Detection of falsified clopidogrel in the presence of excipients using voltammetry.

There has been a recent surge in the amount of substandard and falsified clopidogrel. Pharmacopeial based assays using high performance liquid chromatography and mass spectroscopy are widely used for the measurement of clopidogrel but are not accessible in low to middle income countries. Therefore, our study explored four different techniques (mid-infrared spectroscopy, thin layer chromatography, ultraviolet visible spectroscopy, and differential pulse voltammetry), which could be used in low to middle income countries. Differential pulse voltammetry showed the best performance for accurate and precise determination of clopidogrel in the presence of excipients. Clopidogrel tablets were fully crushed and sonicated in buffer for 30 seconds prior to differential pulse voltammetry measurements using a 3 mm glassy carbon electrode. Measurements were made without removing the excipients and the limit of detection was 0.08 mg ml-1 and the sensitivity was 15.7 µA mg ml-1. When conducting a blinded study, differential pulse voltammetry was able to identify varying types of substandard and falsified samples. Our findings highlight that voltammetry could be a vital analytical technique for the determination of substandard and falsified medicines in low- and middle-income countries.

Comparison of acetylsalicylic acid and clopidogrel non-responsiveness assessed by light transmittance aggregometry and PFA-100® in patients undergoing neuroendovascular procedures.

Objectives: Dual platelet inhibition is commonly used for prevention of cardiovascular events in patients undergoing neuroendovascular procedures. Non-responsiveness to platelet inhibitors may be associated with adverse outcomes. The aim of this study was to evaluate the reliability of the platelet function analyzer PFA-100® in comparison to light transmittance aggregometry (LTA) for monitoring clopidogrel and acetylsalicylic acid (ASA) non-responsiveness in a cohort of patients treated for intracranial aneurysm or cranial artery stenosis. Methods: Non-responsiveness to clopidogrel and ASA was assessed by LTA using adenosine diphosphate (ADP) and arachidonic acid and by PFA-100® with the ADP/prostaglandin E1 (PGE1) and collagen/epinephrine cartridges, respectively. Results: A total of 203 patients (145 females; median age, 57 years) were analyzed. Agreement between the two tests was poor for clopidogrel non-responsiveness (ƙ=0.19) and not better than chance for ASA non-responsiveness (ƙ=0.01). Clopidogrel non-responsiveness by LTA and PFA-100® was associated with higher von Willebrand factor antigen and activity levels. ADP-induced platelet disaggregation was lower in patients with clopidogrel non-responsiveness as assessed by PFA-100®. Clopidogrel non-responsiveness by LTA was associated with a higher prevalence of diabetes and a higher body mass index (BMI). Adverse outcomes (death, thromboembolism, or in-stent thrombosis) occurred in 13% (n=26) of all patients independently of ASA and clopidogrel non-responsiveness as assessed by both devices. Conclusions: Our results show that LTA and PFA-100® are not interchangeable in the assessment of ASA and clopidogrel non-responsiveness in patients undergoing neuroendovascular interventions.

Box-Behnken design directed optimization for sensitivity assessment of anti-platelet drugs.

Optimization of electrospray ionization (ESI) parameters is routinely carried out by one factor at a time (OFAT) or auto-tune software (ATS). Design of experiments (DOE) approach has been reported to be an excellent alternative to OFAT or ATS. Box-Behnken Design (BBD) was successfully used to optimize ESI parameters like nebulizing gas flow rate, desolvation line temperature, heat block temperature, and drying gas flow rate for [M + H]+ intensity of Clopidogrel bisulfate (CLP) and Ticlopidine (TLP). BBD model was found to be significant with p < .0001 for both CLP and TLP. The predicted and optimized (OL) ESI parameters were used for chromatographic analysis and were compared against three levels of ESI parameters, i.e. low level (LL), medium level (ML), and high level (HL). The OL ESI parameters were subjected to chromatographic analysis and its mean peak area was significantly higher than mean peak area for LL, ML, and HL ESI in case of CLP and TLP (p < .001). However, no significant difference was observed between the mean peak area for ML and OL of TLP. Thus, BBD can be considered with 29 trials to optimize four mass spectrometric parameters. The liquid chromatographic parameters percentage of methanol, percentage of formic acid and flow rate were also optimized using BBD. However, the optimized method did not significantly influence the peak response over the non-optimized method.

Silver nanoparticles impregnated chitosan layered carbon nanotube as sensor interface for electrochemical detection of clopidogrel in-vitro.

Continuous periodical monitoring of clopidogrel in physiological body fluids is indispensable in medical diagnosis of heart ailments and cardiovascular diseases. A highly sensitive electrochemical sensor has been fabricated with silver nanoparticles embedded chitosan-carbon nanotube hybrid composite (AgChit-CNT) as sensor interface for detection of the important anti-platelet drug, clopidogrel (CLP). Synthesized AgChit-CNT nanocomposite is examined by x-ray diffraction, Raman spectroscopy and field emission scanning electron microscopy for its chemical and structural characteristics. Crystalline silver nanoparticles of about 35 nm are well distributed in the composite and have formed continuous chain like linkages with CNTs all throughout. Electrochemical responses of the fabricated AgChit-CNT nanocomposite electrode for the determination of CLP have been examined by cyclic voltammetry and electrochemical impedance spectroscopy. The nanoAg patterned CNT nanocomposite interface acts as an excellent electron transfer mediator towards the oxidation of clopidogrel. Electrochemical determination of CLP was investigated by differential pulse voltammetry (DPV) and amperometric analysis under optimized conditions. The limit of detection by DPV and amperometry were 30 nM and 10 nM, respectively, and the time of the analysis is as low as 10 s. Practical applicability for determination in artificially prepared urine and pharmaceutical formulation has been examined with good recovery limits of 95.2 to 102.6%.

Inorganic analysis of falsified medical products using X-ray fluorescence spectroscopy and chemometrics.

Falsified medical products are increasingly prevalent on markets, threatening the health of patients. This study describes the benefits of Energy Dispersive X-Ray Fluorescence (ED-XRF) spectroscopy and chemometrics thus highlighting the importance of conducting inorganic analyses on falsified products. The XRF spectrum is a fingerprint containing the contribution of all chemical substances included in a suspect sample's formulation. Multivariate analysis of XRF spectra, using a properly validated classification model, allows for the authentication of suspect samples. The method is rapid, relying on multi-elemental measurements and involving minimal sample preparation. This methodology provided valuable information about samples inorganic composition and enabled the detection of falsifications of several sample types, including medicine, food supplement and cosmetic samples. Five suspect samples of Plavix® were investigated, and their XRF spectra were studied using chemometrics (Principal Component Analysis and Soft Independent Modelling of Class Analogies). A classification model was validated with positive and negative samples, and four suspect samples were identified as being falsified, whilst the fifth was concluded as an authentic medicine. ED-XRF spectroscopy was also applied on another medicine, a food supplement and three cosmetic samples, and high level of zinc was detected in the second sample and mercury was identified in the last. Estimation of the zinc content was possible using the fundamental parameters method. ED-XRF spectroscopy allows the analyst to conclude on the falsification of the samples and then to assess the harm to patient health.

Development of stability-indicating HPLC method and accelerated stability studies for osmotic and pulsatile tablet formulations of Clopidogrel Bisulfate.

The purpose of the present study is to develop a simple, rapid and sensitive stability-indicating high-performance liquid chromatography (HPLC) method for Clopidogrel Bisulfate (CBS) and further extending it for assessment of CBS stability in osmotic and pulsatile tablet formulations tested under accelerated conditions. A stability-indicating HPLC method for quantitative determination of CBS in gastro-retentive formulations is developed by using a C18 HPLC column, acetonitrile and 0.1% formic acid (60:40 v/v) as mobile phase, with a flow rate of 0.9 mL/min, UV detection at 222 nm and subsequently validated. The key objective was to analyze the stability profile of formulations under accelerated conditions. The retention time (Rt) of CBS was observed as 5.9 min with the linearity range between 0.06-1.95 µg/mL. Forced degradation studies were performed on bulk samples of CBS using acidic, basic, oxidative, thermal (80 °C) and photolytic (under sunlight) conditions. The resulting method was validated as per ICH Q2(R1) guidelines. Moreover, an attempt has been made to identify the degradation products by Liquid chromatography-mass spectrometry (LC-MS) analysis. The proposed method was successfully applied to novel gastro-retentive tablet formulations (osmotic tablet and pulsatile tablet) for assessment of stability under accelerated conditions.