Lohidasan Sathiyanarayanan

Development of Validated HPLC and HPTLC Methods for Simultaneous Determination of Levocetirizine Dihydrochloride and Montelukast Sodium in Bulk Drug and Pharmaceutical Dosage Form

Two chromatographic methods have been described for the simultaneous determination of levocetirizine dihydrochloride and Montelukast sodium in tablets. The fi rst method was a high performance thin layer chromatographic (HPTLC) separation followed by densitometric measurements on normal phase silica gel 60 F 254 . The second method was a high performance liquid chromatographic (HPLC) separation on a BDS Hypersil C 18 column using disodium hydrogen phosphate buffer (0.02 M): Methanol (25: 75, v/v) pH adjusted to 7 with ortho-phosphoric acid as the mobile phase. The proposed methods were validated as per ICH guidelines and successfully applied for the determination of investigated drugs in tablets.

HPTLC Densitometric Evaluation of Tissue Culture Extracts of Nothapodytes foetida Compared to Conventional Extracts for Camptothecin Content and Antimicrobial Activity

Tissue culture technique is becoming popular because of its well-known ability to enhance the content of secondary metabolites in plants. Callus tissue cultures of Nothapodytes foetida were developed using 250 different medium compositions to optimize this procedure. Methanolic extracts of callus (MEC) and of various parts of N. foetida were comparatively analyzed for camptothecin content, and a high performance thin layer chromatography method was developed for its quantitation. Chloroform-ethylacetate-methanol (4 : 5 : 0.5 v/v) was used as the mobile phase. The method was validated for linearity, precision (interday and intraday), repeatability, limit of detection (LOD), limit of quantitation (LOQ), and accuracy. The relationship between the concentration of standard solutions and the peak response was linear within the range of 80 to 480 ng/spot with a correlation coefficient of 0.998 +/- 0.020. Instrumental precision was evaluated as 0.54 (% CV). Repeatability of sample and standard were estimated to be 1.08 and 1.01 (% CV), and LOD and LOQ were found to be 40 and 80 ng/spot, respectively. The accuracy of the method was checked out by a recovery study and the average percentage recovery was calculated as being 99.13 %. The methanolic extract of callus grown in tissue culture with medium composition picloram + thidiazuron + gibberellic acid (1 : 1 : 4; MEC-PTG) showed a higher percentage of camptothecin (5.74 % w/v) than the methanolic extract of fruits (3.56 % w/w), leaves (1.56 % w/w), stem (1.19 % w/w), and root (1.11 % w/w). The results of the antimicrobial screening indicate that MEC-PTG exhibited maximum activity against all microorganisms. Among the fungi tested, MEC-PTG showed maximum activity against A. niger and C. albicans (MIC value 10 microg/mL) whereas among bacteria strains, its activity was highest against B. subtilis and S. lutea (MIC 20 microg/mL).

Method Development and Validation for the Simultaneous Determination of Fexofenadine Hydrochloride and Montelukast Sodium in Drug Formulation Using Normal Phase High-Performance Thin-Layer Chromatography

A simple, precise, specific, and accurate high-performance thin-layer chromatographic method has been developed for the simultaneous determination of fexofenadine hydrochloride (FEX) and montelukast sodium (MTKT) in pharmaceutical dosage form. The separation was carried out on Merck HPTLC aluminum plates of silica gel G60 F254, (20×10 cm) with 250 μm thickness using toluene: ethyl acetate: methanol: ammonia (30%) (0.5: 7: 2: 0.5, v/v/v/v) as mobile phase. HPTLC separation of the two drugs followed by densitometric measurement was carried out in the absorbance mode at 220 nm. The drugs were resolved satisfactorily with 𝑅𝑓 values of 0.21±0.01 and 0.59±0.01 for FEX and MTKT, respectively. The linear regression analysis data for the calibration plots showed good linear relationship with 𝑟2=0.9996 and 0.9998 for FEX and MTKT, respectively, in the concentration range of 2400–10800 ng spot−1 for FEX and 200–900 ng spot−1 for MTKT. The method was validated for precision, robustness, specificity, and accuracy. The limits of detection and quantitation were 100 and 300 ng spot−1, respectively, for FEX and 50 and 100 ng spot−1, respectively, for MTKT. The proposed developed HPTLC method can be applied for identification and quantitative determination of FEX and MTKT in bulk drug and drug formulation.

Comparative antioxidant potential of Withania somnifera based herbal formulation prepared by traditional and non-traditional fermentation processes

Background Ashwagandharishtha is a liquid polyherbal formulation traditionally prepared by fermentation process using the flowers of Woodfordia fruticosa. It contains roots of Withania somnifera as a major crude drug. Alcohol generated during the fermentation causes the extraction of water insoluble phytoconstituents. Yeasts present on the flowers are responsible for this fermentation. Methods Total nine formulations of ashwagandharishtha were prepared by fermentation process using traditional Woodfordia fruticosa flowers (ASG-WFS) and using yeasts isolated from the same flowers. During fermentation, kinetic of alcohol generation, sugar consumption, changes in pH and withanolides extraction were studied. All the formulations were tested for in vitro antioxidant potential by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, hydrogen peroxide scavenging and total reducing power assay. The results were compared with standard ascorbic acid. Results Traditional formulation (ASG-WFS) showed the highest activity (p < 0.001) relative to other formulations and standard ascorbic acid. ASG-WFS showed significant (DPPH) free radical scavenging (78.75%) and hydrogen peroxide scavenging (69.62%) at the concentration of 1000 μg/mL and 100 μg/mL, respectively. Conclusion Traditional process is the best process for preparing ashwagandharishtha to obtain significant antioxidant activity.

Stability-Indicating High-Performance Thin-Layer Chromatographic Method for Quantitative Estimation of Emtricitabine in Bulk Drug and Pharmaceutical Dosage Form

A simple, sensitive, precise, specific and stability indicating high-performance thin-layer chromatographic (HPTLC) method for the determination of emtricitabine both in bulk drug and pharmaceutical dosage form was developed and validated. The method employed aluminium plates precoated with silica gel G60 F254 as the stationary phase. The solvent system consisted of toluene : ethyl acetate : methanol (2 : 8 : 1, v/v/v). This solvent system was found to give compact spots for emtricitabine with 𝑅𝑓 value 0.26±0.01. Densitometric analysis of emtricitabine was carried out in the absorbance mode at 284 nm. Linear regression analysis showed good linearity (𝑟2=0.9997) with respect to peak area in the concentration range of 30–110 ng spot−1. The method was validated for precision, limit of detection (LOD), limit of quantitation (LOQ), robustness, accuracy and specificity. Emtricitabine was subjected to acid and alkali hydrolysis, oxidation, neutral hydrolysis, photodegradation and dry heat treatment. Also the degraded products peaks were well resolved from the pure drug with significantly different 𝑅𝑓 values. Statistical analysis proved that the method is repeatable and specific for the estimation of the said drug. As the method could effectively separate the drugs from their degradation products, it can be employed as a stability indicating method.

Application of HPLC for the Simultaneous Determination of Paracetamol, Chlorzoxazone, and Nimesulide in Pharmaceutical Dosage Form

A simple, precise, and accurate reversed-phase liquid chromatographic method has been developed for the simultaneous determination of paracetamol (PCM), chlorzoxazone (CHZ), and nimesulide (NIM) in pharmaceutical dosage form. The chromatographic separation was achieved on a Thermo Hypersil GOLD C18 column (250 × 4.6 mm i.d., 5 μm particle size). The mobile phase consisted of water : acetonitrile (55 : 45 v/v). The flow rate was set to 1.2 mL min−1 and UV detection was carried out at 275 nm. The retention time () for PCM, CHZ, and NIM was found to be 2.69 ± 0.02, 4.61 ± 0.01, and 9.55 ± 0.02 min, respectively. The validation of the proposed method was carried out for linearity, precision, robustness, limit of detection, limit of quantitation, specificity, and accuracy. The linear dynamic ranges were 32.5–65.0 μg mL−1 for PCM, 37.5–75.0 μg mL−1 for CHZ, and 10.0–20.0 μg mL−1 for NIM. The developed method can be used for routine quality control analysis of titled drugs in pharmaceutical dosage form.

Rapid and sensitive determination of major polyphenolic components in Euphoria longana Lam. seeds using matrix solid-phase dispersion extraction and UHPLC with hybrid linear ion trap triple quadrupole mass spectrometry.

A rapid and sensitive method for the extraction and determination of four major polyphenolic components in Euphoria longana Lam. seeds is presented for the first time based on matrix solid-phase dispersion extraction followed by ultra high performance liquid chromatography with hybrid triple quadrupole linear ion trap mass spectrometry. Matrix solid-phase dispersion method was designed for the extraction of Euphoria longana seed constituents and compared with microwave-assisted extraction and ultrasonic-assisted extraction methods. An Ultra high performance liquid chromatography with hybrid triple quadrupole linear ion-trap mass spectrometry method was developed for quantitative analysis in multiple-reaction monitoring mode in negative electrospray ionization. The chromatographic separation was accomplished using an ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm) column with gradient elution of 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile. The developed method was validated with acceptable linearity (r2 > 0.999), precision (RSD ≤ 2.22%) and recovery (RSD ≤ 2.35%). The results indicated that matrix solid-phase dispersion produced comparable extraction efficiency compared with other methods nevertheless was more convenient and time-saving with reduced requirements on sample and solvent volumes. The proposed method is rapid and sensitive in providing a promising alternative for extraction and comprehensive determination of active components for quality control of Euphoria longana products.

Determination of Major Polyphenolic Components in Euphoria longanaLam. by Validated High Performance Thin-Layer ChromatographyMethod and Direct Analysis in Real Time Mass Spectrometry

In the current study, a simple, sensitive, precise, and specific method has been developed and validated for the determination of four major polyphenolic compounds in Euphoria longana Lam. seeds based on high performance thin-layer chromatography (HPTLC) and confirmed by direct analysis in real time mass spectrometry (DART-MS). The chromatographic separation was accomplished on Merck HPTLC aluminum plates precoated with silica gel G60 F254 (20 cm × 10 cm) with 250 μm thickness as stationary phase using a mobile phase composed of n-butanol: water: methanol: formic acid (7.59: 1.27: 0.13: 1.01, v/v/v/v). Densitometric measurement was performed in the absorbance mode at 280 nm. The compounds were resolved satisfactorily with Rf values of 0.40 ± 0.01, 0.57 ± 0.02, 0.69 ± 0.01, and 0.79 ± 0.01 for corilagin, ellagic acid, epicatechin, and gallic acid, respectively. The method developed was validated with acceptable linearity (r2>0.995), sensitivity, precision (RSD ≤ 1.60%), robustness, and recovery (RSD ≤ 1.77%). The proposed method was successfully applied for the determination of active components for comprehensive quality control of E. longana products. Furthermore, peak identities of compounds from each band were confirmed by direct analysis in real time mass spectrometry.

Characteristic Fingerprint Analysis of Mallotus philippinensis by UltraPerformance Liquid Chromatography Electrospray Ionization MassSpectrometry

The developing countries mostly trust on traditional remedies, which include the use of different plant extracts or the bioactive phytoconstituents. For this purpose, analysis such as chemical fingerprinting strongly represents one of the best possibilities in searching of new economic and therapeutically effective plants for medicine. Mallotus philippinensis Muell. Arg (Euphorbiaceae) is a large genus of the trees and shrubs mainly distributed in the tropical and subtropical regions and are reported to have widespread range of pharmacological activities. A new, simple and rapid ultraperformance liquid chromatography (UPLC) method with photodiode array (PDA) detector has been developed. Further confirmation was performed by electrospray ionization mass spectrometry (ESI-MS) for the chemical fingerprint analysis in extracts of Mallotus philippinensis. The chromatographic separations were obtained on a Waters ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm) column using a gradient elution with 0.1% (v/v) formic acid/water and acetonitrile as mobile phase at a flow rate of 0.4 mL/min. The UPLC-PDA-ESI-MS characteristic fingerprints were established and 7 characteristic peaks were identified along with 5 unknown peaks within 4.5 min by comparing the retention times, λ max (nm), and MS spectra with the literature data. Therefore, this fingerprint analysis method can be applied for the identification and quality control of Mallotus philippinensis.

Investigation of anti-arthritic, antioxidant properties of dichloromethane fraction of Alstonia scholaris Linn. in Freund’s complete adjuvant induced rats and identification of biomarker

Alstonia scholaris (Family: Apocynaceae) is a medicinal plant which has been indicated for the treatment of various diseases including arthritis in folklore medicine. The antiarthritic activity of the ethanolic extract of Alstonia scholaris leaves is reported. The present study was performed to establish the antiarthritic activity of the dichloromethane fraction (DCM) and to identify the constituent(s) responsible for the activity. DCM was tested for antiarthritic activity against Freund’s Complete Adjuvant (FCA) induced arthritic rats. Arthritis assessment, nociceptive threshold and body weight were measured. On day 28, plasma tumor necrosis factor α, synovial leukocyte concentration, synovial tissue myeloperoxide, malonaldehyde, glutathione, glutathione peroxidase and superoxide dismutase were determined. Effect of DCM on ethanol and sodium salicylate induced gastropathy was also studied. DCM was subjected to HPTLC analysis and the phytoconstituent was identified using marker. DCM significantly decreased the signs of arthritis evident with decreased arthritis index, body weight, TNF-α and leukocyte infiltration. DCM significantly reduced gastric lesion indices and gastric juice secretion. It also significantly decreased the levels of lipid peroxidation and myeloperoxide in the articular tissue, whereas significantly increased the antioxidant enzymes glutathione, glutathione peroxidase and superoxide dismutase. Moreover DCM was found to contain ursolic acid, which is one of the biomarkers indicated to have anti-inflammatory activity. The present study is suggestive that DCM has prominent antiarthritic activity which may be attributed to its analgesic, anti-inflammatory, and antioxidant activities. The Ursolic acid may contribute the found antiarthritic activity in part or whole.