Development and Validation of
Analytical methods for Simultaneous Estimation of Ferrous Ascorbate
and Folic Acid in their combined dosage form
Rucha K. Patel*, Nilam M. Patel, Samir K. Shah
Sardar Patel
College of Pharmacy, Vidhyanagar-Vadtal Road, Bakrol, Anand, Gujarat, India
*Corresponding
Author E-mail: ruchamiki1104@gmail.com,
nilampatel2011@gmail.com
ABSTRACT:
A simple, accurate and precise spectroscopic method was
developed for simultaneous estimation of Ferrous Ascorbate
and Folic Acid in their combined dosage form using simultaneous equation and
first order derivative method. In Simultaneous Equation method, Ferrous Ascorbate and Folic Acid were quantified using their absorptivity values of at selected wavelengths, i.e., 265
nm and 344 nm respectively. In First order Derivative method, Ferrous Ascorbate showed zero crossing point at 265.78 nm while
Folic Acid showed zero crossing point at 280.34 nm. The dA/dλ was measured at 280.34 nm for Ferrous Ascorbate and 265.78 nm for Folic Acid and calibration
curves were plotted as dA/dλ
versus concentration, respectively. The method was found to be linear in the
range of 10-80 μg/ml for Ferrous Ascorbate and 1-8 μg/ml for
Folic Acid. The accuracy of the proposed methods was evaluated by recovery
studies and good recovery results were obtained. The method was successfully
applied for simultaneous determination of Ferrous Ascorbate
and Folic Acid in combined dosage form.
KEYWORDS: Ferrous Ascorbate,
Folic Acid, Vierodt’s method, First Order Derivative
Method.
INTRODUCTION:
Anemia is defined as a below normal plasma hemoglobin concentration
resulting from a decreased number of circulating red blood cells or an abnormally
low total hemoglobin content per unit of blood volume.(1) Red blood
cells can be lost through bleeding, which can occur slowly over a long period of time, and can often go undetected.
(2) Ferrous Ascorbate is synthetic molecule of ascorbic acid and iron.
(3) Ferrous Ascorbate and Folic Acid
combination was approved by FDA in year 2011. These both drugs are vital
vitamins that help pregnant women to attain a good health during the entire
pregnancy.
A pregnant woman is likely to become anemic during the entire
stage of conception and taking Ferrous Ascorbate and
Folic Acid regularly or on a daily basis
provides a pregnant woman the sufficient amount of Iron and Folic Acid
(vit.B9).(4)
A. Ferrous Ascorbate: (5-6)
Figure 1: Chemical Structure of
Ferrous Ascorbate
Ferrous Ascorbate is an
anti-anemic agent, and synthetic molecule of Ascorbic acid and Iron. Chemical
name of Ferrous Ascorbate is (2R)-2-[(1S)-1,
2-dihydroxy ethyl]-4, 5-dihydroxyfuran-3-one, iron (2+) (5).
Ascorbic acid converts the ferric form to ferrous form thus making it
absorbable from duodenum and upper jejunum resulting in considerable
enhancement of the absorption of iron. It has been demonstrated that Ferrous Ascorbate is less easily oxidized than Ferrous in ferrous sulphate. (6)
B. Folic Acid: (7-8)
Figure 2: Chemical structure of Folic Acid
Folic Acid is also known as vitamin B9. It is
also known as pteroylglutamic
acid. Chemical name of Folic Acid is (2S)-2-[[4-[(2-amio 4-oxo-1,
4-dihydropteridin-6-yl) methyl amino]benzonyl]amino] pentaidioc acid (7).
Folic Acid, when reduced by enzymes Folate reductase and Di-hydro folate reductase, forms Di-hydro Folic Acid and Tetra-hydro Folic
Acid respectively. (8) The review of literature regarding
quantitative analysis of Ferrous Ascorbate and Folic
Acid revealed that no attempt was made to develop analytical methods for
simultaneous estimation of Ferrous Ascorbate and
Folic Acid. Some spectrometric methods and chromatographic methods have been
reported for the estimation of the individual drugs.
MATERIALS AND
METHODS:
APPARATUS
AND
INSTRUMENT:
A double beam UV/Visible spectrophotometer (Systronics 2203)
with 1 cm quartz cells was
used to measure absorbance
of all the solutions.
Spectra were automatically obtained by Smart
D.B.S software.
An analytical balance (Swisser) was used for weighing the samples.
Volumetric glasswares were used
(Borosillicate)
All instruments
and glass wares
were calibrated.
REAGENTS AND
MATERIAL
Ferrous Ascorbate
(Gift sample from Pride
drug & Pharma Ltd, Gandhinagar.)
Folic Acid (Gift sample from
Redox Industries Pvt. Ltd, Ahmedabad.)
Distilled Water, 0.1 N HCl was
used for development purpose.
PREPARATION OF STANDARD SOLUTIONS:
Standard stock solution
of Ferrous Ascorbate:
Accurately weighed quantity of
Ferrous Ascorbate (100 mg) was transferred to 100 ml
volumetric flask. Small quantity of drug was dissolved in distilled water and sonicated for 10 min then diluted up to mark with Distilled
water to give a stock solution (1000 μg/ml).
Working standard of
Ferrous Ascorbate:
An aliquot (1 ml) from standard
stock solution (1000 μg/ml) was transferred to a
10 ml volumetric flask and diluted up to mark with Distilled water to produce a
working standard solution (100 µg/ml) of Ferrous Ascorbate.
Standard Stock Solution of
Folic Acid:
Accurately
weighed quantity of Folic Acid (10 mg) was transferred into 100 ml volumetric
flask. Small quantity of drug was dissolved in Distilled water and sonicated for 10 min. Diluted up to mark with Distilled
water to give a stock solution (100 μg/ml).
Working standard of Folic
Acid:
An aliquot
(0.1 ml) from standard stock solution (100 μg/ml)
was transferred to a 10 ml volumetric flask and diluted to the mark with
Distilled water to obtain a working standard solution (1 µg/ml) of Folic Acid.
VALIDATION OF PROPOSED METHOD: (9)
Parameters to be considered for
the validation of methods are:
1) LINEARITY
Procedure:
The linearity response was determined by analyzing 5 independent levels of calibration curve in the range of 10-80 μg/ml and 1-8 μg/ml for Ferrous Ascorbate
and
Folic Acid respectively (n=5).
Calibration curves for Ferrous Ascorbate and Folic Acid for both Methods:
From the standard stock solution of
Ferrous Ascorbate and Folic Acid (1000 µg/ml and 100
µg/ml) 1, 2, 4, 6, 8 ml of solution was pipetted out
in series of 10 ml volumetric flasks. The volume was made up to 100 ml with
Distilled water to obtain concentration of 10, 20, 40, 60 and 80 µg/ml of
Ferrous Ascorbate
and 1, 2, 4, 6, 8 µg/ml Folic Acid. Absorbance of above solution was
measured at 265 nm and 344 nm for Ferrous Ascorbate
and Folic Acid of each and 280.34 nm and 265.78 nm ZCP of Folic Acid and
Ferrous Ascorbate respectively. The Calibration curve
were constructed by plotting absorbance Vs concentrations and for linearity,
regression equation were calculated. Each response was average of five
determinations.
2) PRECISION:
I. Intraday
Precision:
Intraday precision of the method was determined by
measuring the responses 3 times on the same day at interval of 1 hr for 3
different concentrations of Ferrous Ascorbate (10, 40
and 80 µg/ml) and Folic Acid (1, 4 and 8 µg/ml) each. The results were reported
in terms of relative standard deviation.
II. Interday
Precision:
Inter-day precisions
of the method was determined by measuring the responses 3 times on 3
consecutive days for 3 different concentrations of Ferrous Ascorbate
(10, 40 and 80 µg/ml) and Folic Acid (1,
4 and 8 µg/ml) each. The results were reported in terms of relative standard
deviation.
3) ACCURACY:
It was determined by
calculating the recovery of Ferrous Ascorbate and
Folic Acid from formulation by standard addition method.
To a fixed amount of
test sample 80 %, 100 % and 120 % amount of standard was added and the amount
of standard added was calculated using regression equation.
Known amount of
standard solutions of Ferrous Ascorbate and Folic
Acid were added to a pre-quantified sample solution of Ferrous Ascorbate and Folic Acid.
Each solution was
injected in triplicate and the percentage recovery was calculated by measuring
the responses and fitting these values into the regression equations of the
respective calibration curves.
4) Lod (Limit of Detection) and Loq (Limit of Quantification):
The Limit of detection and Limit of Quantification of the developed method was assessed by
analyzing five replicates of standard solutions containing concentrations 10
μg/ml for Ferrous
Ascorbate and 1 μg/ml for Folic Acid. The LOD and LOQ may be calculated
as:
Where,
SD =
five replicates of absorbance
Slope = the mean
slope of the 5 calibration
curves
5) ROBUSTNESS:
This parameter was
performed by taking Ferrous Ascorbate (40 µg/ml) and
Folic Acid (4 µg/ml) at different wavelength by measuring the corresponding
responses 3 times. %RSD for area was calculated which should be less than 2%.
ESTIMATION IN MARKED FORMULATION:
Twenty tablets were weighed and
powdered. An accurately weighed powder equivalent
to about 100 mg of Ferrous Ascorbate and 1.5 mg of
Folic Acid was transferred to 100 ml volumetric flask and the volume was made
up to the mark using water as a solvent (1000 µg/ml of Ferrous Ascorbate and 150 µg/ml of Folic Acid). The solution was sonicated for 20min. The solution was filtered through Whattman filter paper no.42. First few ml of filtrate were
discarded. 3 ml of the solution from the
above filtrate was diluted to 100 ml with water to produce 30 µg/ml of
Ferrous Ascorbate and
0.45 µg/ml of Folic Acid).The absorbance of resulting solution was measured at selected wavelength.
RESULTS AND DISCUSSION:
1)
Selection of Wavelength and
Method Development
For
Determination of
Ferrous Ascorbate And Folic Acid by
Simultaneous Equation Method:(10)
The standard solution of Ferrous Ascorbate and Folic Acid
were scanned separately between 200-400nm,
Absorbance maxima were obtained at 265 nm and at 344 nm for Ferrous Ascorbate and Folic Acid respectively.
Figure 3:
Overlay spectra of standard Ferrous Ascorbate (10 μg/ml) and Folic acid
(1 μg/ml)
VALIDATION PARAMETERS:
1. Linearity:
The linearity range
for Ferrous Ascorbate
and Folic Acid was found to be in the range of 10 – 80 μg/ml
and 1-8 μg/ml for both drugs. Correlation
co-efficient, regression line equation of Ferrous Ascorbate and Folic Acid at 265 nm and 344 nm are showed in table no.
1and 2.
Figure 4: Linearity
Spectra of Ferrous Ascorbate (10μg/ml) and Folic
Acid (1 μg/ml)
Table 1: Correlation
co-efficient for calibration curve for Ferrous Ascorbate
and Folic Acid, respectively.
*(n=5)
|
Sr No. |
Wavelength |
Ferrous Ascorbate |
Folic Acid |
|
1 |
265 |
0.998 |
0.994 |
|
2 |
344 |
0.998 |
0.995 |
Table 2:
Regression line equations for Ferrous Ascorbate and Folic Acid
|
Sr No. |
Wavelength |
Ferrous Ascorbate |
Folic Acid |
|
1 |
265 |
y = 0.028x – 0.108 |
y = 0.039x + 0.005 |
|
2 |
344 |
y = 0.001x – 0.108 |
y = 0.039x + 0.006 |
2. Precision:
I. Intraday
precision:
The data for intraday precision for Ferrous Ascorbate
at 265 nm and Folic Acid at 344nm is shown in table 3.The %RSD value was found to be less than ± 2.0 indicated that the method
is precise.
II. Interday
Precision:
The data for interday precision
for Ferrous Ascorbate
at 265 nm and Folic Acid 344 nm is shown in table 3. The %RSD value was found to be less than ± 2.0 indicated that the method is precise.
Table 3: Intraday and Interday precision data
for
estimation of Ferrous Ascorbate and Folic
Acid *(n=3)
|
Drug |
Conc.. (μg/ml) |
Conditions |
||
|
Intra day |
Inter day |
|||
|
%RSD |
||||
|
Ferrous Ascorbate |
10 |
0.92 |
0.89 |
|
|
40 |
0.15 |
0.15 |
||
|
|
80 |
0.17 |
0.12 |
|
|
Folic Acid |
1 |
1.05 |
1.05 |
|
|
4 |
0.61 |
1.21 |
||
|
|
8 |
1.78 |
0.47 |
|
3. Accuracy:
Accuracy of the method was confirmed by recovery study
from marketed formulation at three level of standard addition. Data are shown
in Table 4 and 5. The value of %RSD within the limit indicated that the method is accurate and
percentage recovery.
Table 4: Recovery data of Ferrous Ascorbate *(n=3)
|
Amt of Ferrous Ascorbate(µg/ml) |
% of std added |
Amt of std.added (µg/ml) |
%Recovery (mean ±
SD) |
%RSD |
|
30 |
80 |
54 |
100.65 ± 0.3017 |
0.299 |
|
100 |
60 |
106.36 ± 0.137 |
0.129 |
|
|
120 |
66 |
103.80 ± 0.170 |
0.164 |
Table 5: Recovery data
of Folic Acid*(n=3)
|
Amt of Folic Acid (µg/ml) |
% of std added |
Amt added (µg/ml) |
% Recovery (mean ± SD) |
%RSD |
|
0.45 |
80 |
0.81 |
100.95 ± 0.194 |
0.192 |
|
100 |
0.9 |
101.16 ± 0.145 |
0.144 |
|
|
120 |
0.99 |
100.86 ± 0.152 |
0.151 |
4. Limit of
detection and quantitation:
The LOD for Ferrous Ascorbate
and Folic Acid was
found to be 0.216
µg/ml and 0.069 µg/ml, respectively. The LOQ for Ferrous Ascorbate and Folic Acid was
found to be 0.657 µg/ml and 0.209 µg/ml, respectively.
5. Robustness
and
Ruggedness:
The obtained Robustness results are presented
in table 6
The %RSD value was found to be less than ± 2.0 indicated that the method
is precise.
Table 6: Robustness data of
Ferrous Ascorbate and Folic Acid *(n=3)
|
Condition |
Conc. (µg/ml) |
Different Wavelength |
|||
|
Ferrous Ascorbate |
264.8 (mean ± SD) |
%RSD |
265.2 (mean ± SD) |
%RSD |
|
|
40 |
0.989 ± 0.0058 |
0.592 |
1.004 ± 0.002 |
0.199 |
|
|
343.8 (mean ± SD) |
%RSD |
344.2 (mean ± SD) |
%RSD |
||
|
Folic Acid |
4 |
0.149 ± 0.0011 |
0.771 |
0.165 ± 0.005 |
0.349 |
Analysis of Ferrous Ascorbate and Folic Acid in Combined Dosage form:
Marketed preparation was analyzed by the proposed method.
The amount of Ferrous Ascorbate and Folic Acid was found to be 103.33% and
101.33 % of the labeled amount, respectively. The % assay values are tabulated in Table
7.
Table
7: Analysis data of marketed formulation *(n=3)
|
Drugs |
Result of formulation analysis |
||
|
Amount of drug |
% Assay* ± s.d. |
% RSD |
|
|
Ferrous Ascorbate |
30 µg/ml |
103.33±0.3 |
0.29 |
|
Folic
Acid |
0.45
µg/ml |
101.33±0.80 |
0.792 |
2)
Selection of Wavelength and
Method Development
For
Determination of
Ferrous Ascorbate and Folic Acid by First Order
Derivative Method:(11)
To determine wavelength for measurement, standard spectra of Ferrous Ascorbate
and Folic Acid were scanned between 200 - 400 nm against Distilled water. Spectra
shows of absorbance at 280.34 nm (ZCP of Folic Acid) as wavelengths for
quantification of Ferrous Ascorbate, where no interference due to Folic Acid
was observed similarly absorbance at 265.78 nm (ZCP of Ferrous Ascorbate) is selected for
quantification of Folic Acid, where Ferrous
Ascorbate did not interfere with the
estimation of Folic Acid.
Figure 7: Derivative spectra of standard Ferrous Ascorbate
(10μg/ml) and standard Folic Acid
(1 μg/ml)
VALIDATION PARAMETERS
1. Linearity:
The linearity range
for Ferrous Ascorbate
and Folic Acid was found to be in the range of 10 – 80 μg/ml and 1-8 μg/ml for both drugs. Correlation co-efficient and
regression line equation of Ferrous Ascorbate and
Folic Acid at 280.34 nm and 265.78 nm
are presented in table 8 and 9 respectively.
Table 8 : Correlation co-efficient for calibration curve
for Ferrous Ascorbate and Folic Acid, respectively. *(n=5)
|
Sr No. |
Drug |
Wavelength |
Observation |
|
1 |
Ferrous Ascorbate |
280.34 |
0.997 |
|
2 |
Folic Acid |
265.78 |
0.998 |
Table 9: Regression line
equations for Ferrous Ascorbate
and Folic Acid
|
Sr No. |
Drug |
Wavelength |
Ferrous Ascorbate |
|
1 |
Ferrous Ascorbate |
280.34 |
y = -0.001x +0.003 |
|
2 |
Folic Acid |
265.78 |
y = 0.001x+0.00009 |
2. Precision:
I. Intraday
precision:
The data for intraday precision for Ferrous Ascorbate
and Folic Acid at is shown in table 10. The %RSD value was found to be less than ± 2.0 indicated that the method is precise
II. Interday
Precision:
The data for interday precision
for Ferrous Ascorbate
and Folic Acid is shown in table 10. The %RSD value was found to be less than ± 2.0 indicated that the method is precise.
2.
Accuracy:
Accuracy of the
method was confirmed by recovery study from marketed formulation at three level
of standard addition. Data are shown in table 11 and 12. The value of %RSD within the limit
indicated that the method is accurate and
percentage recovery.
Table 10: Intraday and Interday precision data for estimation of Ferrous Ascorbate and Folic
Acid *(n=3)
|
Drug |
Conc. (μg/ml) |
Conditions |
||
|
Intra day |
Inter day |
|||
|
%RSD |
||||
|
Ferrous Ascorbate |
10 |
0.97 |
1.23 |
|
|
40 |
1.77 |
1.15 |
||
|
|
80 |
0.60 |
0.62 |
|
|
Folic Acid |
1 |
0.50 |
1.92 |
|
|
4 |
0.76 |
1.97 |
||
|
|
8 |
0.76 |
0.62 |
|
Table 11:
Accuracy for Ferrous Ascorbate *(n=3)
|
Amt of Ferrous Ascorbate (µg/ml) |
% of std added |
Amt of std.added(µg/ml) |
% Recovery
(mean* ± SD) |
%RSD |
|
80 |
54 |
102.6± 0.264 |
0.257 |
|
|
30 |
100 |
60 |
103.26 ± 0.208 |
0.201 |
|
120 |
66 |
103.24 ± 0.237 |
0.230 |
Table 12: Accuracy for Folic Acid *(n=3)
|
Amt of Folic Acid
(µg/ml) |
% of std Folic Acid
added |
Amt of std. Added (µg/ml) |
% Recovery (mean* ±
SD) |
%RSD |
|
80 |
0.81 |
100.23 ± 0.251 |
0.25 |
|
|
0.45 |
100 |
0.9 |
101.23 ± 0.111 |
0.110 |
|
120 |
0.99 |
101.13 ± 0.149 |
0.148 |
4. Limit of Detection and Quantitation:
The LOD for Ferrous Ascorbate and Folic Acid was found
to be 0.0104 µg/ml and 0.00164
µg/ml, The LOQ for Ferrous Ascorbate and
Folic Acid was found to be 0.031 µg/ml and 0.0049 µg/ml, respectively.
5. Robustness
and
Ruggedness:
The obtained Robustness results are presented
in table 13
The %RSD value was found to be less than ± 2.0 indicated that the method
is precise.
Table 13:
Robustness for Ferrous Ascorbate and Folic Acid*(n=3)
|
Condition |
Conc. (µg/ml) |
Different Wavelength |
|||
|
Ferrous Ascorbate |
280.14 (mean* ± SD) |
%RSD |
280.54 (mean *± SD) |
%RSD |
|
|
40 |
-0.044± 0.00072 |
1.609 |
-0.042 ± 0.00055 |
1.288 |
|
|
265.58
(mean ± SD) |
%RSD |
265.98 (mean ± SD) |
%RSD |
||
|
Folic Acid |
4 |
0.0073 ± 0.00012 |
1.65 |
0.0001 ± 0.000105 |
1.438 |
Analysis of Ferrous Ascorbate and Folic acid in Combined Dosage
form:
Marketed preparation was analyzed by the proposed method.
The amount of Ferrous Ascorbate and Folic Acid was found to be 102.52 %
and 100.66 % of the labeled amount, respectively. The % assay values are tabulated in Table
14.
Table
14: Analysis data of marketed formulation *(n=3)
|
Drugs |
Result of formulation analysis |
||
|
Amount of drug |
% Assay* ± s.d. |
% RSD |
|
|
Ferrous Ascorbate |
30 µg/ml |
102.52 ±
0.634 |
0.618 |
|
Folic
Acid |
0.45
µg/ml |
100.66 ±
0.536 |
0.533 |
CONCLUSION:
All the parameters are validated as per ICH guidelines for the
method validation and found
to be suitable for
routine quantitative analysis in pharmaceutical dosage forms. The
result of linearity, precision, Robustness
proved to be within limit. Assay
results obtained by proposed
method are in fair agreement. Recovery studies were satisfactory
showed that there is overage of vitamins present in marketed formulation.
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Received on 16.06.2015 Accepted
on 18.08.2015
© Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 5(3): July- Sept. 2015;
Page 126-132
DOI: 10.5958/2231-5675.2015.00020.4