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.Each of the glucuronide isomerselutes separately and can be identified from its NMR spectrum.It hasbeen noted that in general the elution order of transacylated glucuronidesis ( -4-O-acyl-, ) -4-O-acyl-, ) -3-O-acyl-, ( -3-O-acyl-, ( -2-O-acyl- and ) -2-O-acyl-, irrespective of the nature of the carboxylic-acid-containing moiety. 70 LC NMR and Related Techniquesglucuronic acidHOOCOHOOCOHOHO+ RCOOHO ROHHOHOOHOHO-1-O-acylHOOCOHOHOOHOHHOOC HOOCO OHO HOOHHO HOOH OCOR-2-O-acyl -2-O-acyl OHHOOC HOOCO OHO HOOHRCOO RCOOOH OH-3-O-acyl-3-O-acyl OHHOOC HOOCO ORCOO RCOOOHHO HOOH OH-4-O-acyl-4-O-acyl OHFigure 3.4 The reaction scheme depicting acyl migration in glucuronide conjugates.Recently,the back reaction from the ) -2-O-acyl isomer to the ) -1-O-acyl isomer has been identifiedThis directly coupled HPLC NMR method has been used to investigate theacyl migration kinetics of individual isomers of 2-, 3- and 4-fluoro-, and 2- and3-trifluoromethylbenzoyl-d-glucopyranuronic acid separated from an equili-brium mixture of the ( -1-O-acyl isomer, the ) - and ( -2-O-acyl isomers, the Biomedical Applications of HPLC/NMR and HPLC/NMR/MS 712.5 ,2.3 ,2.2 ,2.4 , , 152.5 ,2.3 ,2.1 ,2.2 ,3.4 ,3.3 ,3.1 , 3.2 ,3.3 ,101.1 , 3.5 , ,3.4 ,3.2 ,4.2 ,4.3 ,4.2 ,54.5 ,4.1 ,4.3 ,4.4 , ,4.5 ,4.1 ,Glucuronic acid5.7 5.2 4.7 4.2 3.7(ppm)1Figure 3.5 750 MHz H continuous-flow HPLC NMR spectrum of an equilibrium mixtureof transacylated glucuronides of 4-fluorobenzoic acid.The nomenclature 2,1-) denotes the 1proton in the ) -anomer of the 2-O-acyl glucuronide) - and ( -3-O-acyl isomers, and the ) - and ( -4-O-acyl isomers at pH 7.4 and25 C [49 52].Both continuous-flow HPLC NMR and stop-flow methodshave been used.For detailed kinetic studies, each isomer was separated byusing reversed-phase HPLC and then led into an NMR flow probe in a1600 MHz NMR spectrometer.The flow was stopped and sequential H NMRspectra collected, thus allowing the direct observation of the appearance of theglucuronide positional isomers of that particular glucuronide isomer which hadbeen isolated.This is illustrated in Figure 3.6, which shows the build-up ofother products following the introduction of the ( -4-O-acyl-glucuronide of 2-fluorobenzoic acid into the NMR probe after HPLC separation.The rateconstants for the decomposition of the various isomers were determinedand the acyl migration reactions were simulated by using a mathematicalmodel of the kinetics of the glucuronide rearrangement (incorporating ninefirst-order rate constants determining acyl migration reactions and six first-order rate constants describing the mutarotation of the 2-, 3- and 4-positionalisomers.The acyl migration of the glucuronide metabolite of the model drug 6,11-dihydro-11-oxodibenz (b,e) oxepin-2-acetic acid has also been investigated inurine and pH 7.4 buffer using directly coupled 600 and 750 MHz stop-flowHPLC 1H NMR spectroscopy [53,54].More recently, the acyl migration reaction has been studied for a range ofnon-steroidal anti-inflammatory compounds.For example, a detailed study hasChromatographic separation time (min) 4,3 ,( )4,5 ,( )4,1 ,( ) 4,4 ,( , )2,4,3 ,( ) 2,5 ,( )3,2 ,4,5 ,( )4,2 ,( ) 4,2 ,( )( )3,1 ,( )2,2 ,( )3,5 ,( , ) 2,4, ,( , )3,3 ,( ) 3,3 ,( )3,2 ,( )2,1 ,( ) 2,2 ,( ) 2,5 ,( )302010( , ) 2-O-acyl( ) 2-O-acyl( ) 3-O-acyl( ) 3-O-acyl ( ) 3-O-acyl( ) 3-O-acyl ( ) 2-O-acyl ( , ) 2-O-acyl5.1 4.6 4.1 3.6(ppm)1Figure 3.6 600 MHz H NMR spectrum obtained in stop-flow mode after the ( -4-O-acyl-glucuronide of 2-fluorobenzoic acid had been isolated inthe NMR flow cell.This shows the successive formation of 3-O-acyl- and 2-O-acyl isomers as a function of timeReaction time domain (h) Biomedical Applications of HPLC/NMR and HPLC/NMR/MS 73been made of the reactions of S-naproxen-( -1-O-acyl glucuronide in phosphatebuffer at pH 7.4, purified from human urine after oral administration of atherapeutic dose of approximately 400 mg.In this case, new signals were1observed in the 600 MHz H NMR spectra and these were assigned to the ) -1-O-acyl glucuronide isomer and the structure was confirmed by using 2DTOCSY NMR spectra and parallel HPLC MS studies to provide the molecularweight [55] [ Pobierz całość w formacie PDF ]
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.Each of the glucuronide isomerselutes separately and can be identified from its NMR spectrum.It hasbeen noted that in general the elution order of transacylated glucuronidesis ( -4-O-acyl-, ) -4-O-acyl-, ) -3-O-acyl-, ( -3-O-acyl-, ( -2-O-acyl- and ) -2-O-acyl-, irrespective of the nature of the carboxylic-acid-containing moiety. 70 LC NMR and Related Techniquesglucuronic acidHOOCOHOOCOHOHO+ RCOOHO ROHHOHOOHOHO-1-O-acylHOOCOHOHOOHOHHOOC HOOCO OHO HOOHHO HOOH OCOR-2-O-acyl -2-O-acyl OHHOOC HOOCO OHO HOOHRCOO RCOOOH OH-3-O-acyl-3-O-acyl OHHOOC HOOCO ORCOO RCOOOHHO HOOH OH-4-O-acyl-4-O-acyl OHFigure 3.4 The reaction scheme depicting acyl migration in glucuronide conjugates.Recently,the back reaction from the ) -2-O-acyl isomer to the ) -1-O-acyl isomer has been identifiedThis directly coupled HPLC NMR method has been used to investigate theacyl migration kinetics of individual isomers of 2-, 3- and 4-fluoro-, and 2- and3-trifluoromethylbenzoyl-d-glucopyranuronic acid separated from an equili-brium mixture of the ( -1-O-acyl isomer, the ) - and ( -2-O-acyl isomers, the Biomedical Applications of HPLC/NMR and HPLC/NMR/MS 712.5 ,2.3 ,2.2 ,2.4 , , 152.5 ,2.3 ,2.1 ,2.2 ,3.4 ,3.3 ,3.1 , 3.2 ,3.3 ,101.1 , 3.5 , ,3.4 ,3.2 ,4.2 ,4.3 ,4.2 ,54.5 ,4.1 ,4.3 ,4.4 , ,4.5 ,4.1 ,Glucuronic acid5.7 5.2 4.7 4.2 3.7(ppm)1Figure 3.5 750 MHz H continuous-flow HPLC NMR spectrum of an equilibrium mixtureof transacylated glucuronides of 4-fluorobenzoic acid.The nomenclature 2,1-) denotes the 1proton in the ) -anomer of the 2-O-acyl glucuronide) - and ( -3-O-acyl isomers, and the ) - and ( -4-O-acyl isomers at pH 7.4 and25 C [49 52].Both continuous-flow HPLC NMR and stop-flow methodshave been used.For detailed kinetic studies, each isomer was separated byusing reversed-phase HPLC and then led into an NMR flow probe in a1600 MHz NMR spectrometer.The flow was stopped and sequential H NMRspectra collected, thus allowing the direct observation of the appearance of theglucuronide positional isomers of that particular glucuronide isomer which hadbeen isolated.This is illustrated in Figure 3.6, which shows the build-up ofother products following the introduction of the ( -4-O-acyl-glucuronide of 2-fluorobenzoic acid into the NMR probe after HPLC separation.The rateconstants for the decomposition of the various isomers were determinedand the acyl migration reactions were simulated by using a mathematicalmodel of the kinetics of the glucuronide rearrangement (incorporating ninefirst-order rate constants determining acyl migration reactions and six first-order rate constants describing the mutarotation of the 2-, 3- and 4-positionalisomers.The acyl migration of the glucuronide metabolite of the model drug 6,11-dihydro-11-oxodibenz (b,e) oxepin-2-acetic acid has also been investigated inurine and pH 7.4 buffer using directly coupled 600 and 750 MHz stop-flowHPLC 1H NMR spectroscopy [53,54].More recently, the acyl migration reaction has been studied for a range ofnon-steroidal anti-inflammatory compounds.For example, a detailed study hasChromatographic separation time (min) 4,3 ,( )4,5 ,( )4,1 ,( ) 4,4 ,( , )2,4,3 ,( ) 2,5 ,( )3,2 ,4,5 ,( )4,2 ,( ) 4,2 ,( )( )3,1 ,( )2,2 ,( )3,5 ,( , ) 2,4, ,( , )3,3 ,( ) 3,3 ,( )3,2 ,( )2,1 ,( ) 2,2 ,( ) 2,5 ,( )302010( , ) 2-O-acyl( ) 2-O-acyl( ) 3-O-acyl( ) 3-O-acyl ( ) 3-O-acyl( ) 3-O-acyl ( ) 2-O-acyl ( , ) 2-O-acyl5.1 4.6 4.1 3.6(ppm)1Figure 3.6 600 MHz H NMR spectrum obtained in stop-flow mode after the ( -4-O-acyl-glucuronide of 2-fluorobenzoic acid had been isolated inthe NMR flow cell.This shows the successive formation of 3-O-acyl- and 2-O-acyl isomers as a function of timeReaction time domain (h) Biomedical Applications of HPLC/NMR and HPLC/NMR/MS 73been made of the reactions of S-naproxen-( -1-O-acyl glucuronide in phosphatebuffer at pH 7.4, purified from human urine after oral administration of atherapeutic dose of approximately 400 mg.In this case, new signals were1observed in the 600 MHz H NMR spectra and these were assigned to the ) -1-O-acyl glucuronide isomer and the structure was confirmed by using 2DTOCSY NMR spectra and parallel HPLC MS studies to provide the molecularweight [55] [ Pobierz całość w formacie PDF ]