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LC-MS/MS comprises an important category of chromatographic techniques.This thesis focuses on the use of LC-MS in determination of certain drugs andpesticides. This thesis comprises eight sections.Section IThis section contains a general introduction on (LC-MS/MS), its theory andinstrumentation. After that, an introduction on the pesticides and drugs ispresent.Section IILiquid chromatographic–tandem mass spectrometric method for thequantitation of Fenbucarb, Carbosulfan and carbofuran in human plasma.Carbofuran (CFN), carbosulfan (CSN) and fenobucarb (FBC) are carbamatepesticides that are widely used in gardening and agriculture for the control ofinsects. Human poisoning due to occupational or self-poisoning exposures wasalso reported, so assays are required to quantify the plasma concentration ofthese insecticides. An LC-MS/MS method was developed and validated for thesimultaneous quantification of these three carbamate insecticides in the plasmaof patients with acute intentional self-poisoning. Plasma samples werepretreated by acetonitrile for protein precipitation. Chromatography was carriedout on an a Luna C18(2) analytical column with gradient elution using a mobilephase containing acetonitrile and water with 10mM ammonium acetate.Mass spectrometric analysis was performed by an Applied Biosystems MDSSciex API 2000 triple quadrupole mass spectrometer coupled with electrosprayionization (ESI) source in the positive ion mode. The total run time was 7 min.The assay was validated over a concentration range from 10 to 1000 ng/ml forCSN and FBC and from 20 to 2000 ng/ml for CFN. The precision and accuracyfor both intra- and inter-day determination of all analytes were acceptable(<15%). No significant matrix effect was observed. Stability of compounds was2established for short-term bench and autosampler storage as well as freeze/thawcycles. The method was effectively applied to 270 clinical samples frompatients with a history of acute intentional carbamate self-poisoning.Section IIIDetermination of chlorfluazuron as an example of Benzylurea pesticides inhuman plasma by using liquid chromatography-electrospray ionizationtandem mass spectrometry.Chlorfluazuron (CFZ) is one of most commonly used Benzylurea insecticides.Human poisoning of CFZ has been reported in Sri Lanka, so it is necessary toquantify CFZ in human plasma. We, therefore, developed and validated aLC-MS/MS method for quantitative determination of CFZ in the plasma ofpatients with acute self-poisoning. The plasma samples were first deproteinizedby acetonitrile. Chromatography was performed on a Luna 5 μm C18(2)analytical column with gradient elution using mobile phase containing 0.1%formic acid in water and methanol. An Applied Biosystems MDS Sciex API2000 triple quadrupole mass spectrometer coupled with electrospray ionization(ESI) source was operated in the positive ion mode. The total run time was 5min. The assay was validated over a concentration range from 20 to 2000 ng/mlwith acceptable precision and accuracy (<15%). Matrix effect was notsignificant when tested in six batches of plasma. Stability of CFZ wasestablished for short-term bench and autosampler storage as well as freeze/thawcycles. The method was applied successfully in determination of CFZ level in41 clinical samples from patients with a history of acute intentional CFZ selfpoisoning.3Section IVLiquid chromatography and tandem mass spectrometry method forsimultaneous determination of Atorvastatin and three of its metabolites inhuman plasma by liquid- liquid extraction .There are few published methods for determination of atorvastatin and itsmetabolite in human plasma.. A new method using (LC–MS/MS) has beendeveloped .The method was validated and applied for determination ofatrovastatin(ASN),2-hydroxy atrovaststin (oH-ASN) , atorvastatin lactone(ASN-lac ) and 2-hydroxy atorvastatin lactone(oH-ASN-lac) in human plasma.Chromatography was performed on a Luna 5 μm C18 (2) (Phenomenex)50×2.0mm.Elution with mobile phase consisting of 0.1% formic in water(solvent A) and in acetonitrile: water in the ratio of 95:5 (v/v) (solvent B).The elution was isocratic at a flow rate of 0.25 ml/min and the percent of B was65%. The pump stopped at 4 min.The LC separation and MS/MS optimization were studied to select the mostappropriate operating conditions. The method developed has also beenvalidated. The lowest limits of quantification (LLOQs) were from 0.5ngml−1 for(ASN), 0.1gml−1 for its metabolites .The LC–MS/MS method developed issimple, rapid, accurate, sensitive, and reliable for the quantification andconfirmation of ASN and its metabolites.Section VLiquid chromatography and tandem mass spectrometry method forsimultaneous determination of Atorvastatin and three of its metabolites inhuman plasma by solid phase extraction.Solid phase extraction has been preferred over liquid-liquid extraction becauseof the practical advantages of no emulsions, better recoveries, cleaner extractsachievable, and matrix components selectively. This method was moreconvenient and time saving method for analysis of atrovastatine and itsmetabolites in clinical samples. The assay was validated over a concentration4range from 0.212 to 500 ng/ml for ASN and from 0.564 to 100 ng/ml for itsmetabolites. The precision and accuracy for both intra- and inter-daydetermination of all analytes were acceptable (<15%). No significant matrixeffect was observed. Stability of compounds was established for short termbench and autosampler storage as well as freeze/thaw cycles. The method waseffectively applied to determine clinical from patients on prior statin therapy incritically ill patients with sepsis .Section VILiquid chromatographic–tandem mass spectrometric method for thequantitation of atorvastatin and Gliclazlde in human plasma.A simple, rapid and specific method for simultaneous determination ofatrovastatin and gliclazide in human plasma. By using LC-MS/MS isdescribed. Only 100 μl of plasma and a little sample work-up are required.The atrovastain and gliclazide peaks were separated from endogenous peakson a C18 column by a gradient elution using 0.1% aqueous formic acid and0.1% formic acid in acetonitrile: Water(95:5) . The lowest limit of quantitation(LLOQ) for both analytes in plasma were 5 ng/ml. The method was linear overthe range of 5–200 ng/ml. The method validated and applied to clinical samples.Section VIILiquid chromatographic–tandem mass spectrometric method for thequantitation of piperacillin, tazobactam and cefazolin in human plasma.Antibiotics combinations are given to patient in intensive care unit fortreatment of sepsis. There are few published methods which discus thedetermination of antibiotic combination .The purpose of the present work was todevelop a new, reliable, reproducible, simple and less time-consumingLC-MS/MS in method for the determination of Piperacillin, tazobactam andCefazolin in human plasma. This method involving a simple proteinprecipitation and centrifugal filtration for the simultaneous (LC–MS/MS)quantitation of piperacillin (PPC), tazobactam (TBM) and cefazolin (CZL) in5human plasma. Chromatography was performed on a Luna 5 μm C18(2)Phenomenex 50×2.0mm.The mobile phase was a binary gradient water–acetonitrile with 0.1% Formic acid at flow rate of 0.3 ml min−1. The LCseparation and MS/MS optimization were studied to select the most appropriateoperating conditions.The lowest limits of quantification (LLOQs) were from1.25ugml−1 for (PPC), 0.25ugml−1 for TBM and 0.5ugml−for CZL .All three analytes could be simultaneously quantified in human plasma. Thelinear quantification range comprised 1.25to 500 ugml−1 for (PPC), 0.25 to 100for TBM and 0.5to 200 ngml−1 for CZL .Section VIIICaffeine and its metabolites (theobromine, theophylline and paraxanthine)HPLC assay for CYP1A2 activity in intensive care patientsThis HPLC method is suitable for separation and determination of caffeine and3 of its metabolites in human plasma using a reversed-phase chromatographycolumn, thermo stated to 35 C , with UV detection and isocratic elution.The mobile phase contains a mixture of acetonitrile : Tetra hydro furan : glacialacetic acid : water (20:20:5:950) .The linearity and reproducibility of themethod are validated. the limit of detection is 0.025 ugml−1Finally this thesis contain 183 pages 33 tables ,54 figures and 107 references .
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