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Separation and determination of clopidogrel and its impurities by capillary electrophoresis

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Abstract

Clopidogrel bisulphate, an anti-platelet drug, has been separated from its impurities, namely impurity A, B and C by capillary zone electrophoresis (CZE) using uncoated fused-silica capillary (50.0 microm internal diameter, 31.2cm total length). Four factors affected the separation: buffer concentration, pH of the buffer, concentration of the chiral selector and the applied voltage. Optimization and robustness studies were performed with the aid of reduced central composite experimental design. The buffer used was triethylamine-phosphoric acid and the chosen chiral selector was sulphated beta-cyclodextrin (SCD). The best separation was achieved by using 10mM buffer, pH 2.3, containing 5% (mass/volume (m/v)) SCD. Reversed polarity mode was used with an applied voltage of -12kV and the capillary temperature was maintained at 20 degrees C. The method was validated for quantitative determination of the drug. It offered a limit of detection (LOD) of 0.13 microg/ml, a limit of quantitation (LOQ) of 0.4 microg/ml, and a linearity range of 0.4-300 microg/ml. Commercial bulk samples were analyzed using the developed method.

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... A reduced CCD was used to study the effects of BGE concentration, BGE pH, S-β-CD concentration and voltage on the chiral resolution and migration time. The method was validated and used for S-clopidogrel purity control in bulk commercial samples [80]. ...
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Article
Chirality is one of the major issues in pharmaceutical research and industry. Capillary electrophoresis (CE) is an interesting alternative to the more frequently used chromatographic techniques in the enantioseparation of pharmaceuticals, and is used for the determination of enantiomeric ratio, enantiomeric purity, and in pharmacokinetic studies. Traditionally, optimization of CE methods is performed using a univariate one factor at a time (OFAT) approach; however, this strategy does not allow for the evaluation of interactions between experimental factors, which may result in ineffective method development and optimization. In the last two decades, Design of Experiments (DoE) has been frequently employed to better understand the multidimensional effects and interactions of the input factors on the output responses of analytical CE methods. DoE can be divided into two types: screening and optimization designs. Furthermore, using Quality by Design (QbD) methodology to develop CE-based enantioselective techniques is becoming increasingly popular. The review presents the current use of DoE methodologies in CE-based enantioresolution method development and provides an overview of DoE applications in the optimization and validation of CE enantioselective procedures in the last 25 years. Moreover, a critical perspective on how different DoE strategies can aid in the optimization of enantioseparation procedures is presented.
... Due to the vital importance of this antiplatelet drug several methods have been reported for the determination of CLPB either in pure form, pharmaceutical forms, or biological fluids. It includes spectrophotometric [16][17][18][19], capillary electrophoresis [20,21], potentiometry [22,23], and several chromatographic methods [24][25][26][27]. Major drawbacks of some of these methods are expensive, required multiple sample preparation and qualified operators are required. ...
... In many pharmaceutical formulations, ASA is combined with CLP or PRG to achieve an improved therapeutic effect (39,40). CLP impurity A (CCA) (CLP carboxylic acid) is the major but inactive metabolite that results via hydrolysis of CLP in different conditions, such as the acidic gastric environment (41). Chemical structures are shown in Figure 1. ...
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An isocratic reversed-phase high-performance liquid chromatographic method has been developed and validated for the simultaneous determination of aspirin, prasugrel HCl and clopidogrel bisulfate in the presence of clopidogrel-related compound (impurity-A) in focus on counterfeit. This method was used to determine counterfeited antiplatelet drugs in two substandard Indian pharmaceutical products sold on the market in Yemen and two traditional herbal medicines sold on the market in China. Thin layer chromatography and mass spectrometry of counterfeit herbal medicines have additionally been carried out to verify the identification of adulterants. Chromatographic separation was performed on Inertsil ® ODS-3 C18 (4.6 × 250 mm, 5 μm) with isocratic mobile phase elution containing a mixture of acetonitrile: (25 mM) potassium dihydrogen phosphate buffer, pH 2.7 adjusted with 0.1 M o-phosphoric acid (79: 21, v/v), at a flow rate of 1 mL/min and UV detection at 220 nm. Designs of experiment methodology, Plackett–Burman and Box–Behnken designs were used for the screening and optimization of the mobile phase composition. The method validation was also performed in accordance with the International Council on Harmonization (ICH) guidelines. The method developed for routine analysis was found to be sensitive, simple, accurate and highly robust. The results were statistically compared to reference methods using Student’s t-test and variance ratio F-test at P < 0.05.
... It is also used, along with acetylsalicylic acid [1][2][3][4][5][6] (ASA, aspirin), for the prevention of thrombosis after placement of a coronary stent or as an alternative antiplatelet drug for people intolerant to aspirin. Clopidogrel [7][8][9][10][11][12][13][14][15][16][17][18][19][20] is an inhibitor of platelet activation and aggregation through the irreversible binding of platelets. Clopidogrel is a prodrug and is metabolized to a pharmacologically active metabolite and inactive metabolites. ...
Article
A simple, precise, versatile and a new spectrophotometric method is proposed for the estimation of microgram quantities of the drug Clopidogrel in the presence of Aspirin. It is also known as Clopin-A, The drug forms an Ion – pair complex with Bromo Cresol Green (BCG), the stoichiometry of which is established as 1:1 by Job's continuous variation method. The wavelength of the maximum absorbance of the Ion – pair complex is found to be 440nm. The absorbance values of the Ion – pair complex increased linearly with the increase in the amount of the drug Clopidogrel in the presence of Aspirin. This suggests the suitability of the method for the determination of the drug in the range 10μg/ml to 250μg/ml. This also indicates the verification of the Beer-Lambert’s Law in this range. This method is successfully employed to evaluate the assay of commercial tablets in pharmaceutical formulations for Clopidogrel in the presence of Aspirin and the results agreed very well. The molar absorptivity and Sandell Sensitivity of the method are found to be 1.860×104 lit/mole/cm and 0.0225 μg/ml/cm2 respectively.
... Hence, chiral recognition of both isomers is of utmost importance in pharmaceutical industry. Literature survey reveals chromatographic methods such as liquid chromatography (LC) [15] and capillary zone electrophoresis (CZE) [16] have been employed for enantiomeric separation of CLP in the presence of their antipodes using various chiral selectors in the chiral column. But, the use of these methods require very expensive chiral columns and necessity of presample preparation steps, where as electrochemical techniques provide better alternative in terms of sample preparation, cheaper cost as well as faster detection of chiral analyte. ...
... Triethylamine-phosphoric acid (10mM) was the buffer and -cyclodextrin was the chiral selector used. The temperature was maintained at 20 0 C [73,74]. High Performance Liquid Chromatography method was developed by Roberto Cirilli et al., to quantify of Lansoprazole enantiomers and its impurities [75]. ...
Article
Chiral separation plays a very important role in the modern pharmaceutical analysis and will continue in upcoming years. Separation and identification of chiral impurities are indispensable. According to ICH guidelines, only the active enantiomer of the drug has to be marketed, so there is a focus on separation of the inactive enantiomer which acts as a chiral impurity. The impurities present in the enantiomers also pose various toxic adverse effects on bioavailability and efficacy, hence the need to separate these impurities will forever be trending. This review primarily focuses on the separation techniques like Capillary Electrophoresis (CE), High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), and Supercritical Fluid Chromatography (SFC) followed by the year-wise trend in the separation of selected chiral impurities. In the coming years, researchers should work on using ultra-fast, selective, and sensitive methods for the effective separation of chiral impurities.
... The main side effect of this drug includes headache, nausea, easy bruising, itching, heartburn, bleeding and thrombotic thrombocytopenic purpura [7]. Analytical methods reported in literature for the trace level determination clopidogrel includes UV spectrophotometry [8][9][10], 1H-NMR and a chiral HPLC [11], HPLC [12][13][14], HPTLC [15][16], HPLCMS/MS [17] and capillary electrophoresis [18][19][20]. In comparison to these methods which are expensive and required multiple sample preparation, potentiometric sensors provide a highly selective, sensitive and economical method for the analysis pharmaceuticals. ...
... An optimization design was constructed using CCD as the most efficient surface response designs. CCD has been used successfully and efficiently by several researchers to optimize the experimental conditions for best HPLC and electrophoretic separation (14)(15)(16)(17). Such a design would allow having a model between a response y and number of factors x 1 …x n . ...
Article
In the present decade, great importance has been focused on the development of green analytical methods (GAM) as eco-friendly techniques. Minimizing the wastes, analysis time, hazardous reagents, sample size and energy are the main important principles for development of GAM. This manuscript describes a green, novel, rapid, accurate and reliable capillary zone electrophoresis method (CZE) for the simultaneous separation and determination of zofenopril calcium (ZOF) and hydrochlorothiazide (HCT) in presence of two major impurities of HCT, namely; chlorothiazide (CT) and salamide (DSA). Uncoated fused-silica capillary (50 μm i.d. × 48.5 cm and 40 cm effective length) was used. The main factors affecting the separation were the buffer concentration, pH of the buffer and applied voltage. Optimization of the experimental conditions was performed by applying response surface methodology (RSM). The experiments were designed using central composite face-centered design (CCD). The model obtained from the design described the linear, non-linear and interaction effects of factors on the responses. The optimum conditions given by the design were running buffer of sodium borate (pH 9.15; 10 mM) and 17 kV as positive mode applied voltage. Upon applying these conditions, baseline separation for the four compounds with short analysis time of 5.0 min was achieved. UV detection was performed at 225.0 nm and the capillary temperature was maintained at 25°C. The method was validated and applied for quantitative determination of the studied drugs according to the International Conference on Harmonization (ICH) guidelines. Good linearity was obtained in the range of 10.0-100.0 μg/mL for both ZOF and HCT. As for CT and DSA (HCT impurities), linearity range was 5.0-100.0 μg/mL. The proposed method was successfully applied for the analysis of these drugs in their synthetic mixtures and in their co-formulated pharmaceutical formulations.
... CLO was determined in the presence of its human fluid by mass spectrometry coupled with LC [23,24], and HPLCMS/MS [25]. Also capillary electrophoresis methods were reported [26][27][28], and voltammetry [29]. ...
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The fabrication and the performance response characteristics of a novel sensitive, selective, simple, and rapid sensor for the determination of Clopidogrel bisulphate (CLO-H2SO4) were described. The sensing modified carbon paste sensor comprised of an ion-pair based on Clopidogrel with silico tungastate (CLO-ST) where this study included: composition, usable pH range, response time and temperature. The sensorex hibiteda wide linear dynamic concentration ranging from 1.00x10−7-1.00x10−2 and the usable pH ranges from 1.2-4.8 with the response time ranging from (5-8 sec) which is much faster compared to liquid ISEs with a detection limit equals 0.34 nM. The selectivity of the sensor (CLOH2SO4) was applied with respect to a many of organic and inorganic cations, amino acids and sugars. The application of the sensor (CLO-H2SO4) for its determination was utilized in bulk powder, Plavix Tablet, human (serum-urine) and monitoring Plavix tablet dissolution rates using calibration curve, standard addition, and the potentiometric titration methods. The obtained results were statistically analyzed in both accuracy and precision and were compared using the US pharmacopeia method where there is no significant difference was observed.
... Clopidogrel, (CPG) (+)-(S)-methyl 2-(2-chlorophenyl)-2-(6, 7-dihydrothieno [3, 2-c] pyridin-5(4H)-yl) acetate (Fig. 1A) is a prodrug that is converted in the liver to an active thiol metabolite, which irreversibly inhibits the platelet P2Y12 adenosine diphosphate receptor. Literature survey reveals that few analytical methods have been reported for clopidogrel include RP-HPLC methods [1][2][3][4] , HPTLC method [5,6] , UV method [7] , normal phase HPLC [8] , GC method [9] , LC-MS method [10] , capillary electrophoresis method [11] . ...
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Article
This manuscript describes a simple, sensitive, rapid and precise Ultra fast liquid chromatographic (UFLC) method for the simultaneous determination Clopidogrel and Aspirin in human plasma according to USFDA draft guidelines. In the current study, the analysis was performed on phenomenex C8 (250 × 4.6mm, 5μm) column using phosphate buffer (pH-2.5) and acetonitrile (35: 65 v/v) as mobile phase at flow rate of 1.5 mL/min. In this developed method Clopidogrel and Aspirin eluted at a retention time of 2.697 and 5.337 min respectively. The proposed method is having linearity in the concentration range from 10 to 50μg/mL of Clopidogrel and Aspirin. The current method was validated with respect to linearity; precision, lowest limit of detection (LOD), accuracy and recovery according to the USFDA guidelines. The system consisted of a pump (Shimadzu, prominence, UFLC), with 20µl sample injector, along with a PDA detector at a wavelength of 230 nm and 252 nm for Aspirin and Clopidogrel respectively. Data was compiled using Shimadzu LC Solution software. A good linear relationship over the concentration range of 10-50µg/ml was shown. Validation of the method was carried out as per the USFDA draft guidelines. The method developed was found to be precise, accurate, specific, linear and selective. Statistical analysis shows that the W WO OR RL LD D J JO OU UR RN NA AL L O OF F P PH HA AR RM MA AC CY Y A AN ND D P PH HA AR RM MA AC CE EU UT TI IC CA AL L S SC CI IE EN NC CE ES S S SJ JI IF F I Im mp pa ac ct t F Fa ac ct to or r 2 2. .7 78 86 6 V Vo ol lu um me e 3 3, , I Is ss su ue e 9 9, , 5 51 18 8-5 53 31 1.. R Re es se ea ar rc ch h A Ar rt ti ic cl le e I IS SS SN N 2278 – 4357
... Literature survey reveals that few analytical methods have been reported for clopidogrel include RP-HPLC methods [1][2][3][4], HPTLC method [5,6] , UV method 7 ,normal phase HPLC 8 , GC method 9 Atorvastatin, (3R, 5S, 6E)-7-[4-(4-fluorophenyl)-2-(N-methyl methane sulfonamido)-6-(propan-2-yl) pyrimidin-5-yl]-3, 5dihydroxyhept-6-enoic acid ( fig. 1B) is a 3-hydroxy-3-methylglutaryl coenzyme A-reductase inhibitor, or statin, that has been developed for the treatment of dyslipidemia, atherosclerosis, high cholesterol, hyper lipoproteinemia, elevated LDL, Prevention of Cardiovascular Disease. ...
... CLO was determined in the presence of its human fluid by mass spectrometry coupled with LC (Shin and Yoo, 2007;Takahashi et al., 2008), and HPLCMS/MS (Silvestro et al., 2011). Also capillary electrophoretic (Fayed et al., 2009;Karazniewicz et al., 2010;Serra et al., 2010), and voltammetric (Dermis and Aydogan, 2010) methods were reported. No methods are found in the literature for the determination of CLO by chemical modified carbon paste (CMCP). ...
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Article
The fabrication and the performance response characteristics of a sensitive, selective, simple, and rapid sensor for the determination of clopidogrel bisulphate (CLO-H2SO4) were described. The constructed carbon paste sensor comprised of an ion-pair based on clopidogrel with silicotungastate (CLO-ST) where this study included: composition, usable pH range, response time and temperature. The sensor exhibited a wide linear dynamic concentration ranging from 1.00x10−7-1.00x10−2 and the usable pH ranges from 1.2 – 4.8 with the response time ranging from (5 - 8 s) which is much faster compared to liquid ISEs with a detection limit equals 0.34 nM. The selectivity of the sensor (CLO-H2SO4) was applied with respect to a many of organic and inorganic cations, amino acids and sugars. The application of the sensor was applied in bulk powder, tablets, human (serum-urine) and monitoring Plavix tablets’ dissolution rates. The obtained results were statistically analyzed in both accuracy and precision and were compared using the US pharmacopeial method where there is no significant difference was observed.
... Literature survey reveals that few analytical methods have been reported for clopidogrel include RP-HPLC methods [1][2][3][4], HPTLC method [5,6], normal phase HPLC [7], GC method [8], LC-MS method [9], capillary electrophoresis method [10], UV method [11]. ...
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Objective: To develop simple, sensitive, rapid, robust and reproducible method for the bioanalytical determination of clopidogrel and pantoprazole in rat plasma using reverse phase high performance liquid chromatographic method. Method: The analysis was performed on C8 (250 × 4.6mm, 5μm) column with a mobile phase consisting of 0.03M potassium dihydrogen ortho phosphate buffer (pH 3), acetonitrile in the ratio of 40:60 (v/v) with a flow rate of 1.2 ml/min. The analyte was monitored with UV detector at 240 nm. In the developed method pantaprazole elutes at retention time of 2.6 min and clopidogrel at 8.2 min. Results: The proposed method is having linearity in the concentration range 10-50 μg/ml for both clopidogrel and pantoprazole. The method was validated with respect to system suitability, linearity, precision, limit of detection (LOD) and limit of quantification (LOQ), accuracy (recovery), ruggedness, robustness, stability, forced degradation studies (specificity). The method is extended to animal study. The pharmacokinetic parameters (AUC,Cmax,Tmax) was found statistically significant(p<0.05). Conclusion: The results suggest that concomitant use of clopidogrel and pantoprazole alters pharmacokinetics of clopidogrel.
... CPT, metabolites produced in the presence of 0.3 mM CPT; CPT+GSH, metabolites produced in the presence of 0.3 mM CPT and then treated with 1 mM GSH; 2G6PD, metabolites produced in the absence of G6PD; AM, 1 mM GSH in PRP; GSH, metabolites produced in the presence of 1 mM GSH; NPT, metabolites produced in the presence of 0.3 mM NPT; NPT+ GSH, metabolites produced in the presence of 0.3 mM NPT and then treated with 1 mM GSH; PRP, untreated platelet-rich plasma; 2SH, metabolites produced in the absence of any thiol reductants. known that clopidogrel may decompose to byproducts via nonenzymatic oxidation (Mohan et al., 2008;Fayed et al., 2009). These byproducts do not seem to have any inhibitory effects on platelet aggregation. ...
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In this work we investigated the formation, reactivity and anti-platelet activity of various mixed disulfide conjugates of clopidogrel. Our results showed that the production of the active metabolite (AM) from 2-oxoclopidogrel by human liver microsomes (HLMs) is greatly affected by the thiol reductants used. Among the ten thiol compounds tested, glutathione (GSH) is most efficient in producing the AM at a rate of 167 pmoles AM/min/mg HLM. Interestingly, no AM but only the mixed disulfide conjugates were formed in the presence of 6-chloropyridazine-3-thiol (CPT), 2,5-dimethylfuran-3-thiol (DFT), and 3-nitropyridine-2-thiol (NPT). The MS and MS2 spectra of the conjugates of these thiol compounds confirmed the presence of a mixed disulfide bond linkage between the AM and the thiol reductants. Kinetic studies revealed that the mixed disulfide conjugates were capable of exchanging thiols with GSH to release the AM with second order rate constants ranging from 1.2 to 28 M-1s-1. The mixed disulfide conjugates of CPT and NPT showed potent inhibition of platelet aggregation after pre-treatment with 1 mM GSH, confirming that the AM is responsible for the anti-platelet activity of clopidogrel. Collectively, our results provide strong support for a P450-mediated bioactivation mechanism involving the initial formation of a glutathionyl conjugate followed by thiol-disulfide exchange with another GSH molecule to release the AM. Furthermore, the stable mixed disulfide conjugates identified in this study provide a platform to quantitatively generate the therapeutic AM without the need for P450-mediated bioactivation. This property can be further explored in order to overcome the inter-individual variability in clopidogrel therapy.
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The current work introduces a new nanostructure of Ce³⁺-doped NiO nanodisks fabricated by a facile hydrothermal protocol, whose characteristics were determined using XRD, SEM and EDX techniques. The results confirmed the presence of nanodisk structures, huge surface area and large pore size. Then, the surface of a screen-printed electrode was modified with the as-fabricated nanostructure to achieve a sensitive and selective electrochemical sensor (Ce³⁺-NiO ND/SPE) for determination of Plavix, a cardiovascular drug. Differential pulse voltammetry chronoamperometry and cyclic voltammetry were utilized to monitor the electrochemical behaviors of drug on the surface of modified electrode. The synergetic impact of Ce-doped NiO nanodisks on the Plavix oxidation was due to increased oxidation peak current and decreased oxidation over-potential. Our novel sensor under the optimized experimental and instrumental circumstances could electrochemically determine the study analyte in the range as wide as 0.01 to 700.0 µM and a limit of detection as narrow as 8.3 nM. The practical capability and sensitivity of the proposed sensor were validated by determining the Plavix in real pharmaceutical preparations, with commendable obtains. Graphical abstract
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A rapid and sensitive chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the enantioseparation and determination of clopidogrel bisulfate enantiomers in beagle plasma was established and successfully applied to a stereoselective pharmacokinetic study. The chromatographic separation was achieved on an Ultron ES-OVM column (150 × 2.0 mm, 5 μm) with acetonitrile-ammonium acetate (10 mM, pH 4.5) (22:78, v/v) at a flow rate of 0.3 mL/min. The mass detection was conducted using multiple reaction monitoring mode with the transition of the m/z 322.00→212.00 for clopidogrel and m/z 285.00→193.00 for diazepam (IS), respectively. The calibration curve was linear at the range of 1-800 ng/mL (r²>0.997) for each enantiomer. The intra- and inter-day precision (CV%) was within 10.9% and the accuracy was at the range of 88.5%-101.3% for both enantiomers. The extraction recovery was >90.2% and no obvious matrix effect was observed. The pharmacokinetic results showed that the R-CB have higher Cmax and AUC than that of S-CB. It is first time to find the chiral inversion between S-CB and R-CB in beagle.
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A simple, sensitive, accurate and specific stability-indicating high-performance liquid chromatographic method was developed and validated for the simultaneous estimation of clopidogrel bisulphate and omeprazole in bulk. Extensive testing of clopidogrel bisulphate and omeprazole in different stress conditions were carried out as per the ICH guidelines Q1A (R2) Clopidogrel bisulphate and omeprazole was exposed to various stress conditions like oxidation, hydrolysis, photolysis and neutral decomposition. Clopidogrel bisulphate, which was found to degrade considerably in acidic and photo conditions, was found to be stable in alkaline and neutral conditions, whereas omeprazole was found to be degrading in alkaline, oxidative and photo conditions, but stable in acidic and neutral condition. Apart from the formation of minor degraded products under accelerated conditions, the drugs were reasonably stable in solid state. A good linear relationship over the concentration range of 50-500μg/mL was shown. Validation of the method was carried out as per the ICH guidelines. The method developed was found to be specific, selective, precise and accurate. Clopidogrel bisulphate showed degradation in 5M hydrochloric acid at 80oC, in 3% hydrogen peroxide for 5min the drug showed around 35% of degradation, when exposed to sunlight for 15 min, forming around 25-30% of degradation products. Omeprazole showed 15-18% degradation in alkaline and photo condition.Statistical analysis shows that the method is reproducible and selective for the estimation of clopidogrel bisulphate and omeprazole in dosage form.
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A simple, sensitive, robust and specific Ultra fast liquid chromatographic (UFLC) method was developed and validated for the simultaneous determination Clopidogrel and Pantoprazole in human plasma. In the current study, the analysis was performed on phenomenex C8 (250 × 4.6mm, 5μm) column using potassium dihydrogen orthophosphate buffer (pH-3.0) and acetonitrile (40: 60 v/v) as mobile phase at flow rate 1.2 mL/min. The analyte were monitored with PDA detector at 252nm. In this developed method Clopidogrel and Pantoprazole elutes at a retention time of 5.14 and 2.62 min respectively. The proposed method is having linearity in the concentration range from 5 to 50μg/mL of Clopidogrel and Pantoprazole. The current method was validated with respect to linearity; precision, lowest limit of detection (LOD) and lowest limit of quantification (LOQ), accuracy and recovery according to the USP guidelines. The system consisted of a pump (Shimadzu, prominence, UFLC), with 20μl sample injector, along with a PDA detector at a wavelength of 252nm. Data was compiled using Shimadzu LC Solution software. A good linear relationship over the concentration range of 5-50μg/ml was shown. Validation of the method was carried out as per the USP. The method developed was found to be precise, accurate, specific and selective. Statistical analysis shows that the method is reproducible and selective for the estimation of Clopidogrel and Pantoprazole in dosage form.
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In a hydrophilic droplet three-electrode system, electroactive species within the droplet play very important roles in the electron transfer (ET) process on the solid/electrolyte interface which can then induce an ion transfer (IT) reaction at the liquid/liquid interface. In this work, several redox couples and electroactive species are chosen to study ET-IT coupling processes at the water/1.2-dichloroethane (W/DCE) interface with cyclic voltammetry (CV) and the Osteryoung square wave voltammetry (OSWV). Among them, the redox couple of Ru(NH3)63+/2+ has been found to have the widest useful potential window of about 1.2 V. A hydrophilic droplet three-electrode system using single electroactive molecule instead of redox couple has been confirmed to be stable and has similar functionality of a redox couple. In addition, the lipophilicity of antiplatelet drug clopidogrel at the W/DCE interface is investigated and its ionic partition diagram has been constructed. The protonated clopidogrel is detected in a linear concentration range of 5.0-50 μM and the limit of detection (LOD) is calculated to be 3.0 μM by using the hydrophilic droplet system of Ru(NH3)63+/2+ and the OSWV.
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A simple, sensitive, accurate and specific stability-indicating high-performance liquid chromatographic method was developed and validated for the estimation of clopidogrel bisulphate in bulk. In the present study, extensive testing of clopidogrel bisulphate in different stress conditions were carried out as per the ICH guidelines Q1A (R2). The system consisted of a pump (Shimadzu, prominence, LC20AD), with 20µl sample injector, along with a PDA (Shimadzu, prominence, SPDM20A) detector at a wavelength of 254nm. Data was compiled using Shimadzu LC Solution software. The degraded products formed under various stress conditions were separated successfully from the drug by using a PHENOMENEX C8 Column (150 x 4.6mm, 5µm) with binary gradient conditions. Acetonitrile: phosphate buffer of pH 2.0 was used as mobile phase at flow rate of 1.2ml/min. Clopidogrel bisulphate was exposed to various stress conditions like oxidation, hydrolysis, photolysis and neutral decomposition. Clopidogrel bisulphate, which was found to degrade considerably in acidic, photo and oxidative conditions, was found to be stable in alkaline and neutral conditions. Apart from the formation of minor degraded products under accelerated conditions, the drug was reasonably stable in solid state. A good linear relationship over the concentration range of 150-500µg/ml was shown. Validation of the method was carried out as per the ICH guidelines. The method developed was found to be precise, accurate, specific and selective. Clopidogrel bisulphate showed degradation in 5M Hydrochloric acid at 80 o C, in 3% hydrogen peroxide for 5min the drug showed around 35% of degradation, when exposed to sunlight for 15 min, formed around 25-30% of degradation products. Statistical analysis shows that the method is reproducible and selective for the estimation of clopidogrel bisulphate in dosage form.
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The current work demonstrates a “one-pot” click synthetic procedure for the preparation of triazole bridged cyclodextrin (CD) chiral stationary phases (CSPs) and their application for efficient chiral differentiation of clopridogrel enantiomers under reversed-phase high performance liquid chromatography (HPLC). It was found that native-CD-CSP and methylated-CD-CSP afforded good enantioselectivity towards clopridogrel enantiomers with acetonitrile(ACN) /water and methanol(MeOH) /water as the mobile phases, respectively, while the more versatile phenylcarbamoylated-CD-CSP cannot resolve the enantiomers due to the steric hindrance of the bulky phenylcarbamate moieties. Solvent selection plays an important role in CSPs’ chiral recognition ability towards clopridogrel enantiomers. The 6-hydroxyl moieties on CD rims were found to participate in the chiral recognition process. The optimal chiral resolution (Rs) was improved to 1.95 by transferring the separation from 5 um native-CD-CSP to 3 um native-CD-CSP with ACN/buffer as the mobile phases.
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A novel, rapid and a stability-indicating reverse phase LC method has been developed and validated for the simultaneous estimation of Clopidogrel and Aspirin in tablet dosage form. The chromatographic separation was achieved on a novel core shell technology phenyl hexyl stationary phase of particle size 2.6μ. The method employed a linear gradient elution and the detection wavelength was set at 220nm. The proposed method was extensively validated as per ICH guidelines. The specificity of the method was proved by performing forced degradation studies. Regression analysis shows r value (correlation coefficient) of greater than 0.999 for individual active drug substances. Accuracy was proved in the range of 50% to 150% of working range.
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A simple and rapid TLC method using beta-cyclodextrin as a chiral mobile phase additive (CMPA) was developed for direct separation of S-clopidogrel and its impurity R-clopidogrel. The influence of different factors (stationary phases, organic modifiers, chiral selectors and their concentrations in the mobile phase, and optimal saturation time of the chamber) on enantioseparation was studied. The best resolution of clopidogrel enantiomers was achieved on Polygram (R) cel 300 Ac-10% plates using isopropanol-0.5 mM beta-cyclodextrin (6: 4, v/v) as mobile phase in TLC chamber previously equilibrated with the mobile phase for 20 min. The spots were detected under UV light and using iodine vapours. The method enables rapid separation of clopidogrel enantiomers and can be successfully used in control of stereoselective synthesis of clopidogrel and in control of its purity. Finally, the molecular modelling of the inclusion complexes between the analytes and alpha-, beta-, and gamma-cyclodextrin was performed to investigate the mechanism of the enantiorecognition.
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Objective: To develop simple, sensitive, rapid, robust and reproducible method for the bioanalytical determination of clopidogrel and pantoprazole in rat plasma using reverse phase high performance liquid chromatographic method. Method: The analysis was performed on C8 (250 × 4.6mm, 5μm) column with a mobile phase consisting of 0.03M potassium dihydrogen ortho phosphate buffer (pH 3), acetonitrile in the ratio of 40:60 (v/v) with a flow rate of 1.2 ml/min. The analyte was monitored with UV detector at 240 nm. In the developed method pantaprazole elutes at retention time of 2.6 min and clopidogrel at 8.2 min. Results: The proposed method is having linearity in the concentration range 10-50 μg/ml for both clopidogrel and pantoprazole. The method was validated with respect to system suitability, linearity, precision, limit of detection (LOD) and limit of quantification (LOQ), accuracy (recovery), ruggedness, robustness, stability, forced degradation studies (specificity). The method is extended to animal study. The pharmacokinetic parameters (AUC,Cmax,Tmax) was found statistically significant(p<0.05). Conclusion: The results suggest that concomitant use of clopidogrel and pantoprazole alters pharmacokinetics of clopidogrel. Keywords: Bio-Analytical, Clopidogrel, Pantoprazole, RP-HPLC Method, Rat Plasma, Drug Interaction Study
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A review, with 63 refs., of anal, methods based on capillary electrophoresis used for qual. and quant, detn. of active pharmaceutical substances, inorg. impurities and chiral species.
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A novel stability-indicating reversed phase liquid chromatographic (RP-LC) method was developed for the determination of purity of Nizatidine in Nizatidine oral solutions in the presence of its impurities and degradation products. Two unknown impurities were formed in the formulated drug under the stress conditions (40°C/75% RH for 3 months), with relative retention times (RRTs) 0.93 and 2.14. The two degradant impurities were isolated and characterized using liquid chromatography mass spectrometry (LC-MS). Chromatographic method was developed and validated for Nizatidine United States Pharmacopeia (USP) impurities along with the two degradant impurities. Total 13 impurities were well separated and not interfering with placebo peaks in oral solution. The method was developed using C-18 column in gradient elution mode. The eluted compounds were monitored at 254 nm. Nizatidine was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. The degradation products were well resolved from main peak and its impurities, proving the stability-indicating power of the method. The developed method was validated as per International Conference on Harmonization guidelines with respect to specificity, limit of detection, limit of quantification, precision, linearity, accuracy, robustness, and ruggedness.
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Capillary electrophoresis represents a very powerful separation tool in the area of chiral separations. Cyclodextrin mediated chiral CE is a continuously flourishing technique within the frame of the electromigration methods. In this review a brief overview of the synthetic procedures leading to modified cyclodextrins is provided first. Next, selected aspects related to the utilization of cyclodextrins in chiral CE are discussed specifically in the view of recently published data. Advantages of cyclodextrins and basic principles of chiral CE are remained. The topic of the determination of binding constants is touched. Particular attention is paid to the effort aiming at better understanding of the molecular level of the enantiorecognition between cyclodextrins and the analyte in the solution. Powerful approaches extensively utilized in this field are NMR, molecular modelling, and computer simulations. Then a summary of applications of cyclodextrins in the CE enantioseparations is given, covering years 2008-2013. Finally, the general trend of modified cyclodextrins use in separation science is statistically evaluated. This article is protected by copyright. All rights reserved.
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The review presents a survey of recent applications of high-performance capillary electromigration methods-capillary zone electrophoresis, nonaqueous capillary electrophoresis, capillary isotachophoresis, micellar electrokinetic chromatography, microemulsion electrokinetic chromatography and capillary electrochromatography-for the determination of impurities of pharmaceuticals, including chiral impurities, for the period 2007-2013. In addition, due to the missing evaluation of the determination of counterions of pharmaceuticals by capillary electromigration methods in the last 20 years, the publications dealing with this topic since 1995 are included in this review. General aspects of both these types of applications of capillary electromigration methods in pharmaceutical analysis are discussed and detailed experimental conditions used for determination of various chemical impurities and counterions of many particular drugs are described. This article is protected by copyright. All rights reserved.
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Since the introduction about 30 years ago, CE techniques have gained a significant impact in pharmaceutical analysis. The present review covers recent advances and applications of CE for the analysis of pharmaceuticals. Both small molecules and biomolecules such as proteins are considered. The applications range from the determination of drug-related substances to the analysis of counterions and the determination of physicochemical parameters. Furthermore, general considerations of CE methods in pharmaceutical analysis are described.
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Capillary electromigration techniques are often considered ideal methods for enantioseparations due to their high separation selectivity and flexibility. Thus, numerous methods employing a chiral selector as pseudostationary phase in a background electrolyte have been developed and applied for the chiral analysis of drugs in bulk ware, pharmaceutical formulations and biological matrices. Furthermore, electromigration techniques have been combined with spectroscopic methods such as nuclear magnetic resonance in order to understand the complexation of analytes by chiral selectors. The present review focuses on recent developments and applications of chiral electromigration techniques in pharmaceutical and biomedical analysis including examples illustrating analyte–selector complex formation or mechanistic studies which have been published between January 2009 and July 2011. Selector-mediated chiral separations clearly dominate while no applications of capillary electrochromatography to pharmaceutical or biomedical analysis have been reported during this period of time.
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(S)-(+)-2-Chlorophenylglycine 1 is an important intermediate in the synthesis of Clopidogrel. A recirculating packed bed reactor (RPBR) was constructed for efficient production of (S)-1 by kinetic resolution of racemic N-phenylacetyl-2- chlorophenylglycine 2 using immobilized penicillin G acylase (PGA). The immobilized PGA exhibited maximum activity at 50 °C and pH 8.0 with (R,S)-2 as substrate. The kinetic constants (Km and vmax) of immobilized PGA were calculated to be 20.61 mM and 83.2 mM/min/g, respectively. The substrate displayed inhibitory effect on immobilized PGA with inhibition constant of 221.23 mM. The immobilized PGA showed a strict enantiospecificity for substrate at different temperature, pH and substrate concentration examined. The performance and productivity of RPBR were evaluated by several critical parameters, including immobilized PGA load, substrate feeding rate, height to diameter ratio and so on. The kinetic resolution process shows higher initial reaction rate and conversion by recycling 100 mL of substrate solution (80 mM) through RPBRs packed with 6.0 g immobilized PGA with a feeding rate of 1.5 mL/min while the H/D ratio was 4.0. The immobilized PGA-catalyzed kinetic resolution of (R,S)-2 was successfully operated in the RPBR for 60 batches, with an average productivity of 1.2 g/L/h for (S)-1 in high optical purity (>97% enantiomeric excess) in semi-continuous operation. The residual (R)-2 can be easily racemized and then used as substrate.
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A comprehensive survey of recent developments and applications of capillary electromigration techniques for enantioseparations from January 2006 to June 2010 is presented. The techniques include capillary electrophoresis, chip capillary electrophoresis and capillary electrochromatography. The separation principles and the chiral recognition mechanisms are discussed. Additionally, on-line preconcentrations in chiral capillary electrophoresis are also reviewed.
Article
The development of an RP-HPLC method for the determination of aripiprazole and its nine impurities was performed with the use of partial least squares regression, response surface plot methodology and chromatographic response function. The HPLC retention times and computed molecular parameters of the aripiprazole and its nine impurities were further used for the quantitative structure-retention relationship (QSRR) study. The QSRR model, R(2) : 0.899, Q(2) : 0.832, root mean square error of estimation (RMSEE): 4.761, root mean square error of prediction (RMSEP): 6.614, was developed. Very good agreement between the predicted and observed retention times (tR ) for three additional aripiprazole impurities (TC1-TC3) indicated the high prediction potential of the QSRR model for tR evaluation of other aripiprazole impurities and metabolites. The developed HPLC method is the first reported method for the efficient determination of aripiprazole and its nine impurities, which could be used for the analysis of additional three aripiprazole impurities (TC1-TC3). This article is protected by copyright. All rights reserved.
Article
A simple, accurate and sensitive kinetic method has been developed for the determination of three cardio- vascular drugs: Betaxolol, Clopidogrel, and Imidapril in pharmaceutical formulations. The method is based on the alkaline oxidation of the selected drugs with potassium manganate(VII) at room temperature (25°C) to produce a bluish-green colored product. The reaction is followed spectrophotometrically by measuring the rate of change of absorbance at 610 nm as a function of time. The fixed-time (ΔA) method is adopted for constructing the calibration curves, which were found to be linear over the concentration ranges of 0.40-2.0 μg ml-1 Betaxolol, 1.0-10.0 μg ml-1 Clopidogrel, and 6.0-16.0 μg ml-1 Imidapril. The implemented fixed times were 15 min for Betaxolol and Clopidogrel and 10 min for Imidapril. The proposed method has been successfully applied to pharmaceutical formulations of each drug. The percentage relative error and the coefficient of variation were less than 0.6 and 1.2 respectively. The lower limits of quantitation (LOQ) were 9.75x10-7 M Betaxolol, 3.62x10-6 M Clopidogrel, and 4.22x10-6 M Imidapril. The determination of the investigated drugs by the fixed-concentration and the rate-constant methods is feasible with the regression equations obtained, but the fixed-time method proves to be more applicable.
Article
In this article a review on the recent applications of multivariate techniques for optimization of electromigration methods, is presented. Papers published in the period from August 2007 to February 2013, have been taken into consideration. Upon a brief description of each of the involved CE operative modes, the characteristics of the chemometric strategies (type of design, factors and responses) applied to face a number of analytical challenges, are presented. Finally, a critical discussion, giving some practical advices and pointing out the most common issues involved in multivariate set-up of CE methods, is provided.
Article
Multiwalled carbon nanotubes are evaluated here as solid phase extraction (SPE) sorbent aiming to (±)-chlorpheniramine (CPA) enantioresolution with fluorimetric detection. β-cyclodextrin (CD) was added to the racemate and solutions with HCl and sodium dodecyl sulfate (SDS) in different proportions were assayed as eluents to achieve the separation between both enantiomers. The overall methodology involved a flow injection (FI) strategy enabling high sample throughput and low reagents consumption making it suitable for drug routine quality control. An adequate enantioresolution (2.08) with satisfactory responses for both (R)-CPA (peak area=285) and (S)-CPA (peak area=380) was achieved applying the proposed FI-SPE strategy under the optimized conditions [β-CD]=1.0mmolL(-1), [HCl]=1.0×10(-2)molL(-1), [SDS]=4.0×10(-4)molL(-1) and eluent flow rate=8.0rpm.
Article
This review article provides an overview of the recent advances in enantioanalysis by use of electrophoretic techniques. Due to the big number of publications in the subject mentioned above, this article is focused on chiral method developments and applications published from 2008 until 2011, and it demonstrates chiral selectors used in CE. Numerous chiral selectors have been used over the years, and these include the cyclic and the linear oligo- and polysaccharides, the branched polysaccharides, the polymeric and monomeric surfactants, the macrocyclic and other antibiotics and the crown ethers. Different dual-selector systems are also presented in this article, and the results are compared with those obtained by use of a single chiral selector. Finally, several pharmaceutical and biomedical applications based on chiral recognition are summarized.
Article
Clopidogrel contains a center of dissymmetry, and hence is capable of being resolved into its two mirror image compounds. It has been found that only the (S)-enantiomer, which corresponds to the dextrorotatory form, has antithrombotic activity and that the (R)-enantiomer, which corresponds to the levorotatory form, does not exhibit antithrombotic activity. Moreover, in animal studies, the (R)-enantiomer triggered convulsions at high doses. Consequently, (R)-clopidogrel bisulfate is considered to be one of the impurities in (S)-clopidogrel bisulfate bulk drug substance. Clopidogrel is extensively metabolized in vivo by carboxylesterase hydrolysis on the ester function, resulting in the formation of clopidogrel carboxylic acid (CCA) as the inactive metabolite of clopidogrel. In addition, small amounts of clopidogrel are converted to a pharmacologically active metabolite (AM) via the intermediate metabolite inactive 2-oxoclopidogrel, which is then converted to an AM by a two-step cytochrome P450 oxidation process. Due to the instability of clopidogrel AM and the abundant availability of the more stable CCA in human plasma, CCA is used to indirectly determine the pharmacokinetics of clopidogrel. Furthermore, there is also a possibility that (S)-clopidogrel undergoes an in vivo chiral inversion into the other clopidogrel enantiomer, which becomes hydrolyzed to (R)-CCA. Metabolic pathways and potential in vivo chiral inversions of clopidogrel are described. Until recently, only chromatographic methods were used to determine clopidogrel in biological samples.
Article
This article describes several dual detection methods in microchip and conventional capillary electrophoresis. These dual detection methods could be divided into three classes, which are as follows: dual optical detection, dual electrochemical detection, and combination of optical detection and electrochemical detection. Furthermore, the designs and the basic principles of those dual detectors are described together with the applications and the prospects of the dual detection for microchip and conventional capillary electrophoreses.
Article
This review gives an overview of the applicability of capillary electrophoresis, capillary liquid chromatography, capillary electrochromatography, and their derived techniques to analyze drug impurity mixtures, formulations, biological samples, and chiral compounds. For each application type, a few examples are given to illustrate the potential of the capillary technique. Details are provided about the capillaries used, chiral selectors, and stationary and mobile phases.
Article
Capillary electromigration techniques are often considered ideal methods for the analysis of chiral compounds due to the high resolution power and flexibility of the technique. Therefore, especially capillary electrophoresis using a chiral selector in the background electrolyte, also termed electrokinetic chromatography, has found widespread acceptance in analytical enantioseparations of drug compounds in pharmaceuticals and biological media. Moreover, mechanistic studies on analyte complexation by the chiral selectors have continuously been conducted in an effort to rationalize enantioseparation phenomena. These studies combined capillary electrophoresis with spectroscopic techniques such as nuclear magnetic resonance and/or molecular modeling. The present review focuses on recent examples of mechanistic aspects of capillary electromigration enantioseparations and summarizes recent applications of chiral pharmaceutical and biomedical analysis published between January 2009 and August 2010.
Article
A mixture of 18 neutral UV-active compounds with different characteristics of polarity was determined by capillary electrophoresis using a pseudostationary phase constituted by a microemulsion. The test analytes were volatile fragrance compounds, included in a list of 24 chemicals classified as suspected allergens according to Directive 2003/15/CE. The considered compounds were detected at 195 nm and p-anisaldehyde was chosen as internal standard. The background electrolyte consisted of a standard microemulsion made of 90.95% 10mM borax buffer, pH 9.2, 1.05% n-heptane, 8.00% SDS/n-butanol in 1:2 ratio, to which 40 mM methyl-β-cyclodextrin was added. Temperature and voltage were set at 20 °C and 25 kV, respectively. These experimental conditions allowed separation of the compounds to be obtained in about 20 min. The method was applied to real samples made up of rinse-off scented products. The results obtained using the standard microemulsion as pseudostationary phase showed its high resolution power, capable of effectively separating a complex mixture of analytes. Microemulsion electrokinetic chromatography was confirmed to have a great potential for different analytical challenges, holding up the possibility of using this technique as a good and complementary alternative to HPLC methods for routine analysis.
Article
This review covers recent advances of capillary electrophoresis (CE) in pharmaceutical analysis. The principle, instrumentation, and conventional modes of CE are briefly discussed. Advances in the different CE techniques (non-aqueous CE, microemulsion electrokinetic chromatography, capillary isotachophoresis, capillary electrochromatography, and immunoaffinity CE), detection techniques (mass spectrometry, light-emitting diode, fluorescence, chemiluminescence, and contactless conductivity), on-line sample pretreatment (flow injection) and chiral separation are described. Applications of CE to assay of active pharmaceutical ingredients (APIs), drug impurity testing, chiral drug separation, and determination of APIs in biological fluids published from 2008 to 2009 are tabulated.
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Two simple spectrophotometric methods for the determination of aspirin and clopidogrel in pharmaceutical formulations have been developed. First method is based on the additivity of absorbances. Second method is based on the determination of graphical absorbance ratio at two selected wavelengths, one being the isoabsorptive point for the two drugs (225 nm) and the other being the absorption maximum of hydrolysed aspirin (235.7 nm). Beer Lambert′s law is obeyed for both the drugs in the concentration range 4-18 m g/ml. Both the methods were found to be simple, rapid, accurate and can be adopted in routine analysis of drugs in formulations. The accuracy and reproducibility of the proposed method was statistically validated by recovery studies.
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The thienopyridine derivatives ticlopidine and clopidogrel are inhibitors of ADP-induced platelet aggregation. Pharmacological activity of these prodrugs depends on cytochrome P450 (P450)-dependent oxidation to the active antithrombotic agent. In this study, we investigated the interaction potential of clopidogrel and ticlopidine by using human liver microsomes and recombinantly expressed P450 isoforms. Both clopidogrel and ticlopidine inhibited CYP2B6 with highest potency and CYP2C19 with lower potency. Clopidogrel also inhibited CYP2C9, and ticlopidine also inhibited CYP1A2, with lower potency. Inhibition of CYP2B6 was time- and concentration-dependent, and as shown by dialysis experiments, it was irreversible and dependent on NADPH, suggesting a mechanism-based mode of action. Inactivation was of nonpseudo-firstorder type with maximal rates of inactivation (K(inact)) for clopidogrel and ticlopidine in microsomes (recombinant CYP2B6) of 0.35 (1.5 min(-1)) and 0.5 min(-1) (0.8 min(-1)), respectively, and half-maximal inactivator concentrations (KI) were 0.5 microM (1.1 microM) for clopidogrel and 0.2 microM (0.8 microM) for ticlopidine. Inhibition was attenuated by the presence of alternative active site ligands but not by nucleophilic trapping agents or reactive oxygen scavengers, further supporting mechanism-based action. A chemical mechanism is discussed based on the known metabolic activation of clopidogrel and on the finding that hemoprotein integrity of recombinant CYP2B6 was not affected by irreversible inhibition. These results suggest the possibility of drug interactions between thienopyridine derivates and drug substrates of CYP2B6 and CYP2C19.
Article
Two simple, rapid, precise, highly specific and economical spectrophotometric methods have been developed for the determination of clopidogrel bisulphate in its pharmaceutical dosage forms. Method A is based on the reduction of ferric ions to ferrous ions which produce blue colour with potassium ferricyanide with absorption maximum at 760 nm. The chromogen obeyed linearity over 18-32 μg/ml. Method B is based on the hydrolysis of ester linkage of drug into acid form by heating with sulphuric acid.This acid form of drug has absorption maximum at 217 nm. Beer's law is obeyed in the concentration range of 4-18 μg/ml.
Article
OBJECTIVE: To investigate the enantioseparation of six drugs using capillary electrophoresis (CE) with seven cyclodextrins as the chiral additives. METHODS: The experiment was carried on Beckman P/ACE™ MDQ system. Four kinds of neutral-and three kinds of highly sulfated-cyclodextrins (HS-CDs) were used as the chiral additives in capillary zone electrophoresis (CZE) with normal or reversed-polarity mode. RESULTS: The enantioseparation of the six chiral drugs were obtained successfully under the optimal CE conditions. CONCLUSION: HS-CDs are excellent chiral selectors in CE.
Article
Clopidogrel (SR25990C, PLAVIX) is a potent antiplatelet drug, which has been recently launched and is indicated for the prevention of vascular thrombotic events in patients at risk. Clopidogrel is inactive in vitro, and a hepatic biotransformation is necessary to express the full antiaggregating activity of the drug. Moreover, 2-oxo-clopidogrel has been previously suggested to be the essential key intermediate metabolite from which the active metabolite is formed. In the present paper, we give the evidence of the occurrence of an in vitro active metabolite after incubation of 2-oxo-clopidogrel with human liver microsomes. This metabolite was purified by liquid chromatography, and its structure was studied by a combination of mass spectometry (MS) and NMR experiments. MS results suggested that the active metabolite belongs to a family of eight stereoisomers with the following primary chemical structure: 2-{1-[1-(2-chlorophenyl)-2-methoxy-2-oxoethyl]-4-sulfanyl-3-piperidinylidene}acetic acid. Chiral supercritical fluid chromatography resolved these isomers. However, only one of the eight metabolites retained the biological activity, thus underlining the critical importance of associated absolute configuration. Because of its highly labile character, probably due to a very reactive thiol function, structural elucidation of the active metabolite was performed on the stabilized acrylonitrile derivative. Conjunction of all our results suggested that the active metabolite is of S configuration at C 7 and Z configuration at C 3–C 16 double bound.
Article
A high‐throughput, high performance liquid chromatographic method was developed and validated for the determination of clopidogrel in pharmaceutical dosage forms. The analysis was performed at room temperature using a reversed phase monolithic silica column Chromolith Performance 18e (100 mm×4.6 mm I.D.). The mobile phase consisted of acetonitrile:phosphate buffer (50∶50 v/v, pH 3.0) at a flow rate of 4.0 mL/min. The photodiode array detector was set at 235 nm. The developed method showed a good linear relationship in the concentration range from 1.0 to 40.0 µg/mL with a correlation coefficient of 0.999. The limit of detection and limit of quantification were 0.97 µg/mL and 3.52 µg/mL, respectively.
Article
Dopa and carbidopa, components of the dual therapy for Parkinson's disease treatment, are both provided as single enantiomers, since their D-forms are inactive. To ensure the efficiency and safety of the therapy, these D-enantiomers, therefore, should be considered as impurities. In this paper, the enantioseparation power of different types of cyclodextrins, both neutral and charged ones, on dopa and carbidopa enantiomers was tested. Three methods of simultaneous separation of dopa and carbidopa enantiomers were developed, using highly sulfated β-cyclodextrin and sulfated β-cyclodextrin as chiral selector, in normal and reversed polarity mode. Two methods among these three were found sensitive enough for the quantitation of 0.1% D-enantiomers in L-forms (impurity level). After the optimization study, the best method was selected, using 16 mM sulfated β-cyclodextrin in 15 mM sodium phosphate buffer pH 2.45, an uncoated fused-silica capillary (50 νm inner diameter, 30 cm total length), and an applied voltage of −12 kV. This method is robust and efficient, with very high resolution for all peaks within a short analysis time of 10 min. Quantitatively, the method offers a limit of detection (LOD) of 0.2 νg/mL and a limit of quantitation (LOQ) of 0.5 νg/mL for both D-dopa and D-carbidopa, which is equivalent to 0.02% and 0.05% against the respective L-enantiomers. A linear relationship was found between the concentration of the analyte and the corresponding peak area in a range of 0.5–2.0 νg/mL.
Article
The enantiomeric separation of 37 clinically used racemic basic drugs among 50 drugs was achieved using sulfated β-cyclodextrin (S-β-CD) as chiral selector at pH2.5 and in the reversed polarity mode. The results obtained in this study were different from the one obtained using neutral β-CD and its derivatives as chiral selectors. Using S-β-CD as chiral selector did not require the presence of the substructure 4H to achieve chiral separation as observed with β-Cyclodextrin (β-CD) and its derivatives since among the 37 separated drugs only 7 possess the 4H substructure. The chiral discrimination depends on the appropriate interaction between the analyte and the sulfated β-cyclodextrin.
Article
The aim with this tutorial is to give a simple and easily understandable introduction to experimental design and optimization. The screening methods described in the paper are factorial and fractional factorial designs. Identification of significant variables are performed by normal distribution plots as well as by confidence intervals. Refinements of the models are also discussed. For optimization, the simplex method, central composite designs and the Doehlert design are discussed. The paper also gives an introduction to mixture designs. The paper contains 14 hands-on examples and if anyone needs the answers on these it is just to contact the authors.
Article
A sensitive, selective, precise and stability indicating high-performance thin layer chromatographic method of analysis of clopidogrel bisulphate both as a bulk drug and in formulations was developed and validated in pharmaceutical dosage form. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of carbon tetrachloride-chloroform-acetone (6:4:0.15, v/v/v). This system was found to give compact spots for clopidogrel bisulphate (R(f) value of 0.30+/-0.01). Clopidogrel bisulphate was subjected to acid and alkali hydrolysis, oxidation, photodegradation and dry heat treatment. Also the degraded products were well separated from the pure drug. Densitometric analysis of clopidogrel bisulphate was carried out in the absorbance mode at 230 nm. The linear regression data for the calibration plots showed good linear relationship with r(2)=0.999+/-0.001 in the concentration range of 200-1000 ng. The mean value of correlation coefficient, slope and intercept were 0.999+/-0.001, 0.093+/-0.011 and 8.83+/-0.99, respectively. The method was validated for precision, accuracy, ruggedness and recovery. The limits of detection and quantitation were 40 and 120 ng per spot, respectively. The drug undergoes degradation under acidic and basic conditions, oxidation and dry heat treatment. All the peaks of degraded product were resolved from the standard drug with significantly different R(f) values. This indicates that the drug is susceptible to acid-base hydrolysis, oxidation and dry heat degradation. Statistical analysis proves that the method is reproducible and selective for the estimation of the said drug. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.
This paper describes a GC-MS method for the analysis of the carboxylic acid metabolite (SR26334, II) of methyl (+)-(S)-alpha-(o-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5( 4H)-acetate hydrogensulfate (clopidogrel, SR 25990, I) in plasma and serum. The analytical procedure involves a robotic liquid-liquid extraction with diethyl ether followed by a solid-liquid extraction on C18 cartridges. The derivatization process was performed using n-ethyl diisopropylethylamine and alpha-bromo-2,3,4,5,6-pentafluoro toluene. A structural analogue (III) of II, was used as internal standard. The 1/X2; weighted calibration curve obtained in the range 5-250 ng/ml was well described by a quadratic equation. The extraction efficiency was better than 48% over the range studied; for the internal standard it averaged 51% at 50 ng/ml. Precision ranged from 3.6 to 15.8%, and accuracy was between 92 and 114%. Dilution has no influence on the performance of the method which could then be used to quantitate plasma samples containing up to 25000 ng/ml. The limit of quantification was 5 ng/ml. The method validation results indicate that the performance characteristics of the method fulfilled the requirements for assay methods for use in pharmacokinetic studies.
Article
Like ticlopidine, the ADP receptor antagonist clopidogrel is inactive in vitro and must be administered i.v. or orally to exhibit antiaggregatory and antithrombotic activities. We have previously shown that hepatic metabolism is necessary for activity. This study demonstrates that an active metabolite can be generated from human liver microsomes incubated with clopidogrel. Using several analytical methodologies (LC/MS, NMR, chiral supercritical fluid chromatography), we have identified its structure. In vitro, this highly unstable compound, different from that formed from ticlopidine, exhibited all the biological activities of clopidogrel observed ex vivo: Irreversible inhibition of the binding of 33P-2MeS-ADP to washed human platelets (IC50 = 0.53 µM), selective inhibition of ADP-induced platelet aggregation (IC50 = 1.8 µM) and ADP-induced adenylyl cyclase down-regulation. The irreversible modification of the ADP-receptor site which is responsible for the biological activity could be explained by the formation of a disulfide bridge between the reactive thiol group of the active metabolite and a cysteine residue of the platelet ADP receptor. Abbreviations: ADP: adenosine 5’diphosphate; 2-MeS-ADP: 2-methylthioadenosine-5’-diphosphate; Bmax: maximum binding capacity; IC50: concentration which inhibits 50% of the activity; Kd: dissociation constant; LC/MS: Liquid chromatography coupled to mass spectrometry; NMR: Nuclear magnetic resonance
Article
The macrocyclic antibiotics have recently gained popularity as chiral selectors in CE, HPLC and TLC. The macrocyclic antibiotics used for chiral separations include the ansamycins, the glycopeptides, and the polypeptide antibiotic thiostrepton. Although not strictly considered macrocyclic antibiotics, the aminoglycosides are antibiotics that have been used for chiral separations in CE. More chiral analytes have been resolved using the glycopeptides than with the other macrocyclic antibiotics combined. The glycopeptides vancomycin, ristocetin A and teicoplanin have been used extensively as chiral selectors in CE, with ristocetin A appearing to be the most useful chiral selector followed by vancomycin and teicoplanin, respectively. The macrocyclic antibiotics have also been used as chiral bonded phases in HPLC, and HPLC stationary phases based on vancomycin, ristocetin A and teicoplanin have been commercialized. Ristocetin A seems to be the most useful glycopeptide HPLC bonded phase, but its greater expense can be a drawback. The macrocyclic antibiotics have been used with micelles to improve efficiency, provide unique selectivity, and extend the range of separations to neutral solutes. Changing the macrocyclic antibiotic used in CE or HPLC can significantly alter the enantioselectivity of the separations. In fact, the glycopeptide antibiotics are complementary to one another, where if a partial enantioresolution is obtained with one glycopeptide, there is a high probability that a baseline or better separation can be obtained with another.
Article
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Article
A stability indicating, reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of clopidogrel in pharmaceutical dosage forms. The determination was performed on a semi-micro column, BDS C8 (250 x 2.1 mm i.d., 5 microm particle size); the mobile phase consisted of a mixture of 0.010 M sodium dihydrogen phosphate (pH 3.0) and acetonitrile (35:65, v/v), pumped at a flow rate 0.30 ml min(-1). The UV detector was operated at 235 nm. The retention times for clopidogrel and naproxen, which was used as internal standard, were 3.08 and 6.28 min, respectively. Calibration graphs are linear (r better than 0.9991, n=6), in concentration range 1.00-3.00 microg ml(-1) for clopidogrel. The intra- and inter-day RSD values were less than 1.96%, while the relative percentage error E(r) was less than 2.0% (n=5). Detection and quantitation limits were 0.12 and 0.39 micro ml(-1), respectively. The method was applied in the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.
Article
In this study, 18 copies of PLAVIX tablets containing clopidogrel hydrogensulfate were compared to the innovator drug product for uniformity of mass, impurity profile, content, dissolution properties and stability. In order to be able to separate the R-enantiomer of clopidogrel, an enantiospecific liquid chromatographic method was used to determine the impurities and to perform the assay. The paddle method was used for dissolution testing. Most of the copies were not similar compared to the original drug product: their amount of impurities was higher, the content of clopidogrel lower, the dissolution profiles different and after 3 months under stress conditions in the original packaging, the results for the samples and the reference were significantly different in most of the cases.
Article
A new, sensitive, specific and reproducible method for determination of clopidogrel metabolite (SR26334) in human plasma has been developed. After liquid-liquid extraction on Chem Elut cartridges with dichloromethane, samples were quantified using reversed-phase high performance liquid chromatography with mass detection. The determination was performed on a Luna C18, 3 microm (75 mmx4.6 mm i.d.) column with an acetonitrile-water-formic acid mixture (60:40:0.1, v/v/v) as a mobile phase. The flow rate was set at 0.2 mL/min. Repaglinide was chosen as an internal standard and the time of analysis was 12 min. For SR26334 the limits of detection and quantification were 7.5 ng/mL and 20 ng/mL, respectively, and the calibration curve was linear up to 3000 ng/mL. The extraction recovery of SR26334 from plasma was within the range of 85-90%. The method has been successfully used to study clopidogrel metabolite pharmacokinetics in healthy volunteers.
Article
Clopidogrel (SR25990C, PLAVIX) is a potent antiplatelet drug, which has been recently launched and is indicated for the prevention of vascular thrombotic events in patients at risk. Clopidogrel is inactive in vitro, and a hepatic biotransformation is necessary to express the full antiaggregating activity of the drug. Moreover, 2-oxo-clopidogrel has been previously suggested to be the essential key intermediate metabolite from which the active metabolite is formed. In the present paper, we give the evidence of the occurrence of an in vitro active metabolite after incubation of 2-oxo-clopidogrel with human liver microsomes. This metabolite was purified by liquid chromatography, and its structure was studied by a combination of mass spectometry (MS) and NMR experiments. MS results suggested that the active metabolite belongs to a family of eight stereoisomers with the following primary chemical structure: 2-[1-[1-(2-chlorophenyl)-2-methoxy-2-oxoethyl]-4-sulfanyl-3-piperidinylidene]acetic acid. Chiral supercritical fluid chromatography resolved these isomers. However, only one of the eight metabolites retained the biological activity, thus underlining the critical importance of associated absolute configuration. Because of its highly labile character, probably due to a very reactive thiol function, structural elucidation of the active metabolite was performed on the stabilized acrylonitrile derivative. Conjunction of all our results suggested that the active metabolite is of S configuration at C 7 and Z configuration at C 3-C 16 double bound.
Article
A new, simple, and reproducible method for determination of carboxylic acid metabolite of clopidogrel in human plasma has been developed. After liquid-liquid extraction in acidic medium with chloroform, samples were quantified on a Nova-pak C(8), 5 microm column using a mixture of 30 mM K(2)HPO(4)-THF-acetonitrile (pH = 3, 79:2:19, v/v/v) as mobile phase with UV detection at 220 nm. The flow rate was set at 0.9 mL/min. Ticlopidine was used as internal standard and the total run time of analysis was about 12 min. The method was linear over the range of 0.2-10 microg/mL of clopidogrel metabolite in plasma (r(2) > 0.999). The within-day and between-day precision values were in the range 1.0-4.8%. The limit of quantification of the method was 0.2 microg/mL. The method was successfully used to study the pharmacokinetics of clopidogrel in healthy volunteers.
Article
A fast, sensitive and specific LC-MS/MS bioanalytical method for the determination of unchanged clopidogrel in human plasma has been developed and validated over the range of 10-12,000 pg mL(-1) (r2 0.9993) by the Contract Research group at HFL. Samples (0.3 mL) were buffered (pH 6.8), extracted using diethyl ether and 10 microL of the sample extract was injected onto the LC-MS/MS system. Analysis was performed using a C8 column (temperature controlled to 50 degrees C) by gradient elution at a flow rate of 0.9 mL min(-1) over a 3 min run time. Retention times of 1.61 and 1.59 min were observed for clopidogrel and 2H3-clopidogrel (I.S.), respectively. Detection was achieved using a Sciex API 4000, triple quadrupole mass spectrometer, in positive TurboIonspray (electrospray) ionisation mode. Ion transitions were monitored using MRM (multiple reaction monitoring) for clopidogrel (m/z 322-212) and for 2H3-clopidogrel (m/z 327-217). This validated method was used to support a pharmacokinetic study in healthy volunteers.
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