- *Corresponding Author:
- Elisabeta Antonescu

Lucian Blaga University of Sibiu, Faculty of Medicine, Sibiu 550169, Romania

**E-mail:**[email protected]

Date of Submission | 06 January 2017 |

Date of Revision | 12 May 2017 |

Date of Acceptance | 20 January 2018 |

Indian J Pharm Sci 2018;80(2):268-273 |

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms

## Abstract

The aim of this investigation was to develop a spectrophotometric method in the visible range for quantitative determination of Fe^{3+} in a pharmaceutical formulation based on complexation with methylthymol blue. The reaction occurred at a temperature of 30° and the absorbance remains constant after 25-30 min; the blue-colored resulting complex had a maximum absorption at 628 nm. The combination ratio was established through the Job’s method of continuous variations. In acetate buffer (pH=5), Fe^{3+} forms a complex of 1:1 with methylthymol blue. Lambert-Beer law was obeyed to the concentration area of (2-6) µg/ml; the calibration curve is described by the regression line A= 0.1025° (µg/ml) -0.0013 with a correlation coefficient of R^{2}= 0.9997. For the validation of the method the following parameters were studied, linearity, accuracy, limit of detection, limit of quantification and retrieval. Confidence interval of the average retrieval ranged from 98.79 to 101.13 %. The non-interference of excipients in the solution makes the method suitable for routine dosage of iron in polymaltose complexes. This visible spectrophotometric method was applied to determine Fe^{3+} concentration in polymaltose iron complex. The validation parameters confirmed that this method could serve to determine Fe^{3+}.

## Keywords

Fe^{3+}, methylthymol blue, spectrophotometry, iron polymaltose solution, quantitative

Iron is an essential trace element; the adult human
body contains 3-4 g iron [1,2]. Most of it is found in
hemoglobin. The most important function of this
protein is to transport O_{2} and CO_{2} between the lungs
and different tissues and to regulate blood pH [3,4]. Iron
has various well-established clinical uses, such as the
prevention and treatment of iron-deficiency anaemia.
The occurrence of iron in two oxidation states (Fe2+,
Fe^{3+}), as well as the balance between these two forms are
important for the biological systems that use iron in the
metabolic processes [5]. Also, iron oxides are materials
used as inorganic dyes, pigments for cosmetics and
food additives [6,7]. Although various methods have
been developed for quantitative determination of
iron, spectrophotometry is most frequently used due
to multiple advantages such as sensitivity, fidelity,
rapidness, simplicity and low cost [8-10].

Kinetic methods based on catalytic reactions for iron determination (in traces) have been applied because they present high sensitivity and the procedures and equipment are relatively simple [11,12]. In the field of pharmaceutical analysis, an important element is the determination of metal ions. There are sensitive and selective methods for their determination, but they have the disadvantage of involving expensive instruments [13]. Several analytical methods have been developed for quantitative analysis of iron. These include spectrophotometry [14-16], flux injection analysis [17], voltammetry [6,18], chromatography [12], capillary electrophoresis, atomic absorption and emission spectrometry [11].

## Materials and Methods

Spectrophotometer UV/Vis T80, 190-800 nm. Software
UV Win5, version 5.0.5, pH-meter Mettler Toledo with
combined electrode, Analytical balance MS 105 DU/M,
Mettler Toledo. Standard FeCl_{3} (Merck, Germany),
sample solution of iron (III) polymaltose solution
(50 mg/l), concentrated hydrochloric acid and
concentrated nitric acid (Merck, Germany), methylthymol blue (MTB, Merck, Germany) were
used.

**Preparation of samples**

All solutions were prepared from chemical reagents
of analytical purity. Acetate buffer solution (1 M) was
prepared from glacial acetic acid and sodium acetate
(pH= 5). Stock solution of Fe^{3+} was prepared by
weighing the amount of FeCl_{3} (0.8125 g) corresponding
to 5×10^{-3} mol in a 100 ml volumetric flask, dissolved
in double-distilled water (solution A). Stock solution
B was prepared by diluting 0.4 ml of solution in a
50 ml volumetric flask, then it was filled to the mark
with buffer solution pH= 5.

Stock solution MTB was prepared by weighing
an amount of MTB (3.22389 g) corresponding to
5×10^{-3} mol in a 100 ml volumetric flask, dissolved in
double-distilled water (solution C). 0.4 ml is taken from
this solution and diluted with buffer solution pH= 5 in
a volumetric flask at 50 ml (solution D). Calibration
solutions were prepared through the dilution of stock
solutions in 50 ml volumetric flasks. Sample stock
solutions (0.005 M): Fe^{3+} sample solutions were
prepared from polymaltose iron solution (1 ml sample
contains 50 mg polymaltose iron).

Sample stock solution was prepared by measuring
with precision 1 ml of polymaltose iron solution and
boiling it for 5 min in a water bath in the presence of
concentrated HNO_{3} (1 ml) and concentrated HCl (5 ml).
After cooling, the solution obtained was quantitatively
transferred in a 100 ml volumetric flask and the volume
was made up to the mark with double-distilled water.
An aliquot of 0.4 ml was taken from this solution and
diluted with pH= 5 buffer solution in a volumetric flask
at 50 ml. All solutions were prepared daily.

**Spectral recording**

Spectra were recorded in the spectral range of
200-800 nm (**Figure 1**). The analysis of UV/Vis spectra of
Fe^{3+}, MTB and Fe^{3+}:MTB complex solutions revealed
that only the Fe^{3+}:MTB complex was absorbed at
628 nm (**Figure 2**).

**Combination ratio**

Isomolar solutions were used for establishing the
combination ratio. Job's [10] method was used, in which
the complex combination is formed between Fe^{3+} and
MTB. Eleven series of solutions were prepared, which
contained a volume V_{1} ml of metallic cation solution
(with mole fractions between 0 and 1), a volume V_{2} ml of added solution (with mole fraction between 1 and 0),
where V_{1}+V_{2}= constant.

Sample absorbencies were determined at 628 nm against
a blank solution containing all reagents except for
Fe^{3+} ions. The data obtained were used for the graphic
representation of the absorbance against the mole
fraction. Absorbencies were graphically represented
according to the respective reagent mole fraction of the
metallic cation. The combination ratio was 1:1. Job's
curves are shown graphically-absorbance variation
measured at the maximum of the spectrum for the
complex 1:1, absorbance according to the mole ratio
of the reagent and the mole ratio of the cation (**Figure 3**).

**Formation kinetics of the Fe ^{3+}:MTB complex**

In order to find optimal complex formation conditions,
the influence of pH, buffer concentration, reaction
time and temperature were monitored separately. The
absorption spectre of the complex at different pH levels of 3.6, 5 and 6 was aimed at. Similarly, concentration
of the buffer solution (0.1, 0.5, 1 and 2 M) at different
temperatures (25, 30 and 37°) was monitored. The
optimum conditions for the reaction to occur were, pH
5, buffer solution (1 M), minimum reaction time of
25-30 min and a temperature of 30°. The absorbance
remained constant for 90 min (**Figure 4**).

**Quantitative determination**

After establishing the conditions for the formation
of the complex (pH, buffer concentration, reaction
time, temperature) series of solutions were prepared
containing equal standard/sample volumes and
MTB; they were kept at 30° in water bath for 30 min.
Absorbencies of the sample (A_{s}) and absorbencies
of the standard (A_{st}) were measured at 628 nm. The
quantity of Fe^{3+} in pharmaceutical formulation (M iron)
was calculated according to the dilutions performed
with the following Eqn., M iron = 25 000×A_{s}×C_{st}/A_{st} (mg/ml), where, 25 000 is the dilution coefficient, A_{s}-
absorbance of the sample, C_{st}- concentration of the
standard (mg/ml), A_{st}- absorbance of the standard.

## Results and Discussion

Validation was performed according to the guidelines
of ICH Q2(R1) [19]. The linearity of the visual spectrophotometric method was checked by means
of the calibration line method. The calibration line
consisted of five points and it was computed in
Microsoft Excel. The calibration curve obtained
by applying the spectrophotometric method in the
visual was linear regarding the studied concentration
ranges. Calibration curves were plotted through linear
regression, y= ax+b, where y represents the absorbance
and x is the concentration (μg/ml). The parameters of
linear regression were calculated: a= slope, b= the
intercept, R^{2}= correlation coefficient (**Figure 5**).

In order to study the linearity and accuracy, five samples
were prepared with the following concentration levels
i.e. 60, 80, 100, 120 and 140 %. The calibration curve
obtained by applying the spectrophotometric method in
the Vis is linear on the 2-6 μg/ml concentration range.
Within this interval, the correlation coefficient had the
value of 0.9997. Also, for determining the linearity
of the method, other parameters were evaluated, such
as, the homogeneity of variances, the existence of a
significant slope, the validation of regression lines and
the comparison of intercepts with 0. **Table 1** presents
the statistical parameters that were evaluated for
determining the linearity of the spectrophotometric
method.

Parameter - Fe^{3+} |
Standard values | Pharmaceutical formulation | Theoretical values | Test-imposed conditions |
---|---|---|---|---|

Correlation coefficient | 0.9997 | 0.9981 | - | - |

Intercept | –0.00128 | –0.0018 | - | - |

Slope | 0.1025 | 0.1657 | - | - |

Cohran test-homogeneity variances | C= 0.2803 | C= 0.54 | C_{theoretical}= 0.68 |
C<C_{theoretical} |

Fisher test-significant slope | F= 54150.29 | F= 43625.16 | F_{theoretical}= 4.67 |
F>F_{theoretical} |

Fisher test-regression line validity | F= -3.22 | F= -2.94921 | F_{theoretical}= 3.71 |
F<F_{theoretical} |

Student test-comparison of the intercept with 0 | t= 1.4773 | t= 1.530 | t_{theoretical}= 2.16 |
- |

Student test-comparison of the slopes of regression lines | - | t= 0.321 | t_{theoretical}= 2.056 |
t<t_{theoretical} |

Student test-comparison of the intercepts | - | t= -0.851 | t_{theoretical}= 2.056 |
t<t_{theoretical} |

**Table 1:** The statistical parameters for determining the linearity of the spectrophotometric method

Correlation coefficient is 0.9997 (**Table 1**) and the
Student test-intercept presented t<t_{theoretical} in both cases
(standard and pharmaceutical formulation) for Fe^{3+}.
The variances of the five groups of determinations
(k= 5) need to be homogeneous for a probability of
error of 5 % for the calibration line. This was checked
through the application of the Cohran test. **Table 1** showed that C_{calculated}= 0.280 is lower than C_{theoretical}=
0.540; variances are considered homogeneous. The
existence of a significant slope was verified with the
help of the Fisher statistical test. The value F_{calculated}=
43625.16 is higher than the value F_{theoretical}= 4.67;
the regression line presents significant slope. The
validation of the regression line was verified through
the application of the Fisher test, where F_{calculated}= –
2.94
is lower than F_{theoretical}= 3.71; the regression line for the
analysis of the Fe^{3+} standard is valid.

The comparison of the intercepts with 0 was carried out by means of the Student t test. The calibration line can be used in quantitative calculations if the intercept differs considerably from 0. If it does not differ significantly from 0, the 100 % standard solution will be used as reference (the sample contains the analyte in the expected concentration).

Accuracy shows how close the results obtained are
to the reference values. The accuracy of the method
was verified for the whole range of linearity and for
all the five levels of concentration. The absorbance
represents the average of three determinations for each concentration level. The results presented in **Table 2** showed that accuracy was 98.79-101.13 %.

Parameter - Fe^{3+} |
Calculated values | Theoretical values | Test-imposed conditions |
---|---|---|---|

Cohran test-homogeneity of in-group variances | C= 0.45 | C_{theoretical}= 0.69 |
C<C_{theoretical} |

Fisher test-validity of the average results | F= 0.89 | F_{theoretical}= 3.48 |
F<F_{theoretical} |

Confidence interval of the average found | 98.79-101.13 | - | - |

**Table 2:** The accuracy of the spectrophotometric method

For determining accuracy, the homogeneity of ingroup
variances (the Cohran test) and the validity of
the average found were evaluated. The interpretation
of the values obtained for these parameters is done by
comparing the calculated values to the theoretical ones.
The method is considered exact if in-group variations
are homogenous C_{calculated}<C_{theoretical} (0.45<0.69). The
validation of the average found is done by means of the
Fisher test (the comparison of in-group errors to intergroup
errors). The validation of the repeatability of the
method was performed on different days, on six samples
that had been individually prepared (pharmaceutical
formulation), situated at a concentration level of
100 % (**Table 3**).

Retrieval % | |||
---|---|---|---|

1 d | 2 d | 3 d | |

l= 628 nm | l= 628 nm | l= 628 nm | |

Concentration level : 100 % |
99.105 | 99.231 | 100.087 |

99.180 | 99.368 | 99.889 | |

100.100 | 99.456 | 98.890 | |

99.874 | 99.869 | 99.880 | |

100.120 | 100.062 | 98.988 | |

99.980 | 99.935 | 100.100 | |

Mean |
99.7265 | 99.653 | 99.639 |

Standard deviation |
0.462 | 0.344 | 0.551 |

Relative Standard deviation (%) |
0.463 | 0.345 | 0.553 |

**Table 3:** The fidelity of the spectrophotometric method

**Table 4** presented the fidelity of the spectrophotometric
method-results and statistical evaluation. The Cohran
test was applied for verifying the in-group variances
of results in order to study the fidelity of the method.
For the in-group variances to be homogenous C_{calculated} needs to be lower than C_{theoretical}. In this case, C_{calculated}=
0.39˂C_{theoretical}= 0.68, so in-group variances were
considered homogenous for the spectrophotometric
method. The variation quotients corresponding to the
two precisions were also calculated. The analytical
method could be considered precise if the variation
coefficients values were below 2 %, so the method
could be considered precise, the repeatability variation coefficient CV_{r}= 0.51 % and the reproducibility
variation coefficient CV_{R}= 1.42 % for the error
probability taken into consideration. In conclusion, the
developed UV spectrophotometric method (λ= 628 nm)
was validated and proved to be linear, exact and precise
and it can be used for the quantitative determination
of iron in pharmaceutical formulations. The limit of
detection (LOD) and the limit of quantification (LOQ)
were evaluated on the basis of signal to noise ratio.
The values obtained within the study are presented
in **Table 5**. LOD= 3×S_{cmin}/a, where: S_{cmin}= represents
the standard deviation of ground level concentration,
a= slope of the calibration. LOQ= 10×S_{cmin}/a, where:
S_{cmin}= represents the standard deviation of ground
level concentration, a= slope of the calibration. **Table
5** presents the LOD and the LOQ for determining Fe^{3+}.

Parameter - Fe^{3+} |
Calculated values, % | Test-imposed conditions |
---|---|---|

Cohran test-homogeneity of variances | C= 0.39 | C<C_{theoretical} |

Repeatability/reproducibility variation coefficient | 0.51/2.42 | - |

**Table 4:** The fidelity of the spectrophotometric method statistical evaluation

Analyte | S_{CMIN} |
A | LOD (µg/ml) | LOQ (µg/ml) |
---|---|---|---|---|

Fe^{3+} |
0.0012 | 0.1025 | 0.0351 | 0.1170 |

LOD: limit of detection and LOQ: limit of quantification

**Table 5:** The limit of detection and quantification for determining Fe^{3+}

The non-interference of excipients in the solution
made the method suitable for routine dosage of iron
in polymaltose complexes. Visual spectrophotometry
also allowed for quantitative determination of Fe^{3+} in pharmaceutical formulations, which represented
the main aim of the study performed. The Fe^{3+}:MTB
complex presents a combination ratio of 1:1 and
displays corresponding stability.

The visual spectrophotometric method developed and
validated in this way was applied for Fe^{3+} dosage in
polymaltose iron complexes. The validation of the visual spectrophotometric method for the determination
of Fe^{3+} in iron polymaltose solution was carried
out through the study of the following parameters:
linearity, accuracy, repeatability and reproducibility.
The validation parameters confirm the applicability of
the method with the pursued aim. The method proposed
is simple, adequate for laboratory practice and it has
been successfully used for quantitative determination
of polymaltose iron in commercial pharmaceutical
products.

## Conflicts of interest

There are no conflicts of interest.

## Financial support and sponsorship

Nil.

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