Dhananjay D. Shinde

Two-dimensional LC-MS/MS determination of antiretroviral drugs in rat serum and urine.

A simple, rapid, reliable and highly sensitive on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometric (2D-LC/MS/MS) method to determine antiretroviral drugs viz., abacavir (ABC), nevirapine (NVP) and indinavir (IDV) in rat serum and urine was developed and validated. The analytes were extracted on-line from rat serum and urine by a restricted access material (RAM) column and back-flushed into the reversed-phase C18 column for separation by LC. Detection was carried out by ESI-MS/MS. The developed method showed good selectivity, accuracy and precision for quantification of the antiretroviral drugs in rat serum and urine. Quantification limits for abacavir and nevirapine were 4.0 ng ml(-1), whereas for indinavir 4.7 ng ml(-1). The calibration graphs were linear in the range of 4-50 ng ml(-1)for abacavir, nevirapine and indinavir. The method was successfully applied to study the pharmacokinetics of antiretroviral in rats.

Determination of rat plasma levels of sertraline enantiomers using direct injection with achiral–chiral column switching by LC–ESI/MS/MS

A highly sensitive and selective on-line two-dimensional reversed-phase liquid chromatography/electrospray ionization-tandem mass spectrometric (2D-LC-ESI/MS/MS) method to determine sertraline (SRT) enantiomers in rat plasma was developed and validated. The method was applied to separate and determine the diastereomers and enantiomers of SRT simultaneously. The 2D-LC-ESI/MS/MS system consisted of RAM column in first dimension for trapping proteineous part of plasma and a chiral Cyclobond column as second dimension for separation of enantiomers and diastereomers of SRT using 0.1% aqueous trifluoroacetic acid:acetonitrile (86:14, v/v) as mobile phase in an isocratic elution mode. The linear dynamic range was 0.5-200ng/mL (r(2)>0.999). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in the analysis of SRT enantiomers in rat plasma to support pharmacokinetic studies.

Rapid determination of rifaximin in rat serum and urine by direct injection on to a shielded hydrophobic stationary phase by HPLC

A simple and rapid reversed-phase HPLC method for determination of rifaximin in rat serum and urine was developed. Separation of rifaximin from biological matrix was achieved by direct injection of rat serum and urine onto a restricted-access medium, Supelco LC-Hisep, a shielded hydrophobic stationary phase, using acetonitrile:water:acetic acid (18:82:0.1 v/v/v) as a mobile phase. The linear range was 0.10-20 microg/mL (r(2 )> 0.999, n = 6), intraday and interday variation was <6.10%. The limits of detection and quantification were 0.03 (signal-to-noise ratio >3) and 0.10 microg/mL (signal-to-noise ratio >10), respectively. The method was successfully applied to pharmacokinetic studies of rifaximin after an oral administration to rats.

On‐line 2D‐LC‐ESI/MS/MS determination of rifaximin in rat serum

A highly sensitive and selective on-line two-dimensional reversed-phase liquid chromatography/electrospray ionization-tandem mass spectrometry (2D-LC-ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D-LC-ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C(18 )column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5-10 ng/mL (r(2) > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies.

Development and validation of a reversed phase liquid chromatographic method for separation and determination of related-substances of modafinil in bulk drugs.

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method for determination and evaluation of purity of modafinil in bulk drugs using Kromasil C(18) column with acetonitrile: 0.02M ammonium acetate as a mobile phase in gradient elution mode at 30 degrees C and detection at 225nm using photodiode array detector has been developed. The effects of pH, temperature and the percent of organic modifier on resolution were studied. Related substances, viz, sulphide, sulphoxide, sulphones of the modafinil, acid and ester derivatives, were separated and quantified. The method was found to be simple, rapid, selective and capable of detecting all process related impurities at trace levels in the finished products of modafinil with detection limits of 0.6-2.4x10(-8)g. The method was validated with respect to accuracy, precision, linearity, ruggedness, and limits of detection and quantification. It was found to be suitable not only for monitoring the reactions during the process development but also quality assurance of modafinil.

Precolumn derivatization followed by liquid chromatographic separation and determination of tramiprosate in rat plasma by fluorescence detector: Application to pharmacokinetics

Alzheimer disease (AD) is characterized pathologically by extracellular amyloid deposits composed of amyloid β (Aβ) protein. A simple and rapid method using HPLC with fluorescence detector was developed and validated for determination of tramiprosate in rat plasma. Pre-column derivatization of the deproteinized rat plasma was carried out using o-phthaldialdehyde (OPA) as a fluorescent reagent in presence of 3-mercaptopropionic acid. The liquid chromatographic separation was achieved on a Kromasil C18 column using methanol:acetonitrile: 20 mM phosphate buffer pH 7.5 (8.0:17.5:74.5 v/v/v) as a mobile phase in an isocratic elution mode. The eluents were monitored by a fluorescence detector set at 330 and 450 nm of excitation and emission wavelength respectively. Vigabatrin was used as an internal standard. The method was linear within the range 30.0-1000.0 ng/mL. Design of experiments (DOE) was used to evaluate the robustness of the method. The developed method was applied to study the pharmacokinetics of tramiprosate in rats.

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.

Simultaneous separation and determination of coenzyme Q10 and its process related impurities by NARP-HPLC and atmospheric pressure chemical ionization-mass spectrometry (APCI-MS)

A non-aqueous reversed phase high performance liquid chromatographic (NARP-HPLC) method for determination of coenzyme Q(10) in pharmaceutical preparations has been developed using Kromosil C(8) column with acetonitrile and isopropyl alcohol (84:16, v/v) as a mobile phase. Photodiode array (PDA) detector set at 210 nm was used for monitoring of the eluents. The method is simple, rapid, selective and capable of separating all process impurities at trace level with detection limits <0.1 microg/ml. It has been validated with respect to accuracy, precision, linearity, and limits of detection and quantification. The linearity range was 50-300 microg/ml. The percentage recoveries ranged from 95.10 to 101.02. The method was found to be suitable not only for monitoring the reactions during the process development but also quality assurance of coenzyme Q(10). For identification of related substances atmospheric pressure chemical ionisation-mass spectrometry (APCI-MS) was used.

LC–ESI-MS determination and pharmacokinetics of adrafinil in rats

A highly sensitive and specific liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for investigating the pharmacokinetics of adrafinil in rats was developed. Rat serum pretreated by solid-phase extraction (SPE) was analyzed by LC-MS/MS with an electrospray ionization (ESI) interface. The mobile phase consisted of acetonitrile:water:acetic acid (35:65:0.1, v/v/v) in an isocratic elution mode pumped at 1.0 ml/min. The analytical column (250 mm x 4.6 mm i.d.) was packed with Kromasil C(18) material (5.0 microm). The standard curve was linear from 16.5 to 5000 ng/ml. The assay was specific, accurate (R.S.D.<2.6%), precise and reproducible (within- and between-day precisions R.S.D. <7.0% and <9.0%, respectively). Adrafinil in rat serum was stable over three freeze-thaw cycles at ambient temperature for 6 h. The method had a lower limit of quantitation of 16.5 ng/ml, which offered high sensitivity for the determination of adrafinil in serum. The method was successfully applied to pharmacokinetic studies of adrafinil after an oral administration to rats.

Development of a validated LC method for enantiomeric separation and determination of adrafinil and its related substances on a Chiralcel OJ‐H column connected to PDA and polarimetric detectors in series

A rapid and reliable high-performance liquid chromatographic method for resolution of enantiomers of adrafinil [(±)-ADL], a novel vigilance promoting agent, and its synthetic intermediates was developed. The separation was carried out on a Chiralcel OJ-H using n-hexane-ethanol (62:38 v/v) as a mobile phase. The detection was carried out at 225 nm using a photodiode array (PDA) detector. The optical rotation and order of elution of enantiomers were assigned. The method is suitable not only for process development of ADL but also for quality assurance of bulk drugs and pharmaceuticals.