Babu Rao Chandu

Method Development and Validation of Montelukast in Human Plasma by HPLC Coupled with ESI-MS/MS: Application to a Bioequivalence Study

A simple, sensitive, and specific LC-ESI–MS/MS method for quantification of Montelukast (MO) in human plasma using Montelukast-d6 (MOD6) as an internal standard (IS) is discussed here. Chromatographic separation was performed on YMC-pack pro C18, 50 x 4.6 mm, S-3 μm column with an isocratic mobile phase composed of 10mM ammonium formate (pH 4.0):acetonitrile (20:80 v/v), at a flow-rate of 0.8 mL min−1. MO and MOD6 were detected with proton adducts at m/z 586.2→568.2 and 592.3→574.2 in multiple reaction monitoring (MRM) positive mode respectively. MO and MOD6 were extracted using acetonitrile as precipitating agent. The method was validated over a linear concentration range of 1.0–800.0 ng mL−1 with correlation coefficient (r2) ≥ 0.9996. The intraday precision and accuracy were within 1.91–7.10 and 98.32–99.17. The inter-day precision and accuracy were within 3.42–4.41% and 98.14–99.27% for MO. Both analytes were found to be stable throughout three freeze-thawing cycles, bench top, and autosampler stability studies. This method was utilized successfully for the analysis of plasma samples following oral administration of MO (5 mg) in 31 healthy Indian male human volunteers under fasting conditions.

Development and validation of a Sensitive bioanalytical method for the quantitative estimation of Pantoprazole in human plasma samples by LC–MS/MS: Application to bioequivalence study

The present study aims at developing a simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of pantoprazole sodium (PS) in human plasma using pantoprazole D3 (PSD3) as internal standard (IS). Chromatographic separation was performed on Zorbax SB-C18, 4.6 mm x 75 mm, 3.5 microm, 80 A column with an isocratic mobile phase composed of 10 mM ammonium acetate (pH 7.10): acetonitrile (30:70, v/v), pumped at 0.6 mL/min. PS and PSD3 were detected with proton adducts at m/z 384.2-->200.1 and 387.1-->203.1 in multiple reaction monitoring (MRM) positive mode, respectively. Precipitation method was employed in the extraction of PS and PSD3 from the biological matrix. This method was validated over a linear concentration range of 10.00-3000.00 ng/mL with correlation coefficient (r) > or = 0.9997. Intra- and inter-day precision of PS were found to be within the range of 1.13-1.54 and 1.76-2.86, respectively. Both analytes were stable throughout freeze/thaw cycles, bench top and postoperative stability studies. This method was successfully utilized in the analysis of blood samples following oral administration of PS (40 mg) in healthy human volunteers.

LC-ESI-MS/MS method for the quantification of entecavir in human plasma and its application to bioequivalence study.

Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was used for a quantitative estimation of entecavir (EV) in human plasma using lamivudine (LM) as internal standard (IS). The method herein described is simple, sensitive, and specific. Chromatographic separation was performed on XBridge-C18, 4.6 mm × 50 mm, 5-μm column with an isocratic mobile phase composed of 10 mM ammonium hydrogen carbonate (pH 10.5):methanol (85:15 v/v), pumped at 0.3 ml/min. EV and LM were detected using proton adducts at m/z 278.1→152.1 and 230.2→112.0 in multiple reaction monitoring (MRM) positive mode. Solid phase extraction method was employed in the extraction of EV and LM from the biological matrix. This method was validated over a linear concentration range of 50.0-20000.0 pg/ml with a correlation coefficient (r) ≥0.9983. Intra and inter-day precision of EV was found within the range of 1.2-4.2 for EV and 4.4-4.5 for LM. EV was stable throughout three freeze/thaw cycles, bench top and postoperative studies. This method was successfully used in the analysis of plasma samples following oral administration of EV (0.5 mg) in 26 healthy human volunteers.

Development and Validation of Amisulpride in Human Plasma by HPLC Coupled with Tandem Mass Spectrometry and its Application to a Pharmacokinetic Study

In this study, authors developed a simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for quantification of Amisulpride in human plasma using Amisulpride-d5 as an internal standard (IS). Chromatographic separation was performed on Zorbax Bonus-RP C18, 4.6 × 75 mm, 3.5 μm column with an isocratic mobile phase composed of 0.2% formic acid:methanol (35:65 v/v), at a flow-rate of 0.5 mL/min. Amisulpride, Amisulpride-d5 was detected at m/z 370.1→242.1 and 375.1→242.1. The drug and the IS were extracted by a liquid-liquid extraction method. The method was validated over a linear concentration range of 2.0–2500.0 ng/mL for Amisulpride with a correlation coefficient of (r2) ≥ 0.9982. This method demonstrated intra- and inter-day precision within 0.9 to 1.7 and 1.5 to 2.8 % and intra- and inter-day accuracy within 98.3 to 101.5 and 96.0 to 101.0 % for Amisulpride. Amisulpride was found to be stable at 3 freeze–thaw cycles, bench top and auto sampler stability studies. The developed method was successfully applied to a pharmacokinetic study.

Sildenafil and N-desmethyl sildenafil quantification in human plasma by HPLC coupled with ESI-MS/MS detection: Application to bioequivalence study

The present study aims at developing a simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for simultaneous quantification of sildenafil and its metabolite N-desmethyl sildenafil in human plasma using sildenafil-d8, N-desmethyl sildenafil-d8 as internal standards (IS). Chromatographic separation was performed on Zorbax SB C18, 4.6 × 75 mm, 3.5 μm column with an isocratic mobile phase composed of 10 mM ammonium acetate and acetonitrile (5/95 v/v), at a flow-rate of 0.6 ml min−1. Sildenafil, sildenafil-d8, N-desmethyl sildenafil and N-desmethyl sildenafil-d8 were detected with proton adducts at m/z 475.2 → 283.4, 483.4 → 283.4, 461.3 → 283.4 and 469.4 → 283.4 in multiple reaction monitoring (MRM) positive mode respectively. Both drug, metabolite and internal standards were extracted by liquid–liquid extraction. The method was validated over a linear concentration range of 1.0–1000.0 ng ml−1 for sildenafil and 0.5–500.0 ng ml−1 for N-desmethyl sildenafil with correlation coefficient (r2) ≥ 0.9998 for sildenafil and (r2) ≥ 0.9987 for N-desmethyl sildenafil. This method demonstrated intra and inter-day precision within 1.5 to 5.1 and 2.2 to 3.4% for sildenafil and within 1.3 to 3.1 and 2.8 to 4.3% for N-desmethyl sildenafil. This method demonstrated intra and inter-day accuracy for sildenafil within 97.3 to 98.3 and 96.7 to 97.2% and for N-desmethyl sildenafil within 95.3 to 96.3 and 95.0 to 97.2%. Both analytes were found to be stable throughout three freeze/thaw cycles, bench top and postoperative stability studies. This method was used successfully for the analysis of plasma samples following oral administration of 100 mg in 43 healthy Indian male human volunteers under fasting conditions.

Simultaneous determination of ezetimibe and simvastatin in rat plasma by stable-isotope dilution LC-ESI–MS/MS and its application to a pharmacokinetic study

A simple, sensitive and specific liquid chromatography–tandem mass spectrometry method was developed for simultaneous quantification of ezetimibe and simvastatin in rat plasma. The deuterium isotopes: ezetimibe d4 and simvastatin d6 were used as internal standards for ezetimibe and simvastatin, respectively. MS/MS detection involved a switch of electron spray ionization mode from negative to positive at retention time 3.01 min. Samples were extracted from plasma by liquid–liquid extraction using tertiary butyl methyl ether. Chromatographic separation was achieved with Agilent Eclipse XBD-C18 column using mobile phase that consisted of a mixture of ammonium acetate (pH4.5; 10 mM)–acetonitrile (25:75 v/v). The method was linear and validated over the concentration range of 0.2–40.0 ng/mL for simvastatin and 0.05–15.0 ng/mL for ezetimibe. The transitions selected were m/z 408.3→271.1 and m/z 412.0→275.10 for ezetimibe and ezetimibe d4, and m/z 419.30→285.20 and m/z 425.40→199.20 for simvastatin and simvastatin d6. Intra- and inter-batch precisions for ezetimibe were 1.6–14.8% and 2.1–13.4%; and for simvastatin 0.94–9.56% and 0.79–12%, respectively. The proposed method was sensitive, selective, precise and accurate for the quantification of ezetimibe and simvastatin simultaneously in rat plasma. The method was successfully applied to a pharmacokinetic study by oral co-administration of ezetimibe and simvastatin in SD rats.

Erythrocytes as Carrier for Drugs, Enzymes and Peptides

Erythrocytes are potential biocompatible carriers for different drugs, peptide molecules and different enzymes. Now a day the method that is used for encapsulation of pharmaceuticals into the erythrocytes mainly based on the hypo-osmotic dialysis. Encapsulation of these drugs or enzymes or peptides into erythrocytes significantly changes the pharmacokinetic properties of drugs in both animals and humans, enhancing liver and spleen uptake and targeting the reticuloendothelial system. By the encapsulation of these into erythrocytes can be applied as targeted drug delivery systems. Erythrocytes are successful as carrier systems for different drugs, enzymes and peptide molecules. The result after using drug encapsulated in erythrocytes is more compared to the free form of the drug.

Method development and validation of almotriptan in human plasma by HPLC tandem mass spectrometry: application to pharmacokinetic study.

A simple, sensitive and selective method has been developed for quantification of Almotriptan (AL) in human plasma using Almotriptan-d(6) (ALD6) as an internal standard. Almotriptan and Almotriptan-d(6) were detected with proton adducts at m/z 336.1→201.1 and 342.2→207.2 in multiple reaction monitoring (MRM) positive mode, respectively. The method was linear over a concentration range of 0.5-150.0 ng/mL. The limit of detection (LOD) and limit of quantification (LOQ) for Almotriptan were 0.2 pg/mL and 0.5 ng/mL, respectively. Liquid-liquid extraction was used followed by MS/MS (ion spray). The method was shown to be precise with an average within-run and between-run variation of 0.68 to 2.78% and 0.57 to 0.86%, respectively. The average within-run and between-run accuracy of the method throughout its linear range was 98.94 to 102.64% and 99.43 to 101.44%, respectively. The mean recovery of drug and internal standard from human plasma was 92.12 ± 4.32% and 89.62 ± 6.32%. It can be applied for clinical and pharmacokinetic studies.

Simultaneous Quantification of Mesalamine and Its Metabolite N-Acetyl Mesalamine in Human Plasma by LC-MS/MS and Its Application to a Bioequivalence Study

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used for simultaneous quantification of mesalamine and its metabolite N-acetyl mesalamine in human plasma with N-acetyl mesalamine D3 as an internal standard (IS). Chromatographic separation was performed on a Thermo, HyPURITY C18 (150 x 4.6 mm, 5 m) column with an isocratic mobile phase composed of 10 mM ammonium Original Research Article British Journal of Pharmaceutical Research, 4(13): 1568-1590, 2014 1569 acetate and methanol in the ratio of 85:15 (%v/v), at the flowrate of 0.6 mL/min. The drug, metabolite and internal standard were extracted by liquid-liquid extraction. The method was validated over a linear concentration range of 2-1500 ng/mL for mesalamine and 102000 ng/ml for N-acetyl mesalamine, which demonstrated intra and inter-day precision ranging from 1.60 to 8.63% and 2.14 to 8.67% for mesalamine and 0.99 to 5.67% and 1.72 to 4.89% for N-acetyl mesalamine respectively. Similarly, the intraand inter-day accuracy varied from 102.70 to 105.48% and 100.64 to 103.87% for mesalamine, 99.64 to 106.22% and 100.71 to 104.27% for N-acetyl mesalamine respectively. Both analytes were found to be stable throughout freeze–thawing cycles, bench top and postoperative stability studies. The method was successfully applied to support a bioequivalance study of healthy subjects.

UV - SPECTROPHOTOMETRIC ESTIMATION OF ACYCLOVIR IN BULK AND PHARMACEUTICAL DOSAGE FORMS

Analytical method development being a vital part of pre formulation - formulation research and development obviates the need to develop reliable, effective, e co friendly and cost effective methodologies for routine analysis of active pharmaceutical ingredients. UV spectroscopy is one of the earliest, yet of wide applications in drug analysis in different stages of formulations and quality control; despite the availabilities of sophisti cated chromatographic techniques and other hyphenated techniques. Current research attempts to develop simple, sensitiv e, accurate, precise and economical UV spectrophotometric methods for the routine analysis of acyclovir in bulk and pharmaceutical dosage forms using two separate alkaline medi a , 0.1N NaOH (method A) and 0.1N KOH (method B) and validate them as per ICH gui delines. In both the methods maximum absorbance was observed at 264 nm. Beer’s law was obeyed in the concentration of 2.5 - 40 µg / mL in method A and 2.5 - 30 µg / mL in method B with correlation co efficient of 0.999. The % recovery carried out by adding know n amount of standard drug to pre - analyzed tablet solutions was 98.75 ± 0.52 % to 99.78 ± 0.69 % (method A) and 98.55 ± 0.31 % to 99.78 ± 0.22 % (method B). Intra and interday precision expressed in % RSD were 0.38 ± 0.01 and 0.27 ± 0.02 - 0.44 ± 0.01 respe ctively and the percent purity was 99.85 ± 0.05 %. The methods were validated statistically as per ICH guidelines and the results obtained were within the acceptance criteria for the param eters relating to linearity, accuracy, precision.