Stable Isotope-labeled Internal Standards Research Articles

Protein Biomarker Quantification by Immunoaffinity Liquid Chromatography-Tandem Mass Spectrometry: Current State and Future Vision.

Immunoaffinity-mass spectrometry (IA-MS) is an emerging analytical genre with several advantages for profiling and determination of protein biomarkers. Because IA-MS combines affinity capture, analogous to ligand binding assays (LBAs), with mass spectrometry (MS) detection, this platform is often described using the term hybrid methods. The purpose of this report is to provide an overview of the principles of IA-MS and to demonstrate, through application, the unique power and potential of this technology. By combining target immunoaffinity enrichment with the use of stable isotope-labeled internal standards and MS detection, IA-MS achieves high sensitivity while providing unparalleled specificity for the quantification of protein biomarkers in fluids and tissues. In recent years, significant uptake of IA-MS has occurred in the pharmaceutical industry, particularly in the early stages of clinical development, enabling biomarker measurement previously considered unattainable. By comparison, IA-MS adoption by CLIA laboratories has occurred more slowly. Current barriers to IA-MS use and opportunities for expanded adoption are discussed. The path forward involves identifying applications for which IA-MS is the best option compared with LBA or MS technologies alone. IA-MS will continue to benefit from advances in reagent generation, more sensitive and higher throughput MS technologies, and continued growth in use by the broader analytical community. Collectively, the pursuit of these opportunities will secure expanded long-term use of IA-MS for clinical applications.

The application of isotopically labeled analogues for the determination of small organic compounds by GC/MS with selected ion monitoring.

The study aimed to show the limitations and advantages of the use of stable isotope labeled internal standards (SILISs) for the quantification of small polar compounds (ibuprofen, diclofenac, metoprolol, bisphenol A, 17β-estradiol) in water samples by GC/MS with selected ion monitoring (SIM). The selection of SIM ions shows that for some of the analytes, in the form of trimethylsilylated derivatives, it is problematic to have a minimum of two qualifiers because of the poor mass spectra, high impact of the background or overlapping of the peaks from the analytes and SILISs. The "isotope effect" was observed on GC chromatograms. During the development of the SIM method, special attention was given to the qualifier/quantifier ratio, for which a variety of acceptance criteria can be found in the official guidelines. The values of the solid-phase extraction efficiency, as well as the matrix effect and absolute recovery were the same for the analytes and their corresponding SILISs. The developed method was used for the quantification of analytes in wastewaters.

Liquid chromatography quadrupole-Orbitrap mass spectrometry for the simultaneous analysis of advanced glycation end products and protein-derived cross-links in food and biological matrices

Advanced glycation end products (AGEs) and protein cross-links have been extensively investigated in both food and biomedical fields over the past years. Although there are a few chromatographic and immunological methods for the analysis of selected AGEs, there is no method available for comprehensive simultaneous analysis of major AGEs found in processed foods and biological samples. In the present study, we have reported a validated UHPLC-MS/MS method for simultaneous identification and quantification of 15 different AGEs, furosine (an indicator of Amadori products), 2 protein-derived cross-links (lanthionine and lysinoalanine) and 2 amino acids (Lys and Arg). The analytes were separated on a reversed phase C-18 column and quantified accurately based on the isotope dilution method, where 9 stable isotope-labelled internal standards were used to quantify 20 different analytes using an Orbitrap mass analyzer. The method showed acceptable linearity, accuracy and precision. The LOD and LOQ values in plasma were in the range of 0.30–19.02 and 0.87–57.06 ng/mL, respectively. The recovery values at the three spiked levels were in the range of 71–110%, with some exceptions. The intraday and interday precision were in the range of 1.5–13.2%, however, quantification of N-ɛ-(carboxymethyl)lysine accompanied slightly higher interday precision (30.7%). The applicability of the method was successfully assessed by analyzing AGEs and protein cross-links in six different complex matrices including Ultra-High Temperature (UHT) processed milk, roasted chicken breast meat, roasted chicken skin, roasted pork liver, bovine plasma and perfusion liquid.

Direct Quantification of Natural Moisturizing Factors in Stratum Corneum using Direct Analysis in Real Time Mass Spectrometry with Inkjet-Printing Technique

Proper hydration of the stratum corneum, the skin’s outermost layer, is essential for healthy skin. Water-soluble substances called natural moisturizing factors (NMF) are responsible for maintaining adequate moisture in the skin and are closely associated with a variety of the skin’s functions. Therefore, quantitative analysis methods for NMF are indispensable when attempting to clarify one of the mechanisms of hydration and its effect on the skin. This study sought to develop a quick and simple analytical technique, which can quantify NMF from the skin without the need for extraction or separation, using direct analysis in real time-mass spectrometry (DART-MS). The goal was to deliver a high quantitative capability, so a unique inkjet printing technique was employed to evenly coat a stable isotope-labeled internal standard (SIL-IS) on tape-stripped skin. This technique allowed for the quantification of 26 NMF with established calibration curves and comparatively high linear correlations. The speed of measurement was found to be advantageous as 100 strips of tape can be measured in roughly 2 hours. The effectiveness of the inkjet coating was also verified by comparing its precision with that of conventional pipetting. This new technique can be an alternative method to quantify NMF rapidly and perhaps allow for a clearer elucidation of their function in skin.

Validation of a sensitive UHPLC-MS/MS method for cytochrome P450 probe substrates caffeine, tolbutamide, dextromethorphan, and alprazolam in human serum reveals drug contamination of serum used for research

To evaluate the potential for interactions between botanical dietary supplements and drug metabolism, Phase I clinical pharmacokinetics studies are conducted using an oral cocktail of probe substrates of cytochrome P450 (CYP) enzymes. A sensitive, specific, and fast ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for determination of caffeine (probe of CYP1A2), tolbutamide (probe of CYP2C9), dextromethorphan (probe of CYP2D6), and alprazolam (probe of CYP3A4/5) in human serum. Stable isotope-labelled analogs were used as internal standards, and sample preparation involved only rapid protein precipitation and centrifugation. The method of standard addition was used for the measurement of caffeine, because commercially available pooled human serum contains caffeine. Out of 18 lots of pooled human serum tested, caffeine was detection in all lots, alprazolam was detected in 13 lots, 8 lots contained dextromethorphan, and no tolbutamide was detected. Only serum prepared from the blood of select individuals was determined to be drug-free. The analytical method was validated with respect to linearity, accuracy and precision, recovery, stability, and matrix effects. The calibration curves were linear over the range of 25-12,000 ng/mL for caffeine, 75-36,000 ng/mL for tolbutamide, 0.05−30 ng/mL for dextromethorphan, and 0.1−60 ng/mL for alprazolam. The intra-assay and inter-assay coefficients of variation (%CV) and %Bias were <13 % (<17 % at the lower limit of quantitation). The recovery of each probe substrate ranged from 84.2%–98.5 %. All analytes were stable during sample storage and handling. Matrix effects were minimized by using stable isotope-labeled internal standards. The method was successfully applied to clinical studies investigating the pharmacokinetic alterations of probe substrates caused by chronic consumption of botanical dietary supplements.

Red blood cell metabolism in Rhesus macaques and humans: comparative biology of blood storage.

Macaques are emerging as a critical animal model in transfusion medicine, because of their evolutionary similarity to humans and perceived utility in discovery and translational science. However, little is known about the metabolism of Rhesus macaque red blood cells (RBC) and how this compares to human RBC metabolism under standard blood banking conditions. Metabolomic and lipidomic analyses, and tracing experiments with [1,2,3-13C3]glucose, were performed using fresh and stored RBC (sampled weekly until storage day 42) obtained from Rhesus macaques (n=20) and healthy human volunteers (n=21). These results were further validated with targeted quantification against stable isotope-labeled internal standards. Metabolomic analyses demonstrated inter-species differences in RBC metabolism independent of refrigerated storage. Although similar trends were observed throughout storage for several metabolic pathways, species- and sex-specific differences were also observed. The most notable differences were in glutathione and sulfur metabolites, purine and lipid oxidation metabolites, acylcarnitines, fatty acyl composition of several classes of lipids (including phosphatidylserines), glyoxylate pathway intermediates, and arginine and carboxylic acid metabolites. Species-specific dietary and environmental compounds were also detected. Overall, the results suggest an increased basal and refrigerator-storage-induced propensity for oxidant stress and lipid remodeling in Rhesus macaque RBC cells, as compared to human red cells. The overlap between Rhesus macaque and human RBC metabolic phenotypes suggests the potential utility of a translational model for simple RBC transfusions, although inter-species storage-dependent differences need to be considered when modeling complex disease states, such as transfusion in trauma/hemorrhagic shock models.

Effective quantification of 11 tyrosine kinase inhibitors and caffeine in human plasma by validated LC-MS/MS method with potent phospholipids clean-up procedure. Application to therapeutic drug monitoring

Therapeutic drug monitoring (TDM) help to improve treatment efficacy and safety. Therefore, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous monitoring of 11 tyrosine kinase inhibitors (TKIs) in human plasma. TKIs included in the assay are used in the treatment of chronic myeloid leukemia (CML: imatinib, dasatinib, nilotinib, bosutinib, ponatinib), polycythemia vera (ruxolitinib), chronic lymphocytic leukemia (ibrutinib) and rheumatoid arthritis (filgotinib, tofacitinib, baricitinib, peficitinib). Caffeine was also included in the method. Caffeine increases the acidity of the stomach and decreases its pH as well as is a competitive inhibitor of cytochrome P450 isoenzymes. Thus, it may influence absorption and metabolism of some TKIs, by modifying their plasma levels. The analytes of interest and their stable isotope-labeled internal standards were extracted from 200 μL of human plasma. Microelution-solid phase extraction (μ-SPE) was optimized for method validation and compared to simple protein precipitation (PPT). A gradient elution on a Poroshell 120 EC-C18 column at 60 °C and a flow rate of 0.5 mL/min was applied for analyte separation. The analytical run lasted 8 min and it was followed by a re-equilibration time of 4 min. Dynamic multiple reaction monitoring scan in the positive ionization mode was applied to improve method sensitivity. Endogenous plasma phospholipids can strongly affect MS analysis. Hence, the monitoring of endogenous phospholipids was included in the assay. Full validation of the method was achieved, including tests of precision, accuracy, trueness, linearity, extraction recovery, matrix effect, process efficiency, stability, sensitivity (with excellent LLOQs), selectivity, identity confirmation and carry-over effect. Regarding sample cleanup, more than 91% of early eluting and more than 96% of late eluting endogenous phospholipids were eliminated by μ-SPE when compared to PPT. This method enables the simultaneous plasma monitoring of 11 TKIs and caffeine and ensures high effectiveness in phospholipids elimination. The present approach is currently used in our clinical practice, being applied to TDM of dasatinib, imatinib, nilotinib and ponatinib. TKIs plasma monitoring helps to individualize dose adjustment and manage adverse effects in CML patients.

Quantitative ultra-high-performance liquid chromatography-tandem mass spectrometry for determination of dexmedetomidine in pediatric plasma samples: Correlation with genetic polymorphisms.

Dexmedetomidine is an important sedative agent administered as premedication to achieve procedural sedation in children. To describe the correlation between the genetic state and the concentration of dexmedetomidine, it is necessary to develop a specific, time-saving and economical method for detection of dexmedetomidine in plasma samples. In this work, an ultra-high-performance liquid chromatography (UHPLC)-tandem mass spectrometry method has been established and validated for detection of dexmedetomidine in plasma from pediatric population. After a simple liquid-liquid extraction with an organic solution, the analytes were separated on an ACQUITY BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size) by gradient elution with the mobile phase of acetonitrile and 1‰ aqueous formic acid (flow rate 0.3 mL min-1 ). Mass spectrometry measurements were performed under the positive selected reaction monitoring and the mass transitions monitored were m/z 201.3 → 95.1, 204.2 → 98.0 for dexmedetomidine and deuterated medetomidine (internal standard), respectively. Validation of the method based on China Food and Drug Administration guidelines showed acceptable selectivity. The UHPLC method employed a stable isotope-labeled internal standard, showed good specificity and was successfully used to detect dexmedetomidine in plasma samples from 260 pediatric patients. A subsequent application of this method to a pharmacogenetic study was also described. Importantly, this is the first study to report the correlation between CYP2A6 rs835309 activity and concentration of dexmedetomidine.

Simple Fast Quantification of Cholecalciferol, 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D in Adipose Tissue Using LC-HRMS/MS.

Vitamin D metabolism is actively modulated in adipose tissue during obesity. To better investigate this process, we develop a specific LC-HRMS/MS method that can simultaneously quantify three vitamin D metabolites, i.e., cholecalciferol, 25-hydroxyvitamin D3 (25(OH)D3), and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in a complex matrix, such as mouse adipose tissue and plasma. The method uses pretreatment with liquid–liquid or solid–phase extraction followed by derivatization using Amplifex® reagents to improve metabolite stability and ionization efficiency. Here, the method is optimized by co-eluting stable isotope-labelled internal standards to calibrate each analogue and to spike biological samples. Intra-day and inter-day relative standard deviations were 0.8–6.0% and 2.0–14.4%, respectively for the three derivatized metabolites. The limits of quantification (LoQ) achieved with Amplifex® derivatization were 0.02 ng/mL, 0.19 ng/mL, and 0.78 ng/mL for 1,25(OH)2D3, 25(OH)D3 and cholecalciferol, respectively. Now, for the first time, 1,25(OH)2D3 can be co-quantified with cholecalciferol and 25(OH)D3 in mouse adipose tissue. This validated method is successfully applied to study the impact of obesity on vitamin D status in mice.

Development, Validation, and Application of a New Method To Correct the Nonlinearity Problem in LC-MS/MS Quantification Using Stable Isotope-Labeled Internal Standards.

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become an indispensable tool for bioanalysis. To quantify a small molecule with LC-MS/MS, a stable isotope-labeled analyte is routinely used as the internal standard. However, cross signal contributions between the analyte and its stable isotope labeled internal standard (SIL-IS) could cause problems when the signal response of the LC-MS/MS system is nonlinear. In the present work, we try to illustrate how the "cross talk" between the analyte and its SIL-IS may cause problems for a nonlinear system. We assume that the instrumental responses toward the analyte and its SIL-IS are the same. When the calibration curve is nonlinear, the addition of a SIL-IS would practically move the response of the analyte up along the parabolic line causing a change in the signal strength of the analyte (usually decrease). The more the SIL-IS is added, the larger change the analyte signal would become. Such a problem would only be corrected by making the calibration curve linear. To this end, we proposed a component equation (CE) as the calibration for nonlinearity correction. In this study, we contrasted the accuracy of CE with the common quantitative method using two drugs whose mass spectrometric responses are linear and nonlinear, respectively. The acceptable accuracy results demonstrated that the CE calibration was comparable with the regular quantitative SIL-IS method with a proper weighting factor and much better than that without weighting. Therefore, CE calibration may provide another reliable way for LC-MS/MS quantification.

Comprehensive quantification of sesame allergens in processed food using liquid chromatography-tandem mass spectrometry

Sesame (Sesamum indicum) is a well-recognized food allergen, and sesame-related allergies have been increasingly reported worldwide. Patients with sesame allergy are specifically allergic to different sesame proteins (Ses i 1–7). Because of the increasing burden associated with allergies to sesame, there is an urgent need to develop sensitive and quantitative analytical methods capable of detecting sesame allergen contaminants. Targeted proteomic approach employing liquid chromatography/tandem mass spectrometry (LC-MS/MS) has emerged as a promising technique that offers increased specificity and reproducibility compared to antibody and DNA-based technologies. In this study, we developed a qualitative and absolute quantification method based on the LC-MS/MS-multiple reaction monitoring (MRM) system utilizing stable isotope-labeled internal standard (SIIS) peptides. We optimized extraction and trypsin digestion conditions and selected signature and absolute quantification (AQUA) peptides for seven sesame allergen proteins respectively. This quantitative method produced a linear relationship (R2 > 0.99) in a wide concentration range (0.4–2000 fmol/μL), and the overall coefficient of variation was <5% in multiple tests. Limits of detection (LOD) and Limits of quantification (LOQ) for the seven sesame allergens AQUA peptides were determined to range from 0.1 to 140.0 fmol/μL and 0.4–400 fmol/μL respectively. The results showed that the accuracy of 20, 50, 100 ppm incurred protein Ses i 2 were 90%, 92%, 94% in non-processed material and 92%, 95%,102% in processed material respectively. Overall, this method allowed for rapid and simultaneous absolute quantification of seven sesame allergens. The newly developed LC-MS/MS assay will likely increase the safety of sesame processed foods and add diagnostic value to the sesame allergy testing.

Quantitative analysis of post-translational modifications of histone H3 variants during the cell cycle

Histones participate in epigenetic regulation via dynamic post-translational modifications (PTMs) of histone variants. Comprehensive characterization of histone markers, especially those for the histone variants, could help to decipher the mechanism of epigenetic regulation. However, correctly profiling histone PTMs and its variants using mass spectrometry remains a challenge. Here, we developed an improved, specific and sensitive LC separation in conjunction with a high throughput multiple reaction monitoring combined with stable isotope-labeled internal standards (MRM-SIS) based quantitative method for histone H3 variants in the study of epigenetic regulation in the cell cycle. PTM patterns and the overall abundance of the three main histone H3 variants from Karpas 422 cells were analyzed and quantified simultaneously during different cell stages. The methylation pattern varied between different sites and modification states during the cell cycle. The canonical H3.1 presented regular patterns on K27 and K36, similar to H3.2, albeit differing from variant H3.3. H3.3 K36me2 increased from G1, S to G2 phase, whereas the same marker decreased in both H3.1 and H3.2. This novel discovery inspires more focus and research on the histone variants behavior and function during cell cycle. Moreover, this improved method could be applied to unveil PTMs dynamics of histone variants in several biological processes.

Quantitative and qualitative analysis of pyrrolizidine alkaloids in liqueurs, elixirs and herbal juices

Pyrrolizidine alkaloids (PAs and corresponding N-oxides (PANOs)) are known to have adverse health effects. Their toxic effects on liver cells are especially well-documented. In addition, potential carcinogenic and mutagenic effects in chronic exposure via food and/or herbal medicines have been a subject of vivid discussion in the last decade. Liqueurs and elixirs are traditionally used alcoholic extracts made from parts of plants and herbs. PA cross-contamination of the final products seems likely. Hence, this study aims to detect and quantify the PAs in such products in the light of a possible PA-contamination. The PA content was determined in the form of a single sum parameter using HPLC-ESI-MS/MS and a stable isotope-labeled internal standard. Overall, 56 products available at German pharmacies, drugstores, or internet shops were analyzed, comprising in total 38 samples of liqueurs (mainly bitters), 12 samples of plant elixirs and six different herbal juices. The results showed that 9 out of 38 liqueurs were PA-positive (24%). The total amount of PAs ranged from non-detectable to 9.5 μg/kg. Seven out of ten elixirs were PA-positive (70%) with a maximum PA-content of 3121 μg/kg. Four out of six plant juices were PA-positive (67%) with an average of 4.4 μg/kg (PA-positive samples only).The results and potential risks are discussed in the light of recommended portions for daily consumption or daily doses, in association with the detected PA amounts for individual products and product classes.

A rapid derivatization based LC-MS/MS method for quantitation of short chain fatty acids in human plasma and urine.

Aim: Objective of this study is to develop a robust multi-matrix LC-MS/MS for the quantitation of endogenous short-chain fatty acids (SCFA) biomarkers in human plasma and urine. Methods: Developed method utilizes stable isotope-labeled internal standards, high-throughput derivatization procedure for sample preparation and LC-MS/MS analysis using multiple reaction monitoring transitions in positive electrospray ionization mode. Results: Surrogate matrix method was used for quantitation. Accuracy, precision, parallelism, curve linearity, derivatization efficiency, stability and recovery were all evaluated, and the results were well within the acceptable criteria. Conclusion: SCFA levels in human plasma and urine of inflammatory bowel disease patients versus non-disease subjects were quantified and compared by LC-MS/MS.

Identification of a novel biomarker for pyridoxine-dependent epilepsy: Implications for newborn screening.

Pyridoxine-dependent epilepsy (PDE) is often characterized as an early onset epileptic encephalopathy with dramatic clinical improvement following pyridoxine supplementation. Unfortunately, not all patients present with classic neonatal seizures or respond to an initial pyridoxine trial, which can result in the under diagnosis of this treatable disorder. Restriction of lysine intake and transport is associated with improved neurologic outcomes, although treatment should be started in the first year of life to be effective. Because of the documented diagnostic delay and benefit of early treatment, we aimed to develop a newborn screening method for PDE. Previous studies have demonstrated the accumulation of Δ1 -piperideine-6-carboxylate and α-aminoadipic semialdehyde in individuals with PDE, although these metabolites are unstable at room temperature (RT) limiting their utility for newborn screening. As a result, we sought to identify a biomarker that could be applied to current newborn screening paradigms. We identified a novel metabolite, 6-oxo-pipecolate (6-oxo-PIP), which accumulates in substantial amounts in blood, plasma, urine, and cerebral spinal fluid of individuals with PDE. Using a stable isotope-labeled internal standard, we developed a nonderivatized liquid chromatography tandem mass spectrometry-based method to quantify 6-oxo-PIP. This method replicates the analytical techniques used in many laboratories and could be used with few modifications in newborn screening programs. Furthermore, 6-oxo-PIP was measurable in urine for 4 months even when stored at RT. Herein, we report a novel biomarker for PDE that is stable at RT and can be quantified using current newborn screening techniques.

LC–MS/MS determination of N-acetylaspartic acid in dried blood spot for selective screening of Canavan disease

Canavan disease is a serious rare neurometabolic disorder characteristic of an accumulation of N-acetylaspartic acid (NAA) in biological fluids. Its determination mainly in urine is the key part of the diagnostic procedure. Despite the several advantages of using dried blood spot (DBS) samples for clinical analysis, it has not been reported for this purpose so far. Therefore, this work was focused on the development of a HPLC–MS/MS method for the determination of NAA in a dried blood spot. The method using a stable isotope-labeled internal standard was elaborated and partially validated, with an estimated limit of quantification of 0.06 μmol dm−3, and with intra-day and inter-day precisions up to 4.1 and 8.0%, respectively. The elaborated method was applied for the NAA determination in healthy controls and in two siblings with Canavan disease. There were three quantification approaches evaluated using one-point calibration as well as calibration curves with and without matrix. The results correlated very well and exhibited parallelism of one-point calibration and calibration curve approaches. The mean NAA concentrations determined in DBS of control newborns and older children (10 months–7 years) were 0.9 and 0.5 μmol dm−3, respectively. Patients were clearly distinguished from controls, considering the NAA concentrations were more than 10 times higher than in an aged-matched control group.

Coated direct inlet probe coupled with atmospheric-pressure chemical ionization and high-resolution mass spectrometry for fast quantitation of target analytes

The coated direct inlet probe (CDIP) is a new laboratory-made low-cost technology developed from a direct inlet probe (DIP), which has the advantage of quick enrichment/cleanup of an analyte from liquid samples. A capillary probe is coated with hydroxy-terminated polydimethylsiloxane (OH-PDMS), divinylbenzene (DVB), and β-cyclodextrin (β-CD) by a sol-gel method. This probe can be directly coupled with a commercialized atmospheric-pressure chemical ionization (APCI) ion source and high-resolution mass spectrometry, which are widely applicable, reliable, and durable. The ability to perform quantitative analyses with the use of a stable-isotope-labeled internal standard (SIL-IS) was tested by using different concentrations of acenaphthylene (ACY), acenaphthene (ACP), fluorene (FLR), fluoranthene (FLT), phenanthrene (PHE), and benzo[a]pyrene (B[a]P). Calibration curves with a coefficient of determination of R2 ≥ 0.9982 for different polycyclic aromatic hydrocarbons (PAHs) were obtained. A limit of detection (LOD) of 0.008–0.04 ng mL−1 for PAHs was determined. The entire workflow is solvent-free and can be completed in less than 5 min, which demonstrates the advantages of this technique for quantitative analysis.

Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis.

Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue.

Evaluation of a 24-Hour Caffeine Intake Assessment Compared with Urinary Biomarkers of Caffeine Intake among Young Adults in Canada

BackgroundCaffeine is a widely consumed stimulant, and caffeine-containing products are increasingly available on the market. Few tools are available to capture caffeine intake, particularly among young adults. To estimate caffeine consumption in the previous 24 hours, the 24-Hour Caffeine Intake Recall (CIR-24) was modeled after the Automated Self-Administered 24-Hour Dietary Assessment Tool, using a brand-specific database of caffeine-containing foods, beverages, and supplements. ObjectiveTo evaluate the accuracy of the CIR-24 compared with caffeine concentration biomarkers in urine and a caffeinated beverage intake frequency screener (CBQ) designed to assess usual intake among a young adult population in Canada. Design/participantsIn all, 79 young adults, aged 18 to 29 years, provided 24-hour urine samples and completed the CIR-24 and CBQ. Main outcome measuresExcretion for caffeine and eight caffeine metabolites were quantified from urine samples using high-performance liquid chromatography-polarity switching electrospray ionization-tandem quadrupole mass spectrometry with stable isotope-labeled internal standards. Statistical analyses performedPearson correlations and weighted κ coefficients were calculated for the self-report tools and caffeine biomarkers. ResultsThe CIR-24 was significantly positively associated with all caffeine biomarkers (rp=0.28 to 0.52, κ=0.39 to 0.59), and the CBQ was significantly positively associated with all but one biomarker (rp=0.21 to 0.40, κ=0.32 to 0.45). The CIR-24 yielded a higher mean intake of caffeine than the CBQ. There was strong linear correlation between the CIR-24 and CBQ (rp=0.60, P<0.001), but poor agreement in absolute caffeine consumed (t=2.83, P=0.006); quartile ranking concordance was 0.44 (P<0.001). The CIR-24 performed better than the CBQ across all biomarkers in both linear correlation and quartile ranking. ConclusionsAlthough both the CIR-24 and CBQ performed reasonably well in capturing caffeine intake compared with urinary biomarkers of caffeine consumption, the CIR-24 had stronger agreement than the CBQ. The results suggest that the CIR-24 is a promising tool for evaluating caffeine intake among this population.

A fully automated high-throughput liquid chromatography tandem mass spectrometry method for measuring creatinine in urine

Reliable creatinine measurements are important to evaluate kidney function and for creatinine correction to reduce biological variability of other urinary analytes. A high-throughput, accurate liquid chromatography tandem mass spectrometry method for quantitation of human urinary creatinine has been developed and validated.Sample preparation was fully automated including cryovial decapping, sample ID scanning and two serial dilution steps. Quantitation was performed using a stable isotope-labeled internal standard. Multiplexed chromatographic separation of creatinine was achieved within a one-minute analysis and followed by tandem mass spectrometry in positive electrospray ionization mode. The precursor and product ions of creatinine and D3-creatinine were monitored in selected reaction monitoring mode.Method validation results showed reproducibility with within-run precision of 3.59, 3.49 and 2.84% and between-run precision of 4.01, 3.28 and 3.57% for low, medium and high quality control materials prepared from pooled donor urine, respectively. The method showed excellent accuracy with a bias of −1.94%, −0.78% and −1.07% for three levels of certified reference material. The calibration curve was linear throughout a 7.50–300 mg/dL (0.663–26.5 mmol/L) measurement range (R2 = 0.999), with the mean slope of 0.0115 (95%CI, 0.0108–0.0122) and intercept of 0.0027 (95%CI, 0.0003–0.0051). The limit of detection (LOD) of the method was 3.17 mg/dL (0.280 mmol/L). Analytical specificity was achieved by chromatographically separating creatinine from potentially interfering creatine within a one-minute run and monitoring the Quantitation Ion/Confirmation Ion (QI/CI) ratios in samples.A simple, accurate, high-throughput method was successfully developed for measuring creatinine in human urine samples.