Quantification of modified nucleotides and nucleosides by isotope dilution mass spectrometry.

Epigenetic modifications are closely related to certain disorders of the organism, including the development of tumors. One of the main epigenetic modifications is the methylation of DNA cytosines, 5-methyl-2'-deoxycycytidine. Furthermore, 5-mdC can be oxidized to form three new modifications, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, and 5-carboxy-2'-deoxycytidine. The coupling of liquid chromatography with tandem mass spectrometry has been widely used for the total determination of methylated DNA cytosines in samples of biological and clinical interest. These methods are based on the measurement of the free compounds (e.g., urine) or after complete hydrolysis of the DNA (e.g., tissues) followed by a preconcentration, derivatization, and/or clean-up step. This review highlights the main advances in the quantification of modified nucleotides and nucleosides by isotope dilution using isotopically labeled analogs combined with liquid or gas chromatography coupled to mass spectrometry reported in the last 20 years. The different possible sources of labeled compounds are indicated. Special emphasis has been placed on the different types of chromatography commonly used (reverse phase and hydrophilic interaction liquid chromatography) and the derivatization methods developed to enhance chromatographic resolution and ionization efficiency. We have also revised the application of bidimensional chromatography and indicated significant biological and clinical applications of these determinations.

Evaluation of different isotope dilution mass spectrometry strategies for the characterization of naturally abundant and isotopically labelled peptide standards.

Natural abundance and isotopically labelled tryptic peptides are routinely employed as standards in quantitative proteomics. The certification of the peptide content is usually carried out by amino acid analysis using isotope dilution mass spectrometry (IDMS) after the acid hydrolysis of the peptide. For the validation and traceability of the amino acid analysis procedure, expensive certified peptides must be employed. In this work we evaluate different IDMS alternatives which will reduce the amount of certified peptide required for validation of the amino acid analysis procedure. In this context, the characterization of both natural and isotopically labelled synthetic angiotensin I peptides was carried out. First, we applied a fast procedure for peptide hydrolysis based on microwave-assisted digestion and employed two certified peptide reference materials SRM 998 angiotensin I and CRM 6901-b C-peptide for validation of the hydrolysis procedure. The amino acids proline, leucine, isoleucine, valine, tyrosine, arginine and phenylalanine were evaluated for their suitability for peptide certification by IDMS by both liquid chromatography with tandem mass spectrometry (LC-MS/MS) and gas chromatography with mass spectrometry (GC)-MS/MS. Then, natural angiotensin I and 13C1-labelled angiotensin I were synthesized in-house and purified by preparative liquid chromatography. The concentration of the 13C1-labelled angiotensin I peptide was established by reverse IDMS in its native form using SRM 998 angiotensin I as reference. The concentration of the natural synthesized peptide was determined by IDMS both using the 13C1-labelled peptide in its native form and by amino acid analysis showing comparable results. Finally, the synthetic naturally abundant angiotensin I peptide was employed as "in-house" standard for the validation of subsequent peptide characterization procedures. Therefore, the novelty of this work relies on, first, the development of a faster hydrolysis procedure assisted by focused microwaves, providing complete hydrolysis in 150 min, and secondly, a validation strategy combining GC-MS and LC-MS/MS that allowed us to certify the purity of an in-house-synthesized peptide standard that can be employed as quality control in further experiments.

Comparison between one and two-dimensional liquid chromatographic approaches for the determination of plasmatic stroke biomarkers by isotope dilution and tandem mass spectrometry.

This work presents the evaluation of one- and two-dimensional liquid chromatography for the quantification of three stroke outcome predictors in plasma. Isotopically labelled analogues of L-arginine (L-Arg), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are used to quantify the three analytes by isotope dilution and tandem mass spectrometry. Chromatographic isotope effects were not observed between natural L-Arg and its 15N-labelled analogue but they were observed between natural ADMA and SDMA and their multiple deuterated analogues. Under these conditions, bidimensional chromatography through the use of an automated multiple heart cutting mode provided unsatisfactory results for ADMA and SDMA due to the different amounts of natural and labelled compounds transferred from the first to the second chromatographic dimension. In contrast, using one dimensional liquid chromatography after a derivatization step to esterify carboxylic groups, chromatographic isotope effects did not alter the initial mass balance as full coelution of natural and labelled analogues or baseline resolution between the analytes was not required. This method was successfully validated following the Clinical & Laboratory Standards Institute guidelines and applied to the analysis of plasma samples from patients who had suffered from an intraparenchymal haemorrhagic stroke.

Comparison of GC-NCI MS, GC-ICP-MS, and GC-EI MS-MS for the determination of PBDEs in water samples according to the Water Framework Directive.

The Water Framework Directive (WFD) includes some polybrominated diphenyl ethers (PBDEs) in the list of priority substances that must be measured in surface waters at very low concentrations. The typical approaches applied to the determination of PBDEs in environmental samples might not meet the demanding requirements of the WFD. In this work, the instrumental capabilities of the mass-spectrometry (MS) techniques most frequently used in the determination of PBDEs, namely gas chromatography-negative-chemical-ionisation MS (GC-NCI MS) and GC-electrospray-ionisation tandem MS (EI MS-MS), are evaluated in comparison with highly sensitive GC-inductively-coupled-plasma MS (ICP-MS) for the reliable determination of PBDEs according to the WFD. Three analytical methods based on the liquid-liquid extraction of water samples and measurement of the extracts by GC-NCI MS, GC-EI MS-MS, or GC-ICP-MS are described. The priority PBDEs were quantified in different types of water sample by means of isotope-dilution mass spectrometry (IDMS) using (81)Br-labelled or (13)C-labelled PBDEs spikes, depending on the selected ionisation source. The three proposed methods met the requirements of the European legislation in terms of LOQs and expanded uncertainties. The determination method using (81)Br-labelled PBDEs and GC-ICP-MS had the highest sensitivity and the lowest instrumental limits of detection and expanded uncertainties.

Correction of creatine-creatinine conversion during serum creatinine quantification by two-dimensional liquid chromatography and double-spike isotope dilution tandem mass spectrometry.

BACKGROUND AND AIMS: Development of a candidate reference method based on bidimensional liquid chromatography coupled to ESI-MS/MS and double spike isotope dilution for serum creatinine quantification capable of correcting for creatinine-creatine interconversion during sample pretreatment. Study of the impact of the creatine-creatinine interconversion during the analysis of human serum samples. MATERIALS AND METHODS: 13C1-creatinine and 13C2-creatine are added to the serum sample. Separation carried out by bidimensional liquid chromatography combining reversed phase and a strong cation exchange chromatography. The heart cut, containing creatine and creatinine, is automatically transferred to the second dimension. Quantification carried out by double spike isotope dilution tandem MS/MS. RESULTS: Minimization of spectral interferences and ion suppression due to matrix effects while increasing sample throughput compared to the direct coupling of cation exchange chromatography to the ESI source. Trueness of the method studied with the satisfactory analysis of two certified reference materials. Satisfactory intra- and inter-day precisions obtained analysing a serum pool and control sera. Analysis of 93 serum samples revealed negligible interconversions with no correlation with creatine levels. CONCLUSIONS: The method provides adequate analytical figures of merit for serum creatinine determination according to CSLI guidelines. Negligible creatine-creatinine interconversion is promoted with the applied sample preparation procedure.

Development and evaluation of an electrochemical biosensor for creatinine quantification in a drop of whole human blood.

An electrochemical biosensor for creatinine determination in a drop of whole human blood was developed and applied to the determination of creatinine in real clinical samples. It is based on the modification of a dual carbon working electrode with a combination of three enzymes: creatinine amidohydrolase (CNN), creatine amidinohydrolase (CRN) and sarcosine oxidase (SOX). Electrochemical transduction is performed using horseradish peroxidase (HRP) and potassium hexacyanoferrate(II) as mediator. A drop of human blood is enough to carry out the measurements by differential chronoamperometry where one carbon electrode detects creatine and the other both creatine and creatinine. The integrated differential signal obtained in the biosensor is linear with the concentration of creatinine in blood in the range 0.5-15 mg/dL and the enzyme-modified electrodes are stable for at least 3 months at 4 °C. The biosensor was lined to a reference method based on Isotope Dilution Mass Spectrometry (IDMS) with 50 real human blood samples and the results compared with those obtained by alternative routine techniques based on Jaffé method and an enzymatic method (Cobas 8000 Roche®, Crep2 Roche®). There were no significant differences between the creatinine concentrations found by the routine techniques and the developed biosensor.

Determination of priority polybrominated diphenyl ethers by isotope dilution gas chromatography(electron ionization)MS using 81Br-labeled standards.

A mixture of (81)Br-labeled polybrominated diphenyl ethers (PBDEs), previously synthesized in our laboratory, was separated by liquid chromatography for the individual isolation of different (81)Br-labeled PBDEs containing from 3 to 6 bromine atoms. The different fractions were collected, and a mixed labeled standard was then prepared adequate for the determination of priority PBDEs (congeners 28, 47, 99, 100, 153, and 154) in environmental samples. The spike mixture was then characterized using gas chromatography(electron ionization)MS (GC(EI)MS) both in isotope composition and concentration in combination with multiple least-squares. Contamination from natural abundance BDEs 153 and 154 was detected in the spike mixture, and a new isotope dilution equation was developed to take into account the natural abundance contribution from the spike. The spike mixture was shown to be stable during at least 4 months, and no isotope exchange between natural abundance and labeled PBDEs was detected during this period of time. Finally, the (81)Br-labeled PBDEs standard was used for the determination of congeners 28 (+33), 47, 49, 99, 100, 153, and 154 in a standard reference material (Lake Michigan fish tissue SRM 1947) using three different sample to spike ratios. No methodological calibration needed to be prepared, as no isotopic effects were detected using this labeling mode. Concentrations found were in agreement with the certified concentrations (recoveries between 89% and 116%), and reproducibility was always below 7% RSD. Kragten procedure was used to calculate expanded uncertainties. Very low limits of detection were obtained for all compounds (between 0.02 and 0.9 ng·g(-1)) using the procedure developed here.

Determination of ultra-trace levels of priority PBDEs in water samples by isotope dilution GC(ECNI)MS using 81Br-labelled standards.

A gas chromatography electron capture negative ionization mass spectrometry (GC(ECNI)MS) procedure for the determination of priority polybrominated diphenyl ethers (PBDEs; congeners 28, 47, 99, 100, 153 and 154) in water samples at regulatory EU levels has been developed. The method is based on the use of (81)Br-labelled PBDEs for isotope dilution analysis and the measurement of (79)Br/(81)Br isotope ratios in gas chromatography peaks with the electron capture negative ionization technique. The suitability of this ion source for the precise and accurate measurement of bromine isotope ratios has been demonstrated. The general ECNI-IDMS procedure was evaluated by the analysis of NIST SRM 1947 (Lake Michigan fish tissue) with satisfactory results. For the analysis of water samples, 500 mL of the samples were spiked with the labelled PBDEs and extracted with 10 mL isooctane for 30 min. The extract was evaporated down to ca. 100 µL and injected in the GC(ECNI)MS. Detection limits ranged from 0.014 (-1) to 0.089 pg mL(-1) depending on the congener. Recoveries from real water samples, spiked at a level of 0.5 pg mL(-1), ranged from 77% to 102%.

Synthesis of 81Br-labeled polybrominated diphenyl ethers and their characterization using GC(EI)MS and GC(ICP)MS.

A mixture of different (81)Br-labeled polybrominated diphenyl ethers (PBDEs) was prepared and characterized for its future use as spike for the isotope dilution analysis of PBDEs. The synthesis was carried out by direct bromination of diphenyl ether using (81)Br enriched Br(2) obtained after aqueous oxidation of bromide with potassium peroxymonosulfate and extraction into dichloromethane. The number of bromine atoms introduced in the diphenyl ether molecule depended on the molar ratio between bromine and diphenyl ether. The final mixture prepared contained a mixture of tri-, tetra-, penta-, and hexabrominated PBDEs with a larger concentration of the tetrabrominated congener BDE-47. The isotopic composition of bromine in the resulting PBDEs mixture was determined by GC(ICP)MS and resulted in a 99.53% enrichment of the isotope 81 of bromine. The concentration of three of the PBDE congeners (28, 47, and 99) in the mixture was determined by reverse isotope dilution analysis using a certified, natural abundance, PBDEs mixture and both GC(ICP)MS and GC(EI)MS. For this purpose, the fragmentation and isotope distribution patterns of the different PBDE cogeners in the positive electron ionization source were studied in detail both for natural abundance and labeled compounds. A procedure based on isotope pattern deconvolution was developed which allowed the direct determination of the concentration of the labeled PBDEs in the spike mixture by GC(EI)MS. Finally, the GC(EI)MS isotope pattern deconvolution procedure was applied for the determination of natural abundance congeners 28, 47, and 99 in spiked waters at ng L(-1) levels. Detection limits below 0.5 ng L(-1) could be obtained for all compounds using only 100 mL of sample and liquid-liquid extraction with isooctane.

Isotope dilution LC-ESI-MS/MS and low resolution selected reaction monitoring as a tool for the accurate quantification of urinary testosterone.

A new analytical method for the quantification of testosterone in human urine samples by isotope dilution mass spectrometry is proposed. A standard solution of 13C2-testosterone is added to the samples at the beginning of the sample preparation procedure and then the measurements are carried out by UHPLC-ESI-MS/MS. In the proposed method, the resolution of the first quadrupole of the tandem MS instrument is reduced to transmit the whole precursor ion cluster to the collision cell and measure the isotopic distribution of the in-cell product ions with a small number of SRM transitions. The construction of a methodological calibration graph is avoided using a labelled analogue previously characterised in terms of concentration and isotopic enrichment in combination with multiple linear regression. In this way, the molar fractions of natural and labelled testosterone are calculated in each sample injection and the amount of endogenous testosterone computed from the known amount of labelled analogue. Recovery values between 97 and 107% and precisions between 0.4 and 3.7% (as %RSD) were obtained for testosterone concentrations in urine in the range of 1 to 8 ng g-1. The proposed low resolution SRM methodology was compared for the analysis of human urine samples with the traditional IDMS method based on a calibration graph and the IDMS method based on multiple linear regression combined with standard resolution SRM. A similar accuracy and precision was obtained by the three tested approaches. However, using the low resolution SRM method there was no need to resort to calibration graphs or to specific dedicated software to calculate isotopic distributions by tandem MS and a higher sensitivity was obtained. The proposed low resolution SRM method was successfully applied to the analysis of the certified freeze-dried human urine NMIA MX005.

Evaluation of uncertainty sources in the determination of testosterone in urine by calibration-based and isotope dilution quantification using ultra high performance liquid chromatography tandem mass spectrometry.

Three quantification methodologies, namely calibration with internal standard (Cal-IS, non-weighted), weighted calibration with internal standard (wCal-IS) and isotope pattern deconvolution (IPD) have been used for the determination of testosterone in urine by LC-MS/MS. Uncertainty has been calculated and compared for the three methodologies through intra- and inter-laboratory reproducibility assays. IPD showed the best performance for the intra-laboratory reproducibility, with RSD and combined uncertainty values below 4% and 9% respectively. wCal-IS showed similar performance, while Cal-IS where not constant and clearly worse at the lowest concentration assayed (2ng/mL) reaching RSD values up to 16%. The inter-laboratory assay indicated similar results although wCal-IS RSD (20%) was higher than IPD (10%) and Cal-IS get worse with RSD higher than 40% for the lowest concentration level. Uncertainty budgets calculated for the three procedures revealed that intercept and slope were the most important factors contributing to uncertainty for Cal-IS. The main factors for wCal-IS and IPD were the volumes of sample and/or standard measured.

Potential reference measurement procedures for PBDE in surface water at levels required by the EU Water Frame Directive.

Polybrominated diphenylethers (PBDE), used as flame retardants, are named as priority substances in the Directive 2000/60/EC of the European parliament and of the council establishing a framework for Community action in the field of water policy. An annual average environmental quality standard (EQS) for inland surface waters of 0.0005 µg/L (0.0002 µg/L for other surface waters) for PBDE congeners involved in the technical penta-PBDE mixtures containing PBDE with five bromine atoms has been established. The directives focus especially on the congeners PBDE 28, 47, 99, 100, 153 and 154 contained in the penta-PBDE mixture. Up to now, no reference measurement procedures have been established reaching the limits of quantification (LOQs) and the associated uncertainties as defined in the directives with results traceable to the SI. Within a recent European project on metrology, different approaches for the traceable quantification of PBDE, based on liquid/liquid or solid phase extraction followed by the detection with gas chromatography coupled to either inductively coupled plasma mass spectrometry or triple quadrupole mass spectrometry, were investigated and the related LOQs and expanded uncertainties of the results were compared. A complete uncertainty budget for each method was estimated according to the Guide to the Expression of Uncertainty in Measurement (GUM). All presented analytical procedures can serve as reference measurement procedures regarding the LOQs and their associated expanded uncertainties for monitoring the six priority PBDEs named above. LOQs as low as 0.026 ng/kg with an associated expanded uncertainty of 0.002 ng/kg could be achieved.

Evaluation of the spectral accuracy of mass spectrometers using compounds containing Cl or Br atoms.

Current procedures for the evaluation of spectral accuracy of mass spectrometers are limited by the lack of certified isotopic reference materials and the high uncertainty in the isotopic composition of natural abundance molecules. The calculated uncertainties in the ratio M + 1/M for natural abundance molecules containing any number of C, H, N and/or O atoms are close to 5% relative because of the natural variability of the isotopic composition of carbon. So, we have developed two alternative measurement procedures with much lower theoretical uncertainties for a better evaluation of spectral accuracy in both single and triple quadrupole analysers. The first method is based on the measurement of the M + 2/M, M + 4/M + 2, etc. ratios for halogenated organic compounds containing either Cl or Br. The theoretical uncertainties for these ratios because of natural variability are in the order of 0.3 to 1.0% making them suitable for the evaluation of spectral accuracy with the additional advantage that there is no need to take into account other limitations such as cluster purity or poor mass resolution. This procedure was applied to the evaluation of a single quadrupole GC-MS instruments using natural abundance PCB and PBDE standards with satisfactory results. The second method can be applied to tandem instruments and takes advantage of the loss of two halogen atoms when PCB and PBDE standards are fragmented by Collision Induced Dissociation. Theoretical SRM transition ratios can be calculated as a pure combinatorial probability with theoretical uncertainties lower than 0.1%. By combining PCBs and PBDEs with different number of halogen atoms, a mass range from 100 to 700 u and abundance ratios from 0.1 to 10 can be evaluated. The use of penta-chlorinated PCBs and/or penta-brominated PBDEs is finally recommended for the evaluation of spectral accuracy of mass spectrometers with the EI source. Copyright © 2016 John Wiley & Sons, Ltd.