Validated Stability Indicating Chromatographic Method for the Simultaneous Estimation of Camylofin with NSAID Drugs and a New Approach of Method Transfer from Classical HPLC to a Modern UPLC Instrument

The presented work describes the method development of simultaneous determination of camylofin dihydrochloride (CMF), diclofenac potassium (DCF), and Paracetamol (PCM) using reversed phase high performance liquid chromatography (HPLC-UV) and the method was further transferred to a new generation instrument, ultraperformance liquid chromatography (UPLC-PDA). The detailed validation was carried out for the combination tablet formulation of CMF and DCF by UPLC-PDA. From the method development study, Acquity UPLC HSS C18 (2.1 × 50 mm, 1.8 μm) was finally selected for validation. The satisfactory results were observed for peak shape, retention time, and resolution with a mobile phase of 20 mM ammonium acetate buffer (pH 3.0 with dilute orthophosphoric acid) : methanol (33 : 67 v/v). The isocratic elution of mobile phase was carried out at a flow rate of 0.250 mL/min and detection at 220 nm. Both drugs were efficiently separated out in less than 3.5 min with 1.1 and 3.2 min of retention time of the CMF and DCF with 11.87 of resolution. The linearity was obtained in the 20.0–80.0 μg/mL range of concentration with 0.9998 of correlation coefficients for the substances. The method was analyzed for specificity with detailed force degradation study, which is a simple, precise, and accurate method, as per the International Conference on Harmonization (ICH) guidelines.

Thermodynamic Study of Racemic Ibuprofen Separation by Liquid Chromatography Using Cellulose-Based Stationary Phase

Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID), also known for its significant antipyretic and analgesic properties. This chiral drug is commercialized in racemic form; however, only S-(+)-ibuprofen has clinical activities. In this paper the effect of temperature change (from 288.15 to 308.15 K) on the ibuprofen resolution was studied. A column (250×4.6 mm) packed with tris(3,5-dimethylphenylcarbamate) was used to obtain the thermodynamic parameters, such as enthalpy change (ΔH), entropy change (ΔS), variation enthalpy change (ΔΔH), variation entropy change (ΔΔS), and isoenantioselective temperature (Tiso). The mobile phase was a combination of hexane (99%), isopropyl alcohol (1%), and TFA (0.1%), as an additive. The conditions led to a selectivity of 1.20 and resolution of 4.55. The first peak, R-(−)-ibuprofen, presented an enthalpy change of 7.21 kJ/mol and entropy change of 42.88 kJ/K·mol; the last peak, S-(+)-ibuprofen, has an enthalpy change of 8.76 kJ/mol and 49.40 kJ/K·mol of entropy change.

Development and Validation of RP-HPLC Method for the Determination of Hydrochlorothiazide in Bulk Drug and Pharmaceutical Dosage Form

An HPLC-PDA method was developed and validated for the determination of hydrochlorothiazide in bulk and pharmaceutical formulation. The method was optimized selecting chromatographic conditions of 50 : 50 acetonitrile : water, Inertsil® column (ODS-3 250 mm × 4.6 mm 5 μm), 20 μL injection volume, flow rate of 1 mL/min at ambient temperature (30°C), and 272 nm. Another column of C18Zorbax® (Eclipse Plus, 4.6 × 250 mm, 5 μm) was tested showing no big difference in the method results. The method was validated giving good precision (RSD% < 1), acceptable linearity (R2 ≥ 0.9978), and low LOD and LOQ (0.5 and 1.7 μg/mL, resp.) on both columns. Successful application on pharmaceutical dosage tablet form gave high recovery of 99.93%. The method was compared with official BP and other reported methods. The proposed method is economic, simple, and rapid and hence can be employed for routine analysis in quality control laboratories.

Development and Validation of New RP-HPLC Method for Simultaneous Determination of Methyl and Propyl Parabens with Levetiracetam in Pure Form and Pharmaceutical Formulation

A simple and robust high-performance liquid chromatography (HPLC) method is described for the assay for levetiracetam (LTC), methyl paraben (MHB), and propyl paraben (PHB) either in their pure form or in commercial Levepsy® syrup. The method is selective and stability indicating and all chromatographic conditions were studied to obtain adequate separation of LTC, MHB, and PHB from their degradation products and from excipients. The HPLC separation was carried out on a RP C18 Hypersil BDS analytical column (150 mm × 4.6 mm ID) using gradient elution system. The mobile phase flow rate was 1.5 mLmin−1 and the column temperature was kept at 40°C. Complete separation of the studied components was obtained within a cycle time of 8 min. LTC, MHB, and PHB were eluted at 1.56, 5.86, and 7.85 min, respectively. Detection was carried out at 240 nm using a dual wavelength detector. The method has been validated for linearity, accuracy, precision, specificity, limit of detection, limit of quantitation, robustness, and ruggedness. The proposed method was successfully applied for the determination of LTC in the presence of parabens in Levepsy syrup.

A Simple Analytical Method for High-Throughput Screening of Major Sugars from Soybean by Normal-Phase HPLC with Evaporative Light Scattering Detection

This paper presents a simple analytical method for determining sugars in soybean (Glycine max (L.) Merr.) tissues. Sample preparation was modified from several early published methods. High-performance liquid chromatography (HPLC) equipped with an evaporative light scattering detector (ELSD) was used to separate, identify, and quantify seven sugars, including glucose, galactose, fructose, sucrose, melibiose, raffinose, and stachyose. Two mobile phases were programed into a gradient elution. Mobile phase A is pure water and mobile phase B is a mixture of acetonitrile and acetone 75 : 25 (v/v). Total chromatographic retention time is less than 20 minutes. This method has been validated for detection limit, calibration range, and intraday and interday repeatability. This method has been used analyzing more than 5000 soybean samples in the experiments studying natural genetic variation of sugar contents and components in soybean seeds and other tissues.

Development and Validation of an HPLC Method for the Analysis of Chlorpropham and 3-Chloroaniline in Potato Extracts

Chlorpropham (CIPC) is the main sprout inhibitor used by potato industry. There is concern about the residues of CIPC and its degradation product 3-chloroaniline, 3-CA; hence, analytical methods are required to analyse their residues in potato samples. An HPLC-UV method was developed and validated for the separation and quantification of these compounds using propham (IPC) as an internal standard. The chromatographic conditions required to achieve good separation were 60% mobile phase of methanol, 15-minute run time at a flow rate of 1.5 mL/min, and a detection wavelength of 210 nm using Phenomenex (ODS-2 250 mm × 4.60 mm 5 µm Sphereclone) column at an ambient temperature. The method was validated for precision, linearity, the limit of detection (LOD) and the limit of quantification (LOQ), producing high precision through RSD ≤ 0.03%, and acceptable criteria of the coefficient of determination (R2) of the calibration curves (0.990). LOD values of CIPC and 3-CA were approximately 0.01 µg/mL whereas the LOQ values were approximately 0.04 µg/mL using repeated injection approach. The proposed HPLC method was compared with the standard GC method of the CIPC residues extracted showing good agreement R2=0.99. Despite using the same extract, the recovery results for the proposed HPLC method were 13% higher than GC analysis.

Practical Implication of Chromatographic Method for Estimation of Aceclofenac and Pregabalin in Bulk and Pharmaceutical Dosage Forms

Background. Aceclofenac and Pregabalin in combination significantly reduce pain as compared to individual drug in chronic low back pain. Literature reveals that all the reported spectrophotometric methods either need tedious extraction procedures, do not offer high sensitivity, use nonspecific reagent, or recommend the measurement of absorbance in the near UV region where interference most probably occurs that does not offer suitable linearity range. Result. A selective, sensitive, accurate, and precise, high performance liquid chromatographic method with UV detector analysis of Aceclofenac and Pregabalin was investigated. Good chromatographic separation was achieved using an ODS-BP hypersil C18 column (250 mm × 4.6 mm, i.d., 5 μm) and a mobile phase consisting of 0.05 M phosphate buffer (KH2PO4) (pH 6.0) : methanol (60 : 40, v/v) at a flow rate 1 mL/min. The ultraviolet detector was set at wavelength 218 nm. Retention time for Aceclofenac and Pregabalin was found to be 3.220 and 5.910 min, respectively. Rectilinear relationship with good regression coefficients 0.999 and 0.999 was found over the concentration ranges of 5–25 μg/mL and 3.75–18.75 μg/mL for ACF and PGB, respectively, with detection limits 0.64 and 0.35 μg/mL and quantitation limits 1.95 and 1.06 μg/mL. Conclusion. The mean percentage recoveries were in the range of 98.45–100.08 and 99.69–100.48 for ACF and PGB, respectively. The developed method was successfully applied to the analysis of the drugs in their commercial tablets.

A Validated Stability Indicating RP-HPLC Method Development and Validation for Simultaneous Estimation of Aliskiren Hemifumarate and Amlodipine Besylate in Pharmaceutical Dosage Form

The present study describes the stability indicating RP-HPLC method for simultaneous estimation of aliskiren hemifumarate and amlodipine besylate in pharmaceutical dosage forms. The proposed RP-HPLC method was developed by using waters 2695 separation module equipped with PDA detector and chromatographic separation was carried on C-8 Inertsil ODS (150 × 4.6 mm, 5 µ) column at a flow rate of 1 mL/min and the run time is 10 min. The mobile phase consisted of phosphate buffer and acetonitrile in the ratio of 40 : 60% v/v and pH was adjusted to 3 with orthophosphoric acid and eluents were scanned using PDA detector at 237 nm. The retention time of aliskiren and amlodipine was found to be 3.98 and 5.14 min, respectively. A linearity response was observed in the concentration range of 30–225 µg/mL for aliskiren and 2–15 µg/mL for amlodipine, respectively. Limit of detection and limit of quantification for aliskiren are 0.161 µg/mL and 0.489 µg/mL and for amlodipine are 0.133 µg/mL and 0.404 µg/mL, respectively. The stability indicating method was developed by subjecting the drugs to stress conditions such as acid and base hydrolysis, oxidation, and photo- and thermal degradation and the degraded products formed were resolved successfully from the samples.

Method Development and Validation for Determination of Febuxostat from Spiked Human Plasma Using RP-HPLC with UV Detection

A rapid, simple, selective, and specific reverse phase high performance liquid chromatography (RP-HPLC) method with UV detection (315 nm) was developed and validated for estimation of febuxostat from spiked human plasma. The analyte and internal standard (diclofenac) were extracted using LLE with diethyl ether. The chromatographic separation was performed on Shodex C-18-4E (5 μm; 250×4.6 mm) with a mobile phase comprised of methanol : acetate buffer pH 4, 20 mM (90 : 10 v/v), at a flow rate of 1 mL/min. Febuxostat was well resolved from plasma constituents and internal standard. The calibration curve was linear in the range of 250–8000 ng/mL. The heteroscedasticity was minimized by using weighted least square regression with weighing factor of 1/x. The intraday and interday %RSD was less than 15. Results of recovery studies prove the extraction efficiency. Stability data indicated that febuxostat was stable in plasma after three freeze thaw cycles and upon storage at −20°C for 30 days.

Identification and Quantification of Aldose Reductase Inhibitory Flavonoids in Herbal Formulation and Extract of Gymnema sylvestre Using HPLC-PDA and LC-MS/MS

Adulteration of herbal supplements is a major issue for many countries. A simple and reliable HPLC-PDA method was developed for quantification of aldose reductase inhibitory flavonoids rutin, quercetin, and kaempferol. The chromatographic separation was performed on a Fortis C18 column in gradient mode with detection at 267 nm. The presence of these markers was confirmed through the accurate m/z values and MS/MS data obtained using quadruple time of flight mass spectrometry (QTOF-MS). The proposed method was successfully applied to examine the amount of these active constituents in antiobese polyherbal formulation and plant extract of Gymnema sylvestre.