Simultaneous determination of amlodipine and lisinopril dihydrate using fourth derivative spectroscopy European Journal of Chemistry 14 (1) (2023) 65-71 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2023 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.14.1.65-71.2367 European Journal of Chemistry View Journal Online View Article Online Simultaneous determination of amlodipine and lisinopril dihydrate using fourth derivative spectroscopy Aws Maseer Nejres 1,* and Moath Abdallah Najem 2 1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Mosul, Mosul, 41001, Iraq 2 Department of Pharmaceutical Chemistry, Faculty of Agriculture and Forestry, University of Mosul, 41001, Iraq * Corresponding author at: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Mosul, Mosul, 41001, Iraq. e-mail: aws.m.nejres@uomosul.edu.iq (A.M. Nejres). 10.5155/eurjchem.14.1.65-71.2367 Received: 27 November 2022 Received in revised form: 24 December 2022 Accepted: 12 January 2023 Published online: 31 March 2023 Printed: 31 March 2023 A new fast and simple selective method for the simultaneous determination of lisinopril dihydrate and amlodipine in combined drugs was developed using the fourth derivative spectrum method, based on the zero-crossing-point technique for the determination of compounds in drugs. The wavelength values for lisinopril dihydrate and amlodipine in solvent medium were found to be (203, 207, and 231 nm) and (215, 254, and 277 nm), respectively, with the average obeying Beer’s law in the range of lisinopril dihydrate 2.0 to 45.0 µg/mL and amlodipine 2.0 to 35.0 µg/mL. Lisinopril dihydrate has molar absorptivity regions (9227.76-11700.28 L/mol.cm, 203 nm), (15320.74-20795.59 L/mol.cm, 207 nm), and (2207.60-3311.40 L/mol.cm, 231 nm), while amlodipine (5886.72-10914.96 L/mol.cm, 215 nm), (5518.8-6418.16 L/mol.cm, 254 nm) and (1676.08-1921.36 L/mol.cm, 277 nm). The recovery rate of lisinopril dihydrate in the pharmaceutical dosage forms range was 95.13 to 102.60% and amlodipine 95.14 to 102.80%. The results of the relative error showed that the interferences did not affect the method of estimating these compounds. The proposed method has been successfully applied to estimate pharmaceutical dosage forms. Amlodipine Zero-crossing point Lisinopril dihydrate Simultaneous determination Zero-crossing point technique Fourth derivative spectrum method Cite this: Eur. J. Chem. 2023, 14(1), 65-71 Journal website: www.eurjchem.com 1. Introduction Lisinopril dihydrate (LSD) is one of the long-acting angiotensin-converting enzyme (ACE) inhibitors allowing drugs with the chemical name 1-[6-amino-2-(1-carboxy-3- phenylpropylamino)-hexanoyl]-pyrrolidine-2-carboxylic acid (Scheme 1) [1]. Where it is considered a competitive inhibitor of angiotensin-converting enzyme (ACE) prevents the conversion of angiotensin I to angiotensin II, which works doing decrease sodium and water retention in the body, thus reducing angiotensin II-stimulated aldosterone secretion [2,3]. Amlodipine (ALP), 3, 5-Pyridinedicarboxylic acid, 2-[(2-ami noethoxy)methyl]-4-(2-chlorophenyl)-1, 4-dihydro-6-methyl, 3-ethyl 5-methyl ester, is a calcium channel blocker (Scheme 1). It is used to treat chronic angina, hypertension, and myocardial infarction, as well as a specific type of chronic angina [4-6]. It prevents coronary arterial contraction and narrowing of the arteries, leading to increased blood flow and myocardial oxygenation [7]. Pharmaceutical formulations with the combination of amlodipine as a calcium channel blocker channel blocker and lisinopril as a long-acting ACE inhibitor have been made available on the market to treat hypertension [8]. Several analytical methods for the determination of LSD and ALP are revealed in the literature review, such as high performance thin layer chromatography (HPTLC) for the simultaneous quantification of LSD and ALP with other antihypertensive drugs [9]; Based on the absorption ratio method and the isoabsorptive point, these methods enable the determination of LSD and ALP in the same tablet dosage forms [10,11], at 25 °C, Column C18, 5 µm, 150 mm × 4.6 mm id as stationary phase and mixture solution from acetonitrile: sodium dihydrogen phosphate (50:50, v:v) has been succeeded in estimating simultaneous estimation of LSD and ALP in pharmaceutical dosage forms [12]. Furthermore, the first or second derivative spectrum method was used to determine LSD and ALP, which were prepared in 0.1 M HCl solution and gave accurate results without interference [13]. The derivative spectroscopic techniques are considered simple and direct for the simultaneous determination of mixtures in their pharma- ceutical preparation [14]. Thus, the fourth derivative method achieved the desired purpose and has been utilized to determine LSD and ALP mixtures in their pharmaceutical preparations simultaneously. ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.14.1.65-71.2367 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.14.1.65-71.2367 mailto:aws.m.nejres@uomosul.edu.iq http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.14.1.65-71.2367&domain=pdf&date_stamp=2023-03-31 66 Nejres and Najem / European Journal of Chemistry 14 (1) (2023) 65-71 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.65-71.2367 N H COOH NH2 O N COOH . 2H2O Lisinopril dihydrate (LSD) N H O Cl O O NH2 OO Amlodipine (ALP) Scheme 1. Structures of lisinopril dihydrate (LSD) and amlodipine (ALP). Figure 1. Zero-order order spectrums of (A) LSD, (B) ALP, and (C) overlap LSD and ALP. 2. Experimental 2.1. Apparatus All spectrophotometric absorbances were measured using the Labomed Inc. 2602-UV-vis spectrophotometer using 1-cm quartz cells. pH measurements were taken using a HANNA brand pH211 model pH meter. 2.2. Chemicals All chemical reagents used in this work were of high purity (Lisinopril dihydrate (99% purity, CAS 83915-83-7) supplied by Meryer Company, China and amlodipine (99% purity, CAS 88150-42-9) supplied by Energy Chemical Company, China. 2.3. Derivative spectrophotometry measurement Standard solutions of LSD and ALP were scanned in the wavelength range of 200-300 nm, where distilled water was used as a blank solution. The zero-order and fourth-derivative spectra were measured using UVWin7 software (V5.2.0.1104), and no smoothing was required. 2.4. Preparation of standard solutions Lisinopril dihydrate: A stock solution of LSD (500 μg/mL) was prepared in a 100 mL volumetric flask by dissolving 0.05 g of raw LSD in the appropriate amount of distilled water containing 1 mL of 1 M HCl. The volume was completed in the 100 mL volumetric flask. A standard working solution of LSD (100 μg/mL) was prepared by diluting 20 mL of the stock solution with distilled water in a 100 mL volumetric flask. Amlodipine: A stock solution of ALP (500 μg/mL) was prepared by dissolving 0.05 g of raw ALP in the appropriate volume of distilled water containing 1 mL of 1 M HCl and then transferring it to a 100 mL volumetric flask, completing the volume by distilled water. A standard working solution of ALP (100 g/mL) was prepared by transferring 20 mL of the stock solution in a 100 mL volumetric flask and diluting it with distilled water to mark. 2.5. Assay preparation of pharmaceutical formulations The 10 tablets (Hipril-A; LSD 5 mg/ALP 5 mg) were accurately weighed (1.906 g; for ten tablets) and crushed to a fine powder. The portion equivalent to 0.05 mg for the pharmaceutical formulation was transferred to a 25 mL beaker, an appropriate amount of distilled water was then added containing 1 mL of 1 M HCl, and sonication was performed for 20 minutes with swirling. After that, the resultant solution was filtered through the Whatman filter paper, and the precipitate residue on the filter paper was washed several times with distilled water. Finally, the solution was transferred to a 100 mL volumetric flask and the volume was completed for a mark. 3. Results and discussion When there is convergence in the absorbance spectrum of drugs when using zero-order spectra, and each compound spectrum interferes with the other spectrum, the derivative spectrophotometry technique is used [15]. LSD appears to have one maximum absorbance band at 205 nm. On the contrary, ALP has two maximum absorbance bands at 212 and 238 nm with the shoulder, while the spectrum of the mixture of the two medicinal compounds gave two bands for each of 216 and 235 nm (Figure 1). The derivative spectra technique can resolve compounds with the same or near-wavelength region that generates inseparable interference in zero-order spectro- photometry. 3.1. Fourth derivative spectrum of LSD The fourth derivative spectrum of LSD is shown in Figure 2. LSD has a positive peak at 203, 207, and 231 nm with one negative peak at 221 nm. Nejres and Najem / European Journal of Chemistry 14 (1) (2023) 65-71 67 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.65-71.2367 Table 1. Regression analysis of LSD and ALP individually. λ (nm) Concentration (μg/mL) Regression equation [19] R2 LSD ALP 203 2.0-45 0 y = 0.0216x – 0.0045 0.9997 207 2.0-45 0 y = 0.0331x – 0.0102 0.9993 231 2.0-45 0 y = 0.0068x + 0.0102 0.9970 215 0 2.0-35 y = 0.0049x + 0.0202 0.9988 254 0 2.0-35 y = –0.0145x + 0.0015 0.9996 277 0 2.0-35 y = 0.0049x + 0.0035 0.9971 Figure 2. Fourth derivative spectrum of LSD (2-45 μg/mL). Figure 3. Fourth derivative spectrum of ALP (2-35 μg/mL). These peaks could be estimated when LSD is present alone. LSD has more than one zero crossing point at 215 and 228 nm, where LSD has zero absorbance at any concentration. 3.2. Fourth derivative spectrum of ALP Figure 3 shows the fourth derivative spectrum of ALP concentrations. ALP has appeared at more peaks that could be used to estimate when presenting alone, with three positive peaks at 215, 242, and 277 nm and one negative peak at 205, 223, and 254 nm. These peaks could be used to estimate when presented alone. ALP has more than one zero crossing point at 203, 207, 231, 251, and 271 nm. It has zero absorbance for any concentration. 3.3. Simultaneous determination of LSD and ALP Figure 4 show the simultaneous determination of different concentrations of LSD at 203, 207, and 231 nm in the presence of 5-30 μg/mL ALP, where ALP has a zero-crossing point; therefore, LSD can be determined in the presence of ALP. Also, under the same condition, when the simultaneous deter- mination of the different concentrations of ALP in the presence of 5-45 μg/mL LSD, we selected 215, 254, and 277 nm where LSD has a zero-crossing point at these wavelengths, as shown in Figure 5. The LSD and ALP concentration range was selected according to the zero-crossing point [16]. Based on this, the peak to baseline [17] can be used to measure LSD concent- rations separately or simultaneously at 203, 207, and 231 nm and ALP at 215, 254, and 277 nm. 3.4. Calibration graphs for the analysis of LSD and ALP The calibration curves were constructed using the fourth- order derivative response. Various concentrations can be used to analyze the drug separately or simultaneously. All calibration curve parameters are listed in Figure 6 and Table 1. Describe the compound individually. Table 2 shows the results of the calibration curve at variable concentrations of LSD in the presence of a fixed concentration of ALP, while Table 3 shows the reverse. Based on this, we determine the concentration range that obeys Beer’s law [18]. Other characteristics, such as molar absorptivity and Sandell’s sensitivity values, are given. 3.5. Accuracy and precision of the proposed method To investigate the accuracy and precision of the proposed method, an aliquot (7 and 15 µg/mL) concentration of ALP and LSD, each concentration, was repeated three times individually (Table 4). The recovery% and error% were calculated as accuracy and the relative standard deviation as precision [20]. 68 Nejres and Najem / European Journal of Chemistry 14 (1) (2023) 65-71 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.65-71.2367 (a) (b) Figure 4. Simultaneous determination of LSD in the presence of (a) 5 μg/mL and (b) 30 μg/mL ALP. (a) (b) Figure 5. Simultaneous determination of ALP in the presence of (a) 5 μg/mL and (b) 45 μg/mL LSD. Nejres and Najem / European Journal of Chemistry 14 (1) (2023) 65-71 69 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.65-71.2367 Table 2. Regression analysis of LSD and ALP in a mixture solution. λ (nm) Concentration (μg/mL) Regression equation R2 Molar absorptivity (L/mol.cm) Sandell’s sensitivity (μg/cm2) LSD ALP 203 2-45 5 y = 0.0209x – 0.0085 0.9979 9227.76 0.047 2-45 10 y = 0.0215x – 0.0257 0.9976 9492.68 0.046 2-40 15 y = 0.0224x – 0.0513 0.9888 9890.05 0.044 2-35 20 y = 0.0241x – 0.0548 0.9906 10640.63 0.041 2-30 25 y = 0.0265x – 0.0891 0.9918 11700.28 0.037 2-35 30 y = 0.0258x – 0.0134 0.9877 11391.21 0.038 207 2-45 5 y = 0.0347x – 0.0027 0.9979 15320.74 0.028 2-45 10 y = 0.0380x – 0.0428 0.9963 16777.76 0.026 2-45 15 y = 0.0405x – 0.1294 0.9859 17881.56 0.024 2-35 20 y = 0.0428x + 0.0034 0.9877 18897.06 0.023 2-40 25 y = 0.0399x – 0.1247 0.9901 17616.65 0.025 2-45 30 y = 0.0471x + 0.0904 0.9966 20795.59 0.021 231 2-45 5 y = 0.0063x + 0.0114 0.9969 2781.57 0.158 2-45 10 y = 0.0062x + 0.0184 0.9962 2737.42 0.158 2-45 15 y = 0.0052x + 0.0405 0.9806 2295.90 0.192 2-35 20 y = 0.0058x + 0.0354 0.9678 2560.82 0.172 2-30 25 y = 0.0050x + 0.0370 0.9857 2207.60 0.200 2-35 30 y = 0.0075x + 0.1523 0.9797 3311.40 0.133 Table 3. Regression analysis of LSD and ALP in a mixture solution. λ (nm) Concentration (μg/mL) Regression equation R2 Molar absorptivity (L/mol.cm) Sandell’s sensitivity (μg/cm2) LSD ALP 215 5 2-35 y = 0.0223x + 0.0945 0.9846 9116.24 0.044 10 2-30 y = 0.0248x + 0.0490 0.9839 10138.24 0.040 15 2-35 y = 0.0267x + 0.0057 0.9933 10914.96 0.037 25 2-30 y = 0.0144x + 0.0652 0.9572 5886.72 0.069 254 5 2-35 y = -0.0157x + 0.0139 0.9986 6418.16 0.063 10 2-35 y = -0.0137x + 0.0078 0.9989 5600.56 0.072 15 2-35 y = -0.0142x + 0.0099 0.9991 5804.96 0.070 25 2-35 y = -0.0136x + 0.0103 0.9988 5559.68 0.073 35 2-35 y = -0.0135x + 0.0185 0.9973 5518.8 0.074 45 2-35 y = -0.0140x + 0.0260 0.9958 5723.2 0.071 277 5 2-35 y = 0.0047x – 0.0022 0.9988 1921.36 0.212 10 2-35 y = 0.0044x – 0.0023 0.9983 1798.72 0.227 15 2-35 y = 0.0043x – 0.0057 0.9989 1757.84 0.232 25 2-35 y = 0.0043x – 0.0075 0.9965 1757.84 0.232 35 2-35 y = 0.0043x – 0.0114 0.9912 1757.84 0.232 45 2-35 y = 0.0041x – 0.0151 0.9831 1676.08 0.243 Table 4. The accuracy and precision of the proposed method. λ (nm) Concentration (μg/mL) Found (µg/mL) Precision Accuracy LSD ALP Relative standard deviation (%) Percentage error Percentage recovery 203 7 7 6.95 1.94 -0.72 99.28 15 15 14.85 2.51 -0.86 99.00 207 7 7 6.90 0.52 -1.43 98.57 15 15 14.93 0.21 -0.47 99.53 231 7 7 7.24 3.50 +3.42 103.42 15 15 14.87 2.71 -0.87 99.13 215 7 7 6.87 0.90 -1.86 98.14 15 15 15.97 0.41 -0.20 99.80 254 7 7 6.93 2.04 -1.00 99.00 15 15 14.86 0.25 -0.94 99.06 277 7 7 7.05 2.93 +0.70 100.71 15 15 14.81 2.19 -1.27 98.73 (a) (b) Figure 6. (a) Calibration curve of the LSD at 203, 207, and 231 nm and (b) Calibration curve of the ALP at 215, 254, and 277 nm. 70 Nejres and Najem / European Journal of Chemistry 14 (1) (2023) 65-71 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.65-71.2367 Table 5. The limit of detection LOD and quantification LOQ. λ (nm) SD LOD (µg/mL) LOQ (µg/mL) 203 0.0021 0.1821 0.6073 207 0.0026 0.2373 0.7912 231 0.0008 0.371 1.2371 215 0.0040 0.3942 1.3141 254 0.0008 0.1703 0.5677 277 0.0005 0.3226 1.0756 Table 6. The effect of common excipients on the determination of drugs. Excipients Concentration (μg/mL) λ (nm) / The percentage of error 203 207 231 215 254 277 The relative error percentage ±5.0% Arabic gum 100 0.45 2.23 0.86 2.73 0.75 0 200 1.79 2.86 1.72 2.80 0.76 2.50 Fructose 100 0 1.47 1.72 0.32 2.27 0 200 1.86 1.64 1.73 3.55 3.03 2.50 Sucrose 100 0.99 1.76 1.70 0.21 0.75 2.4 200 0.89 4.71 3.44 1.07 1.51 2.56 Potassium chloride 100 0.96 0.95 2.58 1.50 0 0 200 0.89 1.97 0.86 2.84 0.76 2.50 Glucose 100 0.48 0.33 0 0.53 1.51 2.50 200 1.44 0.65 0.86 0.32 1.51 2.50 Table 7. The results of the proposed method on pharmaceutical drugs and the t-test for LSD and ALP. λ (nm) Hipril-A (5 mg:5 mg) Found Relative standard deviation (%) Percentage error Percentage recovery t-Test LSD ALP 203LSD 7 7 7.18 2.77 +2.57 102.57 ±1.64 15 15 14.27 3.57 -4.87 95.13 ±2.51 207LSD 7 7 6.96 2.06 -0.42 99.42 ±0.37 15 15 14.96 1.35 -0.27 99.73 ±0.27 231LSD 7 7 6.87 1.75 -1.86 98.14 ±1.37 15 15 15.39 0.60 +2.60 102.60 ±0.73 215ALP 7 7 6.66 2.58 -4.86 95.14 ±3.17 15 15 15.42 3.26 +2.80 102.80 ±1.42 254ALP 7 7 7.06 0.56 +0.85 100.85 ±1.73 15 15 14.93 1.79 -0.47 99.53 ±0.30 277ALP 7 7 6.82 1.88 -2.57 97.43 ±0.02 15 15 15.05 3.59 +0.33 100.33 ±0.17 3.6. Limit of detection (LOD) and quantification (LOQ) According to the ICH guidelines, the absorbance of ten samples of the same concentration of a mixed solution containing (2:2 μg/mL) LSD and ALP drugs (2:2 g/mL) was measured [21]. LOD and LOQ were calculated according to the ICH guidelines. Table 5. reveals the acceptable and sensitive values of the method. 3.7. Study of the effect of common excipients The influence of different common excipients added to pharmaceutical drugs on the determination of 10 μg/mL of LSD and 10 μg/mL of ALP with the fourth derivative spectro- photometry method. The method was tolerable for different concentrations (100 and 200 μg/mL) of Arabic gum, fructose, sucrose, potassium chloride, and glucose. The relative error percentage was within ±5.0% (Table 6). 3.8. Applications on pharmaceutical drugs LSD and ALP were determined in a pharmaceutical drug to evaluate the proposed method. The results for the two concentrations are listed in Table 7. The results of the proposed method revealed an acceptable recovery, and the relative standard deviation and a t-test (at three degrees of freedom, which is 3.182 and a 95% confidence level) [22] were appropriate. 4. Conclusions A sensitive spectrophotometric method has been used to determine LSD and ALP in combined drugs simultaneously. Analytical data was based on the zero-crossing point technique using the fourth derivative spectrum method. Determining the accuracy and precision represents the best assessment of the proposed method in routine use. LOD and LOQ reveal the sensitivity of the method. Acknowledgement We thank the University of Mosul, Iraq, for providing instrumental and chemical facilities. The authors also thank Dr. Arkham Alomari for his assistance. Disclosure statement Conflict of interest: The authors declare that they have no conflict of interest. Ethical approval: All ethical guidelines have been adhered to. Sample availability: Samples of the compounds are available from the author. CRediT authorship contribution statement Conceptualization: Aws Maseer Nejres; Methodology: Aws Maseer Nejres, Moath Abdallah Najem; Software: Aws Maseer Nejres; Validation: Aws Maseer Nejres; Formal Analysis: Aws Maseer Nejres; Investigation: Aws Maseer Nejres; Resources: Aws Maseer Nejres, Moath Abdallah Najem; Data Curation: Aws Maseer Nejres; Writing - Original Draft: Aws Maseer Nejres; Writing - Review and Editing: Aws Maseer Nejres; Project Administration: Aws Maseer Nejres, Moath Abdallah Najem. ORCID and Email Aws Maseer Nejres aws.m.nejres@uomosul.edu.iq https://orcid.org/0000-0002-2718-6760 Moath Abdallah Najem moathalhajjar@uomosul.edu.iq https://orcid.org/0000-0002-9933-3774 mailto:aws.m.nejres@uomosul.edu.iq https://orcid.org/0000-0002-2718-6760 mailto:moathalhajjar@uomosul.edu.iq https://orcid.org/0000-0002-9933-3774 Nejres and Najem / European Journal of Chemistry 14 (1) (2023) 65-71 71 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.65-71.2367 References [1]. Zounr, Z. A. Determination of Lisinopril in pure and tablet form by using 2-hydroxynaphthaldehyde as derivatizing reagent. Pak. J. Anal. Environ. Chem. 2021, 22, 115–126. [2]. Shulyak, N.; Budzivula, K.; Kucher, T.; Kryskiw, L.; Poliak, O.; Logoyda, L. Spectrophotometric methods for the determination of lisinopril in medicines. Farmatsiia (Sofia) 2021, 68, 811–818. [3]. Khan, S.; Khan, F. N.; Sadeque, M.; Zainuddin, R.; Zaheer, Z. 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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). 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. Apparatus 2.2. Chemicals 2.3. Derivative spectrophotometry measurement 2.4. Preparation of standard solutions 2.5. Assay preparation of pharmaceutical formulations 3. Results and discussion 3.1. Fourth derivative spectrum of LSD 3.2. Fourth derivative spectrum of ALP 3.3. Simultaneous determination of LSD and ALP 3.4. Calibration graphs for the analysis of LSD and ALP 3.5. Accuracy and precision of the proposed method 3.6. Limit of detection (LOD) and quantification (LOQ) 3.7. Study of the effect of common excipients 3.8. Applications on pharmaceutical drugs 4. Conclusions Acknowledgement Disclosure statement CRediT authorship contribution statement ORCID and Email References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: