Neonatal seizures belong among the most common serious neurological disorders worldwide . Although there are several anti-seizure drugs available, phenobarbital still remains the first-line agent for the treatment of neonatal seizures. The drug has several favorable features including the undisputed efficacy against a broad spectrum of seizure types, low risk of serious acute adverse drug reactions, multiple pathways involved in the drug elimination as well as the availability of parenteral drug formulations and their low cost . Phenobarbital could have synergistic neuroprotective effects when applied with therapeutic hypothermia . There are concerns that phenobarbital may negatively impact psychomotor development and neurological outcomes . A few relatively small studies have indicated the possibility that phenobarbital may affect long-term neurodevelopmental outcomes if the drug was administered either in early childhood for the treatment of febrile seizures or prenatally in gestational medication of their mothers [5–7]. Phenobarbital (5-ethyl-5-phenylpyrimidine-2,4,6 (1H,3H,5H)-trione)  is used to therapy anxiety and drug withdrawal and to help with surgery , as well as to be used in the treatment of all types of seizures except absence seizures . Molecular weight is 232.235g/mol. Several analytical methods have been proposed for determining phenobarbital such as spectrophotometric , chromatographic [11-15] and voltametric [16-18] methods. It is white or almost white, crystalline powder or colorless crystals, very slightly soluble in water, freely soluble in alcohol. It forms water-soluble compounds with alkali hydroxides, carbonates and with ammonia. Figure 1 shows the chemical structure of the drug .
Nanotechnology is a vast and fast emerging area of scientific research studies resolving several complications related to conservative medication therapies, including underprivileged water solubility, lack of capability to target the problematic cancerous cells in individual bodies, common spreading, universal poisonousness as well as weak therapeutic capabilities. Nano-liposomes are nanometer-scale liposomes that are one of the most useful drug delivery systems in the field of drug release and retention. Important reasons for the use of nano-liposomes in the pharmaceutical industry are their similarity to cell membranes and trapping of hydrophobic and hydrophilic substances, drug delivery to the target tissue, control of drug flow in the bloodstream and good biocompatibility. Another important feature of nano-liposomes is the coating of water-soluble drugs in the central aqueous portion and fat-soluble drugs within its bilayer membrane. Nano-liposomes can be effective in reducing drug toxicity and increasing drug efficacy [19-22]. Gold nanoparticles (AuNPs) have several biomedical applications in diagnosis and disease treatment such as targeted chemotherapy and in pharmaceutical drug delivery due to their multifunctionality and unique characteristics .
The aim of the present study was developed a simple and economy method for determining phenobarbital, its raw pure state and in pharmaceutical preparation. The proposed method was based on the chelating complex formation.
Materials and methods
A Shimadzu UV-Visible-1650–Japan double beam spectrophotometer with 1cm matched quartz cells was used for all spectral measurements. pH meter, Jenway-3310- England. Sensitive balance with four digit, Sartorius- Germany, the Ultrasonic water bath, LabTech – Korea.
All the utilized chemicals were used by analytical grade, SDI Samarra-Iraq Donated by a PHB and BARABITAL, DSH from BDH, AuCl3 from Sigma Aldrich, while KOH from GCC.
The standard solution of phenobarbital (PHB) (100 μg/ml)
In volumetric flask, 0.1 g of phenobarbital in a 100 ml was added and dissolve it in 100 ml of deionized water so that the solution concentration is 1000 μg/mL as a stock solution, of which the working solution was prepared at a concentration of 100 μg/mL by withdrawing 10 mL of stock solution and diluting it with deionized water in a 100 mL volumetric flask up to the mark with the same solvent.
Preparation of reagent solution 2,6-dichloroindophenol sodium salt hydrate (DSH) (100 μg/ml)
0.01 g of reagent was dissolved in a 100 mL volumetric flask by deionized water and completed the volume up to the mark with the deionized water.
Preparation of gold trichloride (AuCl3) solution (0.1318M)
0.1 g of gold trichloride was weighed in a 25 mL volumetric flask and dissolved in the deionized water and the volume was completed to the mark.
Preparation of KOH (0.1M)
0.5610 g of KOH was dissolved in a 100 mL volumetric flask with distal water up to the mark with the same solvent.
Preparation of the pharmaceutical preparation BARABITAL (150μg/mL)
10 tablets of the pharmaceutical preparation (supplied from SDI Samarra-Iraq) were weighed and crushed well, taking the equivalent of 0.0681 g weight of one tablet, which contains 15 mg of phenobarbital. The powder was dissolved in 10 mL ethanol in a 100 mL volumetric flask and filled the volume up to mark with deionized water to yield a solution of phenobarbital concentration as 150 μg/mL.
Preparation of gold nanoparticles
20 gm of parsley was weighed after being washed with deionized water and dried, then it was placed in a beaker and immersed in 120 ml of deionized water and boiled for 20 minutes. The extract (brown in color) was filtered and stored in a conical flask. Figure 2 displays the spectrum of parsley extract.
In a 100 mL beaker, 10 mL of AuCl3 gold trichloride solution (0.1318M) as well as 30 ml of parsley extract were added, then the obtained solution placed on a hot plate stirrer at 100 °C, and the magnetic stirrer was set at 400 rpm.
After 10-15 minutes, the color changed from brown to purple, which is evidence for the formation of gold nanoparticles and their reduction by the vital-content of parsley .
The solution was placed in a centrifuge at 3500 rpm for 20 minutes, where the precipitate was collected at the bottom of the test tube and washed several times with deionized water.
The precipitate was placed on a watch glass and dried using a drying oven at 40 °C for 12 hours. The precipitate was stored in a dark place.
0.01 gm of the obtained precipitate was weighed and dissolved with deionized water in a volumetric flask of 100 mL capacity, and completed the volume to the mark so that it was for sonicated for 10 minutes to obtain 100μg/ml from gold nanoparticles solution.
A scan was carried out for wavelengths between 800-200 nm and the absorption was recorded at the maximum wavelength λmax at 556 nm as indicated in Figure 3.
Results and discussion
Diagnostics of prepared gold nanoparticles
TEM technique was used to examine, diagnose and determine the shape and size of gold nanoparticles, as the results show that the particles were spherical in shape, of different sizes and at different power’s magnifications (50,100 nm) they appeared in the form of dark dots and were within the nano-size, as depicted in Figure 4.
The prepared gold nanoparticles was identified using the Energy-dispersive X-ray spectroscopy (EDX) technique to know the contents of the sample from the elements and through the peaks of the ray energy, as shown in Figure 5. The presence of quantities of carbon and oxygen indicates the formation of the oceanic network of gold particles and also of hydrocarbon compounds, and the presence of chlorine is due to the formation of golden chloride.
XRD analysis of (AuNps) Crystalline nanoparticles represented by five our peaks corresponding to standard Bragg reflections 111, 200 ,220, 311 and 222. The intense peak at 38.3 (111). Figures 6 and 7 display XRD diffraction and IR spectrum analysis of (AuNps) Crystalline nanoparticles, respectively.
Preparation of phenobarbital-gold nanoparticles complex (AuNPs)
0.5 mL of KOH (0.1M) was added to 1 mL of PHB (100 μg/mL) and 0.2 mL of DSH reagent (100 μg/mL), and then 1 mL of gold nanoparticle solution (100 μg/mL) was added, as well. The color of the resulting solution was greenish blue and the maximum wavelength of the resulting complex is fixed at 600nm.
Setting optimum conditions
1- Effect of base quantity
Different volumes of KOH solution (0.1M) were added from 0.1-1 mL to 1 mL of PHB (100 μg/mL) and 0.2 mL of the reagent solution and 1 mL of gold nanoparticle solution and the absorbance was measured at 600nm wavelength as shown in Figure 8. It was found that 0.5 mL from KOH exhibited the best absorption.
2- Optimum reagent concentration
A study was conducted to amount of DSH reagent solution giving the greatest absorption of the colored product, as 0.5ml of (0.1M) KOH was added to 1ml of PHB (100 μg/ml) and increasing volumes of 0.1-1 mL of DSH reagent solution (100 μg/mL) and 1ml of gold nanoparticles solution, and as the results indicates, it is evident that 0.5 mL of the reagent is the optimum volume as this volume was used in subsequent experiments, as depicted in Figure 9.
3- Effect of gold nanoparticles concentration
1 mL of the PHB was put into a series of 10 mL volumetric flask, 0.5 mL of (0.1M) KOH and 0.5 mL of (100 μg/mL) DSH reagent solution and the increasing volumes of gold nanoparticles solution (0.5-5 mL) were added, then the absorbance was measured at 600nm. It was found that the volume of 2.5 mL of gold nanoparticle solution gave the highest absorbance, which was adopted in the subsequent experiments, as displayed in Figure 10.
4- The effect of temperature
Using a water bath, the effect of temperatures from 15-60 °C on the formation of the complex was studied. Through the optimal conditions obtained from previous experiments, it is clear in this study and as depicted in Figure 11, that the best absorption is at a temperature of 30-40 °C.
5- Addition sequence effect
It was noted from the obtained results indicated in Table 1 that the following sequence as: Drug + Base + Reagent + Nano-gold solution, respectively gives the highest absorption after heating the complex to a temperature of 40˚C. Therefore, this sequence was followed in subsequent experiments.
6- Measuring of complex stability
It was experimentally found that the absorption of the complex stabilizes after 5 minutes and remains stable for 45 minutes, as shown in Table 2.
Final absorption spectrum
After stabilizing the optimal conditions, 0.5 mL of KOH (0.1M), 0.5 mL of DSH reagent (100μg/mL) and 2.5 mL of gold nanoparticle solution, the absorption of the formed colored product was measured against blank solution in the wavelength range between 200-800 nm, and it was found by the results obtained in Figure 12 that the wavelength of the highest absorption is 600 nm.
The calibration curve
Increasing volumes (0.1 - 6 mL) of PHB (100μg/mL), 0.5 mL of KOH (0.1M), 0.5 mL of DSH reagent solution (100μg/mL) and 2.5 mL of (100μg/mL) of gold nanoparticle solution were added to a series of 10 mL volumetric flask, filled to the mark with deionized water, and after the solution was heated to 40 ˚C, the absorption was measured against the blank at length. The 600 nm wavelength and Figure 13 indicate the linear calibration curve which shows that Beer's law was followed at a range of concentrations (5-45μg/mL) of the drug. The molar absorption of the resulting compound was calculated and found to be 2368.797 L mol-1.cm-1. Sandal’s significance is 0.09803 μg.cm-2.
Accuracy and precision
The accuracy and precision of the method were tested, since the recovery percentage (Rec%) and relative standard deviation (RSD%) values were 100.021-98.971% and 0.1276-0.3215%, respectively. These values demonstrated good accuracy and precision, as indicated in Table 3.
The direct method
The direct method was applied to three concentrations of the pharmaceutical preparation BARABITAL (15mg), which are 15, 25, 35 μg/mL and for six replications for each concentration. The results are indicated in the Table 4.
Multi standard additions method
The drug has been estimated in pharmaceutical preparation (BARABITAL 15mg) by standard additions method as depicted in Figure 14 and Table 5.
Stoichiometry of the reaction
Under the optimum conditions, the stoichiometry of the reaction between PHB and AuNps were investigated by continuous variations methods. The ratio was found to be 1:1, as displayed in Figure 15.
Suggested reaction can be as in the following equation in Scheme 1.
The proposed method was compared with another spectroscopic method, as depicted in Table 6.
The developed method is simple and sensitive which is used to determinate Phenobarbital in its pure raw material form and pharmaceutical product and pharmaceutical form. This method included the reaction of PHB with DSH reagent to form a ligand that reacted with gold nanoparticles in alkaline medium to give blue-green colored chelate complex having maximum absorption at 600nm. The results obtained showed the percentile recovery values, the relative standard deviation, the detection limit, and the quantitative limit that the method is accurate and precision, which indicates the success of the proposed method for determinate PHB.
This research was supported department of Chemistry, College of Education, University of Samarra.
Eman Thiab Al Samarrai: https://www.orcid.org/0000-0002-1970-0889
Othman Rashid Al Samarrai: https://www.orcid.org/0000-0002-1487-4054
How to cite this article: Eman Thiab Al Samarrai, Liqaa H. Alwan, Suha Abdullah Hussein Al-Haddad, Mustafa Hamed Al Samarrai, Muhannad Salim Miteb Al-Obaidi, Othman Rashid Al Samarrai. Spectrophotometric determination of phenobarbital in pharmaceutical preparation using gold nanoparticles. Eurasian Chemical Communications, 2022, 4(9), 812-825. Link: http://www.echemcom.com/article_148641.html
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