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Validation of a Spectrophotometric Analytical Method for the Quantitative Determination of Adrenaline in Injectable Pharmaceutical Formulations

Carlos Montaño-Osorio*, Dalia Bonilla-Martínez, Martha Angélica Villegas-González, Adolfo Eduardo Obaya Valdivia*

Sección de Fisicoquímica, FES Cuautitlán-UNAM, Departamento de Ciencias Químicas. Av. Primero de Mayo s/n, Cuautitlán Izcalli, Edo. Mex. México C.P. 54700Sección de Fisicoquímica, FES Cuautitlán-UNAM, Departamento de Ciencias Químicas. Av. Primero de Mayo s/n, Cuautitlán Izcalli, Edo. Mex. México C.P. 54700

Abstract: A spectrophotometric method is validated for the quantification of adrenaline in injectable pharmaceutical forms by means of the quantitative formation of the iron(III)-adrenaline complex, obtaining the validation parameters at the maximum absorption of the complex (432 nm and 648 nm), determining that the best wavelength to perform the quantification is 648 nm because it satisfies the validation parameters and is the most sensitive. It is shown that the 648 nm method has a linear relationship in an adrenaline concentration range of 1.1704 10–4 M to 1.1704 10–3 M, through the analysis of the confidence intervals for the slope and the ordinate at the origin, as well as the evaluation of the residuals of the regression. The limits of detection and quantification are 3.5267 10–6 M and 2.8200 10–5 M, respectively, for the calibration curves at 648 nm. The method is specific, reproducible, and accurate at 648 nm. The method is shown to be robust in the pH range of 4.70 to 5.15. For the determination of adrenaline at 648 nm, it is possible to use aqueous standards for the construction of calibration curves, without the need to add placebo because a confidence interval of the slope is obtained for the recovery curve of 0.9982–1.0007, so there is no matrix effect.

Keywords: Keywords: adrenaline, validation, spectrophotometry, injectable adrenaline formulations

Received February 21, 2021; Revised April 04, 2021; Accepted April 12, 2021

Citation: Carlos Montaño-Osorio, Dalia Bonilla-Martínez, Martha Angélica Villegas-González, Adolfo Eduardo Obaya Valdivia, Validation of a Spectrophotometric Analytical Method for the Quantitative Determination of Adrenaline in Injectable Pharmaceutical Formulations. American Journal of Pharmacological Sciences, 9 (1). 40-45. January 2021. doi:10.12691/ajps-9-1-4

1. Introduction

In 1894, when Oliver and Schäfer [1] obtained hydroalcoholic extracts from powdered adrenal glands and administered them to several animals, they observed the potent effects on the blood vessels, heart, and skeletal muscle; Fürt took advantage of this and formulated a chelate of this extract with iron, which he named suprarrenine [2]. Shortly after, in 1900, Takamine isolated and elucidated the structure of adrenaline from the hydroalcoholic extracts of Oliver and Shäfer; likewise, he obtained by organic synthesis four grams of adrenaline in crystalline form, a process he patented in 1901 [3].

Early in 1906, the industrial production of adrenaline increased substantially due to the gradual extension of adrenaline use, as in concomitant administration with anesthetics due to the vasoconstrictive action increasing the time of its effect in surgical procedures to prevent bleeding [4], in the prevention and reduction of anaphylactic shock due to its bronchodilator effect [5], and in resuscitation to increase the spontaneous return of blood recirculation [6]. However, adrenaline has potent effects on physiological functions; high levels of concentration can cause intoxication, liver damage, and even heart attacks [7]; that is why the formulations must be strictly careful in the amount they report on the label.

Several instrumental methods are available for the determination of adrenaline in formulations; One of them, the high-resolution liquid chromatography, according to the American Pharmacopeia [8,9] spectrophotometric detection of adrenaline can be used for a quantification limit of 0.003 mg/mL; another one consists of the electrochemical detection that is characterized by the limit of quantification down to 0.001 mg/L, ignoring those chromatographic methods coupled to mass spectrometry for the determination of adrenaline in blood serum [10].

Spectrophotometric methods have been reported for the quantification of adrenaline based on the oxidation of the latter by the Fe³⁺ ion, either in the presence of nitrous salts [11] or by the formation of a complex by the Prussian blue reaction [12], or by the use of proteins as an adrenochrome reaction product [13].

Until 2009 when Gülcin [14] reported that adrenaline reacts with iron(III) to form a colorful complex based on the average pH value of the chemical medium as shown in the Figure 1.

Figure 1. Reaction between adrenaline and iron (III) to form the iron (III)-adrenaline complex

Bonilla et al. implemented a spectrophotometric method for the quantitative determination of adrenaline [15], proposing that the reaction be accomplished in an acetic acid-acetate buffer media of 1 mol/L at pH = 5. In this publication, they suggest that it is not essential to use proteins and salts, which additionally do not require organic solvents such as the HPLC method.

Based on the above, the purpose of this work is to perform the validation of the spectrophotometric method to demonstrate that the method meets the criteria of reproducibility, linearity, specificity, and robustness at two wavelengths where maximum absorption are presented of the complex, as well as to verify the best wavelength through the value of the relative standard deviation of the recovery curves to achieve the appropriate adrenaline quantification. Validation parameters were performed in accordance with the regulations by the Food and Drug Administration (FDA) [16].

2. Materials and Methods

2.1. Reagents

Standard of adrenaline hydrochloride (Sigma) and analytical grade reagents were used: acetic acid (JT Baker), sodium hydroxide (JT Baker), sodium acetate (JT Baker), iron (III) chloride (Fermont), and sodium chloride (JT Baker).

2.2. Instrumentation

The validation parameters are given below, the absorption spectra were performed from 740 nm to 380 nm, at a scanning speed of 480 nm/min in an equipment Perkin Elmer Soft Lambda 18 UV–Vis spectrophotometer, using quartz cells with 1 cm optical path.

2.3. Validation of the analytical method

- The linearity of the system

Calibration curves were prepared in triplicate, preparing adrenaline standard solutions in 1 mol/L acetates to buffer pH = 5. Likewise, an iron (III) solution was prepared in the acetates buffer. Ten systems were prepared as specified in Table 1, with absorbances recorded at the maximum absorption of the complex (432 nm and 648 nm).

Table 1. Systems of the calibration curve for the linearity of the system

- The linearity of the method

A placebo containing 0.8% (w/v) sodium chloride dissolved in 1 mol/L acetates buffer at pH = 5 was prepared. The systems described in Table 1 were carried out.

- Repeatability

The systems corresponding to the concentration of system “e” of the calibration curve of the linearity of the method were prepared in duplicate.

- Specificity

The absorption spectrum of the placebo from 740 nm to 380 nm was performed against a blank of deionized water.

- Robustness

As part of the study of the robustness of the method, the ability to tolerate changes in the pH was evaluated, the calibration curves were prepared at the values of pH = 4.7, 5.15, and 5.30, in triplicate according to Table 1, the systems were filled with the placebo.

3. Results and Discussion

3.1. Specificity

As seen in Figure 2, the iron (III)-adrenaline complex has two maximum absorptions corresponding to 432 nm and 648 nm; consequently, validation was carried out at these two wavelengths.

Figure 2. Absorption spectrum of iron (III)-adrenaline complex

Figure 3. Absorption spectrum of placebo

To ensure that the absorbances are associated with the iron (III)-adrenaline complex, the absorption spectrum of the placebo was performed, and deionized water as a blank.

As shown in Figure 3, in the range of wavelengths under study there is no absorbance, corresponding with placebo it exceeds the value of 0.002 which is located at 720 nm, so at the wavelengths of interest, 648 nm, and 432 nm the absorption value is not associated with a species present in the placebo, therefore the method is specific.

3.2. Repeatability

As part of the evaluation of the repeatability, the percent relative standard deviation (% RSD) at 648 nm was determined by measuring the system “e” of Table 1 in tenfold, obtaining 2.05% while at 432 nm it corresponds to 1.76%, the values being less than 3%, so the method is repeatable.

3.3. The Linearity of the System

Figure 4 shows the evaluation of the linearity of the system corresponding to the calibration curves according to the wavelength under study with the corresponding graph of residuals.

According to the parametric linear regression model applied to the calibration curve at 432 nm, the equation of the line with its following coefficient of determination is obtained:

On the other hand, performing the same analysis to the calibration curve at 648 nm, we obtain:

Figure 4. Calibration curves and residual graphs for evaluation of system linearity A) curve at 432 nm, B) residuals at 432 nm, C) curve at 648 nm, D) residuals at 648 nm

Figure 4 (B and D) shows the residuals are homoscedastic for the linear system in the range in Table 1.

As is known, the confidence intervals applied to the intercept must include zero, while for the slope it must exclude zero, also, it has been determined based on the following equations:

The 95% confidence interval for the origin:

The 95% confidence interval for the slope:

The confidence intervals for the two wavelengths, as well as the conclusion, are shown in Table 2.

Table 2. Confidence intervals for the ordinate to the origin and the slope of the calibration curves for the linearity of the system

Based on these criteria, it is concluded that the system is linear in the range of adrenaline concentrations of 1.1704 10–4 mol/L to 1.1704 10–3 mol/L.

3.4. Method Linearity

The evaluation of this parameter, Figure 5 shows the calibration curves at 648 nm and 432 nm, as well as the graph of the residuals for each curve.

Following the same methodology for the system linearity analysis, we determined the regression model for the linearity of the method, the coefficient of determination, and the confidence intervals for the intercept and the slope, as shown in Table 3.

According to the criteria established by the FDA, the coefficients of determination must be greater than 0.98; as shown in Table 3, the coefficients of determination for the calibration curves at the two wavelengths are 0.9999 and 0.9993, which exceed the value proposed by the FDA; the confidence intervals for the intercept include zero, and the confidence intervals for the slopes exclude it. Likewise, in Figure 4 it is observed that the residuals for the calibration curves are homoscedastic, so the method is linear.

Figure 5. Calibration curves and graph of residuals for method linearity A) curve at 648 nm, B) residuals 648 nm, C) curve at 438 nm, D) residuals at 438 nm

Table 3. Statistics of the linear regression for the method linearity

3.5. Precision, Accuracy, and Influence of Matrix

The recovery curves that are useful for the determination of precision, accuracy, and influence of matrix, are presented in Figure 6.

Figure 6. Charts of the recovery at 432 nm (A), and 648 nm (B)

As observed, the method is accurate at both wavelengths because the relative standard deviation coefficients are 0.3111% at 648 nm and 2.0392% at 432 nm.

The method is accurate at 648 nm according to the confidence interval for the slope of the response curve [0.9982, 1.0007] which includes unity, on the other hand, the method is not exact at 432 nm according to the confidence interval for the slope of the recovery curve [0.9611, 0.9801], which does not include unity.

In addition, the effect of a matrix with confidence intervals was analyzed; at 648 nm there is no matrix influence, while at 432 nm there is matrix influence.

The limits of detection and quantification were calculated based on the standard deviation of regression for both wavelengths using the following equations:

The detection limits are 3.5267 10–6 M and 1.0687 10–5 M at 648 nm and 432 nm respectively, the quantification limits 2.8200 10–5 M (648 nm) and 8.5455 10–5 M (432 nm).

3.6. Robustness

In the determination of the method robustness interval, the calibration curves were performed in triplicate in the concentration range of Table 1, calculating the systems of the curves with the placebo at pH 4.7, 5.15, and 5.30.

For each calibration curve at each pH, a hypothesis test was performed for the slopes with respect to the calibration curve of the linearity of the method being the null hypothesis (H₀) that the slopes are equal and the hypothesis alternates (H₁) that the slopes are different, the null hypothesis is accepted if the calculated Student t-value is less than the 95% t-value from tables (Table 4).

Table 4. Student's t-test for the hypothesis test of the slopes

As shown in Table 4, by applying Student’s t-test for the hypothesis test of the slopes, the method is valid for a range of pH 4.7 to 5.15 because in that interval there are no significant differences at 95% confidence for the slopes of the calibration curves, while at pH of 5.3 the slopes are significantly different. Therefore, the validated method is robust in a pH range of 4.7 to 5.15.

3.7. Application of the Method

Triplicate samples of an injectable adrenaline drug (Pinidrina Laboratories PiSA) were analyzed in which reports that each injectable formulation contains 1 mg of adrenaline per milliliter of the drug, obtained by interpolation with the calibration curves of the method linearity 0.97 ± 0.042 mg of adrenaline per milliliter. Through which it is demonstrated that the method serves to quantitatively determine adrenaline in pharmaceutical formulations.

The validation parameters are summarized in Table 5.

Table 5. Summary of validation parameters

4. Conclusions

The spectrophotometric method for the quantitative determination of adrenaline in pharmaceutical formulations was validated, demonstrating that the method is linear in a range of adrenaline concentrations of 1.1704 10–4 M to 1.1704 10–3 M, demonstrating that there is no matrix effect, so it is possible to use aqueous standards to obtain the calibration curve, being a specific, reproducible, and accurate method. It presents robustness in a pH range from 4.70 to 5.15 for the buffer solution. It is presented that the best wavelength to carry out the determination is 648 nm because it presents the lowest data dispersion, the highest sensitivity, and fulfills all the validation parameters. The adrenaline content was quantified in triplicate in an injectable pharmaceutical formulation (Pinidrina Laboratories PiSA) which reports that the adrenaline content is 1 mg/mL, obtaining 0.97 ± 0.042 mg/mL, so the validated method can be used for the quantification of adrenaline in injectable formulations.

Conflict of Interests

Authors report no conflict of interests.

References

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