Ramisetti Nageswara Rao

RP-HPLC separation and characterization of unknown impurities of a novel HIV-protease inhibitor Darunavir by ESI-MS and 2D NMR spectroscopy

A selective RP-HPLC method for separation and determination of darunavir from its process related substances has been developed and validated. The separation was accomplished on a Hiber, LiChrospher 60, RP-select B, C₈ (250 mm × 4.6 mm, 5 μm) column connected to a photo diode array detector (PDA) using 10 mM phosphate buffer with 0.1% of triethylamine (pH: 4.0 adjusted with orthophosphoric acid)/acetonitrile as a mobile phase under gradient elution. Two unknown impurities of darunavir were isolated and characterized by ¹H, ¹³C, 2D-NMR and mass spectrometry. The method was validated in terms of accuracy, precision, linearity, robustness, LOD and LOQ.

Reversed-phase liquid chromatographic separation of antiretroviral drugs on a monolithic column using ionic liquids as mobile phase additives

The use of 3-methylimidazolium cation-based ionic liquids (ILs) was evaluated as mobile phase additives for separation of antiretroviral drugs on a monolithic column by RP-HPLC. Separation of eight commonly used antiretroviral drugs was achieved on a Chromolith Flash, RP-18e column (25 × 4.6 mm, porous material) using water (pH 4.0 adjusted with acetic acid)/methanol v/v as a mobile phase containing ILs in a gradient elution mode. The effects of concentrations of ILs on retention, resolution and peak shape were studied and a regression equation correlating the interactions between stationary phase and the ILs was established. The retention of all the drugs was decreased notably by using 1-butyl-3-methylimidazolium tetrafluoroborate, while 1-ethyl-3-methylimidazolium methylsulfate reduced gradient drift drastically when compared to triethylamine.

Determination of rifaximin in rat serum by ionic liquid based dispersive liquid-liquid microextraction combined with RP-HPLC.

An efficient and environmental friendly ionic liquid based dispersive liquid-liquid microextraction procedure was optimized for determination of rifaximin in rat serum by reverse phase high-performance liquid chromatography. The effect of ionic liquids, dispersive solvents, extractant/disperser ratio, and salt concentrations on sample recovery and enrichment factors were studied. Among the five ionic liquids studied in the present investigation, 1-butyl-3-methylimidazolium hexafluorophosphate was found to be most effective for extraction of rifaximin. The recovery was found to be more than 98% using 1-butyl-3-methylimidazolium hexafluorophosphate and methanol as extraction and dispersive solvents, at an extractant/disperser ratio of 0.43. The recovery was further enhanced to 99.5% by the addition of 5.0% NaCl solution. A threefold enhancement in detection limit was achieved when compared to protein precipitation. The ionic liquid containing the extracted rifaximin was directly injected into HPLC system. The linear relationship was observed in the range of 0.03-10.0 μg/mL with the correlation coefficient (r(2)) 0.9998. Limits of detection and quantification were found to be 0.01 and 0.03 μg/mL, respectively. The relative standard deviation was 2.5%. The method was validated and applied to study pharmacokinetics of rifaxmin in rat serum.

Enantioselective HPLC resolution of synthetic intermediates of armodafinil and related substances

Armodafinil is a unique psychostimulant recently approved by the US Food and Drug Administration for the treatment of narcolepsy. The chromatographic resolution of its chiral intermediates including related substances in the total synthesis of armodafinil was studied on polysaccharide-based stationary phases, viz. cellulose tris-(3,5-dimethylphenylcarbamate) (Chiralcel OD-H) and amylose tris-(3,5-dimethylphenylcarbamate) (Chiralpak AD-H) by HPLC. The effects of 1-propanol, 2-propanol, ethanol, and trifluoroacetic acid added to the mobile phase and of column temperature on resolution were studied. A good separation was achieved on cellulose-based Chiralcel OD-H column compared to amylose-based Chiralpak AD-H. The effects of structural features of the solutes and solvents on discrimination between the enantiomers were examined. Baseline separation with R(s) >1.38 was obtained using a mobile phase containing n-hexane-ethanol-TFA (75:25:0.15 v/v/v). Detection was carried out at 225 nm with photodiode array detector while identification of enantiomers was accomplished by a polarimetric detector connected in series. The method was found to be suitable not only for process development of armodafinil but also for determination of the enantiomeric purity of bulk drugs and pharmaceuticals.

Isolation and characterization of degradation products of citalopram and process-related impurities using RP-HPLC

A reversed-phase high-performance liquid chromatographic method for simultaneous separation and determination of citalopram hydrobromide and its process impurities in bulk drugs and pharmaceutical formulations was developed. The separation was accomplished on an Inertsil ODS 3V (250x4.6 mm; particle size 5 mum) column using 0.3% diethylamine (pH = 4.70) and methanol/acetonitrile (55:45 v/v) as mobile phase in a gradient elution mode. The eluents were monitored by a photodiode array detector set at 225 nm. The chromatographic behavior of all the related substances was examined under variable conditions of different solvents, buffer concentrations, and pH. The method was validated in terms of accuracy, precision, and linearity. The method could be of use not only for rapid and routine evaluation of the quality of citalopram in bulk drug manufacturing units but also for the detection of its impurities in pharmaceutical formulations. Three unknown impurities were consistently observed during the analysis of different batches of citalopram. Forced degradation of citalopram was carried out under thermal, photo, acidic, alkaline, and peroxide conditions. The degradation products and unknown impurities were isolated and characterized by ESI-MS/MS, (1)H NMR, and FT-IR spectroscopy.

Molecularly imprinted polymer for selective extraction of 3-methylflavone-8-carboxylic acid from human urine followed by its determination using zwitterionic hydrophilic interaction liquid chromatography

A novel water-compatible molecularly imprinted SPE combined with zwitterionic hydrophilic interaction liquid chromatography method for selective extraction and determination of 3-methylflavone-8-carboxylic acid (MFA), the main active metabolite of flavoxate in human urine, was developed and validated. The effects of progenic solvents, pH, cross linker and amount of monomer were studied to optimize the efficiency and selectivity. The molecularly imprinted polymer showed good specific adsorption capacity with an optimum of 200 μmol/g at pH 7.5 and selective extraction of MFA from human urine. The recovery of MFA from human urine was >98%. The lower limit of quantification was 1.20 μg/mL. The proposed method overcomes the matrix effects of endogenous substances generally encountered during direct analysis of urine sample.

LC–MS/MS method for the characterization of the forced degradation products of Entecavir

A rapid, specific, and reliable isocratic LC-MS/MS method has been developed and validated for the identification and characterization of the stressed degradation products of Entecavir (ETV). ETV, an antiviral drug, was subjected to hydrolysis (acidic, alkaline, and neutral), oxidation, photolysis and thermal stress, as per the international conference on harmonization specified conditions. The drug showed extensive degradation under oxidative and acid hydrolysis stress conditions. However, it was stable to thermal, acidic, neutral, and photolysis stress conditions. A total of five degradation products were observed and the chromatographic separation of the drug and its degradation products were achieved on a Waters Symmetry C18 (250 mm × 4.6 mm, id, 5 μm) column using 20 mM ammonium acetate (pH 3)/acetonitrile (50:50, v/v) as a mobile phase. The degradation products were characterized by LC-MS/MS and its fragmentation pathways were proposed. The LC-MS method was validated with respect to specificity, linearity, accuracy, and precision. No previous reports were found in the literature regarding the degradation behavior of ETV.

Rat dried blood spot analysis of (R,S)-(−)- and (S,R)-(+)- enantiomers of emtricitabin on immobilized tris-(3,5-dimethylphenyl carbamate) amylose silica as a chiral stationary phase

An enantioselective high performance liquid chromatography method has been developed and validated by evaluating the suitability of newly introduced immobilized polysaccharide chiral stationary phases, the effect of different organic modifiers and temperature including the entropy and enthalpy on resolution of the (R,S)-(-) & (S,R)-(+) emtricitabine enantiomers on rat dried blood spots. Both the enantiomers were extracted from dried blood spots using ethanol: methanol (80:20 v/v) mixture and separated on an immobilized amylose tris-(3,5-dimethyl phenyl carbamate) chiral stationary phase using n-hexane:ethanol (65:35 v/v) as a mobile phase at a flow rate of 0.8mL/min. The detection was carried out at 280nm using photo diode array detector connected to a polarimeter in series to determine their order of eluton. The method was validated with respect to limits of detection and quantification, linearity, accuracy and precision. The calibration curves were linear over the concentration range of 0.5-500μg/mL for both enantiomers and the correlation coefficient (r(2)) was >0.998. The overall recovery of (R,S)- & (S,R)-enantiomers of emtricitabin from DBS were 90.4 and 90.6%, respectively. The limits of detection and quantification of enantiomers were 0.26, 0.30 and 0.85, 0.92μg/mL for (R,S)- and (S,R)-emtricitabin enantiomers, respectively. The assay was specific and precise (RSD <10%). The stability of emtricitabin was also performed and the results were found to be well within the limits. The effect of hematocrit on extraction of emtricitabin enantiomers from dried blood spots was evaluated and no interference from endogenous substances was observed.

LC-MS/MS characterization of forced degradation products of zofenopril.

A rapid, specific and reliable isocratic LC-MS/MS method has been developed and validated for the identification and characterization of stressed degradation products of Zofenopril. Zofenopril, an anti-hypertensive drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per ICH-specified conditions. The drug showed extensive degradation under oxidative and base hydrolysis stress conditions. However, it was stable to thermal, acid, neutral and photolysis stress conditions. A total of 6 degradation products were observed and the chromatographic separation of the drug and its degradation products were achieved on Phenomenex (Luna) C18 (250mm×4.6mm, i.d., 5μm) column using 20mM ammonium acetate: acetonitrile (50:50, v/v) as a mobile phase. The degradation products were characterized by LC-MS/MS and its fragmentation pathways were proposed. The LC-MS method was validated with respect to specificity, linearity, accuracy and precision. No previous reports were found in the literature regarding the degradation behavior of zofenopril.

Emerging liquid chromatography–mass spectrometry technologies improving dried blood spot analysis

Dried blood spots (DBS), a micro blood sampling technique, has recently gained interest in drug discovery and development due to its inherent advantages over the conventional whole blood, plasma or serum sample collection. Since the regulatory authorities have agreed to the use of blood as an acceptable biological matrix for drug exposure measurements, its applications have been extended not only to therapeutic drug monitoring but also to toxicokinetic and pharmacokinetic studies. The pharmaceutical industry is keen to promote DBS as a prominent tool in bioanalytical applications due to the financial, ethical and organizational issues involved in clinical trials. This could be accomplished due to the latest advances in modern analytical technology, particularly liquid chromatography–mass spectrometry. The present review discusses some of the emerging liquid chromatography–mass spectrometry technologies in improving DBS analysis for its innovative applications in the development of new drugs.