Visible Spectrophotometric Determination of Ferrum Phosphoricum in Homeopathic Formulations

 

Syed Azhar Nizami*, Ashok Kumar D and SC Marihal

Department of Pharmaceutical Chemistry, Dr.HLT College of Pharmacy, Channapatna, Karnataka- 571502.

 

ABSTRACT:

The present study deals with the determination of Ferrum Phophoricum and Ferrum metal in some Homeopathic formulations. The method is based on Fe3+ reduce to Fe2+ with hydroxyl ammonium chloride which react with the 1-10 phenanthroline in the pH range 3-5 to form an orange-red colour complex which shows the maximum absorbance at 518 nm. Beer’s law is obeyed in the concentration range of 0.5-3mg/ml. Results of the analysis were validated statistically and by recovery studies. The Percentage label claim and Percentage recoveries estimated were close to 100% with low value of standard deviation and Percentage coefficient of variation.

 

KEYWORDS:  Ferrum Phosohoricum, Spectrophotometry, Fe2+ 1-10 phenanthroline complex.

 

INTRODUCTION:

Iron deficiency anemia is the most common type of metabolic disorder. This deficiency is due to the negative iron balance occur either due to blood loss, or when tissue stores iron are exhausted1. Hence an adequate supply of iron to the bone marrow is necessary for the synthesis of hemoglobin. Allen’s Biochemic tissue salts and Alferon syrup are the two important Homeopathic preparations containing Ferrum phosphoricum2 and Ferrum metal used therapeutically as a source of iron indicated for iron deficiency anemia, anemia due to chronic blood loss, during pregnancy and lactation. Also in anorexia, vertigo, general debility, weakness and wearness. Literature survey revealed that there is no simple spectrophotometric method available for determination of Ferrum phosphoricum and Ferrum metal in Homeopathic formulations. Therefore in the present study an attempt has been made to develop a simple method to determine the Ferrum phosphoricum and Ferrum metal  in Homeopathic formulations using 1-10 phenanthroline and estimation was done on the principle of UV-Visible spectrophotometry.

MATERIALS AND METHODS

All the absorbance measurements were made on Shimadzu-1700 series UV/Vis spectrophotometer with 1cm matched quartz cell. Whatman filter paper No 42 was used to filter the solutions. The Analytical Grade reagents of Ferric ammonium sulphate, Sodium acetate solution (2 M aqueous solution), Hydroxyl ammonium chloride (10% aqueous solution), 1-10 Phenanthroline (0.25% aqueous solution) (SD fine chemicals, Mumbai) were used. The Double distilled water was used to prepare the reagents and solutions.

 

Preparation of standard iron solution:

The working standard solution was prepared by dissolving ferric ammonium sulphate (0.1mg/ml) in distilled water.

 

In to a series of calibrated 50ml volumetric flask, appropriate aliquots (0.5-3 mg/ml) of standard iron solution were transferred using a microburette and total volume is adjusted to 10ml by adding water. To each flask, 5ml of hydroxyl ammonium chloride was added and stand for 5 minutes, then pH is adjusted to3.5 with the help of 2M sodium acetate and 4ml of 1-10 phenenthroline were added and volume is made up to the mark with distilled water. The absorbance for so formed orange red colored was measured at 518nm within 30 minutes of reaction against the reagent blank prepared in similar manner.

 

Preparation of Test solution:

For the determination of iron content, two Homeopathic formulations namely Allen’s Biochemic tissue salts contains Ferrum phosphoricum(120mg/tab) and Alferon syrup contains Ferrum phosphoricum(200mg/100ml) and  Ferrum metal (100mg/100ml) were  procured from local market. The products selected based on the concentration of iron  present in the formulation and estimated for their percentage of label claim against standard iron solution (ferric ammonium sulphate). Suitable amount of each sample were incinerated in furnace. The ash was then taken with 50ml of 6N HCL and boiled for 30minutes, filtered and volume was made up to 250ml with distilled water.3, 4 From this stock solution aliquots were pipetted out in to 50ml volumetric flask containing 0.5 to 3 mg of iron and same procedure was followed as mentioned in standard preparation.

 

RESULT AND DISCUSSION:

In a replicate analysis (n=5) of two commercial brands by the proposed method, the content of Ferrum phosphoricum in Allen’s Biochemic tissue salts were found to be 199.98mg and Ferrum phosphoricum and Ferrum metal in Alferon syrup were found to be 119.97mg and 100.02 mg respectively. The results obtained by the proposed method were closed to the label claim of Ferrum phosphoricum (120mg/tab) in Allen’s Biochemic tissue salts Ferrum phosphoricum(200mg/100ml) and  Ferrum metal (100mg/100ml) in Alferon syrup  respectively. It indicates that the method is precise and accurate. To confirm the accuracy and precision of the proposed method recovery studies were carried out by standard addition technique.5, 6 A fixed amount of drug was taken from the formulations and standard iron II at three different concentrations was added and each concentration was repeated three times and percentage recovery of the added standard was calculated from the below equation.

 

%Recovery = [CV – CU]/Ca X 100

 

Where CV is the concentration of the analyte measured, CU is concentration of the analyte present in the formulation; Ca is concentration of analyte (pure drug) added to formulation. Results of recovery studies reveal that the method was unaffected by the various excipients present in the formulations. The accuracy of the method is evident from the percentage error lying between 0.044 and 0.061. The RSD values that are less than 3% for three different levels of studies indicate the higher reproducibility of the method in Table 1.

 

Absorption spectra:

Figure 1 shows the absorption spectra of the reaction product of Fe2+and 1-10 phenanthroline against reagent blank. The orange red color formed exhibits absorption maximum at 518 nm, and the respective blanks display only slight absorption at this wavelength. Further neither Fe2+nor 1-10 phenanthroline solution absorbs at this wavelength. Hence, the use of measured volumes of the reagent solution and measurement against corresponding reagent blanks gives linear calibration graph for the reaction product in Figure 2.

 

 

Figure 1. Absorption Spectra of (Iron II) at 518 nm

 

Optimum Hydroxyl Ammonium Chloride and 1-10 Phenanthroline Concentrations:

 

When a study on the effect of 10% Hydroxyl ammonium chloride concentration on the color development was performed, it was observed that in both cases the absorbance increased with increase in the volume of 10% Hydroxyl ammonium chloride solution. It reached maximum when 5ml of the reagent solution was added to 1.0mg of standard iron solution and 4ml of 0.25% solution of 1-10 Phenanthroline in a total volume of 50ml. These results indicate that a maximum absorbance is obtained when the final hydroxyl ammonium concentration is 2%. Larger volumes of hydroxyl ammonium chloride up to 12% had no effect on the sensitivity of the reaction.

 

Similar observation were made when varying volumes of 0.25% of 1-10 Phenanthroline solution were added to fixed amount of Fe2+ solution 1.0mg and 5ml of hydroxyl ammonium chloride (10%). It was diluted to 50ml after full color development. The result of this study reveals that the concentration of Hydroxyl ammonium chloride and 1-10 Phenanthroline reagents are not critical. However, 5ml of Hydroxyl ammonium chloride and 4ml of 1-10 Phenanthroline solution in total volume of 50ml were used to ensure adequate reagent concentration for higher concentration of standard Fe2+solution.

 

Table 1: Accuracy and Precision Data by the Proposed Method

 

Accuracy

Precision

Iron content Present in Formulations (mg)

Standard Iron added (mg)

Iron Recovered* (mg)

%

Recovery

± SD

RSD

Standard Error

A

Ferrum phosphoricum 120

10

130.02

100.3

0.354

0.353

0.034

 

Ferrum phosphoricum 200mg

10

219.97

99.97

0.17

0.156

0.081

B

Ferrum metal 100mg

10

110.03

100.01

0.23

0.342

0.066

 

 

 

 

 

 

 

* Average of three determinations.  A = Allen’s Biochemic tissue salts (Allen’s Laboratories Kolkata), B = Alferon syrup (Allen’s Laboratories Kolkata)

 

Table 2. Optical characteristic, Statistical Regression Data of Reaction Product

Parameters

Values

Absorption maxima (nm).

Beers law limit (mg/ml)

Molar absorptivity L/Mol/cm

Regression equation (Y=a+bx)

Intercept (a)

Slope (b)

Correlation coefficient (r)

518

0.5 – 3

1.43X 104

 

0.0068

0.0022

R2= 0.999

 

 

Effect of reaction time and stability of colored species

The reaction is slow at 32±2o C but the absorbance increases with time and reaches a maximum in 30 min .The developed color remains stable for 2 hrs.

 

Analytical appraisal

Under the experimental conditions described, Beer’s law is obeyed over the concentration ranges 0.5 to 3 mg/ml.  The molar absorptivity at 518 nm was 1.43 X 104  L/mol cm. The limits of detection (LOD) and limits of quantification (LOQ) were found to be 0.160 ng/ml and 0.562 ng/ml. The optical characteristic and regression data are presented in Table 2

 

CONCLUSION:

The above-evaluated parameters in the proposed method revealed that the experimental study signifies simple, accurate, fast, precise and reproducible. It can be used for routine analysis of both the drugs in commercially available Homeopathic formulations.

 

 

ACKNOWLEDGEMENT:

The Authors grateful to Prof. T.V. Narayana, Chairman and Prof. Ravada Ramesh, Principal and Staff of Dr H L T College of pharmacy, Kengal Channapatna, Karnataka for providing the facilities to perform this research work.

 

REFERENCES:

1.        Block J.H. et al, Inorganic Medicinal and Pharmaceutical Chemistry, Varghese Publishing House, Mumbai, 1986; pp. 218-220

2.        Boericke and Dewey, The Twelve Tissue Remedies of Shussler, Sixth edition, B. Jain Publishers Pvt. Ltd.; pp. 61-62.

3.        Paun J.S.Subha and De S. Spectrophotometric determination of Lauha Bhasma and Lauha Bhasma containing preprations. Indian Drugs 2008; 45(2): 141-144.

4.        Subha, Standardization of analytical aspects of Harida Khanda – An Ayurvedic Formulation, M, Pharm. (Ayu) Thesis, Institute of Ayurvedic Pharmaceutical Sciences, Gujrat Ayurvedic University, 2004-05.

5.        Daiel E. Harris. Qualitative chemical analysis.6th edition. W.H. Freemen and company. New York. p.51.

6.        J.M,Green “A Practical guide to analytical method validation” Anal.Chem. 1996; p. 68.

 

Received on 25.01.2010

Accepted on 24.03.2010        

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 2(3): May-June 2010, 217-219