211 This work is licensed under a Creative Commons Attribution 4.0 International License IHJPAS. 37 (1) 2024 Ibn Al-Haitham Journal for Pure and Applied Sciences Journal homepage: jih.uobaghdad.edu.iq PISSN: 1609-4042, EISSN: 2521-3407 1Ali Khalil Mahmood* 2Takleef Dheyab Sallal 3Hasan Mohammed Luaibi 4Khalid Waleed S. Al-Janabi 1,4Department of Chemistry, College of Education for Pure Sciences Ibn Al-Haitham, University of Baghdad, Baghdad, Iraq. 2Ministry of Education, Baghdad, Iraq. 3Department of Environmental Sciences, College of Energy and Environmental Sciences, Al-Karkh University of Science, Baghdad, Iraq. *Corresponding Author: ali.khalil.mahmood@gmail.com Abstract Ciprofloxacin (Cip) and hydrocortisone (Hyd) were simultaneously measured as hydrochloride and sodium succinate, respectively, using the H-point standard addition method (HPSAM). The approach can precisely identify Cip in the presence of Hyd with various analyte-to-interference ratios (5:5, 5:10, 10:5, 10:10) µg.mL-1, in mixed samples containing (1-5µg.ml-1) of Cip, at the wavelengths of (236 and 257) nm. In the same way, Hyd was analyzed in the presence of Cip in different analytes with an interference ratio of (5:5, 5:10, 10:5, 10:10) µg.mL-1, in mixed samples containing (1-5 µg.mL-1) of Hyd, at wavelengths of (266 and 278) nm. The satisfactory results show good reproducibility of the developed method (RSD equals 0.9735-1.6825 and 0.9692- 1.7671 for Cip and Hyd, respectively). The results also show that the excipients had no influence on the assaying of the above drugs (Recovery, 98.87–101.73). The recommended technique has successfully been used to determine the Cip and Hyd in pharmaceutical composites simultaneously with an RSD range of (0.972 to 1.671) and (0.898 to 1.820) for Cip and Hyd, respectively. Keywords: H-Point, Ciprofloxacin, Hydrocortisone, Standard addition, Spectrophotometric methods. 1. Introduction Ciprofloxacin, C17H18FN3O3 [1], is frequently sold as a fluoroquinolone [2]. At very low doses, they exert potent bactericidal actions and a broad antibacterial spectrum [3]. Numerous illnesses, including endocarditis, otorrhea, lower respiratory tract, tissue, gastrointestinal, and urinary disease, have been treated with it. The primary effect is preventing (DNA) replication by inhibiting the gyrase subunit and having an additional impact on chemicals found in cell walls [4]. The Cip’s structure is described in Figure 1. Simultaneous Quantitative Determination of Ciprofloxacin and Hydrocortisone by H-Point Standard Addition Method doi.org/10.30526/37.1.3425 Received 13 April 2023, Received 14 June 2023, Accepted 19 June 2023, Published 20 January 2024 https://creativecommons.org/licenses/by/4.0/ https://jih.uobaghdad.edu.iq/index.php/j/index#1609-4042 https://jih.uobaghdad.edu.iq/index.php/j/index#2521-3407 mailto:ali.khalil.mahmood@gmail.com https://orcid.org/0000-0001-6250-0551 mailto:ali.khakil.mahmood@gmail.com https://orcid.org/0000-0002-7235-8023 mailto:sallaltakleef535@gmail.com https://orcid.org/0000-0001-5240-3774 mailto:hasan.luaibi@gmail.com https://orcid.org/0000-0003-4295-0138 mailto:khalid.Janabi@gmail.com IHJPAS. 37 (1) 2024 212 Figure 1. The chemical structure of ciprofloxacin Corticosteroids like hydrocortisone are frequently used as anti-inflammatory medications [5]. The adrenal cortex produces the hormone hydrocortisone, C21H30O5, which is essential for the immunological and circulatory systems to operate [6]. One of the inexpensive corticosteroids gives available therapy for hospitalized corona patients for respiratory support [7]. It is also used for its anti-inflammatory properties to treat other conditions such as arthritis and colitis [8]. The chemical structure of (Hyd) is provided in Figure 2. Figure 2. The chemical structure of hydrocortisone Some spectrophotometric methods have been reported to determine ciprofloxacin [9–16] and hydrocortisone [17–23]. Due to their intrinsic simplicity, sufficient sensitivity, affordability, and widespread presence in all quality control laboratories, these procedures are the most practical ones. An amendment to the standard addition method called (HPSAM) enables the alteration of an indeterminate error brought on by the existence of an interference in the assaying of an analyte that can be assessed without error [24]. The base of the method lets you find species with spectra that overlap a lot and fix mistakes that happen constantly and proportionally because of interferences and the sample matrix [25]. Also, it can be used in liquid chromatography [26] and spectrophotometry [27]. In addition, time is a variable in the study of kinetic data [28,29]. This research aims to study the effectiveness of the proposed method to assay Cip and Hyd in their pure forms and medicinal composites. 2. Materials and Methods 2.1 Apparatus  Shimadzu 1800 UV-vis spectrophotometer (Japan).  Cip and Hyd pure powders (purity 99.9%) were obtained from (SDI, Iraq). IHJPAS. 37 (1) 2024 213 2.2 Preparations and general procedure Ciprofloxacin (as hydrochloride) standard solution (250 µg.mL-1) was made by combining 25 mg of Cip with 10 mL of distilled water, then diluting the mixture to 100 mL in a volumetric flask. By adding more dilutions, fresh working solutions were created. To make hydrocortisone (as a sodium succinate) standard solution (250 µg.mL-1), 25 mg of Hyd was carefully weighed and then dissolved in 10 mL of distilled water to 100 ml in a volumetric flask. By adding more dilutions, fresh working solutions were created. 2.3 Determination of Ciprofloxacin Cip and Hyd were combined in a series of 2.5 mL aliquots with a ratio of (10:10, 10:20, 20:10, 20:20) µg.mL-1, and 1 mL of various concentrations (5-25) µg.mL-1 of Cip solution was added. Each resultant combination was diluted in a volumetric flask using distilled water to a concentration of 5 mL. The absorbance at (236 and 257) nm was measured against a blank for the reagent using a portion of the solution above that was transferred into a quartz cell. After dilution, the mixture's final ratio is (5:5, 5:10, 10:5, 10:10) µg.mL-1. 2.4 Determination of Hydrocortisone Hyd and Cip were combined in a series of 2.5 mL aliquots with a ratio of (10:10, 10:20, 20:10, 20:20) µg.mL-1, and 1 mL of various concentrations (5-25) µg.mL-1 of Hyd solution was added. Each of the resultant combinations was diluted with distilled water to a concentration of 5ml in a volumetric flask, and the absorbance at (266 and 278) nm was measured in comparison to a blank for the reagent. After dilution, the mixture's final ratio is (5:5, 5:10, 10:5, 10:10) µg.mL-1. 3. Results and Discussion The absorption spectra for Cip and Hyd are shown in Figure 3. Each material inhibits the analytical determination of the other. Therefore, HPSAM was used to determine Cip and Hyd simultaneously. Figure 3. Absorption spectra of (A) 5 µg.mL-1 of (Cip) and (B) 12 µg.mL-1 of (Hyd) 3.1 H-Point standard addition method (HPSAM) The following guidelines were used to choose the proper wavelengths to apply the H-Point Standard Addition Method [27–30]: i. The sample's signal should be linear with the analyte concentration at the two chosen wavelengths. In contrast, the interference signal should stay constant regardless of changes in the analyte concentration. IHJPAS. 37 (1) 2024 214 ii. The analytical signals for the analyte and interference combined in the mixture are equal to the total signals of the two species. iii. The selected wavelengths increment steep slopes to attain the best sensitivity. In this work, for the simultaneous determination of Cip and Hyd, a wavelength pair between (236- 257) and (266-278) nm was used, respectively, as shown in Figures 4 and 5. When one is considered the analyte (Cip) and the other as interference (Hyd), and vers versa, the calibration line plots for the H-Point Standard Addition are shown in (A and B). The concentration of the analyte (CH) was calculated directly from the cross of the two lines. In contrast, the interference concentration was determined by the H-point (AH) coordinate value for a standard of the analyte solution. Figure 4. Graphs of HPSAM at constant concentration of (5μg.mL-1) for both Cip and Hyd, where (A) Cip added to Hyd, (B) Hyd added to Cip. Figure 5. Graphs of HPSAM at constant concentration of (10μg.mL-1) for both Cip and Hyd, where (A) Cip added to Hyd, (B) Hyd added to Cip 3. 2 Application of HPSAM The applicability of the suggested method to estimate Cip and Hyd was clarified after using it on several samples and demonstrating that the analyte concentration (CH) is independent of (AH), which is also independent of (CH), as shown in Figures 6 and 7. IHJPAS. 37 (1) 2024 215 Figure 6. Graphs of HPSAM at (A) constant Conc. of Cip (5 μg.mL-1) and changing Conc. of Hyd (5 and 10 μg.mL-1), (B) constant Conc. of Hyd (5 μg.mL-1) and changing Conc. of Cip (5 and 10 μg.mL-1) Figure 7. Graphs of HPSAM at (A) constant Conc. of Cip (10 μg.mL-1) and changing Conc. of Hyd (5 and 10 μg.mL-1), (B) constant Conc. of Hyd (10 μg.mL-1) and changing Conc. of Cip (5 and 10 μg.mL-1) 3.3 Accuracy and precision Using the suggested technique, several synthetically mixed samples with various Cip and Hyd concentration ratios were examined. The proposed method was checked for accuracy and precision by comparing the Cip and Hyd analysis results with the actual values found in the sample. Tables 1 and 2 demonstrate that the method's accuracy for determining Cip and Hyd is acceptable. On the other hand, the standard deviation value for three duplicate trials was used to measure the approaches' repeatability. Table 1. Accuracy and precision for the analysis of mixture contain Cip (as analyte) and Hyd (as interference) at various concentration ratios Exp. No. CH AH Analyte (Cip)* Interference (Hyd)* Taken Found Rec.% R.S.D Taken Found Rec.% R.S.D 1 5.0106 0.1667 5 5.0106 100.21 1.5981 5 5.1172 102.34 1.7671 2 10.069 0.2848 10 10.069 100.69 0.9735 5 5.0381 100.76 1.8344 3 10.149 0.3788 10 10.149 101.49 1.4817 10 9.9659 99.659 0.9692 * Average of three measurements. * Conc. (µg.mL-1). A = 0.0494x + 0.8338 R² = 0.9998 A = 0.0306x + 0.6430 R² = 0.9993 A = 0.0498x + 0.6664 R² = 0.9999 A = 0.0309x + 0.4761 R² = 0.9999 0 0.2 0.4 0.6 0.8 1 1.2 -10 -5 0 5 10 15 A b so rb a n ce Conc. of Cip add µg/mL A A = 0.0494x + 0.8338 R² = 0.9998 A = 0.0306x + 0.6430 R² = 0.9993 A = 0.0496x + 0.5832 R² = 0.9996 A = 0.0309x + 0.4890 R² = 0.9992 0 0.2 0.4 0.6 0.8 1 1.2 -10 -5 0 5 10 15 A b so rb a n ce Conc. of Cip add µg/mL B A = 0.0163x + 0.9675 R² = 0.9994 A = 0.0247x + 1.0504 R² = 0.9998 A = 0.0170x + 0.5669 R² = 0.9974 A = 0.0251x + 0.6497 R² = 0.9993 0 0.2 0.4 0.6 0.8 1 1.2 1.4 -10 0 10 A b so rb a n ce Conc. of Hyd. add µg/mL A A = 0.0163x + 0.9675 R² = 0.9994 A = 0.0247x + 1.0504 R² = 0.9998 A = 0.0166x + 0.8839 R² = 0.9999 A = 0.0251x + 0.9254 R² = 0.9988 0 0.2 0.4 0.6 0.8 1 1.2 1.4 -10 -5 0 5 10 15 A b so rb a n ce Conc. of Hyd. add µg/mL B IHJPAS. 37 (1) 2024 216 Table 2. Accuracy and precision for the analysis of mixture contain Hyd (as analyte) and Cip (as interference) at various concentration ratios Exp. No. CH AH Analyte (Hyd)* Interference (Cip)* Taken Found Rec.% R.S.D Taken Found Rec.% R.S.D 1 5.1325 0.3983 5 5.1325 102.65 1.7266 5 5.0841 101.08 0.9868 2 10.222 0.3931 10 10.222 102.22 1.5038 5 4.9692 99.384 1.6825 3 9.8690 0.8066 10 9.8690 98.690 1.7298 10 10.1860 101.86 1.4393 * Average of three measurements. * Conc. (µg.mL-1). The proposed method was compared statistically with other methods found in the literature [18,31], and the results are shown in Table 3 below. Table 3. Analytical parameters for simultaneous determination of Cip and Hyd by the Proposed and other methods Methods λmax (nm) Linearity (µg.mL-1) Slope R2 Rec.% RSD Spectrophotometric 278 244 2.0-14 1.0-14 0.0512- 6.3589 0.9999- 1.0000 99.92-100.57 0.32-1.65 HPLC 243 278.6 7.44-52.9 29.7-222 191.14 272.13 0.9988- 0.9995 98.00-101.1 1.20-1.40 Proposed method 236-257 266-278 1.0-5.0 1.0-5.0 0.0166- 0.0486 0.9974- 0.9999 98.690-102.34 0.9692-1.7671 3. 4 Interferences study The findings demonstrated no interferences (Recovery, 98.87-101.73) in determining 10 µg.mL-1 of Cip and Hyd in the presence of 250 µg.mL-1of the investigated excipients, as shown in Table 4. Table 4. Percent recovery for determination of 10 µg.mL-1 of Cip and 10 µg.mL-1of Hyd in the presence of 250 µg.mL-1of Excipients Excipients Cip Hyd Found* Rec. % Found* Rec. % Starch 9.943 99.43 10.129 101.29 Glucose 10.123 101.23 9.924 99.24 Lactose 9.982 99.82 9.887 98.87 Sucrose 9.889 98.89 9.952 99.52 Sodium Citrate 10.173 101.73 10.122 101.22 * Average of three measurements. * Conc. (µg.mL-1). 3.5 Analysis of dosage forms The abovementioned findings suggest the strategy runs well with the tested medications. As a result, the proposed approach was used to analyze the pharmaceutical dosage forms' active component content (HPSAM). The results in Tables 5 and 6 were in line with expectations. IHJPAS. 37 (1) 2024 217 Table 5. Determination of Cip in the presence of Hyd in some pharmaceutical preparations Sample Analyte (Cip)* Interference (Hyd)* Taken Found Rec.% R.S.D Taken Found Rec.% R.S.D BactifloxTM Neo 750 mg Ciprofloxacin as Hydrochloride Tablets, acino Switzerland 5 4.952 99.040 1.628 5 4.944 98.880 1.521 TYFLOX 500 mg Ciprofloxacin as Hydrochloride Tablets, ajanta pharma limited,India 10 10.204 102.05 1.013 5 5.162 103.24 0.898 CIPRODAR Sterile Eye Drops 0.3% Ciprofloxacin as Hydrochloride, Dar Al Dawa, Jordan 10 4.915 98.300 0.972 10 10.197 101.97 1.033 * Average of three measurements. * Conc. (µg.mL-1). Table 6. Determination of Hyd in the presence of Cip in some pharmaceutical preparations Sample Analyte (Hyd)* Interference (Cip)* Taken Found Rec.% R.S.D Taken Found Rec.% R.S.D Hydrocortisone Roussel Tablets 10 mg SANOFI, France 5 5.066 101.32 1.607 5 5.079 101.40 0.975 Hidrkortizone Vial 100mg, Hydrocortisone as sodium succinate, Hemofarm, Serbi 10 9.873 98.730 1.601 5 5.142 102.84 1.492 Hydrocortisone Vial 100mg Hydrocortisone as sodium succinate, EIPICO, Egypt 10 10.262 102.62 1.820 10 10.111 101.11 1.671 *Average of three measurements. * Conc. (µg.mL-1). 4. Conclusion Cip and Hyd were simultaneously measured using the H-point standard addition method in mixed samples containing different analytes with different interference ratios (5:5, 5:10, 10:5, 10:10) µg.mL-1. A wavelength pair between (236-257) and (266-278) nm was used, respectively. The results show that the recommended method is rapid, straightforward, and it is successfully used to determine Cip and Hyd in medicinal composites, with RSD ranging from 0.972 to 1.671 and from 0.898 to 1.820, respectively. The results show that the excipients did not influence the simultaneous assay of the above drugs (Recovery, 98.87-101.73). Acknowledgment The authors thank the staff at the Department of Chemistry/ College of Education for Pure Sciences at Ibn Al- Haitham/ University of Baghdad for their assistance in performing this research. Conflict of Interest The authors declare that they do not have any competing interests. Funding There is no financial support. IHJPAS. 37 (1) 2024 218 Ethical Clearance This work has been approved by the Scientific Committee at the University of Baghdad/ College of Education for Pure Sciences Ibn Al-Haitham. References 1. Al-Omar, M.A. Ciprofloxacin: Analytical Profile. 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