A New Approach for Developing Spectrophotometric Determination of Phenylephrine Drug in Pure, Pharmaceutics and Serum Samples Using Sodium Periodate as Oxidizing Agent via a Green Method of CFIA/Merging Zone Technique

: The research involved a rapid, automated and highly accurate developed CFIA/MZ technique for estimation of phenylephrine hydrochloride (PHE) in pure, dosage forms and biological sample. This method is based on oxidative coupling reaction of 2,4-dinitrophenylhydrazine (DNPH) with PHE in existence of sodium periodate as oxidizing agent in alkaline medium to form a red colored product at ʎ max ) 520 nm( . A flow rate of 4.3 mL.min -1 using distilled water as a carrier, the method of FIA proved to be as a sensitive and economic analytical tool for estimation of PHE.


Introduction:
Phenylephrine hydrochloride C9H14ClNO2 (PHE.HCl) is (1R)-1-(3-Hydroxyphenyl)-2-(methyl amino) ethanol hydrochloride, as one can see in Fig. 1, is a white crystalline powder, belongs to phenethylamine class, with direct action at αadrenergic receptor agonists 1 .It is used topically as a decongestant and given orally, as drops or spray for the nose, in many cases including, for example: allergies, colds, flu, sinusitis, and nasal tumors benign 2,3 .There are different analytical techniques for determination of PHE in bulk form and dosage forms including, ion pair chromatography 4 , electrochemical 5 , titrimetry 6 , capillary zone electrophoresis 7 , high-performance liquid chromatography [8][9] , fluorescence 10 , voltammetry 11 , and spectrophotometry [12][13][14] .Spectrophotometry is paired with Flow Injection Analysis (FIA) and utilized as detectors in FIA system to detect certain samples and elements 15 .The proposed method of flow-injection analysis (FIA) is a technique that has high throughput sample performance with short time for analysis, economical, user-friendliness, green chemistry, accurate with remarkable reproducibility of the outcomes found, does not need further treatment for the samples as well as does not require an expensive or toxic reagent.PHE may be detected in many samples using inexpensive, automated, and user-friendly analytical techniques [16][17][18] .A green approach of CFIA /MZ technique for determining of PHE in pure, pharmaceutical formulations and biological samples is described in this manuscript based on oxidative coupling reaction with DNPH as organic reagent using NaIO4 as oxidizing agent, colored product is measured at λmax )520nm( .

Experimental Material and Reagents
All of the chemicals and reagents used were of analytical grade and they have been used to prepare all of the solutions.
 A stock solution of pure material of PHE.HCl 1000 μg.mL -1 kindly provided from State Company for Drug Industries and Medical Appliance, SDI, Samara, Iraq.It was prepared by dissolving 0.1gm in 100 mL with distilled water in a standard flask.The diluted solutions were made by diluting the stock standard solution with distilled water to the desired concentration. A2,4-dinitrophenylhydrazine solution (DNPH), 1×10 -2 M, M. wt =198.14 g.mol -1 (BDH), was prepared by dissolving 0.19 gm in 1 mL concentrated H2SO4, and stirred, then the compound was completed to 100 mL with distilled water in a standard flask and further dilution was made to these solutions to obtain desired concentrations. An oxidizing agent (NaIO4) solution 9.3×10 -3 M, M. wt =214 g.mol -1 (BDH), was prepared by dissolving 0.19 gm in 100 mL distilled water in a standard flask, more dilute solutions were prepared by dilutions with distilled water. Sodium hydroxide (BDH) 1M solution was prepared by dissolving 4 gm in 100 mL distilled water.More dilute solutions were prepared by dilutions with distilled water.

The Developed CFIA system
All absorbance in the batch procedure was measured by a Shimadzu UV-1800, (Japan) UV-Visible Spectrophotometer doubled ray and quartz cuvette with an optical longitude of 1 cm.The suggested FI manifold was developed as a simple type with one channel numerous in the FIA/merging zones system proposed approach 19 , as shown in Fig. 2. The carrier stream (distilled water) was pumped through the injection valve (six three-way injection valve, handmade) by peristaltic pump (Master flex C/L, two channel, USA), which travels at 90° and three Teflon loops (I.d =0.5 mm) into which the sample (L1), the reagent (L2), and the oxidizing agent and basic medium (L3) were loaded.The reaction coil is made of glass, and it is used to mix the ingredients (2 mm, I.D.).All absorbance and spectrum measurements were performed during the FIA processes using the modified Optima photometer 301-D+ (VIS-Spectro one beam) (Japan).The responses were expressed as peak height mV (n=3) and then were measured using a Kompensograph C1032 (Siemens) or a Chinese optical multimeter (DT9205A, OVA) for measuring absorbance.A flow cell quartz silica (QS, 1 cm) with an inner volume of 80 μL was placed inside in modified detection unit.

Preparation of Pharmaceutical Samples
Four pharmaceutical preparations were taken in the form of (syrup, tablet).1/Tussilet (syrup) 25 μg.mL -1 , which contains 2.5mg of PHE, made by (General Company for the Manufacture of Medicines and Medical Supplies / Samarra -Iraq).2/Astulet (syrup) 25 μg.mL -1 , which contains 2.5 mg of PHE, manufactured by: Aswar AL-khaleej company Samarra -Iraq (SDI).3/ Tussiram (syrup) 50 μg.mL - ,which contains 5mg of PHE, General Company for the Manufacture of Medicines and Medical Supplies / Samarra -Iraq.A stock solution (25,50 μg.mL -1 ) is generated by drawing up a certain volume (5,10 mL) from syrup solution that has been made up to 100 mL with distilled water.Pharmaceutical tablet solution was produced by weighing 20 tablets of drug, then crushed via motor.4/Nogrippin plus (pills) 100 μg.mL -1 (0.01 gm), that contains 10 mg of PHE, manufacturing: Biofarma ilac San.ve Tic.A.S. Sancaktepe / ISTANBUL.It was dissolved in 100 mL distilled water with whisking and then purified.The stock solution (50μg.mL - ) was made in 100 mL volumetric vessel from the stock solution of dosage form and complemented to the marked with D.W.

Serum Samples Preparation
Serum samples were obtained from healthy volunteers and centrifuged at 3000 rpm for 15 minutes to separate the serum from the blood.The serum was then diluted with 1 ml of distilled water and acidified with 1 ml of HNO3 (1M) to precipitate the proteins.For serum sample preparation, 10 μg.mL -1 was tested for accuracy and precision and analyzed thrice 20 .

Result and Discussion: Batch Method
Spectrophotometric determination of PHE based on oxidation of 1 ml 2,4-dinitrophenylhydrazine (1 ×10 - 2 ) M with drug 1ml from 100 μg.mL -1 in presence of sodium periodate (1ml, 9.3×10 -3 M) and 1mL NaOH (1N), were added in 10ml volumetric flask to form colored product (red), measured at λmax520 nm as seen in Fig. 3. and Scheme 1.The complexation ratio of reaction between drug with reagent at consistent sodium periodate and sodium hydroxide concentrations, using two ways was preceded by mole ratio and Job's method, it is clear that the PHE.HCl-DNPH complex link in is 1:1 ratio so the proposed mechanism below is likely to propose as be seen in Scheme1 and Fig. 4.

Figure 4. The complexation ratio between drug with reagent, A\ mole ratio for the complex, B\ job's method for the complex Preliminary Investigation
The effects produced on the absorbance strength of the colored product were observed as each condition was investigated by changing one variable while holding the other constant.The effect of DNPH volume was examined with 10 µg.mL -1 PHE.The volume was monitored that gave the highest absorbance was 2 mL of 1 × 10 -2 M DNPH and this volume was chosen for later adventures.The oxidative agent was examined and the best volume that gives the highest absorbance was 2 mL of 9.3 × 10 -3 M NaIO4, as shown in Fig. 5A-B.The influence of volume and various alkaline solutions type, including KOH, Na2Cr2O7, NH4OH, and NaOH, were tested because the colorful dye product could only be produced in an alkaline environment, when the reaction was conducted in the existence of sodium hydroxide solution did maximum sensitivity and stability occur, as be seen in Fig. 6A-B.

Calibration Curve of Classical Method
In order to estimate PHE, a standard curve with a linear range of 2-50 µg.mL -1 was created, as seen in Fig. 7. Based on the ideal conditions outlined in the established method, these measurements were made using two different PHE levels for precision and accuracy.The results, which are shown in Table 1, demonstrate that the suggested method does have good precision and accuracy (five times determination).

Calculations of Stability Constant
Two groups of solutions were used to determine an observed stability constant 21,22 for the suggested interaction (PHE: DNPH) the first group contained a stoichiometric quantity of PHE to DNPH (As), while the second contained a two-fold excess of DNPH (Am).The proposed mechanism and the drug-toreagent stoichiometry ratio 1:1.According to the following Eq. 1, the interaction between PHE and DNPH takes place: While K is the stability constant, C is the product's molar concentration (M), which is the same as the concentration of PHE (1×10 -2 M), (α) is the degree of dissociation.The absorbance readings of the aqueous solution, which contains an adequate and equivalent amount of reagent, are represented by Am and As. as be seen in Table 2.The spontaneous of complex formation reaction (ΔG value) was obtained based on K evaluation by using the Eq. 2.  = − ----2 (CFIA/ MZ) Spectrophotometric determination After determining the ideal cases for the reaction of PHE with DNPH using the traditional spectrophotometric method, the flow injectionmerging zones methodology was utilized to automate the spectrophotometric reaction in order to analyze the best practical settings and obtain automated spectra to estimate PHE drug.As a result, flow injection analysis technique was developed and employed for determining of PHE.HCl in pure, dosage forms and serum samples

Manifold of the Proposed FI System
The optimal design for a Homemade FIA system was investigated once the system and its linked components were installed, Fig. 2. depicts the developed system which consists of three loops (varying loop lengths with 0.5mm I.D.) that are filled with drug, reagent, oxidizing agent, and base using one line as carrier (distilled water) to a homemade injection valve according to merge materials and chemical reagents and in sequence PHE in L1, DNPH in L2 and NaIO4; NaOH in L3.

Physical Variables
For the reaction, the best loop volumes for the drug, reagent, oxidizing agent and base were (117.7-78.5-117.7)µL equal to 60-40-60 cm as shown in Fig. 9-A, the best reaction coil extent was 50 cm as appears in Fig. 9-B.The flow rates of available system were all examined, and the results indicated that 4.3 mL.min -1 , with an average sample throughput of 62, was the optimal flow rate hour -1 , as displayed in Fig. 9-C.The time it took to inject the solutions into each of the six-three-way valve loops 15 sec and the time it took for the highest peak height to appear 43 sec were used to compute the sampling rate, which came to 62 samples hour -1 .The purge time was calculated using optimal chemical and physical conditions, where different times were chosen, such as 5-10-15-20-25 seconds and an open valve 23 .By adding the sample segment, the purge time was estimated.Fig. 10 shows that the open valve was chosen as the best purge time for completing sample movement between the flow cell and the sample loop with minimal dispersion.

Dispersion of Sample Zone
In the FIA method, the sample interacts with several solutions and becomes scattered throughout the solution, which is a physical phenomenon known as dispersion.The success of the FIA analytical technique is founded on three ideas 24 .(Control of sample zone dispersion, reproducible injection time, and repeatable injection volume) are displayed in Fig. 11 and Table 3.The reaction's dispersion was 1.2 according to the formula D = Co/C, the dispersion was computed reaching the top when performing contact outside of the flow injection system, the peak without dilution is Co, but the peak after dilution is C. (interactivity inside the flow injection system).To get a fixed response stated as a percentage in the first experiment, all of the components were combined in the appropriate beaker, and the final solution was then administered by the flow injection mechanism (as carrier stream) (Co).The second experiment involved the injection of PHE into L1, DNPH into L2, and NaIO4, NaOH into L3.Distilled water is employed in the system as a carrier, and the injected component forces the ingredients into the reaction coil before pushing them toward the detector, creating a response symbolized by C.

Figure 11. Dispersion of PHE in the developed CFIA system
Calibration Curve A series of PHE concentrations from 1-300 µg.mL -1 were made by diluting stock solution (1000)µg.mL - and injecting it into the FI system with DNPH, NaIO4 and NaOH in order to determine the appropriate range of PHE concentration.It shows that the concentration range extends 5-300 µg.mL -1 , as shown in Fig. 12 and Table 4.

Analysis of Variation and Repeatability
The assumed error was calculated, called-for regression, and the sum of the squares of the difference between the response's (yi) and the appraiser's ŷi values to obtain (yi -ŷi) 2 for (n-2) degrees of freedom to yield the sum of squares (S2) 2 .
The sum of squares of the variance was calculated of values ŷi from the average value ӯ (due to regression), and for 1 of degrees of freedom, get sum of squares (S1) 2 , then divide by (S2) 2 , to get value (F) 25, 26 , as shown in Table 5.

Table 5. ANOVA for the developed FIA technique.
The repeatability of the proposed method was acceptable as shown in Table 6.

Methods Validation
The analytical characteristics of each approach, including the detection limit, correlation coefficient (r), relative standard deviation, and linear range, were determined at the optimal condition 27,28 as shown in Table 7. Numerous PHE standard solutions were used to construct calibration curves, and the technique's main analytical figure of deserts was used.Fig. 12. A, statistical treatment of the regression line provided the standard deviation for the residuals (Sy/x), slope (Sb), and intercept (Sa) within 95% confidence intervals for (n-2) degrees of freedom.The proposed CFIA technique superior reproducibility in comparison with batch method was demonstrated to the small subjects because of the current study was completed quickly with high sensitivity (62 samples were analyzed in one hour), as well as simple and easier.

Study of interferences
CFIA/MZ approach was investigated and by using this method, the effects of (cellulose, glucose, sucrose, lactose and sodium citrate) on the accuracy of PHE determination were evaluated.A pure 20 µg.mL -1 PHE sample was spiked with a half, equal, and double excess of a number of interference excipients before analysis was performed on it.between the proposed technique with official method (British pharmacopeia) 1 .A spiked human serum sample's PHE can be calculated with success using the FIA method.A 10 µg.mL -1 PHE tested for accuracy and precision.There were three examinations of each concentration.The serum samples' repeatability is shown in Table 10 to be adequate.

Conclusions:
Through the search in scientific journals specialized in the field of continuous flow injection analysis, no researcher determined the drug PHE in the pure form, pharmaceutical, and biological samples, so the idea of completing the research for the semi-automated determination of phenylephrine hydrochloride using the developed system of CFIA/MZ technology, which is one of the methods of green chemistry.The sensitive spectrophotometric quantification of anti-allergic drugs in medications and serum samples via CFIA design is recommended as a study approach for our work, which is why a research plan was provided for this manuscript.It is distinguished by having a larger calibration range and a high sample rate (s/h).These methods can be used to calculate the amount of PHE in µg.mL - 1 indigence for the prior divorce action, heating or prepping the piece, or extraction using a solid phase.The capital benefits of the CFIA technique include it's a wide working range, reasonable sensitivity, and applicability for routine assessment in pharmaceutical quality control laboratories.This is because, in comparison with other methods and the official standard methods, they reduce reagent waste and the toxicity of organic reagents 30 .

Figure 2 .
Figure 2. The developed of FI system for determination of PHE.HCl in dosage forms and biological samples.

Figure 5 .
Figure 5.Chemical parameter for batch A/ volume of DNPH, B/volume of.NaIO4.

Figure 6 .
Figure 6.Chemical parameter for batch A/ volume of NaIO4, B/ Type of base.

Figure 7 .
Figure 7. Linear calibration curve for the batch method of PHE drug determination.

Figure 9 .
Figure 9.Effect of: A\ Injected volume, B\ Reaction coil, C \ Total flow rate.Purge Time

Figure 12 .
Figure 12.Linear dynamic range for spectrophotometric determination of PHE using the developed CFIA system