Three Simple and Economic Spectrophotometric Methods for Simultaneous Determination of Chitosan and Ascorbic Acid

Three simple, precise, and sensitive UV spectrophotometric methods have been developed and validated for the determination of Chitosan (CH) in the presence of Ascorbic acid (AA) in a binary mixture. Method A is a derivative ratio method ( 1 DD) that measures the peak amplitude of the first derivative for CH and AA at 207.2 and 282 nm; respectively. Method B is a ratio difference method (RDSM) that measures the peak amplitude difference of the ratio spectra (P204.6202.9) and (P268-230) for CH and AA; respectively. Method C is a mean centering of ratio spectra method (MCR) that measures CH and AA at 204.6 and 269.1 nm; respectively. Method A, B, and C were able to successfully determine the studied drugs in a concentration range of 0.5-9 g/mL for CH and 2-20 g/mL for AA. All results were statistically compared with the reported methods, where no significant difference was observed. The proposed methods were applied to the analysis of the studied drugs in pure and pharmaceutical formulations.


INTRODUCTION
Chitosan, is a naturally occurring polymer formed by partial deacetylation of chitin [1]. chitosan consists of (1,4) linked 2-acetamido-2deoxy--D-glucose (N-acetylglucosamine) [2] (Fig. 1a). Chitosan is used in the treatment of wounds by accelerating their healing as the positive charge of chitosan halts bleeding by binding to negatively charged red blood cells resulting in fast coagulation [3]. Also, it enhances the hyaluronic acid synthesis and prevents scar formation [4].
CH is used as an antibacterial against oral bacteria as the negatively charged surface of bacteria interacts with the positively-charged amino groups, NH 3 , of CH, and causes damage to the cell wall of the bacteria. Also, it is used as antifungal in toothpaste as it prevents the formation of plaque and tooth decay [5]. As well as, using CH in the form of nanoparticles improves its antimicrobial activity [1]. As well as, CH accelerates bone formation by increasing osteoblasts formations in bone tissues [6].
As well as, CH is also used as a bio-fertilizer, it has a positive charge so it can be used as a chelating agent for nutrient elements and enhances plant nutrient uptake efficiency so it enhances plant growth [7]. Also, CH improves water retention of the soil [8].
CH plays an important role in weight loss by preventing the absorption of fats, while AA improves CH mixing with fats so it is important to analyze them together.
Our study aims to introduce new simple spectrophotometric methods for the determination of the investigated drugs in pure and pharmaceutical dosage formulations.

Instrumentation
SHIMADZU Dual-beam (Kyoto/Japan) UV-Visible spectrophotometer model UV-1601 PC with 1 cm quartz cuvettes connected to IBM compatible computer fitted with UV-PC personal spectroscopy software version 3.7 Data analysis in the case of the MCR method was performed using Minitab® version 14.12.0 software. pH meter 3510 pH/mV/°C meter (Jenway, UK) with a combined glass electrode was used for pH adjustments.

AA was kindly supplied by Egyptian
International Pharmaceutical Industries Company (EIPICO), Egypt and certified to be 100% while High Mwt CH (600,000-800,000 dalton) was purchased from Acros Organics ( USA) and certified to be ≥98%

Reagents and solvents
All chemicals and reagents used were of analytical grade. Bi-distilled water was used throughout the whole work and indicated by the word "Water". Phosphate buffer solution pH 8 was prepared by using potassium dihydrogen phosphate and disodium hydrogen phosphate (ADWIC, Egypt) with appropriate concentrations (0.1 M potassium dihydrogen phosphate was prepared by dissolving 13.61 g in 1 L and 0.1 M disodium hydrogen phosphate was prepared by dissolving 17.8 g in 1 L, then mixing 25 mL from 0.1 M potassium dihydrogen phosphate solution with 475 mL from 0.1 M disodium hydrogen phosphate solution to prepare 500 mL of phosphate buffer solution pH 8). 1% v/v acetic acid was prepared by using water and acetic acid (ADWIC, Egypt)

Standard solutions
The standard stock solution of AA, (100 g/mL) was prepared in water. CH standard stock solution (100 g/mL) was prepared in 1% v/v acetic acid. These solutions were also used as working standard solutions and diluted with phosphate buffer pH 8 to prepare serial dilutions.

Chitosan binary mixture
Aliquots of standard working solutions of CH and AA were diluted with phosphate buffer pH 8 to prepare a concentration of 5 g/mL. Each was separately scanned over the range 200-400 nm using phosphate buffer pH 8 as a blank

1 DD
The stored spectra of CH were divided by the spectrum of 4 g/mL of AA while the stored spectra of AA were divided by the spectrum of 5 g/mL of CH, then the amplitude of the first derivative of the ratio spectra was recorded at 207.2 nm for CH and 282 nm for AA (scaling factor = 1 and λ = 4). The peak amplitudes were plotted versus the corresponding concentration of CH and AA (0.5-9 g/mL and 2-20 g/mL; respectively) and the regression equations were computed.

RDSM
The stored spectra of CH were divided by the spectrum of 4 g/mL of AA while the stored spectra of AA were divided by the spectrum of 5 g/mL of CH, then the amplitude differences of the ratio spectra (P 204.6-202.9 ) for CH and (P 268-230 ) for AA, were plotted versus the corresponding concentrations of CH and AA (0.5-9 g/mL and 2-20 g/ml; respectively) and the regression equations were computed.

MCR
The stored spectra of CH were divided by the spectrum of 4 g/mL of AA while the stored spectra of AA were divided by the spectrum of 5 g/mL CH, to obtain the ratio spectra which were then mean-centered and measured at 204.6 nm and 269.1 nm; respectively then plotted against the corresponding concentrations of CH and AA (0.5-9 g/mL and 2-20 g/mL; respectively). The regression equations were computed.

Application on laboratory prepared mixtures
Several laboratory prepared mixtures of varying ratios of CH and AA were prepared and the corresponding concentrations were determined by the previously mentioned methods.

2.7.
Application on pharmaceutical formulations

Chitocal® capsule
Twenty capsules of Chitocal® were emptied and weighed. An accurately weighed amount of the fine powder equivalent to 469.704 g CH and 93.94 g AA was weighed and transferred accurately into 100 mL volumetric flask and completed to the mark with 1% acetic acid to prepare 4697.04 g/mL CH and 939.4 g/mL AA stock solutions. Then a suitable dilution with phosphate buffer pH 8 was made from the prepared stocks to obtain 4 g/ml AA sample and 3 g/mL CH sample.
The above-mentioned procedures were followed. To check the validity of the proposed methods, the standard addition technique was adopted.

RESULTS AND DISCUSSION
Three simple and economic spectrophotometric methods were developed for rapid analysis of CH and AA in their binary mixtures, as well as in multi-ingredients formulations (e.g. weight loss formulations), as the amino groups of CH bind to negatively charged molecules like lipids and prevent their absorption and storage by the body [30]. AA reduces the viscosity of CH and improves its ability to mix with the fat in the GIT [31], That is why it was essential to analyze them together in their formulations.
Since the traditional derivative technique failed to resolve the severely overlapped spectra of the studied drugs as in (Fig. 2a and Fig. 2b), so it was crucial to develop new methods able to selectively analyze each of the studied drugs.
Analysis of CH by UV spectrophotometry methods was challenging as it lacks conjugation and did not have significant peaks in the middle and near UV regions, so the proposed methods were the first UV spectrophotometric methods for the analysis of CH in pure and pharmaceutical formulations.
Many solvents and buffered solutions were tried to shift CH wavelength to a longer one. Phosphate buffer pH 8 was the solvent of choice as it made a bathochromic shift to CH-spectrum and allowed it to be measured in the UV region. The reason is that the CH amino group becomes protonated when dissolved in 1% acetic acid and then when phosphate buffer pH 8 is used as a solvent for the preparation of serial dilutions, the protonated amino group becomes free again and regains its lone pair, which leads to bathochromic shift.  The stored spectra of CH were divided by the spectrum of 4 g/mL of AA then the amplitude of the first derivative of the ratio spectra was recorded at 207.2 nm (Fig. 3a).
While the stored spectra of AA were divided by the spectrum of 5 g/mL of CH, then the amplitude of the first derivative of the ratio spectra was recorded at 282 nm (Fig. 3b).

This method was optimized by testing the influence of many variables such as
The wavelength increment over which the derivative was obtained (λ). It was found that λ= 4 and scaling factor= 1 gave the best results in terms of signal to noise ratio, peak resolution, and sensitivity throughout the determination. The effect of divisor concentration was tested, and it was found that 4g/mL AA and 5 g/mL CH as a divisor for determination of CH and AA; respectively were the best in case of sensitivity and response.

RDSM
This method is based on the amplitude difference between two points on the ratio spectra of a mixture, which is directly proportional to the concentration of the component of interest [33,34].
The stored spectra of CH were divided by the spectrum of 4 g/mL of AA then the amplitude difference of the ratio spectra (P 204.6-202.9 ) was computed (Fig. 4a). While the stored spectra of AA were divided by the spectrum of 5 g/mL of CH and then the amplitude difference of the ratio spectra (P 268-230 ) was computed (Fig. 4b).
The method was optimized by testing different divisor concentrations and it was found that 4 g/ml AA and 5 g/mL CH as divisors for determination of CH and AA; respectively were the best in case of sensitivity and response. Also, the choice of the wavelengths, at which the amplitude differences were recorded, was optimized, where the chosen wavelengths exhibited difference amplitudes in ratio spectrum and good linearity is present at each wavelength individually.

MCR
The proposed MCR method is based on the mean centering of the ratio spectra. It eliminates the derivative steps, therefore it enhances the signal to noise ratio. It has been applied for resolving binary and ternary mixtures in complex samples with unknown matrices [35]. The mathematical explanation of the developed method was illustrated by Afkhami and Bahram [36].
After method optimization, CH and AA have been successfully determined at 204.6 nm and 269.1 nm; respectively as shown in (Fig. 5a) and  (Fig. 5b).

Linearity
The linearity of the proposed methods was evaluated using different concentrations of standard solutions where all the methods show good correlation coefficients close to unity, indicating good linearity, as shown in Table 1.

Accuracy
The accuracy of the results was checked by applying the proposed methods for the determination of different samples of CH and AA. The concentrations were obtained from the corresponding regression equations, and the recoveries were calculated, as shown in Table 1.

Precision
Intraday and interday precision of three concentrations of CH (4,5,8 g/mL) by using 1 DD , ( 3.5,4.5,7 g/mL) by using RDSM and ( 2.5,3,4 g/mL) by using MCR, and for AA ( 13,18,19 g/mL) by using 1 DD, RDSM, and MCR were checked. The obtained results for the proposed methods showed accepted results due to low values of % RSD as shown in Table 1.

Limits of detection and quantitation
The limit of detection (LOD) and limit of quantitation (LOQ) were calculated as LOD= 3.3 (σ/S) and LOQ= 10 (σ/S), where 'σ' represents the standard deviation of the intercept and 'S' is the slope of the calibration curve as shown in Table 1.

Selectivity
Selectivity was checked by analyzing of CH with AA in laboratory prepared mixtures with different ratios and was ensured by the results presented in Table 2.

Application to pharmaceutical formulations
The proposed methods were successfully applied to pharmaceutical formulations and the standard addition technique was performed. The concentrations were calculated using the corresponding regression equations as shown in Table 3.

Statistical analysis
A statistical comparison between the results obtained by the proposed methods and those of the reported methods [10,23] of CH and AA was done. The difference between the proposed and reported methods were tested by F-test and t-test as shown in Table 4. The test ascertained that there was no significant difference concerning accuracy and precision between the proposed methods and the reported methods [10,23].

Conclusion
The proposed spectrophotometric methods for the determination of CH and AA could be successfully applied. The results showed good selectivity, accuracy, and precision demonstrating that they are rapid, selective, sensitive, economic and reproducible. So, the proposed methods are of great value for routine efficient analysis of the cited drugs in its pharmaceutical formulations. Also, the proposed methods offer distinct advantages in simplicity, ease of use, and low costs over other analytical methods as shown in Table 5.

Ethics approval and consent to participate
This study does not include any in vitro or in vivo studies (Not applicable).

Availability of data and materials
All data generated or analyzed during this study are included in this published article in the main manuscript.

Acknowledgment
The authors would like to acknowledge all colleagues in the Pharmaceutical analytical Chemistry Department, Ain Shams University for their support.