Multi-analyte Assays For Analysis Of Complex Biological Samples And Their Applications

Analysis of metabolism network of acetaminophen and its application to rat related pharmacokinetic investigation of GanMaoLing granuleAntipyretic and analgesic acetaminophen has been most widely used in the treatment of fever and pain.Although considered safe at therapeutic doses,in overdose,acetaminophen produces a centrilobular hepatic necrosis that can be fatal.The importance of hepatic metabolism in the acetaminophen toxicity has been well established.Acetaminophen is metabolically activated by cytochrome P450 to form the reactive metabolite N-acetyl-p-benzoquinone imine(NAPQI)that covalently binds to cysteine groups on protein,forming acetaminophen-protein adducts.In addition to this P450-mediated metabolism for generating NAPQI,acetaminophen possesses other metabolic pathways that limit the formation of NAPQI.These metabolic pathways are protective ones of acetaminophen and include uridine 5’-diphosphoglucuronosyltrasferase(UGT)-mediated glucuronidation,sulfotransferase(SULT)-mediated sulfation,and P450-mediated hydroxylation and its subsequent conjugations(including methylation,glucuronidation,and sulfation).Meanwhile,the formed NAPQI was detoxified by conjugation with glutathione(GSH)followed by formation of the subsequent metabolites.These metabolic pathways are detoxification ones of acetaminophen.Both protective and detoxification metabolic pathways a metabolism network of acetaminophen,which governs the exposure to NAPQI and factors that affect NAPQI production and detoxification influence susceptibility to acetaminophen toxicity.It is noteworthy that the bulk of the current knowledge of drug-induced liver disease derives from research on acetaminophen hepatotoxicity.To this end,developing assays for measuring acetaminophen and its metabolism network in various bio-samples that could be collected from animals,as well as from human subjects,facilitates toxicological research on acetaminophen.The current investigation was designed to develop such assays and to apply them to analyze plasma,bile,and urine samples from rats orally receiving acetaminophen andGanMaoLing granule,herbal medicine containing acetaminophen.Eleven analytes were selected for analysis of metabolism network of acetaminophen based on literature survey.These analytes were six metabolites[i.e.,acetaminophen-4-O-sulfate(M1),acetaminophen-4-O-glucuronide(M2),3-hydroxy acetaminophen(M3),and the metabolites of M33-hydroxyacetaminophen3-O-sulfate(M3-1),3-hydroxyacetaminophen-3-O-glucuronide(M3-2),and 3-methoxyacetaminophen(M3-3)]from the protective metabolism of acetaminophen,four metabolites from the detoxification of NAPQI[i.e.,acetaminophen glutathione(M4)and its subsequent metabolites 3-cysteinylacetaminophen(M4-1),S-Methyl-3-thioacetaminophen(M4-2),and 3-(N-acetyl-L-cystein-S-yl)acetaminophen(M4-3)],and unchanged acetaminophen.Assay development was based on liquid chromatography/tandem mass spectrometry to achieve high sensitivity and selectivity.Serial plasma,bile,and urine samples were obtained from rats orally receiving acetaminophen and GanMaoLing and analyzed by the newly developed assays.The three types of rat samples were compared with respect to reflecting metabolism network of acetaminophen.In addition,samples after dosing GanMaoLing granule were compared with the respective samples after dosing acetaminophen to access potential for the component herbs-acetaminophen interactions for GanMaoLing.In the current investigation,sensitive and highly selective assays were developed and validated,which could simultaneously measure acetaminophen and its ten metabolites in the rat plasma,urine,and bile samples.In the plasma samples after dosing acetaminophen,unchanged acetaminophen and five protective metabolites were detected with unchanged acetaminophen,M1,and M2 as the major circulating compounds,but the detoxification metabolites were detected at very low levels.In the bile samples,unchanged acetaminophen,nine metabolites of both the productive and the detoxification types were detected with unchanged acetaminophen,M1,M2,M4,and M4-1 as the major circulating compounds.In the urine samples,In the bile samples,unchanged acetaminophen,seven metabolites of both the productive and the detoxification types were detected with unchanged acetaminophen,M1,M2,M4-2,and M4-3 as the major circulating compounds.Although the cumulative amounts of the unchanged acetaminophen and the two detoxification metabolites(the subsequent metabolites of M4)were lower than those of Ml and M2,these circulating compounds were well detected in the urine samples.The preceding results of the rat studies suggested that plasma samples appeared not to be suitable samples for reflecting the metabolism network of acetaminophen.Although bile samples appeared to be good for reflecting the network,such samples are normally difficult to be collected from other species,including human subjects.Urine samples appeared to be an alternative to bile samples.In addition,systemic exposure to unchanged acetaminophen and the both types of metabolites in rats orally receiving acetaminophen appeared to be similar to that in rats orally receiving GanMaoLing,suggesting that potential for component herbs-acetaminophen interactions were low.More investigations are merited to confirm the low interaction potential,including pharmacokinetic investigation of GanMaoLing for its component herbs and clinical and mechanistic investigations of the interaction potential.If it is true that the component herbs-acetaminophen interactions do not likely occur for GanMaoLing,rational use of the granule in clinics for its component acetaminophen does not need to be modified from that of acetaminophen aloneTen coumarins and flavonoids detection methods in plasma together and their application in pharmacokinetic research of Psoralea corylifolia L extractCoumarins and flavonoids are two classes of bioactive constituents present in fruits of Psoralea corylifolia L.To facilitate multi-compound pharmacokinetic research on and safety evaluation of this medicinal herb and its formulated products,a liquid chromatography/mass spectrometry-based assay was developed to quantify simultaneously ten coumarins and flavonoids(i.e.,psoralen,isopsoralen,apigenin,genistein,bavaisoflavone,neobavaisoflavone,bavachin,bavachinin,psoralenoside,and isopsoralenoside)in plasma samples.After being validated with respect to selectivity,accuracy,precision,linear dynamic range,sample preparation recovery,matrix effects,and stability,the assay was applied to a canine pharmacokinetic study by orally dosing Psoralea corylifolia extract at 2 g/kg.The newly developed assay involved using acetonitrile to prepare plasma samples.Chromatographic separation was achieved on a Waters HSS-T3 column with a 8.5 min gradient for elution.The analytes were measured by mass spectrometry in a multi-reaction monitoring mode The assay was demonstrated to be sensitive,reliable,and reproducible for intended use.The content levels of the preceding ten compounds in the herbal extract were 391,288,36.8,3.66,2.27,23.2,8.36,5.09,22800,and 12000 ng/mg,respectively.After dosing the extract in dogs,psoralen,isopsoralen,psoralenoside,and isopsoralenoside were the major circulating compounds detected with Tmax(time to taken to achieve the maximum concentration after dosing)of 1.9-5.7 h,Cmax(maximum concentration)of 383-3613 ng/mL,and t1/2(terminal half-life)of 2.5-4.8 h.Such data for the other six minor circulating compounds were 1.9-5.0 h,1-20 ng/mL,and 3.0-7.3 h,respectively.