Early detection of metabolic changes in drug-induced steatosis using metabolomics approaches.

Steatosis is the accumulation of triglycerides in hepatic cells wherein fats exceed 5% of the entire liver weight. Although steatotic liver damage is reversible due to the liver's regenerative capability, protracted damage often and typically leads to irreversible conditions such as cirrhosis and hepatocellular carcinoma (HCC). Therefore, early steatotic detection is critical for preventing progression to advanced liver diseases. This also becomes particularly important given the higher prevalence of drug usage, as drugs are a frequent cause of liver damage. Currently, the recommendation to diagnose steatosis is using liver enzymes and performing a liver biopsy. Liver biopsy remains the gold standard method of detection, but the procedure is invasive and an unreliable diagnostic tool. Non-invasive, specific and sensitive diagnostic solutions such as biomarkers are therefore needed for the early detection of steatosis. Our aim is to identify changes in urinary metabolites in tetracycline-induced hepatic steatotic rats at different stages of the diseases using metabolomic-based techniques. Sprague Dawley male rats are treated by intraperitoneal injection (I.P.) with either 62.5 mg kg-1 or 125 mg kg-1 tetracycline, an antibiotic previously known to induce steatosis. We analyse the metabolic profile of the urinary tetracycline induced hepatic steatotic rats using 1H nuclear magnetic resonance (NMR), 2D 1H-1H TOCSY (total correlation spectroscopy) and electrospray liquid chromatography-mass spectrometry (ESI-LC-MS/MS) based metabolomics. The combined analysis of haematoxylin & eosin (H&E), oil red O (ORO) and direct measurement of triglyceride content in the liver tissues of the control samples against 125 mg kg-1 and 62.5 mg kg-1 treated samples, reveals that 125 mg kg-1 tetracycline exposure potentially induces steatosis. The combination of 1H NMR, 2D 1H-1H TOCSY and ESI-LC-MS/MS alongside multivariate statistical analysis, detected a total of 6 urinary metabolites changes, across 6 metabolic pathways. Furthermore, lysine concentration correlates with liver damage as tetracycline dose concentration increases, whilst both H&E and ORO fail to detect hepatocellular damage at the lowest dose concentration. We conclude that the combination of 1H NMR and ESI-LC-MS/MS suggests that these are suitable platforms for studying the pathogenesis of steatosis development, prior to morphological alterations observed in staining techniques and offer a more detailed description of the severity of the steatotic disease.

Ultra-fast LC-MS/MS in therapeutic drug monitoring: Quantification of clozapine and norclozapine in human plasma.

A novel approach to high-throughput, targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis has been developed. A single chromatographic system can be used for the analysis of a range of 20 drugs and metabolites with a total analysis time of 36 s (one 96-well plate of prepared samples per hour). To demonstrate the applicability of this approach to quantitative analysis, a method has been validated for the therapeutic drug monitoring of clozapine and norclozapine following automated extraction from human plasma. Chromatographic retention times were 11.4 and 12.4 s for norclozapine and clozapine, respectively (for both analytes the chromatographic peak width was less than 1 s). Comparison with a conventional LC-MS/MS method (5 min analysis time) showed excellent agreement. This new approach offers analysis times more akin to flow-injection analysis, but is likely to be more widely applicable because of chromatographic resolution from residual matrix components and isobaric interferences.

Changes in urinary metabolomic profile during relapsing renal vasculitis.

Current biomarkers of renal disease in systemic vasculitis lack predictive value and are insensitive to early damage. To identify novel biomarkers of renal vasculitis flare, we analysed the longitudinal urinary metabolomic profile of a rat model of anti-neutrophil cytoplasmic antibody (ANCA) vasculitis. Wistar-Kyoto (WKY) rats were immunised with human myeloperoxidase (MPO). Urine was obtained at regular intervals for 181 days, after which relapse was induced by re-challenge with MPO. Urinary metabolites were assessed in an unbiased fashion using nuclear magnetic resonance (NMR) spectroscopy, and analysed using partial least squares discriminant analysis (PLS-DA) and partial least squares regression (PLS-R). At 56 days post-immunisation, we found that rats with vasculitis had a significantly different urinary metabolite profile than control animals; the observed PLS-DA clusters dissipated between 56 and 181 days, and re-emerged with relapse. The metabolites most altered in rats with active or relapsing vasculitis were trimethylamine N-oxide (TMAO), citrate and 2-oxoglutarate. Myo-inositol was also moderately predictive. The key urine metabolites identified in rats were confirmed in a large cohort of patients using liquid chromatography-mass spectrometry (LC-MS). Hypocitraturia and elevated urinary myo-inositol remained associated with active disease, with the urine myo-inositol:citrate ratio being tightly correlated with active renal vasculitis.

Separation and fragmentation study of isocoproporphyrin derivatives by UHPLC-ESI-exact mass MS/MS and identification of a new isocoproporphyrin sulfonic acid metabolite.

Isocoproporphyrin and its derivatives are commonly used as biomarkers of porphyria cutanea tarda, heavy metal toxicity and hexachlorobenzene (HCB) intoxication in humans and animals. However, most are isobaric with other porphyrins and reference materials are unavailable commercially. The structural characterisation of these porphyrins is important but very little data is available. We report here the separation and characterisation of isocoproporphyrin, deethylisocoproporphyrin, hydroxyisocoproporphyrin and ketoisocoproporphyrin, isolated in the faeces of rats fed with a diet containing HCB, by ultra high performance liquid chromatography-exact mass tandem mass spectrometry (UHPLC-MS/MS). Furthermore, we report the identification and characterisation of a previously unreported porphyrin metabolite, isocoproporphyrin sulfonic acid isolated in the rat faeces. The measured mass-to-charge ratio (m/z) of the precursor ion was m/z 735.2338, corresponding to a molecular formula of C36H39N4O11S with an error of 0.3 ppm from the calculated m/z 735.2336. The MS/MS data was consistent with an isocoproporphyrin sulfonic acid structure, derived from dehydroisocoproporphyrinogen by sulfonation of the vinyl group. The metabolite was present in a greater abundance than other isocoproporphyrin derivatives and may be a more useful biomarker for HCB intoxication.

Liquid chromatography-tandem mass spectrometry of porphyrins and porphyrinogens in biological materials: separation and identification of interfering poly(ethylene) glycol by travelling wave ion mobility spectrometry/tandem mass spectrometry.

Biological and clinical samples for porphyrin and porphyrinogen analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS) are often contaminated with poly(ethylene)glycol (PEG), which complicates the interpretation of mass spectra and characterisation of new porphyrin metabolites. Two contaminating PEG molecules (m/z 833 and m/z 835) were completely separated from uroporphyrin I (m/z 831) by travelling wave ion mobility spectrometry and characterised by tandem mass spectrometry. One of the PEG species (m/z 835) also co-eluted with uroporphyrinogen I (m/z 837) and was unresolvable by travelling wave ion mobility spectrometry/MS, therefore contaminating the MS/MS mass spectra owing to isotope distribution. These PEG species, with the [M + H](+) ions at m/z at 833 and/or m/z 835, co-eluted with uroporphyrin I and uroporphyrinogen I by LC-MS/MS and could be wrongly identified as uroporphomethenes.

Direct and simultaneous quantitation of 5-aminolaevulinic acid and porphobilinogen in human serum or plasma by hydrophilic interaction liquid chromatography-atmospheric pressure chemical ionization/tandem mass spectrometry.

Serum/plasma concentrations of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) are elevated in patients with acute hepatic porphyrias, especially during acute attacks. Current assays require lengthy sample pre-treatment and derivatization steps. We report here a rapid, sensitive and specific hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the direct and simultaneous quantitation of ALA and PBG in serum or plasma following simple protein precipitation with acetonitrile and centrifugation prior to injection. ALA and PBG were detected using selected reaction monitoring mode, following positive atmospheric pressure chemical ionization. Calibration was linear from 0.05 to 50 µmol/L for ALA and PBG. For both analytes, imprecision (relative standard deviation) was <13% and accuracy (percentage nominal concentrations) was between 92 and 107%. The method was successfully applied to the measurement of ALA and PBG in serum or plasma samples for the screening, biochemical diagnosis and treatment monitoring of patients with acute hepatic porphyrias.

Liquid chromatography and mass spectrometry of haem biosynthetic intermediates: a review.

This article discusses the separation, analysis and characterisation of intermediates and oxidative by-products of the haem biosynthetic pathway by liquid chromatography and mass spectrometry. Techniques reviewed include high-performance liquid chromatography, ultra-high-performance liquid chromatography, capillary electrophoresis, ion mobility spectrometry, mass spectrometry and tandem mass spectrometry. The emphasis was on the analysis of biological and clinical samples.

Direct and simultaneous determination of 5-aminolaevulinic acid and porphobilinogen in urine by hydrophilic interaction liquid chromatography-electrospray ionisation/tandem mass spectrometry.

Urinary concentrations of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) are elevated in patients with acute hepatic porphyrias, especially during acute attacks. Current assays require lengthy sample pre-treatment and derivatisation steps. We report here a rapid, sensitive and specific hydrophilic interaction liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, for the direct and simultaneous quantitation of ALA and PBG in urine following simple dilution with acetonitrile and centrifugation prior to injection. ALA and PBG were detected using selected reaction monitoring mode, following positive electrospray ionisation. Urine samples (N = 46) from active and latent mutation-confirmed acute hepatic porphyria patients and normal subjects (N = 45) were analysed and the results compared with those of a commercially available spectrophotometric method. The validated calibration range was 3-3000 µmol/L for ALA and 2-2000 µmol/L for PBG. For both analytes, imprecision (relative standard deviation) was less than 5% and accuracy (percentage nominal concentrations) was between 88 and 109%. The lower limit of quantitation was 0.1 µmol/L for both analytes. The calculated LC-MS/MS and spectrophotometric results from patient samples compared well [Pearson correlation (r²) of 0.99 and 0.95, for ALA and PBG, respectively]. The method was successfully applied to the measurement of ALA and PBG in urine samples for the screening, biochemical diagnosis and treatment monitoring of patients with acute hepatic porphyrias.

Variability in the analysis of 25-hydroxyvitamin D by liquid chromatography-tandem mass spectrometry: the devil is in the detail.

BACKGROUND: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly used in clinical laboratories for the analysis of 25-hydroxyvitamin D (25OHD), but measurement is not straightforward. Importantly, LC-MS/MS is not a single technique: variables in sample preparation, chromatography and ionisation/fragmentation should each be considered. METHODS: We analysed results from a survey organised by the international Vitamin D External Quality Assessment Scheme (DEQAS), to determine the influence of such variables on the results for two DEQAS distributions. RESULTS: 65 laboratories returned questionnaires. 346 (57%) individual results were from laboratories using electrospray ionisation (ESI), and 259 (43%) from laboratories using atmospheric pressure chemical ionisation (APCI). Although the mean ratio of results was not significantly different between ESI and APCI (P=0.5828), there was greater variation (P<0.0001) in results obtained by laboratories using ESI. Greater variation (P<0.05) was also observed between results from laboratories monitoring non-specific water-loss transitions. Only 3 laboratories (5%) could resolve the isobaric metabolite 3-epi-25OHD(3) from 25OHD(3). CONCLUSIONS: There are many variables to consider when using LC-MS/MS, including assay standardisation/calibration, chromatography and MS conditions. MS/MS alone cannot distinguish isobaric metabolites such as 3-epi-25OHD(3). Interference can also occur if non-specific transitions are used. Laboratories should always subscribe to an EQA scheme for 25OHD analysis.

Travelling wave ion mobility mass spectrometry of 5-aminolaevulinic acid, porphobilinogen and porphyrins.

RATIONALE: Human porphyrias, diseases caused by enzyme defects in haem biosynthesis, are characterised by the excessive production, accumulation and excretion of porphyrins and/or 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG). A method for the simultaneous separation, detection and identification of ALA, PBG and porphyrins would greatly facilitate the screening and diagnosis of porphyrias. Such a method would also be invaluable for the biochemical study of the haem, chlorophyll and corrin pathways. METHODS: An aqueous mixture containing ALA, PBG and type I isomer porphyrins was diluted with acetonitrile and infused (10 µL/min) into a Waters Synapt G2 high-definition mass spectrometer, equipped with a Z-Spray electrospray ionisation (ESI) source. Mass spectra were acquired in positive ionisation mode and the optimised ion mobility spectrometry (IMS) conditions were as follows: IMS wave height (V), 40; IMS wave velocity (m/s), 648; IMS gas flow (mL/min) 90.40; helium gas flow (mL/min), 182.60. RESULTS: The IMS drift-time increased with increasing ion mass in the order of ALA, PBG, mesoporphyrin, coproporphyrin I, penta-, hexa- and heptacarboxylic acid porphyrin I and uroporphyrin I. The ESI-IMS-MS spectra shows that PBG could form two different positively charged ions by protonation [M+H](+) , m/z 227, or deprotonation [M - H](+) , m/z 225. The protonated PBG (m/z 227) easily eliminated ammonia in source and the fragment ion (m/z 210) was monitored instead. Doubly charged ions of porphyrins having different drift times from the protonated singly charged molecules were observed in high abundance, providing further structural characterisation. CONCLUSIONS: We have shown, for the first time, an analytical method capable of simultaneously separating haem biosynthetic intermediates and metabolites, for a potential rapid clinical screening method for the porphyrias. IMS-MS allowed the separation of doubly charged porphyrin ions, which will be advantageous for the analysis of natural and synthetic tetrapyrrole compounds, while reducing the misinterpretation of contaminants.

Ultra high-performance liquid chromatography of porphyrins in clinical materials: column and mobile phase selection and optimisation.

Ultra high-performance liquid chromatographic (UHPLC) systems on columns packed with materials ranging from 1.9 to 2.7 µm average particle size were assessed for the fast and sensitive analysis of porphyrins in clinical materials. The fastest separation was achieved on an Agilent Poroshell C(18) column (2.7 µm particle size, 50 × 4.6 mm i.d.), followed by a Thermo Hypersil Gold C(18) column (1.9 µm particle size, 50 × 2.1 mm i.d.) and the Thermo Hypersil BDS C(18) column (2.4 µm particle size, 100 × 2.1 mm i.d.). All columns required a mobile phase containing 1 m ammonium acetate buffer, pH 5.16, with a mixture of acetonitrile and methanol as the organic modifiers for optimum resolution of the type I and III isomers, particularly for uroporphyrin I and III isomers. All UHPLC columns were suitable and superior to conventional HPLC columns packed with 5 µm average particle size materials for clinical sample analysis.

Ultra high-performance liquid chromatography of porphyrins.

An ultra high-performance liquid chromatographic (UHPLC) system was developed and optimized for the separation of porphyrins of clinical interest. Optimum conditions for the simultaneous separation of uroporphyrin, hepta-, hexa-, penta-carboxylic acid porphyrins and coproporphyrin and their type I and III isomers on a Thermo Hypersil BDS C18 column (2.4 µm particle size, 100 × 2.1 mm i.d.) using a gradient elution with 10% (v/v) acetonitrile in 1.0 m ammonium acetate buffer (pH 5.16) and 10% (v/v) acetonitrile in methanol at a flow-rate of 0.4 mL/min. The effect of mobile phase buffer molarity on the sensitivity of fluorescence detection and resolution of porphyrin isomers was investigated. The method was successfully applied to the analysis of porphyrins extracted from the urine and faeces of patients with various human porphyrias.

Porphyrinogen fragmentation profiles by ultra-high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry.

An ultra-high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry system is described for the separation and characterisation of uroporphyrinogen, heptacarboxylic acid porphyrinogen, hexacarboxylic acid porphyrinogen, pentacarboxylic acid porphyrinogen and coproporphyrinogen. The separation was carried out on a 100 mm × 2.1 mm Thermo-Hypersil BDS column (2.4 µm average particle size) by gradient elution with a mixture of acetonitrile, methanol and 1 mol/L aqueous ammonium acetate buffer, pH 5.16, as eluent. The fragmentation pattern of each compound was established by collision-induced dissociation tandem mass spectrometry. The most characteristic fragmentation was ring opening at one of the four methylene bridges of the protonated porphyrinogen molecule followed by further cleavages of methylene bridges linking the four pyrrole rings at various points to give product ions with methylenepyrrolenine, methylene-dipyrrolenine and methylene-tripyrrolenine structures.