Direct determination of paracetamol and its metabolites in urine and serum by capillary electrophoresis with ultraviolet and mass spectrometric detection

The use of capillary electrophoresis (CE) for the determination of paracetamol and its main metabolites in urine and serum is described. Due to its high efficacy, CE enables the analysis of drugs directly in complex matrices. Thus, simple, rapid and reliable assays could be developed that made use of some of the main advantages of this analytical technique. In order to prevent the peaks from tailing, a water zone was injected behind the sample. Occasionally occurring peak splittings of paracetamol were investigated and methods to suppress these splittings were developed. Paracetamol, its main metabolites, paracetamol glucuronide, paracetamol sulfate as well as paracetamol cysteinate and paracetamol mercapturate, as metabolites of the oxidative pathway were identified in urine using diode-array detection and coupling of the CE instruments to electrospray–mass spectrometry. The assays were validated. Their usefulness was demonstrated by applying them to the analysis of urine and serum samples of healthy volunteers as well as to urine samples from children under anticancer therapy.     

Determination of nicotine and its metabolites in urine by solid-phase extraction and sample stacking capillary electrophoresis-mass spectrometry

The combination of capillary electrophoresis (CE) and mass spectrometry (MS) with solid-phase extraction (SPE) has been used for the identification of nicotine and eight of its metabolites in urine. The recovery of cotinine from cotinine-spiked urine, by C18 SPE, was found to be 98%. Smokers urine (200 ml) was preconcentrated 200-fold via SPE prior to analysis. The sample stacking mode of CE, when compared to capillary zone electrophoresis, was shown to improve peak efficiency by 132-fold. The combination of hydrodynamic and electrokinetic injection was studied with sample stacking/CE/MS. The on-column limits of detection (LOD) of nicotine and cotinine, by this technique, were found to be 0.11 and 2.25 microg/ml, respectively. Hence, LODs of nicotine and cotinine in urine after 200-fold preconcentration were 0.55 and 11.25 ng/ml, respectively.     

Gas chromatography-mass spectrometry (GC/MS) reveals urine metabolites associated to light and heavy infections by Schistosoma mansoni in mice

High-throughput profiling of metabolites has been used to identify metabolic changes in murine models as a response to the infection by the parasitic trematode Schistosoma. These investigations have contributed to our understanding on the pathogenesis of this tropical neglected disease, with a potential of helping diagnosis. Here, our study aimed to investigate the application of gas chromatography–mass spectrometry (GC/MS) on the profiling of urine metabolites from mice carrying infections by Schistosoma mansoni. Two larval infection doses created distinctive infection intensities in mice, whereby the heavily infected animals were found to release 25 times more eggs in faeces than lightly infected animals. Over 200 urine metabolites were identified from these animals by GC/MS, following two complementary derivatisation methods. A list of 14 individual metabolites with altered relative abundances between groups were identified. Most of the altered metabolites showed a trend of increased abundances in response to infection intensity, indicating host-specific metabolic alterations as a result of the disease. Hippurate, a metabolite which concentration is intimately modulated by the gut microbiota, was found to be highly correlated to infection intensity. Our study showed that urine metabolic profiling by GC/MS can distinguish non-infected animals from those carrying light and heavy infections by S. mansoni, revealing metabolites associated to the infection and providing insights on the pathogenesis of schistosomiasis.     

Metabolic analysis of body fluids by capillary electrophoresis using noncovalently coated capillaries

The potential of capillaries noncovalently coated with charged polymers for the metabolic analysis of body fluids by CE is illustrated. Firstly, the usefulness of a coating consisting of a triple layer of polybrene-dextran sulfate-polybrene for the fast analysis of organic acids is described. The CE system allowed direct injections of CSF, plasma and urine samples, yielding good separation efficiencies. RSDs for migration times and peak areas of organic acids in plasma were <3% and <5%, respectively. The usefulness of the system is illustrated by the profiling of organic acids in plasma and urine samples. Secondly, a CE system comprising a bilayer coating of polybrene-poly(vinylsulfonate), which provides a considerable EOF at low pH is described. This system was combined with TOF-MS and used for the fast analysis of amino acids in cerebrospinal fluid (CSF) and urine with minimal sample pretreatment. RSDs for migration times and peak areas of amino acids in CSF and urine were <2% and <10%, respectively. The applicability of the system is demonstrated by the profiling of endogenous low-molecular weight metabolites in CSF from a healthy individual and a patient with complex regional pain syndrome.     

Chromatographic and capillary electrophoretic methods for the analysis of nicotinic acid and its metabolites

Methods for the assay of nicotinic acid (NiAc) and its metabolites in biological fluids using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are reviewed. Most of the references cited in this review concern HPLC methods. A few CE methods that have been recently reported are also included. As these compounds are relatively polar and have a wide range of physico-chemical properties, the sample pre-treatment or clean-up process prior to analysis is included. Most HPLC methods using an isocratic elution system allow determination of a single or few metabolites, but gradient HPLC methods enable simultaneous determination of five to eight compounds. Simultaneous determination of NiAc including many metabolites in a single run can be achieved by CE. We also discuss the pharmacokinetics of NiAc and some of its metabolites.     

New Metabolic Alterations and A Predictive Marker Pipecolic Acid in Sera for Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC) is a major histological subtype of esophageal cancer with a poor prognosis. Although several serum metabolomic investigations have been reported, ESCC tumor-associated metabolic alterations and predictive biomarkers in sera have not been defined. Here, we enrolled 34 treatment-naive patients with ESCC and collected their pre-and post-esophagectomy sera together with the sera from 34 healthy volunteers for a metabolomic survey. Our comprehensive analysis i...     

Metabolomics study of stepwise hepatocarcinogenesis from the model rats to patients: potential biomarkers effective for small hepatocellular carcinoma diagnosis

The aim of this study is to find the potential biomarkers from the rat hepatocellular carcinoma (HCC) disease model by using a non-target metabolomics method, and test their usefulness in early human HCC diagnosis. The serum metabolic profiling of the diethylnitrosamine-induced rat HCC model, which presents a stepwise histopathological progression that is similar to human HCC, was performed using liquid chromatography-mass spectrometry. Multivariate data analysis methods were utilized to identify the potential biomarkers. Three metabolites, taurocholic acid, lysophosphoethanolamine 16:0, and lysophosphatidylcholine 22:5, were defined as "marker metabolites," which can be used to distinguish the different stages of chemical hepatocarcinogenesis. These metabolites represented the abnormal metabolism during the progress of hepatocarcinogenesis, which could also be found in patients. To test their diagnosis potential 412 sera from 262 patients with HCC, 76 patients with cirrhosis and 74 patients with chronic hepatitis B were collected and studied, it was found that 3 marker metabolites were effective for the discrimination of small liver tumor (solitary nodules of less than 2 cm in diameter) patients, achieved a sensitivity of 80.5% and a specificity of 80.1%,which is better than those of α-fetoprotein (53 and 64%, respectively). Moreover, they were also effective for the discrimination of all HCCs and chronic liver disease patients, which could achieve a sensitivity of 87.5% and a specificity of 72.3%, better than those of α-fetoprotein (61.2 and 64%). These results indicate metabolomics method has the potential of finding biomarkers for the early diagnosis of HCC.     

Characterization of protein-bound gold in rat urine following aurothiomalate administration and of rat and human albumin-gold-thiomalate

The metabolites of gold in the urine of rats given the antiarthritic drug aurothiomalate were investigated by gel permeation chromatography, electrophoresis, and chemical studies. Following a single dose of aurtothiomalate, the excreted gold was protein-bound in the high-molecular-weight (greater than or equal to 150,000 dalton) and serum albumin fractions. Electrophoresis confirmed the presence of albumin, but showed that the other proteins present differ from those in normal or in vitro aurothiomalate-incubated rat sera. The pattern of the proteins establishes that the proteinuria was of the glomerular type. The alterations in the gold distribution produced by incubation of the urine with the low-molecular-weight thiol penicillamine and with exogenously added aurothiomalate indicated the existence of a labile equilibrium of gold among protein binding sites in the urine. Incubation of rat and human sera and commercially prepared serum albumins with aurothiomalate increased the electrophoretic mobility of the albumin. The significance of this change in electrophoretic mobility with respect to two models of gold binding by serum albumin is discussed.     

Standard operating procedures for pre-analytical handling of blood and urine for metabolomic studies and biobanks

(1)H NMR metabolic profiling of urine, serum and plasma has been used to monitor the impact of the pre-analytical steps on the sample quality and stability in order to propose standard operating procedures (SOPs) for deposition in biobanks. We analyzed the quality of serum and plasma samples as a function of the elapsed time (t?=?0-4?h) between blood collection and processing and of the time from processing to freezing (up to 24?h). The stability of the urine metabolic profile over time (up to 24?h) at various storage temperatures was monitored as a function of the different pre-analytical treatments like pre-storage centrifugation, filtration, and addition of the bacteriostatic preservative sodium azide. Appreciable changes in the profiles, reflecting changes in the concentration of a number of metabolites, were detected and discussed in terms of chemical and enzymatic reactions for both blood and urine samples. Appropriate procedures for blood derivatives collection and urine preservation/storage that allow maintaining as much as possible the original metabolic profile of the fresh samples emerge, and are proposed as SOPs for biobanking.     

Urine metabolomics combined with the personalized diagnosis guided by Chinese medicine reveals subtypes of pre-diabetes

The prevalence of type 2 diabetes continuously increases globally. A personalized strategy applied in the pre-diabetic stage is vital for diabetic prevention and management. The personalized diagnosis of Chinese Medicine (CM) may help to stratify the diabetics. Metabolomics is regarded as a potential platform to provide biomarkers for disease-subtypes. We designed an explorative study of 50 pre-diabetic males, combining GC-MS urine metabolomics with CM diagnosis in order to identify diagnostic biomarkers for pre-diabetic subtypes. Three CM physicians reached 85% diagnosis consistency resulting in the classification of 3 pre-diabetic groups. The urine metabolic patterns of groups 1 'Qi-Yin deficiency' and 2 'Qi-Yin deficiency with dampness' (subtype A) and group 3 'Qi-Yin deficiency with stagnation' (subtype B) were clearly discriminated. The majority of metabolites (51%), mainly sugars and amino acids, showed higher urine levels in subtype B compared with subtype A. This indicated more disturbances of carbohydrate metabolism and renal function in subtype B compared with subtype A. No differences were found for hematological and biochemical parameters except for levels of glucose and γ-glutamyltransferase that were significantly higher in subtype B compared with subtype A. This study proved that combining metabolomics with CM diagnosis can reveal metabolic signatures for pre-diabetic subtypes. The identified urinary metabolites may be of special clinical relevance for non-invasive screening for subtypes of pre-diabetes, which could lead to an improvement in personalized interventions for diabetics.     

Quantification of penicillin G during labor and delivery by capillary electrophoresis

In this study, a capillary electrophoresis (CE) method was developed as a means to measure levels of penicillin G (PCN G) in Group B Streptococcus (GBS) positive pregnant women during labor and delivery. Volunteers for this developmental study were administered five million units of PCN G at the onset of labor. Urine, blood, and amniotic fluid samples were collected during labor and post delivery. Samples were semi-purified by solid-phase extraction (SPE) using Waters tC18 SepPak 3 cc cartridges with a sodium phosphate/methanol step gradient for elution. Capillary electrophoresis or reversed-phase high-performance liquid chromatography (RP-HPLC) with diode-array absorbance detection were used to separate the samples in less than 30 min. Quantification was accomplished by establishing a calibration curve with a linear dynamic range. The tC18 SPE methodology provided substantial sample clean-up with high recovery yields of PCN G ( 90%). It was found that SPE was critical for maintaining the integrity of the separation column when using RP-HPLC, but was not necessary for sample analysis by CE where no stationary phase is present. Quantification results ranged from millimolar concentrations of PCN G in maternal urine to micromolar concentrations in amniotic fluid. Serum and cord blood levels of PCN G were below quantification limits, which is likely due to the prolonged delay in sample collection after antibiotic administration. These results show that CE can serve as a simple and effective means to characterize the pharmacokinetic distribution of PCN G from mother to unborn fetus during labor and delivery. It is anticipated that similar methodologies have the potential to provide a quick, simple, and cost-effective means of monitoring the clinical efficacy of PCN G and other drugs during pregnancy.     

Quantitation of a new potent angiotensin II receptor antagonist,TCV-116, and its metabolites in human serum and urine

A sensitive high-performance liquid chromatographic (HPLC) method is described for the determination of a new potent antihypertensive agent, TCV-116, and its two metabolites (M-I and M-II) in human serum or urine. After pre-treatment of the specimens, the analytes were determined using a column switching technique, except for the metabolites in urine which were determined by gradient elution mode HPLC. The quantitation limits for TCV-116, M-I and M-II were all 0.5 ng/ml in serum, and 0.5, 10 and 10 ng/ml in urine, respectively. The methods were applied to clinical trials of TCV-116.     

Five unusual serum protein presentations found by capillary electrophoresis in the clinical laboratory.

Capillary electrophoresis (CE) has been used in our teaching hospital clinical laboratory to assay approximately 13 000 specimens for serum protein electrophoresis (SPE) in 4 1/2 years. During that period we have found several unusual samples, five of which are discussed here. These samples are from separate patients with IgD myeloma, IgG heavy chain disease, a triple IgG(Kappa) monoclonal band, a rapidly changing abnormal/monoclonal band and a mixed type-11 cryoglobulinaemia. Albumin has been used to calibrate the 50-microm fused silica capillary, the quantity of the monoclonal bands being calculated by the software of either the Applied Biosystems 270A-HT or BioFocus instrument. We have found CE for the initial SPE to be a robust, labour-saving, cost effective technique, which is able to determine less than 1 g/l of monoclonal protein. However, because of the expense and time required for immunosubtraction, we prefer isoelectric focusing (IEF) for typing of paraproteins. The only samples which need care in handling are serum containing large amounts of cryoglobulin protein.     

Large-volume sample stacking of selected drugs of forensic significance by capillary electrophoresis

Large-volume sample stacking capillary electrophoresis (LVSS-CE) and conventional capillary electrophoresis (CE) are compared for the separation of drugs of significance to forensic and clinical analyses. LVSS-CE for cations requires the use of an electroosmotic flow (EOF) modifier in conjunction with polarity switching to effect on-column concentration of an analyte and its subsequent migration in the capillary. The run buffer consists of 0.05 mol dm⁻³ disodium tetraborate adjusted to pH 2.2 with orthophosphoric acid, and the EOF modifier is 0.002 mol dm⁻³ cetyltrimethylammonium bromide. CE investigations used an identical run buffer minus the EOF modifier. LVSS-CE and CE investigations used injection times of 30 s and 3 s, respectively. Both modes of capillary electrophoresis are compared in terms of their limits of detection, efficiency, resolution and reproducibility. LVSS-CE is also applied to the analysis of a spiked urine sample.     

Enantioselective determination of zopiclone and its metabolites in urine by capillary electrophoresis

A method has been developed for the stereoselective determination of zopiclone and its main metabolites in urine. After the addition of the internal standard zolpidem the urine samples were extracted at pH 8 with chloroform-isopropanol (9:1). Analyses were carried out using capillary electrophoresis (CE) with β-cyclodextrin as the chiral selector. The analytes were detected using UV laser-induced fluorescence detection with a He-Cd laser operated at 325 nm. Urine samples of two volunteers after oral administration of 7.5 mg zopiclone were investigated. The S-(+)-enantiomers of zopiclone and its metabolites were always excreted in higher amounts than the R-(−)-enantiomers. With the same method the zopiclone enantiomers were quantified in saliva. Compared to high-performance liquid chromatography, the CE method is very fast and simple.     

Gas and liquid chromatography-mass spectrometry studies on the metabolism of the synthetic phenylacetylindole cannabimimetic JWH-250, the psychoactive component of smoking mixtures

Prohibition of some synthetic cannabimimetics (e.g., JWH-018, JWH-073 and CP 47497) in a number of countries has led to a rise in new compounds in herbal mixtures that create marijuana-like psychotropic effects when smoked. The cannabimimetic JWH-250 (1-pentyl-3-(2-methoxyphenylacetyl)indole) was identified in May 2009 by the German Federal Criminal Police as an new ingredient in herbal smoking mixtures. The absence or low presence of the native compound in urine samples collected from persons who had consumed JWH-250 necessitates a detailed identification of their metabolites, which are excreted with urine and present in blood. Using gas and liquid chromatography-mass spectrometry (GC-MS and LC-MS/MS), we identified a series of metabolites in urine samples and serum sample from humans and urine samples from rats that were products of the following reactions: (a) mono- and dihydroxylation of aromatic and aliphatic residues of the parent compound, (b) trihydroxylation and dehydration of the N-alkyl chain, (c) N-dealkylation and (d) N-dealkylation and monohydroxylation. The prevailing urinary metabolites in humans were the monohydroxylated forms, while N-dealkylated and N-dealkyl monohydroxylated forms were found in rats. The detection of the mono- and dihydroxylated metabolites of JWH-250 in urine and serum samples by GC-MS and LC-MS/MS proved to be effective in determining consumption of this drug.     

Synthesis of DEHP metabolites as biomarkers for GC-MS evaluation of phthalates as endocrine disrupters

Phthalates are used primarily as plasticizers to make polyvinyl chloride (PVC) soft and flexible. In recent years the phthalate esters have attracted increasing attention as environmental and biomedical pollutants and, because of their toxicological characteristics. In particular, they are more and more recognized as endocrine disrupters. In this context, we describe herein an efficient synthetic pathway leading to a series of metabolites of di(2-ethylhexyl) phthalate (DEHP). Mono(2-ethylhexenyl) phthalate was used as starting material to obtain these products in good yield, large scale and GC-MS purity. The metabolites of DEHP were synthesized, for the first time, as biomarkers to verify their quantitative determination in human urine and serum by GC-MS analysis for studying the exposure to phthalates and establishing reference values.     

Steroids excreted in urine by neonates with 21-hydroxylase deficiency: Characterization, using GC-MS and GC-MS/MS, of the D-ring and side chain structure of pregnanes and pregnenes

Steroid metabolites in urine from neonates with 21-hydroxylase deficiency are predominantly polyhydroxylated 17-hydroxyprogesterone and androgen metabolites, and most have incompletely defined structure. This study forms part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal types of steroid metabolism.Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra, obtained with constant excitation conditions, were used together to determine the structure of the D-ring and the side chain of 20-oxo and 20-hydroxy pregnane(ene)s without oxo groups on the A-, B-, and C-ring.All possible combinations of D-ring and side chain configuration were considered. Most fragmentations could be interpreted as partial or complete D-ring cleavages with loss of the side chain, aided by comparison with spectra of deuterated derivatives and of borohydride reduced metabolites. Possible rearrangement ions are also discussed. More than 140 endogenous metabolites were characterized.GC-MS/MS was especially beneficial for characterization of compounds with 16,17-dihydroxy-20-oxo structure, interpreted as markers of intra-uterine enzyme induction. It also assisted the differentiation of 16-hydroxy-20-oxo metabolites, present in urine of non-affected neonates, from the diagnostic 17-hydroxy-20-oxosteroids and enabled the detection of 15,17-dihydroxy-20-oxo compounds in low concentrations. The presence of 17,21-dihydroxylated pregnane(ene)s despite the deficit in CYP21A2 is discussed.We conclude that GC-MS combined with GC-MS/MS allows reliable identification of the structure of the D-ring and side chain of pregnane(ene)s without prior isolation, even when in low concentrations in urine.