Simultaneous determination of ginsenoside Rg1, Re, Rd, Rb1 and ophiopogonin D in rat plasma by liquid chromatography/electrospray ionization mass spectrometric method and its application to pharmacokinetic study of 'SHENMAI' injection

A sensitive and rapid liquid chromatography-mass spectrometric method for the simultaneous determination of ginsenoside Rg1, Re, Rd, Rb1 and ophiopogonin D in rat plasma was developed and validated. Chromatographic separation was performed on a C18 column using a step gradient program with the mobile phase of 0.5mmol/L ammonium chloride solution and acetonitrile. The analytes and I.S. were detected using an electrospray negative ionization mass spectrometry in the selected ion monitoring (SIM) mode. The method was linear over the investigated concentration range with a good correlation coefficient higher than 0.997. The lower limits of detection (LLOD) of these analytes were all lower than 2.0ng/mL. The intra- and inter-day precisions were all no more than 7.5% and accuracies were within the range of 97.5-107.0%. The validated method was successfully applied to investigate the pharmacokinetics of ginsenoside Rg1, Re, Rd, Rb1 and ophiopogonin D in rat after intravenous administration of 'SHENMAI' injection.     

In Vitro and In Vivo Metabolism and Inhibitory Activities of Vasicine,a Potent Acetylcholinesterase and Butyrylcholinesterase Inhibitor

Vasicine (VAS), a potential natural cholinesterase inhibitor, exhibited promising anticholinesterase activity in preclinical models and has been in development for treatment of Alzheimer’s disease. This study systematically investigated the in vitro and in vivo metabolism of VAS in rat using ultra performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry. A total of 72 metabolites were found based on a detailed analysis of their ¹H- NMR and ¹³C NMR data. Six key metabolites were isolated from rat urine and elucidated as vasicinone, vasicinol, vasicinolone, 1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-yl hydrogen sulfate, 9-oxo-1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-yl hydrogen sulfate, and 1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-β-D-glucuronide. The metabolic pathway of VAS in vivo and in vitro mainly involved monohydroxylation, dihydroxylation, trihydroxylation, oxidation, desaturation, sulfation, and glucuronidation. The main metabolic soft spots in the chemical structure of VAS were the 3-hydroxyl group and the C-9 site. All 72 metabolites were found in the urine sample, and 15, 25, 45, 18, and 11 metabolites were identified from rat feces, plasma, bile, rat liver microsomes, and rat primary hepatocyte incubations, respectively. Results indicated that renal clearance was the major excretion pathway of VAS. The acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of VAS and its main metabolites were also evaluated. The results indicated that although most metabolites maintained potential inhibitory activity against AChE and BChE, but weaker than that of VAS. VAS undergoes metabolic inactivation process in vivo in respect to cholinesterase inhibitory activity.     

Production of ginsenoside aglycons and Rb1 deglycosylation pathway profiling by HPLC and ESI-MS/MS using Sphingobacterium multivorum GIN723

Using enrichment culture, Sphingobacterium multivorum GIN723 (KCCM80060) was isolated as having activity for deglycosylation of compound K and ginsenoside F1 to produce ginsenoside aglycons such as S-protopanaxadiol (PPD(S)) and S-protopanaxatriol (PPT(S)). Through BLAST search, purified enzyme from S. multivorum GIN723 was revealed to be the outer membrane protein. The purified enzyme from S. multivorum GIN723 has unique specificity for the glucose moiety. However, it has activity with PPD and PPT group ginsenosides such as ginsenosides Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, and F1. From these results, it was predicted that the enzyme has activity on several ginsenosides. Therefore, the biotransformation pathway from Rb1, which is a major, highly glycosylated compound of ginseng, was analyzed using high-performance liquid chromatography and electrospray ionization mass spectrometry/mass spectrometry. The dominant biotransformation pathway from Rb1 to PPD(S) was determined to be Rb1 → Gp-XVII → Gp-LXXV → CK → PPD(S). S. multivorum GIN723 can be used as a whole cell biocatalyst because its activity as whole cells is nine times higher than its activity as cell extracts. The specific activity of whole cells is 2.89 nmol/mg/min in the production of PPD(S). On the other hand, the specific activity of cell extracts is 0.32 nmol/mg/min. The productivity of this enzyme in whole cell form is 500 mg/1 l of cultured cell. Its optimum reaction condition is 10 mM of calcium ions added to a phosphate buffer with a pH of 8.5.     

In vivo rat metabolism and pharmacokinetic studies of ginsenoside Rg3

Metabolism of an anti-tumor active component of Panax ginseng, ginsenoside (20R)-Rg(3), was studied for better understanding its pharmacokinetics in rat. LC-MS was used to determine Rg(3) and its metabolites in rat plasma, urine and feces samples. An average half-life of 18.5 min was obtained after the ginsenoside was intravenously dosed at 5 mg/kg. However, Rg(3) was not detected in rat plasma collected after oral administration at 100 mg/kg. Only 0.97-1.15% Rg(3) of the dosed amount was determined in feces. Hydrolysis and oxygenated metabolites were detected and identified in feces collected after oral administration by using LC-MS and MS-MS.     

Measurement of angiotensin metabolites in organ bath and cell culture experiments by liquid chromatography - electrospray ionization - mass spectrometry (LC-ESI-MS).

The metabolism of renin-angiotensin system (RAS) is more complicated than previously expected and understanding the biological phenomena regulated by variety of angiotensin metabolites requires their precise and possibly comprehensive quantitation. Physiological concentrations of angiotensins (Ang) in biological fluids are low, therefore their accurate measurements require very sensitive and specific analytical methods. In this study we developed an accurate and reproducible method of quantitation of angiotensin metabolites through coupling of liquid chromatography and electrospray ionization - mass spectrometry (LC-ESI-MS). With this method main angiotensin metabolites (Ang I, II, III, IV, 1-9, 1-7, 1-5) can be reliably measured in organ bath of rat tissues (aorta, renal artery, periaortal adipose tissue) and in medium of cultured endothelial cells (EA.hy926), exposed to Ang I for 15 minutes, in the absence or in the presence of angiotensin converting enzyme inhibitor, perindoprilat. Presented LC-ESI-MS method proved to be a quick and reliable solution to comprehensive analysis of angiotensin metabolism in biological samples.     

High performance liquid chromatographic-mass spectrometric determination of ginsenoside Rg3 and its metabolites in rat plasma using solid-phase extraction for pharmacokinetic studies

To support pharmacokinetic studies of ginsenosides, a novel method to quantitatively analyze ginsenoside Rg3 (Rg3), its prosapogenin ginsenoside Rh2 (Rh2) and aglycone 20(S)-protopanaxadiol (ppd) in rat plasma was developed and validated. The method was based on gradient separation of ginsenosides present in rat plasma using high performance liquid chromatography (HPLC), followed by detection with electrospray ionization(ESI) mass spectrometry (MS) in negative ion mode with the mobile phase additive, ammonium chloride (500 microM). Differentiation of ginsenosides was achieved through simultaneous detection of the [M(+)Cl(-)] adduct of ginsenoside Rg3 and [M(+)Cl(-)] adducts of its deglycosylated metabolites Rh2 and ppd, and other ions after solid phase extraction (SPE). The /specific ions monitored were m/z 819.50 for Rg3, m/z 657.35 for Rh2, m/z 495.40 for ppd and m/z 799.55 for the internal standard (digitoxin). The mean recoveries for Rg3, Rh2 and ppd were 77.85, 82.65 and 98.33%, respectively using 0.1 ml plasma for extraction. The lower limits of quantification were 10.0, 2.0 and 8.0 ng/ml (equivalent to 0.1, 0.02 and 0.08 ng in each 10 microl injection onto the HPLC column) for Rg3, Rh2 and ppd, respectively. The method has been demonstrated to be highly sensitive and accurate for the determination of Rg3 and its metabolites in rat plasma.     

Formation of monoclonal antibody against a major ginseng component, ginsenoside Rb1 and its characterization

The ratio of hapten and bovine serum albumin in an antigen conjugate was determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibody against ginsenoside Rb1 was produced by fusing splenocytes immunized with a ginsenoside Rb1- bovine serum albumin conjugate with HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A very small cross-reaction appeared with ginsenoside Rc and ginsenoside Rd. The full measuring range of the assay extends from 20 ngml-1 to 400 ngml-1 of ginsenoside Rb1. This revised version was published online in July 2006 with corrections to the Cover Date.     

Application of ultra-performance LC-TOF MS metabolite profiling techniques to the analysis of medicinal <Emphasis Type="Italic">Panax</Emphasis> herbs

The morphological appearance and some ingredients of Panax ginseng, Panax notoginseng and Panax japonicus of the Panax genus are similar. However, their pharmacological activities are obviously different due to the significant differences in the types and quantity of saponins in each herb. In the present study, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) was used to profile the abundances of metabolites in the three medicinal Panax herbs. Multivariate statistical analysis technique, that is, principle component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used to discriminate between the Panax samples. PCA of the analytical data showed a clear separation of compositions among the three medicinal herbs. The critical markers such as chikusetsusaponin IVa, ginsenoside R0, ginsenoside Rc, ginsenoside Rb1, ginsenoside Rb2 and ginsenoside Rg2 accountable for such variations were identified through the corresponding loading weights, and the tentative identification of biomarkers is completed by the accurate mass of TOFMS and high resolution and high retention time reproducibility performed by UPLC. The proposed analytical method coupled with multivariate statistical analysis is reliable to analyze a group of metabolites present in the herbal extracts and other natural products. This method can be further utilized to evaluate chemical components obtained from different plants and/or the plants of different geographical locations, thereby classifying the medicinal plant resources and potentially elucidating the mechanism of inherent phytochemical diversity.     

Hepatocyte metabolism of coenzyme Q1 (ubiquinone-5) to its sulfate conjugate decreases its antioxidant activity

Previous studies on the metabolism of coenyzme Q (CoQ) have focused on products found in the urine, bile or feces. However, the metabolites found in these samples were end products from a multitude of catabolic processes which did not necessarily reflect CoQ intracellular metabolism (e.g. in the liver, the major site of CoQ synthesis or metabolism). Using isolated rat hepatocytes, we have found that the sulfation of coenzyme Q1 (CoQ1) was the initial and dominant step following its reduction to the hydroquinone. This metabolic process is important as conjugation may occur on the hydroquinone metabolites of any coenzyme10 scission product retaining the quinone ring. By using rat liver cytosol, we were able to identify the monosulfated metabolite of CoQ1. The CoQ1 sulfate conjugate was identified by mass spectrometry followed by tandem mass spectrometry. The rate of formation of the CoQ1 sulfate conjugate was markedly increased by the addition of NADH and was prevented by dicumarol, a DT-diaphorase (NQO1) inhibitor. CoQ1 sulfate conjugate formation catalysed by cytosol was inhibited by the sulfotransferase 1A (SULT1A) inhibitor, pentachlorophenol (PCP) suggesting that sulfation was carried out by the SULT 1A isoform. CoQ1 sulfation in isolated hepatocytes and inversely CoQ1 hydroquinone formation were dependent on the concentration of inorganic sulfate in the media. Intracellular sulfation also decreased CoQ1 antioxidant and cytoprotective activity towards cumene hydroperoxide (CHP) induced cell death. Sulfotransferases may therefore play a significant role in endogenous CoQ metabolism following its degradation to short chain products.     

Liquid chromatography–tandem mass spectrometry analysis of cocaine and its metabolites from blood, amniotic fluid, placental and fetal tissues: study of the metabolism and distribution of cocaine in pregnant rats

The ability to simultaneously quantitate cocaine and its 12 metabolites from pregnant rat blood, amniotic fluid, placental and fetal tissue homogenates aids in elucidating the metabolism and distribution of cocaine. An efficient extraction method was developed to simultaneously recover these 13 components using underivatized silica solid-phase extraction (SPE) cartridges. The overall recoveries for cocaine and its metabolites were studied from pregnant rat blood (47–100%), amniotic fluid (61–100%), placental homogenate (31–83%), and fetal homogenate (39–87%). Extraction of the samples using silica is not classical SPE, but rather allows for the concentration of the sample into a small volume prior to injection and the removal of the proteins due to their strong interaction with the active silica surface. A positive ion mode electrospray ionization liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was used and validated to simultaneously quantitate cocaine and 12 metabolites from these four biological matrices. A gradient elution method with a Zorbax XDB C₈ reversed-phase column was used to separate the components. Multiple reaction monitoring (MRM) of a product ion arising from the corresponding precursor ion was used in order to enhance the selectivity and sensitivity of the method. Low background noise was observed from the complex biological matrices due to efficient SPE and the selectivity of the MRM mode. Linear calibration curves were generated from 0.01 to 2.50 ppm. The method also showed high intra-day (n=3) and inter-day (n=9) precision (% RSD) and accuracy (% error) for all components. The limits of detection (LODs) for the method ranged from 0.15 to 10 ppb. The LODs of cocaine and its major metabolites were less than 1 ppb from all four biological matrices. This method was applied to the study of the metabolism and distribution of cocaine in pregnant rats following intravenous infusion to a steady state plasma drug concentration. The following results were observed in the pregnant rat study: (1) the observations correlated strongly with the previous literature data on cocaine metabolism and distribution, (2) cocaine and norcocaine accumulated in the placenta, (3) arylhydroxylation of cocaine was a major metabolic pathway, (4) para-arylhydroxylation of cocaine was favored over meta-arylhydroxylation in rats and (5) accumulation of cocaine and its major metabolites was observed in the amniotic fluid.     

Transformation of verapamil by Cunninghamella blakesleeana

A filamentous fungus, Cunninghamella blakesleeana AS 3.153, was used as a microbial model of mammalian metabolism to transform verapamil, a calcium channel antagonist. The metabolites of verapamil were separated and assayed by the liquid chromatography-ion trap mass spectrometry method. After 96 h of incubation, nearly 93% of the original drug was metabolized to 23 metabolites. Five major metabolites were isolated by semipreparative high-performance liquid chromatography and were identified by proton nuclear magnetic resonance and electrospray mass spectrometry. Other metabolites were characterized according to their chromatographic behavior and mass spectral data. The major metabolic pathways of verapamil transformation by the fungus were N dealkylation, O demethylation, and sulfate conjugation. The phase I metabolites of verapamil (introduction of a functional group) by C. blakesleeana paralleled those in mammals; therefore, C. blakesleeana could be a useful tool for generating the mammalian phase I metabolites of verapamil.     

Metabolic analysis of four phenolic acids in rat by liquid chromatography-tandem mass spectrometry

A liquid chromatography-diode array detection-electrospray ionization ion trap mass spectrometry (LC-DAD-ESI-MS(n)) method was established for the analysis of danshensu, caffeic acid, ferulic acid and isoferulic acid in rat plasma, bile, urine and feces after oral administration or intravenous injection. Liquid-liquid extraction was employed for the preparation of biosamples, and the chromatographic separation was carried out using an Agilent Zorbax Extend C(18) reversed phase column and acetonitrile-0.1% formic acid as the mobile phase. Totally nineteen metabolites were detected and identified as prototype, methylated, hydroxylated, sulfated and glucuronized conjugates. The metabolism of the individual phenolic acids in biosamples was investigated, and the metabolic pathway was proposed. By comparing the metabolism of different compounds which shared similar structures, we were able to find that methylation was the main pathway of danshensu metabolism, and the double bond on the side chain was critical for the drug excretion via bile and the formation of glucuronized conjugates. The results proved that the established method was simple, sensitive and reliable, which could be used to detect and identify the structures of metabolites and to better understand their in vivo metabolism.     

Simultaneous determination of three Panax notoginseng saponins at sub-nanograms by LC-MS/MS in dog plasma for pharmacokinetics of compound Danshen tablets

Compound Danshen tablets are composed of Panax notoginseng, Salvia miltiorrhiza and Borneol. The tablets are prescribed for treatment of cardiovascular diseases in China. The present study aimed at developing a specific and sensitive LC-MS/MS method to simultaneously determine three bioactive P. notoginseng saponins, i.e., notoginsenoside R1, ginsenoside Rg1 and Rb1, in dogs after a single oral administration of the compound tablets in order to obtain the clinically relevant saponin-related pharmacodynamics of the tablets in patients. The R1, Rg1 and Rb1 were extracted from dog plasma with acetone-methanol (80:20, v/v), separated by reversed phase liquid chromatography and determined by tandem mass spectrometry (LC-MS/MS) with positive electrospray ionization (ESI). The developed method reached lower limit of quantitation (LLOQ) at 0.10 ng/ml for the three saponins. The method was validated in terms of selectivity, matrix effects, linearity, precision and accuracy, and then was applied to a pharmacokinetic study of the three bioactive saponins simultaneously in dogs after a single oral administration of compound Danshen tablets at a clinical equivalent dose. The C(max) and AUC((0-∞)) for R1, Rg1 and Rb1 were 1.91, 3.34 and 28.6 ng/ml, and 7.5, 11.0, and 1712 (h ng/ml), respectively.     

Liquid chromatography-tandem mass spectrometry analysis of protocatechuic aldehyde and its phase I and II metabolites in rat

A method using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) analysis was established for the identification of metabolites in rat after oral administration of protocatechuic aldehyde, a major bioactive phenolic acid in the roots of Salvia miltiorrhiza. Eleven metabolites in rat plasma and urine were firstly identified as protocatechuic aldehyde, protocatechuic acid and their methylated, glucuronized or glycine conjugates on the basis of their MS fragmentation behaviors, while nine of these metabolites (except protocatechuic aldehyde and protocatechuic acid) were detected in rat bile. In addition, the possible metabolic pathway was proposed for the first time. In the phase I metabolism, protocatechuic aldehyde could be oxidized to protocatechuic acid. The conjugates would be formed in rat intestine, liver and kidney and excreted from rat urine and bile. Enthrohepatic circulation played an important role in the metabolism of protocatechuic aldehyde. The results proved that the established method was simple, reliable and sensitive, revealing that it could be used to rapid screen and identify the structures of active components responsible for pharmacological effects of protocatechuic aldehyde and to better understand its in vivo metabolism.     

Molecular mass spectrometry imaging in biomedical and life science research

This review describes the current state of mass spectrometry imaging (MSI) in life sciences. A brief overview of mass spectrometry principles is presented followed by a thorough introduction to the MSI workflows, principles and areas of application. Three major desorption-ionization techniques used in MSI, namely, secondary ion mass spectrometry (SIMS), matrix-assisted laser desorption ionization (MALDI), and desorption electrospray ionization (DESI) are described, and biomedical and life science imaging applications of each ionization technique are reviewed. A separate section is devoted to data handling and current challenges and future perspectives are briefly discussed at the end.     

Rapid analysis of metabolic stability of dopamine receptor antagonists and detection of their metabolites by liquid chromatography/tandem mass spectrometry

In vitro metabolic stability of dopamine D(3)/D(4) receptor antagonists and identification of their metabolites by high-performance liquid chromatography (HPLC) coupled with ion-trap mass spectrometry (ITMS) were assessed in rat liver microsomes. The compounds were divided into three cassette groups for rapid quantitative analysis of multiple drugs and simultaneous detection of their metabolites. The samples from incubation with rat liver microsomes were pooled into designed cassette groups and analyzed by HPLC/electrospray ITMS in full-scan mode. The metabolic stability of the drugs was determined by comparing their signals after incubation for 0 and for 30min. The metabolic stability of the examined dopamine receptor antagonists was in the range of 9.9-84.4%. In addition, the present cassette analysis allowed the simultaneous detection of metabolites formed during the same incubation without having to reanalyze the samples. The metabolites were first characterized by nominal mass measurement of the corresponding protonated molecules. Subsequent multistage tandem mass spectrometry on the ion-trap instrument allowed characterization of the structure of the detected metabolites. N,O-dealkylation and ring hydroxylation reactions were identified as major metabolic reactions in piperazinylalkylisoxazole derivatives. These results suggested that the present approach is useful for the rapid evaluation of metabolic stability and structural characterization of metabolites within a short period in new drug discovery.     

Quantitative determination of helicid in rat biosamples by liquid chromatography electrospray ionization mass spectrometry

A simple liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS) method with highly improved sensitivities for the determination of helicid in rat bile, urine, feces and most tissues was developed. The tissues and feces were firstly homogenized mechanically using deionized water as the media. Bile, urine, tissues and feces homogenates were extracted by liquid–liquid extraction with n-butyl alcohol for sample preparation. The subsequent analysis procedures were performed on a Shimadzu LCMS2010A system (electrospray ionization single quadrupole mass analyzer). A Luna C₁₈ column (150 mm × 2.00 mm, 5 μm) was used as the analytical column, while a mixture of acetonitrile and ammonium chloride water solution was used as the mobile phase. The proportions of mobile phase were changed timely according to gradient programs. Chlorinated adducts of molecular ions [M+Cl]^? at m/z 319.00 and 363.05 were used to quantify helicid and bergeninum (internal standard), respectively. The method was validated to be accurate, precise and rugged with good linearity. The proposed method was successfully applied to the preclinical tissue distribution and excretion studies of helicid in rats.     

Identification of YH439 and its metabolites in rat urine by gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry

YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.     

Microbial conversion of ginsenoside Rb1 to minor ginsenoside F2 and gypenoside XVII by Intrasporangium sp. GS603 isolated from soil

A new strain, GS603, having beta-glucosidase activity was isolated from soil of a ginseng field, and its ability to convert major ginsenoside Rb(1) to minor ginsenoside or gypenoside was studied. Strain GS603 was identified as an Intrasporangium species by phylogenetic analysis and showed high ginsenoside-converting activity in LB and TSA broth but not in nutrient broth. The culture broth of the strain GS603 could convert ginsenoside Rb(1 )into two metabolites, which were analyzed by TLC and HPLC and shown to be the minor ginsenoside F(2) and gypenoside XVII by NMR.     

In Vitro Metabolic Stability of Exendin-4: Pharmacokinetics and Identification of Cleavage Products

The aim of this study was to investigate the metabolic stability and cleavage sites of exendin-4 in rat tissue homogenates, as well as to identify the types of proteases involved in exendin-4 degradation. The stability of exendin-4 in kidney and liver homogenates from rats was evaluated using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) with gradient elution. Furthermore, we used a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and LC-ESI-MS/MS to identify the structures of the major degradation products of exendin-4, and peptidase inhibitors were used to characterize exendin-4 degradation in rat liver and kidney homogenates and to identify the proteases involved in exendin-4 metabolism. Exendin-4 had a half-life of 7.8 and 100.9 min in the kidney and liver homogenate, respectively. The enzymes most likely to be involved in the degradation of exendin-4 were aminopeptidases, serineproteases, and metalloproteases. Exendin-4(15-39) and exendin-4(16-39) were the predominant direct exendin-4 metabolites in the kidney, and the main product of exendin-4 metabolism in the liver was exendin-4(12-39). Our results indicated that the metabolism of exendin-4 involved an initial endoproteolytic cleavage and subsequent exoproteolytic digestion. The degradation of exendin-4 in the kidney and liver homogenates followed distinct patterns, and the primary cleavage sites of exendin-4 degradation in rat kidney homogenates were located after AA-14, and -15, whereas those in rat liver homogenates were located after AA-11.