1. INTRODUCTION:

Phenylephrine HCl (PHE), as known as [(R)-1-3-(hydroxyl phenyl)-2-(methyl amino) ethanol hydrochloride, C₉H₁₃O₂N.HCl¹. PhenylephrineHCl drugs are white crystalline powder or light yellow salt². It represents the type of drugs called sympathomimetics that directly affect the adrenergic receptor³. It behaves as a stimulator for alpha receptors. It is used widely as a decongestant for sinusitis and nasopharyngitis, non-specific and allergic conjunctivitis⁴. The drug PHE is found in vasopressor medicines as eyewashes, nasal decongestant, and syrup⁵^. Many of methods have suggested to determination of PHE in pharmaceutical formulations⁶^_⁷ such as High-performance liquid chromatography (HPLC)⁸^-⁹, voltammetry¹⁰, titmetry¹¹^,fluorescence¹², flow injection¹³, UV-Vis Spectrophotometry¹⁴^-¹⁵^-¹⁶.

The dispersive liquid liquid microextraction (DLLME) has many advantages in the estimation of pharmaceutical preparation like rapid, safety and low cost¹⁷^-¹⁸. It has applied as one of the determination and pre-concentration methods in analytical chemistry¹⁹^-²⁰. In this work, the proposed procedure is based ion-pair reaction of PHE with alizarin yellow reagent in the acidic media, then assessment and pre-concentration the usage dispersive liquid liquid microextraction (DLLME). The aim of this research is to classify, investigate, and determine the best conditions for quantifying PHE drugs using two different methods. One of them is directs extraction method at λ_max430nm and the second one is dispersive liquid liquid microextraction (DLLME) and then compare between the two methods.

2. MATERIALS AND METHODS:

2.1. Materials:

Double beam UV-Vis spectrophotometer was used for all spectral and absorption intensity measurements with corresponded 1cm quartz cells. All the pH measurements were carried out by a Metrohm 780 digital pH meter(Switzerland) with a combined glass electrode.An IKA genius 3 vortex mixer (Staufen, Germany) was employed in extraction procedure. A Hermle Z-300 centrifuge (Wehingen, Germany) was used to separate the organic from aqueous phase.

2.2. Methods:

The chemicals PhenylephrineHCl (PHE) from Sigma –Aldrich, hydrochloric acid from Scharla, alizarin yellow from sigma-Aldrich, acetic acid, chloroform, ethanol, methanol, carbon tetrachloride were purchased from BDH(England).Double distilled water was used throughout the experiments.A 500μg/mL stock solution of PHE was prepared by dissolving 0.05gm of PHE in10 mL of 0.1N HCl and making up to 100ml with double distilled water in a volumetric flask. Acetate buffer solution was prepared by the addition of (9mL) of 0.1M sodium acetate to (41mL) of acetic acid 0.1M to adjust the pH at 4.0 and the mixture is brought up to 100ml with double distill water.

2.3. Pharmaceutical preparations procedure:

Three concentration of PhenylephrineHCl (eye drops 10% w/v) (European)5, 10, 15µg.mL^-1 were taken, they were treated in the same way as direct and DLLME extraction in pure drugs, and the absorbance was measured at a wavelength of 430nm.Phenylephrine Hydrochloride BP (Dolo-cold 10mg PEP). Tablets (India) 50 mg, are carefully weighed, the pills are milled an Dissolved in 0.1N HCl and the filtered it complete the volume up to in a 100ml volumetric flask. They were treated in the same way as direct and DLLME extraction in pure drugs, and the absorbance was measured at a wavelength of 430nm.

2.4. General procedure of direct extraction for PHE medications:

A 0.5mL standard PHE drug solution was moved to a 5mL volumetric flask stoppered tube containing 2mL alizarinyellow reagent solution, and 1.2mL of acetate buffer (PH=4). The volume was adjusted to 5mL with 0.1N Hydrochloric acid. Chloroform 5mL was added and mixed well for one minute using vortex. The drug-reagent ion pair complex was extracted in chloroform. The chloroform layer was separated out. The absorbance of the colored solution was scanned on spectrophotometer in the range of 300-700nm against reagent blank. The blank was prepared similarly in which volume of standard solution of amino drug was replaced by an equal volume of 0.1N Hydrochloric acid.

2.5. General procedure of dispersive liquid liquid microextraction (DLLME) for amino medications:

In a 15mL glass centrifuge tube, add 0.5mL of medication (20µg.mL^-1), 1mL of alizarin yellow reagent (20µg.mL^-1), and 0.8 mL of acetate buffer (PH=4), then complete the volume to 5mL with 0.1N HCl, and then complete to 10mL with distilled water. Volume of 400µL chloroform as an extraction solvent and ethanol 700µL as dispersive solvent were rapidly injected into the solution using micro syringe to produce the formation of a cloudy solution. The mixture was centrifuged at 2000rpm for 6 minutes. The yellow ion pair complex was obtained with a micro syringe, put in a 1cm Quartz cell, and the absorbance at 430nm was measured against a blank.

3. RESULTS AND DISCUSSION:

The ion-pair complex is formed when the cation of phenylephrine HCl (PH⁺) binds to the anion of theAlizarinyellow reagent(A^-) to form an intense yellow-colored ion-pair complex (A^- _ PH+). The absorbance of the yellow complex can be measured by spectrophotometer analysis in pH 4 at 430nm against blank; the results were illustrated in the (figure 1).

Figure. 1. Absorption Spectra of the Resulting complex.

3.1. Optimization of direct extraction:

The direct extraction combine with the UV-Vis. Spectrophotometer was used to select the suitable conditions for the complexity of phenylephrine HCl drug and a alizarin yellow reagent at wavelength 430nm, the most critical factor in this complex formation process is the acidic function (pH), type pH and volume of pH, reagent volume and type solvent. The range of pH for the phosphate buffer was used between (1-8), and it was noticed that the best acidity function for the complex formation was at pH = 4. Since the type of buffer has an effect on the formation of complexes, a variety of buffer solutions such as phosphate, acetate, and citrate buffer were used, and it was noticed that the acetate buffer gave the highest absorption value.

The different volumes of acetate were eximinated, and it was observed that a volume of 1.2mL had the highest absorption value at 430nm. This volume was considered the best for the complex formation between the phenylephrine HCl and an alizarin yellow reagent. The volume of reagent needed for complex formation was determined to be 2 mL.

The influence of several solvents (chloroform, carbon tetrachloride, benzene, and hexane) on complex formation was also investigated; Chloroform was showed to be the optimum solvent for obtaining the best absorption. Two methods were used to denote the ratio of phenylephrine HCl and alizarin reagent, continuous variation and the molar ratio techniques, it was noticed that the ratio for both techniques was 1:1 (phenylephrine HCl drug: alizarin yellow reagent) (fig.2).

Fig.2: Continuous variation method and Mole-ratio method of Phenylephrine HCl

The interferences effect from several compounds such as starch, glucose, fructose, and others was investigated, and it was found that they had no influence on the extraction recovery, as shown in the Table.1.

Table.1: Extraction recovery with different interference compound

Compound	Recovery%
Maltose	99.4
Starch	97.8
Glucose	98.3
Lactose	96.6
Fructose	99.5
Glysine	96.6

3.2. Calibration curve:

After the identification of the optimal conditions for complex formation between phenylephrine HCl and an alizarin yellow reagent. The calibration curve was constructed using the absorbance against concentration of Phenylephrine HCl. The concentration range was ranges (1-20µg.mL^-1). The regression equation of calibration curve HCl is Y=0.071X+0.061 and R²=0.997 of the linear calibration (Fig.3)

Figure. 3 : Standard calibration curve of complex formation

3.3. Optimization of dispersive liquid liquid Microextraction (DLLME):

At wavelength 430nm, the DLLME combine with a UV-Vis. spectrophotometer was used to select the best conditions for the complexity of phenylephrine HCl drug and an alizarin yellow reagent. The effect of the extraction solvent (chloroform, tetra chlorocarbon, and benzene), as well as the dispersed solvent (ethanol, methanol, acetone, and acetonitrile). was investigated, the result was showed that chloroform and ethanol were the best extraction and dispersion solvents for complex formation.

The acidity function, type of buffer solution, and Volume of buffer solution were also tested, the range of pH of phosphate buffer which used is between (1-8). It was also noticed that the best acidity function for the formation of the complex was at pH = 4. Since the type of buffer has an effect on the forming of complexes, a variety of buffer solutions (phosphate, acetate, and citrate) were used, and it was noticed that the acetate buffer gave the highest absorption value.

The different volumes of acetate were studied, and it was noticed that the volume of 0.8 ml had the highest absorption value at 430nm. This volume was considered the best for the complex formation between the phenylephrine HCl and an alizarin yellow reagent. The volume of Alizarin yellow reagent needed for complex formation was determined to be 1mL.

The best volumes for both the extraction and dispersion solvents were found to be 400µL and 700µL, respectively. The effect of speed and time in the centrifuge plays an important role in the formation of complexes. The best speed and extraction time were 6 minutes and 2000rpm.

The effect of several interferences such as starch, glucose, fructose, and others was investigated, and it was found that they had no influence on the estimated substance's absorbance value, as shown in the Table.2.

Table.2: Interference study

Compound	Recovery%
Maltose	96.8
Starch	95.3
Glucose	96
Lactose	97.8
Fructose	97.7
Glysin	99

3.4. Calibration curve:

After the identification of the optimal conditions for complex formation between phenylephrine HCl and an alizarin yellow reagent. The calibration curve was constructed using the absorbance against concentration of Phenylephrine HCl. The concentration range in was shown to be (0.5-13µg.mL^-1). The regression equation of phenylephrine HCl is Y=0.061X-0.032 and R²=0.996 of the linear calibration (fig.4).

Table.3 Analytical parameter of direct extraction and DLLME method.

*Parameters*	Direct extraction	DLLME
λ_max nm	430
Color	yellow
Regression equation	Y=0.071X+ 0.061	Y=0.061X- 0.032
Linearty range(µg/mL^-1)	1-20	1-13
Correlation Cofficient (R²)	0.997	0.996
Ɛ(L.mol^-1.cm^-1)	14459.9	12423.3
Sandell^,ssensivity (µg. cm^-2)	0.0141	0.0164
Slope (b)	0.071	0.061
Intercept(a)	0.061	0.032-
Limit of detection(µg/mL^-1)	0.34	0.094
Limit of quantification(µg/mL^-1)	1.12	0.31
C.L.for the slope(b±ts_b) at 95%	0.071± 0.275	0.061±0.266
C.L.for the intercept(a±ts_a) at 95%	0.061±2.96	0.032 ± 1.42
Standard error for regression line (S_y/x)	0.45	0.176
*C.L for Conc.X₁μg ml^-1at 95%	5.09±0.1241	2.8±0.0015
*C.L for Conc.X_2μg ml^-1at 95%	9.82± 0.099	5.12±0.0025
*C.L for Conc.X₃μg ml^-1at 95%	14.04±0.174	6.85±0.0025
Direct extraction (X₁=5, X₂=10, X₃=15) DLLME (X₁=3, X₂=5, X₃=7)

Table.4: Application of the proposed direct extraction and DLLME for the evaluation of phenylephrine HCl

drug	direct extraction
	Conc. of drug mg.L^-1		Relative Error%	Recov. %	Average Recov%	RSD% (n=3)
	Taken	Found	Relative Error%	Recov. %	Average Recov%	RSD% (n=3)
eye drops	5	5.2	-4	104	100	0.02
	10	10.2	-2	102		0.98
	15	14.1	6	94		0.015
Dolo-cold (tablet)	5	4.91	1.8	98.2	97.9	0.024
	10	9.35	6.5	93.5		0.021
	15	15.3	-2	102		0.04
DLLME
eye drops	3	2.6	13.3	86.7	95.5	0.02
	5	5.2	-4	104		0.01
	7	6.7	4.3	95.7		0.04
Dolo-cold (tablet)	3	2.64	12	88	96.3	0.08
	5	5.1	-2	102		0.04
	7	6.92	1.1	98.8		0.01

Figure. 4 : Standard calibration curve of complex formation(DLLME)

Table.5: Comparison the values of Linearity, LOD and Recovery of the direct extraction and DLLME method with various methods reported in literature.

Ref.	Recovery	LOD mg\L	Linearityμg/ml	Method
11	100.113	2.5	8.0-50	Conductometric titration
21-22	98.70 ± 1.494	0.30	1.00 -10.00	Thin-layer chromatography
23-24	101.1 ± 0.3	0.76	100–800	voltammetry
25	ــــــــ	20	0.03–8	Flow injection
26	99.90	0.200	2 -30	UV-Vis Spectrophotometry
27-28	100.27-100.31	0.0557	10-70	UV-Vis Spectrophotometry
29	101.20	0.892	10-100	UV-Vis Spectrophotometry
Present work	97.88	0.34	1-20	Direct extraction
Present work	99.89	0.094	0.5-13	DLLME

3.5. Accuracy and precision for direct extraction and DLLME method:

The calibration curve for phenylephrine HCl was used to determine the concentrations in the proposed procedure. In order to obtain accuracy in direct extraction and DLLME, the average drug was obtained, proving that this process was very accurate. Relative standard deviation was calculated and the recoveries predicted accuracy and reproducibility, by using three different concentrations. The results were appropriate for the proposed method.

3.6. Comparison with literature studies:

The DLLME method and the direct extraction method were compared to previously used phenylephrine HCl determination methods. In terms of RSD, Recovery, LOD, and other aspects, Table 5 demonstrates that the proposed method may be compared to and approved with previous procedures.

4. CONCLUSION:

Simple direct and DLLME extraction based on the extraction of an intense yellow bicolor ionic compound between phenylephrine hydrochloride and Alizarin yellow reagent, combined with UV –Vis. Spectrophotometry. The suggested direct extraction and DLLME method have been successfully used to extract and quantify phenylephrine hydrochloride in both pure and pharmaceutical preparations.

5. ACKNOWLEDGMENTS:

The authors express their gratitude to the Pharmaceutical Chemistry Branch of the College of Pharmacy as well as the Chemistry Department of the College of Science, University of Anbar, for providing all laboratory equipment.

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Received on 13.06.2021 Modified on 29.07.2021

Research J. Pharm.and Tech 2022; 15(4):1648-1652.

DOI: 10.52711/0974-360X.2022.00275