Flow Injection Analysis-tandem Mass Spectrometry Research Articles

The determination of mycotoxins using isotope-coded labeling and dispersive liquid–liquid microextraction by high throughput flow injection analysis coupled with tandem mass spectrometry

A novel and rapid method for the determination of ochratoxin A (OTA), funmonisin B1 (FB1) and zearalenone (ZEN) in legumes samples by high throughput flow injection analysis-tandem mass spectrometry (FIA-MS/MS) in conjunction with isotope-coded labeling (ICD) coupled with dispersive liquid–liquid microextraction (DLLME) was developed. The samples were extracted by a known QuEChERS method (Quick, Easy, Cheap, Effective, Rugged, Safe). The extracted components and mycotoxin standards were, respectively, labeled by d0-10-methyl-acridone-2-sulfonyl chloride and its deuterated counter part (d3-MASC). Subsequently, the mixed light-/heavy-labeled solutions were enriched and purified by DLLME method and corresponding extracts were directly used for FIA-MS/MS analysis. The FIA-MS/MS method coupled with ICD-coded labeling technique for the determination of mycotoxins in legume samples could be achieved within 2 min, which was much suitable for high throughput screening analysis. Meanwhile, the labeled mycotoxins with d3-MASC as internal standards could effectively eliminate matrix interference and ensure the accuracy and precision of mass spectrometry analysis. The limits of detection (LODs) and the limits of quantification (LOQs) ranged from 0.29 to 0.42 μg/L and 0.85 to 1.30 μg/L, respectively. The recoveries were 66.9 %–85.2 %, and the daily and inter day RSDs were within the range of 2.0 %–4.9 % and 2.4 %–5.0 %, respectively. The established method could be feasibility applied to the determination of mycotoxins in legume samples with satisfactory results.

Multiplexing Homocysteine into First-Tier Newborn Screening Mass Spectrometry Assays Using Selective Thiol Derivatization.

Classical homocystinuria (HCU) results from deficient cystathionine β-synthase activity, causing elevated levels of Met and homocysteine (Hcy). Newborn screening (NBS) aims to identify HCU in pre-symptomatic newborns by assessing Met concentrations in first-tier screening. However, unlike Hcy, Met testing leads to a high number of false-positive and -negative results. Therefore, screening for Hcy directly in first-tier screening would be a better biomarker for use in NBS. Dried blood spot (DBS) quality control and residual clinical specimens were used in analyses. Several reducing and maleimide reagents were investigated to aid in quantification of total Hcy (tHcy). The assay which was developed and validated was performed by flow injection analysis-tandem mass spectrometry (FIA-MS/MS). Interferents of tHcy measurement were identified, so selective derivatization of Hcy was employed. Using N-ethylmaleimide (NEM) to selectively derivatize Hcy allowed interferent-free quantification of tHcy by FIA-MS/MS in first-tier NBS. The combination of tris(2-carboxyethyl)phosphine (TCEP) and NEM yielded significantly less matrix effects compared to dithiothreitol (DTT) and NEM. Analysis of clinical specimens demonstrated that the method could distinguish between HCU-positive, presumptive normal newborns, and newborns receiving total parenteral nutrition. Here we present the first known validated method capable of screening tHcy in DBS during FIA-MS/S first-tier NBS.

Age and gender-specific reference intervals for a panel of lysophosphatidylcholines estimated by tandem mass spectrometry in dried blood spots

Background and objectivesVery long-chain fatty acyl-lysophosphatidylcholines (VLCFA-LysoPCs) are measured in dried blood spots (DBS) for identifying X-linked adrenoleukodystrophy (X-ALD) and other inherited peroxisomal disorders. Our study aimed to establish age- and gender-specific reference intervals for a panel of LysoPCs measured by tandem mass spectrometry in DBS. MethodsLysoPCs (26:0-, 24:0-, 22:0- and 20:0-LysoPCs) were estimated by flow injection analysis-tandem mass spectrometry (FIA-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods in 3.2 mm blood spots of 2689 anonymized, putative normal subjects (1375 males, and 1314 females) aged between 2 days and 65 years. Samples were divided into groups: Neonates (0–1month), Infants (>1m-1year), Children and Adolescents (>1–18years), and Adults (>18years). Reference intervals were determined using the percentile approach and represented as the median with the 1st and 99th percentile lower and upper limits. ResultsThe percentage coefficient of variation (CV) for repeatability assays of internal and external quality control samples were within acceptable limits. Significant differences (P <0.0001, P <0.01) were observed in the concentrations of 26:0-, 24:0-, 22:0- and 20:0-LysoPCs and their ratios, 26:0/22:0-, 24:0/22:0-, 26:0/20:0-and 24:0/20:0-LysoPC in neonates and infants when compared to children, adolescents, and adults. Levels of 26:0-, 24:0- and 22:0-LysoPCs decreased, whereas 20:0-LysoPC increased with age. There were no significant gender-based differences in the concentration of LysoPCs. ConclusionWe established age- and gender-specific reference intervals for a panel of LysoPCs in DBS. These reference values would be helpful when interpreting LysoPC values in DBS during screening for X-ALD and other peroxisomal disorders.

Improving Harmonization and Standardization of Expanded Newborn Screening Results by Optimization of the Legacy Flow Injection Analysis Tandem Mass Spectrometry Methods and Application of a Standardized Calibration Approach.

Newborn screening (NBS) laboratories in the United Kingdom adhere to common protocols based on single analyte cutoff values (COVs); therefore, interlaboratory harmonization is of paramount importance. Interlaboratory variation for screening analytes in UK NBS laboratories ranges from 17% to 59%. While using common stable isotope internal standards has been shown to significantly reduce interlaboratory variation, instrument set-up, sample extraction, and calibration approach are also key factors. Dried blood spot (DBS) extraction processes, instrument set-up, mobile-phase composition, sample introduction technique, and calibration approach of flow injection analysis-tandem mass spectrometry (FIA-MS/MS) methods were optimized. Inter- and intralaboratory variation of methionine, leucine, phenylalanine, tyrosine, isovaleryl-carnitine, glutaryl-carnitine, octanoyl-carnitine, and decanoyl-carnitine were determined pre- and postoptimization, using 3 different calibration approaches. Optimal recovery of analytes from DBS was achieved with a 35-min extraction time and 80% methanol (150 μL). Optimized methodology decreased the mean intralaboratory percentage relative SD (%RSD) for the 8 analytes from 20.7% (range 4.1-46.0) to 5.4% (range 3.0-8.5). The alternative calibration approach reduced the mean interlaboratory %RSD for all analytes from 16.8% (range 4.1-25.0) to 7.1% (range 4.1-11.0). Nuclear magnetic resonance analysis of the calibration material highlighted the need for standardization. The purities of isovaleryl-carnitine and glutaryl-carnitine were 85.13% and 69.94% respectively, below the manufacturer's stated values of ≥98%. For NBS programs provided by multiple laboratories using single analyte COVs, harmonization and standardization of results can be achieved by optimizing legacy FIA-MS/MS methods, adopting a common analytical protocol, and using standardized calibration material rather than internal calibration.

A Simple Flow Injection Analysis-Tandem Mass Spectrometry Method to Reduce False Positives of C5-Acylcarnitines Due to Pivaloylcarnitine Using Reference Ions.

Flow injection analysis–tandem mass spectrometry (FIA-TMS) has been applied in a first-tier test of newborn screening (NBS). Although isovalerylcarnitine (i-C5), which is a diagnostic indicator of isovaleric acidemia (IVA), is isobaric with pivaloylcarnitine (p-C5), 2-methylbutyrylcarnitine, and n-valerylcarnitine, these isomers cannot be distinguished by the FIA-TMS. There are many reports of false positives derived from p-C5 due to the use of pivalate-conjugated antibiotics. In this study, we developed a new FIA-TMS method to distinguish i-C5 and p-C5. We found that the intensity ratio of product ions for i-C5 and p-C5 was different in a certain range even under the same analytical conditions. The product ions with the most distinct differences in ionic intensity between the isomers and the collision energies that produce them were determined to be m/z 246.2 > 187.1 and −15 V, respectively. In addition to the quantification ion, a reference ion was defined, and the similarity of the i-C5 and p-C5 reference ion ratios (i-C5 score and p-C5 score, respectively) were used to estimate which isomer (i-C5 and p-C5) was responsible for elevated C5 acylcarnitine in dried blood spots (DBSs). As a result of analyses of 11 DBS samples derived from pivalate-conjugated antibiotics and four DBS samples from IVA patients using our method, it was found that our method was able to correctly determine the type of C5-acylcarnitine (i-C5 or p-C5) in the DBS samples. Implementation of this new FIA-TMS method into the current NBS protocol will allow for a reduction in false positives in IVA.

A Preliminary Metabolomic Study of Yorkshire Terrier Enteropathy

Perturbations of metabolite profiles in human and canine enteropathies have been reported before. However, data in dogs are scarce and inconsistent. Currently, the metabolite profile in Yorkshire Terrier enteropathy (YTE) and the impact of treatment is unknown. The objective of this study was to investigate the plasma metabolome of 13 Yorkshire Terriers with YTE and compare it to 20 healthy Yorkshire Terriers. Furthermore, we studied the impact of treatment on the metabolome. In this prospective observational study, plasma metabolite profiles were analyzed by flow injection analysis-tandem mass spectrometry (FIA-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a targeted metabolomics kit. Metabolite analysis revealed that YTE is accompanied by changes in lipid and bile acid metabolism. YTE was associated with a significant decrease of long-chain fatty acids (octadecenoic acid, eicosadienoic acid, eicosatrienoic acid) and lower levels of long-chain acylcarnitines (tetradecanoylcarnitine, hexadecanoylcarnitine, hexadecenoylcarnitine, octadecenoylcarnitine) compared with healthy controls. Furthermore, taurodeoxycholic acid, a secondary bile acid, was decreased in plasma from YTE patients. These changes might be breed-specific and might be involved in the pathogenesis of YTE. Interestingly, changes in metabolite levels were not recovered after treatment and differed considerably from healthy controls.

High throughput and very specific screening of anabolic-androgenic steroid adulterants in healthy foods based on stable isotope labelling and flow injection analysis-tandem mass spectrometry with simultaneous monitoring proton adduct ions and chloride adduct ions

In this work, a stable isotope labelling-flow injection analysis-tandem mass spectrometry (SIL-FIA-MS/MS) with simultaneous monitoring [M+H]+ and [M+Cl]− method was developed for very specific and high throughput screening of anabolic-androgenic steroids (AAS) illegally added to healthy foods. Initially, a simple centrifugation step was carried out for liquid samples, and for solid samples, a solid-liquid extraction step was conducted. Afterwards, batch chemical derivatization was carried out. After adding a certain amount of 13C6-3-NPH labelled AAS standards as the internal standards, it can be directly transferred for FIA-MS/MS analysis based on the no MS response characteristics of 3-NPH. The 3-NPH labelled AAS showed dual-polarity property, observing chloride adduct ion ([M+Cl]−) in negative ion mode and proton adduct ion ([M+H]+) in positive ion mode. The average time cost for pretreatment of each sample was less than 1 min by carrying out batch processing. The subsequent FIA-MS/MS detection enabled rapid and high throughput detection. The addition of 13C6-3-NPH-labelled AAS as internal standards can correct the matrix effect to achieve accurate quantitative analysis. The detection sensitivity was also improved by 2–5 folds after 3-NPH labelling. The limits of detection (LODs) in positive MRM mode were in ranges of 0.1–0.3 ng/mL. The validated method with simultaneous monitoring [M+H]+ and [M+Cl]− was validated in the range of 6.0–1000 ng/mL with the linear coefficient (R2) greater than 0.997. Satisfactory recoveries were found to be in ranges of 93.0–108.7%. The intra-day and inter-day RSDs were in the range of 3.5–9.9% and 5.1–14.1%, respectively. No changes in detection sensitivity of the mass spectrometry and no carry-over effects were found after numerous consecutive injections of AAS derivates. Compared with previously reported methods, the proposed method proved accurate, very specific, high throughput with good sensitivity.

Fragmentation of Cannabinoids by Flow Injection Analysis Tandem Mass Spectrometry (FIA-MS/MS).

The analysis of plant material from Cannabis sativa L. has long been targeted on its main psychologically active metabolite, Δ9-tetrahydrocannabinol (THC). In addition to the diverse plant composition and medicinal interest in several cannabinoids, these compounds may also be related to the different characteristics of samples sold illegally. Currently, it is indisputable that other cannabinoids should also be considered in cannabis assays. Mass spectrometry has been used to identify and characterize substances in the most different scenarios, and knowing the analyte fragmentation profile is essential for characterizing samples of diverse origin. In this work, flow injection analysis-tandem mass spectrometry with electrospray ionization (FIA-ESI-MS/MS) in positive and negative modes was used to evaluate the fragmentation profiles of eight cannabinoids commonly found in cannabis samples: THC, tetrahydrocannabinolic acid, Δ8-tetrahydrocannabinol, cannabidiol, cannabidiolic acid, cannabigerol, cannabigerolic acid and cannabinol. By exploring the fragmentation data from mass spectrometry, the samples were classified using a chemometric model of partial least squares discriminant analysis (PLS-DA). When ESI in negative mode is used with adequate collision energies, it is possible to identify differences in the fragmentation of isomers. Based on that, chemometric tools were employed to classify different samples. The PLS-DA applied to FIA-ESI-MS/MS data yielded satisfactory classification. Thus, the results presented can be applied as a preliminary tool in the analysis of unknown samples, guiding more accurate investigations in terms of chemical composition. This study of the cannabinoid fragmentation pattern by flow injection MS showed that cannabinoids can be distinguished by their fragmentation spectra after negative electrospray ionization. Multivariate data analysis (PLS-DA) allowed classification of different cannabis samples.

Development, validation, and clinical application of an FIA-MS/MS method for the quantification of lysophosphatidylcholines in dried blood spots.

BackgroundLysophosphatidylcholine (LPC) plays pivotal roles in several physiological processes and their disturbances are closely associated with various disorders. In this study, we described the development and validation of a reliable and simple flow injection analysis–tandem mass spectrometry (FIA‐MS/MS)‐based method using dried blood spots (DBS) for quantification of four individual LPC (C20:0, C22:0, C24:0, and C26:0).MethodsLysophosphatidylcholines were extracted from 3.2 mm DBS with 85% methanol containing 60 ng/ml internal standard using a rapid (30 min) and simple procedure. The analytes and the internal standard were directly measured by triple quadrupole tandem mass spectrometry in multiple reactions monitoring mode via positive electrospray ionization.ResultsMethod validation results showed good linearity ranging from 50 to 2000 ng/ml for each LPC. Intra‐ and inter‐day precision and accuracy were within the acceptable limits at four quality control levels. Recovery was from 70.5% to 107.0%, and all analytes in DBS were stable under assay conditions (24 h at room temperature and 72 h in autosampler). The validated method was successfully applied to assessment of C20:0‐C26:0LPCs in 1900 Chinese neonates. C26:0‐LPC levels in this study were consistent with previously published values.ConclusionWe propose a simple FIA‐MS/MS method for analyzing C20:0‐C26:0LPCs in DBS, which can be used for first‐tier screening.

Differences of Phenylalanine Concentrations in Dried Blood Spots and in Plasma: Erythrocytes as a Neglected Component for This Observation.

Monitoring phenylalanine (Phe) concentrations is critical for the management of phenylketonuria (PKU). This can be done in dried blood spots (DBS) or in EDTA plasma derived from capillary or venous blood. Different techniques are used to measure Phe, the most common being flow-injection analysis tandem mass spectrometry (FIA-MS-MS) and ion exchange chromatography (IEC). Significant differences have been reported between Phe concentrations in various sample types measured by different techniques, the cause of which is not yet understood. We measured Phe concentrations in 240 venous blood samples from 199 patients with hyperphenylalaninemia in dried blood spots, EDTA plasma and erythrocytes by FIA-MS-MS and IEC. Phe concentrations were significantly lower in erythrocytes than in plasma leading to about 19% lower Phe DBS concentrations compared with plasma independent from the method used for quantification. As most therapy recommendations for PKU patients are based on plasma concentrations reliable conversion of DBS into plasma concentrations is necessary. Variances of Phe concentrations in plasma and DBS are not linear but increases with higher concentrations indicating heteroscedasticity. We therefore suggest the slope of the 75th percentile from quantile regression as a correction factor.

Detection of 3-O-methyldopa in dried blood spots for neonatal diagnosis of aromatic L-amino-acid decarboxylase deficiency: The northeastern Italian experience

ObjectiveAromatic L-amino acid decarboxylase (AADC) deficiency is a rare inherited autosomal recessive disorder of biogenic amine metabolism. Diagnosis requires analysis of neurotransmitter metabolites in cerebrospinal fluid, AADC enzyme activity analysis, or molecular analysis of the DDC gene. 3-O-methyldopa (3-OMD) is a key screening biomarker for AADC deficiency. MethodsWe describe a rapid method for 3-OMD determination in dried blood spots (DBS) using flow-injection analysis tandem mass spectrometry with NeoBase™ 2 reagents and 13C6-tyrosine as an internal standard, which are routinely used in high-throughput newborn screening. We assessed variability using quality control samples over a range of 3-OMD concentrations. ResultsWithin-day and between-day precision determined with quality control samples demonstrated coefficients of variation <15%. 3-OMD concentrations in 1000 healthy newborns revealed a mean of 1.33 μmol/L (SD ± 0.56, range 0.61–3.05 μmol/L), 100 non-AADC control subjects (age 7 days – 1 year) showed a mean of 1.19 μmol/L (SD ± 0.35–2.00 μmol/L), and 81 patients receiving oral L-Dopa had a mean 3-OMD concentration of 14.90 μmol/L (SD ± 14.18, range 0.4–80.3 μmol/L). A patient with confirmed AADC was retrospectively analyzed and correctly identified (3-OMD 10.51 μmol/L). In April 2020, we started a pilot project for identifying AADC deficiency in DBSs routinely submitted to the expanded newborn screening program. 3-OMD concentrations were measured in 21,867 samples; no patients with AADC deficiency were identified. One newborn had a high 3-OMD concentration due to maternal L-Dopa treatment. DiscussionWe demonstrated a rapid new method to identify AADC deficiency using reagents and equipment already widely used in newborn screening programs. Although our study is limited, introduction of our method in expanded neonatal screening is feasible and could facilitate deployment of screening, allowing for early diagnosis that is important for effective treatment.

Utility of measuring very long-chain fatty-acyl carnitines in dried blood spots for newborn screening of X-linked Adrenoleukodystrophy

We measured C24:0 and C26:0-carnitines in dried blood spots by flow injection analysis-tandem mass spectrometry method to evaluate whether they can be used as markers for newborn screening of X-linked Adrenoleukodystrophy (X-ALD). We found that C26:0-carnitine was 95.1% and 44.7% sensitive for identifying male X-ALD cases and heterozygous females, respectively. False negatives were found for C24:0-carnitine (11/82) and C26:0-carnitine (4/82). We conclude that C24:0 and C26:0-carnitines may not be reliable markers for X-ALD screening.

Impact of Treatment with RUTF on Plasma Lipid Profiles of Severely Malnourished Pakistani Children.

(1) Background: Little is known on impacts of ready-to-use therapeutic food (RUTF) treatment on lipid metabolism in children with severe acute malnutrition (SAM). (2) Methods: We analyzed glycerophospholipid fatty acids (FA) and polar lipids in plasma of 41 Pakistani children with SAM before and after 3 months of RUTF treatment using gas chromatography and flow-injection analysis tandem mass spectrometry, respectively. Statistical analysis was performed using univariate, multivariate tests and evaluated for the impact of age, sex, breastfeeding status, hemoglobin, and anthropometry. (3) Results: Essential fatty acid (EFA) depletion at baseline was corrected by RUTF treatment which increased EFA. In addition, long-chain polyunsaturated fatty acids (LC-PUFA) and the ratio of arachidonic acid (AA)/linoleic acid increased reflecting greater EFA conversion to LC-PUFA, whereas Mead acid/AA decreased. Among phospholipids, lysophosphatidylcholines (lyso.PC) were most impacted by treatment; in particular, saturated lyso.PC decreased. Higher child age and breastfeeding were associated with great decrease in total saturated FA (ΣSFA) and lesser decrease in monounsaturated FA and total phosphatidylcholines (ΣPC). Conclusions: RUTF treatment improves EFA deficiency in SAM, appears to enhance EFA conversion to biologically active LC-PUFA, and reduces lipolysis reflected in decreased ΣSFA and saturated lyso.PC. Child age and breastfeeding modify treatment-induced changes in ΣSFA and ΣPC.

Relationship between the plasma acylcarnitine profile and cardiometabolic risk factors in adults diagnosed with cardiovascular diseases

The development of cardiovascular diseases (CVDs) is often asymptomatic. Identification of initial indicators of cardiometabolic disruption may assist in its early detection. The objective was to determine the relationships between plasma acylcarnitines (ACs) and cardiometabolic risk factors in adults with and without CVDs. The AC profile in human plasma of healthy controls [non-CVD group, n = 13)] and individuals diagnosed with CVDs (CVD group, n = 34) were compared. A targeted analysis of 29 ACs was performed using flow injection analysis-tandem mass spectrometry. There were significant direct correlations (p < 0.05) between ACs and cardiometabolic risk factors. Comparing the groups after adjustment for covariates, showed that the ACs that were best differentiated (p < 0.05) between the two groups and that presented “good” diagnostic accuracy were carnitine [30.7 (25.5–37.7) vs. 37.7 (32.3–45.0) µM], the short-chain ACs: acetylcarnitine [8.9 (7.4–10.2) vs. 11.9 (9.2–14.4) µM] and isovalerylcarnitine [0.10 (0.06–0.13) vs. 0.13 (0.10–0.16) µM], and the medium-chain ACs: hexanoylcarnitine [0.04 (0.03–0.05) vs. 0.06 (0.05–0.07) µM] and decenoylcarnitine [0.18 (0.12–0.22) vs. 0.22 (0.17–0.32) µM]. This assessment contributes to the identification of the unique metabolic features exhibited in association with cardiometabolic risk in adults diagnosed with CVD. The altered metabolites have the potential to be used as biomarkers for early detection of CVD.

Performance of laboratory tests used to measure blood phenylalanine for the monitoring of patients with phenylketonuria.

Analysis of blood phenylalanine is central to the monitoring of patients with phenylketonuria (PKU) and age-related phenylalanine target treatment-ranges (0-12 years; 120-360 μmol/L, and >12 years; 120-600 μmol/L) are recommended in order to prevent adverse neurological outcomes. These target treatment-ranges are based upon plasma phenylalanine concentrations. However, patients are routinely monitored using dried bloodspot (DBS) specimens due to the convenience of collection. Significant differences exist between phenylalanine concentrations in plasma and DBS, with phenylalanine concentrations in DBS specimens analyzed by flow-injection analysis tandem mass spectrometry reported to be 18% to 28% lower than paired plasma concentrations analyzed using ion-exchange chromatography. DBS specimens with phenylalanine concentrations of 360 and 600 μmol/L, at the critical upper-target treatment-range thresholds would be plasma equivalents of 461 and 768 μmol/L, respectively, when a reported difference of 28% is taken into account. Furthermore, analytical test imprecision and bias in conjunction with pre-analytical factors such as volume and quality of blood applied to filter paper collection devices to produce DBS specimens affect the final test results. Reporting of inaccurate patient results when comparing DBS results to target treatment-ranges based on plasma concentrations, together with inter-laboratory imprecision could have a significant impact on patient management resulting in inappropriate dietary change and potentially adverse patient outcomes. This review is intended to provide perspective on the issues related to the measurement of phenylalanine in blood specimens and to provide direction for the future needs of PKU patients to ensure reliable monitoring of metabolic control using the target treatment-ranges.

The North Carolina Experience with Mucopolysaccharidosis Type I Newborn Screening

To evaluate the performance of a 2-tiered newborn screening method for mucopolysaccharidosis type I (MPS I) in North Carolina. The screening algorithm included a flow injection analysis-tandem mass spectrometry assay as a first-tier screening method to measure α-L-iduronidase (IDUA) enzyme activity and Sanger sequencing of the IDUA gene on dried blood spots as a second-tier assay. The screening algorithm was revised to incorporate the Collaborative Laboratory Integrated Reports, an analytical interpretive tool, to reduce the false-positive rate. A medical history, physical examination, IDUA activity, and urinary glycosaminoglycan (GAG) analysis were obtained on all screen-positive infants. A total of 62 734 specimens were screened with 54 screen-positive samples using a cut-off of 15% of daily mean IDUA activity. The implementation of Collaborative Laboratory Integrated Reports reduced the number of specimens that screened positive to 19 infants. Of the infants identified as screen-positive, 1 had elevated urinary GAGs and a homozygous pathogenic variant associated with the severe form of MPS I. All other screen-positive infants had normal urinary GAG analysis; 13 newborns had pseudodeficiency alleles, 3 newborns had variants of unknown significance, and 2 had heterozygous pathogenic variants. An infant with severe MPS I was identified and referred for a hematopoietic stem cell transplant. Newborn IDUA enzyme deficiency is common in North Carolina, but most are due to pseudodeficiency alleles in infants with normal urinary GAG analysis and no evidence of disease. The pilot study confirmed the need for second-tier testing to reduce the follow-up burden.

Rapid Quantitative Analysis of Multiple Explosive Compound Classes on a Single Instrument via Flow-Injection Analysis Tandem Mass Spectrometry.

A flow-injection analysis tandem mass spectrometry (FIA MSMS) method was developed for rapid quantitative analysis of 10 different inorganic and organic explosives. Performance is optimized by tailoring the ionization method (APCI/ESI), de-clustering potentials, and collision energies for each specific analyte. In doing so, a single instrument can be used to detect urea nitrate, potassium chlorate, 2,4,6-trinitrotoluene, 2,4,6-trinitrophenylmethylnitramine, triacetone triperoxide, hexamethylene triperoxide diamine, pentaerythritol tetranitrate, 1,3,5-trinitroperhydro-1,3,5-triazine, nitroglycerin, and octohy-dro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine with sensitivities all in the picogram per milliliter range. In conclusion, FIA APCI/ESI MSMS is a fast (<1 min/sample), sensitive (~pg/mL LOQ), and precise (intraday RSD < 10%) method for trace explosive detection that can play an important role in criminal and attributional forensics, counterterrorism, and environmental protection areas, and has the potential to augment or replace several of the existing explosive detection methods.

FIA-MS/MS determination of creatinine in urine samples undergoing butylation

Flow injection analysis-tandem mass spectrometry has become widely used for analysis of many biomarkers in various biological matrices. To improve the sensitivity, the compounds are often determined as their butylesters. Since the concentration of urinary excreted compounds are generally reported after normalization to creatinine, the aim of this study was to investigate the possibility of creatinine determination in urine samples which underwent butylation. The impact of derivatization on urinary creatinine determination was investigated by measuring of underivatized and derivatized samples. The 10% creatine to creatinine conversion was observed during butylation, what above 700 μmol creatine/mmol creatinine caused significant creatinine overestimation. In that case, correction for creatine conversion rate was done. QC samples at six concentration levels were examined and precision and accuracy values fulfill the European Medicine Agency validation requirements. The elaborated method was applied for determination of creatinine in 41 real human urine samples. Determined creatinine concentrations were in the range of 0.27–22.3 mmol/L, linearity was confirmed within the concentration range of 0.27–31.7 mmol/L. Obtained results highly correlated with routinely used enzymatic assay for all tested samples and proposed method provide reliable determination of creatinine in butylated urine in a single run with butylesters of other analytes of interest.

Oxidized phosphatidylcholines suggest oxidative stress in patients with medium-chain acyl-CoA dehydrogenase deficiency

Inborn errors of metabolism encompass a large group of diseases caused by enzyme deficiencies and are therefore amenable to metabolomics investigations. Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is a defect in β-oxidation of fatty acids, and is one of the most well understood disorders. We report here the use of liquid chromatography–mass spectrometry (LC–MS) based untargeted metabolomics and targeted flow injection analysis–tandem mass spectrometry (FIA–TMS) that lead to discovery of novel compounds of oxidative stress. Dry blood spots of controls (n=25) and patient samples (n=25) were extracted by methanol/water (1/1, v/v) and these supernatants were analyzed by LC–MS method with detection by an Orbitrap Elite MS. Data were processed by XCMS and CAMERA followed by dimension reduction methods. Patients were clearly distinguished from controls in PCA. S-plot derived from OPLS-DA indicated that medium-chain acylcarnitines (octanoyl, decenoyl and decanoyl carnitines) as well as three phosphatidylcholines (PC(16:0,9:0(COOH))), PC(18:0,5:0(COOH)) and PC(16:0,8:0(COOH)) were important metabolites for differentiation between patients and healthy controls. In order to biologically validate these discriminatory molecules as indicators for oxidative stress, a second cohort of individuals were analyzed, including MCADD (n=25) and control (n=250) samples. These were measured by a modified newborn screening method using FIA–TMS (API 4000) in MRM mode. Calculated p-values for PC(16:0,9:0(COOH)), PC(18:0,5:0(COOH)) and PC(16:0,8:0(COOH)) were 1.927×10−14, 2.391×10−15 and 3.354×10−15 respectively. These elevated oxidized phospholipids indeed show an increased presence of oxidative stress in MCADD patients as one of the pathophysiological mechanisms of the disease.

Development and validation of fast and simple flow injection analysis–tandem mass spectrometry (FIA–MS/MS) for the determination of metformin in dog serum

A simple, fast and sensitive quantification method for the drug metformin in dog serum was developed using flow injection analysis (FIA)–tandem mass spectrometry (MS/MS). The method was fully validated according to industry standards. It is the first time that FIA–MS/MS for metformin was developed surpassing all existing methods in terms of time of analysis. The quantification method was dependent on the formation of [M+H]+ using electrospray ionization (ESI) and employing multiple reaction monitoring (MRM) using quadrupole-linear ion trap (4000 QTRAP®) instrument. A deuterated internal standard (IS) of metformin bearing six deuterium atoms was used to compensate for matrix effects and for variation in ion current within the ESI source. The ion transitions that were monitored were m/z 130.1→m/z 71.0 and m/z 130.1→m/z 60.1 for metformin and m/z 136.0→m/z 77.0 for the internal standard. A linear response (r=0.9966) was established for a range of concentrations of 5–2340ng/mL. The inter- and intra-day variations were within the acceptable criteria for all quality control samples. The method was successfully applied for measurement of serum metformin concentration in dogs after intravenous injection.