Nejal M. Bhatt

Surface plasmon resonance based selective and sensitive colorimetric determination of azithromycin using unmodified silver nanoparticles in pharmaceuticals and human plasma

In this article we report a novel method for colorimetric sensing and selective determination of a non-chromophoric drug-azithromycin, which lacks native absorbance in the UV-Visible region using unmodified silver nanoparticles (AgNPs). The citrate-capped AgNps dispersed in water afforded a bright yellow colour owing to the electrostatic repulsion between the particles due to the presence of negatively charged surface and showed surface plasmon resonance (SPR) band at 394nm. Addition of positively charged azithromycin at a concentration as low as 0.2μM induced rapid aggregation of AgNPs by neutralizing the negative charge on the particle surface. This phenomenon resulted in the colour change from bright yellow to purple which could be easily observed by the naked eye. This provided a simple platform for rapid determination of azithromycin based on colorimetric measurements. The factors affecting the colorimetric response like pH, volume of AgNPs suspension and incubation time were suitably optimized. The validated method was found to work efficiently in the established concentration range of 0.2-100.0μM using two different calibration models. The selectivity of the method was also evaluated by analysis of nanoparticles-aggregation response upon addition of several anions, cations and some commonly prescribed antibiotics. The method was successfully applied for the analysis of azithromycin in pharmaceuticals and spiked human plasma samples with good accuracy and precision. The simplicity, efficiency and cost-effectiveness of the method hold tremendous potential for the analysis of such non-chromophoric pharmaceuticals.

Manipulating Ratio Spectra for the Spectrophotometric Analysis of Diclofenac Sodium and Pantoprazole Sodium in Laboratory Mixtures and Tablet Formulation

Objective. Three sensitive, selective, and precise spectrophotometric methods based on manipulation of ratio spectra, have been developed and validated for the determination of diclofenac sodium and pantoprazole sodium. Materials and Methods. The first method is based on ratio spectra peak to peak measurement using the amplitudes at 251 and 318 nm; the second method involves the first derivative of the ratio spectra (Δλ = 4 nm) using the peak amplitudes at 326.0 nm for diclofenac sodium and 337.0 nm for pantoprazole sodium. The third is the method of mean centering of ratio spectra using the values at 318.0 nm for both the analytes. Results. All the three methods were linear over the concentration range of 2.0–24.0 μg/mL for diclofenac sodium and 2.0–20.0 μg/mL for pantoprazole sodium. The methods were validated according to the ICH guidelines and accuracy, precision, repeatability, and robustness are found to be within the acceptable limit. The results of single factor ANOVA analysis indicated that there is no significant difference among the developed methods. Conclusions. The developed methods provided simple resolution of this binary combination from laboratory mixtures and pharmaceutical preparations and can be conveniently adopted for routine quality control analysis.

Simultaneous analysis of aliskiren and hydrochlorothiazide in pharmaceutical preparations and spiked human plasma by HPTLC

Abstract A simple, selective and precise method based on HPTLC has been developed for the simultaneous determination of aliskiren and hydrochlorothiazide in a fixed-dose tablet formulation and human plasma. The chromatography was performed on silica gel 60 GF254 plates, with a mobile phase consisting of methanol–chloroform (6:4, v/v). Densitometric analysis of the analytes was carried out at 225 nm. Under optimized conditions, the Rf values were 0.26 ± 0.02 and 0.71 ± 0.02, and the resulting regression plots were linear (r2 ≥ 0.9997) in the concentration ranges of 1.00–10.0 and 0.10–1.00 μg band−1 for aliskiren and hydrochlorothiazide. The limit of detection and limit of quantitation of the validated method were 0.206 and 0.624 μg band−1 for aliskiren and 0.015 and 0.046 μg band−1 for hydrochlorothiazide, respectively. The % expected content of aliskiren and hydrochlorothiazide in the commercial tablet formulation was 99.2% and 101.3%, respectively. For spiked plasma sample preparation, the analytes and nebivolol internal standard were extracted from 500 μL of plasma sample by solid-phase extraction on LiChrosep® DVB-HL cartridges. The mean extraction recovery of aliskiren and hydrochlorothiazide from human plasma was 87.2% and 76.5%, respectively. In addition, the stability of the analytes in plasma was established under different storage conditions.

Combining simplicity with cost-effectiveness: Investigation of potential counterfeit of proton pump inhibitors through simulated formulations using thin-layer chromatography

A simple, accurate and precise high-performance thin-layer chromatographic method has been developed and validated for the analysis of proton pump inhibitors (PPIs) and their co-formulated drugs, available as binary combination. Planar chromatographic separation was achieved using a single mobile phase comprising of toluene: iso-propranol: acetone: ammonia 5.0:2.3:2.5:0.2 (v/v/v/v) for the analysis of 14 analytes on aluminium-backed layer of silica gel 60 FG254. Densitometric determination of the separated spots was done at 290nm. The method was validated according to ICH guidelines for linearity, precision and accuracy, sensitivity, specificity and robustness. The method showed good linear response for the selected drugs as indicated by the high values of correlation coefficients (≥0.9993). The limit of detection and limit of quantiation were in the range of 6.9-159.2ng/band and 20.8-478.1ng/band respectively for all the analytes. The optimized conditions afforded adequate resolution of each PPI from their co-formulated drugs and provided unambiguous identification of the co-formulated drugs from their homologous retardation factors (hRf). The only limitation of the method was the inability to separate two PPIs, rabeprazole and lansoprazole from each other. Nevertheless, it is proposed that peak spectra recording and comparison with standard drug spot can be a viable option for assignment of TLC spots. The method performance was assessed by analyzing different laboratory simulated mixtures and some marketed formulations of the selected drugs. The developed method was successfully used to investigate potential counterfeit of PPIs through a series of simulated formulations with good accuracy and precision.

Optimization of a novel high-performance thin-layer chromatographic method for the concurrent estimation of three proton-pump inhibitors and diclofenac in their binary combinations

In the present study, a simple and efficient high-performance thin-layer chromatographic (HPTLC) method was developed for the separation and quantitation of three proton-pump inhibitors, omeprazole, pantoprazole, and rabeprazole, from their binary combinations with diclofenac. Using a “quality by design” approach, preliminary trials were performed on pre-coated silica gel HPTLC plates using toluene together with various alcohols (methanol, ethanol, iso-propanol, n-butanol) as the mobile phase. For better peak symmetry, ammonia was added in different volumes, and its effect on analyte retention and separation was also assessed. The mobile phase consisting of toluene-n-butanol-25% ammonia (3:7:0.2, v/v) afforded excellent separation of proton-pump inhibitors from diclofenac as well as from each other. The retardation factor (RF) for all the separated compounds was between 0.20 and 0.80. The developed method was successfully validated as per the International Conference on Harmonization (ICH) guidelines, and the selected drugs were determined simultaneously from dosage forms without any interference from the excipients.

Influence of organic modifier and separation modes for lipophilicity assessment of drugs using thin layer chromatography indices

Lipophilicity constitutes one of the most important physicochemical properties in the design and development of drug molecules. In the present work thin layer chromatography (TLC) has been utilized to evaluate lipophilicity of 11 representative drugs, which included six proton pump inhibitors (omeprazole, pantoprazole, rabeprazole, lansoprazole, ilaprazole, and tenatoprazole), an anti-vertigo drug, betahistine, nonsteroidal anti-inflammatory drug, ibuprofen, anti-malarial drug, atovaquone, an anti-HIV agent, atazanavir and a hormonal drug, calcitriol. Normal as well as reversed-phase separation modes were evaluated to study the effect of different organic modifiers for the estimation of lipophilicity. The quantitative descriptor of lipophilicity, the partition coefficient (logP) was estimated by suitably optimizing the solvent systems for both the modes. The best mobile phase pairs for NPTLC and RPTLC were toluene-acetonitrile and water-methanol respectively. Principal component analysis, hierarchical cluster analysis, as well as non-parametric methods like sum of ranking differences and generalized pair wise correlation revealed the dominant pattern in the data. The results obtained from both the separation modes were comparable and were in good agreement with the computational data for all the drugs.

The first normal-phase high-performance thin-layer chromatographic method for the simultaneous determination of the antimalarial drugs atovaquone and proguanil from Malarone® tablets

Atovaquone (ATQ, trans-2-[4-(4-chlorophenyl)cyclohexyl]-3hydroxy-1,4-naphthalenedione) is chemically similar to ubiquinone, which plays an essential role in mediating electron transfer between mitochondrial respiratory enzyme complexes. It is a selective inhibitor of parasite mitochondrial electron transport [1], and it is suggested that ATQ disturbs the mitochondrial membrane potential of Plasmodium falciparum. Proguanil hydrochloride (PRG) is a prodrug which is converted by the hepatic cytochrome P-450 isoenzyme to its active metabolite, cycloguanil (CG), and the inactive metabolite, 1-(4-chlorophenyl)biguanide [2]. PRG was found to markedly enhance the ability of ATQ to collapse mitochondrial transmembrane potential. ATQ and the active metabolite of PRG (cycloguanil) are effective against the erythrocytic and exo-erythrocytic stages of Plasmodium and thereby provide causal and suppressive prophylaxis. The fixed dose combination of ATQ and PRG has been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency for the treatment of acute, uncomplicated P. falciparum malaria. In addition, it has been shown that the combination is of great importance in areas where the drugs chloroquine, halofantrine, mefloquine, and amodiaquine may have unacceptable failure rates, presumably due to drug resistance [3, 4].

Determination of cilostazol and its active metabolite 3,4-dehydro cilostazol from small plasma volume by UPLC−MS/MS

A simple, rapid and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC−MS/MS) method has been developed for the simultaneous determination of cilostazol and its pharmacologically active metabolite 3,4-dehydro cilostazol in human plasma using deuterated analogs as internal standards (ISs). Plasma samples were prepared using solid phase extraction and chromatographic separation was performed on UPLC BEH C18 (50 mm×2.1 mm, 1.7 µm) column. The method was established over a concentration range of 0.5–1000 ng/mL for cilostazol and 0.5–500 ng/mL for 3,4-dehydro cilostazol. Intra- and inter-batch precision (% CV) and accuracy for the analytes were found within 0.93–1.88 and 98.8–101.7% for cilostazol and 0.91–2.79 and 98.0–102.7% for the metabolite respectively. The assay recovery was within 95–97% for both the analytes and internal standards. The method was successfully applied to support a bioequivalence study of 100 mg cilostazol in 30 healthy subjects.