Available online on 15.06.2022 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2011-2022 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access   Full Text Article                                                                                                                                                                            Research Article 

New Smartphone Based Colorimetric Method Development and Validation of Drugs Containing Nitrogen, Phosphorus and Sulphur

Tejasbharthi Pravinbharthi Goswami*  , Apeksha Kadam  , Rajashree Mashru  

The Maharaja Sayajirao University of Baroda, G.H Patel Pharmacy building, Donor’s Plaza, Fatehgunj, Vadodara 390001, Gujarat, India

INTRODUCTION:

Introduction of Amoxicillin (Sulphur atom containing drug): 

Amoxicillin is a semisynthetic, acid stable antibiotic that belongs to the Penicillin (beta lactam antibiotics) class of medicines. 

In both humans and animals, it has been found to be effective against a wide range of illnesses caused by a wide spectrum of Gram positive and Gram-negative bacteria. 

It is a congener of ampicillin semisynthetic amino penicillin) that differs only in the hydroxylation of the phenyl side chain from the parent medication. 

After oral administration, it has found a niche in the treatment of ampicillin-resistant infections.

Figure 1: Chemical structure of amoxicillin ¹

Amoxicillin is a chemical compound that is used to treat infections (2S,5R,6R)-6-[[(2R)-2-amino-2 -(4-hydroxyphenyl) acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid is a kind of heptane-2-carboxylic acid.^1,2Amoxicillin trihydrate have various HPLC methods^3–6, UV spectroscopic methods^7,8, colorimetric methods^9–11 and HPTLC methods^12–14 have been reported for estimation alone or in combination of other drugs. 

Introduction of silodosin (Nitrogen atom containing drug):

Figure 2: Structure of silodosin⁴¹

Silodosin is a 1A-adrenoceptor antagonist that has been licenced to treat the signs and symptoms of benign prostatic hyperplasia.¹⁵

The three a1-adrenoceptor subtypes have quite different functions: Prostate contraction is mediated mostly by a1A receptors, whereas the human urethra has exclusively a1A receptors, whereas a1A, a1B, and a1D all mediate blood vessel dilation. 

As a result, compared to less selective α-adrenoceptor blockers, silodosin's strong selectivity for the a1A subtype should result in greater cardiovascular tolerance without a loss of efficacy on urinary tract symptoms. Indeed, both in the animal model and in investigations with healthy individuals, insignificant effects on the cardiovascular system have been found. In dogs given oral dosages of silodosin up to 200 times the normal therapeutic dose indicated for humans, there were moderate effects on blood pressure but no effects on cardiac repolarisation. In healthy individuals given silodosin at a dose three times larger than the normal therapeutic dose, no significant changes in heart rate, pulse rate, or QRS interval duration were found (24 mg).

Silodosin have various HPLC methods^16–18 UV spectroscopic methods^19,20, colorimetric methods and HPTLC methods²¹ have been reported for estimation alone or in combination of other drugs. 

Introduction of sofosbuvir (Phosphorus atom containing drug):

Sofosbuvir is a phosphoramidate prodrug that is converted into the potent antiviral agent 2'-deoxy-2'-a-fluoro-b-C-methyluridine-5'- monophosphate in the liver.

Although phosphoramidate prodrugs have been researched as a technique of boosting nucleoside potency in cell culture by raising the concentration of active nucleotide, at the time of sofosbuvir's discovery, no phosphoramidate prodrug technology had been applied in the treatment of HCV. The triphosphate derivative of b-D2'-deoxy-2'-R-F-2'-b-C-methyluridine, a uridine nucleoside, was discovered to be a particularly effective inhibitor of HCV protease NS5B in previous research. 

Figure 3: Structure of Sofosbuvir²²

The monophosphate derivative of b-D-2'-deoxy-2'-R-F-2'-b-C-methyluridine can be converted to the triphosphate derivative enzymatically in HCV replicon cells.

Intracellular monophosphorylation of b-D-2'-deoxy-2'-R-F-2'-b-C-methyluridine, on the other hand, does not happen. As a result, the monophosphate derivative of b-D-2'-deoxy-2'-R-F-2'-b-C-methyluridine was chosen as a candidate for therapeutic development.

The development of the phosphoramidate prodrug sofosbuvir was based on the hypothesis that first-pass metabolism would produce the required triphosphate antiviral nucleotide at the targeted site of action, the liver²².

Sofosbuvir have various HPLC methods^23–25 UV spectroscopic methods^26,27, colorimetric methods and HPTLC methods^28–30 have been reported for estimation alone or in combination of other drugs. 

For work in the visual region the type of instrument largely used consists of a light source, a monochromator, a photometer, an eyepiece for observing the photometric field, and a holder for the sample. The holder is a cell for transmission measurements of a liquid, or a device for supporting ran opaque object on which reflection measurements are to be made. Spectrophotometric measurements are not limited to coloured systems, unlike chemists' "colorimetric" results. For many years, photographic methods were used to determine absorption spectra in the ultraviolet and infrared parts of the spectrum.

A spectrophotometer's fundamental data shows the proportion of light incident on a sample that is reflected or transmitted by it. For a particular wave length, a single value may be obtained, or values may be determined to cover the entire visible range. The results in the latter situation are usually displayed as a curve, with transmission or reflection as the ordinates and wave length as the abscissas. When creating a curve that covers part or all of the visible range, the question of what wave-length interval to employ to define individual points and what spectral band width to use for the light source arises. If somehow the curve is steep and has small sharp absorption bands, the points may need to be taken at every millimicron with the narrowest feasible spectral band.³¹

Colorimetric analysis is a handy method for determining the concentration of coloured substance in a solution. Light in the visible spectrum is absorbed by coloured substances, and the amount of light absorbed is proportional to the concentration of the substance in solution.³²

Due to their ease of use and adaptability to portable equipment, colour shifts recorded using Smartphone-based sensors are attracting significant interest in chemical investigation. Smartphones have gained popularity as analytical instruments because they are widely available at a low cost and allow data gathering, storage, and processing all in one device. The mobile camera is used as a detector in smartphone colorimetry.³³

A variety of smartphone-based colorimetric applications are available. One of them is Photo Metrix-PRO. Photo Metrix PRO was free to download from the Windows Phone Store and the Google Play Store. For univariate analysis, this programme uses basic linear correlation, and for multivariate exploratory analysis, it uses principal components analysis (PCA). The image data is taken by the smartphone camera and transformed into RGB histograms (red, green, and blue).³⁴

The RBG colour model is based on a colour perception hypothesis in which the human eye has various sensitivity peaks located around red, green, and blue. Multivariate analysis (e.g., partial least squares, PLS) could be used in this software to improve Colorimetry's RGB colour system applicability.³⁵

The colour intensity is proportional to the concentration of the substance that is being measured. The visible band of light in the electromagnetic spectrum has a wavelength of 400 nm to 800 nm. A colorimeter/visible spectrophotometer is a device that measures the absorbance of a given wavelength of light to determine the concentration of a solution. When choosing a reagent for colorimetric analysis, consider its specificity and sensitivity.³⁶

This procedure necessitated the use of sophisticated tools. The goal of this study is to establish a simple, cost-effective method for estimating amoxicillin trihydrate. 

The method uses ammonium metavanadate as a colouring ingredient, which reacts with sulphur containing amoxicillin trihydrate³⁷Nitrogen containing Silodosin and Phosphorus containing Sofosbuvir^38–40 to produce a green colour. Photo Metrix-PRO application captured and analysed the data image.

MATERIAL AND METHOD:

Chemicals and reagents:

5% Ammonium Metavanadate solution, amoxicillin trihydrate, silodosin, Sofosbuvir, double distilled water, 0.1M H₂SO₄, 40% H₂SO₄.

Apparatus and Applications:

The API samples were weighed on an electronic balance (A×120) (Shimadzu). Smartphone camera and uploaded to the mobile (photometrix Pro) Application.

Preparation of 0.1M H₂SO₄:

To make 0.1M solution, slowly add 0.136ml of 98% H₂SO₄ to around 6.25ml of double distilled water. Adjust the final volume of solution to 25ml with double distilled water.

Preparation of 5% ammonium metavanadate reagent:

Weigh about 5gm of ammonium metavanadate reagent in 100ml of 40% H₂SO₄ and heat on water bath until solid residue dissolve.

Preparation of standard stock solution:

Weigh about 10mg of Amoxicillin trihydrate, silodosin and sofosbuvir then transferred all of them into previously calibrated 10ml volumetric flasks respectively. The final volume was made up to the mark using 0.1M H₂SO₄ to obtain the standard stock solution of 1000µg/ml concentration each.

Method development:

UV-Vis. spectroscopy:

Selection of wavelength:

Using ammonium metavanadate as a blank, the drug solution was scanned across the range 400-800nm. Amoxicillin trihydrate, silodosin and sofosbuvir was found to have an absorbance of 762nm. Prepare a calibration curve using the working solution, ranging from 30-150μg/ml for amoxicillin trihydrate, 20-100 μg/ml for silodosin and sofosbuvir.

Reaction mechanism:

Ammonium metavanadate is inorganic oxidizing agent. The vanadate has oxidation states in its compound of +5, +4, +3 and +2. The usual source of vanadium in the +5-oxidation state is ammonium metavanadate. The reaction for oxidation of Amoxicillin trihydrate, silodosin and sofosbuvir were done in acidic medium. Heat is given during chemical reaction to prevent reoxidation. Ammonium metavanadate is orange red color complex but when it reacts with Amoxicillin trihydrate, silodosin and sofosbuvir individually it forms green color complex.

Oxidation state: From +5 it comes to +3 of vanadium.

Method optimization:

Optimization of regent concentration for the drugs:

Ammonium metavanadate was allowed to react with Amoxicillin trihydrate, silodosin, sofosbuvir to form a green colour with absorption maxima at 762nm, by keeping another parameter constant. The optimization of the experiment was established by varying the concentration of reagent in the range of 1.25%–10%, where, maximum absorbance of reagent was found at 5%; as shown in Table 1.                                                              

Table 1: Optimization of reagent concentration

Optimization of reagent volume for Amoxicillin trihydrate:

The effect of reagent volume was carried out in range from 0.25-6ml. From green colour complex and absorbance maxima optimized volume was selected. A linear increment in absorbance was observed with the increase in volume of reagent up to 4ml. However, above its linearity was disturbed and hence 4ml was selected as optimum reagent volume. 

Table 2: Optimization of reagent volume for Amoxicillin trihydrate

Optimization of reagent volume for Silodosin: 

The effect of reagent volume was carried out in range from 2-6ml.Optimized volume was selected taking into consideration green colour complex & absorbance maxima. 4ml of reagent volume was selected for method.

Table 3: Optimization Of volume reagent for silodosin

Optimization of reagent volume for sofosbuvir:

The effect of reagent volume was carried out in range from 3-7ml. Optimized volume was selected taking into consideration green colour complex & absorbance maxima. 5ml of reagent volume was found to be optimum for method. 

Table 4: Optimization Of volume reagent for Sofosbuvir

Optimization of reaction time for amoxicillin trihydrate:

The effect of reaction time was carried out in from 10-50 min. From colour complex reaction observed between 10- 50 min, slight increase in colour intensity was observed at 30 minutes (Figure 4).

Figure 4: Optimization graph of reaction time for amoxicillin trihydrate

Optimization of reaction time for silodosin:

The effect of reaction time carried out in from 10-50 min. From colour complex reaction observed between 10- 50 min, slight increase in colour intensity was observed at 30 minutes (Figure 5).

Figure 5: Optimization graph of reaction time for silodosin

Optimization of reaction time for sofosbuvir:

The effect of reaction time carried out in from 20-60 min. From colour complex reaction observed between 10- 50 min, slight increase in colour intensity was observed at 50 minutes (Figure 6).

Figure 6: Optimization graph of reaction time for sofosbuvir

Preparation of calibration graph: 

Aliquots of standard solution of Amoxicillin trihydrate corresponding to 30-150 µg/ml, Silodosin and Sofosbuvir corresponding to 20-100 µg/ml were taken into 10ml volumetric flask. To each flask 4 ml of 5% Ammonium metavanadate reagent was added and solutions were heated on water bath for respective time for each as mentioned above. The solution was allowed to cool at room temperature and then volume was made up to 10 ml with distilled water. The absorbance of the solution was measured at 762 nm against blank.

Estimation of Amoxicillin Trihydrate Using Smartphone Application: 

Experimental Setup: 

The coloured solution was transfer into slandered glass cuvette which was placed in 18cm×18cm of white box and 6W LED (Light Emitting Diode) bulb was connected to control the intensity throughout the experiment shown in Figure 7.

Figure 7: Experimental set up

The image of a colour complex solution was taken with a smart phone and analysed using a photometric application to determine the red-green-blue intensities (RGB scale) of the image. The concentration of the image taken by PhotoMetrix PRO was estimated using a linear regression equation. PhotoMetrix creates and analyses colour histograms on RGB scales, which it then converts into a calibration curve. 

Figure 8: Steps for run the photometrix pro application

Using univariate and multivariate analysis, this programme processes and displays the results. For the best results, many smartphone types were used. Figure 8 depicts the steps for utilising the PhotoMetrixPRO application.

Method validation: 

According to validation requirements, the UV–visible spectrophotometry and PhotoMetrix applications were separately validated in terms of linearity and robustness. For both approaches, a formulation assay was carried out. Under optimal conditions, excellent linearity of Amoxicillin trihydrate was reported in the range of 30-150 µg/ml, Silodosin and sofosbuvir was reported in the range of 20-100 µg/ml. In the case of UV-vis spectrophotometry, the concentration of tablet formulation was calculated using a regression equation, while photometrix was calculated within the programme.

RESULT AND DISCUSSION:

Method Validation: 

Amoxicillin trihydrate was linear with the concentration range of 30-150 µg/ml at 762 nm, Silodosin and sofosbuvir were linear with the concentration range of 20-100 µg/ml at 762 nm, by obeying Beer’s law (Figure 9). A calibration curve was plotted between concentration Vs absorbance. The plot was found to be linear (Figure 10).

Figure 9: linearity of Amoxicillin trihydrate(a), Silodosin(b), Sofosbuvir(c)

Figure 10: Calibration graph for Amoxicillin trihydrate(a), Silodosin(b), Sofosbuvir(c)                                  

The precision of an analytical method refers to the degree of agreement between a set of measurements acquired by sampling the same homogeneous sample many times under the method's specified circumstances. The intraday (Repeatability) and interday precision were calculated here. Three-concentration samples with lowest, upper, and middle limits of both medicines were taken and analysed three times at the same concentration level on the same day for intra-day precision and three times on three different days for inter-day precision for three drugs. The percent RSD of Amoxicillin trihydrate( Table 5), Silodosin(Table 6) and Sofosbuvir(Table 7) was calculated and determined to be less than 2.

Table 5: Intraday and Interday precision of Amoxicillin trihydrate

Table 6: Intraday and Interday precision of Silodosin

Table 7: Intraday and Interday precision of Sofosbuvir

Recovery tests were used to determine the method's accuracy. At 80 percent, 100 percent, and 120 percent, a known quantity of the pure drug was added to the pre-analysed sample formulation. The percentage recovery and percentage relative standard deviation of the percentage recovery of different formulations were determined and are shown in Table 8 (Amoxicillin trihydrate), Table 9 (Silodosin) and Table 10 (Sofosbuvir).

Table 8: Accuracy data for marketed formulations of Amoxicillin trihydrate

Table 9: Accuracy data for marketed formulations of Silodosin

Table 10: Accuracy data for marketed formulation of Sofosbuvir

The assay of different formulations was determined which falls within the acceptance criteria (98-102%) for Amoxicillin trihydrate (Table 11), Silodosin (Table 12) and Sofosbuvir (Table 13).

Table 13                         

Table 11: Assay results for different marketed formulations of Amoxicillin trihydrate

Table 12: Assay results for different marketed formulations of Silodosin

Table 13: Assay results for marketed formulation of Sofosbuvir

The specificity was done by using the blank and marketed formulation which having excipients and 100μg/ml solution was prepared from the marketed formulation. The specificity of the method is demonstrated in following Figure 11 for all the three drugs in which graphs shows the specific absorbance at 762 nm for each. Hence, it can be concluded that this method is specific.

Figure 11: Specificity indicating graph of Amoxicillin trihydrate(a), Silodosin(b), Sofosbuvir

The ruggedness of the developed method was studied in two labs as well as with the use of two different smartphones. The %RSD of both these parameters was found to be less than 2 as shown in the Table 14(Amoxicillin trihydrate),Table 15(Silodosin) and Table 16(Sofosbuvir).

Table 14: Method Ruggedness Result for Amoxicillin trihydrate

Table 15: Method Ruggedness Result for Silodosin

Table 16: Method Ruggedness Result for Sofosbuvir

Estimation of Amoxicillin trihydrate using Smartphone application: 

By using PhotoMetrix PRO application the image was captured and according to concentration which shows the gradients of colours for all the three drugs (Figure 12). The linear regression equation was observed (Figure 13). Regression equation data of both methods for all the three drugs shown in Table 17. 

Figure 12: Chart of colour intensity corresponding to the concentration of amoxicillin trihydrate(a), Silodosin(b), Sofosbuvir(c)

Figure 13: Calibration curve of the amoxicillin trihydrate(a), Silodosin(b), Sofosbuvir(c) by photometrix pro application

Table 17: Regression data for both UV and photometrix application

The linearity of the standard amoxicillin trihydrate was taken in the range of 30-150 µg/ml, Silodosin and Sofosbuvir 20-100 µg/ml. The calibration curve and regression equation generated by the application was shown in the Figure 13. The % assay was found to be within the acceptance criteria. 

Assay of formulation: 

The assay was performed on the different marketed formulations by both the methods. Sample solutions were analysed and concentration was estimated as a % Recovery from linear regression equation. Assay results were found to be in acceptable range and significant for both the methods. Results of assays are shown in Table 18.

Table 18: Assay results of different formulation for both the methods

Statistical Comparison of two methods: 

The obtained assay results from the PhotoMetrix application and the UV technique were compared using a paired t-test (two tails). Using a t-test, it was discovered that tstat values were lower than tcritical values and P values were higher than the applied alpha value (*P>0.05). It signifies that there is no discernible difference between the approaches means. As a result, the PhotoMetrix application can be used to identify Amoxicillin trihydrate, Silodosin as well as Sofosbuvir using colorimetry. Table 19 displays the information.

 Table 19: Applied Pair t-Test Result

CONCLUSION: 

Ammonium metavanadate is reagent used as an oxidizing agent and it converts +5 to +3 oxidation state, here we utilizing its oxidation power as a colorimetric reagent which shows the significance colour change in presence of acidic medium and heat it gives the colour when reacting with compound containing sulphur, nitrogen and phosphorus atom in its structure. The smartphone-based PhotoMetrix PRO application is used to develop a novel and cost-effective colorimetric detection method for Amoxicillin trihydrate, Silodosin and Sofosbuvir. The approach relied on a basic colouring ingredient and a quick operation. The main aim of this work was to make the colorimetric measurement of drug content easier with the use of such smartphone-based applications. The approach was also compared to a UV method created using the same reagent and protocol, and no significant differences in assay results were identified. In quantitative drug estimate in pharmaceutical dosage forms, this unique method can be utilised as an alternative to analytical science.

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