Novel direct high-performance liquid chromatographic method for determination of salicylate glucuronide conjugates in human urine

A novel direct high-performance liquid chromatographic (HPLC) assay for the simultaneous determination of three salicylate glucuronide conjugates and other salicylate metabolites in human urine has been developed. Salicylate glucuronide conjugates were purified by HPLC from the urine of a volunteer after oral administration of aspirin and identified by selective hydrolysis with beta-glucuronidase and with sodium hydroxide. This method gave high reproducibility with coefficients of variation less than 10%. The total urinary recovery of salicylic acid after a single 1.2-g dose of soluble aspirin was greater than 90%. This assay has been successfully used to re-evaluate the capacity-limited pharmacokinetics of salicylic acid in humans.     

Mass spectrometric analysis of cyclofenil and its metabolites in human urine

Using gas chromatography/electron impact-mass spectrometry (GC/EI-MS) and high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS), the structures of cyclofenil metabolites in human urine have been assigned. The hydroxyl metabolites liberated from the glucuronide conjugates after acid hydrolysis were characterized as the trimethylsilyl (O-TMS) derivatives using GC/MS. The conjugate glucuronide forms were detected without hydrolysis by HPLC/MS. Cyclofenil was not observed in urine. Tentative structures for the two metabolites are proposed.     

Simultaneous determination of inulin and p-aminohippuric acid (PAH) in human plasma and urine by high-performance liquid chromatography

Inulin and p-aminohippuric acid (PAH) clearances are used for the estimation of glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). A simple and rapid high-performance liquid chromatography (HPLC) method with UV detection is described for the simultaneous determination of inulin and PAH in the same chromatogram in the plasma and urine of humans. Plasma and urine samples were hydrolyzed with perchloric acid (0.7%) in boiling water. The mobile phase consisted of 0.01 M potassium dihydrogenphosphate with 0.02 M tetramethylammonium chloride and o-phosphoric acid (pH 3)-acetonitrile (94:6, v/v), pumped at a rate of 1.2 ml min-1 on a C8 reversed-phase column. Tannic acid was used as the internal standard and UV detection at 285 nm was employed. The calibration curves were linear over the concentration range of 12.5-100 mg l-1 for inulin and 6.25-50 mg l-1 for PAH with determination coefficients greater than 0.997. The method is accurate (bias < 13%) and reproducible (intra- and inter-day relative standard deviation less than 11%), with a limit of quantitation of 12.5 mg l-1 and 6.25 mg l-1 for inulin and PAH, respectively. Analytical recoveries from urine and plasma were ranged from 81 to 108% for both compounds. This fully validated method, which allows the simultaneous determination of inulin and PAH clearances, is simple, rapid (total run time < 10 min) and requires only a 200 microliters plasma or urine sample.     

Conventional and thermal lens spectrophotometric determination of p-aminobenzoic acid and arylamine diuretics previous azodye formation in a micellar medium

The determination of the diuretics hydrochlorothiazide, bendroflumethiazide and furosemide by both conventional and thermal lens spectrophotometry (TLS, 100 mW of pump power at 514.5 nm) following previous hydrolysis, diazotization and coupling with N-(naphthyl)ethylenedine (NED) in a sodium dodecyl sulphate (SDS) micellar medium of pH approximately 1 was studied. p-Aminobenzoic acid (PABA) was used as a model compound to optimize the derivatization procedures. 3-Substituted indoles, such as 5-hydroxyindole-3-acetic acid and tryptophan, gave N-nitroso derivatives which interfered with the determination of the diuretics in urine. The derivatized diuretics in urine were separated using HPLC with a Spherisorb ODS-2 C(18) column, and a 0.1M SDS mobile phase containing 5% n-propanol and 0.001M sodium dihydrogen phosphate (pH 3). The diuretics gave limits of detection (LODs) of ca. 5 x 10(-9)M for the TLS procedure. The LODs were 20-50-fold higher for the corresponding spectrophotometric procedure.     

Analysis of p-aminobenzoic acid and its metabolites in urine and plasma by ion pair HPLC in the NBT-PABA pancreatic function test

An ion pair HPLC method which can simultaneously detect the major metabolites of an exocrine pancreatic function testing agent (NBT-PABA) in plasma and urine has been developed. This assay has been applied to a pharmacokinetic study of PABA and its metabolites in 3 healthy adult volunteers following the oral administration of 1 g NBT-PABA. The 6 h testing period currently used to collect urine following the NBT-PABA ingestion is adequate for the recovery of PABA and its metabolites. Plasma determination of these compounds may provide an improved evaluation of the pancreatic performance in patients with abnormal liver or kidney function.     

Determination of trimethoprim metabolites including conjugates in urine using high-performance liquid chromatography with combined ultraviolet and electrochemical detection

A high-performance liquid chromatographic method for the determination of trimethoprim metabolites in pig urine was developed. The metabolites-glucuronic acid and sulphuric acid conjugates of phenolic metabolites formed by demethylation of trimethoprim-were quantitated after treatment of urine with beta-glucuronidase (Escherichia coli). The sulphuric acid conjugate was not susceptible to enzymatic hydrolysis and was therefore assayed as the conjugate by use of ion-pair chromatography on the reversed-phase column. In order to find suitable conditions for enzymatic hydrolysis of the glucuronides, the conjugates were obtained by gel chromatography of urine from a [14C] trimethoprim-treated pig.     

Free and total para-aminobenzoic acid analysis in plants with high-performance liquid chromatography/tandem mass spectrometry

para-Aminobenzoic acid (PABA), a precursor in the synthesis of folates in plants, is determined by liquid chromatography/tandem mass spectrometry (LC/MS/MS). In plants PABA can be converted into its beta-D-glucopyranosyl ester (PABA-Glc) and can also exist in its free form. In this work, we developed and validated a quantitative method to study free and total PABA in plants. The total PABA (free PABA plus PABA-Glc) can be evaluated after acid hydrolysis at 80 degrees C for 2 hours. The plant material is homogenized and the PABA content is quantified using the standard addition procedure. The validated method is selective, sensitive, simple, accurate, has a recovery between 99.6 to 102.5%, is reproducible (RSD between 1.4 and 4.4%), and is linear between 2.5 and 1538 ng/mL. Free and total PABA determinations in five vegetables showed that different plant species had different amounts of free and total PABA, and that the ratios of total versus free PABA were also variable. This new method could be valuable for studies of folate synthesis in plants.     

LC determination and pharmacokinetic study of vitexin-4″-O-glucoside in rat plasma after oral administration

A simple and specific high-performance liquid chromatography (HPLC) method was developed for the pharmacokinetic study of vitexin-4″-O-glucoside (VOG) in rats after oral administration. The plasma samples were deproteinised with methanol after the addition of an internal standard, hesperidin. HPLC analysis was performed on a Diamonsil C(18) analytical column, using methanol -0.5% aqueous phosphoric acid (45:55, v/v) as the mobile phase with ultraviolet detection at 330?nm. The calibration curve was linear over the range of 5-450?μg?mL(-1) in rat plasma. The average extraction recovery of VOG was 98.74%?±?0.44%, and the relative standard deviations of the intra- and inter-day precisions were not greater than 4.1% and 2.0%, respectively. The validated method was successfully applied during a pharmacokinetic study in rats after oral administration of VOG at different doses, and all the results indicated that the pharmacokinetics of VOG in rats obeyed nonlinear processes.     

Determination of halofuginone in bovine plasma by competing-ion high performance liquid chromatography after solid phase extraction

A high performance liquid chromatography (HPLC) method for the determination of the anticoccidial and antitheilerial drug halofuginone in bovine plasma was developed. Samples were diluted with acetic acid (10%, v/v) and cleaned up on a Bond Elut C8 column. The analyte was eluted from the extraction column and chromatographed by reversed-phase HPLC using decylamine as a competing-ion reagent. Detection was by UV at 243 nm. Recovery from plasma was 75%, and within-day and between-day coefficients of variation were 5.23 and 6.35% respectively. The specificity and sensitivity of this method (limit of detection in plasma, 1 ng/mL) were sufficiently high to enable us to characterize the time course of the drug in plasma after oral administration of therapeutic doses to cattle.     

High‐performance liquid chromatography assay using ultraviolet detection for urinary quantification of milrinone concentrations in cardiac surgery patients undergoing cardiopulmonary bypass

An analytical assay using liquid–liquid extraction and high‐performance liquid chromatography with ultraviolet detection was developed for the quantification of total (conjugated and unconjugated) urinary concentrations of milrinone after the inhalation of a 5 mg dose in 15 cardiac patients undergoing cardiopulmonary bypass. Urine samples (700 μL) were extracted with ethyl‐acetate and subsequently underwent acid back‐extraction before and after deconjugation by mild acid hydrolysis. Milrinone was separated on a strong cation exchange analytical column. The mobile phase consisted of a constant mixture of acetonitrile:tetrahydrofurane–NaH₂PO₄ buffer (40:60 v/v, pH 3.0). Thirteen calibration curves were linear in the concentration range of 31.25–4000 ng/mL, using olprinone as the internal standard (r² range 0.9911–0.9999, n = 13). Mean milrinone recovery and accuracy were respectively 85.2 ± 3.1% and ≥93%. Intra‐ and inter‐day precisions (coefficients of variation) were ≤5% and ≤8%, respectively. Over a 24 h collection period, the cumulative urinary milrinone recovered from 15 patients was 26.1 ± 7.7% of the nominal 5 mg dose administered. The relative amount of milrinone glucuronic acid conjugate was negligible in the urine of patients undergoing cardiopulmonary bypass This method proved to be reliable, specific and accurate to determine the cumulative amount of total milrinone recovered in urine after inhalation. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparative study on the excretion of vitexin‐4′′‐O‐glucoside in mice after oral and intravenous administration by using HPLC

The aim of the present study was to characterize the excretion of pure vitexin‐4”‐O‐glucoside (VOG) in mice following oral and intravenous administration at a dose of 30 mg/kg. A sensitive and specific HPLC method with hespridin as internal standard, a Diamonsil C₁₈ column protected with a KR C₁₈ guard column and a mixture consisting of methanol–acetonitrile–tetrahydrofuran–0.1% glacial acetic acid (6:2:18:74, v/v/v/v) as mobile phase was developed and validated for quantitative analysis in biological samples. VOG could be excreted as prototype in excreta including urine and feces after both routes of administration, and the cumulative excretion of VOG was 24.31 ± 11.10% (17.97 ± 5.59% in urinary excretion; 6.34 ± 5.51% in fecal excretion) following oral dosing and 5.66 ± 3.94% (4.78 ± 3.13% in urinary excretion; 0.88 ± 0.81% in fecal excretion) following intravenous dosing. The results showed that the elimination of VOG after the two routes was fairly low, which meant that VOG was metabolized as other forms and the elimination after oral dosing was almost 4.3‐fold that after intravenous dosing. For both routes of administration, VOG excreted as prototype in urine was much more than that in feces, nearly 2.83‐fold for oral administration and 5.43‐fold for intravenous administration, which should be attributed to enterohepatic circulation. Taken together, renal excretion was the dominant path of elimination of VOG for oral and intravenous administration in mice and biliary excretion contributed less. Copyright © 2013 John Wiley & Sons, Ltd.     

Allan J. Bard and Cynthia G. Zoski (Eds): Electroanalytical Chemistry

Coupled with evaporative light scattering detection, a high-speed counter-current chromatography (HSCCC) method was applied to the separation and purification of three tauro-conjugated cholic acids of taurochenodeoxycholic acid (TCDCA), taurohyodeoxycholic acid (THDCA) and taurohyocholic acid (THCA) from Pulvis Fellis Suis (Pig gallbladder bile) for the first time. The two-phase solvent system composed of chloroform-methanol-water-acetic acid (4:4:2:0.3, v/v/v/v) was selected for the one-step separation where the lower phase was used as the mobile phase in the head to tail elution mode. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature were 800 rpm, 1.5 ml/min and 25°C respectively. From 100 mg of the crude extract, 10.2 mg of TCDCA, 11.8 mg of THDCA and 5.3 mg of THCA were obtained with the purity of 94.6%, 96.5% and 95.4%, respectively. in one step separation The HSCCC fractions were analyzed by high-performance liquid chromatography (HPLC) and the structures of the three tauro-conjugated cholic acids were identified by ESI-MS, (1)H NMR and (13)C NMR.     

Simultaneous determination of albiflorin and paeoniflorin in rat urine by solid-phase extraction and high-performance liquid chromatography following oral administration of Si-Wu decoction

A high performance liquid chromatographic method (HPLC), together with solid phase extraction (SPE), was developed for simultaneous determination of albiflorin and paeoniflorin in rat urine after oral administration of Si-Wu decoction. The samples were pretreated with solid phase extraction using Extract-Cleantrade mark cartridges. Analysis of the extract was performed on a reversed-phase C18 column and a mobile phase made up of acetonitrile and 0.03% formic acid (17:83, v/v). UV detection was set at 230 nm. The assay was linear over the range 2.625-52.50 mg/mL for albiflorin and 3.875-77.50 microg/mL for paeoniflorin. The average percentage recoveries of three spiked urines were 97.01 +/- 3.32 and 102.32 +/- 6.97 for albiflorin and paeoniflorin, respectively. The intra-day precision (RSD) ranged from 0.21 to 1.79% at concentrations of 4.20, 10.50, 26.25 and 39.375 microg/mL of albiflorin and 0.12 to 2.92% at concentrations of 3.875, 10.85, 23.25 and 58.125 microg/mL of paeoniflorin, and inter-day precision (RSD) was from 1.02 to 1.86% for albiflorin and 0.94 to 3.30% for paeoniflorin, at the same four concentrations. This method was applied in order to analyze albiflorin and paeoniflorin in rat urine following oral administration of traditional Chinese medicinal preparation of Si-Wu decoction.     

Synthesis and in vitro Property Study of Polyaspartamides

Cationic polyaspartamides including poly-α,β-[N＇-（2-aminoethyl）-L-aspartamide] （PAEA）, poly-α,β-[N＇-（4- aminobutyl）-L-aspartamide] （PABA）, poly-α,β-[N＇-（6-aminohexyl）-L-aspartamide] （PAHA）, poly-α,β-[N＇-（5-amino- 3-azapentyl）-L-aspartamide] （PAAPA） and poly-α,β-[N＇-（8-amino-3,6-diazaoctyl）-L-aspartamide] （PADAOA） were synthesized from polysuccinimide. Their properties were evaluated by ^1H NMR, IR, GPC, fluorescence measurement and in vitro cytotoxicity assays. The molecular weights per primary amine charge group of PAEA（1） （Mn= 2229）, PAAPA and PADAOA are 212, 279, and 226. Polyaspartamides including PAEA（1）, PAAPA, PADAOA and low molecular weight PAHA are markedly less toxic than poly（ethyleneimine） and poly（L-lysine）, however, PABA and higher molecular weight PAHA are slightly less toxic than poly（L-lysine）. Cell cytotoxicity of PAHA was seen to decrease with increasing molecular weight of PAHA, due to water solubility reduction. The negatively charged plasmid DNA has been found to be completely neutralized and complexed by the cationic polyaspartamides at an N/P ratio of 5 ： 1 to 10 ： 1, forming self-assembled polyplexes via ionic interactions. These polyaspartamide/DNA complexes possess stable zeta potentials and mean particle diameters of about 180 nm for PAEA （1）/DNA and PAAPA/DNA complexes and 280 nm for PADAOA/DNA complexes.     

Effects of pH and phosphate on the adsorptive fractionation of purified Aldrich humic acid on kaolinite and hematite

The molecular weight (MW) fractionation of purified Aldrich humic acid (PAHA) resulting from adsorption on kaolinite and hematite was investigated for different solution pH and phosphate conditions. Adsorption was highly pH-dependent, with higher uptake at lower pH values. For all pH conditions, the weight-average MW (MWw) of residual PAHA remaining in solution after adsorption deviated from the original MWw, indicating that preferential adsorption of certain MW components occurred. The extent of preferential adsorption depended on the percent carbon adsorption at a given pH condition. For similar percent carbon adsorption ranges, a greater extent of preferential adsorption of the higher MW PAHA components was observed with higher pH values as demonstrated by the lowest residual MWw value occurring at pH 9. Detailed analyses of selected residual PAHA samples clearly showed that adsorption selectivity for particular MW components was strongly influenced by solution pH. The extent of preferential adsorption of lower MW PAHA components decreased in the presence of a small amount of phosphate. This effect was more evident for hematite than kaolinite, and became greater with lower solution pH irrespective of the mineral type. The different fractionation patterns observed for PAHA were reasonably well explained by the physicochemical trends occurring in its MWw fractions and the underlying sorption processes.     

Investigation of phenolic acids in yacon (Smallanthus sonchifolius) leaves and tubers

Thin-layer chromatographic (TLC) screening of crude extracts of dried leaves and tubers of yacon (Smallanthus sonchifolius, Asteraceae) and products of acid hydrolysis of tubers on the silica gel HPTLC plates using the developing solvents ethyl acetate-formic acid-water (85:10:15, v/v/v) and n-hexane-ethyl acetate-formic acid (20:19:1, v/v/v) proved the presence of chlorogenic, caffeic and ferulic acid. These phenolic acids were isolated from the crude extract of yacon leaves by preparative TLC, and identified after elution by HPLC/MS, as well as by direct injection of the crude extract into the HPLC/MS system. Acid hydrolysis of tubers released the increased amount of phenolic acids (e.g. caffeic acid and ferulic acid), flavonoid quercetin and an unidentified flavonoid, which was detected by TLC analysis. Ferulic acid, isomers of dicaffeoylquinic acid and still an unidentified derivative of chlorogenic acid (Mr = 562) as constituents of yacon leaves and ferulic acid as constituent of yacon tubers are reported here for the first time. These acids gave significant contribution to the radical scavenging activity detected directly on the TLC plate sprayed with 1,1-diphenyl-2-picrylhydrazyl (DPPH).     

Chromatographic purification and identification of polar metabolites of benazolin-ethyl from soybean

The metabolism of benazolin-ethyl (4-chloro-2-oxobenzothiazolin-3-ylacetic acid ethyl ester), a post emergence herbicide, has been studied in soybean using (14C)-phenyl labelled compound. Preliminary studies were performed on excised soybean leaves. Following hydrolysis of the ethyl ester to benazolin acid (4-chloro-2-oxobenzothiazolin-3-ylacetic acid), extensive metabolism to polar conjugates was observed. The polar fraction from a Bligh-Dyer extraction was purified by solvent partitioning, preparative TLC and reverse phase HPLC with ion suppression. The two major metabolites were characterised by fast atom bombardment mass spectrometry with accurate mass determination as an aspartate conjugate and a malonyl-beta-glucose ester of benazolin acid. Subsequent experiments were performed by spraying intact plants at growth stage V4. The major polar metabolite isolated one month after treatment was identified as the aspartate conjugate by mass spectrometry and high resolution nuclear magnetic resonance spectroscopy.     

Quantitative determination of BMS-378806 in human plasma and urine by high-performance liquid chromatography/tandem mass spectrometry

BMS-378806 is a human immunodeficiency virus (HIV) entry inhibitor that is being developed for the oral treatment of HIV infection. Human plasma and urine LC/MS/ MS methods have been developed and validated for the quantitation of BMS-378806. For human plasma method, methyl t-butyl ether was used to extract BMS-378806 from plasma in a 96-well format, and the organic layers were dried down and then reconstituted for the injection, while a dilute-and-shoot approach was used for human urine method in a 96-well format. Chromatographic separation was achieved isocratically on a Phenomenex C18 (2) Luna column (2 x 50 mm2, 5 microm). The mobile phase contained 60:40 v/v of 0.1% formic acid in water and ACN. Detection was by positive ion electrospray MS/MS. The standard curves ranged from 1.25 to 1000 ng/mL for the plasma assay and from 10 to 5000 ng/mL for the urine assay. The curves were fitted to a 1/x2 weighted quadratic regression model for both methods. The validation results demonstrated that both methods had satisfactory precision and accuracy across the calibration ranges. The methods were applied to the analysis of human plasma and urine samples from a single ascending dose clinical study to assess the pharmacokinetics of the drug. The pharmacokinetic analysis results indicated the absorption and disposition of the drug was rapid. The systemic exposure of BMS-378806 was generally dose proportional among the doses from 100 to 1200 mg, but not dose proportional to 1600 mg. There were modest increases in the systemic exposure when the drug was given with food or given as a solution formulation. Renal excretion was not a substantial elimination pathway of the drug. BMS378806 was safe and well tolerated over a dose range of 100-1600 mg administered as a single oral dose.     

Simultaneous determination of three polyphenols in rat plasma after orally administering hawthorn leaves extract by the HPLC method

A simple and sensitive HPLC method was developed to simultaneously determine three active compounds, vitexin-4″-O-glucoside (VG), vitexin-2″-O-rhamnoside (VR) and hyperoside (HP), in rat plasma after administering the hawthorn leaves extract (HLE). An HPLC assay with baicalin as the internal standard was carried out using a Phenomsil C?? analytical column with UV detection at 332?nm. The mobile phase consisted of methanol-acetonitrile-tetrahydrofuran-1% glacial acetic acid (6?:?1.5?:?18.5?:?74, v/v/v/v). The calibration curves were linear over the range of 2.5-500, 0.2-25 and 0.25-12.5?μg?mL?1 for VG, VR and HP, respectively. The method was reproducible and reliable, with relative standard deviations of the intra- and inter-day precision between 1.2% and 13.2% for the analysis of the three analytes. The validated HPLC method herein described was successfully applied to the pharmacokinetic study of VG, VR and HP after oral administration of HLE to rats over the dose range of 2.5-10 mL?kg?1.     

Extractionless HPLC Method for the Determination of Naproxen in Human Plasma and Urine

Abstract

A simple and rapid (extractionless) high-performance liquid chromatographic method with ultraviolet detection, at 278 nm, is described for the determination of naproxen in human plasma and urine. Niflumic acid is used as internal standard. The chromatographic system consists of a reversed-phase C18-Spherisorb column with acetonitrile/0.1 M sodium acetate (35:65 v/v, pH 6.14) as the mobile phase. The retention time is 3.0 min for naproxen and 3.8 min for niflumic acid. The total run time is 5 min and the typical assay time is 10 min. The method is sufficiently sensitive for biopharmaceutical studies, after the oral administration of a single sustained release dose.