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Elevated plasma homocysteine has been identified as a strong and independent risk factor for cardiovascular diseases, and recently, it has been associated with the development of dementia in older adults. Selected ion-monitoring isotope-dilution LC/MS (electrospray) has been developed and evaluated as a reference method for the accurate determination of total homocysteine in human plasma. Homocysteine is quantitatively isolated from plasma via the use of anion-exchange resins and then detected and quantified in stabilized plasma extracts with selected ion-monitoring LC/MS. This method is shown to be highly comparable to LC/MS/MS determinations in terms of its analytical accuracy and precision, yet this alternative measurement approach does not necessitate the enhanced instrumentation or added expense required of tandem MS/MS determinations. LC/MS detection of homocysteine was linear (standard error of the estimate for the regression line was 0.0323) over 3 orders of magnitude, and the calculated limits of detection and quantification were 0.06 [micro]mol/L (0.12 ng on column) and 0.6 [micro]mol/L (1.2 ng on column), respectively. Independent calibration curves showed excellent linearity ([r.sup.2] [greater than or equal to] 0.996) between 0 and 25 [micro]mol/L homocysteine over a 3-day period. The accuracy and precision of total homocysteine measurements for patient samples and quality control pools using LC/MS were compared to total homocysteine measurements using LC/MS/MS, GC/MS, FPIA, and LC-FD. LC/MS performed well in relation to the other homocysteine methods in terms of its capability to accurately quantify plasma homocysteine over the normal range (5-15 [micro]mol/L).

Unless repaired, DNA damage can drive mutagenesis or cell death. DNA repair proteins may therefore be used as biomarkers in disease etiology or therapeutic response prediction. Thus, the accurate determination of DNA repair protein expression and genotype is of fundamental importance. Among DNA repair proteins involved in base excision repair, apurinic/apyrimidinic endonuclease 1 (APE1) is the major endonuclease in mammals and plays important roles in transcriptional regulation and modulating stress responses. Here, we present a novel approach involving LC-MS/MS with isotope-dilution to positively identify and accurately quantify APE1 in human cells and mouse tissue. A completely 15N-labeled full-length human APE1 was produced and used as an internal standard. Fourteen tryptic peptides of both human APE1 (hAPE1) and 15N-labeled hAPE1 were identified following trypsin digestion. These peptides matched the theoretical peptides expected from trypsin digestion and provided a statistically significant protein score that would unequivocally identify hAPE1. Using the developed methodology, APE1 was positively identified and quantified in nuclear and cytoplasmic extracts of multiple human cell lines and mouse liver using selected-reaction monitoring of typical mass transitions of the tryptic peptides. We also show that the methodology can be applied to the identification of hAPE1 variants found in the human population. The results describe a novel approach for the accurate measurement of wild-type and variant forms of hAPE1 in vivo, and ultimately for defining the role of this protein in disease development and treatment responses. [ABSTRACT FROM AUTHOR]

One primary challenge in nanotoxicology studies is the lack of well-characterised nanoparticle reference materials which could be used as positive or negative nanoparticle controls. The National Institute of Standards and Technology (NIST) has developed three gold nanoparticle (AuNP) reference materials (10, 30 and 60 nm). The genotoxicity of these nanoparticles was tested using HepG2 cells and calf-thymus DNA. DNA damage was assessed based on the specific and sensitive measurement of four oxidatively-modified DNA lesions (8-hydroxy-2´-deoxyguanosine, 8-hydroxy-2´-deoxyadenosine, (5´ S)-8,5´-cyclo-2´-deoxyadenosine and (5´ R)-8,5´-cyclo-2´-deoxyadenosine) using liquid chromatography/tandem mass spectrometry. Significantly elevated, dose-dependent DNA damage was not detected at concentrations up to 0.2 μg/ml, and free radicals were not detected using electron paramagnetic resonance spectroscopy. These data suggest that the NIST AuNPs could potentially serve as suitable negative-control nanoparticle reference materials for in vitro and in vivo genotoxicity studies. NIST AuNPs thus hold substantial promise for improving the reproducibility and reliability of nanoparticle genotoxicity studies. [ABSTRACT FROM AUTHOR]

Background: Vitamin B12 and folate measurements in serum show wide inter-methodology variability. This variability appears to be due in part to the lack of standardisation against internationally accepted reference materials. Pooled human serum, lyophilised in ampoules and designated 03/178, was therefore evaluated by 24 laboratories in seven countries for its suitability to serve as an International Standard (IS) for B12 and folate. Methods: IS 03/178 was assayed using a range of commercial analysers, microbiological assays and, for folate, candidate reference methods based on liquid chromatography coupled to isotope-dilution tandem mass spectrometry (LC/MS/MS). Results: Mean vitamin B12 and folate values for reconstituted 03/178 across all laboratories and methods were 480 pg/mL [coefficient of variation (CV) 12.8%] and 5.52 ng/mL (CV 17.1%), respectively. The total folate content of reconstituted 03/178, determined using LC/MS/MS, was 12.1 nmol/L (equivalent to 5.33 ng/mL), made up of 9.75 nmol/L 5-methyl tetrahydrofolic acid (5MeTHF; CV 5.5%), 1.59 nmol/L 5-formyl tetrahydrofolic acid (5FoTHF; CV 4.2%) and 0.74 nmol/L folic acid (FA; CV 31.6%). The inclusion of three serum samples in the study with different B12 and folate levels demonstrated a considerable reduction in inter-laboratory variability when the B12 and folate content of the samples was determined relative to the IS 03/178 rather than to the analyser calibration. IS 03/178 demonstrated satisfactory long-term stability in accelerated degradation studies. Conclusions: Use of IS 03/178 to standardise serum B12 and folate assays reduced inter-laboratory variability. The World Health Organization (WHO) Expert Committee on Biological Standardisation established 03/178 as the first IS for serum vitamin B12 and serum folate, with assigned values of 480 pg/mL of vitamin B12 and 12.1 nmol/L folate when the lyophilised contents of the ampoule are reconstituted with 1 mL of water. Clin Chem Lab Med 2007;45:380–6. [ABSTRACT FROM AUTHOR]

Abstract: Two different isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods for the quantitative determination of folic acid (FA) in multivitamin/multielement tablets are reported. These methods represent distinct improvements in terms of speed and specificity over most existing microbiological and chromatographic methods for the determination of FA in dietary supplements. The first method utilizes an aqueous/organic-based extraction solvent combined with positive-ion mode LC/MS/MS detection of protonated [M+H]+ FA molecules and the second method utilizes a pure aqueous-based extraction solvent combined with negative-ion mode LC/MS/MS detection of deprotonated [M−H]− FA molecules. The LC/MS/MS methods exhibit comparable linear dynamic ranges (≥3 orders of magnitude), limits of detection (0.02ng on-column) and limits of quantification (0.06ng on-column) for FA. Two methods employing different extraction and different MS detection modes were developed to allow method cross-validation. Successful validation of each measurement procedure supports the use of either method for the certification of FA levels in dietary supplements. The accuracy and precision of each measurement procedure were evaluated by applying each method to the quantitative determination of FA in a NIST standard reference material (NIST SRM 3280 multivitamin/multielement tablets). The FA measurement accuracy for both methods was ≥95% (based on the manufacturer''s assessment of the FA level in SRM 3280) with corresponding measurement precision values (% RSD) of approximately 1%. [Copyright &y& Elsevier]

Total homocysteine (tHCY) and folate are interrelated biomarkers for arteriosclerosis and coronary heart disease. Although many different methods for both tHCY and folate are clinically available, the intermethod and interlaboratory results are often poor, resulting in the need for a matrix reference material and reference methods. The National Institute of Standards and Technology (NIST) has developed isotope dilution liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/ tandem mass spectrometry (LC/MS/MS) methods for determination of tHCY and several folate forms including 5-methyltetrahydrofolic acid (5MT) and folic acid (FA). Additionally, a method for simultaneous measurement of tHCY, 5MT, and FA has been developed and validated. In collaboration with the Centers for Disease Control and Prevention (CDC), mass spectrometric methods and methods used in clinical laboratories have been applied to characterize a new Standard Reference Material (SRM), SRM 1955, “Homocysteine and Folate in Human Serum,” containing low, medium, and high levels of tHCY and 5MT. Additionally, FA, 5-formyltetrahydrofolic acid (5FT), vitamin B12, and total folate values are provided. Use of the new SRM should improve clinical measurements and will permit traceability to internationally recognized certified reference materials, as described by European Directive 98/79/EC on in vitro diagnostic medical devices. [ABSTRACT FROM AUTHOR]

A unified extraction and quantification procedure based on stable isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the simultaneous determination of total homocysteine and folate (5-methyltetrahydrofolic acid and folic add) levels in human serum and plasma. This is the first report documenting the simultaneous extraction and quantification of these structurally dissimilar analytes. Analytes are quantitatively isolated from samples (500 μL) prior to LC/ MS/MS analysis using a two-step stabilization process combined with C18 solid-phase extraction. The method exhibits excellent linearity over 4 orders of magnitude for each analyte. Measurement repeatability (RSD, N 2) ranged from 0.3% to 3% for all analytes over 1 day of analysis. Total method variability (RSD, N 6) ranged from 0.7% to 10% for all analytes over three independent days of analysis. The accuracy and practical applicability of the method were demonstrated by applying the method to the quantitative determination of each analyte in a new NIST serum Standard Reference Material (NIST SRM 1955 Homocysteine and Folate in Frozen Human Serum) and in a small subset of normal donor plasma samples. [ABSTRACT FROM AUTHOR]

The measurement of 5-methyltetrahydrofolic acid (5MT) blood levels is one of several factors used to diagnose folate deficiency in humans. 5MT can be selectively purified from either human plasma or human serum via solid-phase extraction procedures and specifically detected and quantified in the extracts with liquid chromatography/isotope-dilution electrospray-ionization mass spectrometry. Two different, yet complementary, solid-phase extraction–liquid chromatography/mass spectrometry methods have been developed and applied to the quantification of 5MT from such extracts. One method utilizes the high-affinity folate-binding protein from cow’s milk coupled with multiple-reaction-monitoring-mode tandem mass spectrometry while the other method utilizes reversed-phase C18 extraction followed by selected-ion-monitoring-mode mass spectrometry. The accuracy of each method is assessed through a comparative determination of 5MT levels in homogenous plasma and serum pools. Additionally, each method is compared and evaluated against the “total folate” results provided by routine radioassay and microbiological assay determinations. On the basis of the experimental data presented in this report, it is suggested that both methods have the capacity to serve as potential reference methods for the quantification of circulating 5MT in plasma or serum. [Copyright &y& Elsevier]

The predominant circulating folate monoglutamate in human plasma (>90%), and thus the most significant folate for accurately diagnosing folate deficiency, is 5-methyltetrahydrofolic acid (5MT). Folate deficiency is typically indicated when circulating folate levels are ⩽3 ng/mL. The quantitative determination of plasma folates in general, and of 5MT in particular, is complicated by their naturally low levels (pg/mL to ng/mL), their instability, and their tendency to interconvert. Highly specific and sensitive analytical methods are needed to accurately quantify endogenous 5MT in human plasma. A method that utilizes the specific high-affinity binding sites of bovine folate binding protein (FBP) and the selectivity and sensitivity of selected ion monitoring mode isotope-dilution liquid chromatography/mass spectrometry (LC/MS) to quantify plasma 5MT has been developed. The method is based on the solid-phase affinity extraction (SPAE) of 5MT and its stable isotopically labeled analogue ([13C5]5MT) from plasma (1 mL) using FBP immobilized to polymeric beads. The excess high-affinity binding sites on the affinity columns enable quantitative extraction of 5MT from plasma under optimized sample pH conditions. Additionally, it is demonstrated that plasma proteins do not hinder the determination of 5MT; therefore, protein precipitation is not required before the affinity extraction step. Detection and quantification of the extracted 5MT is provided by positive-ion mode LC/MS in which the protonated molecular ions [M+H]+ of the analyte and the internal standard are monitored. The method shows linearity over three orders of magnitude (0.04–40 ng/mL) and has limits of detection and quantification of 0.04 and 0.4 ng/mL, respectively. Calibration curves obtained by spiking 5MT into plasma exhibited good linearity between 0 and 25 ng/mL and both the plasma calibration standards and the plasma samples were stable for at least 48 h at room temperature. The recovery (average ± % RSD) of 5MT spiked into plasma from 5 to 25 ng/mL was 98.0%±1.6% (n=15). 5MT levels determined by SPAE-LC/MS compared to “total folate” levels determined by radioassay and microbiological assay were discordant. Reasons for the discordancy are theorized, but it is clear that there exists an urgent need for clinical reference materials containing certified folate levels. [Copyright &y& Elsevier]

Examines the separation and identification of catechins in tea using liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry. Detection of protonated molecular ions; Establishment of the identities of major catechins and caffeine in tea; Determination of polyphenols in natural products.

Abstract: Oxidatively induced DNA lesions (5′R)- and (5′S)-8,5′-cyclo-2′-deoxyadenosines (R-cdA and S-cdA) are detectable and accumulate in vivo due to disease states and defects in DNA repair. They block transcription and inhibit gene expression, and may play a role in disease processes. Accurate measurement of these lesions in DNA in vivo is necessary to understand their biological effects. We report on a methodology using liquid chromatography/isotope-dilution tandem mass spectrometry to measure R-cdA and S-cdA in DNA. This methodology permitted the detection of these compounds at a level of 0.1fmol on-column. Levels of R-cdA and S-cdA in mouse liver DNA amounted to 0.133±0.024 and 0.498±0.065 molecules/107 DNA 2′-deoxynucleosides, respectively. The successful measurement of R-cdA and S-cdA in DNA in vivo suggests that this methodology will be used for understanding of their repair and biological consequences, and that these compounds may be used as putative biomarkers for disease states. [Copyright &y& Elsevier]