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LC method for determination of prasugrel and mass spectrometry detection for thermal and alkaline degradation products
Abstract
A stability-indicating RP-LC method for the determination of prasugrel in tablets was developed and validated. Stress testing of prasugrel was carried out in accordance with ICH guidelines, where the drug was submitted to acidic and basic hydrolysis, oxidative, thermal and photolytic conditions. Prasugrel was unstable under all the conditions and the degradations products were analyzed by HPLC-UV. Furthermore, two main degradation products found under alkaline and thermal conditions were investigated by LC-MS. Based on the fragmentation patterns, two products resulted from hydrolysis of the acetate ester moiety of prasugrel were observed. Due the chemical equilibrium, tautomerism occurs between the ketone and alcohol functions justifying the similar molecular weight and fragment pattern obtained in degradation products analysis. Successful separation was achieved on a RP-18 octadecyl silane column using acetonitrile and triethylamine 0.5% mixture (50:50, v/v) as the mobile phase at 25 °C. The flow rate was 1.0 mL/min and the detector wavelength was 263 nm. The method proposed in this work was successfully applied to quality control of prasugrel and contribute to stability assessment of pharmaceutical products containing this drug.
... Prasugrel is a prodrug of piperazine derivative ( Figure 1) that inhibits platelet aggregation in vivo by antagonism of the P2Y12 class of platelet purinergic receptors [1][2][3] . Subsequent studies showed that the conversion of active metabolite is mediated largely by Cytochrome (CYP) 3A4 and CYP2B6 and, to a lesser extent, by CYP2C9 and CYP2C19 [4,5] . ...
... Prasugrel was administered orally at a dose of 30 mg/kg in 0.25% sodium Carboxy Methyl Cellulose (CMC) suspension. Blood samples were collected from the retro-orbital plexus of rats under light ether anesthesia into microfuge tubes containing heparin as an anti-coagulant at 0.5, 1,2,3,4,5,8,10,12 and 24 h post-dosing. Plasma was harvested by centrifuging the blood at 2500×g for 5 min at 20˚C and stored frozen at −80 ± 10˚C until analysis. ...
... The retention time for prasugrel HCl was 11.5 min. Rigobello et al. [48] a stability indicating that RP-LC method for the determination of prasugrel in tablets was developed and validated. Stress testing of prasugrel was carried out in accordance with ICH guidelines, where the drug was submitted to acidic and basic hydrolysis, oxidative, thermal, and photolytic conditions. ...
A review is presented on different analytical techniques used for quantitative analysis of selected anticoagulants dabigatran, rivaroxaban, and prasugrel. Efforts have been made to collate all the relevant references to the extent possible. The review discusses the advantages and disadvantages of the cited analytical techniques, which will help to give insights into the methods used for estimation of selected anticoagulants such as dabigatran, rivaroxaban, and prasugrel, from clinical isolates, and its dosage forms. The review highlights the basic as well as advanced techniques performed for estimating dabigatran, rivaroxaban, and prasugrel. The techniques illustrated here have been demonstrated to be useful for quantitative determination of selected anticoagulants and may find application in analyzing other related properties.
Current study develops and validates a dissolution test for Prasugrel hydrochloride 10 mg in coated tablets. After sink condition, filters and drug stability were evaluated, the discriminatory dissolution conditions were achieved with a USP apparatus 1 (basket) at 50 rpm stirring speed and 900 mL of 0.01 M HCl as dissolution medium. The UV spectrometric method at 220 nm was performed and validated for the determination of Prasugrel. The parameters specificity, linearity, accuracy, precision and robustness were evaluated according to international protocols. UV method and dissolution test proposed in current analysis may be applied for quality control of coated tablets containing Prasugrel since there is no official monograph for this drug. © 2014, Eduem - Editora da Universidade Estadual de Maringa. All rights reserved.
Forced degradation is a degradation of new drug substance and drug product at conditions more severe than accelerated conditions. It is required to demonstrate specificity of stability indicating methods and also provides insight into degradation pathways and degradation products of the drug substance and helps in elucidation of the structure of the degradation products. Forced degradation studies show the chemical behavior of the molecule which in turn helps in the development of formulation and package. In addition, the regulatory guidance is very general and does not explain about the performance of forced degradation studies. Thus, this review discusses about the current trends in performance of forced degradation studies by providing strategy for the conduct of studies, degradation mechanisms and also describes the analytical methods helpful for development of stability indicating method.
A simple, sensitive, specific, accurate, and stability-indicating reversed phase high performance liquid chromatographic method was developed for the simultaneous determination of aspirin and prasugrel, using a Kromasil 100 C18 (150×4.6 mm, 5 μ) column and a mobile phase composed of acetonitrile:methanol:water (30:10:60, v/v), pH 3.0 adjusted with o-phosphoric acid. The retention times of aspirin and prasugrel were found to be 3.28 min and 6.61 min, respectively. Linearity was established for aspirin and prasugrel in the range of 15-150 and 2-20 μg/ml, respectively. The percentage recoveries of aspirin and prasugrel were found to be in the range of 99.34-100.32% and 98.92-102.09%, respectively. Both the drugs were subjected to acid, alkali and neutral hydrolysis, oxidation, dry heat, and UV degradation. The degradation studies indicated aspirin to be more susceptible to alkaline hydrolysis, while prasugrel to be more susceptible to neutral hydrolysis. The degradation products were well resolved from the pure drug with significant differences in their retention time values. This method can be successfully employed for simultaneous quantitative analysis of aspirin and prasugrel in bulk drugs and formulations.
A simple, sensitive and reproducible gradient reverse-phase ultra performance liquid chromatographic (UPLC) method was developed for the quantitative determination of Prasugrel and all possible process-related impurities (positional isomers, degradants and byproducts) at the level of 0.5 µ g mL to 2.0 µ g mL . The main objective of this study is to control the impurities at crude and final stages of prasugrel hydrochloride. This method is applicable to Drug substance and pharmaceutical dosage forms. Six potential impurities were formed during the process development including the degradation products and the desacetyl impurity is existing in its keto and enol form was stabilized by using acidic pH. All these impurities were well separated primarily with a gradient HPLC method, based on their selectivity differences by using a polar embedded C8 column, monobasic potassium buffer, a basic organic modifier and acetonitrile in the mobile phase. Initially structures of all these impurities formed were identified by liquid chromatography equipped with mass spectrometer and further confirmed by spiking with the characterized impurities. The drug was subjected to International Conference on Harmonization (ICH) prescribed hydrolytic, oxidative, photolytic and thermal stress conditions. The performance of the method was validated according to the present ICH guidelines.
Drug impurity profiling, i.e. identification, structure elucidation and quantitative determination of impurities
and degradation products in bulk drug materials and pharmaceutical formulations is one of the most important fields of activities in modern pharmaceutical analysis. The reason for the increased importance of this area is that unidentified, potentially toxic impurities are health hazards and in order to increase the safety of drug therapy, impurities should be identified and determined by selective methods.
The number of papers dealing with drug impurity profiling is high and the rate is continuously increasing. The aim of this review is to characterise the state-of-the-art in the field of impurity profiling of drugs based mainly on papers published in the last 5 years.
The main areas, discussed in this review are separation and determination of impurities with known structure, off-line and on-line chromatographic – spectroscopic methods for the structure elucidation of impurities and degradation products as well as some selected topics such as impurity issues in compendia, genotoxic impurities and analytical aspects of enantiomeric purity of chiral drugs.
The points of view of the selection of papers for the review and their discussion are
• papers with interesting chemical background i.e. where the impurities are products of interesting side reactions of the synthesis or products of interesting degradation reactions, and
• papers where up to date on-line methods (HPLC-MS, HPLC-NMR, etc.) played predominant role in the structure elucidation.
A novel stability-indicating ultra-performance liquid chromatographic (UPLC) assay method was developed and validated for prasugrel and its degradation products. The UPLC separation was performed on Acquity® UPLC BEH C18 column (1.7 µm, 2.1 mm × 150 mm) using isocratic mode (acetonitrile:water, 80:20 v/v) at flow rate of 0.1 mL/min and the high performance liquid chromatography (HPLC) separation was achieved on Phenomenex® C8 column using isocratic mode (acetonitrile:10 mM ammonium acetate, 85:15 v/v) at flow rate of 0.9 mL/min. Prasugrel was found to degrade significantly in hydrolytic (acid, alkali, and neutral), and oxidative stress conditions and was stable in thermal and photolytic stress conditions. The RSD (%) values calculated for the AUC of UPLC and HPLC are 0.0039 and 0.0015, respectively. The UPLC and HPLC linearity of the proposed method were investigated in the range of 10–60 µg/mL. The r2 value of UPLC and HPLC were found to be 0.9980 and 0.9983, respectively. Method detection limit (MDL) and method quantification limit (MQL) were found to be 0.20 µg/mL and 1.00 µg/mL for UPLC and 0.50 µg/mL and 1.80 µg/mL for HPLC, respectively. The RSD (%) values for intra-day and inter-day precision were <1.0%, confirming that the method is sufficiently precise. The validation studies were carried out fulfilling ICH requirements.
A simple, rapid and precise method was developed for the quantitative estimation of prasugrel hydrochloride in pharmaceutical dosage form. A chromatographic separation of prasugrel and its degradants was achieved with Zorbax XDB C(8), 150 × 4.6 mm, 3.5μm analytical column using aqueous solution of 0.05 M ammonium acetate pH 4.5 with acetic acid-acetonitrile (40:60 v/v). The instrumental settings include flow rate of 1.0 ml/min, column temperature at 30°C and detector wavelength of 254 nm using a photodiode array detector. Theoretical plates for prasugrel were 7023. Tailing factor for prasugrel was 1.11. Prasugrel was exposed to thermal, photolytic, hydrolytic and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. Peak homogeneity data of prasugrel was obtained using photodiode array detector in the stressed sample chromatograms, which demonstrated the specificity of the method for the estimation in presence of degradants. The described method showed excellent linearity over a range of 10-300 μg/ml for prasugrel. The correlation coefficient is 0.999. The relative standard deviation of peak area for six measurements is always less than 2%. Overall, the proposed method was found to be suitable and accurate for quantitative determination and stability study of prasugrel in pharmaceutical dosage form.
Platelet adhesion, activation and aggregation play a pivotal role in atherothrombosis. Intracoronary atherothrombosis is the most common cause of the development of acute coronary syndrome (ACS), and plays a central role in complications occurring around percutaneous coronary intervention (PCI) including recurrent ACS, procedure-related myocardial infarction or stent thrombosis. Inhibition of platelet aggregation by medical treatment impairs formation and progression of thrombotic processes and is therefore of great importance in the prevention of complications after an ACS or around PCI. An essential part in the platelet activation process is the interaction of adenosine diphosphate (ADP) with the platelet P2Y12 receptor. The P2Y12 receptor is the predominant receptor involved in the ADP-stimulated activation of the glycoprotein IIb/IIIa receptor. Activation of the glycoprotein IIb/IIIa receptor results in enhanced platelet degranulation and thromboxane production, and prolonged platelet aggregation. The objectives of this review are to discuss the pharmacological limitations of the P2Y12 inhibitor clopidogrel, and describe the novel alternative P2Y12 inhibitors prasugrel and ticagrelor and the clinical implications of the introduction of these new medicines.
2-Acetoxy-5-(alpha-cyclopropylcarbonyl-2-fluorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (prasugrel) is a novel thienopyridine prodrug with demonstrated inhibition of platelet aggregation and activation. The biotransformation of prasugrel to its active metabolite, 2-[1-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid (R-138727), requires ester bond hydrolysis, forming the thiolactone 2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone(R-95913), followed by cytochrome P450-mediated metabolism to the active metabolite. The presumed role of the human liver- and intestinal-dominant carboxylesterases, hCE1 and hCE2, respectively, in the conversion of prasugrel to R-95913 was determined using expressed and purified enzymes. The hydrolysis of prasugrel is at least 25 times greater with hCE2 than hCE1. Hydrolysis of prasugrel by hCE1 showed Michaelis-Menten kinetics yielding an apparent K(m) of 9.25 microM and an apparent V(max) of 0.725 nmol product/min/microg protein. Hydrolysis of prasugrel by hCE2 showed a mixture of Hill kinetics at low substrate concentrations and substrate inhibition at high concentrations. At low concentrations, prasugrel hydrolysis by hCE2 yielded an apparent K(s) of 11.1 microM, an apparent V(max) of 19.0 nmol/min/microg, and an apparent Hill coefficient of 1.42, whereas at high concentrations, an apparent IC(50) of 76.5 microM was obtained. In humans, no in vivo evidence of inhibition exists. In vitro transport studies using the intestinal Caco-2 epithelial cell model showed a high in vivo absorption potential for prasugrel and rapid conversion to R-95913. In conclusion, the human carboxylesterases efficiently mediate the conversion of prasugrel to R-95913. These data help explain the rapid appearance of R-138727 in human plasma, where maximum concentrations are observed 0.5 h after a prasugrel p.o. dose, and the rapid onset of action of prasugrel.
A new visible spectrophotometric method has been developed and validated for the estimation of Prasugrel hydrochloride in bulk, pharmaceutical dosage form and in biological fluids. The method makes use of ion pair complex formation between drug and bromophenol blue (BPB). The complex of Prasugrel showed λ max at 416 nm. A good linearity with correlation coefficient within the limit was observed for the drugs at the concentration range of 50-100 μg/ml for Prasugrel. The reagents used were optimized. The developed methods were assessed for precision, accuracy, sensitivity. Thus a simple, easy to perform, economical, precise and accurate visible spectrophotometric method has been developed for the estimation of Prasugrel in bulk, biological and solid oral dosage form.
A new, simple, specific, accurate, precise, and rapid reverse phase high performance liquid chromatographic method was developed and validated for the determination of prasugrel Hydrochloride in pure and tablet dosage forms. The HPLC separation was carried out by reverse phase chromatography on Kromasil C18 (100 x 4.6mm; 5μm) with a mobile phase consist of methanol buffer (680mg potassium dihydrogen phosphate in 500 ml water, pH-2.1 adjusted with ortho phosphoric acid) in the ratio of 70:30 v/v delivered in isocratic mode at a flow rate of 0.8 ml/min. The prasugel Hydrochloride was quantified at 220nm. The retention time of Prasugrel Hydrochloride was 1.9 min. The developed method was validated according to ICH guidelines. The interday and intraday precision were found to be within limits. The developed method has adequate sensitivity and specificity for the determination of prasugrel Hydrochloride in bulk and its tablet dosage forms. Accuracy (recoveries: 98.92-101.19%) and reproducibility were found to satisfactory. The developed method was found to be cost effective and was successfully employed for the determination of Prasugrel Hydrochloride in various pharmaceutical preparations.
A stability indicating HPLC assay method has been developed and validated for the estimation of prasugrel hydrochloride in bulk and pharmaceutical dosage form. A RP-HPLC isocratic separation was achieved on C18 column (250×4.6 mm, 5μm) utilizing a mobile phase comprising of methanol and acetonitrile in the ratio of 90: 10 (v/v) and the eluents from the column were detected using a variable wavelength detector at 240 nm. The stress testing of prasugrel hydrochloride was carried out under acidic, alkaline, neutral hydrolysis, oxidation, photolytic and thermal degradation (dry heat) conditions and prasugrel hydrochloride was well resolved from its degradation products. The proposed method has permitted the quantification of prasugrel hydrochloride in the linearity range of 10-160 μg/ml and the flow rate was maintained at 1ml/min. The column was maintained at ambient temperature and the complete separation was achieved for prasugrel hydrochloride with all degradation products in an overall analytical run time of approximately 15 min. The retention time of prasugrel hydrochloride was found to be 3.78 min. The method was validated as per ICH guidelines.
To develop and validate a simple, sensitive, precise and specific reverse phase high performance liquid chromatographic method for the determination of prasugrel in bulk and tablet dosage forms. The HPLC separation was carried out by reverse phase chromatography on inertsil ODS-3V column (5μm; 250x4.6mm) with a mobile phase composed of 0.02M potassium dihydrogen orthophosphate, 0.02M dipotassium hydrogen orthophosphate in water: Acetronitrile (30:70 v/v) in isocratic mode at a flow rate of 1ml/min. The detection was monitored at 210nm. The calibration curve for prasugrel was linear from 100 to 600ng/ml. The interday and intraday precision was found to be within limits. The proposed method has adequate sensitivity, reproducibility and specificity for the determination of prasugrel in bulk and its tablet dosage forms. LOD and LOQ for prasugrel were found to be 0.25 μg/ml and 0.75 μg /ml respectively. Accuracy (recoveries: 99.8-101.2%) and reproducibility were found to satisfactory. The proposed method is simple, fast, accurate and precise for the simultaneous quantification of Prasugrel in dosage form, bulk drugs as well as for routine analysis in quality control.
A reverse-phase liquid chromatographic (RP-LC) method was developed for the assay of Prasugrel in bulk. The Chromatography was performed on Kromasil C18 column. The eluted compounds were monitored by UV detection at 257nm using mobile phase methanol-potassium dihydrogen orthophosphate (pH 2.2; 10mM) (70:30, v/v). The method was statistically validated for linearity, accuracy, precision and repeatability. The linearity of prasugrel was demonstrated in concentration range 15-75µg/mL. The limit of detection and quantitation were 10 and 50ng/mL. The method developed was precise, accurate and specific for estimation of prasugrel in bulk.
Aim:
To characterize the pharmacokinetics (PKs), pharmacodynamics (PDs), and tolerability of different dose regimens of prasugrel in healthy Chinese subjects.
Methods:
This was a single-centered, open-label, parallel-design study. Subjects received a single loading dose (LD) of prasugrel followed by once-daily maintenance dose (MD) for 10 d. They were enrolled into 3 groups: 60 mg LD/10 mg MD; 30 mg LD/7.5 mg MD; 30 mg LD/5 mg MD. Blood samples were collected after the first and last dose. The serum concentration of the active metabolite of prasugrel was determined using a LC/MS/MS method. Platelet aggregation was assessed using the VerifyNow-P2Y12 assay.
Results:
Thirty-six healthy native Chinese subjects (19 males) aged 18–45 were enrolled; mean age and body weight were similar across the treatment groups (n=12 for each). The metabolite AUC0–4 and Cmax increased dose-proportionally across the dose range of 5 mg to 60 mg. The median Tmax was 0.5 h in all groups. The PD parameters, indicated by the inhibition of ADP-induced platelet aggregation, were met more rapidly in the 60 mg group than the 30 mg group after the LD (94%–98%). This high degree of inhibition of platelet aggregation was maintained following the 10 mg MD (87%–90%) and was lower in the 7.5 mg and 5 mg MD groups (79%–83% and 64%–67%, respectively). Prasugrel was well tolerated in healthy Chinese subjects for single doses up to 60 mg and a MD of 10 mg for 10 d.
Conclusion:
The PKs and PDs of the active metabolite of prasugrel were similar to those in Chinese subjects reported by a previous bridging study, which demonstrated that the exposure to the active metabolite in Chinese subjects was higher than in Caucasians.
In this review, the set-up and data interpretation of experimental designs (screening, response surface, and mixture designs) are discussed. Advanced set-ups considered are the application of D-optimal and supersaturated designs as screening designs. Advanced data interpretation approaches discussed are an adaptation of the algorithm of Dong and the estimation of factor effects from supersaturated design results. Finally, some analytical applications in separation science, on the one hand, and formulation-, product-, or process optimization, on the other, are discussed.
Despite recent advances in the treatment of acute coronary syndromes (ACS), including dual antiplatelet therapy with aspirin and a thienopyridine during the acute phase and for secondary prevention, this condition remains a leading cause of morbidity and mortality. The limitations of the currently available antiplatelet agents have triggered the development of newer drugs. In this review we summarize the mechanisms of actions and results of current clinical trials of novel antiplatelet agents. These include prasugrel, a thienopyridine prodrug which has a mechanism similar to that of clopidogrel but superior pharmacokinetic features; ticagrelor, a non-thienopyridine that binds reversibly to the platelet P2Y(12) receptor; cangrelor, an intravenously administered analog of ticagrelor; the thrombin receptor antagonist SCH 53048; and terutroban (S18886), a thromboxane A(2) receptor inhibitor.
Two fast and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based bioanalytical assays were developed and validated to quantify the active and three inactive metabolites of prasugrel. Prasugrel is a novel thienopyridine prodrug that is metabolized to the pharmacologically active metabolite in addition to three inactive metabolites, which directly relate to the formation and elimination of the active metabolite. After extraction and separation, the analytes were detected and quantified using a triple quadrupole mass spectrometer using positive electrospray ionization. The validated concentration range for the inactive metabolites assay was from 1 to 500 ng/mL for each of the three analytes. Additionally, a 5x dilution factor was validated. The interday accuracy ranged from -10.5% to 12.5% and the precision ranged from 2.4% to 6.6% for all three analytes. All results showed accuracy and precision within +/-20% at the lower limit of quantification and +/-15% at other levels. The validated concentration range for the active metabolite assay was from 0.5 to 250 ng/mL. Additionally, a 10x dilution factor was validated. The interbatch accuracy ranged from -7.00% to 5.98%, while the precision ranged from 0.98% to 3.39%. Derivatization of the active metabolite in blood with 2-bromo-3'-methoxyacetophenone immediately after collection was essential to ensure the stability of the metabolite during sample processing and storage. These methods have been applied to determine the concentrations of the active and inactive metabolites of prasugrel in human plasma.
To assure the quality of drugs, impurities must be monitored carefully. It is important to understand what constitutes an impurity and to identify potential sources of such impurities. Selective analytical methods need to be developed to monitor them. It is generally desirable to profile impurities to provide a yardstick for comparative purposes. New impurities may be observed as changes are made in the synthesis, formulation, or production procedures, albeit for improving them. At times it is necessary to isolate and characterize an impurity when hyphenated methods do not yield the structure or when confirmation is necessary with an authentic material. Availability of an authentic material can also allow toxicological studies and provide a standard for routine monitoring of the drug product.
A liquid chromatography-tandem mass spectrometry method was developed to chromatographically separate the four stereoisomers of the active metabolite of prasugrel, R-138727, in human plasma after derivatization with bromomethoxyacetophenone to stabilize the molecule. This technique was designed to determine the relative contribution of each stereoisomer, based on statistical analyses of each stereoisomer's chromatographic peak areas. The methodology was validated and used for the analysis of clinical samples in which R-138727 had been derivatized at the time of blood collection. This technique can be useful to determine the ratios of stereoisomers in biological samples (e.g., plasma) especially in situations in which authentic standards of each individual stereoisomer are scarce or unavailable. In humans, the metabolic formation of R-138727 from prasugrel was found to be stereoselective, where 84% of R-138727 was present as RS and RR, the two most pharmacologically potent isomers, whereas the SR and SS enantiomers accounted for approximately 16%. The ratios of the R-138727 stereoisomers were consistent among subjects, regardless of the dose or time of sample collection or whether the blood was sampled after the first dose or after 4 weeks of therapy.