Simultaneous determination of sulpiride and mebeverine by HPLC method using fluorescence detection: application to real human plasma
© Walash et al 2012
Received: 3 October 2011
Accepted: 14 February 2012
Published: 14 February 2012
A new simple, rapid and sensitive reversed-phase liquid chromatographic method was developed and validated for the simultaneous determination of sulpiride (SUL) and mebeverine Hydrochloride (MEB) in the presence of their impurities and degradation products. The separation of these compounds was achieved within 6 min on a 250 mm, 4.6 mm i.d., 5 m particle size Waters®-C18 column using isocractic mobile phase containing a mixture of acetonitrile and 0.01 M dihydrogenphosphate buffer (45:55) at pH = 4.0. The analysis was performed at a flow rate of 1.0 mL/min with fluorescence-detection at excitation 300 nm and emission at 365 nm. The concentration-response relationship was linear over a concentration range of 10- 100 ng/mL for both MEB and SUL with a limit of detection 0.73 ng/mL and 0.85 ng/mL for MEB and SUL respectively. The proposed method was successfully applied for the analysis of both MEB and SUL in bulk with average recoveries of 100.22 ± 0.757% and 99.96 ± 0.625% respectively, and in commercial tablets with average recoveries of 100.04 ± 0.93% and 100.03 ± 0.376% for MEB and SUL respectively. The proposed method was successfully applied to the determination of MEB metabolite (veratic acid) in real plasma simultaneously with SUL. The mean% recoveries (n = 3) for both MEB metabolite (veratic acid) and SUL were 100.36 ± 2.92 and 99.06 ± 2.11 for spiked human plasma respectively. For real human plasma, the mean% recoveries (n = 3) were and respectively.
Mebeverine undergoes rapid and extensive presystemic (first-pass) hydrolysis in the gut and/or the liver into mebeverine alcohol and veratic acid .
British Pharmacopoeia described a non aqueous titrimetric method for determination of MEB in pure form .
Sulpiride (SUL) 5-(Aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxy-benzamide (Figure 1) is the most widely prescribed anti-psychotic drug. It is a selective dopamine D2 antagonist with antidepressant activities it exerts a mood elevating effect and antiemetic action .
Regarding SUL, British Pharmacopoeia described a non aqeous titrimetric method for determination of SUL in pure form .
Combination of MEB and SUL is used for treatment gastrointestinal and colic spasms which are a consequence of psychosomatic manifestation of nervous tention, mental stress or anxiety. MEB and SUL were determined in their binary mixture via derivative spectroscopy [3, 4], first derivative synchronous fluorescence spectroscopy . TLC- densitometry , HPLC [4–6] and chemometric techniques .
The aim of the present study is to establish and develop a novel, sensitive and selective HPLC method for simultaneous determination of MEB and SUL either per se or in pharmaceutical preparations and in real human plasma as SUL and MEB metabolite (veratic acid). However, to the best of our knowledge, no analytical methods have been reported for the simultaneous determination of all compounds in the presence of their impurities and degradation products, which might interfere with the analysis of the active ingredients
HPLC experiments were performed with a Merck Hitachi L- 7100 chromatograph equipped with a Rheodyne injector valve with a 20 μL loop. The detection of analytes was monitored at emmision 365 nm after excitation at 300 nm by L-7485 fluorescense-detector (Darmstadt, Germany). Chromatograms were recorded on a Merck Hitachi D-7500 integrator. Mobile phases were degassed using Merck L 7612 solvent degasser. The separation of these compounds was achieved on Waters®-C18 column (250 mm, 4.6 mm i.d., 5 m particle size) using isocractic mobile phase containing a mixture of acetonitrile and 0.01 M dihydrogenphosphate buffer 45:55 (v/v). The analysis was performed at a flow rate of 1 mLmin-1. The pH of the buffer was adjusted with orthophosphoric acid. Prior to any analysis, the mobile phase was filtered using 0.45 μm filters. The system was equilibrated with the mobile phase before injection.
Reagents and chemicals
Working reference standard of SUL and MEB were kindly supplied by RAMEDA (The Tenth of Ramadan) Co. for Pharmaceutical Industries & Diagnostic Reagents, 6th of October City, Egypt.
Colona® tablets (Batch No. 09425, labelled to contain 25 mg SUL and 100 mg MEB per tablet) are from RAMEDA (The Tenth of Ramadan) Co. for Pharmaceutical Industries & Diagnostic Reagents, 6th of October City, Egypt.
Acetonitrile- HPLC grade (Riedel-de Haen, Germany), Phosphoric acid 85% (Riedel-de Haen, Germany). Analytical grade potassium dihydrogenphosphate and orthophosphoric acid were obtained from Prolabo (Paris, France). HPLC grade methanol (MeOH) was obtained from Prolabo (Paris, France).
SUL impurities, 2-methoxy-5-sulfamoyl benzoic acid methyl ester (sules) (CAS No. 33045-52-2) and 2-aminomethyl-1-ethylpyrrolidine (sulam) (CAS No. 26116-12-1) were imported from Wuhan Yuancheng Technology Development Co. Ltd., China.
Preparation of standard solutions
Preparation of stock standard solutions
Stock standard solutions of MEB, SUL and SUL impurities (200 μg/mL each) were prepared by accurately weighed 10.0 mg of each of MEB and SUL and transferred to 50 ml measuring flasks. The compounds were separately dissolved in 50 ml of methanol. The stock solutions were further diluted with the mobile phase to obtain the working concentration ranges. The standard solutions were kept in the refrigerator and were found to be stable for 10 days.
Preparation and separation of the hydrolytic degradation products of MEB
Stock standard solution of veratic acid (200 μg/mL each) was prepared by accurately weighed 10.0 mg of veratic acid and transferred to 50 ml measuring flask. The compound was dissolved in 50 mL of methanol. The stock solution was further diluted with the mobile phase to obtain the working standard solution.
Construction of the calibration curves
Aliquots of MEB and SUL covering the working concentration range 10-100 ng/mL for each of MEB and SUL were quantitatively transferred into a series of 10 mL volumetric flasks, after being diluted to 10 mL with the mobile phase (pH = 4). 20 μL aliquots were injected (in triplicate) and calibration curve was constructed by plotting the peak area ratio between a drug and the other drug as internal standard against the final concentration of drugs in ng/mL, alternatively, the corresponding regression equations were derived.
Procedure for the synthetic mixture
Aliquot volumes of MEB and SUL standard solutions in the ratio of 4:1 were transferred into a series of 10 mL volumetric flasks. The flasks completed to the volume with the mobile phase. The procedure described under section 2.4 was performed.
Procedure for commercial tablets
10 film coated tablets were bleached with methanol swab and pulverized well. A weighed quantity of the powder equivalent to 100 mg MEB and 25 mg of SUL (in their pharmaceutical ratio of 4:1) was transferred into a small conical flask and extracted with 3 × 30 mL of methanol. The extract was filtered into a 100 mL volumetric flask. The conical flask was washed with few milliliters of methanol. The washings were passed into the same volumetric flask and completed to the volume with the same solvent. Aliquots covering the working concentration range were transferred into 10 mL volumetric flasks. The recommended procedure under "Calibration Curve" was performed. The nominal content of the tablets were determined either from a previously plotted calibration graph or using the corresponding regression equation.
Procedure for spiked human plasma
Suitable aliquots of mixture of MEB metabolite (veratic acid) and SUL stock solutions containing 10.0-50.0 ng/mL and 10.0-100.0 ng/mL respectively were transferred into centrifugation tubes. 1.0 ml of human plasma was added to each tube. Then, alkalinization was achieved by addition of 0.1 mL of NaOH (1.0 M) and the tubes were shaken for 1 min. The samples were mixed well using a vortex mixer and then extracted with 3 × 3 mL of ethyl acetate/dichloromethane (5:1 v/v) by centrifugation for 5 min at 4000 rpm. The organic layer was transferred into an evaporating dish and evaporated till dryness. The residue was reconstituted in 3 mL methanol . The procedure described under calibration curve was followed. The nominal content of the drug was determined from the previously plotted calibration graph or using the corresponding regression equation.
Procedure for real human plasma
Three healthy volunteers (women around 35 years old) were administered Colona® tablets after 8 hours fasting. Further, 5 mL blood samples were drawn at different time intervals at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 and 6.0 hr into tubes containing 0.5 ml of 2% EDTA solution to prevent blood coagulation. The blood was processed to plasma by centrifugation at 4000 rpm and 15 min. the supernatant plasma samples transferred into test tubes and the procedure mentioned above was followed for the monitoring of drug concentration in plasma.
Results and discussion
In an attempt to optimize the separation of MEB and SUL and their impurities and degradation products in bulk and plasma, the effects of some chromatographic parameters such as mobile phase composition and pH of the buffer solution were investigated.
Effect of the mobile phase composition
Type of Organic Modifier
Acetonitrile was replaced by methanol but it did not give good resolved peaks. Acetonitrile was the organic modifier of choice giving symmetrical narrow peaks.
Ratio of Organic Modifier
The effect of changing the ratio of organic modifier on the selectivity and
Effect of experimental parameters on the number of theoretical plates of the studied compounds
No. of theoretical plates (N)
Ratio of organic modifier A/B
Ionic strength of phosphate buffer [M]
Flow rate mL/min
acetonitrile was the best one giving well resolved peaks and highest number of theoretical plates. Ratios less than 45% resulted in peaks with very long unacceptable retention times, whereas ratios higher than 75% resulted in precipitation in the mobile phase.
Effect of pH
The effect of changing the pH of the mobile phase on the selectivity and retention times of the test solutes was investigated using mobile phases of pH ranging from 3.0-5. Table 1 illustrated that a pH of 4.0 was the most appropriate one giving well-resolved peaks and highest number of theoretical plates. pHs higher than 5.0 produced precipitation in the mobile phase.
Ionic Strength of Buffer
The effect of changing the ionic strength of phosphate buffer on the selectivity and retention times of the test solutes was investigated using mobile phases containing concentration of 0.0075- 0.04 M of phosphate buffer. Table 1 shows that 0.01 M phosphate buffer was found to be the most suitable giving best resolution and highest number of theoretical plates.
Effect of Flow Rate
The effect of flow rate on the formation and separation of peaks of the studied compounds was studied and a flow rate of 1 mL/min was optimal for good separation in a reasonable time (Table 1).
Validation of the Method
Concentration Ranges and Calibration Graphs
Performance data of the proposed method
Concentration range (ng/mL)
Limit of detection (LOD) (ng/mL)
Limit of quantification (LOQ) (ng/mL)
Correlation coefficient (r)
standard deviation of the residuals (Sy/x)
standard deviation of the intercept (Sa)
standard deviation of the slope(Sb)
2.23 × 10-5
7.0 × 10-7
Limit of Quantitation and Limit of Detection
The limits of quantification (LOQ) was calculated according to ICH Q2B recommendations . The limits of detection (LOD) was also calculated according to ICH Q2B recommendations . The results of LOD and LOQ of MEB and SUL respectively are abridged in Table 2.
LOQ and LOD were calculated according to the following equations :LOQ = 10 σ/S
LOD = 3.3 σ/S
Application of the proposed method to the determination of the studied drugs in the pure form
Reference method 
The reference method is based on HPLC separation of the two drugs on a reversed phase, Bondapak CN column and UV detection was done at 243 nm using buclizine hydrochloride as internal standard .
Accuracy and Precision
Application of the proposed method for determination of the studied drugs in their synthetic mixtures and in co-formulated tablets
Concentration found (ng/mL)
1-MEB and SUL mixture
2-Colona ® tablets a
(MEB 100 mg +SUL 25 mg/tab.)
Batch # 09425.
Intermediate precision was performed through replicate analysis of MEB and SUL in pure form. The results are shown in Table 4, for a period of 3 successive days.
Application of the method
Validation of the proposed method for determination of MEB and SUL raw materials using the proposed method
Concentration added (ng/mL)
100.85 ± 0.636
100.37 ± 0.490
100.2 ± 0.854
99.75 ± 0.921
99.08 ± 0.589
100.53 ± 0.650
100.23 ± 0.737
101.03 ± 1.023
100.06 ± 0.573
98.96 ± 0.824
99.99 ± 0.961
100.78 ± 0.762
Degradation product of MEB (veratic acid) and impurity of SUL (sules) of is easily detectable and can be determined quantitatively as shown Figure 4. Therefore, the proposed method can be used for the quality control of the tablets.
The high sensitivity of the proposed method allowed the determination of MEB metabolite and SUL in biological fluids by HPLC method. The method was further applied to the in-vivo determination of both drugs in real human plasma. Sulpiride is absorbed from gastro-intestinal tract. Following oral ingestion of a single oral dose of 50 mg of SUL, a peak plasma concentration of 0.03-0.60 mg/L (mean 0.18 mg/L) is attained in about 4-7 hours . This value lies within the working concentration range of the proposed method.
Application of the proposed method for determination of the studied drugs in spiked and real human plasma
Concentration taken (ng/mL)
Concentration found (ng/mL)
1-Spiked plasma sample
a- Intra-day precision
b- Inter- day precision
2- Real plasma sample
A new simple and sensitive method was explored for the simultaneous determination of MEB and SUL in their combined tablets. The HPLC method using florescence detection, by virtue of its high sensitivity, could be applied to the analysis of both drugs in their co-formulated dosage forms and biological fluids; it was possible to measure concentrations as low as 0.85 and 0.73 ng/mL for MEB and SUL respectively with good accuracy Moreover, the proposed method allowed therapeutic monitoring of MEB metabolite (veratic acid) in real human plasma without interference with SUL. Moreover, the proposed method is simple, time saving.
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