Abstract
Background
Taurine, known as 2-aminoethanesulphonic acid, is a sulfur-containing β-amino acid not incorporated into proteins. However, it takes part in biochemical reactions of significant importance, such as conjugation with bile acids to form bile salts essential for fat absorption, cell membrane stabilization, antioxidation, detoxification, osmoregulation, neuromodulation, and brain and retinal development.
Objective
To increase its dissolution and absorption rate for early onset of action, we developed a medicated gel formulation of taurine. Chewable gels are suitable dosage forms for pediatric, geriatric, and dysphagic patients. The current research aims to develop a simple, sensitive, and reproducible spectrofluorometric method for determining taurine in chewable gel.
Method
A spectrofluorimetric method was developed, and the determination of taurine was based on Hantzsch condensation synthesis, and the fluorescence intensity was measured at emission wavelength (λem) of 474 nm after excitation (λex) at 416 nm. The spectrofluorimetry method for taurine was validated according to the Q2B(R1) ICH guideline for method validation and validated for linearity, LOD, LOQ, interday and intraday precision, accuracy, and robustness.
Results
The calibration curve was linear in a 1.5–50 µg/ml concentration range. The correlation coefficient (r2) was found to be 0.994. The % RSD values of intra-day precision lie between 0.11 and 0.09; for inter-day precision, it was found to be between 0.09 and 0.11; in both cases, it is less than 2 %. The percentage accuracy study was between 91.81 and 99.93 with a % RSD value of 0.078, less than 2 %. LOD & LOQ values have been mathematically calculated to 4.03 µg/ml and 12.23 µg/ml, respectively. The method was precise, accurate, and robust.
Conclusion
All validation parameters were found to be within their acceptable limits. Thus, the developed spectrofluorimetric method of taurine is economical, fast, and robust. Further, this method was utilized to estimate taurine content uniformity in chewable gel for in-vitro release studies.
Keywords: ASD, Spectrofluorimetric method, Taurine, Chewable gel, Hantzsch reaction
1. Introduction
Taurine, 2-aminoethanesulfonic acid, is a derivative of the sulfur-containing amino acid cysteine (Wang et al., 2011). It is the only known natural sulfonic acid (Guerrero-Esteban et al., 2019). It is one of the most abundant free amino acids in the human body and is widely distributed in biological fluids and tissue. Taurine (C2H7NO3S) (molecular weight 125.14 g/mol) is soluble in water. It is a weak organic acid (Omar, Hammad, et al., 2015). Taurine is a neutral β-amino acid; both the amine and sulphonic groups can undergo ionisation, and the dissociation constant of the latter confers its biological and functional specificity (Wang et al., 2011). Taurine plays a vital role in several essential biological processes, such as the development of the eye and brain, reproduction, and immune function, including anti-inflammatory activity, antioxidant activity, membrane stabilisation, and osmoregulation (Jakaria et al., 2019). Changes in taurine levels in physiological fluids and tissues occur in various diseases or pathological conditions such as schizophrenia, inflammation, hepatic damage, sepsis, retinitis pigmentosa, and cancer (Ghandforoush-Sattari et al., 2010, Hussy et al., 2000) Taurine is a valuable biomarker of some diseases, such as Autism Spectrum Disorders (ASD) (Hussy et al., 2000). Autism Spectrum Disorder is a neurodevelopmental disorder that affects the brain in the developmental stage (Vénat-Bouvet et al., 2012). ASD is characterized by core symptoms like loss of social interaction, non-social approach, and impairment of communication as well as by associated symptoms like irritable nature, anxiety, aggression, epilepsy, and sensory processing disorder (Bhandari and Kuhad, 2015). Supplementation of taurine has been shown to prevent oxidant-induced damage while deficiency of taurine leads to immune activation in the CNS with Purkinje cell loss, microglial activation, and astrocytosis, similar to that observed in the ASD brain (Vénat-Bouvet et al., 2012). Taurine protects neurons against glutamate-induced excitotoxicity by reducing the glutamate-induced increase in intracellular calcium levels (Jakaria et al., 2019). Our in-silico study has demonstrated the potential of Taurine in ASD (Bhandari et al., 2023).
Albeit clinical effectiveness and patient success in their treatments depend on patient compliance (Lin et al., 2008). Dysphagia and off-taste, which frequently happen when active pharmaceutical ingredients (APIs) for oral administration are formulated as hard tablets, may lead to non-compliance. Patients often break, chew, or crush hard tablets and mix them with food or water to make swallowing more manageable, even though doing so may alter the API's release profile and affect the rate or degree of drug absorption and, consequently, bioavailability (Liu et al., 2014, T et al., 2012) The off-taste of APIs is significantly enhanced by tablet crushing, which thus raises the possibility of incorrect dosing (T et al., 2012).
Increasing user-friendly dosage forms that are simple to swallow and have barely perceptible API off-taste would benefit the pediatric and geriatric subpopulations, which need to enhance drug dose acceptance and compliance (T et al., 2012). By keeping the API's solubility in the formulation to a minimum, suspension-based formulations offer appropriate taste-masking qualities and chemical stability (Lin et al., 2008; Patient Non-Adherence Costs Underestimated | Healthcare Packaging). Different destabilizing mechanisms, like the sedimentation and flocculation of the suspended particles, might affect suspension formulations (Adherence to Long-Term Therapies: Evidence for Action). To combat the destabilizing process, an unique drug formulation has been created that permits API delivery as a suspension contained in a dispersed form within a chewable gel matrix within a unit dose. Using the well-known API taurine as a model ingredient, the unique pharmacological formulation was developed as a consistent soft-chew dose with taste-masking qualities (Taylor and Aulton, 2013).
Generally, chewable gels are transparent or translucent, nongreasy semisolid solutions or suspensions of small inorganic particles or large organic molecules interpenetrated by a liquid. These are meant for oral or external application to mucous membranes and may be ingested without water. Medicated gel formulations are a more suitable dosage form for pediatric, geriatric, and dysphagic patients, offering rapid dissolution and absorption of medicine, thereby allowing for the early onset of action. The dysphagic patients are often choked by water while consuming liquid formulations, and this problem will be eliminated by administering liquid formulations with high viscosity within the kind of chewable gels (Dille et al., 2018, Prakash et al., 2014).
A novel chewable gel of taurine was developed by our lab for children having ASD as an add-on therapy targeted glutamate-induced excitotoxicity, oxidative stress, neuroinflammation and gut-brain dysbiosis (Patent no. 202211035087: “A pharmaceutical formulation, its method of preparation and use thereof”). This is a first-of-a-kind formulation being developed for children suffering from ASD as an add-on therapy. It is impertive from a quality control point of view that a suitable analytical method be developed for each formulation. Several methods have been devised for the evaluation of taurine in literature. Determination or quantification of taurine is difficult as its structure lacks a chromophoric group and hence, most methods involve derivitization and are complex. Gas chromatography (GC) (Mou et al., 2002), high-performance liquid chromatography (HPLC) ((PDF) Simultaneous Analysis of Taurine and Caffeine in Energy Drinks Using Hydrophilic Interaction Chromatography with UV and Evaporative Light Scattering Detection on Line), ion chromatography (IC), thin layer chromatography (TLC) (Mou et al., 2002), ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS) (Alam et al., 2021), electrochemiluminescence and UV-spectrophotometric techniques have been used (B. Draganov, 2014). However, the above methods involve complex sample prepration as well as complex and sophisticated instrumentation along with some of the above techniques being less sensitive and specific. Most of the methods developed till date either require pre-column, post-column or on-column derivitization. The post-column derivitization requires ion-exchange chromatography and an exclusive instrument whereas the pre-column derivitization methods are time-consuming. Hence, the need of the hour is a simple, robust and fast method of analysis of taurine which is economical and do not require a sophisticated or exclusive instrument and can be developed as quality-control tool for analysis of any batch to batch variation.
Fluorescence is the molecular absorption of light energy at one wavelength, and its nearly instantaneous re-emission is at another, usually at a longer wavelength. Some molecules fluoresce naturally, and others can be modified to make fluorescent compounds. Fluorescent compounds have two characteristic spectra: an excitation spectrum (the wavelength and amount of light absorbed) and an emission spectrum (the wavelength and amount of light emitted). These spectra are often called a compound's fluorescence signature or fingerprint. No two compounds have the same fluorescence signature. It is this principle that makes fluorimetry a highly specific analytical technique. Fluorometry is the measurement of fluorescence. The instrument used to measure fluorescence is called a fluorometer or fluorimeter. A fluorometer generates the wavelength of light required to excite the analyte of interest; it selectively transmits the wavelength of light emitted and then measures the intensity of the emitted light. The emitted light is proportional to the measured analyte concentration (up to a maximum concentration) (Naresh, 2023)
The present study aims to develop and validate a simple, robust, and economical spectrofluorimetric method for determining taurine in chewable gel formulation, the other methods being less economical and requiring complex and exclusive instrumentation. The developed chewable gel formulation will be commercialized and a simple analytical method can help in accurately determining the content uniformity, in-vitro drug release, and the analysis during stability studies at industrial scale. The Hantzsch reaction, which includes reagents like acetylacetone and formaldehyde, was used to detect taurine in chewable gel. Our work is novel as we propose development of a simple, robust, fast, economical and reproducible method for detection of taurine in novel chewable gel which is being developed for first time for autistic children. Hence, this method can be easily developed even in a simple lab with less funding and instrumentation as well as at an industrial scale lab and also can be scaled for other industrial scale formulations containing taurine as API.
2. Materials and methods
2.1. Equipment
All the measurements were carried out by using a Hitachi F 2500, Japan spectrofluorimeter with a 1 cm quartz cell, and the slit width for both monochromators was set at 10 nm. The results were processed by FL 2.0 Solution® software with other equipment types, such as a BS223S (Sartorius, USA) analytical balance and hot plate from Macro Scientific Works Pvt.Ltd, pH meter from (Eutech instruments pH 510, India).
2.2. Chemicals and reagents
All materials were of analytical reagent grade, and all solutions were prepared with double-distilled water. Taurine was purchased from SRL chem, Formaldehyde from Thermo Fisher Scientific India Pvt. Ltd., Acetylacetone from SRL chem, and sodium acetate trihydrate was purchased from Fisher Scientific.
2.3. Formulation of taurine chewable gel
The prototype of chewable gel was optimized using Design Experts Software 7.0. as well as OFAT analysis (Patent no. 202211035087: “A pharmaceutical formulation, its method of preparation and use thereof”) filed. The ingredients of the gel are taurine, sorbitol, dextrose, HPMC, PEG 4000, Sodium citrate, citric acid (INS 330), pectin, FOS, preservative, sucralose, flavor, color, and water. The amount of sucrose: pectin and acid-regulator along with buffering agent sodium citrate was optimized as it was critical for the gelation. The formulation was also optimized for pH and temperature which are also critical factors responsible for congealing. The chewable gels were formed after cooling and were removed from silicone molds for further analysis (Fig. 1).
Fig. 1.
A Novel Chewable Gel.
2.4. Preparation of standard and reagent solutions
2.4.1. Stock solution of taurine
Stock solution (100 mg/ml) of taurine was daily prepared in distilled water and further diluted with the same solvent as appropriate.
2.4.2. Acetylacetone solution
A standard solution of acetylacetone (10 % v/v) was daily prepared by mixing 2 ml of the reagent with 20 ml distilled water (El-Hamd et al., 2017).
2.4.3. Formaldehyde solution
A standard solution of formaldehyde (20 %, v/v) was daily prepared by mixing 10.8 ml of the reagent with 20 ml distilled water.
2.4.4. Buffer solution
Solution of acetate buffer of a pH of 4.1 was prepared 0.1 M buffer by mixing 0.150 g of sodium acetate trihydrate in water and 1.950 µl of acetic acid, adjusting pH 4.1 and dilute to 100 ml with distilled water (Hammad et al., 2017).
2.5. Spectrofluorimetric method development using Hantzsch dihydropyridine reaction
Due to its low cost and high sensitivity, the Hantzsch dihydropyridine reaction has been widely applied for drug analysis in different matrices. This study's proposed method for determining taurine was based on Hantzsch condensation synthesis to give the yellow fluorescent product. This reaction proceeds via the formation of dihydropyridine derivatives by condensation of formaldehyde acetylacetone in the presence of the studied drugs, which act as the source of a primary amine moiety to form highly fluorogenic condensation products (Vijesh et al., 2011; Hammad et al., 2017).
2.5.1. Procedure
A volume of 1.0 ml of standard solution was transferred into a test tube. 1.0 ml of sodium acetate buffer (0.1 M, pH 4.1), acetylacetone (10 % v/v), and formaldehyde (20 % v/v) were added, and the reaction was allowed to stand for about 70 min in a water bath previously heated to 100°C. After cooling, the reaction sample was diluted with 6 ml of distilled water.
2.5.2. Optimisation
Different experimental factors affecting reaction development and fluorescent product stability were thoroughly investigated and optimised. Individually, these parameters were altered, while the others remained unchanged. Taurine standard solution of 100 µg/ml was used for optimization. These parameters include temperature, heating time and volume of reagents (Hammad et al., 2017, Omar et al., 2015).
2.5.2.1. Effect of temperature and heating time
The optimum heating time for the reaction was studied by measuring the fluorescent intensity at different temperatures (40–100°C) and different intervals (5–70 min) in a thermostatically controlled water bath.
2.5.2.2. Effect of volume of acetylacetone, formaldehyde and buffer
The effect of acetylacetone, formaldehyde and buffer volumes on RFI was investigated. Different volumes ranging from 0.1 to 2.5 ml of acetylacetone, 0.1–2.5 ml of formaldehyde, and from 0.1–2.5 ml of sodium acetate buffer were studied.
2.5.2.3. Effect of pH
The effect of pH was studied using sodium acetate buffer from 4.1 to 6.0. The fluorescence intensity was highly dependent on pH.
2.6. Validation of the analytical method
The spectrofluorimetric method developed to assay Taurine was validated according to International Council for Harmonisation (ICH) guidelines. The following validation parameters were evaluated such as linearity, LOD, LOQ, precision, accuracy, robustness. The analytical parameters which were set for validation of the method are listed in Table 1.
Table 1.
Analytical parameters for the determination of taurine using a spectrofluorimetric method.
| λex | 416 |
| λem | 474 |
| Concentration range (µg/ml) | 1.5–50 µg/ml |
| R2 | 0.994 |
| STEYX (standard error of Y given X for a least- squares linear) | 82.3533 |
| Slope | 67.32 |
| LOD | 4.036928 |
| LOQ | 12.23311 |
2.6.1. Linearity
The linear range for taurine was evaluated within the 1.5 – 50 µg/ml range. The stock solution of taurine was diluted to six different concentrations in the range of 1.5–50 µg/ml, and a calibration curve was obtained by plotting RFI versus the taurine concentration (µg/ml).
2.6.2. LOD and LOQ
To determine the method's sensitivity, LOD and LOQ were employed as metrics in compliance with the Guidance for Industry Q2B Validation of Analytical Procedures Techniques (1996). The residual standard deviation from the calibration curves' linear regression analysis was used to calculate the LOQ and LOD of gabapentin and capsaicin for the analysis.
Here's how to compute LOD and LOQ LOD = 3.3 σ / S and LOQ = 10 σ /S where σ is the standard deviation of the response S is the slope of the calibration curve
2.6.3. Accuracy
The accuracy of the analytical method was determined in the range of 2.5, 5, 25, 100, and 300 μg/ml. These were within the range of the calibration plot and were analyzed in triplicate (n = 3) with three samples of each concentration and were prepared from independent stock solutions. Assessment of accuracy was done as the percentage accuracy and mean % recovery. The percentage relative standard deviation (RSD) was also calculated.
2.6.4. Precision
The precision (% RSD) of the developed analytical was evaluated by calculating the intra-day and inter-day coefficient of variation. Precision was determined for an analytical method by studying the intra-day variability or repeatability as well as inter-day variability ranging from 100 µg/ml, 150 μg/ml, and 300 µg/ml at all three concentrations of taurine in triplicate (n = 3)
2.6.5. Robustness
The robustness of the proposed procedure was assessed by evaluating the influence of minor variations in the experimental variables on the analytical performance (effect of heating and heating time, concentration of reagents, and pH of acetate buffer). For robustness, 100 µg/ml concentrations were given in triplicate for each parameter. The percentage of RSD was calculated to observe any deviation in the relative fluorescence intensity (RFI) based on variation in these parameters.
3. Application of the method
3.1. In-vitro release & drug release kinetics
United States Pharmacopoeia dissolution apparatus II (Lab India DS 8000) with a paddle rotation speed of 50 rpm was used to monitor the in-vitro dissolution profile of taurine from chewable gel.The dissolution vessel was filled with 900 ml of the release media (phosphate buffer 7.2 pH USP 2020). The temperature was maintained at 37°±0.5°C. The dissolution studies were carried out in triplicate. 5 ml of the media was removed at each time interval and replaced with the fresh media maintained at 37°C for 6 min. The fluorescence intensity was measured at an emission wavelength (λem) of 474 nm after excitation (λex) at 416 nm. The calculations were done accordingly to calculate the percentage release of the drug at various time intervals. For comparison, 150 mg of the drug was taken in a capsule, dissolution was performed, and the fluorescence intensity was recorded at both 474 nm and 416 nm. The calculation was done accordingly to calculate the percentage release of the drug at various time intervals and the drug release kinetics of the batch were determined as data was fit in different kinetic models such as zero order, first order, Korsmeyer peppas, Higuchi model.
3.2. Content uniformity
The formulations were designed to contain 150 mg of Taurine. The optimized chewable gels (n=6) were weighed on an analytical balance and then dissolved in 100 ml of deionized water. Content uniformity was quantified by the developed and validated spectroflurometric method of Taurine using Hantzsch dihydropyridine reaction.
4. Results
4.1. Spectrofluorimetric method development using Hantzsch dihydropyridine reaction
The method was developed using Hantzsch dihydropyridine reaction where yellow coloured fluorescent product was formed. The fluorescence intensity of the resultant solution was measured at an emission wavelength (λem) of 474 nm after excitation (λex) at 416 nm (Musa et al., 2019; Tian and Li, 1205).
4.2. Optimisation of spectrofluorimetric method
Method was optimized with respect to temperature, heating time and volume of reagents.
4.2.1. Effect of temperature and heating time
The results revealed that the heating step is vital for the completeness of the reaction. The maximum RFI was reached at the boiling water bath (100°C) after 70 min (Fig. 3 & Fig. 4).
Fig. 2.
Excitation (λex 416) and emission (λem474) spectra of Hantzsch reaction products for Taurine standard solution.
Fig. 3.
Effect of temperature on the Hantzsch reaction with taurine standard solution(100 µg/ml) indicating that maximum fluorescence intesity was observed at 100 °C after 70 minutes.
Fig. 4.
Effect of the heating time on Hantzsch reaction with taurine standard solution (100 µg/ml) indicated that maximum fluorescence intesity was observed at 70 minutes.
4.2.2. Effect of volume of acetylacetone, formaldehyde and buffer
Effect of different volumes ranging from 0.1 to 2.5 ml of acetylacetone (Fig. 5) and 0.1–2.5 ml of formaldehyde (Fig. 6), and from 0.1–2.5 ml of sodium acetate buffer (Fig. 7) indicated that the best results were obtained with 1 ml of 10 % v/v acetylacetone, 1 ml of 20 % v/v formaldehyde and 1 ml of 0.1 M sodium acetate buffer.
Fig. 5.
Effect of volume of acetylacetone on Hantzsch reaction with standard solution of taurine (100 µg/ml) indicated maximum fluorescence intestity at 1 ml.
Fig. 6.
Effect of volume of formaldehyde on Hantzsch reaction with standard solution with taurine (100 µg/ml) indicated maximum fluorescence intensity at 1 ml.
Fig. 7.
Effect of volume of sodium acetate buffer on Hantzsch reaction with standard solution of taurine (100 µg/ml) indicated maximum fluorescence intensity at 1 ml.
4.2.3. Effect of pH
The highest relative fluorescent intensity (RFI) was attained at pH 4.1 (Fig. 8).
Fig. 8.
Effect of pH on Hantzsch reaction with standard solution of taurine (100 µg/ml)indicated that highest relative fluorescence intensity at 4.1 pH.
4.3. Validation of analytical method
The spectrofluorimetric method developed to assay Taurine was validated in accordance with International Council for Harmonisation (ICH) guidelines. The following validation parameters were evaluated such as linearity, LOD, LOQ, precision, accuracy, robustness.
4.3.1. Linearity
A straight line was obtained over the entire concentration range of 1.5–50 µg/ml (Fig. 9). Multiple linear regression analysis was performed using MS-Excel Data Analysis Tool-Pack. The regression statistics showed the value of the coefficient of determination (R2) to be 0.994701418. Using analysis of variance (ANOVA) on the regression line yielded very small F and p values (F = 1.05468 × 10−13, p = 0.0046 × 10−5) at 95 % confidence level. Residual analysis in regression shows a random pattern of the residuals as indicated by the residual plot (Fig. 10). The regression statistics showed the value of the coefficient of determination (R2) to be 0.993376772
Fig. 9.
Calibration curve of taurine indicating linearity over a concentration range of 1.5–50 µg/ml with R2 value as0.9947.
Fig. 10.
Residual plot indicating the appropriateness of the linear regression model. Xvariable 1 indicates the concentration.
4.3.2. LOD and LOQ
The limit of detection (LOD) and limit of quantification (LOQ) of taurine were obtained using calibration curve analysis; LOD and LOQ were found to be 4.03692 μg/ml and 12.23311 μg/ml, respectively.
4.3.3. Accuracy
The excellent mean recovery values close to 100 % and their low values of relative standard deviation (% RSD) < 2 % depict the high accuracy of the analytical method (Table 2).
Table 2.
Accuracy data for the developed analytical method for Taurine.
| Conc (µg/ml) | Mean±SD | % RSD | % Accuracy |
|---|---|---|---|
| 2.5 | 2.29±1.91 | 0.46 | 91.81022 |
| 5 | 4.7±1.2 | 0.28 | 95.90511 |
| 25 | 24.7±1.1 | 0.13 | 99.90511 |
| 100 | 99.7±1.5 | 0.02 | 99.79526 |
| 300 | 299.7±0.5 | 0.02 | 99.93175 |
4.3.4. Precision
The precision values was found to follow the ICH guidelines as indicated by the mean recovery values and low values of % RSD, which were within the acceptable limits (Table 3).
Table 3.
Intra-day and Inter-day precision for taurine using the Hantzsch reaction.
| Precision | Concentration (µg/ml) | Mean±SD | % RSD | % Accuracy |
|---|---|---|---|---|
| 100 | 99.7±0.01 | 0.11 | 99.79 | |
| Intraday | 150 | 149.7±0.06 | 0.09 | 99.86 |
| 300 | 299.7±0.06 | 0.07 | 99.93 | |
| Interday | 100 | 99.8±0.01 | 0.11 | 99.79 |
| 150 | 149.6±0.06 | 0.09 | 99.86 | |
| 300 | 299.7±0.06 | 0.07 | 99.93 |
4.3.5. Robustness
The percentage of RSD was found to be in the range of 1.2–2 %. As indicated in the table, small variable variations did not significantly affect the results. This showed the reliability of the proposed method during routine work (Table 4).
Table 4.
Robustness of the proposed method for determination of the Taurine.
| Variation | RFI (Relative fluorescence intensity) | SD | %RSD |
|---|---|---|---|
| pH (Sodium acetate buffer) | |||
| 4 | 4364.8 | 1.90 | 0.07 |
| 4.1 | 4354.8 | 1.61 | 0.04 |
| Heating time | |||
| 65 min | 5412 | 0.68 | 0.012 |
| 75 min | 5420 | 1.15 | 0.021 |
| Acetylacetone volume (ml) | |||
| 0.9 | 4486 | 0.81 | 0.018 |
| 1.1 | 4583 | 0.81 | 0.017 |
| Formaldehyde volume (ml) | |||
| 0.9 | 4496 | 0.98 | 0.021 |
| 1.1 | 4589 | 0.74 | 0.016 |
| Sodium acetate buffer volume (ml) | |||
| 0.9 | 4481 | 1.09 | 0.024 |
| 1.1 | 4591 | 0.81 | 0.017 |
4.4. Application of the developed spectrofluorometric method
4.4.1. In-vitro release & drug release kinetics
Results indicated that 100 % of the drug was released in 120 minutes (Fig. 11) and it shows zero order release as the best fit model as indicated by the value of regression coefficient (Fig. 12).
Fig. 13.
First-order profile of chewable gel formulation of taurine.
Fig. 14.
Korsmeyer-Peppas profile of chewable gel formulation of taurine.
Fig. 15.
Higuchi profile of chewable gel formulation of taurine.
Fig. 11.
Dissolution profile of chewable gel formulation of taurine indicating 100 % release from chewable gel in 120 minutes.
Fig. 12.
Zero-order profile of chewable gel formulation of taurine.
From the above graph it has been concluded that the drug shows zero order release kinetics as it was the best fit model as observed after data fitting (Table 5).
Table 5.
Release-kinetics and R² value of chewable gel of taurine.
| Release kinetics | R² value |
|---|---|
| Zero order | 0.9581 |
| First order | 0.7075 |
| Korsmeyer peppas | 0.9402 |
| Higuchi | 0.5543 |
4.4.2. Content-uniformity
The drug content in the chewable gel was found in the range of 97.63 %±0.63 % - 99.23 %±0.66 %, which conforms with the pharmacopoeial specification of 95–105 %.
5. Discussion
To increase the dissolution and absorption rate for early onset of action, taurine-based medicated gel formulation was developed. Chewable gels are suitable dosage forms for pediatric, geriatric, and dysphagic patients. This work aimed to develop a simple, sensitive, and reproducible spectrofluorimetric method for the determination of taurine in chewable gel. The Hantzsch reaction uses reagents such as acetylacetone and formaldehyde to determine Taurine. The developed spectrofluorimetric method was validated in terms of linearity, accuracy, precision, and robustness.
Further, this method was utilized to carry out an estimation of taurine for in-vitro release studies, and content uniformity in chewable gel. The method was linear within the range of 1.5–50 μg/ml, as indicated by the value of the determination factor, which was found to be 0.994, yielding the best-fit line. Multiple linear regression analysis was performed using MS Excel Data Analysis Tool-Pack. The regression statistics showed the value of the coefficient of determination (R2) to be 0.994701418. The precision and accuracy were found to follow the ICH guidelines as indicated by the mean recovery values and low values of % RSD, which were within the acceptable limits. The method was also found to be robust as % RSD values obtained after varying various parameters were within acceptable limits. The system suitability parameters were within the permissible limits laid down by ICH Q2B guidelines and USP 32-NF-27, indicating that the developed analytical method for taurine was accurate, precise, and robust. When this developed and validated method was further applied for the quantification of
Taurine in the chewable gel, there was no shift in the excitation and emission spectra of taurine, indicating the sensitivity as well as the robustness of the developed and validated spectrofluorimetric method of taurine. In-vitro drug dissolution studies showed that ∼100 % of the drug was released in 120 minutes with the best model as zero order release kinetics. Content uniformity was found in the 97.63 %±0.63 % - 99.23±0.66 %, which conformed with the pharmacopoeial specification of 95–105 %. This method was also further utilized during formulation stability studies and in-vivo pharmacokinetic studies. The developed spectroflurometric method is advantageous over other available methods as it has high sensitivity which allows for the detection of low concentrations of taurine as well as selectivity leading to negligible interference from other substances. It is typically faster than some chromatographic methods, allowing for quicker sample throughput. It also requires minimum sample prepration compared to other techniques. Whereas, the HPLC method has a longer run time and requires more sophisticated instrumentation and setup as well as involves more extensive sample preparation. GC-MS is also an alternative which provides detailed structural information through mass spectrometry. However, it requires derivatization of taurine, which adds complexity and is more time-consuming and requires specialized equipment. There are other colourimetric methods which are less sensitive than spectrofluorometric and chromatographic methods and are more susceptible to interference from other substances in the sample. Hence, the choice of method depends on the specific requirements of the analysis, such as sensitivity, specificity, sample type, and available resources. Considering that, our developed spectrofluorometric detection is particularly advantageous for rapid, sensitive analyses where interference is minimal, making it a strong choice for taurine detection in various sample matrices. It can be easily developed at a lab and is also reproducible at an industrial scale for QC purposes for determination of batch to batch variation in formulations containing taurine.
6. Conclusion
To satisfy the fast-growing need for new analytical methods for the newly approved and existing drugs, analytical methods have to be developed continuously or modified for the existing methods. Thus, creating any new or improved method for analyzing analytes usually depends on tailoring the existing analytical approaches and instrumentation.
All validation parameters were found to be within their acceptable limits. Thus, the developed spectrofluorimetric method of taurine is economical, fast, and robust. Further, this method was utilized to estimate taurine content uniformity and for in-vitro release studies. Hence, a simple and economical method like this could be easily used as a quality control tool during assay determination of Taurine in large scale batches at an industrial scale. It doesn’t require complicated and exclusive instrumentation.
CRediT authorship contribution statement
Yuvraj Patidar: Writing – original draft, Validation, Project administration. Pritiman Pothal: Writing – original draft, Formal analysis. Jyoti K. Paliwal: Writing – review & editing, Investigation, Data curation, Conceptualization. Anurag Kuhad: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Ranjana Bhandari: Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. Niharika Goswami: Writing – original draft, Investigation, Formal analysis, Data curation.
Acknowledgements
The author wants to acknowledge the funding received as an Early Career Award-2020 to Dr. Ranjana Bhandari from the International Brain Research Organization (IBRO) and the BIG-BIRAC award (20th call ) (BIRAC/FITT01149/BIG-20/22) from Department of Biotechnology, Govt. of India.
Contributor Information
Ranjana Bhandari, Email: akb10in@yahoo.co.uk, rbhandari@pu.ac.in.
Anurag Kuhad, Email: anurag_pu@yahoo.com, anurag.kuhad@pu.ac.in.
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