Giuseppe Paglia

Development and validation of a fast quantitative method for plasma dimethylarginines analysis using liquid chromatography-tandem mass spectrometry.

OBJECTIVESnThe aim of this work was to implement a fast, accurate and simple method to quantify plasma ADMA and SDMA, in a run time suitable for routine analysis.nnnDESIGN AND METHODSnWe developed and validated a hydrophilic interaction chromatographic method coupled to tandem mass spectrometry (HILIC-MS/MS) for separation and simultaneous quantification of Arginine (Arg) and its dimethylarginines, ADMA and SDMA, with a short run time (less than 5 min) using a small volume of human plasma (0.02 mL).nnnRESULTSnCorrelation coefficients (r) of the calibration curves ranged from 0.9926 to 0.9984. Within-day and between-day imprecision (CV%) and inaccuracy (%), carry-over and recovery were also evaluated for validation. Preliminary data of Arg, ADMA and SDMA from 30 apparently healthy subjects and type 2 diabetic patients (n=33) with and without kidney dysfunction were calculated and some statistical differences occurred among them (p<0.05).nnnCONCLUSIONSnData from calibration curves and quality controls reveal that the method is accurate and precise. Healthy subjects and diabetic patients values are in agreement with those reported in other studies.

A powerful couple in the future of clinical biochemistry: in situ analysis of dried blood spots by ambient mass spectrometry

Since the early 1960s, dried blood spots (DBS) on filter paper have been used in clinical applications. The first key milestone in the use of DBS was the screening of phenylketonuria and other inborn errors of metabolism using microbiological and enzymatic analytical methods. 20 years after its introduction, advanced mass spectrometers and new soft ionization techniques have permitted the coupling of liquid chromatography with MS and tandem MS (MS/MS) and since the 1990s, DBS analysis by LC-MS/MS expanded screening to many inborn errors of metabolism simultaneously. Recently, DBS-LC-MS/MS analysis has been used in other fields such as pharmacology, toxicology and forensic sciences. Today, new ambient ionization techniques, coupled to MS, directly desorb/ionize molecules from solid samples. This presents new opportunities for the in situ analysis of DBS. Most likely, ambient MS methods will be used to analyze DBS, increasing the clinical applications of MS within the next 10 years.

Direct analysis of sterols from dried plasma/blood spots by an atmospheric pressure thermal desorption chemical ionization mass spectrometry (APTDCI-MS) method for a rapid screening of Smith-Lemli-Opitz syndrome

Here is proposed a rapid and sensitive method involving atmospheric pressure thermal desorption chemical ionization mass spectrometry (APTDCI-MS) for specific laboratory screening of the Smith-Lemli-Opitz syndrome (SLOS), an inherited defect of cholesterol biosynthesis. Biochemical findings in the blood of SLOS patients are low cholesterol (Chol), high 7- and 8-dehydrocholesterol (DHCs) levels and high DHCs/Chol ratios. The APTDCI proposed method is able to ionize sterols for qualitative and quantitative analysis directly from dried plasma/blood spots. Critical APTDCI parameters--desolvation gas flow and temperature--were optimized analyzing Chol, 7-DHC and cholesteryl stearate standards spotted onto a glass slide acquiring the full scan spectra in positive ion mode. Chol levels in dried plasma spots of unaffected controls (n = 23) obtained by the proposed method were compared with those of the enzymatic method (y = 0.9166x + 0.3811; r = 0.8831) while Chol and DHCs of SLOS patients (n = 9) were compared with the gas chromatography flame ionization detection (GC-FID) method (y = 0.8214x + 0.7388; r = 0.8288). The APTDCI-MS method is also able to differentiate normal from SLOS samples directly analyzing whole blood and washed red cells spotted on paper. In conclusion, the intrinsic analytical high-throughput of APTDCI-MS method for sterol analysis could be useful to screen SLO syndrome.

Evaluation of mobile phase, ion pairing, and temperature influence on an HILIC‐MS/MS method for L‐arginine and its dimethylated derivatives detection

Asymmetric N(G),-N(G)-dimethylarginine (ADMA) increases in diseases such as renal failure, diabetes mellitus, and hypercholesterolemia. The feasibility and utility of a hydrophilic interaction chromatography (HILIC) method for the separation of free L-arginine (Arg), ADMA, and symmetric N(G),-N(G)-dimethylarginine (SDMA) on a typical silica column were explored and the impact of some experimental parameters on the chromatographic behavior of these analytes was investigated. The effect of water and TFA content in mobile phase and of column temperature was investigated during the development of a fast and simple HILIC-MS/MS method that might be suitable for the quantification of free Arg, ADMA, and SDMA in plasma for routine analysis. Our results show that a good compromise between efficiency and peak shape with acceptable retention and total chromatographic run time is achieved using an ACN/water (90:10) mobile phase with TFA% as additive ranging from 0.015 to 0.025% and column temperature ranging from 25 to 30 degrees C.

Precursor ion scan profiles of acylcarnitines by atmospheric pressure thermal desorption chemical ionization tandem mass spectrometry

The fatty acyl esters of L-carnitine (acylcarnitines) are useful biomarkers for the diagnosis of some inborn errors of metabolism analyzed by liquid chromatography/tandem mass spectrometry. In this study the acylcarnitines were analyzed by atmospheric pressure thermal desorption chemical ionization using a commercial tandem mass spectrometer (APTDCI-MS/MS). The method is based on the precursor ion scan mode determination of underivatized acylcarnitines desorbed from samples by a hot desolvation gas flow and ionized by a corona pin discharge. During desorption/ionization step the temperature induces the degradation of acylcarnitines; nevertheless, the common fragment to all acylcarnitines [MH-59](+) is useful for analyzing their profile. APTDCI parameters, including angle of collection and incidence, gas flows and temperatures, were optimized for acylcarnitines. The experiments were performed drying 2 microL of an equimolar mixture of acylcarnitine standards on a glass slide. The specificity was evaluated by comparing product ion spectra and the precursor ion spectra of 85 m/z of acylcarnitines obtained by the APTDCI method and by electrospray ionization flow injection analysis (ESI-FIA). The method was also employed to analyze acylcarnitines extracted from a pathological dried blood spot and a control. The method enables analysis of biological samples and recognition of some acylcarnitines that are diagnostic markers of inherited metabolic diseases. The intrinsic high-throughput analysis of the ambient desorption ionization methods offers a new opportunity either for its potential application in clinical chemistry and for the expanded screening of some inborn errors of metabolism.

Orotic acid quantification in dried blood spots and biological fluids by hydrophilic interaction liquid chromatography tandem mass spectrometry

Orotic acid (ORA) is an intermediate metabolite in the pathway of pyrimidine nucleotides; its urinary excretion is useful to diagnose the hereditary orotic aciduria and some hyperammonemic inherited defects of urea cycle enzymes and amino acid transporters. ORA analysis is based on stable isotope dilution by GC-MS or LC-MS/MS methods. We developed a fast assay that measures the ORA in dried blood spots (DBS), plasma and urine using hydrophilic interaction LC-MS/MS. Within- and between-day analytical imprecision (CV%) of three quality control levels, in plasma, DBS and urine, ranged from 0.8 to 14.1%, while the inaccuracy ranged from -13.5 to 9.4%. In healthy children (n=20), ORA concentrations were less than 0.69 microM in plasma, less than 0.82 microM in DBS and from 0.2 to 1.4 mmol/mol of creatinine in urine. A patient with citrullinemia showed ORA levels of 133 microM in plasma and 39 microM in DBS. A patient with hyperammonemia-hyperornithinemia-homocitrullinemia (HHH) syndrome presented a urinary ORA level of 9.1 mmol/mol of creatinine. The method is potentially able to discriminate affected patients from reference subjects; the clinical validation should be expanded on a higher number of patients.

Profiling of acylcarnitines and sterols from dried blood or plasma spot by atmospheric pressure thermal desorption chemical ionization (APTDCI) tandem mass spectrometry

Free carnitine and acylcarnitines play an important role in the metabolism of fatty acids. Sterols are structural lipids found in the membranes of many eukaryotic cells, and they also have functional roles such as the regulation of membrane permeability and fluidity, activity of membrane-bound enzymes and signals transduction. Abnormal profiles of these compounds in biological fluids may be useful markers of metabolic changes. In this review, we describe the subset of the lipidome represented by acylcarnitines and sterols, and we summarize how these compounds have been analyzed in the past. Over the last 50years, lipid mass spectrometry (MS) has evolved to become one of the most useful techniques for metabolic analysis. Today, the introduction of new ambient ionization techniques coupled to MS (AMS), which are characterized by the direct desorbing/ionizing of molecules from solid samples, is generating new possibilities for in situ analysis. Recently, we developed an AMS approach called APTDCI to desorb/ionize using a heated gas flow and an electrical discharge to directly analyze sterols and indirectly investigate acylcarnitines in dried blood or plasma spot samples. Here, we also describe the APTDCI method and some of its clinical applications, and we underline the common complications and issues that remain to be resolved.

Determination of dimethylarginine levels in rats using HILIC-MS/MS: An in vivo microdialysis study

Nitric oxide (NO) is one of the most important mediators and neurotransmitters and its levels change under pathological conditions. NO production may be regulated by endogenous nitric oxide synthase (NOS) inhibitors, in particular asymmetric dimethylarginine (ADMA). Most of the interest is focused on ADMA, since this compound is present in plasma and urine and accumulation of ADMA has been described in many disease states but little is known about cerebrospinal fluid (CSF) concentrations of this compound and of its structural isomer symmetric dimethylarginine (SDMA). To determine the levels of methylarginines, we here present a new hydrophilic interaction chromatography (HILIC)-MS/MS method for the precise determination of these substances in CSF from microdialysis samples of rat prefrontal cortex (PFC). The method requires only minimal sample preparation and features isotope-labelled internal standards.

Evaluation of Seasonal Variability of Toxic and Essential Elements in Urine Analyzed by Inductively Coupled Plasma Mass Spectrometry

Human exposure to elements is a process difficult to control and monitor. Studies on this topic usually rely on single spot urine sample to assess exposure, with the risk of ignoring variability over a longer period. In this work, we measured the urinary excretion of toxic and essential elements during 1xa0year with the overall goal of exploring the variability caused by seasonality on their concentration. Seven participants were recruited, and first morning urines were collected three times for each season, during November, January, April and July. Participants followed the same balanced diet during the week of collection. We then monitored nineteen essential and toxic elements in urines by inductively coupled plasma mass spectrometry. Unsupervised multivariate statistical analysis separated samples collected during summer from the ones collected during other seasons. Twelve elements had a significant seasonal variation (ANOVA test, pxa0<xa00.05) and their levels resulted increased during summer. These elements were both contaminants, such as Ni, Hg, Cd and Tl, and essential elements such as Se and Cu. However, none of these elements was detected at toxic concentration. In this study, we point out for the first time the variability of urine element concentration due to seasonality and we propose that the season of collection should be considered when providing urinary reference values of elements.

Unbiased Lipidomics and Metabolomics of Human Brain Samples

Mass spectrometry (MS)-based lipidomics and metabolomics approaches have been used to discover new diagnostic and therapeutic targets of neurodegenerative disorders. Here, we describe a protocol to conduct an integrated metabolomics and lipidomics profiling of postmortem brains of frozen tissue samples from clinically characterized patients and age-matched controls. Metabolites and lipids can be extracted from each brain tissue sample, using a biphasic liquid/liquid extraction method. An unbiased liquid chromatography MS-based lipidomics and metabolomics workflows allows to screen for the content and composition of lipids and polar metabolites for each brain tissue. Data processing and statistical analysis are then used to compare the molecular content of all the samples, grouping them into cluster based on molecular similarities. The final results highlight classes of metabolites and biochemical pathways that are altered in brain samples from diseased brains compared to those from healthy subjects, helping to generate novel hypotheses on their mechanistic and functional significance.