Summary Chapter 8 CHAPTER 8. Summary. Page 173
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1 CHAPTER 8 Summary Page 173
2 Chapter 2: Liquid Chromatography/Tandem Mass Spectrometry Method for Quantitative Estimation of PEG 400 and its Applications A rapid sensitive and selective MRM based method for the determination of polyethylene glycol 400 (PEG 400) in rat plasma was developed using liquid chromatography/tandem mass (LC-MS/MS). PEG 400 and telmisartan (internal standard) were extracted from rat plasma with acetonitrile and analyzed on C18 column (Waters Xbridge, 50 x 4.6 mm, 3.5µm) with the mobile phase (A-0.1% formic acid in water; B-methanol). A generic gradient method with a short run time of 3.5 min was developed for the analysis of PEG 400. A total of nine oligomers were identified for PEG 400. The most abundant ions corresponding to PEG 400 oligomers at m/z 327, 371, 432, 476, 520, 564, 608, 652 and 696 with daughter ion at m/z 89 were selected for multiple reaction monitoring (MRM) in electrospray mode of ionisation. Analyte peak area of the oligomers was summed up to calculate the plasma concentrations of total PEG 400. The standard curve was linear (0.9954) over the concentration range of 1.01 to µg/ml. The developed method was successfully applied to the absolute bioavailability study of PEG 400 in male SD rats. Plasma concentrations of PEG 400 were measured after administration through oral and intravenous routes in male SD rats at a dose of 3.38 g/kg. Pharmacokinetic (PK) parameters were characterized by performing the analysis using Phoenix Winnonlin software (v 6.3). Plasma concentration profile/pk parameters of PEG 400 were established in both intravenous and oral routes, which help to qualify the analytical batch of NCEs having spiky plasma concentration profiles/erratic results. Purity of the PEG 400 oligomers was estimated using ELSD detection. Differences in pharmacokinetics of oligomers were studied. It was found that with increase in molecular weight of the oligomer, a decrease in absolute bioavailability was observed. Page 174
3 Chapter 3: Identification and Reduction of Matrix Effects caused by PEG 400 in bioanalysis using Liquid chromatography/tandem mass Ion suppression effect of dosing vehicle excipient polyethyleneglycol 400 on the accuracy of liquid chromatography/tandem mass (LC-MS/MS) measurements was studied. Ion suppression cause significant errors in accuracy of the pharmacokinetic results. Plasma concentrations of PEG 400 ranging from 3-10 mg/ml in the initial sampling points caused 2-10 fold ion supression on most of the analytes studied. This can result in false rejection of compounds in a drug discovery screen. Several sample preparation methods, enhanced chromatographic selectivity, and alternative ionization methods were investigated as means to avoid or minimize ion suppression effects. The elimination of ion suppression effects was achieved by liquid liquid Extraction (LLE) with hexane as sample preparation method. The mechanism of ion suppression caused by polyethylene glycol 400 in relation to both liquid and gas phase reactions was proposed. Chapter 4: Liquid chromatography/tandem Mass Spectrometry Method for the Quantitative Estimation of CrEL and its Applications A rapid sensitive and selective MRM based method for the determination of Cremophor EL (CrEL) in rat plasma was developed using liquid chromatography/tandem mass (LC-MS/MS). CrEL and polypropylene glycol (internal standard) were extracted from rat plasma with acetonitrile and analysed on C18 column (XBridge, 50 x 4.6 mm, 3.5 µm) with the mobile phase (A- 0.1 % formic acid in water; B- acetonitrile with 20% tetrahydrofuran). A generic gradient method with a short run time of 3.5 min was developed for the analysis of CrEL. The most abundant molecular ions corresponding to PEG oligomers at m/z 828, 872, 916 and 960 with daughter ion at m/z 89 were selected for multiple reaction monitoring (MRM) in electrospray mode of ionisation. Plasma concentrations of CrEL were quantified after administration through oral and intravenous routes in male sprague dawley rats at a dose of 0.26 g/kg. The standard curve was linear (0.9972) over the concentration range of 1.00 to 200 µg/ml. Pharmacokinetic parameters were Page 175
4 characterized by performing the analysis using phoenix winnonlin software (v 6.3). Plasma concentration profile/pk parameters of CrEL in male sprague dawley rats was established in both intravenous and oral routes, which help to qualify the analytical batch of NCEs having spiky plasma concentration profiles/erratic results. Chapter 5: Identification and Reduction of Matrix Effects caused by CrEL in bioanalysis using Liquid chromatography/tandem mass Ion suppression effect of dosing vehicle excipient Cremophor EL (CrEL) on the accuracy of liquid chromatography/tandem mass (LC-MS/MS) measurements was studied. Ion supression cause significant errors in accuracy of the pharmacokinetic results. Plasma concentrations of CrEL ranging from mg/ml in the initial sampling points caused 2-10 fold ion supression on most of the analytes studied. This can result in false rejection of compounds in a drug discovery screen. Various sample preparation methods, enhanced chromatographic selectivity, and alternative ionization methods were investigated as means to avoid or minimize ion suppression effects. The elimination of ion suppression effects was achieved by Liquid-Liquid Extraction (LLE) with hexane, TBME in electrospray ionisation (ESI) mode as sample preparation method. In contrast to ESI mode that had severe suppression effects from CrEL, atmospheric pressure chemical ionisation (APCI) mode is totally free of suppression effects. The mechanism of ion suppression caused by CrEL in relation to both liquid and gas phase reactions was proposed. Chapter 6: Liquid chromatography/tandem Mass Spectrometry Method for the Quantitative Estimation of Solutol HS15 and its Applications A rapid sensitive and selective pseudomrm (pmrm) based method for the determination of solutol HS15 (SHS15) in rat plasma was developed using liquid chromatography/tandem mass (LC-MS/MS). SHS 15 and telmisartan (internal standard) were extracted from rat plasma with acetonitrile and analysed on C18 column (Thermo, 50 x 4.6 mm, 2.5 µm) with the mobile phase (A-0.1 % formic acid in water; B- Acetone). A generic gradient method with a short run time of 5 min was developed for the analysis of SHS15. The most abundant ions corresponding to Page 176
5 SHS15 free polyethyleneglycol (PEG) oligomers at m/z 481, 525, 569, 613, 657, 701, 745, 789, 833, 877, 921 and 965 were selected for pseudo multiple reaction monitoring (pmrm) in electrospray mode of ionisation. Purity of the lipophilic and hydrophilic components of SHS15 was estimated using ELSD detector. Plasma concentrations of SHS15 were measured after oral administration at 2.50 g/kg dose and intravenous administration at 1.00 g/kg dose in male SD rats. The standard curve was linear (0.9978) over the concentration range of 0.40 to µg/ml. Pharmacokinetic parameters were characterized by performing the analysis using phoenix winnonlin software (v 6.3). Differences in pharmacokinetics of oligomers were studied. With the increase in molecular weight of the oligomer, a decrease in absolute bioavailability was observed. Plasma concentration profile/pk parameters of SHS15 in male sprague dawley rats was established in both intravenous and oral routes, which help to qualify the analytical batch of NCEs having spiky plasma concentration profiles/erratic results. Chapter 7: Identification and Reduction of Matrix Effects caused by SHS 15 in bioanalysis using Liquid chromatography/tandem mass Ion suppression effect of dosing vehicle excipient Solutol HS15 (SHS15) on the accuracy of liquid chromatography/tandem mass (LC-MS/MS) measurements was studied. Ion supression cause significant errors in accuracy of the pharmacokinetic results. Plasma concentrations of SHS15 ranging from 1-2 mg/ml in the initial sampling points caused 2-10 fold ion supression on most of the analytes studied. This can result in false rejection of compounds in a drug discovery screen. Several sample preparation methods, enhanced chromatographic selectivity, and alternative ionization methods were investigated as means to avoid or minimize ion suppression effects. The elimination of ion suppression effects was achieved by Liquid-Liquid Extraction (LLE) with hexane, tert-butyl-methyl ether (TBME) as sample preparation method. The mechanism of ion suppression caused by SHS15 in relation to both liquid and gas phase reactions was proposed. Page 177
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