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 method for the determination of rosiglitazone on rat dried blood spots and rat urine: Application to pharmacokinetics.

A bioanalytical method for the quantification of rosiglitazone on rat dried blood spots (DBS) and rat urine using liquid chromatography, electrospray ionization coupled with tandem mass spectrometry (LC-ESI-MS/MS) was developed and validated. The chromatographic separation was achieved on a Nova-Pak C18 Column (150 mm × 4.6 mm i.d., 4 µm), using 30 mM ammonium acetate (pH 4.0 adjusted with acetic acid) and acetonitrile (75:25, v/v) as a mobile phase at ambient temperature. LC-MS detection was performed with selected ion monitoring using target ions at m/z 358 and m/z 356 for rosiglitazone and pioglitazone respectively. The calibration curve showed a good linearity in the concentration range of 0.05-100 ng/mL. The effect of hematocrit, blood volume and punch location for DBS samples was studied. The mean recoveries of rosiglitazone from DBS and urine were 93.30% and 92.49% respectively. The intra and inter-day precisions of RSD were less than 4.82% in DBS as well as urine. The limit of detections and quantifications were 0.015 and 0.052 ng/mL in DBS and 0.023 and 0.075 ng/mL in urine samples respectively. The method was validated as per FDA guidelines and successfully applied to a pharmacokinetic study of rosiglitazone in rats.

Development of LC-MS/MS method for the determination of dapiprazole on dried blood spots and urine: application to pharmacokinetics.

A rapid and highly sensitive liquid chromatography­tandem mass spectrometric (LC-MS/MS) method for determination of dapiprazole on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse-phase C18 column (250 × 4.6 mm i.d., 5 µm), using 20 mm ammonium acetate (pH adjusted to 4.0 with acetic acid) and acetonitrile (80:20, v/v) as a mobile phase at 25 °C. LC-MS detection was performed with selective ion monitoring using target ions at m/z 326 and m/z 306 for dapiprazole and mepiprazole used as internal standard, respectively. The calibration curve showed a good linearity in the concentration range of 1­3000 ng/mL. The effect of hematocrit on extraction of dapiprazole from DBS was evaluated. The mean recoveries of dapiprazole from DBS and urine were 93.88 and 90.29% respectively. The intra- and inter-day precisions were <4.19% in DBS as well as urine. The limits of detection and quantification were 0.30 and 1.10 ng/mL in DBS and 0.45 and 1.50 ng/mL in urine samples, respectively. The method was validated as per US Food and Drug Administration guidelines and successfully applied to a pharmacokinetic study of dapiprazole in rats.

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.

HPLC-PDA-ORD bioassay of S-(+) and R-(-) clopidogrel on rat dried blood spots.

A simple and rapid chiral high-performance liquid chromatography (HPLC) method was developed and validated for bioanalysis of clopidogrel enantiomers on rat dried blood spots (DBS). Clopidogrel enantiomers were extracted from DBS using ethanol: methanol (80:20, v/v) and separated on a Chiralcel OJ-H column containing cellulose tris (4-methly benzoate) as a polysaccharide stationary phase using n-hexane-ethanol-diethylamine (70:30, 0.1 v/v) as a mobile phase at a flow rate of 1.0 mL/min. The detection was carried out at 220 nm using a photodiode array (PDA) detector while the elution order of the enantiomers was determined by a polarimeter connected to PDA in series. The effect of hematocrit on extraction of clopidogrel enantiomers from DBS was evaluated and no interference from endogenous substances was noticed. The overall accuracy of (R) and (S) enantiomers of clopidogrel from DBS were 91.6 and 89.2%, respectively. The calibration curves were linear over the concentration range of 1-500 µg/mL for both enantiomers. The results show that the method is specific, precise, and reproducible (intra- and interday precision relative standard deviations (RSDs) <10.0%). The stability of racemic clopidogrel was performed under all storage conditions and the results were found to be well within the acceptance limits.

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.

RP-HPLC separation and ESI-MS, 1H, and 13C NMR characterization of forced degradants including process related impurities of carisbamate: method development and validation.

A stability indicating reversed phase HPLC method was developed and validated for determination of process related impurities and forced degradants of carisbamate (CRS) in bulk drugs. Carisbamate when subjected to acid/base hydrolysis, H2O2 oxidation, photolysis and thermal stress significant degradation was observed during acid/base hydrolysis and the degradants were isolated and characterized by ESI-MS, (1)H and (13)C NMR. MS/MS and 2D-NMR (COSY and HSQC) studies revealed the possible isomerization of CRS under stress conditions. The optimum separation was accomplished on Agilent XDB C18 column (150mm×4.6mm; 5µm) using 0.02M KH2PO4 (pH=3.5) and CH3CN as a mobile phase in a gradient elution mode at a flow rate of 1.0mL/min. The eluents were monitored by PDA detector at 211nm and quantitation limits were obtained in the range of 0.1-0.3µg/mL for CRS, degradants and other impurities. The LC method was validated with respect to accuracy, precision, linearity, robustness and limits of detection and quantification as per ICH guidelines.

Liquid chromatographic separation and thermodynamic investigation of stereoisomers of darunavir on Chiralpak AD-H column.

Liquid chromatographic separation of stereoisomers of darunavir on Chiralpak AD-H, a column containing the stationary phase coated with amylose tris(3,5-dimethylphenylcarbamate) as a chiral selector, was studied under normal-phase conditions at different temperatures between 20 and 50°C. The effect of quality and quantity of different polar organic modifiers viz: methanol, ethanol, 1-propanol, and 2-propanol in the mobile phase as well as column temperature on retention, separation, and resolution was investigated and optimized. The optimum separation was accomplished using a mobile phase composed of n-hexane/ethanol/diethyl amine (80:20:0.1 v/v/v) at 40°C. Apparent thermodynamic parameters ΔH(0) and ΔS* were derived from the Van't Hoff plots (lnk' versus 1/T) and used to explain the strength of interactions between the stereoisomers and amylose tris(3,5-dimethylphenylcarbamate) coated chiral stationary phase.

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.

Liquid chromatographic separation of darunavir enantiomers on coated and immobilized amylose tris(3, 5-dimethylphenylcarbamate) chiral stationary phases.

Liquid chromatographic separation of darunavir enantiomers on covalently bonded and physically adsorbed polysaccharide chiral stationary phases was studied at different temperatures. The separations were accomplished under normal-phase conditions by using different combinations of hexane, organic modifiers (2-propanol, 1-propanol and ethanol), and diethylamine as mobile phase solvents. The effect of organic modifiers and the column temperature on retention, separation, and resolution was investigated. The observed differences were explained in terms of the coated and immobilized nature of the two columns. Van't Hoff plots (ln k' vs. 1/T, ln α vs. 1/T) and apparent thermodynamic parameters were derived to understand the effect of temperature on separation.

Evaluation of (R)-(-)-α-methoxy phenyl acetic acid as a chiral shift reagent for resolution and determination of R and S enantiomers of modafinil in bulk drugs and formulations by 1H NMR spectroscopy.

(R)-(-)-α-Methoxy phenyl acetic acid, (S)-(-)-1,1'-(2-naphthol), and (R)-(+)-α-methoxy-α-trifluoromethyl phenyl acetic acid were evaluated as chiral shift reagents (CSRs) for (1)H NMR spectroscopic resolution and determination of R and S enantiomers of modafinil (MDL) in bulk drugs and formulations. Effects of the nature of CSR and the weight ratio of substrate to shift reagent on enantiomeric discrimination were investigated. Intramolecular and intermolecular hydrogen bonding interactions between the drug and the CSR seem to be the driving force for desired resolution. A mechanism was proposed to explain the interactions between (R, S)-enantiomers of MDL and (R)-(-)-α-methoxy phenyl acetic acid. The method was validated and applied successfully to determine the enantiomeric purity of MDL in tablet formulations.

LC-MS of novel narcoepileptics and related substances.

Modafinil, adrafinil and their related substances were synthesized and analyzed by RP-LC with ESI-MS/MS. The ionization mode, polarity, cone voltage, and chromatographic conditions were evaluated. The optimum LC-MS conditions to obtain fragment ions indispensable for identification of the structures were described. The bulk drugs purity of modafinil and adrafinil was evaluated on Kromasil C(18) column with ACN/0.02 M ammonium acetate as mobile phase in gradient elution mode at 30 degrees C. The method was found to be suitable not only for monitoring the reactions during the process development but also for quality assurance of modafinil and adrafinil.

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.

Isolation and characterization of process related impurities of olanzapine using HPLC and ESI-MS/MS.

An RP-HPLC method for the simultaneous separation and determination of olanzapine (OLZ) and its process impurities in bulk drugs and pharmaceutical formulations was developed. The separation was accomplished on Inertsil ODS 3V (4.6 mm x 250 mm; particle size 5 microm) column using 0.2 M ammonium acetate (pH = 4.50) and ACN as mobile phase in gradient elution mode. The analytes were monitored by a photo diode array (PDA) detector set at 254 nm and the flow rate was kept at 1.0 mL/min. The chromatographic behavior of all the compounds was examined under variable compositions of different solvents, buffer concentrations, and pH. The method was validated in terms of accuracy, precision, and linearity. Four unknown process impurities observed consistently during the analysis of different batches of OLZ were isolated and characterized by ESI-MS/MS, (1)H NMR, and FT-IR. The proposed RP-HPLC method was successfully applied to the analysis of commercial formulations. The method could be of use not only for rapid and routine evaluation of the quality of OLZ in bulk drug manufacturing units but also for the detection of its impurities in pharmaceutical formulations.

Development and validation of a reversed-phase HPLC method for monitoring of synthetic reactions during the manufacture of a key intermediate of an anti-hypertensive drug.

A reversed-phase high-performance liquid-chromatographic method for monitoring of reactions involved in process development of a key intermediate of antihypertensive drugs, e.g, doxazosin mesylate, prazosin, alfuzosin, terazosin, etc., has been developed and validated. The HPLC profiles of impurities of 4-amino-2-chloro-6,7-dimethoxyquinazoline were used as fingerprints to follow the synthetic procedures in the manufacturing unit. The separation was accomplished on an Inertsil ODS-3 column with isocratic elution using acetonitrile-ammonium acetate (10 mM; pH 4.0; 50:50 v/v) as mobile phase and a photodiode array detector set at 240 nm at ambient temperature. The method was validated with respect to accuracy, precision, linearity, and limits of detection and quantification. The method could detect the impurities at a level of 0.01 to 0.20 microg/mL and it was found to be suitable not only for monitoring of reactions but also for quality assurance of 4-amino-2-chloro-6,7-dimethoxyquinazoline.

Separation and determination of synthetic impurities of sildenafil (Viagra) by reversed-phase high-performance liquid chromatography.

A simple and rapid high-performance liquid chromatographic method for the separation and determination of process-related impurities of sildenafil was developed. The separation was achieved on a reversed-phase C18 column using acetonitrile-0.05 M potassium dihydrogen orthophosphate (70:30 v/v) as a mobile solvent at a flow rate of 1.0 ml/min and UV detection at 230 nm. The method was used not only for quality assurance, but also for monitoring the chemical reactions during the synthesis of sildenafil. It was found to be specific, precise and reliable for the determination of all process-related impurities of sildenafil in bulk drugs and formulations.