Universal procedures for spectrophotometric determination of anticoccidial drugs; application to multi-ingredient veterinary formulation and computational investigations for multivariate analysis European Journal of Chemistry 12 (4) (2021) 368-376 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2021 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. https://dx.doi.org/10.5155/eurjchem.12.4.368-376.2144 European Journal of Chemistry View Journal Online View Article Online Universal procedures for spectrophotometric determination of anticoccidial drugs; application to multi-ingredient veterinary formulation and computational investigations for multivariate analysis Mahmoud Mohamed Abbas *, Amira Mabrouk El-Kosasy , Lobna Abd El-Aziz Hussein and Nancy Magdy Hanna Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, 11566, Egypt mahmoud_abbas@pharma.asu.edu.eg (M.M.A.), kosasy2030@yahoo.com (A.M.E.), lobna.analytical@yahoo.com (L.A.E.H.), nancynassif@hotmail.com (N.M.H.) * Corresponding author at: Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, 11566, Egypt. e-mail: mahmoud_abbas@pharma.asu.edu.eg (M.M. Abbas). 10.5155/eurjchem.12.4.368-376.2144 Received: 10 July 2021 Received in revised form: 05 August 2021 Accepted: 07 August 2021 Published online: 31 December 2021 Printed: 31 December 2021 Simple, accurate, and eco-friendly spectrophotometric procedures were proposed and implemented for simultaneous determination of anticoccidial drugs from three different classes namely, amprolium hydrochloride (AMP), sulfaquinoxaline sodium (SQX) and diaveridine hydrochloride (DVD). Dual wavelength in ratio spectra procedure was proposed where the difference in amplitudes (ΔP) in the ratio spectra at 264 nm and 301.9 nm (ΔP264&301.9 nm) corresponded to AMP with mean percentage recovery 100.00±0.923%, while (ΔP250.9&279 nm) and (ΔP218&243.5 nm) corresponded to SQX and DVD with mean percentage recoveries 99.31±1.083 and 100.64±1.219%, respectively. The dual wavelength in ratio spectra procedure was validated according to the ICH guidelines and accuracy, precision and repeatability were found to be within the acceptable limit. Multivariate chemometric approaches, namely, partial least-squares (PLS-2) and principal component regression (PCR) were also proposed with mean percentage recoveries 99.31±0.769, 98.91±1.192 and 99.04±1.245% for AMP, SQX and DVD, respectively, in PLS-2 and 99.63±1.005, 99.11±1.272 and 98.93±1.338% for AMP, SQX and DVD, respectively, in PCR. These procedures were successfully applied to the multi-ingredient veterinary formulation with mean percentage recoveries 100.75±1.238, 99.29±0.875 and 99.34±0.745% for AMP, SQX and DVD, respectively, in dual wavelength in ratio spectra procedure and 101.03±1.261, 101.48±0.984 and 101.10±1.339% for AMP, SQX and DVD, respectively, in PLS-2 and 100.22±1.204, 101.10±0.546 and 100.91±0.677% for AMP, SQX and DVD, respectively, in PCR. Amprolium Diaveridine Ratio spectra Sulfonamides Green chemistry Multi-ingredient veterinary formulation Cite this: Eur. J. Chem. 2021, 12(4), 368-376 Journal website: www.eurjchem.com 1. Introduction Coccidiosis is a term sometimes applied to infections with protozoa of the order Eucoccidiorida. Coccidian protozoa, primarily Eimeria, cause economically important infections in domesticated animals [1]. Anticoccidial drug combinations proved to be very effective as prophylactic and treatment of coccidiosis in poultry because of the different mechanisms of action of the drugs being used in combination. A multi-ingredient veterinary formulation composed of three anticoccidial drugs of three different classes was investigated. Amprolium hydrochloride (1-[(4-amino-2-pro pyl-5-pyrimidinyl)methyl]-2-methylpyridinium chloride hydro chloride) (Figure 1) is a thiamine analogue which inhibits the uptake of thiamine by second-generation schizonts of Eimeria tenella and so prevents formation of thiamine coenzyme which is required for many essential metabolic reactions [2]. Sulfaquinoxaline sodium (4-amino-N-2-quinoxalinyl benzene sulfonamide monosodium salt) (Figure 1) is an important member of the sulfonamides class and it interferes with the early phases of folate synthesis. Sulfonamides are often used in combination with dihydrofolate reductase inhibitors (DHFRI) such as diaveridine hydrochloride (2,4-diamino-5-(3,4-dimet- hoxybenzyl) pyrimidine hydrochloride) (Figure 1) which belongs to diaminopyrimidines class because of the observed synergistic effects due to activity at two places in folate biosynthesis [3]. Literature survey revealed many reported methods for the determination of AMP, SQX, and DVD either alone or with other drugs in different matrices such as veterinary formulation, surface water, eggs, chicken muscles, chicken plasma, chicken liver and chicken feed. These methods include HPLC/UV [4-6], LC/MS [7-12], spectroscopic methods [13-20] and electro- chemical method [21]. To the best of our knowledge, the ternary mixture of AMP, SQX, and DVD has not been investigated spectrophotometrically. So, the main point of the current work was to develop simple, accurate and eco-friendly spectrophotometric procedures for the simultaneous determination of the three drugs in their multi-ingredient veterinary formulation. ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.12.4.368-376.2144 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.12.4.368-376.2144 mailto:mahmoud_abbas@pharma.asu.edu.eg mailto:kosasy2030@yahoo.com mailto:lobna.analytical@yahoo.com mailto:nancynassif@hotmail.com mailto:mahmoud_abbas@pharma.asu.edu.eg http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.12.4.368-376.2144&domain=pdf&date_stamp=2021-12-31 Abbas et al. / European Journal of Chemistry 12 (4) (2021) 368-376 369 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.368-376.2144 N+ N N NH2 Cl - H Cl (a) Na+ N - N N S O O NH2 (b) N N O O NH2NH2 H Cl (c) Figure 1. Structural formulas for (a) amprolium hydrochloride, (b) sulfaquinoxaline sodium, and (c) diaveridine hydrochloride. 2. Experimental 2.1. Instrumentation The UV absorption spectra were recorded using a Shimadzu UV-1601 dual beam UV-visible spectrophotometer using 1-cm matched quartz cells. The spectral bandwidth was 0.1 nm with wavelength scanning speed of 2800 nm/min. Instrument software (version 3.91) was used to process the absorption. Matlab® (version 7.0.1.24704) was used to process the spectral data for multivariate analysis. 2.2. Materials 2.2.1. Standards Amprolium hydrochloride, sulfaquinoxaline sodium, and diaveridine hydrochloride were kindly supplied by Pharma Swede Pharmaceutical Company, 10th of Ramadan City, Egypt. Their purities were found to be 99.22, 99.77, and 99.26%, respectively, according to the reported spectrophotometric methods [13-15], respectively. Analytical grade methanol was used throughout the work (ADWIC, Egypt). Standard stock solutions of AMP, SQX, and DVD (100 µg/mL) were prepared in methanol. 2.2.2. Veterinary formulation A multi-ingredient veterinary formulation (Tricure®, Batch No. 0821/15) is a premix with 20 g amprolium hydrochloride, 25 g sulfaquinoxaline sodium and 6.4 g diaveridine hydro chloride per 100 g, produced by Arabcomed Co. S.A.E. Obour City, Egypt. 2.3. Procedures 2.3.1. Methods 2.3.1.1. Dual-wavelength-in-ratio spectra method 2.3.1.1.1. For determination of AMP Various aliquots equivalent to 10-200 µg of AMP were taken from its stock standard solution (100 µg/mL) and completed to volume with methanol in a series of 10-mL volumetric flasks to obtain final concentrations of 1-20 µg/mL. The spectra of these solutions were scanned from 200-400 nm using methanol as a blank. AMP spectra are divided by the spectrum of 2 µg/mL of DVD. The amplitudes of the ratio spectra were obtained at 264 nm and 301.9 nm. A calibration curve was constructed that relates the differences in the amplitudes in the chosen wavelength couple ΔP264&301.9 nm to the corresponding concent- ration of AMP [22,23]. 2.3.1.1.2. For determination of SQX and DVD Various aliquots equivalent to 5-100 µg of SQX and 5-190 µg of DVD were taken from their stock standard solutions (100 µg/mL) and completed to volume with methanol in two separate series of 10-mL volumetric flasks to obtain final concentrations of 0.5-10 µg/mL and 0.5-19 µg/mL, respect- tively. The spectra of these solutions were scanned from 200- 400 nm using methanol as a blank. The stored spectra were divided by the spectrum of 3 µg/mL of AMP. The amplitudes of the ratio spectra were obtained at 250.9, 279.0, 218.0, and 243.5 nm. Calibration curves for both SQX and DVD were constructed by plotting the differences in the amplitudes at the chosen wavelength couple ΔP250.9&279 nm and ΔP218&243.5 nm for SQX and DVD, respectively, versus the corresponding concent- ration [22,23]. 2.3.1.2. PLS-2 and PCR For each of PLS-2 and PCR, a calibration set consisting of twenty laboratory-prepared mixtures of AMP, SQX, and DVD in different proportions (Table 1) was obtained by the use of a multilevel multifactor experimental Brereton design [24] where levels (L) were the concentrations used and the experiments’ number was L2. The mixtures were prepared by taking various aliquots from AMP, SQX and DVD stock standard solutions (100 µg/mL) into a group of 10 mL volumetric flasks with ranges of concentrations 3-11, 1-9, and 3-15 µg/mL for AMP, SQX and DVD, respectively. The spectra of the prepared mixtures were recorded in the range 210-400 nm with 0.2 nm intervals, transferred to MATLAB software producing 951 data points/spectrum, so the produced spectral data matrix composed of 25 rows repre- senting various samples and 951 columns representing wavelengths. For each of PLS-2 and PCR, the calibration model was made after mean centering as a pre-processing step, while the cross-validation method was “random” for PLS-2 and “leave one out” for PCR. 370 Abbas et al. / European Journal of Chemistry 12 (4) (2021) 368-376 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.368-376.2144 Table 1. Calibration and validation set for the PLS-2 and PCR methods. Mixture no AMP SQX DVD 1 7 5 9 2 7 1 3 3 3 1 15 4 3 9 6 5 11 3 15 6 5 9 9 7 11 5 6 8 7 3 6 9 5 3 12 10 5 7 15 11 9 9 12 12 11 7 9 13* 9 5 15 14 7 9 15 15 11 9 3 16 11 1 12 17 3 7 3 18 9 1 9 19* 3 5 12 20* 7 7 12 21* 9 7 6 22 9 3 3 23 5 1 6 24 3 3 9 25* 5 5 3 * Validation set. The suitable selection of the factors’ number to be employed for constructing the model was essential for accomplishing the right quantification in both PLS-2 and PCR calibrations. To choose the ideal number of significant latent variables, F statistics were applied [24]. The validation set is composed of 5 laboratory-prepared mixtures containing different proportions of AMP, SQX, and DVD (Table 1). In addition, the concentrations of AMP, SQX, and DVD in the veterinary formulation extract were calculated using the optimized PLS-2 or PCR calibration model after being recorded in the same specified lambda range [25-33]. 2.3.2. Application of the proposed procedures for determination of AMP, SQX, and DVD in laboratory- prepared mixtures Solutions containing various proportions of the ternary mixture of AMP, SQX, and DVD were prepared. Zero-order absorption curves of these mixtures were recorded using methanol as a blank. By applying the proposed procedures, the concentrations of the drugs in the prepared mixtures were deduced. 2.3.3. Application of the proposed procedures for the determination of AMP, SQX, and DVD in veterinary formulation A portion of the Tricure® premix containing 2 mg of AMP, 2.5 mg of SQX, and 0.64 mg of DVD was accurately weighed, sonicated in 25 mL methanol for 2 min and filtered into a 100 mL volumetric flask. The residue was washed three times, each with 5 mL of methanol, and then completed to volume with methanol. The procedures mentioned above in Sections 2.3.1.1 and 2.3.1.2 were applied and the concentrations of the drugs were deduced. The validity of the methods was tested using the standard addition technique. 3. Results and discussion Three anticoccidial drugs, namely, amprolium hydro chloride, sulfaquinoxaline sodium, and diaveridine hydro chloride from three different classes; thiamine analogues, sulfonamides and dihydrofolate reductase inhibitors, respect- tively, were investigated to be analyzed in their multi- ingredient veterinary formulation by simple, accurate and eco- friendly spectrophotometric procedures. Zero-order absorp- tion spectra (D0) of the ternary mixture (AMP, SQX, and DVD) show severe overlap, which made the analysis of each drug in the presence of the other challengeable (Figure 2). By applying the proposed procedures, this overlapping can be resolved, allowing the quantitative analysis of the components of the mixture. 3.1. Method development and optimization 3.1.1. Dual-wavelength-in-ratio spectra method This method can be applied to determine the ternary mixture of the investigated compounds whose spectra show a severe overlap [22,23]. It is based on the principles of both the ratio difference method [34-36] and the dual wavelength method [37-40]. The proposed method can determine a ternary mixture, such that every compound can be selectively deter- mined after total elimination of the impediment caused by the other two compounds. The developed method can be regarded as a dual wavelength in the ratio spectrum where the difference should be considered at certain two wavelengths showing equal peak amplitudes for the interfering component in its ratio spectrum [38,39]. After dividing by the spectrum of 2 µg/mL DVD, DVD was cancelled, while SQX showed equal amplitudes at 264.0 and 301.9 nm. Therefore, the difference in amplitudes at these two wavelengths ΔP264&301.9 nm corresponded to AMP concentration (Figure 3). Similarly, after dividing by the spectrum of 3 µg/mL AMP, SQX, and DVD were determined in the ratio spectra at ΔP250.9&279 nm and ΔP218&243.5 nm, respectively, as shown in Figure 4. The linear regression equations were found to be the following: ΔPAMP = 1.4271×C - 1.1461 r = 0.9998 (1) ΔPSQX = 0.7811×C - 0.0569 r = 0.9998 (2) ΔPDVD = 0.8451×C + 0.0751 r = 0.9998 (3) where C is the µg/mL concentration of AMP, SQX or DVD, ΔP is the difference in amplitudes at the two wavelengths and r is the correlation coefficient. Abbas et al. / European Journal of Chemistry 12 (4) (2021) 368-376 371 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.368-376.2144 Figure 2. Zero order absorption spectra of 10 µg/mL AMP (-), 10 µg/mL SQX (- - -), and 10 µg/mL DVD (……) using methanol as blank. Figure 3. Ratio spectra of 10 µg/mL AMP (—), 10 µg/mL SQX (- - -), and 10 µg/mL DVD (…..) using 2 µg/mL DVD as a divisor and methanol as blank. Figure 4. Ratio spectra of 10 µg/mL AMP (—), 10 µg/mL SQX (- - -) and 10 µg/mL DVD (…..) using 3 µg/mL AMP as a divisor and methanol as blank. 3.1.2. Multivariate chemometric methods: (PLS-2) and (PCR) The optimum number of latent variables (LVs) described by the constructed models was found to be five factors for both PLS-2 and PCR as shown in Figures 5 and 6. The predictive ability of the developed models was assessed using the validation set by plotting known versus predicted concentrations for each drug. A good linearity was observed for each drug, as indicated by the correlation coefficients 0.9997 (y = 0.9987x - 0.0380), 0.9981 (y = 0.9437x + 0.2559) and 0.9999 (y = 0.9803x + 0.0646) for AMP, SQX and DVD, respectively, in PLS-2, and 0.9995 (y = 1.0122x - 0.0965), 0.9964 (y = 0.9531x + 0.2082) and 0.9999 (y = 0.9804x + 0.0552) for AMP, SQX and DVD, respectively, in PCR. 372 Abbas et al. / European Journal of Chemistry 12 (4) (2021) 368-376 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.368-376.2144 Figure 5. RMSECV plot of the cross-validation results of the training set as a function of the number of principal components used to construct the PLS-2 calibration, using zero-order absorption spectra of AMP, SQX and DVD. Figure 6. RMSECV plot of the cross-validation results of the training set as a function of the number of principal components used to construct the PCR calibration, using zero-order absorption spectra of AMP, SQX and DVD. Figure 7. Residual versus actual concentration (μg/mL) plot for AMP, SQX and DVD in the validation set, using the PLS-2 method. For both PLS-2 and PCR, the concentration residuals were plotted against the actual concentrations of the prepared mixtures (Figures 7 and 8), and the residuals for all samples were found to be randomly distributed around zero [25-33]. The average recoveries of each drug using PLS-2 and PCR methods are summarized in Table 2. The following equation gives the RMSEP: RMSEP= �𝜀𝜀�𝑦𝑦𝑟𝑟−𝑦𝑦𝑝𝑝� 2 𝑛𝑛 (4) where yr and yp are the true and predicted values, respectively, and (n) is the number of samples used in validation. Statistical parameters of each drug, using the optimized PLS-2 and PCR methods are shown in Table 3. The procedures were applied for determination of the studied drugs in their laboratory-prepared mixtures with average percentage recovery as given in Table 4. The proposed procedures were found to be valid and applicable for the analysis of the drugs in their multi-ingredient veterinary formulation with acceptable mean percentage recoveries. Furthermore, the standard addition technique was performed to assess the accuracy of the proposed methods. The obtained results revealed that there was no interference from excipients as shown in Table 5. -0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0 2 4 6 8 10 12 14 16 Co nc en tra tio n re si du al s Actual concentrations AMP SQX DVD Abbas et al. / European Journal of Chemistry 12 (4) (2021) 368-376 373 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.368-376.2144 Table 2. Percent recoveries of AMP, SQX and DVD in the validation set, using PLS-2 and PCR methods. Mixture number Using zero order spectra Found, % AMP SQX DVD PLS-2 PCR PLS-2 PCR PLS-2 PCR 13 99.32 99.81 98.10 98.00 98.37 98.26 19 99.97 99.74 99.77 100.54 99.07 99.12 20 99.51 99.84 98.11 98.45 98.36 98.33 21 99.73 100.77 98.00 98.12 98.23 97.80 25 98.00 98.00 100.59 100.44 101.19 101.17 Mean 99.31 99.63 98.91 99.11 99.04 98.93 Table 3. Statistical parameters for simultaneous determination of AMP, SQX, and DVD, using optimized PLS-2 and PCR methods. Parameters Using zero-order spectra AMP SQX DVD PLS-2 PCR PLS-2 PCR PLS-2 PCR Concentration range (μg/mL) 3-11 3-11 1-9 1-9 3-15 3-15 Number of factors 5 5 5 5 5 5 Root mean square error of calibration 0.1831 0.2751 0.1627 0.1966 0.1459 0.1629 Root mean square error of prediction 0.06057 0.060526 0.098217 0.096624 0.157454 0.166605 Root mean square error of cross-validation 0.8633 0.4075 0.6176 0.2622 0.5189 0.2276 Intercept a -0.0380 -0.0965 0.2559 0.2082 0.0646 0.0552 Slope a 0.9987 1.0122 0.9437 0.9531 0.9803 0.9804 (r2) a 0.9997 0.9995 0.9981 0.9964 0.9999 0.9999 a Data of the straight line plotted between predicted concentrations of each component versus actual concentration. Table 4. Determination of amprolium hydrochloride, sulfaquinoxaline sodium, and diaveridine hydrochloride in laboratory prepared mixtures by the proposed dual wavelength in ratio spectra spectrophotometric method. No of mixtures Claimed concentration taken (µg/mL) Dual wavelength in ratio spectra method % Recovery a AMP SQX DVD AMP SQX DVD 1 4 5 1.3 100.72 101.97 98.57 2 11 3 15 100.32 98.98 99.95 3 3 9 6 99.77 99.19 99.29 4 5 7 15 101.57 100.41 98.95 5 7 3 6 99.96 98 100.72 Mean 100.47 99.71 99.50 SD 0.715 1.527 0.852 RSD% 0.712 1.531 0.856 a Average of three determinations. Figure 8. Residual versus actual concentration (μg/mL) plot for AMP, SQX, and DVD in the validation set, using the PCR method. 3.2. Method validation Dual wavelength in ratio spectra method validation has been performed according to ICH guidelines [41]. 3.2.1. Linearity It was assessed by analysing different concentrations of standard solutions of each drug. The values of the correlation coefficients were close to unity indicating good linearity, the characteristic parameters for the constructed equations are summarized in Table 6. 3.2.2. Range The calibration range was established depending on the practical range according to adherence to Beer’s law and the concentration of the tested compounds present in their multi- ingredient veterinary formulation to obtain accurate, precise and linear results (Table 6). 3.2.3. Specificity Laboratory-prepared mixtures of the tested drugs were analysed. Satisfactory results were obtained and presented in Table 4. 3.2.4. Accuracy The accuracy of the results was checked by applying the proposed procedures for determination of different concent- rations for each drug within its linearity range (Tables 2 and 6). To assure the accuracy of the proposed method, a standard addition technique was applied (Table 5). -0.10 -0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0 2 4 6 8 10 12 14 16 Re si du al c on ce nt ra tio n Actual concentration AMP SQX DVD 374 Abbas et al. / European Journal of Chemistry 12 (4) (2021) 368-376 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.368-376.2144 Table 5. Determination of amprolium hydrochloride, sulfaquinoxaline sodium, and diaveridine hydrochloride in multi-ingredient veterinary formulation by the proposed procedures. Dosage form Drug Dual-wavelength-in-ratio spectra method Taken (µg/mL) Found±S.D. (%) a Added (µg/mL) Found (µg/mL) % Recovery a Tricure® contains 20 g AMP, 25 g SQX and 6.4 g DVD per 100 g AMP 3.2 100.21±0.815 4 4.068 101.70 5 5.060 101.2 6 5.961 99.35 Mean±S.D. 100.75±1.238 SQX 4.0 101.12±1.290 3 2.963 98.77 4 3.952 98.80 5 5.015 100.30 Mean±S.D. 99.29±0.875 DVD 4.0 99.61± 1.600 4 3.956 98.90 5 5.010 100.20 6 5.935 98.92 Mean±S.D. 99.34±0.745 PLS-2 method Tricure® contains 20 g AMP, 25 g SQX and 6.4 g DVD per 100 g AMP 3.2 99.43±0.154 4 4.096 102.40 5 5.038 100.76 6 5.995 99.92 Mean±S.D. 101.03±.261 SQX 4.0 97.97±0.866 3 3.076 102.53 4 4.053 101.33 5 5.029 100.58 Mean±S.D. 101.48±0.984 DVD 4.0 101.09±0.333 4 4.090 102.25 5 5.071 101.42 6 5.978 99.63 Mean±S.D. 101.10±1.339 PCR method Tricure® contains 20 g AMP, 25 g SQX and 6.4 g of DVD per 100 g AMP 3.2 98.67±0.065 4 3.954 98.85 5 5.055 101.10 6 6.043 100.72 Mean±S.D. 100.22±1.204 SQX 4.0 101.18±0.448 3 3.040 101.33 4 4.060 101.50 5 5.024 100.48 Mean±S.D. 101.10±0.546 DVD 4.0 100.37±0.282 4 4.007 100.18 5 5.076 101.52 6 6.061 101.02 Mean±S.D. 100.91±0.677 a Average of three determinations. Table 6. Assay parameters and method validation for the determination of pure samples of the studied drugs using the proposed dual wavelength in ratio spectra method. Parameters Dual-wavelength-in-ratio spectra method AMP SQX DVD λ (nm) ΔP 264 and 301.9 ΔP 250.9 and 279 ΔP 218 and 243.5 Concentration range (µg/mL) 1-20 0.5-10 0.5-19 Linearity Slope Intercept Correlation coefficient (r) 1.4271 0.7811 0.8451 -1.1461 -0.0569 0.0751 0.9998 0.9998 0.9998 Accuracy (mean±S.D.) 100.00±0.923 99.31±1.083 100.64±1.219 Specificity 100.47±0.715 99.71±1.527 99.50±0.852 Precision (%RSD) Repeatability a Intermediate precision b 1.261 0.365 0.630 0.643 0.257 0.334 LOD (µg/mL) c 0.210 0.102 0.184 LOQ (µg/mL) c 0.635 0.309 0.557 a The intraday (n = 3), average of three different concentrations repeated three times within day. b The interday (n = 3), average of three different concentrations repeated three times in three successive days. c Limit of detection and limit of quantitation. 3.2.5. Precision It was tested by determining three concentrations for each compound within its linearity range, where the concentrations were analyzed three times, each intraday (for repeatability) and on three successive days (for intermediate precision). The concentrations were obtained from the corresponding regres- sion equation then the percentage recoveries and %RSD values are calculated (Table 6). 3.2.6. Detection and quantitation limits They were calculated from the standard deviation (σ) of the response and the slope of the calibration curve (S) according to the following equations: LOD = 3.3 (σ/S) and LOQ = 10 (σ/S). Results presented in Table 6 indicate that the proposed proce- dure is sensitive for determination of the studied drugs. 3.3. Statistical analysis Results obtained by the proposed procedures for deter- mination of the studied drugs were statistically compared with those obtained by applying the reported spectrophotometric methods [13-15]. The calculated t- and F-values were found to be less than the theoretical ones, confirming accuracy and precision at 95% confidence level, as shown in Table 7. Abbas et al. / European Journal of Chemistry 12 (4) (2021) 368-376 375 2021 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.12.4.368-376.2144 Table 7. Statistical comparison of the results obtained by applying the proposed method and the reported methods for the analysis of pure AMP, SQX and DVD. Value Dual wavelength in ratio spectra method PLS-2 PCR Reported methods AMP SQX DVD AMP SQX DVD AMP SQX DVD AMP [13] b SQX [14] c DVD [15] d Mean 100.00 99.31 100.64 99.31 98.91 99.04 99.63 99.11 98.93 99.22 99.77 99.26 SD 0.923 1.083 1.219 0.769 1.192 1.245 1.005 1.272 1.338 0.992 0.548 1.701 RSD% 0.923 1.091 1.211 0.774 1.205 1.257 1.009 1.283 1.352 1.000 0.549 1.714 N 5 5 5 5 5 5 5 5 5 5 5 6 Variance 0.852 1.173 1.486 0.591 1.421 1.550 1.010 1.618 1.790 0.984 0.300 2.893 Student’s t-test a 1.284 (2.306) 0.851 (2.306) 1.508 (2.262) 0.144 (2.306) 1.456 (2.306) 0.241 (2.262) 0.643 (2.306) 1.064 (2.306) 0.351 (2.262) _ _ _ F value a 1.153 (6.388) 3.907 (6.388) 1.948 (6.256) 1.661 (6.388) 4.732 (6.388) 1.865 (6.256) 1.028 (6.388) 5.390 (6.388) 1.616 (6.256) _ _ _ a The values in parentheses are the corresponding theoretical values of t and F at p = 0.05. b Ratio difference method for determination of AMP at 239 nm and 310 nm. c First derivative spectrophotometry at 268.2 nm for SQX. d Colorimetric determination of DVD at 460 nm via charge transfer complex formation with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) reagent. The proposed work has many points of strength in accordance with the principles of green chemistry [42] such as the usage of methanol as an environmentally preferable green solvent throughout the whole work [43]. Also, the proposed procedures offer better sensitivity and simpler data manipu- lation as compared to the tedious colorimetric methods [15,44,45], i.e. all the proposed methods avoid unnecessary colorimetric derivatization because such steps require addi- tional reagents and can generate waste. Moreover, the adoption of chemometrics spectrophotometry (calculation spectro- photometry) which is a combination of chemometrics with analytical chemistry for the reduction of data dimensionality, grouping of variables, and processing of analytical signals. In this way, the analysis time, consumption of solvents or reagents, can be minimized [46]. The proposed procedures have been applied successfully on the multi-ingredient veterinary formulation and the good recovery and accuracy make them applicable in QC laboratories without the difficulties of HPLC. As compared to some recently published research articles [17-20], the proposed work shows better sensitivity for the determination of the three anticoccidial drugs as shown in Tables 3 and 6. 4. Conclusion The proposed methods are simple, accurate, sensitive, and specific. They can be used for the routine analysis of the chosen drugs in their available multi-ingredient veterinary formulation in quality control labs lacking HPLC instruments. In addition, the proposed work conforms to the principles of green chemistry because of the use of a green solvent, simple data manipulation, and the elimination of time-consuming deriva- tization steps. Acknowledgements We would like to thank Pharma Swede Pharmaceutical Company, 10th of Ramadan City, Egypt, for their cooperation and supply of pure standards of the investigated anticoccidial drugs. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. CRediT authorship contribution statement Conceptualization: Amira Mabrouk El-Kosasy; Methodology: Mahmoud Mohamed Abbas; Software: Mahmoud Mohamed Abbas; Validation: Mahmoud Mohamed Abbas; Formal Analysis: Mahmoud Mohamed Abbas; Investigation: Mahmoud Mohamed Abbas; Resources: Lobna Abdel-Aziz Hussein; Data Curation: Mahmoud Mohamed Abbas; Writing - Original Draft: Mahmoud Mohamed Abbas; Writing - Review and Editing: Nancy Magdy Hanna; Visualization: Mahmoud Mohamed Abbas, Nancy Magdy Hanna; Funding acquisition: Mahmoud Mohamed Abbas, Amira Mabrouk El-Kosasy, Lobna Abdel-Aziz Hussein, Nancy Magdy Hanna; Supervision: Amira Mabrouk El-Kosasy; Project administration: Lobna Abdel-Aziz Hussein. ORCID Mahmoud Mohamed Abbas https://orcid.org/0000-0001-6941-7865 Amira Mabrouk El-Kosasy https://orcid.org/0000-0001-7521-5805 Lobna Abdel-Aziz Hussein https://orcid.org/0000-0002-0447-1710 Nancy Magdy Hanna https://orcid.org/0000-0001-5824-5228 References [1]. Sweetman, S. C.; Martindale, W. 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The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Instrumentation 2.2. Materials 2.2.1. Standards 2.2.2. Veterinary formulation 2.3. Procedures 2.3.1. Methods 2.3.1.1. Dual-wavelength-in-ratio spectra method 2.3.1.1.1. For determination of AMP 2.3.1.1.2. For determination of SQX and DVD 2.3.1.2. PLS-2 and PCR 2.3.2. Application of the proposed procedures for determination of AMP, SQX, and DVD in laboratory-prepared mixtures 2.3.3. Application of the proposed procedures for the determination of AMP, SQX, and DVD in veterinary formulation 3. Results and discussion 3.1. Method development and optimization 3.1.1. Dual-wavelength-in-ratio spectra method 3.1.2. Multivariate chemometric methods: (PLS-2) and (PCR) 3.2. Method validation 3.2.1. Linearity 3.2.2. Range 3.2.3. Specificity 3.2.4. Accuracy 3.2.5. Precision 3.2.6. Detection and quantitation limits 4. Conclusion Acknowledgements Disclosure statement CRediT authorship contribution statement ORCID PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField18: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: PrintField28: