Development and Validation of Liquid Chromatography Method for Determination of Glimepiride in Presence of (Vimto®) Soft Drinks in Rats: Application to Pharmacokinetics Studies.

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 (C max) was 126.01 ng/mL and was reached within 1 h (T max) of administration. Mean area under curve (AUC) was 964.70 ng/mL and was reached within 24 h of administration. The Vimto soft drink significantly (P < 0.050) reduced glimepiride peak serum concentration to 57.87 ng/mL and was reached within 2 h of administration. AUC was significantly reduced to 335.04 ng*h/mL (P < 0.050).
CONCLUSION: Glimepiride pharmacokinetic parameters such as C max 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.

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.[1] 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.[2] 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.[3]

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[4] 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.[5]

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].[6]

Figure 1

Glimepiride chemical structure

Nutritional status and food components may alter the pharmacokinetics or pharmacodynamics of a drug.[7] The interaction between glimepiride and food components may alter its absorption,[8] 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.[910]

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.[11] Also, after oral administration, glimepiride is completely (100%) absorbed from the gastrointestinal tract.[12] A significant absorption of glimepiride within 1 h after administration and peak drug levels (Cmax) at 2–3 h was reported.[13] 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.[14] 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.[15]

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.[16] 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,[17] ultraviolet spectrophotometric,[18] reversed-phase high-performance liquid chromatography (RP-HPLC),[19] liquid chromatography–mass spectrometry (LC-MS/MS),[1220] liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS),[21] and recently high-performance liquid chromatography–mass spectrometry (HPLC-MS).[14]

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.

Instrumentation

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 × 2.1 mm), 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 (0.5 mM ammonium chloride and 0.04% formic acid) and 75% of methanol. ACE C8 column particle size of 5 µm and dimensions of 50 × 2.1 mm was used. Detailed information about mass spectrometric conditions are shown in Table 1.

Table 1

Summary of chromatographic conditions and mass spectrometric conditions

HPLC conditions	Pump flow rate 0.5 mL/min	Autosampler injection volume 2 μL		Auto sampler temp. 12°C		Column oven temp. 20°C
Chromatography	Total run time	1.0 min
	Mobile phase (isocratic elution)	Step	Total time (min)	Flow rate (μL/ min)	A	B
					(%) 0.5 mM ammonium chloride and 0.04% formic acid	(%) Methanol
		0	0.00	500	25	75
		1	1.00	500	25	75
	Column type			ACE C8, (50 × 2.1) mm, 5 μm
	Expected retention times (minutes)		Glimepiride		Clarithromycin
		0.65		0.41
MRM detection conditions	Analytes and IS	Q1 mass	Q3 mass	DP	EP	CE	CXP
	Glimepiride	491.198	352.200	76	10	19	10
	Clarithromycin	748.501	158.300	51	10	39	10
MS conditions	CUR	CAD	IS voltage		TEM	GC1	GC2
Positive	20	10	5500	400		45	40

CAD = Collision associated dissociation gas, CE = Collision energy, CUR = Curtain gas, CXP = Collision cell exit potential, DP = Declustering potential, EP = Entrance potential, GC1 = Nebulizing gas, GC2 = Drying gas, MRM = Multiple Ion Monitoring, MS = Mas spectrometer, TEM = Temperatur, IV = Ionspray voltage

Preparation of stock and working solutions of clarithromycin (internal standard)

A stock solution of clarithromycin (100 µg/mL) was prepared by dissolving 10 mg in 100 mL methanol. Working solutions of internal standard (IS) (500 ng/mL) was prepared by diluting 1.0 mL of the stock solutions in 200 mL methanol.

Preparation of stock and working solutions of glimepiride

Stock solution of glimepiride (25 µg/mL) was prepared by dissolving 12.5 mg in 500 mL 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

	Serial solutions				Serum spiking
	Solution no.	Volume from stock (μL)	Total volume (μL)	Working solutions (μg/mL)	Cal ID	Volume from working solution (μL)	Total volume (μL)	Final concentration (ng/mL)
Calibration points	1	8	1000	0.2	C1	25	1000	5
	2	16	1000	0.4	C2	25	1000	10
	3	80	1000	2.0	C3	25	1000	50
	4	160	1000	4.0	C4	25	1000	100
	5	320	1000	8.0	C5	25	1000	200
	6	640	1000	16.0	C6	25	1000	400
	7	960	1000	24.0	C7	25	1000	600
QC points	8	24	1000	0.6	QCL	25	1000	15
	9	480	1000	12.0	QCM	25	1000	300
	10	800	1000	20.0	QCH	25	1000	500

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 600 ng/mL) and three quality control (QC) concentrations (15, 300, and 500 ng/mL) were prepared in serum. Samples were divided in Eppendorf tubes and stored at –30°C and were used daily during analysis.

Method validation

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.

Linearity

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.

Stability

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%.

Recovery

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.

Preclinical study

Preparation of glimepiride solution and Vimto juice

A total of 15 mg of glimepiride was dissolved in 1000 mL of 20 mM NaOH solution (0.015 mg/mL) (the added NaOH is required to increase the solubility of glimepiride). Vimto juice was prepared by mixing 50 mL of Vimto juice with 150 mL 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 200 g. Rats were placed in air-conditioned environment (20–25°C) and exposed to a photoperiod cycle (12 h light/12 h dark) daily. Rats were fasted for 24 h before experiment day.

Rats were labeled and grouped as control (n = 8) (glimepiride only) and test (n = 8) (glimepiride and Vimto) groups. Glimepiride 0.1 mg/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 0.1 mg/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 24.0 h of drug administration and were immediately centrifuged at 5000 rpm for 10 min; 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 500 ng/mL clarithromycin by vortexing for 15 s, then 600 μL of methanol was added and vortexed vigorously for 1.0 min, followed by centrifugation at 14000 rpm for 7 min. A total of 250 μL of the supernatant was transferred to flat bottom insert and then 2 μL was injected.

Pharmacokinetic analysis

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 24 h (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.

Statistical analysis

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 1 h are shown in Figures 2–4.

Figure 2

Glimepiride blank chromatogram

Figure 4

Glimepiride rat unknown chromatogram at 1 h

Glimepiride zero chromatogram

Precision

Intraday

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.

Table 3

Results of inter- and intraday precision and accuracy

	LLOQ	QCL	QCM	QCH
Day 1
Target conc.	5 ng/mL	15 ng/mL	300 ng/mL	500 ng/mL
Calculated conc. ± SD	4.73 ± 0.10	15.04 ± 0.52	303.10 ± 15.31	507.66 ± 25.53
Accuracy ± SD	94.69 ± 2.08	100.30 ± 3.47	101.00 ± 5.10	101.53 ± 5.11
CV%	2.19	3.461	5.05	5.03
Range	4.95–4.87	14.51–15.86	287.34–324.32	471.51–524.35
Day 2
Target conc.	5 ng/mL	15 ng/mL	300 ng/mL	500 ng/mL
Calculated conc. ± SD	5.07 ± 0.28	15.58 ± 0.50	296.22 ± 11.65	513.68 ± 17.20
Accuracy ± SD	101.32 ± 5.52	103.87 ± 3.36	98.74 ± 3.88	102.74 ± 3.44
CV%	5.45	3.23	3.93	3.35
Range	4.58–5.31	14.70–16.03	280.99–308.77	492.27–533.42
Day 3
Target conc.	5 ng/mL	15 ng/mL	300 ng/mL	500 ng/mL
Calculated conc. ± SD	5.09 ± 0.12	15.39 ± 0.60	308.06 ± 5.16	501.95 ± 16.31
Accuracy ± SD	101.79 ± 3.87	102.62 ± 3.98	102.69 ± 1.72	100.39 ± 3.26
CV%	3.81	3.88	1.67	3.25
Range	4.79–5.32	14.76–16.23	299.94–312.37	482.93–521.89
Average for the three days
Target conc.	5 ng/mL	15 ng/mL	300 ng/mL	500 ng/mL
Calculated conc. ± SD	4.96 ± 0.25	15.34 ± 0.59	302.46 ± 11.91	507.76 ± 19.52
Accuracy ± SD	99.27 ± 3.97	102.60 ± 1.285	102.60 ± 4.86	101.55 ± 1.18
CV%	3.82	3.64	3.93	3.84

Inter-day

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.

Accuracy

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

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)

Stability

Autosampler stability

Autosampler stability was examined using 15 ng/mL (QCL) and 500 ng/mL (QCH) samples. Samples were injected directly at 0.00 h and reinjected after 24.0 h. Results were less than 15% [Table 4].

Table 4

Glimepiride quality control samples stability at 10°C (Autosampler stability)

Time	AUC (drug)	AUC (IS)	Ratio	Measured concentration (ng/mL)	Mean measured	Accuracy %	Stability
QCL (15 ng/mL)
00.00 h	16,267	390,147	0.042	15.144	15.22	100.96	102.06
	15,473	376,586	0.041	14.924		99.49
	16,626	386,703	0.043	15.615		104.10
24.00 h	16,519	411,224	0.040	14.592	14.20	97.28	97.97
	16,588	398,425	0.042	15.122		100.81
	15,893	383,748	0.041	15.043		100.29
QCH (500 ng/mL)
00.00 h	16,267	390,147	0.042	15.144	15.22	100.96	102.06
	15,473	376,586	0.041	14.924		99.49
	16,626	386,703	0.043	15.615		104.10
24.00 h	16,519	411,224	0.040	14.592	14.20	97.28	97.97
	16,588	398,425	0.042	15.122		100.81
	15,893	383,748	0.041	15.043		100.29

QCL = 15 ng/mL, QCH = 500 ng/mL

Short-term stability

QCL and QCH samples were spiked and kept at room temperature for 24 h and then processed with freshly prepared QC samples [Table 5].

Table 5

Glimepiride quality control samples stability at 10°C (Autosampler stability)

Time	AUC (drug)	AUC (IS)	Ratio	Measured concentration (ng/mL)	Mean measured	Accuracy %	Stability
QCL (15 ng/mL)
00.00 h	16,267	390,147	0.042	15.144	15.22767	100.96	101.59
	15,473	376,586	0.041	14.924		99.49
	16,626	386,703	0.043	15.615		104.10
24.00 h	30,816	414,042	0.074	15.325	14.98867	102.16	98.43
	27,822	386,797	0.072	14.776		98.50
	29,194	403,593	0.072	14.865		99.10
QCH (500 ng/mL)
00.00 h	574,571	403,533	1.424	515.925	513.5013	103.19	102.66
	581,429	410,402	1.417	513.345		102.67
	577,878	409,580	1.411	511.234		102.25
24.00 h	948,204	416,593	2.276	499.207	500.1593	99.84	97.40
	936,898	415,144	2.257	494.968		98.99
	974,939	422,348	2.308	506.303		101.26

QCL = 15 ng/mL, QCH = 500 ng/mL

Freeze and thaw stability

Three cycles of freezing and thawing of the concentrations, 15 ng/mL (QCL) and 500 ng/mL (QCH), were used. The mean stability was 97.72%–102.32%, [Table 6].

Table 6

Glimepiride quality control samples freeze and thaw stability

Time	AUC (drug)	AUC (IS)	Ratio	Measured concentration (ng/mL)	Mean measured	Accuracy %	Stability
QCL (15 ng/mL)
00.00 h	16,267	390,147	0.042	15.144	15.22767	100.96	101.95
	15,473	376,586	0.041	14.924		99.49
	16,626	386,703	0.043	15.615		104.10
Cycle #3	31,286	405,612	0.077	14.946	14.93633	99.64	98.08
	32,532	427,029	0.076	14.760		98.40
	32,340	414,973	0.078	15.103		100.69
QCH (500 ng/mL)
00.00 h	574571	403533	1.424	515.925	513.5013	103.19	102.32
	581,429	410,402	1.417	513.345		102.67
	577,878	409,580	1.411	511.234		102.25
Cycle #3	1,071,343	420,382	2.548	499.528	501.8187	99.91	97.72
	1,092,881	427,368	2.557	501.241		100.25
	110,1627	427,848	2.575	504.687		100.94

QCL = 15 ng/mL, QCH = 500 ng/mL

Absolute recovery

Serum samples containing concentrations of QCL (15 ng/mL), QCM (300 ng/mL), and QCH (500 ng/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

Concentration	AUC (drug)	AUC (IS)	Mean (drug)	CV% (drug)	Mean (IS)	CV% (IS)
Mobile phase
15 ng/mL QCL	12,395	420,368	12,526	1.70	420,263	0.42
	12,771	421,975
	12,412	418,446
300 ng/mL QCM	263,349	422,043	256,675	2.26	424,373	0.83
	252,856	422,656
	253,819	428,419
500 ng/mL QCH	415,490	423,249	417,262	0.37	425,134	0.51
	418,067	427,520
	418,229	424,632
Serum
15 ng/mL QCL	12,152	412,926	12,281	1.70	407,857	1.08
	12,521	405,152
	12,169	405,493
300 ng/mL QCM	257,185	415,511	250,975	2.14	416,127	1.00
	247,898	420,564
	247,842	412,305
500 ng/mL QCH	417,223	406,114	411,479	1.25	414,423	1.76
	407,343	419,700
	409,870	417,454

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].

Table 8

Absolute recovery of glimepiride and internal standard

Concentration	Glimepiride			Internal standard
	Mean serum	Mean mobile phase	Absolute recovery %	Mean serum	Mean mobile phase	Absolute recovery %
15 ng/mL (QCL)	12,281	12,526	98.04	407,857	420,263	97.05
300 ng/mL (QCM)	250,975	256,675	97.78	416,127	424,373	98.06
500 ng/mL (QCH)	411,479	417,262	98.61	414,423	425,134	97.48

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 126.01 ng/mL after an hour of administration and the minimum concentration was 7.63 ng/mL after 24 h. In addition, glimepiride after drinking Vimto reached its maximum concentration of 57.87 ng/mL after 2 h and decreased gradually to a minimum concentration of 5.63 ng/mL after 24 h [Table 9].

Figure 6

Glimepiride concentration–time profile. Data shown as mean ± standard deviation (SD) (n = 8)

Table 9

Serum glimepiride drug concentration at selected time after oral dose of glimepiride and glimepiride in the presence of Vimto

Time (hour)	Concentration ± STD (ng/mL) Glimepiride	Concentration ± STD (ng/mL) Glimepiride + Vimto	P values
0.0	0.00	0.00
0.5	120.05 ± 73.20	52.41 ± 33.55	0.032
1.0	126.01 ± 64.17	57.05 ± 28.89	0.015
2.0	119.50 ± 51.71	57.87 ± 22.59	0.008
3.0	102.69 ± 47.69	54.32 ± 21.55	0.020
4.0	70.68 ± 36.85	45.00 ± 12.76	0.084
6.0	41.66 ± 10.93	35.40 ± 6.71	0.189
8.0	42.24 ± 13.36	32.86 ± 6.17	0.093
10.0	32.59 ± 7.67	30.16 ± 5.98	0.491
24.0	7.63 ± 3.15	5.63 ± 0.84	0.150

STD = standard deviation

Also, pharmacokinetic parameters for both groups were compared as in Table 10. Both Cmax and AUC (126.01 ng/mL and 964.70 ng*h/mL) were significantly (P < 0.05) reduced (57.87 ng/mL and 665.91 ng*h/mL).

Table 10

Comparison in major pharmacokinetic parameters between glimepiride alone and glimepiride with Vimto

Parameter	Glimepiride	Glimepiride + Vimto	P value
AUC0–24	964.70	665.91	0.039
Tmax	1.00	2.00	0.109
Cmax	126.01	57.87	0.016
Kel	0.106	0.114	0.350
Half-life	6.55	6.09	0.284

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.[7] 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.[1422]

Glimepiride is metabolized by CYP450 and it is a substrate for p-GP OATP2B1 transporter in human.[23] 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[11] 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.[14] 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 1 h, Cmax for glimepiride alone is 126.01 ng/mL, Cmax for glimepiride with Vimto is 57.87 ng/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.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.