Metabolomics of the diabetic nephropathy: behind the fingerprint of development and progression indicators. / Metabolómica de la nefropatía diabética: tras la huella de indicadores de desarrollo y progresión.

Current diagnostic methods are not very sensitive to detect the initial stages diabetic nephropathy of type 2. In this work, a review of metabolomic approximation studies for the identification of biomarkers of this disease with potential to differentiate between early stages, evaluate and direct treatment and help slow kidney damage. Using public (Pubmed and Google Scholar) and private (Scopus and Web of Knowledge) databases, a systematic search of the information published related to metabolomics of diabetic nephropathy in different biospecimens (urine, serum, plasma and blood) was made. Later, the MetaboAnalyst 4.0 software was used to identify the metabolic pathways associated with these metabolites. Groups of potential metabolites were identified for monitoring diabetic nephropathy with the available literature data. In the urine, oxide-3-hydroxyisovalerate, TMAO, aconite and citrate and hydroxypropionate derivatives are highlighted; meanwhile, in the serum: citrate, creatinine, arginine and its derivatives; and in the plasma: amino acids such as histidine, methionine and arginine has a potential contribution. Using MetaboAnalyst 4.0 the metabolic pathways related to these metabolites were related. The search for biomarkers to measure the progression of diabetic nephropathy, together with analytical strategies for their detection and quantification, are the starting point for designing new methods of clinical chemistry analysis. The association between the metabolic pathway dysfunction could be useful for the overall assessment of the treatment and clinical follow-up of this disease.

Valeric acid lowers arterial blood pressure in rats.

Valeric acid (VA) is a short-chain fatty acid produced by microbiota and herbs such as Valeriana officinalis. Moreover, VA is released from medicines such as estradiol valerate by esterases. We evaluated the concentrations of endogenous VA in male, 14-week-old rats in the liver, heart, brain, kidneys, lungs, blood and in the colon, a major site of microbiota metabolism, using liquid chromatography coupled with mass spectrometry. In addition, the tissue distribution of VA D9-isotope (VA-D9) administered into the colon was assessed. Finally, we investigated the effect of exogenous VA on arterial blood pressure (BP) and heart rate (HR) in anesthetized rats, and the reactivity of mesenteric (MA) and gracilis muscle (GMA) arteries ex vivo. Physiological concentration of VA in the colon content was ≈650 µM, ≈ 0.1-1 µM in the investigated tissues, and ≈0.4 µM in systemic blood. VA-D9 was detected in the tissues 5 min after the administration into the colon. The vehicle did not affect BP and HR. VA produced a dose-dependent decrease in BP, and at higher doses lowered HR. The hypotensive effect of VA was inhibited by 3-hydroxybutyrate, an antagonist of GPR41/43-receptors but not by the subphrenic vagotomy. Hexamethonium prolonged the hypotensive effect of VA while atropine did not influence the hypotensive effect. VA dilated GMA and MA. In conclusion, the exogenous VA produces vasodilation and lowers BP. The colon-derived VA rapidly penetrates to tissues involved in the control of BP. Further studies are needed to evaluate the effects of endogenous and exogenous VA on the circulatory system.

Profiling Vaccinium macrocarpon components and metabolites in human urine and the urine ex-vivo effect on Candida albicans adhesion and biofilm-formation.

The aim of this work was to profile, by using an HPLC-MS/MS method, cranberry compounds and metabolites found in human urine after ingestion of a highly standardized cranberry extract (Anthocran®). Two different strategies were adopted for the data analysis: a targeted and an untargeted approach. These strategies allowed the identification of 42 analytes including cranberry components, known metabolites and metabolites hitherto unreported in the literature, including six valerolactones/valeric acid derivatives whose presence in urine after cranberry consumption has never been described before. Absolute concentrations of 26 over 42 metabolites were obtained by using pure available standards. Urine collected at different time points after the last dosage of Anthocran® were tested on the reference strain C. albicans SC5314, a biofilm-forming strain. Fractions collected after 12 h were found to significantly reduce the adhesion and biofilm formation compared to the control (p < 0.05). A similar effect was then obtained by using Anthocran™ Phytosome™, the lecithin formulation containing 1/3 of standardized cranberry extract and formulated to enhance the absorption of the cranberry components. The urinary profile of cranberry components and metabolites in the urine fractions collected at 1 h, 6 h and 12 h after the last capsule intake were then reproduced by using the pure standards at the concentration ranges found in the urine fraction, and tested on C. albicans. Only the mixture mimicking the urinary fraction collected at 12 h and containing as main components, quercetin and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone was found effective thus confirming the ex-vivo results.

Metabolism of Nine Synthetic Cannabinoid Receptor Agonists Encountered in Clinical Casework: Major in vivo Phase I Metabolites of AM-694, AM-2201, JWH-007, JWH-019, JWH-203, JWH-307, MAM-2201, UR-144 and XLR-11 in Human Urine Using LC-MS/MS.

BACKGROUND: `Herbal mixtures` containing synthetic cannabinoid receptor agonists (SCRAs) are promoted as legal alternative to marihuana and are easily available via the Internet. Keeping analytical methods for the detection of these SCRAs up-to-date is a continuous challenge for clinicians and toxicologists due to the high diversity of the chemical structures and the frequent emergence of new compounds. Since many SCRAs are extensively metabolized, analytical methods used for urine testing require previous identification of the major metabolites of each compound. OBJECTIVE: The aim of this study was to identify the in vivo major metabolites of nine SCRAs (AM- 694, AM-2201, JWH-007, JWH-019, JWH-203, JWH-307, MAM-2201, UR-144, XLR-11) for unambiguous detection of a drug uptake by analysis of urine samples. METHOD: Positive urine samples from patients of hospitals, detoxification and therapy centers as well as forensic-psychiatric clinics were analyzed by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time-of-flight mass spectrometry (LCqToF- MS) for investigation of the major in vivo metabolites. RESULTS: For all investigated SCRAs, monohydroxylation, dihydroxylation and/or formation of the Nhexanoic/ pentanoic acid metabolites were among the most abundant metabolites detected in human urine samples. Substitution of the fluorine atom was observed to be an important metabolic reaction for compounds carrying an N-(5-fluoropentyl) side chain. N-Dealkylated metabolites were not detected in vivo. CONCLUSION: The investigated metabolites facilitate the reliable detection of drug uptake by analysis of urine samples. For distinction between uptake of the fluorinated and the non-fluorinated analogs, the N-(4-hydroxypentyl) metabolite of the non-fluorinated analog was identified as a useful analytical target and consumption marker.

Excretion of metabolites of the synthetic cannabinoid JWH-018 in urine after controlled inhalation.

Each year, synthetic cannabinoids are occurring in high numbers on the illicit drug market but data obtained after controlled application are rare. The present study on pharmacokinetics in urine is part of a pilot study on adverse effects of JWH-018, which is one of the oldest and best known synthetic cannabinoids. Six subjects inhaled smoke from 2 and 3mg JWH-018. The drug and ten potential metabolites were analyzed in urine samples collected during 12h after inhalation by liquid chromatography-mass spectrometry (LC-MS/MS) without and with conjugate cleavage. The parent compound was not detectable, but 13 of its metabolites, all of which were conjugated. Concentrations of the predominant metabolite, JWH-018 pentanoic acid, were less than 5ng/ml, but in two subjects it was still detected up to 4 weeks after ingestion. Other major metabolites were 5- and 4-HOpentyl-JWH-018, JWH-073 butanoic acid and a hypothetically dihydroxylated and dehydrogenated metabolite of JWH-018. Occasionally, further hydroxylated metabolites were found. Generally, hydroxylated metabolites were detected in concentrations lower than 1ng/ml already 10h after inhalation. All concentrations were much lower than reported for urine samples of authentic JWH-018 users. The formation of the metabolite JWH-018 pentanoic acid was found to be slightly delayed, but its rather high concentrations and detection over several weeks after single dosing makes it a useful target for urine analysis. The different excretion of carboxylic acid and hydroxylated metabolites may aid in evaluation of time of use.

A discovery-driven approach to elucidate urinary metabolome changes after a regular and moderate consumption of beer and nonalcoholic beer in subjects at high cardiovascular risk.

SCOPE: The aim of this work was to study the urinary metabolomics changes of participants that consumed beer, nonalcoholic beer (na-beer), and gin. METHODS AND RESULTS: Thirty-three males at high cardiovascular risk between 55 and 75 years old participated in an open, randomized, crossover, controlled trial with three nutritional interventions consisting of beer, na-beer, and gin for 4 wk. Diet and physical activity was monitored throughout the study and compliance was assessed by measurement of urinary isoxanthohumol. Metabolomic analysis was performed in urine samples by LC coupled to an LTQ-Orbitrap mass spectrometer combined with univariate and multivariate statistical analysis. Ten metabolites were identified. Eight were exogenous metabolites related to beer, na-beer, or gin consumption, but two of them were related to endogenic changes: hydroxyadipic acid linked to fatty acid oxidation, and 4-guanidinobutanoic acid, which correlated with a decrease in urinary creatinine. Plasmatic acylcarnitines were quantified by targeted MS. A regular and moderate consumption of beer and na-beer decreased stearoylcarnitine concentrations. CONCLUSION: Humulinone and 2,3-dihydroxy-3-methylvaleric acid showed to be potential biomarkers of beer and na-beer consumption. Moreover, the results of this trial provide new evidence that the nonalcoholic fraction of beer may increase fatty oxidation.

Metabolic syndrome increases dietary α-tocopherol requirements as assessed using urinary and plasma vitamin E catabolites: a double-blind, crossover clinical trial.

Background: Vitamin E supplementation improves liver histology in patients with nonalcoholic steatohepatitis, which is a manifestation of the metabolic syndrome (MetS). We reported previously that α-tocopherol bioavailability in healthy adults is higher than in those with MetS, thereby suggesting that the latter group has increased requirements.Objective: We hypothesized that α-tocopherol catabolites α-carboxyethyl hydroxychromanol (α-CEHC) and α-carboxymethylbutyl hydroxychromanol (α-CMBHC) are useful biomarkers of α-tocopherol status.Design: Adults (healthy or with MetS; n = 10/group) completed a double-blind, crossover clinical trial with four 72-h interventions during which they co-ingested 15 mg hexadeuterium-labeled RRR-α-tocopherol (d6-α-T) with nonfat, reduced-fat, whole, or soy milk. During each intervention, we measured α-CEHC and α-CMBHC excretions in three 8-h urine collections (0-24 h) and plasma α-tocopherol, α-CEHC, and α-CMBHC concentrations at various times ≤72 h.Results: During the first 24 h, participants with MetS compared with healthy adults excreted 41% less α-CEHC (all values are least-squares means ± SEMs: 0.6 ± 0.1 compared with 1.0 ± 0.1 µmol/g creatinine, respectively; P = 0.002), 63% less hexadeuterium-labeled (d6)-α-CEHC (0.04 ± 0.02 compared with 0.13 ± 0.02 µmol/g creatinine, respectively; P = 0.002), and 58% less d6-α-CMBHC (0.017 ± 0.004 compared with 0.041 ± 0.004 µmol/g creatinine, respectively; P = 0.0009) and had 52% lower plasma d6-α-CEHC areas under the concentration curves [area under the curve from 0 to 24 h (AUC0-24h): 27.7 ± 7.9 compared with 58.4 ± 7.9 nmol/L × h, respectively; P = 0.01]. d6-α-CEHC peaked before d6-α-T in 77 of 80 paired plasma concentration curves. Urinary d6-α-CEHC 24-h concentrations were associated with the plasma AUC0-24 h of d6-α-T (r = 0.53, P = 0.02) and d6-α-CEHC (r = 0.72, P = 0.0003), and with urinary d6-α-CMBHC (r = 0.88, P < 0.0001), and inversely with the plasma inflammation biomarkers C-reactive protein (r = -0.70, P = 0.0006), interleukin-10 (r = -0.59, P = 0.007), and interleukin-6 (r = -0.54, P = 0.01).Conclusion: Urinary α-CEHC and α-CMBHC are useful biomarkers to noninvasively assess α-tocopherol adequacy, especially in populations with MetS-associated hepatic dysfunction that likely impairs α-tocopherol trafficking. This trial was registered at clinicaltrials.gov as NCT01787591.

Validation of JWH-018 and its metabolites in blood and urine by UPLC-MS/MS: Monitoring in forensic cases.

The herbal products referred to as 'Spice' have been used as 'legal alternatives' to cannabis worldwide since 2004. The first synthetic cannabinoid JWH-018 was detected in 'Spice' products in 2008, and has been banned by many legal authorities since the beginning of 2009. In order to prove use of JWH cannabinoids (JWHs), specific and robust methods were needed. We have developed a specific and reliable method for the detection and quantification of JWH-018, JWH-018 N-pentanoic acid, and JWH-018 N-(5-hydroxypentyl) in blood and urine using solid-phase extraction followed by UPLC-MS/MS analysis. The method has been validated in terms of linearity (0.1-50ng/mL), selectivity, intra-assay and inter-assay accuracy and precision (CV<15%), recovery (85-98%), limits of detection (LOD) (0.08-0.14ng/mL), and quantification (LOQ) (0.10-0.21ng/mL). Matrix effects, stability, and process efficiency were also assessed. The method has been applied to 868 authentic samples received by the Department of Chemistry (Istanbul) in the Council of Forensic Medicine of the Ministry of Justice.

Pink urine.

A 55-year-old man was admitted after a suspected hypnotic overdose of valerian extracts. In addition to altered consciousness, the first clinical symptoms included not only diffuse rash on the face, trunk, and limbs, but also an inspiratory dyspnea with a marked hypoxemia. A major laryngeal edema was noted during orotracheal intubation. After correction of hypoxemia, the patient became agitated and propofol was administered by continuous infusion. In addition, the patient passed pink urine staining the urine collection bag. The presence of an unidentified toxic substance was suspected.

Identification and pharmacokinetics of novel alkylresorcinol metabolites in human urine, new candidate biomarkers for whole-grain wheat and rye intake.

Biomarkers of dietary intake are prominent tools in nutritional research. The alkylresorcinol metabolites 3,5-dihydroxybenzoic acid (3,5-DHBA) and 3-(3,5-dihydroxyphenyl)propanoic acid (3,5-DHPPA) have been proposed as exposure biomarkers of whole-grain (WG) wheat and rye intake. However, the profile of alkylresorcinol metabolites is not fully understood. The aim of this study was to investigate the metabolism of alkylresorcinols in mice and in humans, while further determining urinary pharmacokinetics of the novel alkylresorcinol metabolites to explore their potential as biomarkers of WG wheat intake. Utilization of the liquid chromatography-mass spectrometry approach resulted in 10 alkylresorcinol metabolites identified in mice and in humans, including 3 phenolic acids and 7 of their phase II conjugates. Among them, 2 novel metabolites were discovered: 5-(3,5-dihydroxyphenyl)pentanoic acid (3,5-DHPPTA) and 2-(3,5-dihydroxybenzamido)acetic acid (3,5-DHBA glycine). The structures of these 2 metabolites were confirmed by comparing with authentic standards synthesized in-house. In the pharmacokinetic study, a group of 12 volunteers consumed a polyphenolic-restricted diet for 4 d before ingesting WG wheat bread containing 61 mg of alkylresorcinols. Urine samples were collected for 32 h, and alkylresorcinol metabolites were quantified with HPLC-coulometric electrode array detection. The mean urinary excretion rates and mean apparent half-life of 3,5-DHPPTA, 3,5-DHBA glycine, 3,5-DHBA, and 3,5-DHPPA at each time point were determined. Our results suggest that 3,5-DHPPTA and 3,5-DHBA glycine may be used in combination with 3,5-DHBA and 3,5-DHPPA as potential biomarkers to increase the accuracy of recording WG wheat and rye intake in epidemiologic studies. Further validation of 3,5-DHPPTA and 3,5-DHBA glycine as potential biomarkers is warranted.

Monitoring of urinary metabolites of JWH-018 and JWH-073 in legal cases.

Due to their cannabis-like effects, synthetic cannabinoids have attracted much public attention since 2008. Thus, elucidation of the metabolic pattern and the detection of the intake of these drugs have been of major concern. In order to suggest appropriate urinary biomarkers to prove JWH-018 or JWH-073 intake, we selected the major metabolites of JWH-018 and JWH-073, namely (ω)-, (ω-1)-hydroxy, carboxy and 6-hydroxyindole metabolites, and validated a method for the quantification of these metabolites using solid-phase extraction based on LC-MS/MS analysis. Authentic urine specimens obtained from drug offenders were screened via a synthetic cannabinoid ELISA kit and were analyzed by LC-MS/MS for confirmation. Twenty-one out of a total of 52 samples (40%) were found positive for at least one metabolite of JWH-018 or JWH-073. N-pentyl hydroxy metabolites of JWH-018 and carboxy metabolites of JWH-018 and JWH-073 were detected in all positive samples. However, the rest of the metabolites were either not detected or only a small amount of them were found. A considerable variation was observed in the concentration ratio of (ω) and (ω-1)-hydroxy metabolites of JWH-018. Based on the results, it may have some pitfalls to determine the ingestion of specific synthetic cannabinoids by detecting a few metabolites, considering the continuous emergence of structurally related synthetic cannabinoids. Thus, use of synthetic cannabinoids should be proven carefully through comprehensive investigation of analytical results of biological specimens.

Gas chromatography-mass spectrometry of JWH-018 metabolites in urine samples with direct comparison to analytical standards.

JWH-018 (1-pentyl-3-(1-naphthoyl)indole) is one of numerous potential aminoalkylindoles contained in products marketed as 'K2' or 'Spice'. Investigation of the urinary metabolites from consumption of these compounds is important because they are banned in the United States and many European countries. An efficient extraction procedure and gas chromatography-mass spectrometry (GC-MS) method were developed for detection of 'K2' metabolites in urine from individuals suspected of using these products. Analytical standards were used to elucidate the structure-specific mass spectral fragmentations and retention properties to confirm proposed identifications and support quantitative studies. A procedure for the synthesis of one of these metabolites (5-hydroxypentyl JWH-018) was also developed. Results are comparable to existing LC-MS/MS methods, with the same primary metabolites detected. The specific metabolite hydrolysis products include 4-hydroxpentyl, 5-hydroxypentyl, and N-pentanoic acid derivatives.

Investigation of a wide spectrum of inherited metabolic disorders by 13C NMR spectroscopy.

High-resolution (1)H NMR spectroscopy of body fluids has proved to be very useful in diagnostics of inherited metabolic diseases, whereas (13)C NMR remains almost unexploited. In this paper the application of (13)C NMR spectroscopy of fivefold concentrated urine samples for diagnosis of selected metabolic diseases is reported. Various marker metabolites were identified in test urine samples from 33 patients suffering from 10 different diseases, providing information which could be crucial for their diagnoses. Spectra were accumulated for 2 h or overnight when using spectrometers operating at 9.4 or 4.7 T magnetic fields, respectively. Interpretation of the measurement results was based on a comparison of the peak positions in the measured spectrum with reference data. The paper contains a table with (13)C NMR chemical shifts of 73 standard compounds. The method can be applied individually or as an auxiliary technique to (1)H NMR or any other analytical method.

A sensitive liquid chromatographic method for the analysis of isovaleric and valeric acids in urine as fluorescent derivatives.

A simple and sensitive isocratic liquid chromatographic method was developed for the analysis of isovaleric and valeric acids in human urine as biomarkers in metabolic acidosis. The method is based on the derivatization of isovaleric and valeric acids with a fluorescent reagent 2-(2-naphthoxy)ethyl-2-(piperidino)ethanesulfonate for labeling the analytes with the naphthoxy fluorophore. The resulting fluorescent derivatives of isovaleric and valeric acids were separated on a phenyl-hexyl column, using a mixed solvent of methanol-water-tetrahydrofuran (55:31:14, v/v) as the mobile phase. The separated derivatives were monitored with a fluorimetric detector (excitation at 225 nm and emission at 360 nm). The linear range of the method for the determination of isovaleric acid or valeric acid derivative was over 0.2 approximately 8.0 microM. The detection limit (signal to noise ratio=3 with 10 microl injected) of isovaleric acid or valeric acid was about 0.04 microM. Application of the method to the analysis of isovaleric acid in the urine of a patient with isovaleric acidemia proved feasible.

Amino-acid depletion induced by abnormal amino-acid conjugation and protein restriction in isovaleric acidemia.

BACKGROUND: Previously, we detected 19 'new' amino-acid conjugates in the urine of patients with isovaleric acidemia. There is currently a poor understanding of the relationship between the clinical symptoms and the excreted metabolites occurring in these patients, owing to insufficient metabolite characterization and quantification. Consequently, controversial treatment protocols exist, particularly pertaining to dietary protein restriction. OBJECTIVE: To determine the effect of the previously identified amino-acid conjugates and conventional dietary protein restriction therapy, on the free amino-acid concentrations in isovaleric acidemia patients, to better explain the clinical symptoms and develop more effective therapy. DESIGN: Free amino-acid quantification via liquid chromatography mass spectrometry (LC-MS-MS) was performed on pre- and post-treatment urine or serum samples collected from six isovaleric acidemia patients, previously investigated for the presence of new induced N-isovaleryl and N-acetyl-amino-acid conjugates. RESULTS: Depleted amino-acid concentrations were detected in varying degrees in all six patients and did not recover after conventional treatment. CONCLUSIONS: The 19 potentially toxic metabolites previously identified and the consequent amino-acid depletions detected in this study, may explain many of the clinical symptoms associated with isovaleric acidemia. Furthermore, the occurrence of amino-acid depletions in these patients, steers away from the controversial dietary protein restriction treatment protocols, and towards dietary leucine restriction alone with essential amino-acid supplementation, in combination with glycine and L-carnitine supplementation.

'Classical' organic acidurias, propionic aciduria, methylmalonic aciduria and isovaleric aciduria: long-term outcome and effects of expanded newborn screening using tandem mass spectrometry.

'Classical organic acidurias' comprise isovaleric aciduria, propionic aciduria and methylmalonic aciduria. Available data from the literature suggest that the use of 'new' therapeutic strategies has improved survival but has not modified neurodevelopment. Progressive neurocognitive deterioration is almost invariably present in propionic and methylmalonic acidurias, while large-scale studies on the long-term outcome of patients with isovaleric aciduria are still lacking. In order to answer to some of the questions suggested by Wilson and Jungner in 1968 about the criteria of disease screening, we compared the natural history of patients with 'classical' organic acidurias diagnosed on clinical bases to those diagnosed through neonatal mass screening using tandem mass spectrometry. Decreased early mortality, less severe symptoms at diagnosis, and more favourable short-term neurodevelopmental outcome were recorded in patients identified through expanded newborn screening. The short duration of follow-up so far does not allow us to draw final conclusions about the effects of newborn screening on long-term outcome. The evaluation of the effect of neonatal screening on the detection rate of these three diseases showed that the incidence of isovaleric aciduria was significantly higher in the screening population than in clinically detected cases, with no changes for propionic and methylmalonic acidurias. Further multicentre longitudinal studies are needed to assess the usefulness of expanded newborn screening for 'classical' organic acidurias and to better understand the clinical spectrum of these diseases. This paper describes the long-term outcome and the impact of expanded newborn screening on the so-called 'classical' organic acidurias (propionic aciduria, methylmalonic aciduria and isovaleric aciduria).

Simultaneous retention index analysis of urinary amino acids and carboxylic acids for graphic recognition of abnormal state.

Simultaneous profiling analysis of urinary amino acids (AAs) and carboxylic acids (CAs) was combined with retention index (I) analysis for graphic recognition of abnormal metabolic state. The temperature-programmed I values of the AA and CA standards measured as ethoxycarbonyl (EOC)/methoxime (MO)/tert-butyldimethylsilyl (TBDMS) derivatives were used as the reference I values. Urine samples were subjected to the sequential EOC, MO and TBDMS reactions for the analysis by gas chromatography (GC) and GC-mass spectrometry. The complex GC profiles were then transformed into their respective I patterns in bar graphic forms by plotting the normalized peak area ratios (%) of the identified AAs and CAs against their reference I values as the identification numbers. When the present method was applied to infant urine specimens from normal controls and patients with inherited metabolic diseases such as phenylketonuria, maple syrup urine disease, methylmalonic aciduria or isovaleric aciduria, each I pattern of bar graph more distinctly displayed quantitative abundances of urinary AAs and CAs in qualitative I scale, thus allowing graphic discrimination between normal and abnormal states.

Quantitative analysis of dipeptidyl peptidase inhibitor P32/98 and its main metabolite in rat, dog, mouse, monkey, human plasma and human urine using liquid chromatography-tandem mass spectrometry: application to pharmacokinetic evaluation.

A sensitive, specific and robust assay was developed for the simultaneous determination of the oral antidiabetic drug candidate P32/98 and its main metabolite P57/99 in different biological fluids using LC-MS/MS in the atmospheric pressure chemical ionization (APCI) positive mode. Both analytes were isolated from the biological matrices by solid phase extraction using a strong cation exchanger. This assay was successfully cross-validated for rat, dog, mouse, monkey, human plasma and human urine. The pre-study validation results, as well as the in-study quality control (QC) data obtained, demonstrate the feasibility of the assay for pharmacokinetic evaluation of the compounds in different species and confirm the robustness of the assay for routine use.

Branched-chain organic acidurias.

Branched chain organic acidurias are a group of disorders that result from an abnormality of specific enzymes involving the catabolism of branched chain amino acids (leucine, isoleucine, valine). Maple syrup urine disease (MSUD), isovaleric acidaemia (IVA), propionic aciduria (PA) and methylmalonic aciduria (MMA) represent the most commonly encountered abnormal organic acidurias. All these four disorders present in neonates as a neurologic distress of the intoxication type with either ketosis or ketoacidosis and hyperammonaemia. There is a free interval between birth and clinical symptoms. MMA, PA and IVA present with a severe dehydration, leuconeutropenia and thrombopenia which can mimic sepsis. All these disorders can be diagnosed by identifying acylcarnitine and other organic acid compounds in plasma and urine by gas chromatography mass spectrometry or tandem MS-MS. These disorders are amenable to treatment by removing toxic compounds and by using special diets and carnitine.