorbate whereas phosphate buffer: methanol (88:12, v/v) for folic acid. Mobile phase was delivered at flow rate of 1.0 ml/min for ferrous ascorbate and folic acid. The detection wavelength was 264 nm for ferrous ascorbate and 280 nm for folic acid. The retention time of ferrous ascorbate and folic acid was found to be 2.7 and 6.8 min, respectively. The linearity was obeyed in the concentration range of 10-50 µg/mL for ferrous ascorbate (r² = 0.999) and 10-50 µg/mL for folic acid (r²= 0.999). Tablet content of ferrous ascorbate and folic acid was found 100.44 % and 98.69 %, respectively.

INTRODUCTION

Iron deficiency and sickle cell is the most common cause of anemia worldwide, accounting for about half of all anemia cases. Prevalence of anemia in India is high because of low dietary intake, poor availability of iron and chronic blood loss due to hookworm infestation and malaria. An exogenous source of folic acid is necessary for the maintenance of normal erythropoiesis. Study was carried out to assess efficacy, safety and tolerability of combination of ferrous ascorbate and folic acid (Phosfomin-XT) in patients with iron deficiency anemia, the report sho wn the increase in bioavailability of ferrous in presence of ascorbate salt. ferrous ascorbate (Fig. 1.A) is newly introduced salt of ferrous having more bioavailability than other salts of ferrous ascorbate [1]. But till date no more literature available for estimation of ferrous ascorbate. The use of therapeutic multivitamins are indicated in cases of deficiency in pathological conditions in which the nutritional requirements are greatly increased or in conditions in which absorption, utilization, or excretion of vitamins are abnormal [2]. Folic acid (Fig. 1.B) deficiency is the result of megaloblastic anemia [3].

Ion pair chromatographic method was reported for simultaneous determination of seven water soluble vitamins nicotinamide, thiamine, riboflavin, pyridoxine, pyridoxal, pyridoxamine, cyanocobalamine and folic acid [2]. HPLC method was developed for estimation of folic acid [2-4]. RP-HPLC method for estimation of folic acid was also reported in Indian pharmacopoeia (2010) [5]. But till date, no report is available for estimation of ferrous ascorbate in combination with folic acid in bulk and pharmaceutical formulation. An attempt has made to develop and validate a new, rapid and sensitive method for estimating the content of ferrous ascorbate and folic acid.

Experimental Conditions

Instrumentation

The HPLC chromatographic system Agilent Technologies 1200 series, assembled with a G1315D quaternary pump, a G1315D diode array detector, a rheodyne injector fitted with a 20 μL loop and EZ Chrome Elite software.

Reagents and chemicals

Ferrous ascorbate and folic acid were obtained as a gift samples from Curex Pharmaceutical Pvt. Ltd., Jalgaon (MS) and S. kant Healthcare Ltd, Wapi (gujrat). HPLC grade acetonitrile, and methanol was purchased from Rankem Ltd., India. Double distilled water was generated in house. IPHARED tablets (ferrous ascorbate equivalent to elemental iron and folic acid in ratio of 100:1.5 mg) were purchased from a local market.

Standard stock solution of ferrous ascorbate (100 µg/mL)

An accurately weighed quantity of about 10 mg ferrous ascorbate was dissolved in 100.0 mL of phosphate buffer (pH 7).

Working standard solution of ferrous ascorbate

Working standard solution of ferrous ascorbate in the concentration range of 10–50 μg/mL was prepared by diluting an aliquot portion of about 1– 5 mL ferrous ascorbate stock solution (100 μg/mL) to 10 mL with mobile phase.

Standard stock solution of folic acid (100 µg/mL)

An accurately weighed quantity of about 10 mg folic acid was mixed with 0.5 mL 2.86% solution of sodium carbonate and diluted it upto 100 ml with mobile phase [5].

Working standard solution of folic acid

Similarly working standard solution of folic acid in the concentration range of 10–50 μg/mL was prepared by diluting an aliquot portion of about 1–5 mL folic acid stock solution (100 μg/mL) to 10 mL with mobile phase.

Sample preparation for Tablet analysis

An accurately weight 20 tablets were crushed to obtained fine powder.

Sample preparation of ferrous ascorbate

A quantity of tablet powder equivalent to 10 mg of ferrous ascorbate (11.31 mg) was dissolved in 100 mL phosphate buffer pH 7, sonicated for 30 minutes. The solution was filtered. The aliquot portion of filtrate (2.0 mL) was diluted to 10.0 mL with mobile phase.

Sample preparation of folic acid

A quantity of tablet powder equivalent to 5 mg of folic acid (2.72 g) was mixed with 0.5 mL of 2.86% of sodium carbonate solution and then dissolved in 100 mL mobile phase. The mixture was sonicated for 30 minutes. The solution was filtered. An aliquot portion of filtrate (4.0 mL) was further diluted to 10.0 mL with mobile phase.

Chromatographic condition ferrous ascorbate and folic acid

Chromatographic condition ferrous ascorbate

The optimized chromatographic conditions for ferrous ascorbate estimation were Gemini-NX- C18 (250× 4.60, 5µm) as a stationary phase, mixed phosphate buffer (pH 7): methanol (98:2, v/v) as mobile phase, flow rate 1 mL/min with detection wavelength 264 nm. The chromatogram for ferrous ascorbate is as shown in (Fig. 2).

The optimized chromatographic conditions for folic acid estimation were Gemini-NX- C18 (250× 4.60, 5µm) as a stationary phase, phosphate buffer: methanol (88:12, v/v) as mobile phase, flow rate 1 mL/min with detection wavelength 280 nm. The chromatogram for ferrous ascorbate is as shown in (Fig. 3).

Results and discussion

Ferrous ascorbate and folic acid combination was used in management of anemia [6]. In literature various HPLC methods for estimation of folic acid was reported [7-9]. But no method was reported for the estimation of ferrous ascorbate and simultaneous estimation of ferrous ascorbate and folic acid combination. A numbers of trials were carried out for simultaneous estimation of ferrous ascorbate and folic acid. Initially, therefore attempt was done for simultaneous estimation of both drugs by reversed-phase, HPLC separation was tried using phosphate buffer pH 3 and methanol (98:2) as the mobile phase, in which ferrous ascorbate eluted at 2.4 min which is nearer to dead time of column and folic acid was not eluted, the resolution and numbers of theroetical plates was also poor. The organic content of mobile phase was also investigated in different ratio to optimize the separation of ferrous ascorbate and folic acid simultaneously. But no mobile phase was found suitable for analysis. Therefore, attempt was made to estimate ferrous ascorbate and folic acid in bulk and in pharmaceutical formulation separately using different chromatographic condition which was optimized separately for ferrous ascorbate and folic acid (Table I). Therefore attempt was made to estimate ferrous ascorbate and folic acid by using two different mobile phases and sample preparation method. The mobile phase phosphate buffer pH 7- Methanol (98:2) was selected for ferrous ascorbate estimation. The buffer-methanol (88:12, v/v) was selected for folic acid estimation as per Indian pharmacopoeia [5]. The constant flow rate 1mL/min was maintained for both analysis. Detection wavelength 264 nm for ferrous ascorbate and 280 nm were selected for folic acid. Retention time was found to be 2.7 min for ferrous ascorbate and 6.8 min for and folic acid.

VALIDATION PARAMETERS [10, 11].

System suitability

System suitability parameters, such as number of theoretical plates, HETP, and peak tailing, were determined. The results obtained are shown in Table I the number of theoretical plates for ferrous ascorbate and folic acid was found 3219 and 2535 respectively with separate mobile phase.

Linearity and range

Ferrous ascorbate and folic acid follows a linearity of response between 10–50 µg/mL. Linearity study was performed separately by analyzing five different concentrations of ferrous ascorbate and folic acid. The Solution were diluted appropriately with mobile phase to obtain concentrations in the range of 10-50 μg/ml. All measurements were repeated three times for each concentration.

Y = 23397x + 28729 is the equation for ferrous ascorbate with R² value 0.999

Y = 188287x +150052 is the equation for folic acid with R² value 0.999

Precision

The repeatability was studied by multiple injections of a homogenous sample of 20 µg/mL of ferrous ascorbate and folic acid separately. The results of repeatability study of ferrous ascorbate and folic acid are as follows.

The result of intra-day precision study, % RSD for ferrous ascorbate was found 1.31 and for folic acid was found to be 1.61.

The result of Inter-day precision study, the % RSD for ferrous ascorbate was found 1.60 and for folic acid was found to be 1.54 respectively.

The results of the precision study indicate that the method is reliable and reproducible, with a relative standard deviation less than 2.0%.

Accuracy

Recovery study was performed to validate the accuracy of the developed method. Recovery study was performed at 80%, 100% and 120 % level. The known amount of standard ferrous ascorbate was added to preanalyzed sample and subjected for HPLC analysis.

Procedure for ferrous ascorbate

Sample solution

A quantity of tablet powder equivalent to 10 mg ferrous ascorbate (11.35 mg) was dissolved in 100 mL phosphate buffer, sonicated for 30 minutes. The solution was filtered.

Standard stock solution of ferrous ascorbate (100 µg/mL)

An accurately weighed quantity of about 10 mg ferrous ascorbate was dissolved in 100.0 mL of phosphate buffer pH 7.

Working solution of ferrous ascorbate

Three sets containing 10.0 ml volumetric flask were taken. To each set, 2 ml of sample solution was added. To the first set (80%) of three volumetric flasks, a quantity of about 1.6 mL of standard stock solution of ferrous ascorbate was added. To the second set (100%) of three volumetric flasks, a quantity of about 2.0 mL of standard stock solution ferrous ascorbate was added. To the third set (120%) of three volumetric flasks, a quantity of about 2.4 mL of standard stock solution of ferrous ascorbate was added. The volume was made up to the mark with mobile phase in all flasks.

The sample and standard solution were analyzed as per optimized chromatographic conditions for ferrous ascorbate.

Procedure for folic acid

Sample solution of folic acid

A quantity of tablet powder equivalent to 10 mg folic acid (5360 mg) was dissolved in 1mL of 2.86% of solution of ferrous sodium carbonate and volume was made with 100 mL buffer, sonicated for 30 minutes. The solution was filtered through whatman filter paper No.41.

Standard stock solution of folic acid (100 µg/mL)

Weighed accurately about 10 mg folic acid mixed with 0.5 ml 2.86% solution of sodium carbonate solution and diluted with 100 ml of mobile phase.

Working solution

Three sets containing 10.0 ml volumetric flask were taken. To each set, 2 ml of sample solution was added. To the first set (80%) of three volumetric flasks, a quantity of about 1.6 mL of standard stock solution of folic acid was added. To the second set (100 %) of three volumetric flasks, a quantity of about 2.0 mL of standard stock solution folic acid was added. To the third set (120 %) of three volumetric flasks, a quantity of about 2.4 mL of standard stock solution of folic acid was added. Then, the volume was made up to the mark with mobile phase in all flasks.

The sample and standard solution were analyzed as per optimized chromatographic conditions given for folic acid. These results of accuracy are as summarized in Table no: II

Determination of the limit of detection and quantitation (LOD and LOQ)

The limit of detection and limit of quantitation is determined by using following formulae and result is given below.

,

Where

SD = Standard deviation of response, S = Slope of linearity curve equation

The result of LOD (μg/ml) of ferrous ascorbate and folic acid was found to be 0.259 and 0.213 respectively.

The result of LOQ (μg/ml) of ferrous ascorbate and folic acid was found to be 0.784 and 0.645 respectively.

Robustness

As per ICH norms, small but deliberate variations, by altering the flow rate of the mobile phase, were made to check the method’s capacity to remain unaffected (method stability). The change was made in flow rate by altering flow rate 0.9 mL /min and 1.1 mL/min Results of analysis are summarized in Table no: III

Tablet analysis

Contents of ferrous ascorbate and folic acid found in the tablets by the proposed method are shown in Table VI the low RSD values indicate that the method is precise and accurate.

Specificity study

Specificity study was conducted for ferrous ascorbate and folic acid in presence of excipients present in tablet.

Preparation of physical mixture for specificity study

The average weight of 20 tablets was found to be 804 mg. Therefore, as per labelled claim, tablet formulation excipients in tablet were 75 mg.

Mixture of drug and excipients {728 µg/mL ferrous ascorbate, 1.5 µg/mL folic acid and 75 µg/mL excipients in equal parts (like lactose monohydrate, microcrystalline cellulose, magnesium stearate, starch pregelatinised etc.)} was prepared. The solution was analyzed by optimized chromatographic conditions given for ferrous ascorbate and folic acid.

The peak purity value of ferrous ascorbate and folic acid was found to be more than the input threshold value. It shows that the peak of ferrous ascorbate and folic acid was free from any impurity or co-elution in presence excipients present in tablet.

The VU-spectrum of ferrous ascorbate and folic acid is as shown in Fig. 4 and Fig. 5

Also the peak purity spectrum of ferrous ascorbate and folic acid was shown in Fig. 6 and Fig. 7

Conclusion

RP-HPLC method for estimation ferrous ascorbate and folic acid in bulk and in pharmaceutical formulation was successfully developed as per ICH guidelines. The method was found accurate, precise and sensetive hence it can be used for routine analysis by any analyst.