RP-HPLC Method Development and Validation for Metformin and Cefuroxime Interaction Studies in Human Plasma

M. A Garba et al., ISSN: 2814-1423 70 Nigerian Journal of Pharmaceutical and Biomedical Research Vol. 8 Issue.1 April, 2024. p-ISSN: 2579-1419 e-ISSN: 2814-1423 RP-HPLC Method Development and Validation for Metformin and Cefuroxime Interaction Studies in Human Plasma 1*Musa. A Garba,1Danmusa. M. Umar., 2Abdulkadir H., 3Abdulkadir Nasiratu., 4Abdulfatai. Khadijat., and 5Makama Musa 1Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Kaduna State University, Kaduna, Nigeria. 2Department of Computer Science, Faculty of computing , Kaduna State University, Kaduna , Nigeria. 3Department of Biological Sciences, Nigerian Defence Academy, Kaduna 4Department of Medical Laboratory Science, Faculty of Allied Health Sciences, Kaduna State University, Kaduna, Nigeria 5Department of Statistics, Shehu Idris Institute of Health Makarfi Campus, Kaduna State University, Kaduna Nigeria. *Corresponding Author: [email protected], doi.org.10.55639/njpbr.1061.006 Abstract BackgroundMetformin is usually prescribed with cefuroxime in comorbid diabetic infections involving the lungs (tonsillitis), nose, ear, skin, bladder, and Neisseria gonorrhea infections. The interaction of the two drugs is yet known. Aim: This study was aimed at developing and validating a simple and reproducible RP-HPLC method for the interaction Studies of Metformin and Cefuroxime in HumanPlasma Method: The study was designed into two phases. In phase one, all volunteers received 1 g of metformin, while in phase two metformin 1g was administered along with 500 mg of Cefuroxime. In each phase, blood samples were collected at intervals within 24 hours post-drug administration. 0.5 ml of HCl was used to acidify the plasma samples, which were then deproteinized with acetonitrile and centrifuged. The supernatant was then rinsed with dichloromethane and introduced into the HPLC system using a Poros hell 120 EC-C18 column. An isocratic mobile phase was employed, which consisted of 90:10% v/v acetonitrile and 0.03M dibasic ammonium phosphate at a flow rate of 0.8 ml/min, a detection wavelength of 236 nm, and temperature-controlled. Results: Chromatographic separation was achieved in 10 minutes with metformin and phenytoin having retention times of 2.230 and 4.407 minutes respectively. The method was precise (3.43% RSD), accurate (% Er of 2.24 and % recovery 96.52%), with a linear calibration curve (r = 0.995.). LOD and LOQ of the developed method are 0.02 and 0.05 μg/mL respectively. All the parameters were within the acceptable limits. Conclusion: From the result, the developed and validated method was accurate and suitable for routine analysis of metformin in human plasma. it was also decided that metformin and cefuroxime may be given together to individuals with type 2 diabetes with caution to avoid toxicity and or therapeutic failure Keywords: Cefuroxime, Drug-drug interaction, Human plasma, RP-HPLC method. Introduction Metformin (1-dimethyl biguanide hydrochloride) is the first-line drug of choice for type 2 diabetes and the most commonly prescribed drug for this condition worldwide, either alone or in combination with insulin or other oral antidiabetes (Flory et al., 2015). Metformin inhibits hepatic glucose production, reduces intestinal glucose absorption, and improves glucose uptake and utilization. Besides lowering blood glucose levels, metformin M. A Garba et al., ISSN: 2814-1423 71 may have additional health benefits, including weight reduction, lowering plasma lipid levels, and prevention of some vascular complications (Gong et al.,2012). It is also indicated for other conditions i.e. polycystic ovary syndrome (PCOS). Metformin is increasingly recognized as a potential anticancer agent due to a reduced cancer incidence in diabetic patients treated with the drug, and recently, patients taking metformin were associated with a reduced risk of COVID-19-related mortality (Jeong and Jusko 2021). Metformin is a highly ionized, water-soluble drug that is absorbed, distributed, and eliminated by transporters (Duong et al.,2013). It is actively secreted by the kidney's tubules and eliminated unchanged in the urine. Many high-performance chromatographic (HPLC) methods for the analysis of metformin in plasma were reported in the literature, most of the methods used were either ion pair reagent or cation exchange columns (Liu and Coleman 2019), while some require elaborate sample preparation (Lilly et al.,2009). Also, other methods of determination of metformin by gas chromatography and UV-visible spectrophotometer were reported respectively (El-Bardicy et al.,1989, Tache et al., 2001, Lin et al., 2001, Ashor and Kabbani 2003). Though these methods are sensitive and reproducible, the reverse phase-HPLC (RP-HPLC) method for the estimation of metformin in human plasma was found to be more suitable (Lilly et al., 2009, Fatema et al., 2010). Previously published techniques had many drawbacks, such as expense and the need for laborious, time-consuming sophisticated extraction processes. Ultra-filtration and columnswitching techniques have been suggested to improve specificity and selectivity (Lilly et al., 2009). The research aims to create and validate an affordable, straightforward, and accurate method for determining the presence of metformin in human plasma. Fig 1: Chemical Structure of Metformin Fig 2: Chemical Structure of Cefuroxime M. A Garba et al., ISSN: 2814-1423 72 Material and method Materials Equipment and reagents Digital weighing balance OHAUS model EP 64, Switzerland, U.V. detector + U.V/Vis spectrometer by PG instrument Ltd U.K, Centrifuge: Heroes (labafuge300), HPLC column: Zobrax SB-Aq. (C18 4.6 X150 nm id 5nm (particle size), Acetonitrile analytical grade, Methanol Sigma – Aldrich U.K, HPLC Agilent technologies Model 1260 Infinity Series. Dipotassium hydrogen phosphate Buffer by J.T Baker USA, metformin HCL (Reference Standard), phenytoin (internal standard –Ranbaxy Pharmaceutical), Acetic acid analytical grade, Hydrochloric acid Analytical grade, Dichloromethane, HPLC sample bottles 1.5 mL Thermo Electron Corporation Central CL2 centrifuge, HPLC grade methanol, HPLC grade water, Tetrahydrofuran THF (Analytical), Sodium acetate, Hydrochloric Acid (Analytical). Methods Preparation of stock standards and working solutions Metformin (analyte) and phenytoin (internal standard) were prepared as stock solutions (1.0 mg/mL) in HPLC-grade water. After that, they were diluted with mobile phase and blank human plasma, respectively, to yield working solutions containing 25 µ/mL and 150 µ/mL. Six calibration standards in the range of 0.05 – 5.0 µ/mL were prepared in human plasma and vortexed for 1 min, 0.5 aliquots were transferred into a glass tube and stored at -200 C before use. Precision Real and reference samples were used to assess the method's precision. Metformin and phenytoin were determined at standard concentration levels of 0.05, 2.50, and 5.00 µgmL-1, respectively, for intraday and interday fluctuations. Six consecutive iterations of the same process were carried out on the same day to ensure intraday precision and method repeatability. The same process was carried out under identical experimental settings on different days to verify the method's intermediate (interday) precision. Accuracy and recovery The accuracy of this method was checked by standard addition method, where 80, 100 and 120 % of a pre-analyzed 18 μg/mL solution of metformin containing internal standard (IS) and serum was added to the same (18 μg/mL solution) to obtain 32.4, 36 and 39.6 μg/mL solutions of metformin. The mixtures were centrifuged as described under preparation of the calibration curve before finally injecting into the HPLC machine. After obtaining the chromatograms, the metformin content was determined by Subtracting the peak area ratio of metformin/phenytoin (IS) of the pre-analyzed unspiked solution (16 μg/mL) from that found in each of the spiked solutions (32.4, 36 and 39.6 μg/mL) and interpolating the final concentrations from the calibration curve. Accuracy was expressed as percentage relative error (% Er) and percentage recovery. Limit of detection and limit of quantification (LOD and LOQ) M. A Garba et al., ISSN: 2814-1423 73 The limit of detection (LOD) was determined by studying the calibration curve using samples containing the drug in the range of LOD. The standard deviation of y-intercepts of the regression lines was used as the standard deviation. LOD is expressed as: 𝐿𝑂𝐷 = 3.3𝑄 4𝑆 While the limit of quantitation (LOQ) was determined using the expression: 𝐿𝑂𝑄 = 10𝑄 4𝑆 Where Q in each case is the standard deviation of y-intercepts of the regression lines determined through the LINEST function in Microsoft Office Excel 2016, and S is the slope of the calibration curve. Calibration Curve of Metformin Standard Solution The calibration curve of metformin was prepared using a blank plasma sample (3.0 mL) spiked with 1.0 ml of each of the different concentrations (0.05 - 5.0 µ/mL) and the standard metformin and phenytoin in a separate plain collection tube and 1 ml of the thawed plasma was added to each tube and shaken and finally subjected to the extraction procedure earlier developed. The calibration curve was constructed using peak height ratio versus concentrations of metformin. The coefficient of Variation and correlation coefficient R2 (0.998) were calculated with a statistical data package. The findings demonstrated that the detector responded well at the employed concentration. Preparation of Mobile Phase Mobile phase A 1.36 g of sodium acetate was dissolved in 500 ml of HPLC water to form a 20 M solution. 90 ml triethylamine was added, the pH of the solution was adjusted to 7.2 with the addition of 1% acetic acid, and 1.5ml tetrahydrofuran (THF) was added to the mixture. The solution was filtered by vacuum and sonicated for degassing. Mobile Phase B 1.36 g of sodium acetate trihydrate was dissolved in 100 ml of HPLC water and adjusted to PH 7.2 with 1% acetic acid. 200 ml of acetonitrile and methanol each were added. The mixture was filtered through a membrane filter and degassed assed before being used for HPLC analysis Volunteers and Ethical Approval For this study, patients with diabetes mellitus were recruited from the Comprehensive Health Center (primary health care) and Yusuf Dantsoho General Hospital, Tudun-Wada, Kaduna State, Nigeria. The presence of the characteristic hyperglycemia symptoms and a fasting plasma glucose concentration of ≥ 130 mg/dL were used to establish diabetes mellitus. The study received ethical clearance after it was properly presented to and discussed with the Kaduna State Ministry of Health's human ethics committee. The committee's reference number is MOH/ADM/744/VOL.1/1160NHREC/17/0 3/2018, dated 7th MARCH, 2023. Written informed consent was obtained from each volunteer, and it was saved and recorded. Study Design and Blood Collection The selection process was carried out by clinicians using criteria based on the 1989 recommendation of the National Diabetes Data Group. The study involved twelve newly diagnosed diabetic patients, whose ages ranged from 29.0 ± 4.9 years, weights of 60 ± 7 kg, and heights of 162.8 ± 10.6 M. A Garba et al., ISSN: 2814-1423 74 cm. A two-period, one-way, single-dose cross-over study was the adopted protocol. The subjects act as their control. The research was split into two phases, each separated by a week-long washout interval. After an overnight fast, all patients received metformin (1 g) alone in phase one (Marathe et al., 2000, PattanaSripalakit et al., 2006, ADA, 2013). In phase two, individuals also received metformin (1 g) concomitant with cefuroxime (500 mg). Blood samples were drawn at various intervals after the drugs were administered: 0, 0.5, 1.5, 3.0, 4.0, 6.0, 8.0, 12.0, 16.0, and 24.0 hours. They were then kept in an EDTA vacutainer at -4 0C until analysis. Using a Poroshell 120 EC-C18 4.6 mm X 50 mm 2.7 microns column, mobile phase acetonitrile (A) / Methanol (B) (10:90), and a UV detector at 236 nm, 2 μL of the deproteinized supernatant liquid was injected into the RP-HPLC to measure the concentration of metformin hydrochloride. Sample preparation and extraction Volunteer samples in screw-capped glass were allowed to acclimate to room temperature, along with aliquots of 0.5 mL of the calibration curve. After adding 50 µl of 1M HCl, 2 mL of acetonitrile, and 100µL of the IS working solution to each tube, was vortexed for 15 seconds. The samples were cleaned after the centrifugation process by vortexing the supernatant for 15 seconds in a cleaned tube filled with 2 mL dichloromethane. The combination was centrifuged at room temperature for five minutes at 4000 rpm. The supernatant was then introduced into the HPLC apparatus to get the metformin and phenytoin (I.S.) chromatogram. Pharmacokinetic Parameters From the best-fit slope of the terminal loglinear decrease in plasma concentrations versus time profile, the elimination rate constant (Ke) was calculated using linear regression. The half-life (t1/2) was calculated as 0.693/Ke. Using linear trapezoidal integration, the area under the plasma concentration curve to the last measurable concentration (Ct) at time t (AUC0-t) was determined. The formula for the AUC extrapolated to infinity (AUC0-∞) AUC0-t + Ct/Ke was used. Other Pharmacokinetic parameters i.e. maximum plasma concentration (Cmax), Time to reach maximum plasma concentration (Tmax), Volume of distribution (VD), Total body clearance (Cl), Area under the curve (AUC0-t) from zero hours to last concentration, (AUC0-∞) from zero hours to infinity and Area under the Moment curve were obtained with the aid of the Software – Pharm PK software (Joel et al., 2012, Melmed et al., 2012, Sambo et al., 2019). Statistical analysis Data were expressed as mean ± SEM. Graph Pad Prism Version 7.02 software Windows (San Diego California, USA) was used for data analysis using Wilcoxon (matched-pairs) signed rank test with p<0.05. Results Table 1 represents the Optimized chromatographic conditions of the method while Table 2 represents the validation parameters of the developed method. Chromatograms obtained are presented in Figures 3, 4, and 5 with the retention time of 2.230 and 4.407 respectively. The calibration curve for the RP-HPLC method is shown in Figure 6. Table 3 displays the findings of the RP-HPLC method's validation parameters as well as the comparison of the pharmacokinetics of metformin when given separately and concomitant with clindamycin in healthy volunteers. M. A Garba et al., ISSN: 2814-1423 75 Table 1: Optimized Chromatographic Conditions Table 2: Validation parameters of the developed method Parameters Values Limit of detection (LOD) 0.02µ/ml Limit of quantification 0.05 Accuracy(percentage recovery) 96.52% Accuracy(% ER ) 2.24 % Precision ( % CV) 3.43 % Specificity (percentage recovery) 96.52 % Robustness (percentage deviation) 6.21 % Mobile phase : A B Ratio : 10 90 Column Type Poroshell 120 ECC18. Colum Dimension (4.6mm x 50 mm 2.7 Microns) Wavelength : 236 Temperature : Ambient Flow rate : 0.8 ml/min Run time : 10 MIN Injection volume : 2 µl pH : 7.0 Chromatogram : Methanol Phenytoin Retention time (min) : 2.230 4.407 Signal Rate 0.1 min M. A Garba et al., ISSN: 2814-1423 76 Figure 3: HPLC chromatogram of phenytoin alone Figure 4: HPLC chromatogram of metformin alone Figure 5: HPLC chromatogram of metformin and Phenytoin spiked with human plasma M. A Garba et al., ISSN: 2814-1423 77 Figure 6: Calibration curve of developed RP-HPLC method for the quantitative analysis of metformin in plasma. Table 3: Comparison of pharmacokinetics of metformin (mean, n = 6) alone and when coadministered with cefuroxime in healthy volunteers (Mean ± S.D, N=6) Metformin alone Metformin + cefuroxime Ke(h-1) 0.30 ±0.01 0.15±0.12 Cmax (ng/ml) 1,880.25 ±0.45 1,282.35±0.4 Tmax(min) 3.0 ±0.19 1.5±0.17 AUC0-8 (h ngmLh-1) 6,770±0.52 4,377±0.80 Vd (ml) 1,470.59±0.27 3,45.27±0.02 CL (mlh-1) 4,425.76±0.24 5,252.75 ±0.26 t 1/2β(h) 0.301±0.13 0.152±0.14 t1/2α(h) 2.30 ±0.52 4.54±0.32 *Significant difference (p<0.05) Discussion Metformin and cefuroxime tablets were subjected to quality control, and the outcome was within an acceptable range. Figure 1 and 2 represents the molecular structure of metformin and cefuroxime. Many Columns were used for trial and error for this method to get the best resolutions, Zorbax X DB-C18, (4.6x150 nm,3.5nm), ZOBRAX SB-Aq. C18 (4.6 x150 nm id 5nm), Zorbax X SB-C18, 6x150 nm,3.5nm), was found to elute metformin at a longer time and did not adequately resolve metformin from the internal standard. However, Poroshell 120 EC-C18 4.6 mm X 50 MM 2.7 Microns was found to elute the analyte in less than two minutes and resolved metformin from the internal standard under the optimized chromatographic conditions Table 1, consisting of a mobile phase A (Sodium acetate, triethylamine, 1% acetic acid, and tetrahydrofuran) and B (Sodium acetate y = 1.288x + 0.6412 R² = 0.9948 0 10 20 30 40 50 60 70 80 90 010 20 30 40 50 60 70 Peak area ratio Concentraion(µg mL-1) M. A Garba et al., ISSN: 2814-1423 78 trihydrate, acetic acid, acetonitrile, and methanol) (90:10, v: v), the mobile phase was adjusted to (pH 7.2) at a flow rate of 0.8 ml/min and ambient temperature. Multiple drug prescription is often recommended in patients suffering from diabetes with complications (AL-Mohamadi and Ibrahim 2015). Despite this growing phenomenon, the influence of diabetes on drug metabolism in the administration of several drugs has not been fully investigated (AL-Mohamadi and Ibrahim 2015). This study evaluated the effect of 500 mg cefuroxime co-administered with 1 g metformin in healthy volunteers. The changes in pharmacokinetic parameters were not statistically significant when metformin was administered alone and with cefuroxime (Eileen et al., 2009), also reported insignificant changes in the pharmacokinetics of metformin when coadministered with amoxicillin. The changes in pharmacokinetic parameters were not statistically significant (Garba et al., 2018). Although cefuroxime does not alter most of metformin's pharmacokinetic characteristics, certain alterations were noted. The Cmax dropped from 1,880.25 ±0.45 to 1,282.35±0.4 and AUC0-8 from 6,770±0.52 to 4,377±0.80. Although a clear theoretical mechanism is not obvious, and metformin and cefuroxime interact with different transporters, thiazides could affect transporter activity through altered ionic balance (Bello et al., 2017). When metformin and cefuroxime were given together, the mean postprandial glucose level changes were insignificant. This may be a result of non-interaction when metformin was co-administered with cefuroxime. The developed and validated method reverse-phase high-performance chromatography (RP-HPLC) method in the interaction studies of cefuroxime and metformin in human plasma was very effective and efficient. The results of the findings indicated pharmacokinetic changes when metformin was administered alone and concomitantly with cefuroxime, though not statistically significant (p>0.05). It is, therefore, recommended that metformin be co-administered with cefuroxime to type II diabetic patients without the risk of side effects. Acknowledgment The Authors thank the management of Kaduna State University for providing a conducive atmosphere for the research and appreciate the contributions of the Health Research Ethical Committee of the Department of Health and Planning Ministry of Health. Conflict of Interest No conflict of interest is associated with this work References ADA (American Diabetes Association) (2013). Nutritional Recommendations and Principles for an Individual with Diabetes Mellitus. Diabetes care .10:126 – 132. AL-mohamadi AA, Ibrahim DA (2015). Possible Study of Drug-Drug Interactions between Lisinopril and Gliclazide in Experimental Animals. Journal of Drug Discovery and Therapeutics 3(33):04-12 Retrieved from http://jddt.in/index.php/jddt/article/vie w/344