HPLC-electrospray Tandem Mass Spectrometry Research Articles

Simultaneous quantification of 24 aldehydes and ketones in oysters (Crassostrea gigas) with different thermal processing procedures by HPLC-electrospray tandem mass spectrometry

Aldehydes and ketones are secondary oxidation products resulting from lipid oxidation that occurs during food processing. These small molecule compounds not only have an impact on the quality, odor and flavor of food, but also play a role in the pathogenesis of many human diseases. In this study, a HPLC-MS/MS analytical method was developed and validated for the simultaneous determination of 24 aldehydes and ketones. The coefficients of determination (R2) for all aldehydes and ketones were higher than 0.9975 at the range of 0.2–2000 ng/mL. The recoveries were in the range 71.20–108.13% with RSD < 10%. The method was tested by analyzing lipids from oysters with different thermal processing (boiling, frying, roasting and air frying) procedures; the highest concentration for saturated aldehydes and ketones while the highest content of unsaturated aldehydes in boiling treatment. Meanwhile, fatty acid oxidative decomposition was in agreement with aldehydes and ketones formation. Moreover, principal component analysis, orthogonal partial least-squares discriminant analysis and variable importance in projection value showed that lipid oxidation is positively related to the formation of a variety of aldehydes and ketones.

Serum and acute phase protein changes in laying hens, infested with poultry red mite.

The poultry red mite (PRM) is one of the most economically important ectoparasites of laying hens globally. This mite can have significant deleterious effects on its fowl host including distress, anemia, reduced egg production, and reduced egg quality. This study was conducted to evaluate the influence of PRM on the serum protein profile in laying hens and its effect on the acute phase proteins (APPs) to assess their potential as biomarkers for mite infestation. Three APPs: alpha-1 acid glycoprotein (AGP), serum amyloid-A (SAA), and ceruloplasmin (CP) were measured in serum samples collected from laying hens at 12 and 17 wk of age, and then for up to 4 mo after a challenge with PRM (starting at 18.5 wk of age). The serum protein profile (SDS-PAGE/nanoflow HPLC electrospray tandem mass spectrometry) and concentration of individual serum proteins (SDS-PAGE-band densitometry) were also compared. Post challenge there was a positive correlation (r = 0.489; P < 0.004) between the levels of SAA and the PRM numbers. The levels of SAA steadily increased after the PRM challenge and were significantly different than the pre-challenge levels at 28, 32, and 36 wk of age (P < 0.01). The PRM numbers also peaked around 31-33 wk of age. The results for AGP and CP in comparison were inconsistent. Proteomics revealed the presence of 2 high molecular weight proteins in the serum between 12 and 17 wk of age. These were identified as Apolipoprotein-B and Vitellogenin-2, and their increase was commensurate with the onset of lay. No other major differences were detected in the protein profiles of blood sera collected pre and post challenge. We conclude that SAA could be used as a useful biomarker to monitor PRM infestation in commercial poultry flocks and that PRM infestation does not disrupt the production of the major proteins in the serum that are associated with egg formation.

Development of an LC-MS/MS method for studying migration characteristics of acetaldehyde in polyethylene terephthalate (PET)-packed mineral water

During storage, acetaldehyde migration from polyethylene terephthalate (PET) bottles can affect the quality of mineral water even in the low µg l−1 range negatively, as it features a fruity or plastic-like off-flavour. For a sensitive and fast analysis of acetaldehyde in mineral water, a new analysis method of 2,4-dinitrophenylhydrazine (DNPH) derivatisation followed by HPLC-electrospray tandem mass spectrometry (ESI-MS/MS) was developed. Acetaldehyde was directly derivatised in the mineral water sample avoiding extraction and/or pre-concentration steps and then analysed by reversed-phase HPLC-ESI-MS/MS using multiple reaction monitoring mode (MRM). Along with method development, the optimum molar excess of DNPH in contrast to acetaldehyde was studied for the mineral water matrix, because no specific and robust data were yet available for this critical parameter. Best results were obtained by using a calibration via the derivatisation reaction. Without any analyte enrichment or extraction, an LOD of 0.5 µg l−1 and an LOQ of 1.9 µg l−1 were achieved. Using the developed method, mineral water samples packed in PET bottles from Germany were analysed and the correlation between the acetaldehyde concentration and other characteristics of the samples was evaluated illustrating the applicability of the method. Besides a relationship between bottle size and CO2 content of the mineral water and acetaldehyde migration, a correlation with acetaldehyde migration and the material composition of the bottle, e.g. recycled PET, was noted. Investigating the light influence on the acetaldehyde migration with a newly developed, reproducible light exposure setup, a significant increase of the acetaldehyde concentration in carbonated mineral water samples was observed.

Study on Characterization of the Degradation Products and Dynamics of Valtrate in the Artificial Gastrointestinal Fluid by HPLC- ESI MS/MSn

The present study developed a systematic method for the degradation products and dynamics of valtrate in artificial intestinal fluid and gastric fluid. HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MSn) was applied to separate the degradation products of valtrate and identify structures of the main degradation products. Samples were separated on an Agilent Extend-Cl8 by gradient elution using acetonitrile and water–acetic acid (0.25%, v/v). Analysis was performed in positive ion mode with 3000 V of capillary voltage, 40 psi of atomization pressure, 10 L min−1 dry nitrogen gas velocity, and m/z 100–1000 of scan range. Six degradation products were all identified to own the stem-nucleus structures of baldrinals. Two of them were identified through the reference substances. An HPLC method with isocratic elution using acetonitrile and water (68:32, v/v) at 254 nm was established to determine content change of valtrate in artificial intestinal fluid and gastric fluid at different time points. The method was validated with good linearity in wide linear ranges (r2 = 0.9999), and good intra-day precision (RSD = 1.0%). The degradations of valtrate in artificial intestinal fluid and gastric fluid follow pseudo-first-order kinetic process, the half-life period in artificial gastric fluid is 1.73 hr, and in artificial intestinal fluid it is 1.61 hr. This study enriched the knowledge on degradation products of valtrate and may reveal the mechanism of the degradation of valtrate.

Human serum levels of perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) in Uyghurs from Sinkiang-Uighur Autonomous Region, China: background levels study.

Perfluorinated compounds (PFCs), such as perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA), are a family of commonly used industrial chemicals whose persistence and ubiquity in blood samples of humans and wildlife have become a growing concern. Despite PFOS and PFOA having been found in human blood and tissue samples from occupationally exposed workers and the general worldwide population, little systematic knowledge has accrued with respect to exposure levels in Uyghurs in the Sinkiang-Uighur Autonomous Region of China, which is predominantly agricultural and pastoral. Our goal was to provide background data for biological monitoring in the general population of this region. In this study, 110 self-reported healthy human serum samples were collected from nonoccupationally exposed Uyghurs volunteers and analyzed by microbore HPLC-electrospray tandem mass spectrometry. Among the 110 blood specimens, PFOS was detected in 102 samples (93%) and ranged from the lower limit of quantification of 0.01 to 22.63 μg/L with a median of 1.93 μg/L (interquartile range 1.00-3.43 μg/L). The median was higher among males (2.39 μg/L; interquartile range 1.23-4.40 μg/L) than that among females (1.20 μg/L; interquartile range 0.83-2.77 μg/L). No significant difference was observed with respect to age. The concentration of PFOA was lower than that of PFOS and was found only in seven samples (6%) at concentrations above the limit of quantification. This study is the first investigation to reveal serum PFOS and PFOA levels in the general population of Uyghurs. PFOS and PFOA concentrations found in the present investigation were lower than those found in recent studies consisting of subjects from different geographic locations (PFOS 5.0-44.7 μg/L, PFOA 1.5-10 μg/L).

Developmental Truncations of Connexin 50 by Caspases Adaptively Regulate Gap Junctions/Hemichannels and Protect Lens Cells against Ultraviolet Radiation

The gap junction-forming connexin (Cx) 50 is truncated gradually during lens development. Premature cleavage of lens connexins is thought to be associated with cataract formation. We have shown previously that Cx50 is likely to be cleaved by caspase-3 like protease during chick lens development. Here, using HPLC-electrospray tandem mass spectrometry, we mapped two cleavage sites at the C terminus of Cx50 after Glu-368 and Asp-379 and identified caspase-3 and caspase-1 as the responsible proteases, respectively. The activity of caspase-1, like caspase-3, was detected in the outer cortex increased during lens development, which coincided with the accumulation of the truncated fragments of Cx50 in the core region of the lens. The truncated Cx50 fragments present in older lenses were reproduced in the younger lens after treatment with UV radiation; this cleavage could be partially blocked by caspase-1/3-specific inhibitors. Interestingly, as compared with full-length Cx50, caspase-truncated Cx50 showed a dramatic decrease in gap junction coupling and a loss of hemichannel function. Furthermore, expression of caspase-truncated Cx50 fragments increased cell viability against UV radiation as compared with full-length Cx50. Together, these results suggest that both caspase-1 and -3 are responsible for the cleavage at the C terminus of Cx50 during lens development. The reduction of gap junction coupling and closure of hemichannels formed by truncated Cx50 are likely to adaptively protect cells against elevated oxidative stress associated with lens aging.

Arsenic speciation in freshwater snails and its life cycle variation

Terrestrial snails are consumed by humans occasionally and they are an important food source for many creatures including fish and birds. Little is known about arsenic speciation in these gastropods, let alone life cycle variations. Here we report on the arsenic speciation in freshwater snails from Pender Island and Vancouver Island, B.C., Canada, which was determined on methanol/water extracts (43-59% extraction efficiency) by using high performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) and HPLC-electrospray tandem mass spectrometry. The tetramethylarsonium ion, oxo-arsenosugars and thio-arsenosugars are the main arsenic species encountered. Arsenobetaine, which is commonly found in the marine environment, is minor. Live bearing snails Viviparidae sp. from Pender Island were maintained in aquaria and the arsenic speciation in the unborn, newly born, and adult animals was monitored. Oxo-arsenosugars predominate in the adults, whereas thio-arsenosugars seem to predominate in juveniles, suggesting that these arsenicals are snail metabolites.

Therapeutic drug monitoring of everolimus using the dried blood spot method in combination with liquid chromatography–mass spectrometry

An assay of everolimus based on finger prick sampling and consecutive application as a blood spot on sampling paper has been developed. We explored several methods [K. Hoogtanders, J. van der Heijden, M. Christiaans, P. Edelbroek, J. van Hooff, L. Stolk, J. Pharm. Biomed. Anal. 44 (2006) 658–664; A. Allanson, M. Cotton, J. Tettey, et al., J. Pharm. Biomed. Anal. 44 (2007) 963–969] and developed a new method, namely the impregnation of sampling paper with a solution of plasma–protein, formic acid and ammonium acetate, in combination with the extraction of the blood spot by filter filtration. This kind of sample preparation provides new possibilities for blood spot sampling especially if analytes are adsorbed to the paper. The dried blood spot was analysed using the HPLC–electrospray-tandem mass spectrometry method, with 32-desmethoxyrapamycin as the internal standard. The working range of our study was 2–30 μg/l. Within this range, intra-and inter-assay variability for precision and accuracy was <15%. Everolimus blood spot samples proved stable for 3 days at 60 °C and for 32 days at 4 °C. Everolimus concentrations of one stable out-patient were compared after both blood spot sampling and conventional venous sampling on various occasions. Results indicate that this new method is promising for therapeutic drug monitoring in stable renal transplant patients.

HPLC–electrospray tandem mass spectrometry for simultaneous quantitation of eight plasma aminothiols: Application to studies of diabetic nephropathy

It was reported that Hcy was related to the development of kidney disease, but it remains unknown whether Hcy is an independent biomarker for diabetic nephropathy. Analytical method for simultaneous determination of aminothiols among the Hcy metabolic cycle is desirable to discover other potential biomarkers. A high-performance liquid chromatography–electrospray tandem mass spectrometric (HPLC–ESI-MS/MS) method was established for simultaneous quantitation of Cysteine (Cys), total homocysteine (tHcy), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), cystathionine (Cysta), methionine (Met), glutathione (GSH) and cysteinylglycine (Cys-gly) in plasma with N-(2-mercaptopropionyl)-glycine (MPG) as internal standard. The method had simple pretreatment without derivatization and the chromatograms show better separation of the eight aminothiols and the analytic time was 20 min. The results demonstrated that it provided an excellent linearity for all analytes over their respective concentration ranges and illustrated excellent precision and plasma recovery as well. Then, the method was applied in the case–control study of patients with diabetes mellitus (DM) and diabetic nephropathy (DN). In conclusion, it is an effective method to quantitate the concentrations of aminothiols in the human plasma. SAH and SAM were suggested as better potential biomarkers of DM and DN.

Simultaneous quantification of 11 pivotal metabolites in neural tube defects by HPLC–electrospray tandem mass spectrometry

One-carbon metabolism that involves folate metabolism and homocysteine metabolism plays a powerful role in embryonic development. Any impairment to this metabolism during the neurulation process would trigger the occurrence of neural tube defects (NTDs). The great importance of one-carbon metabolism necessitates the establishment of methodology to determine the relative compounds involved in the metabolic cycles. We have developed a sensitive method for measurement of 11 pivotal compounds by using high-performance liquid chromatography coupled to mass spectrometry (HPLC–MS/MS) in sera of pregnant women. Use of an aqueous chromatography column increased retention time and separation of the polar compounds in the system, resulting in fewer co-elution and interference from the other compounds that can lead to ion suppression. Calibration curves suitable for the analysis of maternal serum were linear ( r 2 > 0.997) with limits of detection from 0.05 to 1 ng/mL. Intra-day coefficients of variation (CVs) and inter-day CVs were both lower than 11%. With the developed method, 96 serum samples including 46 cases and 50 controls were analyzed. The established method provided a reliable method for quantifying most of the compounds involved in the one-carbon metabolism simultaneously, thus made it possible to elucidate NTDs with multiple factors instead of one single and provided a solid foundation for the diagnosis and prevention of NTDs as well as some other one-carbon metabolism related diseases.

A HPLC–mass spectrometric method suitable for the therapeutic drug monitoring of everolimus

We report here the validation of an HPLC–electrospray–tandem mass spectrometry method for the quantification of everolimus, an immunosuppressant drug. Whole blood samples (100 μl) were extracted by protein precipitation which involved sample pre-treatment with zinc sulphate followed by acetonitrile (containing internal standard, 40- O-(3′-hydroxy)propyl-rapamycin). HPLC was performed using a step-gradient at a flow rate of 0.6 ml/min on a Waters TDM C 18 column (10 mm × 2.1 mm I.D.) with a resultant chromatographic analysis time of 2 min. Mass spectrometric detection by selected reaction monitoring (everolimus m/ z 975.5 → 908.3; internal standard m/ z 989.5 → 922.3). The assay was linear from 0.5 to 40 μg/l ( r 2 > 0.994, n = 11). The inter- and intra-day analytical recovery and imprecision for quality control samples (1.25, 12.5 and 30 μg/l) were 93.4–98.2% and <10.7%, respectively ( n = 10). At the lower limit of quantification (0.5 μg/l) the inter- and intra-day analytical recovery was 94.4–95.8% with imprecision of <14.1% ( n = 10). The absolute recovery of everolimus (6.5 μg/l) and internal standard (12.5 μg/l) was 96.5 and 88.3%, respectively ( n = 3). A comparison of our method against the mean of all HPLC methods for a series of samples from an external proficiency testing scheme revealed good correlation as shown by the regression analysis: y = 0.973 x + 0.301 ( r 2 = 0.986, n = 71). In conclusion, the method described is suited to the current requirements for therapeutic drug monitoring of everolimus.

Fast and Efficient Determination of Arginine, Symmetric Dimethylarginine, and Asymmetric Dimethylarginine in Biological Fluids by Hydrophilic-Interaction Liquid Chromatography–Electrospray Tandem Mass Spectrometry

Nitric oxide is synthesized from the amino acid Arg by the enzyme endothelial nitric oxide synthase, which is competitively inhibited by the arginine metabolite asymmetric dimethylarginine (ADMA). In this way, increased concentrations of ADMA lead to reduced nitric oxide production associated with a range of cardiac diseases. Research in this field requires the measurement of Arg and of ADMA and its closely related substance, symmetric dimethylarginine (SDMA). We quantified Arg, ADMA, and SDMA in human plasma, human urine, and cell culture supernatant by HPLC-electrospray tandem mass spectrometry. Sample preparation required only protein precipitation. Separation was by liquid chromatography on a 150 x 3 mm silica column with an isocratic mobile phase consisting of water-acetonitrile-trifluoroacetic acid-propionic acid (10:90:0.025:1 by volume). The chromatographic run time was 7 min. The chromatograms were interference-free in all matrices. In the low-concentration quality-control samples, the interassay CVs in plasma were 4.7% for Arg, 7.7% for ADMA, and 4.9% for SDMA. Similar values were obtained in urine and cell culture supernatants. The calibration functions were linear and covered the ranges of healthy and pathologic samples. The new method requires neither derivatization nor complete chromatographic separation between ADMA and SDMA for quantification of the 3 metabolites, has calibration functions that are independent of the sample matrix, and provides measured concentrations that agree with those reported in the literature.

Sialic acid concentrations in plants are in the range of inadvertent contamination

The long held but challenged view that plants do not synthesize sialic acids was re-evaluated using two different procedures to isolate putative sialic acid containing material from plant tissues and cells. The extracts were reacted with 1,2-diamino-4,5-methylene dioxybenzene and the fluorescently labelled 2-keto sugar acids analysed by reversed phase and normal phase HPLC and by HPLC-electrospray tandem mass spectrometry. No N-glycolylneuraminic acid was found in the protein fraction from Arabidopsis thaliana MM2d cells. However, we did detect 3-deoxy-D: -manno-octulosonic acid and trace amounts (3-18 pmol/g fresh weight) of a compound indistinguishable from N-acetylneuraminic acid by its retention time and its mass spectral fragmentation pattern. Thus, plant cells and tissues contain five orders of magnitude less sialic acid than mammalian tissues such as porcine liver. Similar or lower amounts of N-acetylneuraminic acid were detected in tobacco cells, mung bean sprouts, apple and banana. Yet even yeast and buffer blanks, when subjected to the same isolation procedures, apparently contained the equivalent of 5 pmol of sialic acid per gram of material. Thus, we conclude that it is not possible to demonstrate unequivocally that plants synthesize sialic acids because the amounts of these sugars detected in plant cells and tissues are so small that they may originate from extraneous contaminants.

Purine Metabolites in Gout and Asymptomatic Hyperuricemia: Analysis by HPLC–Electrospray Tandem Mass Spectrometry

Hyperuricemia, a serum urate concentration >0.45 mmol/L (7.0 mg/dL) in men and 0.36 mmol/L (6.0 mg/dL) in women, is the biochemical hallmark of gout (1)(2)(3), but many individuals with life-long hyperuricemia do not develop gouty arthritis (4). Conversely, serum urate concentrations may be within reference values in some patients with acute gout, particularly during the early phases of the disorder. We hypothesized that gout patients may have a different profile of purine precursors than do asymptomatic people with hyperuricemia and that concentrations of these compounds may reflect disorders of purine metabolism. To our knowledge, purine metabolites have not been studied and compared in gout and asymptomatic hyperuricemia. With established methods for measuring purine compounds in blood and urine (5)(6)(7)(8)(9), retention time is not always adequate for identification of every peak because urine and blood usually contain many interfering compounds. Quantification by tandem mass spectrometry (MS/MS) (10)(11) may require an internal standard, which often is hard to obtain. In this study of 10 purine compounds in the blood of gout patients and hyperuricemia patients with no gout symptoms, we used HPLC for serum sample separation, MS/MS for peak identification, and ultraviolet (UV) detection for quantification. Purines were purchased from Fluka and Sigma. The HPLC-MS experiment was performed on an Agilent 1100 series liquid chromatograph with a mass spectrometric detector trap. Separation was carried out on a Supelcosil™ LC-18-DB column [25 cm × 4.6 mm (i.d.); 5-μm film thickness]. The column temperature was maintained at 25 °C. The mobile phases were as follows: 10 mmol/L ammonium acetate, adjusted to pH 6.5 with glacial acetic acid (eluant A), and methanol (eluant B). The elution gradient was as follows (flow rate, 1 mL/min): 0 to 10 …

Identification of the major selenium compound, Se-Methionine, in three yeast ( Saccharomyces cerevisiae) dietary supplements by on-line narrowbore liquid chromatography–electrospray tandem mass spectrometry

On-line monitoring of six Se-compounds was accomplished by using an XTerra MS C 18 column coupled to electrospray tandem mass spectrometry (ES–MS–MS). In view of the nature of the compounds, the positively charged ion pairing agent tetraethylammoniumchloride (TEACl) was added to the mobile phase. The HPLC–ES–MS–MS method was optimized with six commercially available Se-compounds. Substitution of the analytical column by the narrowbore type significantly enhanced the sensitivity of the method. We were able to detect the m/ z of these six molecules on-line. Furthermore, all product ions could be monitored. The method was applied to three different yeast-based supplements. They were submitted to proteolytic digestion and screened for their Se-content by HPLC–HG–AFS (hydride generation-atomic fluorescence spectrometry). By application of on-line narrowbore HPLC–electrospray tandem mass spectrometry, the main compound present in these three supplements, Se-Methionine, could be measured on its m/ z and its product ions. The method can be further extended for on-line measurement of different Se-species in complex matrices

Analysis of pyrimidine synthesis "de novo" intermediates in urine and dried urine filter- paper strips with HPLC-electrospray tandem mass spectrometry.

The concentrations of the pyrimidine "de novo" metabolites and their degradation products in urine are useful indicators for the diagnosis of an inborn error of the pyrimidine de novo pathway or a urea-cycle defect. Until now, no procedure was available that allowed the analysis of all of these metabolites in a single analytical run. We describe a rapid, specific method to measure these metabolites by HPLC-tandem mass spectrometry. Urine or urine-soaked filter-paper strips were used to measure N-carbamyl-aspartate, dihydroorotate, orotate, orotidine, uridine, and uracil. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry, and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled reference compounds were used as internal standards. All pyrimidine de novo metabolites and their degradation products were measured within a single analytical run of 14 min with lower limits of detection of 0.4-3 micromol/L. The intra- and interassay variation for urine with added compounds was 1.2-5% for urines and 2-9% for filter-paper extracts of the urines. Recoveries of the added metabolites were 97-106% for urine samples and 97-115% for filter-paper extracts of the urines. Analysis of urine samples from patients with a urea-cycle defect or pyrimidine degradation defect showed an aberrant metabolic profile when compared with controls. HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders affecting the pyrimidine de novo pathway. The use of filter-paper strips could facilitate collection, transport, and storage of urine samples.

Quantification of free sialic acid in urine by HPLC-electrospray tandem mass spectrometry: a tool for the diagnosis of sialic acid storage disease.

Sialic acid storage diseases (SSDs) are severe autosomal recessive neurodegenerative disorders caused by a transport defect across the lysosomal membrane, which leads to accumulation of sialic acid in tissues, fibroblasts, and urine. Defective free sialic acid transport can be established by quantification of free sialic acid in urine. Urine sample size was adjusted to the equivalent of 100 nmol of creatinine. After addition of 2-keto-3-deoxy-d-glycero-d-galactonononic acid as internal standard, samples were diluted with water to an end volume of 250 microL. We used 10 microL for HPLC-tandem mass spectrometric analysis in the negative electrospray ionization mode, monitoring transitions m/z 308.3-->m/z 86.9 (sialic acid) and m/z 267.2-->m/z 86.9 (internal standard). The overall method was validated and studied for ion suppression, interfering compounds, and pH effects. Samples from controls (n = 72) and SSD patients (n = 3) were analyzed. The limit of detection was 3 micromol/L. Intraassay imprecision (CV; n = 10) was 6%, 3%, and 2% at 30, 130, and 1000 mmol/mol creatinine, respectively; corresponding interassay CV (n = 10) were 5%, 5%, and 2%. Recovery was 109% (100-1000 mmol/mol creatinine). The mean (SD) [range] excretion rates (mmol/mol creatinine) were 31.3 (16.6) [0.7-56.9] at 0-1 year (n = 20), 21.2 (9.8) [6.3-38.3] at 1-3 years (n = 15), 14.4 (8.2) [1.7-32.9] at 3-10 years (n = 25), and 4.6 (2.6) [0-9.8] above age 10 years (n = 12). SSD patients 1.2, 3.9, and 12 years of age had concentrations of 111.5, 54.2, and 36.1 mmol/mol creatinine, respectively. The HPLC-tandem MS method for free sialic acid in urine is more rapid, accurate, sensitive, selective, and robust than earlier methods and may serve as a candidate reference method for free sialic acid in diagnosis of SSD.

Quantification of 5,6-dihydrouracil by HPLC-electrospray tandem mass spectrometry.

In humans, the pathway for the catabolism of uracil and thymine consists of three consecutive steps. Dihydropyrimidine dehydrogenase catalyzes the reduction of uracil and thymine to 5,6-dihydrouracil and 5,6-dihydrothymine, respectively. The second step is catalyzed by dihydropyrimidinase and consists of reversible hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to N -carbamyl-β-alanine and N -carbamyl-β-aminoisobutyric acid, respectively. Finally, β-ureidopropionase catalyzes the conversion of N -carbamyl-β-alanine and N -carbamyl-β-aminoisobutyric acid to β-alanine and β-aminoisobutyric acid, respectively, ammonia, and CO2. Patients with a defect in one of the enzymes of the pyrimidine degradation pathway can be diagnosed by an aberrant excretion profile of the pyrimidine bases and their degradation products in urine (1). For example, in patients with a complete deficiency of dihydropyrimidinase, highly increased concentrations of 5,6-dihydrouracil and 5,6-dihydrothymine and moderately increased concentrations of uracil and thymine can be detected in urine. It has also …

Perfluorooctanesulfonate and other fluorochemicals in the serum of American Red Cross adult blood donors.

Perfluorooctanesulfonyl fluoride-based products have included surfactants, paper and packaging treatments, and surface protectants (e.g., for carpet, upholstery, textile). Depending on the specific functional derivatization or degree of polymerization, such products may degrade or metabolize, to an undetermined degree, to perfluorooctanesulfonate (PFOS), a stable and persistent end product that has the potential to bioaccumulate. In this investigation, a total of 645 adult donor serum samples from six American Red Cross blood collection centers were analyzed for PFOS and six other fluorochemicals using HPLC-electrospray tandem mass spectrometry. PFOS concentrations ranged from the lower limit of quantitation of 4.1 ppb to 1656.0 ppb with a geometric mean of 34.9 ppb [95% confidence interval (CI), 33.3-36.5]. The geometric mean was higher among males (37.8 ppb; 95% CI, 35.5-40.3) than among females (31.3 ppb; 95% CI, 30.0-34.3). No substantial difference was observed with age. The estimate of the 95% tolerance limit of PFOS was 88.5 ppb (upper limit of 95% CI, 100.0 ppb). The measures of central tendency for the other fluorochemicals (N-ethyl perfluorooctanesulfonamidoacetate, N-methyl perfluorooctanesulfonamidoacetate, perfluorooctanesulfonamidoacetate, perfluorooctanesulfonamide, perfluorooctanoate, and perfluorohexanesulfonate) were approximately an order of magnitude lower than PFOS. Because serum PFOS concentrations correlate with cumulative human exposure, this information can be useful for risk characterization.

Analysis of Carnitine Biosynthesis Metabolites in Urine by HPLC–Electrospray Tandem Mass Spectrometry

We developed a method to determine the urinary concentrations of metabolites in the synthetic pathway for carnitine from N(6)-trimethyllysine and applied this method to determine their excretion in control individuals. In addition, we investigated whether newborns are capable of carnitine synthesis from deuterium-labeled N(6)-trimethyllysine. Urine samples were first derivatized with methyl chloroformate. Subsequently, the analytes were separated by ion-pair, reversed-phase HPLC and detected online by electrospray tandem mass spectrometry. Stable-isotope-labeled reference compounds were used as internal standards. The method quantified all carnitine biosynthesis metabolites except 4-N-trimethylaminobutyraldehyde. Detection limits were 0.05-0.1 micromol/L. The interassay imprecision (CV) for urine samples with added compounds was 6-12%. The intraassay imprecision (CV) was 1-5% (3-10 micromol/L). Recoveries were 94-106% at 10-20 micromol/L and 98-103% at 100-200 micromol/L. The mean (SD) excretions of N(6)-trimethyllysine and 3-hydroxy-N(6)-trimethyllysine were 2.8 (0.8) and 0.45 (0.15) mmol/mol creatinine, respectively. gamma-Butyrobetaine and carnitine excretions were more variable with values of 0.27 (0.21) and 15 (12) mmol/mol creatinine, respectively. After oral administration of deuterium-labeled N(6)-trimethyllysine, all urines of newborns contained deuterium-labeled N(6)-trimethyllysine, 3-hydroxy-N(6)-trimethyllysine, gamma-butyrobetaine, and carnitine. HPLC in combination with electrospray ionization tandem mass spectrometry allows rapid determination of urinary carnitine biosynthesis metabolites. Newborns can synthesize carnitine from exogenous N(6)-trimethyllysine, albeit at a low rate.