Spectrophotometric Assay for the Determination of Phenylephrine-HCl using Sodium Nitroprusside and Hydroxylamine Hydrochloride as a reagents

 

Maysam H. Al-Fakhry, Omar A. Sheej Ahmad*

Department of Chemistry, College of Education for Pure Science, University of Mosul, Mosul, Iraq.

 

ABSTRACT:

This study addressed a straightforward, precise and easy spectrophotometric method to determine phenylephrine hydrochloride (PHP) in bulk and pharmaceuticals dosage (drops and syrup). This method involves the interaction of sodium nitroprusside (SNP) with phenylephrine hydrochloride and hydroxylamine hydrochloride (HAH) in a phosphate buffer solution (pH 12) to produce a green color product. The overlain spectra of the product showed maximum PHP absorption at 714nm. The absorbance was started to increase linearly with the increased concentration of PHP. Under the optimal conditions, Beer’s law has been obeyed within the concentration range 0.2 – 8µg/mL. The developed method has good accuracy (average recovery) of 101.04% and precision (RSD) is less than 1.2%.  with molar absorptivity 4.4610x10⁴ L.mol^-1.cm^-1. The detection limit (LOD) and limit of quantification limit (LOQ) were 0.047 and 0.155ng/mL respectively. The suggested approach was applied practically to the determination of PHP in drug products (drops and syrup) with good precision and sensitivity. In addition, the validity of the suggested procedure was confirmed by the standard addition method. The advanced spectrophotometric procedure for assay of PHP is precise, simple, and reproducible with inexpensive and quite safe solvents. It could be applied in principles and practice for routine work analysis of PHP in bulk and pharmaceutical dosages.

 

 

 

1. INTRODUCTION: 

The Phenylephrine hydrochloride (PHP) is a crystalline (dust-like) powder form with white color. It belongs to a group of medications known as the sympathomimetic^1-3. The PHP (Nec-synephrine) is (R)-1-(3-hydroxyphenyl)-2-methyl-aminoethanol hydrochloride. Chemically, the PHP quite similar to epinephrine (EPP). It's a convenient long-acting vasoconstrictor with only a minor influence on the heart of the central nervous system (CNS). It was applied topically in the form of nose drops. Phenylephrine is rarely used as a vasopressor to boost blood pressure in unstable patients with hypertension. It stimulates α-receptors in a specific region of the organism (human).

 

 

The local uses of PHP as congestion, for allergic and unspecified conjunctivitis, sinusitis, and pharyngeal and nasopharyngitis⁴. For people who have a sinus infection, PHP nasal drops and spray are utilized to treat symptoms for instance heavy nasal secretion, nasal hechin, sneezing, and larynx^2,5,6.

 

PHP syrup can be used to relieve coughs, congestion, and prevent or treat signs of infection such as runny nose, nasal congestion, sneezing, and throat, eye discharge caused by flu, cold or high fever⁵.

 

Different analysis methods for PHP have been described in the literature, including UV-Vis spectrophotometry^7-12, voltammetry^13,14, high-pressure liquid chromatography (HPLC)^15-18, FTIR¹⁹, HPTLC^12,20, RP-HPLC^21-23, liquid chromatography (LC)²⁴, and capillary zone electrophoresis (CZE)²⁵ which also has been used as an indicator for PHP values.

 

The goal of this project is to create a responsive and simple analytical method for determining PHP in bulk formula and pharmaceutical dosages.

 

The analytical procedure was relied on the PHP reaction with hydroxylamine and sodium nitroprusside in a medium of phosphate buffer (pH 12) to form a colored product (green). As a result, for simplicity, the suggested method could be used for routine analysis of PHP in bulk formula and formulation.

 

 

C₉H₁₃NO₂. HCl               M. wt 203.7

Figure 1. Chemical structure of phenylephrine hydrochloride.

 

2. EXPERIMENTAL:

A Shimadzu spectrophotometer 1800 double beam with a fixed slit width (1 cm) coupled with Shimadzu UV software was used for all spectral measurements and analysis of all data.

 

2.1 Reagents:

All chemical compounds used in the experiments were in analytical grade without any further purifications.

PHP was provided from SDI-Iraq, sodium-nitroprusside, hydroxylamine, and sodium hydroxide were purchased from Fluka.

A standard solution of PHP (100 µg/ml) was prepared freshly by the dissolution of 0.01g of pure drug in 100 mL of DW in α volumetric flask²⁶.

Sodium nitroprusside solution (0.1M) was prepared through a mix of 2.9705g of material in 100 mL distilled water and kept far away from light.

Hydroxylamine hydrochloride solution (0.04M) was prepared through the dissolution of 0.6949 g of material in 250 mL of DW and kept in the refrigerator⁸.

Buffer solution for (pH 12) was freshly prepared through a mix of 100 mL of 0.1 M hydrogen sodium phosphate with 3.5 mL from sodium hydroxide (6.0 M) in 200 mL volumetric flask, after that the volume was filled precisely up the mark with DW.

 

2.3 Procedure for pharmaceutical preparations:

2.3.1 Nasal drops:

Three nasal drops bottles contents (0.25% of PHP) were mixed. The results match up with 50 milligrams of PHP (10 ml) after which distilled water is used for adulteration to 50 mL in a volumetric flask to get 500 μg/mL of PHP. Using simple dilution with distilled water to make appropriate additives from pharmaceuticals for analytical procedures.

 

2.3.2 Syrup:

The suitable volume of syrup that is containing PHP that equals 5 mg was poured in a 100 mL flask, diluted and mixed with DW. The solution has been studied as mentioned in the previous research²⁷.

 

3. RESULTS AND DISCUSSION:

3.1 Spectral features:

A green dye forms when PHP reacts with sodium nitroprusside and hydroxylamine in an alkaline medium (pH 12) with a high value of absorption at 714nm compared to the reagent blank as shown in Figure 2

 

 

Figure 2. Absorption spectra of 24 µg of (A) PHP /25 mL treated as described procedure and measured versus blank and (B) the blank measured versus DW.

 

The developed method is based on an aromatic electrophilic reaction, as illustrated in Figure 4. Phenol could be converted to phenolate in high alkaline mediums. As a result, the electrophilic reaction may occur without problems because the benzene ring becomes very active owing to the presence of negative oxygen (O). According to the theory of pure organic chemistry, the nitroso group (⁺NO) of the sodium nitroprusside attacks the phenolate molecule at meta or para positions and in the PHP case from meta-site. The electrophobic and electrophilic properties of the substituent within the benzene ring affect the response expansion and subsequently the sensitivity of the suggested method^28,29. This shows that the creation of the green to blue color by the suggested method could be owing either to the creation of the blue indophenol complex or to producing a complex of charge transfer type.

 

 

Figure 3. Suggested Reaction

3.2 Optimization of reaction conditions:

The factors affecting the stability and sensitivity of the colored complex produced by the reaction of PHP with sodium nitroprusside (SNP) in the presence of hydroxylamine (HA) in an alkaline medium have been precisely considered and verified. In the current study, various parameters such as (Effect of pH and buffer solution, volume and concentration of SNP, HA, Effect of temperature, time, and temperature variation) were investigated.

 

3.3 Effect of pH:

The green color could be produced in an alkaline medium as mentioned previously³⁰. So, the effects of various alkaline buffers (pH 9-12) and sodium hydroxide (pH > 12) were examined. Consequently, the influence of different types of basic and buffer solutions was examined. The highest stability and sensitivity were achieved by using phosphate buffer solution as presented in.

 

 

 

Figure 4.  Effect of the volume of 0.1 M sodium nitroprusside (A) 0.04 M hydroxylamine-hydrochloride (B) and phosphate buffer (C) on 4 µg/mL PHP

 

Table 1. Effect of the potential of hydrogen (pH) on the colored product's sensitivity.

 

3.4 Effect of reagents concentration:

Different volumes (0.1 to 4mL) of 0.1M of SNP solution Have been added to test the effect of the SNP on the formation of the colored products. The absorbance value of the reaction product was raised when using up to 0.5mL of SNP; however, when the volume was increased, the absorbance of the reaction product decreased, therefore 0.5mL of 0.1 M of SNP was used to achieve high absorbance. At the same time, 0.04 M of hydroxylamine-hydrochloride (from 0.1 to 3mL) has been applied to check the effect of HAH on the absorbance intensity. It was found that the 1.0mL of the solution was sufficient to achieve the highest absorbance intensity, and it is to be used for the next experiments. Therefore, the volume of phosphate buffer solution has been tested (0.5 to 4mL). The results show that the 3.0 mL of phosphate buffer maintenance solution gives the brightest colored product of 4µg/mL for PHP Figure 4.

 

3.5 Effect of Temperature, Time and Color stability:

Despite the color enhancement (formed right away), the color intensity peaked when PHP solution was reacted with HAH and SNP in phosphate buffer for 10 minutes, consequently, 30 minutes was chosen as the best development time in the following studies. The color achieved has stability for more than 24hours. Temperature effects color density of the dye was studied. The lab results show, that higher absorption was obtained when the color was applied at a temperature of (25^oC) than when volumetric flasks were ice-bathed in (0^oC) as shown in Figure 5.

 

 

Figure 5. Effect of temperature and developing time on the absorbance of 4 µg/mL PHP

 

3.6 Effect of the order of addition of the reaction components:

To find the effect of the order of addition on the color intensity of the product, different orders of addition of the reaction components were tried. It should be flowed the order No. I (PHP+SNP+HAH+B). The absorbance was found to be increased compared to others. It was utilized in all subsequent experiments Table 2.

 

Table 2. Effect of addition sequence on product absorption intensity

 

3.7 Procedure and Calibration Curve:

A PHP solution (100μg/mL) was transferred to the series of calibrated flasks of 25ml between 0.05-2.0mL then pouring 0.5mL of sodium nitroprusside 0.1M and then 1.0mL of HA solution 0.4mL and 3.0mL of maintenance solution (pH 12), add distilled water to complete the volume to the mark, by using the reagent blank the absorbance volumes were measured at 714nm. Beers law was used for many concentrations ranging from 0.2 to 8µg PHP/mL Figure (1). any concentration beyond 2 gives negative results from Beers law. The molar absorbance was 4.4610 x 10⁴ L.mol^-1.cm^-1.

 

A PHP solution 100μg/ml was transferred to a series transferred to the series of calibrated flasks of 25 mL between 0.05-2.0ml levels than 0.5mL of sodium nitroprusside 0.1 M and then 1.0mL of HA solution 0.4 ml and 3.0mL of maintenance solution (pH 12) was added, volumes were completed to the mark by adding DW, volumes of absorbance were measured at 714nM compared to reagent blank. using Beer’s law to measure concentrations between 0.2 to 8μg PHP/mL. The concentrations above 2 µg/ml show negative results than those shown in Beer's law. A molar absorption was 4.4610x10⁴ L.mol^-1.cm^-1.

 

3.8 Validation of assay procedure:

Under the optimized experimental conditions, a straight-line Calibration curve was obtained over the calibration range of 0.2-8µg/mL of PHP with molar absorptivity 4.4610 x10⁴ L.mol^-1.cm^-1 and Sandell sensitivity 4.5662 ng.cm^-2, indicating the method is sensitive. The calculations of dictation limits and quantitative limits were according to the value of the standard deviation of the absorbance measurements that are taken from a series of solutions containing the lower concentration of PHP in the Calibration curve³¹.

 

 

Figure 6. Calibration curve for the determination of PHP

 

The limits of detection µg/ml) and quantification (0.23 µg/mL) were below the lower limit of Beer's law range. Regression analysis revealed a very small intercept (0.015) and perfect linearity (correlation coefficient, r =0.998) between the absorbance and concentration of drug in the Beer's law studied.

 

3.9 Precision and Accuracy:

The solution containing four different concentrations of drug (0.1, 0.6, 1.8, µg/mL) was prepared and analyzed in six replicates to validate the precision and accuracy of the suggested method. It can be considered that mean recovery (101.04%) and relative standard deviation (<1.2%) to be a good enough adopted to be valid for application. These respectable limits of precision were appropriate for quality control analysis (QCA) of PHP in its pharmaceutical dosages.

 

3.10 Interference:

To investigate the efficiency and selectivity of the suggested analytical method, an efficient study of additives and excipients such as glucose, starch, Arabic gum, and lactose that are frequently present in pharmaceutical dosage forms. Results revealed that there was no interference from foreign or excipients materials up to 250μg/25ml for the developed method.

 

3.11 Analytical applications:

The present method was evaluated by commercial formulations of PHP and comparing the results obtained with those obtained by the standard addition procedure, Figure 7. A satisfactory agreement between results was obtained with an acceptable range.

 

4. COMPARISON OF THE METHODS:

The results shown in Table 5 indicate that the developed method is more sensitive and has high stability than the published manuscripts. In addition, the published methods require heating and a long time of analysis. 

Table 3 and Table 4.

 

 

 

 

 

Table 3.  Assay of PHP in pharmaceutical preparations

*Average of three determinations, ** RE: Relation error of reaction versal standard addition value.

 

   

Figure 7. Standard addition graphs of PHP in pharmaceutical preparations. A: PHP syrup for concentrations 1 and 2.4 µg/mL

 

 

Table 4. Assay of PHP in pharmaceutical preparations using standard addition procedure and the proposed method

 

 

Table 5. Comparison of the developed method with published methods

 

There are no interferences that have been noticed for the suggested method and it can be applied using an aqueous medium to determine the drug in pharmaceutical dosages^{9, 32}. The results point out that the suggested method is highly sensitive than the published methods. However, the color is much more stable than published work^{3, 7, 9, 33}. The suggested method has an advantage over the reported visible spectrophotometric methods concerning, high wavelength, reproducibility, precision and accuracy. It has been applied to determine PHP in more than one pharmaceutical formulation.

 

5. CONCLUSION:

The suggested method for the essay of PHP using the spectrophotometric method is simple, rapid, and sensitive, the green dye product formed is fairly soluble and it has high stability. The proposed method was useful for many methods concerning its high sensitivity, which allows determining of up to 0.2 µg/mL. The method is cost-effective and does not require any expensive and complicated instruments compare with the chromatographic methods. In addition, there is no need for any extraction steps. Hence, it can be successfully adopted for QCA   and  routine analysis of PHP in bulk drugs. Its advantages are the low cost of reagents, stability of colorful species for > 24, simplicity of sample treatment, and acceptable precision and accuracy. This method is recommended as a useful tool to analyze PHP in pharmaceuticals.

 

6. ACKNOWLEDGEMENTS:

This work was supported by the University of Mosul. So, the Authors would like to thank the University of Mosul for its financial support.

 

7. CONFLICT OF INTEREST:

The authors certify that there is no conflict of interest concerning the publication of this manuscript.

 

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Accepted on 21.06.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(1):169-174.