Development and Validation of HPLC Method for Simultaneous Estimation of Ondansetron Dihydrochloride, Famotidine, and Drotaverine Hydrochloride in Rabbit Plasma

Development and Validation of HPLC Method for Simultaneous Estimation of Ondansetron Dihydrochloride, Famotidine, and Drotaverine Hydrochloride in Rabbit Plasma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development and Validation of HPLC Method for Simultaneous Estimation of Ondansetron Dihydrochloride, Famotidine, and Drotaverine Hydrochloride in Rabbit Plasma Rao Khurram Ayoub, Syed Nisar Hussain Shah, Asma Aslam, Romana Riaz, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6133930/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Several methods have been reported for the individual determination of Ondansetron (OND), Famotidine (FAM), and Drotaverine (DRO); however, they are not suitable for simultaneously estimating these drugs. Hence, a simple and accurate high-performance liquid chromatographic (HPLC) method was developed and validated for the simultaneous estimation of the three drugs. The method was optimized using the central composite design. The effect of independent variables was optimized while applying the central composite design approach. The quadratic effect of independent variables was significant on the percentage recovery and the retention time ( p < 0.0001) of the studied drugs. The chromatographic separations were carried out at a 1.5 mL/min flow rate using a 40:60 (v/v) ratio mixture of acetonitrile and phosphate buffer pH 4.8 as optimized parameters. The retention time of OND, FAM, and DRO was observed to be 1.93, 1.45, and 5.46 min, respectively. Similarly, the percentage recoveries of OND, FAM, and DRO were found within the range of 99.16–103.8%, 92.1–101.3%, and 97.6–105.6%, respectively. All the experiments were performed in six replicates (p < 0.05). Hence, the suggested method can be used for the simultaneous determination of OND, FAM, and DRO method can be used for the simultaneous determination of these drugs. HPLC Famotidine Drotaverine Ondansetron Central composite design Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Ondansetron (OND), chemically known as (RS)-9-methyl-3-[(2-methylimidazol-1-yl) methyl]-2,3-dihydro-1 H -carbazol-4-1 acts as a selective antagonist of 5-hydroxytryptamine type 3 (5-HT3) receptor. OND is used to alleviate nausea and vomiting associated with radiotherapy, chemotherapy and surgery at doses ranging from 4–24 mg [ 1 ]. Drotaverine (DRO) is an isoquinoline derivative with the chemical name of 1-[(3, 4-diethoxyphenyl) methylidene]-6, 7-diethoxy-3, 4-dihydro-2 H -isoquinoline. It is an antispasmodic drug that produces its effect on smooth muscles by inhibiting the phosphodiesterase (PDE) enzyme system[ 2 ], [ 3 ]. DRO is used to treat spasmodic discomfort caused by smooth muscle illnesses such as irritable bowel syndrome as well as biliary, intestinal, or ureteric colic. The recommended dose of DRO ranges from 40–80 mg [ 4 ] [ 5 ]. Famotidine (FAM) is chemically designated as 3-[2-[(aminoiminomethyl)amino]-4-thiazolyl]methyl]thio]- N - (aminosulfonyl) propanimidamide. It is classified as an antagonist of H2 receptor and indicated for the management of peptic ulcer and gastroesophageal reflux disease [ 6 , 7 ]. Typically, it is used at doses ranging from 20–40 mg [ 8 ]. Structural formulas of OND, DRO, and FAM are revealed in Fig. 1 . Due to their recommended pharmacological dose, this combination of drugs is formulated with a variable quantity of active ingredients i.e. 8 mg of OND, 69 mg of DRO hydrochloride, 151 mg of FAM. This variable amount of active ingredients in a multidrug formulation complicates the process of routine analysis. Whereas, differing polarity and chromatographic behavior of these active moieties further complicate the analysis. Several methods have been described in literature for the quantitative determination of these drugs in individual dosage forms. For example, OND has been reported to be analyzed using high-performance thin-layer chromatography (HPTLC), HPLC, and spectrophotometry [ 9 – 11 ]. For example, Patel et al . established a simultaneous equation method for the simultaneous estimation of OND and omeprazole in bulk and pharmaceutical preparations. This method used two separate wavelenthgs for ompeprazole and ondancertron. Methanol was used as a solvent and the % recovery of OND was found to be 99.49–99.66% [ 12 ]. In another study, Dedandia et al . have developed an HPLC method for the simultaneous determination of OND and omeprazole in combined doasahe form. A methanol and acetonitrile (90:10) was used as mobile phase. The flow rate was kept to 0.5 ml/min and the retention time of OND was found to be 11.08 min. However, the % recovery of OND was recorded to be 99.54 to 100.2% [ 13 ]. Similarly, the analytical methods reported for the estimation of DRO hydrochloride are spectrophotometry, voltammetry, thin-layer chromatography (TLC) and HPLC [ 2 , 14 – 17 ]. For example, Azhlwar et al. developed a stability indicating HPLC method for simultaneous estimation of DRO and aceclofenac. The mobile phase composed of acetonitrile and 0.1% trifluoro acetic acid (55:45%v/v) and the % recovery of DRO was reported to be 99.97 ± 0.3250 [ 18 ]. Another reverse phase HPLC method was developed by Panigrahi & Shrama for the simultaneous estimation of DRO and omeprazole in tablet dosage forms. This method was developed using a mixture of 0.1 M ammonium acetate and methanol as mobile phase for tablet dosage forms. The flow rate was maintained at 1.5 ml/min. DRO was eluted with aretention time of 7.969 min while the % recovery of DRO was found to be within the range of 100.66–100.94% [ 2 ]. Similarly, Dahivelkar et al have established an HPLC method for simultaneous determination DRO hydrochloride and mefenamic acid in human plasma. The compounds were separated in an isocratic mode while using a mixture of 20 mM ammonium acetate buffer and acetonitrile (55:45) as mobile phase. DRO was eluted with a retention time of 8.21 min and its % percent recovery was reported to be 94.51% [ 19 ]. Abdellatef et al. developed spectrophotometric and spectrodensitometric techniques for simultaneous determination of paracetamol and DRO and reported the % recovery of DRO to be 100.11% ± 1.91 [ 20 ] . Different methods developed for the analysis of FAM include capillary electrophoresis, HPLC, TLC, spectrophotometry, spectrofluorimetry, and voltammetry [ 21 – 23 ]. In this regard, Peikova et al developed an isocratic reverse phase HPLC method for the simultaneous estimation of FAM and ibuprofen in tablet formulation. The mobile phase consisting of 0.5 M KH 2 PO 4 (pH 2.2) and acetonitrile was pumped ata flow rate of 1.2 ml/min. The chromatographic separation was carried out using a C 8 column at room temperature. The retention time and % recovery of FAM were found to be The retention time and % recovery of FAM were found to be 8.37 and 99.24%, respectively [ 24 ]. In another study, simultaneous estimation of FAM and ibuprofen was carried out using HPLC in isocratic mode The mobile phase was composed of 70:20:10 (v/v/v) Methanol: Water: Phosphate buffer in the ratio of 70:20:10 (v/v/v) and pumped at a flow rate was 1.0 ml/min. The retention time of FAM was 7.8 min, whereas the % recovery was found to be in the range of 99.703-100.433% [ 25 ]. Similarly, an HPLC method was developed for simultaneous estimation of FAM, paracetamol and ibuprofen by Panchale et al . The chromatographic separation was performed on a C 18 column using a mobile pahse made up of water and methanol at a ratio of 17:83. The flow rate was maintained at 0.7 ml/min. The retention time of famotine was found to be 8.58 min. whereas the % recovery was reported to be 100.90 ± 1.99 [ 26 ]. Recently Bharati et al. have developed an HPLC method to detect FAM in pre-clinical sample in rats. A combination of water, methanol and acetonitrile (70:20:10, v/v) was used as a mobile phase and pumped at a rate of 1 ml/min. The retention time of FAM was found to be 5.7 min while, the % recovery was reported to be within the range of 98.06 to 103.56% [ 27 ]. However, no single chromatographic method has been reported to quantitatively determine these components in a combined form. The above-mentioned drugs (OND, FAM, and DRO) might be co-administered to treat different gastrointestinal conditions. Since the co-administration of drugs leads to altered biological functions, the methods for their simultaneous in vivo estimation are helpful in comprehending their interactions. Therefore, the development of new analytical methods that allow simultaneous determination of multiple drugs is essential for the study of pharmacokinetics, therapeutic monitoring, and bioequivalence [ 28 ]. Hence, the present study aimed to develop a simple, accurate, and rapid method for the simultaneous estimation of OND, FAMO, and DRO that will further help in understanding their drug-drug interactions and pharmacokinetic studies. To the best of our knowledge, the simultaneous estimation of OND, FAM, and DRO in combined tablet dosage form has not been described so far. Considering the scarcity of data in this particular aspect, this study was designed to establish a method for the simultaneous estimation of OND, FAM, and DRO. Moreover, the method of drug analysis should be sufficiently sensitive, accurate, and robust. Hence, the central composite design (CCD) approach was incorporated to evaluate the impact of various factors both individually and collectively. The CCD approach operates on the fundamentals of mathematical models, experimental outlines, and the output of these factors. Hence, current study reports the optimization, development, and validation of an HPLC technique with CCD for the concurrent estimation of OND, FAM, and DRO. Experimental Reagents and Materials Potassium dihydrogen phosphate (KH 2 PO 4 , analytical grade) and HPLC grade of methanol, and acetonitrile were purchased from Merck (Darmstadt, Germany). The working standards of OND, FAM, and DRO were received as a gift by Mediceena Pharma (Lahore, Pakistan) and used without any modification. Double distilled water filtered through 0.45 µm was used to prepare the solutions. Instrumentation The chromatographic separations were performed using a Waters Alliance e2695 HPLC system coupled to a 2998 PDA detector with an Agilent EclipseXDB-C18 analytical column (250 mm ×4.6 mm, with a particle size of 5µm). The mobile phase was composed of acetonitrile and KH 2 PO 4 buffer (0.02M at pH 4.8) in the ratio 40:60 (v/v), and it was pumped isocratically at a flow rate of 1.5 mL/min. The mobile phase and all the solutions were used after filteration through a 0.22 µM filter (Millipore, Bedford, MA USA). A thermostatically controlled oven was used to maintain the column temperature at 35°C. The samples were analyzed at a wavelength of 240 nm and each chromatographic analysis was completed in approximately 10 min. The chromatographic data were analyzed using the Empower™ Program (Waters, USA). Preparation of Solutions Preparation of Potassium Dihydrogen Phosphate buffer The buffer was prepared at a concentration of 0.02M by dissolving 2.72g of KH 2 PO 4 in sufficient deionized water. The pH of the resulting solution was maintained at 4.8 while adjusting the final volume up to 1000 mL with deionized water. Preparation of Diluent The diluent was prepared by mixing 50 mL of KH 2 PO 4 (pH 4.8) with 50 mL of acetonitrile. Standard Stock Solution Reference standards of OND (32 mg), FAM (30 mg), and DRO (14 mg) were dissolved in a sufficient amount of diluent consisting of 0.02M KH 2 PO 4 (pH 4.8) and acetonitrile (50:50). Then the volume of this solution was adjusted to 100 mL to achieve the standard stock solutions of OND (32 mg/100 mL), FAM (30 mg/100 mL), and DRO (14 mg/100 mL). Working Solutions An aliquot (5 mL) of the standard stock solution was diluted to make the final concentrations of 16 µg/mL, 15 µg/mL, 7 µg/mL for OND, FAM and DRO, respectively. Sample Solution of Tablet Twenty tablets of optimized formulation (F4) were weighed and ground into a fine powder. In addition to active ingredients, the formulation was composed of excipients including primojel (5mg), croscarmellose sodium (1.74 mg), sodium bicarbonate (65 mg), citric acid (65 mg), saccharine (2 mg), orange flavor (1.5 mg), lactose (143.26 mg), hydroxypropyl methylcellulose (110 mg), guar gum (16.5 mg), and magnesium stearate (12 mg). The powder containing 8 mg of OND, 150 mg of FAM and 70 mg of DRO was weighed, dissolved in a sufficient amount of diluent and sonicated for 15 min. The diluent was composed of 0.02M KH 2 PO 4 (pH 4.8) and acetonitrile (50:50). After adjusting the final volume (100 mL), the solution was filtered and used to prepare the working solution of FAM, DRO and OND at a concentration of 15µg/mL, 7 µg/mL and 16 µg/mL. Treatment of Plasma Samples A volume of 100 µL of rabbit plasma was taken in a centrifuge tube and 10 µL each of OND (84 µg/mL), DRO (723 µg/mL), and FAM (1024 µg/mL) were included in it. Drugs were extracted from plasma using the solvent precipitation technique. Briefly, acetonitrile was added to the above mentioned mixture and centrifugation was performed at 10,000 rpm for 10 min. The resulting organic layer was separated, dried using a nitrogen gas evaporator (25 psi, 4℃), and reconstituted to form a solution. This solution was filtered and an aliquot (20 µL) was injected into the HPLC system. The retention time of OND, DRO, and FAM samples was noted. The plamsa samples were investigated consistently throughout the study. Experimental Design The HPLC method was optimized using the CCD technique. The independent factors (including pH of phosphate buffer, composition and flow rate of mobile phase) were investigated to determine their impact on the percentage recovery and retention time of OND, FAM, and DRO. A list of these factors and responses along with their levels and symbols is shown in Table 1. The variables and their levels were selected based on initial experiments as well as a literature survey [29, 30] 31,32 . The quadratic model was established between the dependent and independent variables using the following equation. Y i = b 0 + b 1 X 1 + b 2 X 2 + b 3 X 3 + b 12 X 1 X 2 + b 13 X 1 X 3 + b 23 X 2 X 3 + b 11 X 1 2 + b 22 X 2 2 + b 33 X 3 2 where Y i represents the computed response b 0 represents intercept b 1 -b 33 represent the regression coefficient X 1 , X 2 and X 3 represent the coded value of factors X a , X b Xi 2 = interactive and quadratic standings Method Validation Standard Calibration Curve To validate the linearity of the method, a series of working solutions of FAM, OND, and DRO were prepared at five different concentrations (5-25 µg/mL) and injected into the HPLC system for analysis. Standard calibration curves for these drugs were generated by plotting the drug concentration vs. average peak area ( Figure 2 ). The intercept (b), slope (m), and correlation coefficient (r 2 ) were determined from the regression analysis. Specificity The specificity of the method was analyzed to assure that there was no interference from excipients, degradation, or other impurities. The specificity of the method was investigated by injecting a solution containing a standard drug (FAM/DRO/OND), placebo, and mobile phase. A formulation containing only excipients was designated as a placebo. The resulting peak purity was used as a parameter to evaluate the specificity of the method ( Figure 5 ). Precision Intermediate precision and repeatability were applied to assess the method's precision. The intermediate precision was evaluated by inter- and intraday variation studies. The intra-day precision was assessed by repeatedly injecting the three different concentrations of OND (8, 16, 24 µg/mL), FAM (7.5, 15, 22.5 µg/mL) and DRO (3.5, 7, 10.5 µg/mL) by two different analysts (Table 3), whereas the inter-day precisions was analyzed by injecting the three dilutions in triplicate on three consecutive days (Table 4). To check the repeatability, 10 injections of the standard drugs i.e. OND, DRO, and FAM (15 µg/mL) were injected. As a result, a linear, similar, and optimum response was obtained (Table 5). Accuracy The accuracy of the proposed method was determined by performing recovery studies (Table 6). The peak area of the sample (containing a predefined quantity of drug) was compared with that of the standard solution (spiked with the same amount). The standard solutions were prepared at a concentration of 8, 16, and 24 µg/mL (for OND), 7.5, 15, and 22.5 µg/mL (for FAM), and 3.5, 7, and 10.5 µg/mL (for DRO). Detection and Quantification Limits As per ICH guidelines [31] 33 , the theoretical limit of detection (LOD) and limit of quantification (LOQ) were computed using the following equations: LOD = 3σ/S ..................Eq. [2] LOQ = 10σ/S ..................Eq. [3] σ represents the standard deviation of the y-intercept of the regression line. S represents the slope of the standard curve. Robustness The robustness of the method is established by deliberately changing the experimental conditions. In the present study, different parameters including room temperature (±5 ℃), pH of the mobile phase (±1), and detection wavelength (±0.1) were varied to determine the robustness of the method. System Suitability It was evaluated by analyzing various parameters, including resolution, retention time, peak area, and number of theoretical plates (Table 7). Stability of Drugs The stability of samples was determined by freeze-thaw cycles, where test solutions were removed from the freezer and thawed at room temperature. The sample was again stored in the freezer for 24 h. and the procedure was repeated thrice before the final stability assessment. These solutions were then analyzed by HPLC for three consecutive days (Table 8). Results and Discussion Current study deals with the development of an HPLC method for the simultaneous estimation of OND, FAM and DRO. These drugs might be co-administered under certain gastrointestinal conditions, therefore development of an analytical method for concurrent determination of these drugs is critical for pharmacokinetics and therapeutic monitoring. Given that no single method is available for the simultaneous estimation of these drugs, the present study was designed to develop and validate an HPLC method that can be used for the concurrent determination of these drugs. Different combination of solvents were tested to select the mobile phase. The best results were procued when a comibtaion of acetonitrile and 0.02 M KH 2 PO 4 buffer(40:60) was used as a mobile phase. The proposed method was optimized using external calibration standard and has been validated in terms of precision, accuracy, specificity, robustness, the limit of detection and quantification, system suitability, and the stability of drug molecules. The stated parameters ensured the reproducibility of the reported method. A detailed discussion of each step is given below. HPLC Method Optimization The CCD approach was adopted to optimize the chromatographic conditions and a quadratic model was built. The obtained responses (Y 1 -Y 6 ) were best fitted to this model and a regression equation was developed. It was observed that the factors X 1 , X 2, and X 3 produced a variable effect on the retention time and percentage recovery of OND, FAM, and DRO. A mobile phase made up of (40:60) ratio of acetonitrile and phosphate buffer with pH 4.8, injected at a flow rate of 1 mL/min, resulted in a varied retention time for OND (1.8-2.2 min.), FAM (1.3-1.7 min.), and DRO (5-6 min.). This variation was also observed when the flow rate was adjusted to 1.5 mL/min. while keeping the composition of the mobile phase the same. However, all the injected concentrations possessed a similar retention time (Y 1 , Y 2 , Y 3 ) when the mobile phase composed of acetonitrile and phosphate buffer with pH 4.8 (40:60) was injected at 1.5 mL/min. Moreover, the percentage recovery of three drugs (Y 4 , Y 5 , Y 6 ) was found to be within the range of 90-110%. Hence, it can be suggested that the design showed an insignificant association between the variables X 1 and X 2 . The results indicate that the composition and flow rate of the mobile phase play an important role while performing the simultaneous estimation of OND, FAM, and DRO. Moreover, the quadratic effects of X 1 , X 2 , and X 3 (independent variables) on the percent recovery ( p = 0.002) and retention time ( p < 0.001) of OND, FAM, and DRO were found to be significant. Validation of the Optimized Factors The effects X 1 , X 2 , and X 3 were validated by making a comparison between predicted and obtained results. As shown in Table 2, a difference between the predicted and observed data for OND, FAM, and DRO was found to be within the range of ±6%. Chromatographic Method Validation Analytical Curve The linearity was determined for OND, FAM, and DRO at various concentrations ranging from 5-25 µg/mL. The calibration curves were generated by plotting the mean peak areas against respective concentrations. The linear regression equation for OND was y= 24053x-8118.5 whereas the value of square of correlation coefficient (R 2 ) was 1. The linear regression equation for FAM was y= 12769x-5037.6 and the value of R 2 was 1. Similarly, the linear regression equation for DRO was y= 32208x-16417 and the value of R 2 was found to be 0.9999. These results indicate the linearity of the developed method (Figure 2, 3 and 4). Moreover, the ANOVA analysis was also carried out and the p-value (<0.05) established that the results are statistically significant. Specificity The specificity of the method was analyzed and no interference was observed between the active drug ingredient and excipients. As shown in Figure 5, the retention times of OND, FAM, and DRO were found to be 2.1 min, 1.48 min, and 6.4 min, respectively. Precision Intra-day precision of the method was investigated by analyzing the three dilutions of each drug (OND, FAM, DRO) in triplicates. However, the inter-day precision was assessed by analyzing the three dilutions of OND, FAM, and DRO on three different days. As shown in Table 3 and 4, all the RSD values were determined to be within the acceptable range i.e. less than 2%, hence reflecting the precision of the proposed method. In order to assess the repeatability, 10 injections of standard solutions (FAM, OND, and DRO) were injected at a concentration of 15 µg/mL (Figure 6). The RSD value for peak area was found to be 0.3%, 0.4%, and 0.4% for OND, FAM, and DRO, respectively (Table 5). These results indicate the precision of the developed HPLC method. Accuracy It was established by computing the percent recovery for OND, FAM, and DRO. Percent recoveries were found to be 99.87-103.8%, 92.1-101.33%, and 97.6-105.6% for OND, FAM, and DRO, respectively (Table 6). These results indicate the accuracy of the suggested method. LOD and LOQ The LOD for OND, FAM, and DRO was determined to be 0.421, 0.534, and 2.178 µg/mL, respectively. While, the LOQ for OND, FAM, and DRO was found to be 1.364, 1.747, and 7.061 µg/mL, respectively. These are the lowest values on the calibration curve that were obtained with adequate accuracy and precision under the optimized chromatographic conditions. Hence, these results indicate the sensitivity of the proposed method. Robustness It was observed that altering the robustness parameters (room temperature, pH of mobile phase, and detection wavelength) had no considerable variation in the retention time of OND, FAM and DRO. System Suitability System suitability parameters were analyzed to ensure the adequacy and feasibility of the proposed method. As shown in Table 7, OND, FAM, and DRO yielded a %RSD value of less than 2% for retention time and peak area, suggesting that there is little variation in measured values. The tailing factors for OND, FAM, and DRO were 1.42, 1.73, and 1.48, respectively, indicating the symmetry of all peaks. The efficiency of the column is reflected in several theoretical plates that were determined to be 4247, 4802, and 6629 for OND, FAM, and DRO, respectively. A resolution value of 1 represents an adequate degree of separation, and the results showed that OND (R = 5.778) and DRO (R = 18.034) possessed a high degree of peak resolution. Hence, proposed method reflects the satisfactory results in terms of %RSD values, tailing factor, theoraticla plates and resolution for all the drugs. These results confirm the faesiblity of developed method. Stability of Drugs in Rabbit Plasma The stability of drugs in rabbit plasma was evaluated by freeze-thaw cycles and average degradation for selected concentrations of OND, FAM, and DRO was determined to be 97%. Hence a simple, accurate, precise method was developed method for the simulatneus estimation of OND, FAM and DRO. Moreover, the system suitability parameters were studied using rabbit plasma and method was foun to be appropriate for the simultaneous estimation of these drugs in rabbit plasma. Eventually, the work could be extended to determine the human pharmacokinectis of these drug. Conclusions In the present study, chromatographic conditions were optimized employing the CCD, and a simple HPLC technique was developed for the concurrent estimation of OND, FAM, and DRO. The chromatographic parameters (flow rate, composition, and pH of the mobile phase) were optimized to achieve reproducible peaks with high resolution. Preliminary studies revealed that using a 60:40 (v/v) mixture of acetonitrile and phosphate buffer (pH 4.8) as a mobile phase, flowing through the column at a flow rate of 1.5 mL/min. produced the best results. The method was further validated as per ICH recommendations and was found appropriate for the simultaneous estimation of OND, FAM, and DRO with excellent linearity, sensitivity, accuracy, and precision. Hence, the proposed method can be used for the routine analysis of OND, FAM, and DRO in combined dosage forms. Declarations Author contributions; All authors have been involved in study design, data collection, formal analysis, and manuscript writing. All authors approved the final version of the manuscript and agreed to submit it. Acknowledgments: The authors would like to extend their sincere appreciation to the Department of Pharmaceutics and DTL Multan for providing necessary facilities during this study. Data Availability: All data generated or analyzed during this study are included in this published article Declaration of Interest; The authors declare that there are no conflicts of interest. References Kannappan V, Kanthiah S (2017) Chromatographia 80:229-236. Panigrahi D, Sharma R (2008) Acta chromatographica 20:439-450. Azmi SNH, Al Rawahi WAN, Al Yahyai AI, Al Qasimi AA, Al Fuliti KS, Al Qalhati OS, Rahman N, Ahmed QU (2024) Journal of Chromatography B 1234:124035. Rajput JM. Rai RR, Nijhawan S (2021) Saudi Journal of Gastroenterology 27:136-143. Zarghi A, Shafaati A, Foroutan S, Khoddam A (2005) Journal of pharmaceutical and biomedical analysis 39:677-680. El-Shaheny R, Radwan MO, Belal F, Yamada K (2020) Journal of Pharmaceutical and Biomedical Analysis 186:113305. Jaimini M, Rana A, Tanwar Y (2007) Current drug delivery 4:51-55. Balint A, Silvia I, Bondoroi-Papp Z, Muntean D-L (2018) The Medical-Surgical Journal 122:207-212. 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Tables Table 1 : List of independent and dependent variables selected for Central Composite Design Variables Level Constraints -1 0 +1 Independent X 1 (Flow rate in mL/min) 1.0 1.5 2.0 Within the range X 2 (Mobile Phase) 1 3 5 Within the range X 3 (pH of Phosphate Buffer) 4.8 5.8 6.8 Within the range Dependent Y 1 (Retention Time of OND) 1.8 to 2.2 min Y 2 (Retention Time of FAM) 1.3 to 1.7 min Y 3 (Retention Time of DRO) 5.0 to 6.0 min Y 4 (Recovery of OND) 90% to 110% Y 5 (Recovery of FAM) 90% to 110% Y 6 (Recovery of DRO) 90% to 110% Table 2 : Validating the optimized factors for simultaneous estimation of OND, FAM and DRO by the proposed HPLC method Response Predicted Value Observed Value % Residual OND FAM DRO OND FAM DRO OND FAM DRO Retention Time (min) 1.82 1.52 5.43 1.93 1.45 5.46 -6.04 +4.60 -0.55 % Recovery 99.63 100.34 101.65 98.46 101.76 100.49 +1.17 -1.41 +1.14 Table 3 : Result of intra-day precision for estimation of OND, FAM and DRO by proposed HPLC method (n=3) Ondansetron Famotidine Drotaverine Conc. (µg mL -1 ) Mean Conc. (µg mL -1 ) RSD (%) Conc. (µg mL -1 ) Mean Conc. (µg mL -1 ) RSD (%) Conc. (µg mL -1 ) Mean Conc. (µg mL -1 ) RSD (%) 8.0 8.77 ±0.462 0.712 7.5 7.23±0.452 0.762 3.5 3.02±0.468 0.128 16.0 16.27±0.156 1.213 15.0 15.87±0.176 0.189 7.0 7.67±0.741 0.389 24.0 24.76±0.278 0.128 22.5 22.75±0.487 0.264 10.5 10.29±0.498 0.045 RSD= Relative standard deviation Table 4 : Result of inter-day precision for estimation of OND, FAM and DRO by proposed HPLC method (n=3) Ondansetron Famotidine Drotaverine Conc. (µg mL -1 ) Mean Conc. (µg mL -1 ) RSD(%) Conc. (µg mL -1 ) Mean Conc. (µg mL -1 ) RSD(%) Conc. (µg mL -1 ) Mean Conc. (µg mL -1 ) RSD(%) 8.0 8.53 ±0.671 0.476 7.5 7.52±0.154 0.709 3.5 3.51±0.571 0.253 16.0 16.04±0.341 0.719 15.0 15.73±0.657 1.298 7.0 7.69±0.856 0.572 24.0 24.21±0.456 0.128 22.5 22.19±0.942 0.109 10.5 10.51±0.308 1.148 RSD= Relative standard deviation Table 5 : Result of repeatability for simultaneous estimation of OND, FAM and DRO by proposed HPLC method Parameter Peak area Ondansetron Famotidine Drotaverine Mean 352998.750 186547.193 466055.356 SD ± 1126.557 777.309 1811.782 %RSD 0.3 0.4 0.4 SD= Standard deviation, RSD= Relative standard deviation Table 6: Result of percent recovery of OND, FAM, DRO in rabbit plasma by proposed HPLC method (n=6) Actual Conc. (µg mL -1 ) OND FAM DRO Recovered Conc. (µg mL -1 ) RSD (%) Recovery (%) Recovered Conc. (µg mL -1 ) RSD (%) Recovery (%) Recovered Conc. (µg mL -1 ) RSD (%) Recovery (%) 5.0 5.19 ±0.573 0.961 103.8 4.92±0.254 0.931 98.40 5.28±0.068 0.873 105.6 10.0 10.07±0.056 0.265 100.7 9.21±0.783 0.298 92.11 9.76±0.341 0.275 97.61 15.0 14.98±0.429 0.572 99.87 15.2±0.512 0.679 101.3 15.34±0.998 0.548 102.2 20.0 20.31±0.857 0.761 101.5 19.4±0.956 0.317 97.00 19.78±0.254 0.769 98.91 25.0 24.79±0.155 0.347 99.16 24.7±0.487 0.528 98.80 25.45±0.053 0.741 101.8 RSD= Relative standard deviation Table 7: System suitability parameters determined for simultaneous estimation of OND, FAM and DRO in rabbit plasma by proposed HPLC method (n=5) Parameter FAM OND DRO Mean RSD(%) Limit Mean RSD(%) Limit Mean RSD(%) Limit Retention Time (min.) 1.48 0.01 - 2.2 0.3 - 6.6 0.8 - Peak Area 186547.193 0.3 < 2 352998.750 0.3 < 2 466055.356 0.4 < 2 Tailing factor 1.73 0.01 < 2 1.42 0.5 < 2 1.48 1.6 1 18.034 7.0 > 1 RSD= Relative standard deviation Table 8 : Result of stability study for OND, FAM and DRO in rabbit plasma evaluated via freeze- thaw cycles (n=6) Time Concentration (µg/mL) Area OND FAM DRO At start (First day) 15 µg/mL 353877.809 186632.882 469027.938 % Recovery 100%±0.58 100%±0.89 100%±0.93 After 24 Hours 15 µg/mL 364876.232 182056.209 4710923.446 % Recovery 103.11%±0.91 97.54%±0.36 100.44%±0.67 After 48 Hours 15 µg/mL 350578.342 188987.329 461005.375 % Recovery 99.07%±0.66 101.26%±1.05 98.29%±0.69 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6133930","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":423835437,"identity":"b95da148-52d7-47c9-ba97-86ee7cfca2d5","order_by":0,"name":"Rao Khurram Ayoub","email":"","orcid":"","institution":"Government of Punjab","correspondingAuthor":false,"prefix":"","firstName":"Rao","middleName":"Khurram","lastName":"Ayoub","suffix":""},{"id":423835438,"identity":"6f610e0d-e901-424a-92bc-d37446b2ce81","order_by":1,"name":"Syed Nisar Hussain Shah","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYDACZiBmbGCAkB/BDAYDIE4gTgvjTKK0MMC1ALXzEqPFnJ394efKHYft5dsPtz223VGX2MDevE2CcUcaTi2WzTzGkmfPHE7ccCax3Tj3DFtiA8+xMgnGMzk4tRgc5mGQbGw7nGDAkNgmndvGk9ggkWMmwdhWgUcL++OfQC328v0P26Qt2yQSG+TfENLCYAayhbHhBtAWxjYDoC08IC14HWZm2diWnrjhxsM2yd4zCcZtPGnFFolncHvf4Pzxxzcb26yBDkt/JvFzR51sP/vhjTc+7kjGqQUTsIGIxAYSdEAALHJHwSgYBaNgFAABAPeSVSirbw3DAAAAAElFTkSuQmCC","orcid":"","institution":"Bahauddin Zakariya University","correspondingAuthor":true,"prefix":"","firstName":"Syed","middleName":"Nisar Hussain","lastName":"Shah","suffix":""},{"id":423835439,"identity":"8aacf265-694a-4376-8b10-cb42d3f6efd8","order_by":2,"name":"Asma Aslam","email":"","orcid":"","institution":"Bahauddin Zakariya University","correspondingAuthor":false,"prefix":"","firstName":"Asma","middleName":"","lastName":"Aslam","suffix":""},{"id":423835440,"identity":"c728cf30-066d-4370-9c46-c88f7ac4a24a","order_by":3,"name":"Romana Riaz","email":"","orcid":"","institution":"Bahauddin Zakariya University","correspondingAuthor":false,"prefix":"","firstName":"Romana","middleName":"","lastName":"Riaz","suffix":""},{"id":423835441,"identity":"eec1d425-f445-4a1a-a79d-41b6031a9f36","order_by":4,"name":"Naveed Nisar","email":"","orcid":"","institution":"Bahauddin Zakariya University","correspondingAuthor":false,"prefix":"","firstName":"Naveed","middleName":"","lastName":"Nisar","suffix":""},{"id":423835442,"identity":"5e818e90-ddc4-4541-bf93-330e374475d6","order_by":5,"name":"Shakeel Ijaz","email":"","orcid":"","institution":"Quaid-e-Azam College of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Shakeel","middleName":"","lastName":"Ijaz","suffix":""},{"id":423835443,"identity":"eb168c79-93b0-405e-a94e-64176417c097","order_by":6,"name":"Aousaf Ahmad","email":"","orcid":"","institution":"Quaid-e-Azam College of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Aousaf","middleName":"","lastName":"Ahmad","suffix":""},{"id":423835444,"identity":"c6347e35-ea03-474b-b507-40bd3650bc7d","order_by":7,"name":"Muneeb Ahmad","email":"","orcid":"","institution":"Quaid-e-Azam College of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Muneeb","middleName":"","lastName":"Ahmad","suffix":""}],"badges":[],"createdAt":"2025-03-01 09:53:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6133930/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6133930/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77950574,"identity":"5e3fef94-e3f0-4260-9d99-d2c07c736249","added_by":"auto","created_at":"2025-03-07 07:22:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8096,"visible":true,"origin":"","legend":"\u003cp\u003eThe chemical structures of OND, FAM, and DRO\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6133930/v1/4f84faad809e692a77f78b3f.png"},{"id":77950586,"identity":"6af1ee3a-6b2d-48b7-b0c1-8dfd78e0beb9","added_by":"auto","created_at":"2025-03-07 07:22:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":32524,"visible":true,"origin":"","legend":"\u003cp\u003eStandard calibration curve generated for OND with a concentration range of 5-25 µg/mL. The R\u003csup\u003e2\u003c/sup\u003e value depicts the linearity of proposed method.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6133930/v1/371e1738088e96526afa0918.png"},{"id":77950571,"identity":"e3d0a006-923b-46ce-aa0e-d0c95f72d89f","added_by":"auto","created_at":"2025-03-07 07:22:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54680,"visible":true,"origin":"","legend":"\u003cp\u003eStandard calibration curve generated for FAM with a concentration range of 5-25 µg/mL. The R\u003csup\u003e2\u003c/sup\u003e value depicts the linearity of proposed method.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6133930/v1/0eff2992ce7a52147c4135c1.png"},{"id":77952109,"identity":"bf5715e9-ee62-49f1-9e50-669fa016ba70","added_by":"auto","created_at":"2025-03-07 07:30:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":59331,"visible":true,"origin":"","legend":"\u003cp\u003eStandard calibration curve generated for DRO with a concentration range of 5-25 µg/mL. The R\u003csup\u003e2\u003c/sup\u003e value depicts the linearity of proposed method.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6133930/v1/3ff9c1e63f2974e130bef582.png"},{"id":77952536,"identity":"9f27a348-8b32-4fac-919e-b38380e2c11c","added_by":"auto","created_at":"2025-03-07 07:38:25","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":75705,"visible":true,"origin":"","legend":"\u003cp\u003eRetention time of OND (2.1 min.), FAM (1.48 min.), and DRO (6.4 min.) indicating the specificity of the proposed method as no interference was observed.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6133930/v1/ac840421874009257af02c2e.jpeg"},{"id":77950576,"identity":"f891a628-a85c-47b9-8aef-70fadf59c996","added_by":"auto","created_at":"2025-03-07 07:22:25","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":115454,"visible":true,"origin":"","legend":"\u003cp\u003eRepeatability of the proposed method was achieved by injecting 10 injections of standard solutions of OND, FAM, and DRO at a concentration of 15 µg/mL.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6133930/v1/346d467f530d6b342198147f.jpeg"},{"id":77954611,"identity":"c605451d-90aa-4d0a-841d-897addde139c","added_by":"auto","created_at":"2025-03-07 07:54:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1765343,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6133930/v1/996a6049-5587-4cc2-8059-7fdcc42dc53e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and Validation of HPLC Method for Simultaneous Estimation of Ondansetron Dihydrochloride, Famotidine, and Drotaverine Hydrochloride in Rabbit Plasma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOndansetron (OND), chemically known as (RS)-9-methyl-3-[(2-methylimidazol-1-yl) methyl]-2,3-dihydro-1\u003cem\u003eH\u003c/em\u003e-carbazol-4-1 acts as a selective antagonist of 5-hydroxytryptamine type 3 (5-HT3) receptor. OND is used to alleviate nausea and vomiting associated with radiotherapy, chemotherapy and surgery at doses ranging from 4\u0026ndash;24 mg [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Drotaverine (DRO) is an isoquinoline derivative with the chemical name of 1-[(3, 4-diethoxyphenyl) methylidene]-6, 7-diethoxy-3, 4-dihydro-2\u003cem\u003eH\u003c/em\u003e-isoquinoline. It is an antispasmodic drug that produces its effect on smooth muscles by inhibiting the phosphodiesterase (PDE) enzyme system[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. DRO is used to treat spasmodic discomfort caused by smooth muscle illnesses such as irritable bowel syndrome as well as biliary, intestinal, or ureteric colic. The recommended dose of DRO ranges from 40\u0026ndash;80 mg [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Famotidine (FAM) is chemically designated as 3-[2-[(aminoiminomethyl)amino]-4-thiazolyl]methyl]thio]-\u003cem\u003eN\u003c/em\u003e- (aminosulfonyl) propanimidamide. It is classified as an antagonist of H2 receptor and indicated for the management of peptic ulcer and gastroesophageal reflux disease [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Typically, it is used at doses ranging from 20\u0026ndash;40 mg [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Structural formulas of OND, DRO, and FAM are revealed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDue to their recommended pharmacological dose, this combination of drugs is formulated with a variable quantity of active ingredients i.e. 8 mg of OND, 69 mg of DRO hydrochloride, 151 mg of FAM. This variable amount of active ingredients in a multidrug formulation complicates the process of routine analysis. Whereas, differing polarity and chromatographic behavior of these active moieties further complicate the analysis.\u003c/p\u003e \u003cp\u003eSeveral methods have been described in literature for the quantitative determination of these drugs in individual dosage forms. For example, OND has been reported to be analyzed using high-performance thin-layer chromatography (HPTLC), HPLC, and spectrophotometry [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For example, Patel \u003cem\u003eet al\u003c/em\u003e. established a simultaneous equation method for the simultaneous estimation of OND and omeprazole in bulk and pharmaceutical preparations. This method used two separate wavelenthgs for ompeprazole and ondancertron. Methanol was used as a solvent and the % recovery of OND was found to be 99.49\u0026ndash;99.66% [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In another study, Dedandia \u003cem\u003eet al\u003c/em\u003e. have developed an HPLC method for the simultaneous determination of OND and omeprazole in combined doasahe form. A methanol and acetonitrile (90:10) was used as mobile phase. The flow rate was kept to 0.5 ml/min and the retention time of OND was found to be 11.08 min. However, the % recovery of OND was recorded to be 99.54 to 100.2% [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSimilarly, the analytical methods reported for the estimation of DRO hydrochloride are spectrophotometry, voltammetry, thin-layer chromatography (TLC) and HPLC [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For example, Azhlwar \u003cem\u003eet al.\u003c/em\u003e developed a stability indicating HPLC method for simultaneous estimation of DRO and aceclofenac. The mobile phase composed of acetonitrile and 0.1% trifluoro acetic acid (55:45%v/v) and the % recovery of DRO was reported to be 99.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3250 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Another reverse phase HPLC method was developed by Panigrahi \u0026amp; Shrama for the simultaneous estimation of DRO and omeprazole in tablet dosage forms. This method was developed using a mixture of 0.1 M ammonium acetate and methanol as mobile phase for tablet dosage forms. The flow rate was maintained at 1.5 ml/min. DRO was eluted with aretention time of 7.969 min while the % recovery of DRO was found to be within the range of 100.66\u0026ndash;100.94% [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Similarly, Dahivelkar \u003cem\u003eet al\u003c/em\u003e have established an HPLC method for simultaneous determination DRO hydrochloride and mefenamic acid in human plasma. The compounds were separated in an isocratic mode while using a mixture of 20 mM ammonium acetate buffer and acetonitrile (55:45) as mobile phase. DRO was eluted with a retention time of 8.21 min and its % percent recovery was reported to be 94.51% [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Abdellatef \u003cem\u003eet al.\u003c/em\u003e developed spectrophotometric and spectrodensitometric techniques for simultaneous determination of paracetamol and DRO and reported the % recovery of DRO to be 100.11% \u0026plusmn; 1.91 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] .\u003c/p\u003e \u003cp\u003eDifferent methods developed for the analysis of FAM include capillary electrophoresis, HPLC, TLC, spectrophotometry, spectrofluorimetry, and voltammetry [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this regard, Peikova \u003cem\u003eet al\u003c/em\u003e developed an isocratic reverse phase HPLC method for the simultaneous estimation of FAM and ibuprofen in tablet formulation. The mobile phase consisting of 0.5 M KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (pH 2.2) and acetonitrile was pumped ata flow rate of 1.2 ml/min. The chromatographic separation was carried out using a C\u003csub\u003e8\u003c/sub\u003e column at room temperature. The retention time and % recovery of FAM were found to be The retention time and % recovery of FAM were found to be 8.37 and 99.24%, respectively [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In another study, simultaneous estimation of FAM and ibuprofen was carried out using HPLC in isocratic mode The mobile phase was composed of 70:20:10 (v/v/v) Methanol: Water: Phosphate buffer in the ratio of 70:20:10 (v/v/v) and pumped at a flow rate was 1.0 ml/min. The retention time of FAM was 7.8 min, whereas the % recovery was found to be in the range of 99.703-100.433% [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Similarly, an HPLC method was developed for simultaneous estimation of FAM, paracetamol and ibuprofen by Panchale \u003cem\u003eet al\u003c/em\u003e. The chromatographic separation was performed on a C\u003csub\u003e18\u003c/sub\u003e column using a mobile pahse made up of water and methanol at a ratio of 17:83. The flow rate was maintained at 0.7 ml/min. The retention time of famotine was found to be 8.58 min. whereas the % recovery was reported to be 100.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Recently Bharati \u003cem\u003eet al.\u003c/em\u003e have developed an HPLC method to detect FAM in pre-clinical sample in rats. A combination of water, methanol and acetonitrile (70:20:10, v/v) was used as a mobile phase and pumped at a rate of 1 ml/min. The retention time of FAM was found to be 5.7 min while, the % recovery was reported to be within the range of 98.06 to 103.56% [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, no single chromatographic method has been reported to quantitatively determine these components in a combined form.\u003c/p\u003e \u003cp\u003eThe above-mentioned drugs (OND, FAM, and DRO) might be co-administered to treat different gastrointestinal conditions. Since the co-administration of drugs leads to altered biological functions, the methods for their simultaneous in vivo estimation are helpful in comprehending their interactions. Therefore, the development of new analytical methods that allow simultaneous determination of multiple drugs is essential for the study of pharmacokinetics, therapeutic monitoring, and bioequivalence [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Hence, the present study aimed to develop a simple, accurate, and rapid method for the simultaneous estimation of OND, FAMO, and DRO that will further help in understanding their drug-drug interactions and pharmacokinetic studies.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, the simultaneous estimation of OND, FAM, and DRO in combined tablet dosage form has not been described so far. Considering the scarcity of data in this particular aspect, this study was designed to establish a method for the simultaneous estimation of OND, FAM, and DRO. Moreover, the method of drug analysis should be sufficiently sensitive, accurate, and robust. Hence, the central composite design (CCD) approach was incorporated to evaluate the impact of various factors both individually and collectively. The CCD approach operates on the fundamentals of mathematical models, experimental outlines, and the output of these factors. Hence, current study reports the optimization, development, and validation of an HPLC technique with CCD for the concurrent estimation of OND, FAM, and DRO.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cp\u003e\u003cstrong\u003eReagents and Materials\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePotassium dihydrogen phosphate (KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, analytical grade) and HPLC grade of \u0026nbsp;methanol, and acetonitrile were purchased from Merck (Darmstadt, Germany). The working standards of OND, FAM, and DRO were received as a gift by Mediceena Pharma (Lahore, Pakistan) and used without any modification. Double distilled water filtered through 0.45 \u0026micro;m was used to prepare the solutions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstrumentation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chromatographic separations were performed using a Waters Alliance e2695 HPLC system coupled to a 2998 PDA detector with an Agilent\u0026nbsp;EclipseXDB-C18 analytical column (250 mm \u0026times;4.6 mm, with a particle size of 5\u0026micro;m). The mobile phase was composed of acetonitrile and KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e buffer (0.02M at pH 4.8) in the ratio 40:60 (v/v), and it was pumped isocratically at a flow rate of 1.5 mL/min. The mobile phase and all the solutions were used after filteration through a 0.22 \u0026micro;M filter (Millipore, Bedford, MA USA). A thermostatically controlled oven was used to maintain the column temperature at 35\u0026deg;C. The samples were analyzed at a wavelength of 240 nm and each chromatographic analysis was completed in approximately 10 min. The chromatographic data were analyzed using the\u0026nbsp;Empower\u0026trade; Program (Waters, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of Solutions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of Potassium Dihydrogen Phosphate buffer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe buffer was prepared at a concentration of 0.02M by dissolving 2.72g of KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e in sufficient deionized water. The pH of the resulting solution was maintained at 4.8 while adjusting the final volume up to 1000 mL with deionized water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of Diluent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe diluent was prepared by mixing 50 mL of KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (pH 4.8) with 50 mL of acetonitrile.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStandard Stock Solution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReference standards of OND (32 mg), FAM (30 mg), and DRO (14 mg) were dissolved in a sufficient amount of diluent consisting of 0.02M KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (pH 4.8) and\u003csub\u003e\u0026nbsp;\u003c/sub\u003eacetonitrile (50:50). Then the volume of this solution was adjusted to 100 mL to achieve the standard stock solutions of OND (32 mg/100 mL), FAM (30 mg/100 mL), and DRO (14 mg/100 mL).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWorking Solutions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn aliquot (5 mL) of the standard stock solution was diluted to make the final concentrations of 16 \u0026micro;g/mL, 15 \u0026micro;g/mL, 7 \u0026micro;g/mL for OND, FAM and DRO, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Solution of Tablet\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty tablets of optimized formulation (F4) were weighed and ground into a fine powder. In addition to active ingredients, the formulation was composed of excipients including primojel (5mg), croscarmellose sodium (1.74 mg), sodium bicarbonate (65 mg), citric acid (65 mg), saccharine (2 mg), orange flavor (1.5 mg), lactose (143.26 mg), hydroxypropyl methylcellulose (110 mg), guar gum (16.5 mg), and magnesium stearate (12 mg). The powder containing 8 mg of OND, 150 mg of FAM and 70 mg of DRO was weighed, dissolved in a sufficient amount of diluent and sonicated for 15 min. The diluent was composed of 0.02M KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (pH 4.8) and\u003csub\u003e\u0026nbsp;\u003c/sub\u003eacetonitrile (50:50).\u0026nbsp;After adjusting the final volume (100 mL), the solution was filtered and used to prepare the working solution of FAM, DRO and OND at a concentration of 15\u0026micro;g/mL, 7 \u0026micro;g/mL and 16 \u0026micro;g/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment of Plasma Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA volume of 100 \u0026micro;L of rabbit plasma was taken in a centrifuge tube and 10 \u0026micro;L each of OND (84 \u0026micro;g/mL), DRO (723 \u0026micro;g/mL), and FAM (1024 \u0026micro;g/mL) were included in it. Drugs were extracted from plasma using the solvent precipitation technique. Briefly, acetonitrile was added to the above mentioned mixture and centrifugation was performed at 10,000 rpm for 10 min. The resulting organic layer was separated, dried using a nitrogen gas evaporator (25 psi, 4℃), and reconstituted to form a solution. This solution was filtered and an aliquot (20 \u0026micro;L) was injected into the HPLC system. The retention time of OND, DRO, and FAM samples was noted. The plamsa samples were investigated consistently throughout the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe HPLC method was optimized using the CCD technique. The independent factors (including pH of phosphate buffer, composition and flow rate of mobile phase) were investigated to determine their impact on the percentage recovery and retention time of OND, FAM, and DRO. \u0026nbsp;A list of these factors and responses along with their levels and symbols is shown in Table 1. The variables and their levels were selected based on initial experiments as well as a literature survey [29, 30]\u003csup\u003e31,32\u003c/sup\u003e. The quadratic model was established between the dependent and independent variables using the following equation.\u003c/p\u003e\n\u003cp\u003eY\u003csub\u003ei\u003c/sub\u003e = b\u003csub\u003e0\u0026nbsp;\u003c/sub\u003e+ b\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e1\u003c/sub\u003e+ b\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e + b\u003csub\u003e3\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e + b\u003csub\u003e12\u003c/sub\u003eX\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e + b\u003csub\u003e13\u003c/sub\u003eX\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e + b\u003csub\u003e23\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e + b\u003csub\u003e11\u003c/sub\u003eX\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e+ b\u003csub\u003e22\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e + b\u003csub\u003e33\u003c/sub\u003eX\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003ewhere\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eY\u003csub\u003ei\u003c/sub\u003e represents the computed response\u003c/p\u003e\n\u003cp\u003eb\u003csub\u003e0\u0026nbsp;\u003c/sub\u003erepresents intercept\u003c/p\u003e\n\u003cp\u003eb\u003csub\u003e1\u003c/sub\u003e-b\u003csub\u003e33\u003c/sub\u003e represent the regression coefficient\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eX\u003csub\u003e1\u003c/sub\u003e, X\u003csub\u003e2\u003c/sub\u003e and X\u003csub\u003e3\u003c/sub\u003e represent the coded value of factors X\u003csub\u003ea\u003c/sub\u003e, X\u003csub\u003eb\u003c/sub\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eXi\u003csup\u003e2\u003c/sup\u003e = interactive and quadratic standings\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Method Validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eStandard Calibration Curve\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the linearity of the method, a series of working solutions of FAM, OND, and DRO were prepared at five different concentrations (5-25 \u0026micro;g/mL) and injected into the HPLC system for analysis. Standard calibration curves for these drugs were generated by plotting the drug concentration vs. average peak area (\u003cstrong\u003eFigure 2\u003c/strong\u003e). The intercept (b), slope (m), and correlation coefficient (r\u003csup\u003e2\u003c/sup\u003e) were determined from the regression analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecificity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe specificity of the method was analyzed to assure that there was no interference from excipients, degradation, or other impurities. The specificity of the method was investigated by injecting a solution containing a standard drug (FAM/DRO/OND), placebo, and mobile phase. A formulation containing only excipients was designated as a placebo. The resulting peak purity was used as a parameter to evaluate the specificity of the method (\u003cstrong\u003eFigure 5\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntermediate precision and repeatability were applied to assess the method\u0026apos;s precision. The intermediate precision was evaluated by inter- and intraday variation studies. The intra-day precision was assessed by repeatedly injecting the three different concentrations of OND (8, 16, 24 \u0026micro;g/mL), FAM (7.5, 15, 22.5 \u0026micro;g/mL) and DRO (3.5, 7, 10.5 \u0026micro;g/mL) by two different analysts (Table 3), whereas the inter-day precisions was analyzed by injecting the three dilutions in triplicate on three consecutive days (Table 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo check the repeatability, 10 injections of the standard drugs i.e. OND, DRO, and FAM (15 \u0026micro;g/mL) were injected. As a result, a linear, similar, and optimum response was obtained (Table 5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe accuracy of the proposed method was determined by performing recovery studies (Table 6). The peak area of the sample (containing a predefined quantity of drug) was compared with that of the standard solution (spiked with the same amount). The standard solutions were prepared at a concentration of 8, 16, and 24 \u0026micro;g/mL (for OND), 7.5, 15, and 22.5 \u0026micro;g/mL (for FAM), and 3.5, 7, and 10.5 \u0026micro;g/mL (for DRO).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection and Quantification Limits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs per ICH guidelines [31]\u003csup\u003e33\u003c/sup\u003e, the theoretical limit of detection (LOD) and limit of quantification (LOQ) were computed using the following equations:\u003c/p\u003e\n\u003cp\u003eLOD = 3\u0026sigma;/S \u0026nbsp; ..................Eq. [2]\u003c/p\u003e\n\u003cp\u003eLOQ = 10\u0026sigma;/S ..................Eq. [3]\u003c/p\u003e\n\u003cp\u003e\u0026sigma; represents the standard deviation of the y-intercept of the regression line.\u003c/p\u003e\n\u003cp\u003eS represents the slope of the standard curve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe robustness of the method is established by deliberately changing the experimental conditions. In the present study, different parameters including room temperature (\u0026plusmn;5 ℃), pH of the mobile phase (\u0026plusmn;1), and detection wavelength (\u0026plusmn;0.1) were varied to determine the robustness of the method.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSystem Suitability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt was evaluated by analyzing various parameters, including resolution, retention time, peak area, and number of theoretical plates (Table 7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStability of Drugs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe stability of samples was determined by freeze-thaw cycles, where test solutions were removed from the freezer and thawed at room temperature. The sample was again stored in the freezer for 24 h. and the procedure was repeated thrice before the final stability assessment. These solutions were then analyzed by HPLC for three consecutive days (Table 8).\u0026nbsp;\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eCurrent study deals with the development of an HPLC method for the simultaneous estimation of OND, FAM and DRO. These drugs might be co-administered under certain gastrointestinal conditions, therefore development of an analytical method for concurrent determination of these drugs is critical for pharmacokinetics and therapeutic monitoring. Given that no single method is available for the simultaneous estimation of these drugs, the present study was designed to develop and validate an HPLC method that can be used for the concurrent determination of these drugs. Different combination of solvents were tested to select the mobile phase. The best results were procued when a comibtaion of acetonitrile and 0.02 M KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e buffer(40:60) was used as a mobile phase. The proposed method was optimized using external calibration standard and has been validated in terms of precision, accuracy, specificity, robustness, the limit of detection and quantification, system suitability, and the stability of drug molecules. The stated parameters ensured the reproducibility of the reported method. A detailed discussion of each step is given below.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHPLC Method Optimization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CCD approach was adopted to optimize the chromatographic conditions and a quadratic model was built. The obtained responses (Y\u003csub\u003e1\u003c/sub\u003e-Y\u003csub\u003e6\u003c/sub\u003e) were best fitted to this model and a regression equation was developed. It was observed that the factors X\u003csub\u003e1\u003c/sub\u003e, X\u003csub\u003e2,\u003c/sub\u003e and X\u003csub\u003e3\u003c/sub\u003e produced a variable effect on the retention time and percentage recovery of OND, FAM, and DRO. A mobile phase made up of (40:60) ratio of acetonitrile and phosphate buffer with pH 4.8, injected at a flow rate of 1 mL/min, resulted in a varied retention time for OND (1.8-2.2 min.), FAM (1.3-1.7 min.), and DRO (5-6 min.). This variation was also observed when the flow rate was adjusted to 1.5 mL/min. while keeping the composition of the mobile phase the same. However, all the injected concentrations possessed a similar retention time (Y\u003csub\u003e1\u003c/sub\u003e, Y\u003csub\u003e2\u003c/sub\u003e, Y\u003csub\u003e3\u003c/sub\u003e) when the mobile phase composed of acetonitrile and phosphate buffer with pH 4.8 (40:60) was injected at 1.5 mL/min. Moreover, the percentage recovery of three drugs (Y\u003csub\u003e4\u003c/sub\u003e, Y\u003csub\u003e5\u003c/sub\u003e, Y\u003csub\u003e6\u003c/sub\u003e) was found to be within the range of 90-110%. \u0026nbsp; Hence, it can be suggested that the design showed an insignificant association between the variables X\u003csub\u003e1\u003c/sub\u003e and X\u003csub\u003e2\u003c/sub\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results indicate that the composition and flow rate of the mobile phase play an important role while performing the simultaneous estimation of OND, FAM, and DRO. Moreover, the quadratic effects of X\u003csub\u003e1\u003c/sub\u003e, X\u003csub\u003e2\u003c/sub\u003e, and X\u003csub\u003e3\u003c/sub\u003e (independent variables) on the percent recovery (\u003cem\u003ep\u003c/em\u003e = 0.002) and retention time (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) of OND, FAM, and DRO were found to be significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation of the Optimized Factors \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects X\u003csub\u003e1\u003c/sub\u003e, X\u003csub\u003e2\u003c/sub\u003e, and X\u003csub\u003e3\u003c/sub\u003e were validated by making a comparison\u0026nbsp;between predicted and obtained results. As shown in Table 2, a difference between the predicted and observed data for OND, FAM, and DRO was found to be within the range of \u0026plusmn;6%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatographic Method Validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalytical Curve\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe linearity was determined for OND, FAM, and DRO at various concentrations ranging from 5-25 \u0026micro;g/mL. The calibration curves were generated by plotting the mean peak areas against respective concentrations. The linear regression equation for OND was y= 24053x-8118.5 whereas the value of square of correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e) was 1. The linear regression equation for FAM was y= 12769x-5037.6 and the value of \u0026nbsp;R\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e was 1. Similarly, the linear regression equation for DRO was y= 32208x-16417 and the value of \u0026nbsp;R\u003csup\u003e2\u0026nbsp;\u003c/sup\u003ewas found to be 0.9999. These results indicate the linearity of the developed method (Figure 2, 3 and 4). Moreover, the ANOVA analysis was also carried out and the p-value (\u0026lt;0.05) established that the results are statistically significant.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecificity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe specificity of the method was analyzed and no interference was observed between the active drug ingredient and excipients. As shown in Figure 5, the retention times of OND, FAM, and DRO were found to be 2.1 min, 1.48 min, and 6.4 min, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntra-day precision of the method was investigated by analyzing the three dilutions of each drug (OND, FAM, DRO) in triplicates. However, the inter-day precision was assessed by analyzing the three dilutions of OND, FAM, and DRO on three different days. As shown in Table 3 and 4, all the RSD values were determined to be within the acceptable range i.e. less than 2%, hence reflecting the precision of the proposed method.\u003c/p\u003e\n\u003cp\u003eIn order to assess the repeatability, 10 injections of standard solutions (FAM, OND, and DRO) were injected at a concentration of 15 \u0026micro;g/mL (Figure 6). The RSD value for peak area was found to be 0.3%, 0.4%, and 0.4% for OND, FAM, and DRO, respectively (Table 5). These results indicate the precision of the developed HPLC method.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt was established by computing the percent recovery for OND, FAM, and DRO. Percent recoveries were found to be 99.87-103.8%, 92.1-101.33%, and 97.6-105.6% for OND, FAM, and DRO, respectively (Table 6). These results indicate the accuracy of the suggested method.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLOD and LOQ\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe LOD for OND, FAM, and DRO was determined to be 0.421, 0.534, and 2.178 \u0026micro;g/mL, respectively. While, the LOQ for OND, FAM, and DRO was found to be 1.364, 1.747, and 7.061 \u0026micro;g/mL, respectively. These are the lowest values on the calibration curve that were obtained with adequate accuracy and precision under the optimized chromatographic conditions. Hence, these results indicate the sensitivity of the proposed method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt was observed that altering the robustness parameters (room temperature, pH of mobile phase, and detection wavelength) had no considerable variation in the retention time of OND, FAM and DRO.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSystem Suitability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSystem suitability parameters were analyzed to ensure the adequacy and feasibility of the proposed method. As shown in Table 7, OND, FAM, and DRO yielded a %RSD value of less than 2% for retention time and peak area, suggesting that there is little variation in measured values. The tailing factors for OND, FAM, and DRO were 1.42, 1.73, and 1.48, respectively, indicating the symmetry of all peaks. The efficiency of the column is reflected in several theoretical plates that were determined to be 4247, 4802, and 6629 for OND, FAM, and DRO, respectively. A resolution value of 1 represents an adequate degree of separation, and the results showed that OND (R = 5.778) and DRO (R = 18.034) possessed a high degree of peak resolution. Hence, proposed method reflects the satisfactory results in terms of %RSD values, tailing factor, theoraticla plates and resolution for all the drugs. These results confirm the faesiblity of developed method.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStability of Drugs in Rabbit Plasma\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe stability of drugs in rabbit plasma was evaluated by freeze-thaw cycles and average degradation for selected concentrations of OND, FAM, and DRO was determined to be \u0026lt; 3% (Table 8). The results showed that all the \u0026nbsp;samples were stable for a period of 48 h. with a recovery of \u0026gt;97%.\u003c/p\u003e\n\u003cp\u003eHence a simple, accurate, precise method was developed method for the simulatneus estimation of OND, FAM and DRO. Moreover, the system suitability parameters were studied using rabbit plasma and method was foun to be appropriate for the simultaneous estimation of these drugs in rabbit plasma. Eventually, the work could be extended to determine the human pharmacokinectis of these drug.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn the present study, chromatographic conditions were optimized employing the CCD, and a simple HPLC technique was developed for the concurrent estimation of OND, FAM, and DRO. The chromatographic parameters (flow rate, composition, and pH of the mobile phase) were optimized to achieve reproducible peaks with high resolution. Preliminary studies revealed that using a 60:40 (v/v) mixture of acetonitrile and phosphate buffer (pH 4.8) as a mobile phase, flowing through the column at a flow rate of 1.5 mL/min. produced the best results.\u003c/p\u003e\n\u003cp\u003eThe method was further validated as per ICH recommendations and was found appropriate for the simultaneous estimation of OND, FAM, and DRO with excellent linearity, sensitivity, accuracy, and precision. Hence, the proposed method can be used for the routine analysis of OND, FAM, and DRO in combined dosage forms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions;\u0026nbsp;\u003c/strong\u003eAll authors have been involved in study design, data collection, formal analysis, and manuscript writing. All authors approved the final version of the manuscript and agreed to submit it.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe authors would like to extend their sincere appreciation to the Department of Pharmaceutics and DTL Multan for providing necessary facilities during this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eAll data generated or analyzed during this study are included in this published article\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest;\u0026nbsp;\u003c/strong\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKannappan V, Kanthiah S (2017) Chromatographia 80:229-236.\u003c/li\u003e\n\u003cli\u003ePanigrahi D, Sharma R (2008) Acta chromatographica 20:439-450.\u003c/li\u003e\n\u003cli\u003eAzmi SNH, Al Rawahi WAN, Al Yahyai AI, Al Qasimi AA, Al Fuliti KS, Al Qalhati OS, Rahman N, Ahmed QU (2024) Journal of Chromatography B 1234:124035.\u003c/li\u003e\n\u003cli\u003eRajput JM.\u003c/li\u003e\n\u003cli\u003eRai RR, Nijhawan S (2021) Saudi Journal of Gastroenterology 27:136-143.\u003c/li\u003e\n\u003cli\u003eZarghi A, Shafaati A, Foroutan S, Khoddam A (2005) Journal of pharmaceutical and biomedical analysis 39:677-680.\u003c/li\u003e\n\u003cli\u003eEl-Shaheny R, Radwan MO, Belal F, Yamada K (2020) Journal of Pharmaceutical and Biomedical Analysis 186:113305.\u003c/li\u003e\n\u003cli\u003eJaimini M, Rana A, Tanwar Y (2007) Current drug delivery 4:51-55.\u003c/li\u003e\n\u003cli\u003eBalint A, Silvia I, Bondoroi-Papp Z, Muntean D-L (2018) The Medical-Surgical Journal 122:207-212.\u003c/li\u003e\n\u003cli\u003eRaza A, Ijaz AS, Rasheed U (2007) Journal of the Chinese Chemical Society 54:223-227.\u003c/li\u003e\n\u003cli\u003eDeshmukh T, Deo S, Inam F, Lambat T, Gurubaxani S, Choudhari A (2015) Int J Adv Sci Eng Technol 1:15-20.\u003c/li\u003e\n\u003cli\u003ePatel SA, Patel KN, Patel MR (2022) International Journal of Pharmaceutical Sciences Review and Research 74:150-154.\u003c/li\u003e\n\u003cli\u003eDedania Z, Dedania R, Karkhanis V, Sagar GV, Baldania M, Sheth N (2009) Asian Journal of Research in Chemistry 2:108-111.\u003c/li\u003e\n\u003cli\u003eSharma S, Sharma MC (2017) Arabian Journal of Chemistry 10:S397-S403.\u003c/li\u003e\n\u003cli\u003eYegorova A, Leonenko I, Aleksandrova D, Scrypynets Y, Aleksandrova A (2013) Journal of Applied Pharmaceutical Science 3:006-011.\u003c/li\u003e\n\u003cli\u003eAyad MM, Youssef NF, Abdellatif HE, Soliman SM (2006) Chemical and pharmaceutical bulletin 54:807-813.\u003c/li\u003e\n\u003cli\u003eMetwally FH, Abdelkawy M, Naguib IA (2006) Journal of AOAC International 89:78-87.\u003c/li\u003e\n\u003cli\u003eAzhlwar S, Ravi TK (2011) Int J Pharm Pharm Sci 3:245-250.\u003c/li\u003e\n\u003cli\u003eDahivelkar PP, Bari SB, Bhoir S, Bhagwat AM (2009).\u003c/li\u003e\n\u003cli\u003eAbdellatef HE, Ayad MM, Soliman SM, Youssef NF (2007) Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 66:1147-1151.\u003c/li\u003e\n\u003cli\u003eBasavaiah K, Zenita O (2011) Qu\u0026iacute;mica Nova 34:735-742.\u003c/li\u003e\n\u003cli\u003eSalehniya H, Amiri M, Nekoueian K (2022) Analytical and Bioanalytical Chemistry Research 9:59-71.\u003c/li\u003e\n\u003cli\u003eMyhal AV, Marksa M, Golovchenko OS, Georgiyants VA, Ivanauskas L (2017) News of Pharmacy:21-24.\u003c/li\u003e\n\u003cli\u003ePeikova L, Georgieva M, Tsvetkova B (2014) Pharmacia 61:4-5.\u003c/li\u003e\n\u003cli\u003eNyola N, Jeyabalan GS (2012) Journal of Applied Pharmaceutical Science:79-83.\u003c/li\u003e\n\u003cli\u003ePanchale WA, Suroshe R, Rathod MS, Pandhare YL (2019) Research Journal of Pharmacy and Technology 12:231-236.\u003c/li\u003e\n\u003cli\u003eBharati S, Gaikwad V, Chellampillai B (2023) Analytical Chemistry Letters 13:202-212.\u003c/li\u003e\n\u003cli\u003eCelia C, Di Marzio L, Locatelli M, Ramundo P, D\u0026rsquo;Ambrosio F, Tartaglia A (2020) Separations 7:29.\u003c/li\u003e\n\u003cli\u003eSubramanian V, Nagappan K, Mannemala SS (2015) Acta Chimica Slovenica 62.\u003c/li\u003e\n\u003cli\u003eXu H, Paxton J, Lim J, Li Y, Wu Z (2014) Journal of Pharmaceutical and Biomedical Analysis 98:371-378.\u003c/li\u003e\n\u003cli\u003eICH I (2005) Harmonised Tripartite Guideline: validation of analytical procedure: text and Methodology Q2 (R1), 2005.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e: List of independent and dependent variables selected for Central Composite Design\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eVariables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLevel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eConstraints\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e+1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIndependent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003eX\u003csub\u003e1\u0026nbsp;\u003c/sub\u003e(Flow rate in mL/min)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eWithin the range\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003eX\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e(Mobile Phase)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eWithin the range\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003eX\u003csub\u003e3\u0026nbsp;\u003c/sub\u003e(pH of Phosphate Buffer)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e4.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e5.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e6.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eWithin the range\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"6\" valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDependent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003eY\u003csub\u003e1 \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/sub\u003e(Retention Time of OND)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e1.8 to 2.2 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003eY\u003csub\u003e2 \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/sub\u003e(Retention Time of FAM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e1.3 to 1.7 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003eY\u003csub\u003e3 \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/sub\u003e(Retention Time of DRO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e5.0 to 6.0 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003eY\u003csub\u003e4 \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/sub\u003e(Recovery of OND)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e90% to 110%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003eY\u003csub\u003e5 \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/sub\u003e(Recovery of FAM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e90% to 110%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003eY\u003csub\u003e6 \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/sub\u003e(Recovery of DRO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e90% to 110%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e: Validating the optimized factors for simultaneous estimation of OND, FAM and DRO by the proposed HPLC method\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eResponse\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePredicted Value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eObserved Value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Residual\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOND\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFAM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDRO\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOND\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFAM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDRO\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOND\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFAM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDRO\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRetention Time (min)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e1.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e1.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e5.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e1.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e5.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-6.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e+4.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Recovery\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e99.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e100.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e101.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e98.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e101.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e100.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e+1.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-1.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e+1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e: Result of intra-day precision for estimation of OND, FAM and DRO by proposed HPLC method (n=3)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 32px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOndansetron\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 32px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFamotidine\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDrotaverine\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Conc.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean Conc.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRSD (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConc.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean Conc. (\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;RSD (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConc.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean Conc.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;RSD (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e8.77 \u0026plusmn;0.462\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.712\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7.23\u0026plusmn;0.452\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.762\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e3.02\u0026plusmn;0.468\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.128\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e16.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e16.27\u0026plusmn;0.156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e1.213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e15.87\u0026plusmn;0.176\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.189\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7.67\u0026plusmn;0.741\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.389\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e24.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e24.76\u0026plusmn;0.278\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e22.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e22.75\u0026plusmn;0.487\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e0.264\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e10.29\u0026plusmn;0.498\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.045\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eRSD= Relative standard deviation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e: Result of inter-day precision for estimation of OND, FAM and DRO by proposed HPLC method (n=3)\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 32px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Ondansetron\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 32px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFamotidine\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 34px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDrotaverine\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConc.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean Conc.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRSD(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConc. (\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean Conc.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;RSD(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConc.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean Conc.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRSD(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e8.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e8.53 \u0026plusmn;0.671\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.476\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7.52\u0026plusmn;0.154\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.709\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e3.51\u0026plusmn;0.571\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.253\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e16.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e16.04\u0026plusmn;0.341\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.719\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e15.73\u0026plusmn;0.657\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e1.298\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e7.69\u0026plusmn;0.856\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.572\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e24.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e24.21\u0026plusmn;0.456\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e22.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e22.19\u0026plusmn;0.942\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10.5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e10.51\u0026plusmn;0.308\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e1.148\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eRSD= Relative standard deviation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e: Result of repeatability for simultaneous estimation of OND, FAM and DRO by proposed HPLC method\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePeak area\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOndansetron\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFamotidine\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDrotaverine\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e352998.750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e186547.193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e466055.356\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSD\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e1126.557\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e777.309\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e1811.782\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%RSD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eSD= Standard deviation, RSD= Relative standard deviation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6:\u0026nbsp;\u003c/strong\u003eResult of percent recovery of OND, FAM, DRO in rabbit plasma by proposed HPLC method (n=6)\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eActual Conc.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOND\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFAM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 30px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDRO\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecovered Conc.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRSD\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecovery\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecovered Conc.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRSD\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecovery\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecovered Conc.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRSD\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecovery\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e5.19 \u0026plusmn;0.573\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.961\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e103.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e4.92\u0026plusmn;0.254\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.931\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e98.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e5.28\u0026plusmn;0.068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.873\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e105.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e10.07\u0026plusmn;0.056\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.265\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e100.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e9.21\u0026plusmn;0.783\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.298\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e92.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e9.76\u0026plusmn;0.341\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.275\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e97.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e14.98\u0026plusmn;0.429\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.572\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e99.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e15.2\u0026plusmn;0.512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.679\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e101.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e15.34\u0026plusmn;0.998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.548\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e102.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e20.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e20.31\u0026plusmn;0.857\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.761\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e101.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e19.4\u0026plusmn;0.956\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e97.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e19.78\u0026plusmn;0.254\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.769\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e98.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e25.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e24.79\u0026plusmn;0.155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.347\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e99.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e24.7\u0026plusmn;0.487\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.528\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e98.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e25.45\u0026plusmn;0.053\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 7px;\"\u003e\n \u003cp\u003e0.741\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e101.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eRSD= Relative standard deviation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 7:\u003c/strong\u003e System suitability parameters determined for simultaneous estimation of OND, FAM and DRO in rabbit plasma by proposed HPLC method (n=5)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eParameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFAM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOND\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDRO\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRSD(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLimit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRSD(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLimit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRSD(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLimit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRetention Time (min.)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e6.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePeak Area\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e186547.193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026lt; 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e352998.750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026lt; 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e466055.356\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026lt; 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTailing factor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026lt; 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026lt; 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e1.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026lt; 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTheoretical Plates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e4802\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e4247\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e6629\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width: 41px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eResolution\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e5.778\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026gt; 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e18.034\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u0026gt; 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;RSD= Relative standard deviation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 8\u003c/strong\u003e: Result of stability study for OND, FAM and DRO in rabbit plasma evaluated via freeze- thaw cycles (n=6)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration (\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eArea\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOND\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFAM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDRO\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAt start (First day)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e15 \u0026micro;g/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e353877.809\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e186632.882\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e469027.938\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Recovery\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e100%\u0026plusmn;0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e100%\u0026plusmn;0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e100%\u0026plusmn;0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAfter 24 Hours\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e15 \u0026micro;g/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e364876.232\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e182056.209\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e4710923.446\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Recovery\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e103.11%\u0026plusmn;0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e97.54%\u0026plusmn;0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e100.44%\u0026plusmn;0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAfter 48 Hours\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e15 \u0026micro;g/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e350578.342\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e188987.329\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e461005.375\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 42px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Recovery\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e99.07%\u0026plusmn;0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20px;\"\u003e\n \u003cp\u003e101.26%\u0026plusmn;1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e98.29%\u0026plusmn;0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HPLC, Famotidine, Drotaverine, Ondansetron, Central composite design","lastPublishedDoi":"10.21203/rs.3.rs-6133930/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6133930/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSeveral methods have been reported for the individual determination of Ondansetron (OND), Famotidine (FAM), and Drotaverine (DRO); however, they are not suitable for simultaneously estimating these drugs. Hence, a simple and accurate high-performance liquid chromatographic (HPLC) method was developed and validated for the simultaneous estimation of the three drugs. The method was optimized using the central composite design. The effect of independent variables was optimized while applying the central composite design approach. The quadratic effect of independent variables was significant on the percentage recovery and the retention time (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) of the studied drugs. The chromatographic separations were carried out at a 1.5 mL/min flow rate using a 40:60 (v/v) ratio mixture of acetonitrile and phosphate buffer pH 4.8 as optimized parameters. The retention time of OND, FAM, and DRO was observed to be 1.93, 1.45, and 5.46 min, respectively. Similarly, the percentage recoveries of OND, FAM, and DRO were found within the range of 99.16\u0026ndash;103.8%, 92.1\u0026ndash;101.3%, and 97.6\u0026ndash;105.6%, respectively. All the experiments were performed in six replicates (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Hence, the suggested method can be used for the simultaneous determination of OND, FAM, and DRO method can be used for the simultaneous determination of these drugs.\u003c/p\u003e","manuscriptTitle":"Development and Validation of HPLC Method for Simultaneous Estimation of Ondansetron Dihydrochloride, Famotidine, and Drotaverine Hydrochloride in Rabbit Plasma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-07 07:22:20","doi":"10.21203/rs.3.rs-6133930/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e437d852-d112-4513-b405-84e5b1e288de","owner":[],"postedDate":"March 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-07T07:22:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-07 07:22:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6133930","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6133930","identity":"rs-6133930","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}