|Year : 2019 | Volume
| Issue : 1 | Page : 49-59
Development and validation of liquid chromatography method for determination of glimepiride in presence of (Vimto®) soft drinks in rats: application to pharmacokinetics studies
Mohammed Hamad1, Areej Rahhal2, Wael Abu Dayyih2, Eyad Mallah2, Alice Abu Dayyih3, Zainab Zakaria2, Tawfiq Arafat2
1 Department of Basic Sciences, College of Science and Health Professions, King Saud bin Abdulaziz University for Health Sciences, Jeddah, Kingdom of Saudi Arabia
2 Department of Pharmaceutical Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan
3 School of Chemistry, Biology and Pharmacy, Bio- and Pharmaceutical Analysis Department Hochschule Fresenius University of Applied Sciences, Idstein, Germany
|Date of Web Publication||12-Feb-2019|
Dr. Wael Abu Dayyih
Department of Pharmaceutical Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy and Medical Sciences University of Petra, Queen Alia International Road, P.O. Box 961343, Amman
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: Diet and beverages are thought to have notable effects on drugs. Recently, this relationship has received significant consideration. Aims: To develop and validate a simple, rapid, and sensitive method for the determination of glimepiride in rat serum. This will be performed using high-performance liquid chromatography–mass spectrometry (HPLC-MS/MS). Potential pharmacokinetic interactions between glimepiride and the soft drink, Vimto, will also be investigated in the serum of experimental rats. Materials and Methods: HPLC-MS/MS was constructed and clarithromycin was used as an internal standard. Results: The method was validated in terms of linearity, precision, accuracy, stability, and system suitability parameters. The method was found to be satisfactory and suitable for the determination of glimepiride. The precision of glimepiride was high (coefficient of variation, CV% <15%), the accuracy over all 3 days of validation was within the accepted criteria. Glimepiride peak serum concentration (Cmax) was 126.01ng/mL and was reached within 1h (Tmax) of administration. Mean area under curve (AUC) was 964.70ng/mL and was reached within 24h of administration. The Vimto soft drink significantly (P < 0.050) reduced glimepiride peak serum concentration to 57.87ng/mL and was reached within 2h of administration. AUC was significantly reduced to 335.04 ng*h/mL (P < 0.050). Conclusion: Glimepiride pharmacokinetic parameters such as Cmax and AUC were significantly affected by the Vimto soft drink. Therefore, this study developed a simple, rapid, and sensitive method for validation and determination of the effects of soft drinks on drugs using the LC-MS/MS method.
Keywords: Glimepiride, liquid chromatography–mass spectrometry, pharmacokinetics, Vimto
|How to cite this article:|
Hamad M, Rahhal A, Dayyih WA, Mallah E, Dayyih AA, Zakaria Z, Arafat T. Development and validation of liquid chromatography method for determination of glimepiride in presence of (Vimto®) soft drinks in rats: application to pharmacokinetics studies. J Pharm Bioall Sci 2019;11:49-59
|How to cite this URL:|
Hamad M, Rahhal A, Dayyih WA, Mallah E, Dayyih AA, Zakaria Z, Arafat T. Development and validation of liquid chromatography method for determination of glimepiride in presence of (Vimto®) soft drinks in rats: application to pharmacokinetics studies. J Pharm Bioall Sci [serial online] 2019 [cited 2019 May 25];11:49-59. Available from: http://www.jpbsonline.org/text.asp?2019/11/1/49/252089
| Introduction|| |
Diabetes mellitus (DM) is a chronic disease that necessitates continuing therapies and regular self-care. It is considered as universally prevalent and millions worldwide are living with diabetes. DM is a heterogeneous group of metabolic disorders characterized by hyperglycemia and abnormalities in carbohydrate, fat, and protein metabolism, and may result in chronic complications including microvascular, macrovascular, and neuropathic disorders. Different approaches for DM management are applied to reduce the risk for microvascular and macrovascular disease complications, to ameliorate symptoms, to reduce mortality, and to improve quality of life.
Glimepiride is an oral antidiabetic drug, which belongs to the sulfonylurea group and is usually used for patients with type 2 DM. Glimepiride acts to lower blood glucose by stimulating the release of insulin from pancreatic β cells and also by increasing the sensitivity of peripheral tissues to insulin, but the mechanism by which glimepiride lowers blood glucose during long-term administration has not been clearly established.
Glimepiride is chemically designed as: 3-ethyl-4-methyl-N-(4[N-((1r,4r)-4-methylcyclohexylcarbamoyl)sulfamoyl]phenethyl)-2-oxo-2,5-dihydro1H-pyrrole-1-carboxamide, with a molecular formula of C24H34N4O5S and a molecular weight of 490.6 [Figure 1].
Nutritional status and food components may alter the pharmacokinetics or pharmacodynamics of a drug. The interaction between glimepiride and food components may alter its absorption, leading to either decreasing or increasing its plasma levels, causing failure of treatment or increasing the risk of side effects and toxicity. Glimepiride is processed in liver by cytochrome p450 (CYP450) enzyme and any drug that increase or decrease the activity of such enzyme may alter the action of glimepiride.,
When glimepiride was given with meals, the mean Tmax was slightly increased (12%) and the mean Cmax and area under curve (AUC) were slightly decreased by 8% and 9%, respectively. Also, after oral administration, glimepiride is completely (100%) absorbed from the gastrointestinal tract. A significant absorption of glimepiride within 1h after administration and peak drug levels (Cmax) at 2–3h was reported. In addition, recently, it was found that licorice juice significantly (P < 0.05) increased the AUC0–6, Cmax, and t1/2 of glimepiride and significantly (P < 0.05) decreased the clearance and elimination rate constant, whereas grapefruit juice found to have no significant (P > 0.05) effect on glimepiride pharmacokinetics. Moreover, the risk of hypoglycemia may be increased or prolonged when moderate or large amounts of alcohol have been consumed concurrently with sulfonylurea antidiabetic agents.
Vimto is a soft drink that is a local favorite drink, especially during Ramadan in Arab world. It was first manufactured and registered as a health tonic in cordial form in 1908 in Manchester, England, and then decades later as a carbonated drink. It contains the juice of grapes, raspberries, and black currants (in a 3% concentration), flavored with herbs and spices and sugar. It is considered as one of the most popular drinks during the holy month of Ramadan in some Arab countries. Although, the Vimto drink is consumed in a limited population, we used it as an example of high-sugar content drinks that were consumed by a wide range of populations including diabetic without knowing the effect of these drinks on their medications.
Several methods were applied for the determination of glimepiride separately or in combination with other drugs in pharmaceutical formulations and biological samples, such as spectrophotometry, ultraviolet spectrophotometric, reversed-phase high-performance liquid chromatography (RP-HPLC), liquid chromatography–mass spectrometry (LC-MS/MS),, liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS), and recently high-performance liquid chromatography–mass spectrometry (HPLC-MS).
This study aimed to develop and validate a simple, rapid, and sensitive method for the determination of glimepiride in rat serum using HPLC-MS/MS. Also, to study the possible pharmacokinetic interactions between glimepiride and Vimto soft drink when they are taken together in experimental rats without studying the exact molecular mechanism resulting from the constituents that make such effect. Also, we aimed to make patients aware about their drug effects when combined with some diets or beverages.
| Materials and Methods|| |
Chemicals and reagents
Both glimepiride (B#WS/07/272) and clarithromycin (B#0040310095) were obtained from the Jordanian Pharmaceutical Manufacturing, Jordan. Rats for this study were obtained from animal house in the University of Petra. Deionized water (Nanopure, Fisher Scientific), methanol and acetonitrile of advanced gradient grade (Fisher Scientific, USA), and formic acid and sodium hydroxide of advanced gradient grade (GPR Supplies Ltd, United Kingdom) were used in this study.
An API mass spectrometer (HITACHY, Japan) composed of degasser (Agilent 1260), solvent delivery systems pump (Agilent 1200), autosampler (Agilent 1200), Thermostat column compartment (Waters, USA) (Agilent 1200), and API 4000 mass spectrometer, ACE 5, C18 (50×2.1mm), 5 μm; computer system, Windows XP, SP3, data management software 1.5.1; sonicator, crest model-175 (Ultrasonic), Sartorius balance BP 2215, Sartorius PH meter (professional meter PP-25) (Goettingen, Germany), and centrifuge (Eppendorf 5417C) (Hamborg, Germany) were used.
Chromatographic conditions for glimepiride
A mobile phase consisting of 25% of water (0.5mM ammonium chloride and 0.04% formic acid) and 75% of methanol. ACE C8 column particle size of 5 µm and dimensions of 50×2.1mm was used. Detailed information about mass spectrometric conditions are shown in [Table 1].
|Table 1: Summary of chromatographic conditions and mass spectrometric conditions|
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Preparation of stock and working solutions of clarithromycin (internal standard)
A stock solution of clarithromycin (100 µg/mL) was prepared by dissolving 10mg in 100mL methanol. Working solutions of internal standard (IS) (500ng/mL) was prepared by diluting 1.0mL of the stock solutions in 200mL methanol.
Preparation of stock and working solutions of glimepiride
Stock solution of glimepiride (25 µg/mL) was prepared by dissolving 12.5mg in 500mL methanol and stored at –20°C until used. Working solutions (0.2, 0.4, 2.0, 4.0, 8.0, 16.0, 24.0, 0.6, 12.0, and 20.0 μg/mL) were prepared as in [Table 2].
|Table 2: Preparation of glimepiride serial solutions and serum spiking samples|
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Preparation of calibration curve and quality control samples in serum
As in [Table 2], seven spiked concentrations (calibration curve) (5, 10, 50, 100, 200, 400, and 600ng/mL) and three quality control (QC) concentrations (15, 300, and 500ng/mL) were prepared in serum. Samples were divided in Eppendorf tubes and stored at –30°C and were used daily during analysis.
Precision and accuracy
The intraday precision and accuracy of the method was determined by the analysis of six replicates of the lower limit of quantification (LLOQ) and QC levels in the same day. The inter-day variability was determined by the analysis of three runs of the LLOQ and QC levels in three different days. The relative standard deviation (RSD) values or coefficient of variation (CV%) were calculated from the ratios of the standard deviation (SD) to the mean and expressed as percentage.
The accuracy of the method was determined by comparing practical amounts recovered from the control samples with actual values present in the samples (theoretical values).
The acceptable limits of intraday and inter-day accuracy and precision were below 15% except at the LLOQ, for which accuracy and precision should be below 20% as per the European medicines agency’s scientific guidelines.
The calibration curve of glimepiride is a plot of the peak area ratio (PAR) of the drug to the IS as a function of the drug concentration (C). This gives the following equation: (PAR = Slope × C + Intercept).
Linearity of the plotted curve is evaluated through the value of the correlation coefficient (R2), which should be more than 0.98.
A short-term stability, freeze–thaw stability, and autosampler stability tests were performed. Stability was carried out using low and high concentrations of QC samples. The analyte was considered stable if the assay values were within the acceptable limit of accuracy ±15%.
The absolute recovery was calculated by comparing the AUCs for serum extracted samples with un-extracted samples (solution), those represent 100% recovery. However, extent of the recovery of the glimepiride and IS should be consistent and reproducible.
Preparation of glimepiride solution and Vimto juice
A total of 15mg of glimepiride was dissolved in 1000mL of 20mM NaOH solution (0.015mg/mL) (the added NaOH is required to increase the solubility of glimepiride). Vimto juice was prepared by mixing 50mL of Vimto juice with 150mL water.
Animal handling and study protocol
The study protocol was approved by the ethical committee of the High Research Council, Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan. Adult Sprague Dawley laboratory rats with an average weight of 200g. Rats were placed in air-conditioned environment (20–25°C) and exposed to a photoperiod cycle (12h light/12h dark) daily. Rats were fasted for 24h before experiment day.
Rats were labeled and grouped as control (n = 8) (glimepiride only) and test (n = 8) (glimepiride and Vimto) groups. Glimepiride 0.1mg/kg was given by oral gavage to the control group. Vimto juice was pre-administered for half an hour to the second group before glimepiride dose of 0.1mg/kg.
Sample collection and processing
Blood samples were collected from the rat’s tail at zero, 0.5, 1, 2, 3, 4, 6, 8, 10, and 24.0h of drug administration and were immediately centrifuged at 5000rpm for 10min; serum obtained was placed into labeled Eppendorf tubes and stored at –30°C till analysis.
Sample extraction of the rat samples, calibrator, and QC samples was carried out by taking 100 µL of blank, zero, standards, QC low (QCL), QC mid (QCM), QC high (QCH) or rat samples in tubes and mixed with 50 µL of IS solution containing 500ng/mL clarithromycin by vortexing for 15s, then 600 μL of methanol was added and vortexed vigorously for 1.0min, followed by centrifugation at 14000rpm for 7min. A total of 250 μL of the supernatant was transferred to flat bottom insert and then 2 μL was injected.
Plasma level–time profiles of glimepiride and glimepiride with Vimto juice were assembled by drawing a curve between average plasma levels (in ng/mL) and time (in hours). The parameters: area under the curve to 24h (AUClast), area under the curve to infinity (AUCinf), maximum concentration of drug in serum (Cmax), time to achieve Cmax (Tmax), half-life (t0.5), and elimination rate constant (Kel) were calculated by non-compartmental analysis model using WinNonlin software, version 5.1. Tripos, L.P, St. Louis, MO, USA.
The statistical significance of difference in the mean of the variables, such as Cmax, Tmax and AUClast, between two groups was assessed using the independent samples Student’s t-test used to identify significance in pharmacokinetic parameters using 95% confidence interval. The Statistical Package for the Social Sciences (SPSS), version 21, (SPSS Inc., Chicago, IL, USA) computer software was used, and P < 0.05 was considered significant.
| Results|| |
A validation process was applied for 3 days to determine glimepiride levels in rat’s plasma as per international council for harmonisation (ICH) of technical requirements for pharmaceuticals for human use and the European medicines agency’s scientific guidelines using LC-MS/MS method. The chromatograms of glimepiride blank, zero, and rat plasma sample after 1h are shown in [Figure 2], [Figure 3], [Figure 4]., ,
The precision or coefficient of variation (CV%) of mean predicted concentrations (LLOQ, QCL, QCM, and QCH) during the 3 days of validation ranged between 1.67% and 5.45%. Detailed results are shown in [Table 3].
Inter-day precision was evaluated over the 3 days. CV% was less than 5.12% (LLOQ). CV% for QCL, QCM, and QCH were 3.64, 3.93, and 3.84%, respectively [Table 3].
The precision (CV %) did not exceed 20% for LLOQ and 15% for the other concentrations, which prove the closeness of the measurements.
The accuracy of mean predicted concentration compared to target concentrations (LLOQ, QCL, QCM, and QCH) are shown in [Table 3], the minimum value was 94.68% and the maximum was 104.87% during validation. Also, the mean accuracy for LLOQ and QCL was 101.79% and 102.62%, respectively.
Inter-day accuracy over the concentration range was between 99.26% and 102.26% as shown in [Table 3]. Compared with the accepted criteria, which is 85%–115% for all concentrations except for LLOQ, which is 80%–120%, the accuracy obtained is within the required range according to ICH guidelines.
Linearity is determined by calculating the regression line using a mathematical treatment of the results (i.e., least mean squares) versus glimepiride concentrations. Equation of linear regression was used for estimating glimepiride levels at each validation day, using target concentration for getting the “D area/IS area” at each of the validation days and for stability testing.
During validation, the coefficient (R2) was greater than 0.99. The linearity for the mean six calibration curves and correlation, slope, R2, and intercept is shown in [Figure 5]. Therefore, validation results of the 3 days are passed within the required criteria in terms of linearity.
|Figure 5: The plot of linearity for mean six calibration curves (correlation = 0.999927, slope = 0.003679, R2 = 0.999854, intercept = 0.005945)|
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Autosampler stability was examined using 15ng/mL (QCL) and 500ng/mL (QCH) samples. Samples were injected directly at 0.00h and reinjected after 24.0h. Results were less than 15% [Table 4].
|Table 4: Glimepiride quality control samples stability at 10°C (Autosampler stability)|
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QCL and QCH samples were spiked and kept at room temperature for 24h and then processed with freshly prepared QC samples [Table 5].
|Table 5: Glimepiride quality control samples stability at 10°C (Autosampler stability)|
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Freeze and thaw stability
Three cycles of freezing and thawing of the concentrations, 15ng/mL (QCL) and 500ng/mL (QCH), were used. The mean stability was 97.72%–102.32%, [Table 6].
Serum samples containing concentrations of QCL (15ng/mL), QCM (300ng/mL), and QCH (500ng/mL) were prepared in triplicate. The coefficient of variation for glimepiride was between (0.37%–2.26%) in mobile phase, whereas in serum was ranged between 1.25% and 2.14% as shown in [Table 7].
|Table 7: Absolute mean peak area and precision for glimepiride and internal standard per quality control level in mobile phase and serum|
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The absolute recovery for glimepiride was ranged from 97.78% to 98.61% and for IS were ranged from 97.05% to 98.06 [Table 8].
Effect of pre-administration of Vimto on glimepiride pharmacokinetics
As shown in [Figure 6], glimepiride concentrations were lower after concomitant administration with Vimto. The maximum serum concentration (Cmax) (without Vimto) was 126.01ng/mL after an hour of administration and the minimum concentration was 7.63ng/mL after 24h. In addition, glimepiride after drinking Vimto reached its maximum concentration of 57.87ng/mL after 2h and decreased gradually to a minimum concentration of 5.63ng/mL after 24h [Table 9].
|Figure 6: Glimepiride concentration–time profile. |
Data shown as mean ± standard deviation (SD) (n = 8)
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|Table 9: Serum glimepiride drug concentration at selected time after oral dose of glimepiride and glimepiride in the presence of Vimto|
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Also, pharmacokinetic parameters for both groups were compared as in [Table 10]. Both Cmax and AUC (126.01ng/mL and 964.70 ng*h/mL) were significantly (P < 0.05) reduced (57.87ng/mL and 665.91 ng*h/mL).
|Table 10: Comparison in major pharmacokinetic parameters between glimepiride alone and glimepiride with Vimto|
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| Discussion|| |
Validated analytical method of glimepiride was developed to evaluate the consistency of measuring the drug level in rat plasma.
Drug–food relations may alter the pharmacokinetics or pharmacodynamics of a drug or nutritional constituent or nutritional status. Nutrients may activate or suppress some enzymes in the gut, which will affect the bioavailability of certain drugs. The interaction between drugs and nutrients may cause elevation of drug concentration in plasma, which increases the risk of side effects and toxicity, or decrease in plasma levels of drug, which may lead to failure of treatment. Also, nutrients and ingredients in beverages may increase or decrease the bioavailability of the drug.,
Glimepiride is metabolized by CYP450 and it is a substrate for p-GP OATP2B1 transporter in human. The lower pharmacokinetics values of glimepiride when administered with Vimto may be due to the induction of the CYP450 enzyme by the components of Vimto. This is in accordance with the results of McCall who revealed lower Cmax and AUC when drug taken after a meal. On contrast, in a previous work of our group, we showed that licorice juice elevated the bioavailability of the glimepiride, whereas grape juice showed no significant effect. The mechanism behind the decrease in glimepiride bioavailability may be due to the induction of metabolic enzymes required for its metabolism. Lower glimepiride concentrations affect its hypoglycemic effect. For that, physicians should advise the patients with diabetes to take care of the food they consume when they take glimepiride.
| Conclusion|| |
A simple, rapid, and sensitive method for validation and determination of glimepiride in the presence of Vimto juice has been developed by using HPLC-MS/MS. Serum glimepiride level was affected by the concomitant administration of Vimto.
Glimepiride reaches its maximum serum level within 1h, Cmax for glimepiride alone is 126.01ng/mL, Cmax for glimepiride with Vimto is 57.87ng/mL, the difference between Cmax (single administration vs. combination with Vimto) is significant, and the difference in AUC is significant as well (P < 0.05). The difference between Cmax (single administration vs. combination with Vimto) is significant and the difference in AUC is significant (P < 0.05).
This study can lead to many possible future studies such as studying the effect of this combination on human serum.
We would like to express our sincere thanks to the Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan, for giving us an opportunity to complete this research successfully.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]