Adaptive Modelling of Mutated FMO3 Enzyme Could Unveil Unexplored Scenarios Linking Variant Haplotypes to TMAU Phenotypes.

BACKGROUND: Trimethylaminuria (TMAU) is a rare genetic disease characterized by the accumulation of trimethylamine (TMA) and its subsequent excretion trough main body fluids, determining the characteristic fish odour in affected patients. We realized an experimental study to investigate the role of several coding variants in the causative gene FMO3, that were only considered as polymorphic or benign, even if the available literature on them did not functionally explain their ineffectiveness on the encoded enzyme. METHODS: Mutational analysis of 26 TMAU patients was realized by Sanger sequencing. Detected variants were, subsequently, deeply statistically and in silico characterized to determine their possible effects on the enzyme activity. To achieve this goal, a docking prediction for TMA/FMO3 and an unbinding pathway study were performed. Finally, a TMAO/TMA urine quantification by 1H-NMR spectroscopy was performed to support modelling results. RESULTS: The FMO3 screening of all patients highlighted the presence of 17 variants distributed in 26 different haplotypes. Both non-sense and missense considered variants might impair the enzymatic kinetics of FMO3, probably reducing the interaction time between the protein catalytic site and TMA, or losing the wild-type binding site. CONCLUSIONS: Even if further functional assays will confirm our predictive results, considering the possible role of FMO3 variants with still uncertain effects, might be a relevant step towards the detection of novel scenarios in TMAU etiopathogenesis.

Quality consideration for the validation of urine TMA and TMAO measurement by nuclear magnetic resonance spectroscopy in Fish Odor Syndrome.

OBJECTIVES: Trimethylaminuria, also known as Fish Odor Syndrome (FOS), is a condition characterized by the presence of high concentrations of trimethylamine (TMA) in urine, sweat and expired air of affected patients. Diagnosis of this benign but unpleasant disease is mainly based on clinical presentation and assessment of TMA and its metabolite, TMAO (trimethylamine-N-oxide), concentrations in urine of patients. MATERIAL AND METHODS: We here described the validation of an analytical method for measurement of TMA and TMAO in urine using nuclear magnetic resonance (NMR) according to the specifications of the ISO 15189 norm. We used a fast validation protocol, based exactitude profile method, enabling to determine accuracy, intra and inter-day precision from a limited number of samples. RESULTS: The linearity was established from 2.5 to 100 mg/L for TMA measurement and from 10 to 1000 mg/L for TMAO measurement, with good analytical performances i.e. accuracy, intra and inter-day precision. We also report a case diagnose for FOS from this method. CONCLUSIONS: This method validation ensures the robustness of NMR in routine use for diagnosis of trimethylaminuria, as part of the reference center for inherited metabolic diseases at the Tours hospital.

Targeted urine metabolomics with a graphical reporting tool for rapid diagnosis of inborn errors of metabolism.

The current diagnostic work-up of inborn errors of metabolism (IEM) is rapidly moving toward integrative analytical approaches. We aimed to develop an innovative, targeted urine metabolomics (TUM) screening procedure to accelerate the diagnosis of patients with IEM. Urinary samples, spiked with three stable isotope-labeled internal standards, were analyzed for 258 diagnostic metabolites with an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) configuration run in positive and negative ESI modes. The software automatically annotated peaks, corrected for peak overloading, and reported peak quality and shifting. Robustness and reproducibility were satisfactory for most metabolites. Z-scores were calculated against four age-group-matched control cohorts. Disease phenotypes were scored based on database metabolite matching. Graphical reports comprised a needle plot, annotating abnormal metabolites, and a heatmap showing the prioritized disease phenotypes. In the clinical validation, we analyzed samples of 289 patients covering 78 OMIM phenotypes from 12 of the 15 society for the study of inborn errors of metabolism (SSIEM) disease groups. The disease groups include disorders in the metabolism of amino acids, fatty acids, ketones, purines and pyrimidines, carbohydrates, porphyrias, neurotransmitters, vitamins, cofactors, and creatine. The reporting tool easily and correctly diagnosed most samples. Even subtle aberrant metabolite patterns as seen in mild multiple acyl-CoA dehydrogenase deficiency (GAII) and maple syrup urine disease (MSUD) were correctly called without difficulty. Others, like creatine transporter deficiency, are illustrative of IEM that remain difficult to diagnose. We present TUM as a powerful diagnostic screening tool that merges most urinary diagnostic assays expediting the diagnostics for patients suspected of an IEM.

Rare genetic variants affecting urine metabolite levels link population variation to inborn errors of metabolism.

Metabolite levels in urine may provide insights into genetic mechanisms shaping their related pathways. We therefore investigate the cumulative contribution of rare, exonic genetic variants on urine levels of 1487 metabolites and 53,714 metabolite ratios among 4864 GCKD study participants. Here we report the detection of 128 significant associations involving 30 unique genes, 16 of which are known to underlie inborn errors of metabolism. The 30 genes are strongly enriched for shared expression in liver and kidney (odds ratio = 65, p-FDR = 3e-7), with hepatocytes and proximal tubule cells as driving cell types. Use of UK Biobank whole-exome sequencing data links genes to diseases connected to the identified metabolites. In silico constraint-based modeling of gene knockouts in a virtual whole-body, organ-resolved metabolic human correctly predicts the observed direction of metabolite changes, highlighting the potential of linking population genetics to modeling. Our study implicates candidate variants and genes for inborn errors of metabolism.

[Recommendations for aminoacids chromatography analysis]. / Recommandations concernant l'analyse de la chromatographie des acides aminés.

Biochemical diagnosis of hereditary metabolic diseases requires the detection and simultaneous identification of a large number of compounds, hence the interest in metabolic profiles. Amino acid chromatography allows the identification and quantification of more than forty compounds. As part of the accreditation process for medical biology examinations according to standard NF EN ISO 15189, the group from SFEIM recommends an approach to accredit amino acid chromatography. Validation parameters and recommendations are discussed in this specific framework.

[Recommendations for acylcarnitine profile analysis]. / Recommandations concernant l'analyse du profil des acylcarnitines.

Biochemical diagnosis of hereditary metabolic diseases requires the detection and simultaneous identification of a large number of compounds, hence the interest in metabolic profiles. Acylcarnitine profile allows the identification and quantification of more than thirty compounds. As part of the accreditation process for medical biology examinations according to standard NF EN ISO 15189, the group from SFEIM recommends an approach to accredit acylcarnitine profile. Validation parameters and recommendations are discussed in this specific framework.

Coincidence of 3-methylglutaconic aciduria and duplication 5q - a case report and literature review.

3-methylglutaconic aciduria includes a heterogeneous group of inborn errors of metabolism. The disease may have various clinical presentations, as can duplication 5q. We present the case of a 13-year-old boy with 3-methylglutaconic aciduria and duplication 5q. The main symptoms included myopathy, weakness, spastic paresis intensified mostly in the lower limbs, and intellectual disability. Additional studies showed elevated levels of 3-methylglutaconic acid in urine and ammonia in plasma. A duplication in region 5q23.3q31.1 was found in array-based comparative genomic hybridization. Next-generation sequencing did not reveal any pathological mutation. On the basis of the clinical picture and the results of biochemical and genetic tests 3-methylglutaconic aciduria type IV with duplication 5q was diagnosed.

Delayed appearance of 3-methylglutaconic aciduria in neonates with early onset metabolic cardiomyopathies: A potential pitfall for the diagnosis.

Infantile onset cardiomyopathies are highly heterogeneous with several phenocopies compared with adult cardiomyopathies. Multidisciplinary management is essential in determining the underlying etiology in children's cardiomyopathy. Elevated urinary excretion of 3-methylglutaconic acid (3-MGA) is a useful tool in identifying the etiology in some metabolic cardiomyopathy. Here, we report the delayed appearance of 3-MGA-uria, between 6 and 18 months in three patients (out of 100 childhood onset cardiomyopathy) with neonatal onset cardiomyopathy, secondary to TMEM70 mutations and TAZ mutations (Barth syndrome), in whom extensive metabolic investigations, performed in the first weeks of life, did not display 3-MGA-uria. Serial retrospective evaluations showed full characteristic features of TMEM70 and TAZ mutations (Barth syndrome) in these three patients, including a clearly abnormal monolysocardiolipin/cardiolipin ratio in the two Barth syndrome patients. Serially repeated metabolic investigations finally discovered the 3-MGA-uria biomarker in all three patients between the age of 6 and 18 months. Our observation provides novel insights into the temporal appearance of 3-MGA-uria in TMEM70 and TAZ mutations (Barth syndrome) and focus the importance of multidisciplinary management and careful evaluation of family history and red flag signs for phenocopies in infantile onset cardiomyopathies.

NMR-based newborn urine screening for optimized detection of inherited errors of metabolism.

Inborn errors of metabolism (IEMs) are rare diseases produced by the accumulation of abnormal amounts of metabolites, toxic to the newborn. When not detected on time, they can lead to irreversible physiological and psychological sequels or even demise. Metabolomics has emerged as an efficient and powerful tool for IEM detection in newborns, children, and adults with late onset. In here, we screened urine samples from a large set of neonates (470 individuals) from a homogeneous population (Basque Country), for the identification of congenital metabolic diseases using NMR spectroscopy. Absolute quantification allowed to derive a probability function for up to 66 metabolites that adequately describes their normal concentration ranges in newborns from the Basque Country. The absence of another 84 metabolites, considered abnormal, was routinely verified in the healthy newborn population and confirmed for all but 2 samples, of which one showed toxic concentrations of metabolites associated to ketosis and the other one a high trimethylamine concentration that strongly suggested an episode of trimethylaminuria. Thus, a non-invasive and readily accessible urine sample contains enough information to assess the potential existence of a substantial number (>70) of IEMs in newborns, using a single, automated and standardized 1H- NMR-based analysis.

Urinary 2,8-dihydroxyadenine excretion in patients with adenine phosphoribosyltransferase deficiency, carriers and healthy control subjects.

BACKGROUND: Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder of adenine metabolism that results in excessive urinary excretion of the poorly soluble 2,8-dihydroxyadenine (DHA), leading to kidney stones and chronic kidney disease. The purpose of this study was to assess urinary DHA excretion in patients with APRT deficiency, heterozygotes and healthy controls, using a recently developed ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) assay. METHODS: Patients enrolled in the APRT Deficiency Registry and Biobank of the Rare Kidney Stone Consortium (http://www.rarekidneystones.org/) who had provided 24-h and first-morning void urine samples for DHA measurement were eligible for the study. Heterozygotes and healthy individuals served as controls. Wilcoxon-Mann-Whitney test was used to compare 24-h urinary DHA excretion between groups. Associations were examined using Spearman's correlation coefficient (rs). RESULTS: The median (range) 24-h urinary DHA excretion was 138 (64-292) mg/24 h and the DHA-to-creatinine (DHA/Cr) ratio in the first-morning void samples was 13 (4-37) mg/mmol in APRT deficiency patients who were not receiving xanthine oxidoreductase inhibitor therapy. The 24-h DHA excretion was highly correlated with the DHA/Cr ratio in first-morning void urine samples (rs = 0.84, p < .001). DHA was detected in all urine samples from untreated patients but not in any specimens from heterozygotes and healthy controls. CONCLUSIONS: High urinary DHA excretion was observed in patients with APRT deficiency, while urine DHA was undetectable in heterozygotes and healthy controls. Our results suggest that the UPLC-MS/MS assay can be used for diagnosis of APRT deficiency.

Quantification of Underivatized Amino Acids on Dry Blood Spot, Plasma, and Urine by HPLC-ESI-MS/MS.

Enzyme deficiencies in amino acid metabolism may increase the levels of a single or several compounds in physiological fluids becoming diagnostically significant biomarkers for one or a group of metabolic disorders. Therefore, it is important to monitor a wide range of free amino acids simultaneously and to quantify them. This is time consuming if we use the classical methods and, especially now that many laboratories have introduced Newborn Screening Programs for the semiquantitative analysis, the detection and quantification of some amino acids need to be performed in a short time to reduce the rate of false positives.We have modified the stable isotope dilution HPLC-ESI-MS/MS method previously described by Qu (Qu et al., 2002) for a more rapid, robust, sensitive, and specific detection and quantification of underivatized amino acids. The modified method reduces the time of analysis to 10 min with very good reproducibility of retention times and a better separation of the metabolites and their isomers.The omission of the derivatization step, enabled to achieve some important advantages: fast and simple sample preparation, exclusion of artifacts, and interferences. The use of this technique is highly sensitive and specific and allowed to monitor 40 underivatized amino acids including the key isomers and quantification of some of them, in order to cover many diagnostically important intermediates of metabolic pathways.We propose this HPLC-ESI-MS/MS method for underivatized amino acids as a support for the newborn screening as secondary test using the same dried blood spots for a more accurate and specific examination in case of suspected metabolic diseases. In this way we avoid plasma collection from the patient as it normally occurs, reducing anxiety for the parents and further costs for analysis.The same method was validated and applied also to plasma and urine samples with good reproducibility, accuracy, and precision. The fast run time, the feasibility of high sample throughput, and the small amount of sample required make this method very suitable for routine analysis in the clinical setting.

Trimetilaminuria: tres mutaciones distintas en una sola familia / Trimethylaminuria: three different mutations in a single family

Introducción: La trimetilaminuria primaria (TMAP) o síndrome de olor a pescado es una metabulopatía genética caracterizada por acumulo en secreciones corporales de un compuesto muy volátil, la trimetilamina.Caso clínico: paciente sana de 8 meses de edad que tras la introducción del pescado inicia mal olor corporal que no desaparece con el baño, la madre acude repetidamente al pediatra sin identificar el trastorno, el diagnóstico se retrasa hasta los 3 años de edad en que por insistencia materna, es derivada a nuestra Unidad Hospitalaria, realizándose pruebas genéticas diagnósticas y posibilitando el diagnostico paterno que había pasado desapercibido durante 35 años, detectándose 3 mutaciones distintas en la familia.Discusión: la trimetilaminuria primaria es una enfermedad de causa genética con sintomatología concreta de mal olor corporal que puede pasar desapercibida durante muchos años. Una sospecha clínica adecuada y la solicitud de pruebas complementarias, permite su diagnóstico y facilita su manejo clínico

Background: primary trimethylaminuria or fish odor syndrome is a genetic metabolopathy characterized by the accumulation of trimethylamine, a very volatile compound in body secretions. Case report: we present the case of a healthy 8-month-old patient who, after the introduction of fish in the diet, starts a bad body odor that does not disappear with bathing. The mother visits the pediatrician repeatedly but no disorder is identified. The diagnosis is delayed until the patient is three years old. Due to maternal insistence, the patient is referred to our hospital unit, where genetic diagnostic tests are performed, enabling the paternal diagnosis that had gone unnoticed for 35 years and detecting three different mutations in the family. Discussion: primary trimethylaminuria is a genetic disease with specific symptomatology of bad body odor that can go unnoticed for many years. An adequate clinical suspicion and the request of adequate complementary tests allow its diagnosis and facilitate its clinical management

[Trimethylaminuria: three different mutations in a single family]. / Trimetilaminuria: tres mutaciones distintas en una sola familia.

INTRODUCTION: Background: primary trimethylaminuria or fish odor syndrome is a genetic metabolopathy characterized by the accumulation of trimethylamine, a very volatile compound in body secretions. Case report: we present the case of a healthy 8-month-old patient who, after the introduction of fish in the diet, starts a bad body odor that does not disappear with bathing. The mother visits the pediatrician repeatedly but no disorder is identified. The diagnosis is delayed until the patient is three years old. Due to maternal insistence, the patient is referred to our hospital unit, where genetic diagnostic tests are performed, enabling the paternal diagnosis that had gone unnoticed for 35 years and detecting three different mutations in the family. Discussion: primary trimethylaminuria is a genetic disease with specific symptomatology of bad body odor that can go unnoticed for many years. An adequate clinical suspicion and the request of adequate complementary tests allow its diagnosis and facilitate its clinical management.

INTRODUCCIÓN: Introducción: La trimetilaminuria primaria (TMAP) o síndrome de olor a pescado es una metabulopatía genética caracterizada por acumulo en secreciones corporales de un compuesto muy volátil, la trimetilamina.Caso clínico: paciente sana de 8 meses de edad que tras la introducción del pescado inicia mal olor corporal que no desaparece con el baño, la madre acude repetidamente al pediatra sin identificar el trastorno, el diagnóstico se retrasa hasta los 3 años de edad en que por insistencia materna, es derivada a nuestra Unidad Hospitalaria, realizándose pruebas genéticas diagnósticas y posibilitando el diagnostico paterno que había pasado desapercibido durante 35 años, detectándose 3 mutaciones distintas en la familia.Discusión: la trimetilaminuria primaria es una enfermedad de causa genética con sintomatología concreta de mal olor corporal que puede pasar desapercibida durante muchos años. Una sospecha clínica adecuada y la solicitud de pruebas complementarias, permite su diagnóstico y facilita su manejo clínico.

A case of dihydropyrimidinase deficiency incidentally detected by urine metabolome analysis.

Dihydropyrimidinase deficiency is a rare autosomal recessive disease affecting the second step of pyrimidine degradation. It is caused by mutations in the DPYS gene. Only approximately 30 cases have been reported to date, with a phenotypical variability ranging from asymptomatic to severe neurological illness. We report a case of dihydropyrimidinase deficiency incidentally detected by urine metabolome analysis. Gas chromatography-mass spectrometry-based urine metabolomics demonstrated significant elevations of dihydrouracil and dihydrothymine, which were subsequently confirmed by a quantitative analysis using liquid chromatography-tandem mass spectrometry. Genetic testing of the DPYS gene revealed two mutations: a novel mutation (c.175G > T) and a previously reported mutation (c.1469G > A). Dihydropyrimidinase deficiency is probably underdiagnosed, considering its wide phenotypical variability, nonspecific neurological presentations, and an estimated prevalence of 2/20,000. As severe 5-fluorouracil-associated toxicity has been reported in patients and carriers of congenital pyrimidine metabolic disorders, urinary pyrimidine analysis should be considered for those who will undergo 5-fluorouracil treatment.

Xanthine urolithiasis: Inhibitors of xanthine crystallization.

OBJECTIVE: To identify in vitro inhibitors of xanthine crystallization that have potential for inhibiting the formation of xanthine crystals in urine and preventing the development of the renal calculi in patients with xanthinuria. METHODS: The formation of xanthine crystals in synthetic urine and the effects of 10 potential crystallization inhibitors were assessed using a kinetic turbidimetric system with a photometer. The maximum concentration tested for each compound was: 20 mg/L for 3-methylxanthine (3-MX); 40 mg/L for 7-methylxanthine (7-MX), 1-methylxanthine (1-MX), theobromine (TB), theophylline, paraxanthine, and caffeine; 45 mg/L for 1-methyluric acid; 80 mg/L for 1,3-dimethyluric acid; and 200 mg/L for hypoxanthine. Scanning electron microscopy was used to examine the morphology of the crystals formed when inhibitory effects were observed. RESULTS: Only 7-MX, 3-MX, and 1-MX significantly inhibited xanthine crystallization at the tested concentrations. Mixtures of inhibitors had an additive effect rather than a synergistic effect on crystallization. CONCLUSION: Two of the inhibitors identified here-7-MX and 3-MX-are major metabolites of TB. In particular, after TB consumption, 20% is excreted in the urine as TB, 21.5% as 3-MX, and 36% as 7-MX. Thus, consumption of theobromine could protect patients with xanthinuria from the development of renal xanthine calculi. Clinical trials are necessary to demonstrate these effects in vivo.

Hereditary xanthinuria is not so rare disorder of purine metabolism.

Hereditary xanthinuria (type I) is caused by an inherited deficiency of the xanthine oxidorectase (XDH/XO), and is characterized by very low concentration of uric acid in blood and urine and high concentration of urinary xanthine, leading to urolithiasis. Type II results from a combined deficiency of XDH/XO and aldehyde oxidase. Patients present with hematuria, renal colic, urolithiasis or even acute renal failure. Clinical symptoms are the same for both types. In a third type, clinically distinct, sulfite oxidase activity is missing as well as XDH/XO and aldehyde oxidase. The prevalence is not known, but about 150 cases have been described so far. Hypouricemia is sometimes overlooked, that´s why we have set up the diagnostic flowchart. This consists of a) evaluation of uric acid concentrations in serum and urine with exclusion of primary renal hypouricemia, b) estimation of urinary xanthine, c) allopurinol loading test, which enables to distinguish type I and II; and finally assay of xanthine oxidoreductase activity in plasma with molecular genetic analysis. Following this diagnostic procedure we were able to find first patients with hereditary xanthinuria in our Czech population. We have detected nine cases, which is one of the largest group worldwide. Four patients were asymptomatic. All had profound hypouricemia, which was the first sign and led to referral to our department. Urinary concentrations of xanthine were in the range of 170-598 mmol/mol creatinine (normal < 30 mmol/mol creatinine). Hereditary xanthinuria is still unrecognized disorder and subjects with unexplained hypouricemia need detailed purine metabolic investigation.

[Trimethylaminuria: 'Help, my child smells of fish!'] / Trimethylaminurie: 'Help, mijn kind ruikt naar vis!'.

BACKGROUND: Trimethylaminuria is caused by a functional enzyme defect and is usually congenital. This metabolic disease is characterised by body odour resembling fish. CASE DESCRIPTION: A 7-year-old boy was referred with abnormal body odour, which his mother described as resembling fish. This odour caused mainly social problems. Because of the characteristic odour trimethylaminuria was considered. Further metabolic investigations showed a high concentration of trimethylamine in the urine, consistent with this diagnosis. CONCLUSION: Trimethylaminuria is rare, but due to its psychological and social impact it is important that it is recognised. Although bad body odour is seldom a manifestation of a metabolic disease, it should always be included in the differential diagnosis.

Urinary sulphatoxymelatonin as a biomarker of serotonin status in biogenic amine-deficient patients.

Melatonin is synthesized from serotonin and it is excreted as sulphatoxymelatonin in urine. We aim to evaluate urinary sulphatoxymelatonin as a biomarker of brain serotonin status in a cohort of patients with mutations in genes related to serotonin biosynthesis. We analized urinary sulphatoxymelatonin from 65 healthy subjects and from 28 patients with genetic defects. A total of 18 patients were studied: 14 with autosomal dominant and recessive guanosine triphosphate cyclohydrolase-I deficiency; 3 with sepiapterin reductase deficiency; and 1 with aromatic L-amino acid decarboxylase deficiency. Further 11 patients were studied after receiving serotoninergic treatment (serotonin precursors, monoamine oxidase inhibitors, selective serotonin re-uptake inhibitors): 5 with aromatic L-amino acid decarboxylase deficiency; 1 with sepiapterin reductase deficiency; 3 with dihydropteridine reductase deficiency; and 2 with 6-pyruvoyltetrahydropterin synthase deficiency. Among the patients without therapy, 6 presented low urinary sulphatoxymelatonin values, while most of the patients with guanosine triphosphate cyclohydrolase-I deficiency showed normal values. 5 of 11 patients under treatment presented low urine sulphatoxymelatonin values. Thus, decreased excretion of sulphatoxymelatonin is frequently observed in cases with severe genetic disorders affecting serotonin biosynthesis. In conclusion, sulphatoxymelatonin can be a good biomarker to estimate serotonin status in the brain, especially for treatment monitoring purposes.

Screening Mentally Retarded Children for Inborn Errors of Metabolism.

BACKGROUND: Most inborn errors of metabolism result in mental retardation and death due to accumulation of abnormal metabolites in the tissues. The presence of abnormal metabolites in the urine of mentally retarded individuals has been used worldwide for detection of inborn errors of metabolism. The purpose of the study is to determine the prevalence of inborn error of metabolism in mentally retarded children. METHODS: Random urine samples were collected from mentally retarded children at two institutes in Kathmandu, and also from 60 normal children from Duwakot, Nepal after obtaining consent from their parents. Urine was then tested for the presence of amino acids, keto-acids, mucopolysaccharides, fructose, glucose and protein using simple qualitative color reactions in the laboratory. RESULTS: The tests detected eight cases of Phenylketonuria, which turned out to be false positive on paper chromatography. Three cases of presence of ketone bodies (acetoacetate), ten cases of α-ketoaciduria, two cases of mucopolysaccharidosis and twelve cases of fructosuria amongst the sixty-two urine samples were also found. CONCLUSIONS: Certain aminoacidurias, ketoacidurias and mucopolysaccharidoses might be present in the Nepalese population. Within consideration of errors, the samples tested positive should be evaluated by a higher end method to confirm the utility of these simple and cheap chemical tests.

Molecular identification in metabolomics using infrared ion spectroscopy.

Small molecule identification is a continually expanding field of research and represents the core challenge in various areas of (bio)analytical science, including metabolomics. Here, we unequivocally differentiate enantiomeric N-acetylhexosamines in body fluids using infrared ion spectroscopy, providing orthogonal identification of molecular structure unavailable by standard liquid chromatography/high-resolution tandem mass spectrometry. These results illustrate the potential of infrared ion spectroscopy for the identification of small molecules from complex mixtures.