Liquid Chromatography-isotope Dilution Mass Spectrometry Research Articles

Quantification of SARS-CoV-2 monoclonal IgG mass fraction by isotope dilution mass spectrometry.

The availability of serology assays to measure antibodies against the SARS coronavirus 2 (SARS-CoV-2) expanded rapidly during the Covid-19 pandemic. The interchangeable use of such assays to monitor disease progression and immune protection requires their standardization, for which suitably characterized monoclonal antibody materials can be useful. The methods, based on isotope dilution mass spectrometry, to value assign the mass fraction of such a material in solution within the context of an international interlaboratory comparison study (CCQM-P216) are described. The mass fraction in solution of a humanized IgG monoclonal antibody (mAb) against the SARS-CoV-2 Spike glycoprotein in the study sample has been value assigned through a combination of liquid chromatography, isotope dilution mass spectrometry (LC-ID-MS) methods and size exclusion chromatography with UV detection (SEC-UV). The former were developed for the quantification of amino acids and proteotypic peptides as surrogate analytes of the mAb while the latter was applied for the determination of the relative monomeric mass fraction. High-resolution mass spectrometry (hrMS) allowed the molecular weight evaluation and ruled out the presence of significant impurities. Method trueness was assessed using a subclass homologous IgG1 material value assigned by amino acid analysis. The assigned mass fraction of monomeric SARS-CoV-2 IgG in solution was 390 ± 16mg/g. The associated expanded uncertainty originated mainly from acid hydrolysis variability and Trypsin/Lys-C digestion variability and efficiency.

Traceable value of immunoglobulin G against receptor-binding domain of SARS-CoV-2 confirmation and application to point-of-care testing system development.

A highly purified and bioactive immunoglobulin G monoclonal antibody against receptor-binding domain of SARS-CoV-2 (RBD-IgG-MAb) has been accurately quantified by amino acid determination using isotope dilution liquid chromatography-mass spectrometry. Absolute quantification of RBD-IgG-MAb was achieved by averaging 4 amino acid certified reference materials, which allows the quantitative value (66.1 ± 5.8 μg/L) to be traced to SI unit (mol). Afterwards, the RBD-IgG-MAb was employed as control and calibrationcompound for the development of a point-of-care testing (POCT) system based on colloidal gold lateral flow immunoassay, which aimed to rapidly and accurately detect the level of protective RBD-IgG after vaccination. Under the detection parameters, a sigmoidal curve has been plotted between signal intensity and the logarithmic concentration for quantitative detection with the limit of detection of about 0.39μg/mL. The relative standard deviations of intra-assay and inter-assay were lower than 2.3% and 14%, and the recoveries ranged from 87 to 100%, respectively. Fingertip blood samples from 37 volunteers after vaccination were analyzed by the POCT system; results showed that levels of RBD-IgG in 33 out of 37 samples ranged from 0.45 to 2.46μg/mL with the average level of 0.91μg/mL. The developed POCT system has been successfully established with the quantity-traceability RBD-IgG-MAb as control and calibration compound, and the scientific contribution of this work can be promoted to other areas.

A potential primary method for peptide purity analysis by gas chromatography-isotope dilution infrared spectrometry.

Here we proposed a method for peptide purity analysis using gas chromatography-isotope dilution infrared spectroscopy. The principle and feasibility of the proposed measurement method were investigated. The derivatization, separation, and infrared detection conditions for amino acids were optimized, and the performance of the method was investigated. Then, the proposed method was used for assessment of [Glu1]-fibrinopeptide B purity, and the results were compared with those obtained by high performance liquid chromatography-isotope dilution mass spectrometry. The average purity of six sub-samples using the proposed method was (0.755 ± 0.017)g/g, which agreed well with that obtained by isotope dilution mass spectrometry (0.754 ± 0.012) g/g. The repeatability of the proposed method was 2.2%, which was similar to that of isotope dilution mass spectrometry (1.7%). The proposed method has a similar principle and had similar accuracy, precision, and linearity to isotope dilution mass spectrometry; however, the developed method had higher limit of detection (LOD) and limit of quantitation (LOQ) values because of the low sensitivity of infrared detection. The results were also Système International d'Unités (SI) traceable. The developed method has the advantage of lower cost compared with isotope dilution mass spectrometry because only one isotope-labeled atom in an analog is required, and several infrared spectra can be extracted, averaged, and used for an amino acid calculation during one run, potentially leading to higher accuracy. This method could be easily expanded to the accurate quantitation of other organic compounds, including proteins. It is expected that the proposed method will be widely used in chemical and biological measurements as a new primary method.

MP06-19 ETHNICITY AND RACE SPECIFIC TOTAL TESTOSTERONE NORMS - TIME FOR CHANGE?

You have accessJournal of UrologyCME1 Apr 2023MP06-19 ETHNICITY AND RACE SPECIFIC TOTAL TESTOSTERONE NORMS - TIME FOR CHANGE? William Shyr, Jonathan Davilla, and Darius Paduch William ShyrWilliam Shyr More articles by this author , Jonathan DavillaJonathan Davilla More articles by this author , and Darius PaduchDarius Paduch More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003217.19AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Normal total testosterone (TT) levels are an area of significant controversy and are critically important in clinical care. Anecdotal evidence suggests that ethnicity may play a role in TT. We analyzed the National Health and Nutrition Examination Survey (NHANES) data to study the effects of ethnicity on TT levels using the recommended method of TT measurement. METHODS: Data from the 2013-2016 NHANES survey were included. TT was measured using isotope dilution liquid chromatography-mass spectrometry certified by CDC Hormone Standardization Program. CDC's definition of race and ethnic groups was used. Only men 18-79 years old were included in the study. Men taking testosterone and aromatase inhibitors were excluded. Mean TT levels in each of the ethnic groups were compared using multivariate linear regression adjusting for the effect of ethnicity, age, BMI, DM, HTN, CAD, hypercholesterolemia, and smoking. RESULTS: Of the 4,772 men, there were 1,824 Non-Hispanic Whites (NHW), 1,323 Hispanics (H), 606 NH Asians (NHA), and 1,019 NH Blacks (NHB). The median age for all groups was 46, with no significant differences in age between groups. The mean TT and 95% CI were 421 ng/dl (411-431) for NHW, 416 ng/dl (402-430) for H, 418 ng/dl (404-432) for NHA, and 457 ng/dl (444-471) for NHB. When adjusted for smoking, DM, hypercholesterolemia (HCL), BMI, and HTN, multivariate linear regression showed that NHB men had higher TT with an effect size of 30.63 (p<0.001) while NHA men have lower TT with an effect size of -43.33 (p<0.001) compared to NHW men. The differences in TT were more pronounced in men with normal BMI (18.5-24.9) aged 40-49; TT of NHB men was 637 ng/dL, compared to the TT of NHW men at 469 ng/dL (p<0.01) (Figure 1). CONCLUSIONS: This is the largest study to date evaluating the effects of ethnicity on TT using the CDC-recommended method of testosterone measurements. NH Blacks have significantly higher TT levels even when controlling for age and comorbidities, while NH Asians have the lowest TT. The difference is most evident in Black men with a normal BMI age less than 50. This finding has important implications in clinical care and epidemiological studies. We recommend using ethnicity-specific TT levels, especially for NH Black men. Source of Funding: Fracchia Research Scholarship © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e60 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information William Shyr More articles by this author Jonathan Davilla More articles by this author Darius Paduch More articles by this author Expand All Advertisement PDF downloadLoading ...

Liquid Chromatography–Mass Spectrometry-Based Qualitative Profiling of mRNA Therapeutic Reagents Using Stable Isotope-Labeled Standards Followed by the Automatic Quantitation Software Ariadne

mRNA-based medicines are a promising modality for preventing virus-caused illnesses, including COVID-19, and treating various types of cancer and genetic diseases. To develop such medicines, methods to characterize long mRNA molecules are needed for quality control and metabolic analysis. Here, we developed an analytical platform based on isotope-dilution liquid chromatography-mass spectrometry (LC-MS) that quantitatively characterizes long, modified mRNAs by comparing them to a stable isotope-labeled reference with an identical sequence to that of the target medicine. This platform also includes database searching using the mass spectra as a query, which allowed us to confirm the primary structures of 200 to 4300 nt mRNAs including chemical modifications, with sequence coverage at 100%, to detect/identify defects in the sequences, and to define the efficiencies of the 5'-capping and integrity of the polyadenylated tail. Our findings indicated that this platform should be valuable for quantitatively characterizing mRNA vaccines and other mRNA medicines.

Perfluorooctanesulfonate Can Cause Negative Bias in Creatinine Measurement in Hemodialysis Patients Using Polysulfone Dialysis Membranes.

Serum creatinine is an important clinical marker for renal clearance. However, two conventional methods (Jaffe and enzymatic) are prone to interferences with organic compounds as compared to the standard method (isotope dilution–liquid chromatography–mass spectrometry) and can cause a significant negative bias. Perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) are two common perfluorochemicals (PFCs) that can easily be accumulated in humans. We aimed to verify whether this bias is the result of an accumulation of PFCs. The serum creatinine values of 124 hemodialysis patients were analyzed using the three methods. We also aimed to evaluate which biochemical parameters will influence the difference between the conventional methods and the standard method. We found that a significant underestimation occurred when using the conventional methods. Albumin is an independent factor associated with negative bias, but it loses this correlation after dialysis, likely due to the removal of protein-bound uremic toxins. PFOS can cause negative bias when using the enzymatic method. Furthermore, this linear correlation is more significant in patients who used polysulfone-based dialysis membranes, possibly due to the better clearance of other uremic toxins. The serum creatinine of uremic patients can be significantly underestimated when using conventional methods. PFCs, as well the type of dialysis membrane being used, can be influencing factors.

Development of Certified Reference Material for Amino Acids in Dried Blood Spots and Accuracy Assessment of Disc Sampling.

To achieve the measurement reliability of amino acids used as diagnostic markers in clinical fields, establishing a reference measurement system is required, in which certified reference materials (CRMs) are an essential step in the hierarchy of measurement traceability. This study describes the development of dried blood spot (DBS) CRMs for amino acid analysis with complete measurement traceability to the International System of Units (SI). Six essential amino acids—proline, valine, isoleucine, leucine, phenylalanine, and tyrosine—were analyzed using isotope-dilution liquid chromatography–mass spectrometry (ID-MS). For minimizing measurement bias and uncertainty overestimation, whole spots with 50 μL of whole blood were adopted in the certification. The between-spot homogeneities by whole spot sampling were lower than 2.1%. The relative expanded uncertainties of the six amino acids in the developed DBS CRMs were lower than 5.7% at 95% confidence. The certified values are traceable to SI through both gravimetric preparation and the primary method in certification, ID-MS. Comparison among DBS testing laboratories revealed discrepancies between the whole spot and disc sampling methods. The actual sampling volume was accurately estimated by weighing, which revealed the possibility of underestimation in routine DBS testing. The candidate CRMs can support the standardization of DBS testing for amino acids through the qualification and validation of many kinds of measurement procedures to compensate the measurement bias caused by matrix-specific sampling error.

A new method for the evaluation of measurement uncertainty in strict accordance with measurement model: Determination of total thyroxine in human serum by triple isotope dilution mass spectrometry

The rigorous and reasonable evaluation of uncertainty is crucial for the reliability of the results of a reference measurement procedure. A new uncertainty budget is proposed for uncertainty evaluation of a reference measurement procedure for total thyroxine in human serum based on isotope dilution liquid chromatography-mass spectrometry. In this work, a measurement model for uncertainty evaluation of triple isotope dilution-mass spectrometry was established. The propagation of uncertainties assigned to various input quantities was expressed in strict accordance with the measurement model. A detailed description of the uncertainty evaluation process for the measurement result of a specific serum is given, including the estimate of the input quantities and the determination of the combined standard uncertainty. The expanded uncertainty of the specific human serum (at coverage probability of 95%) was 4.8 nmol/L at the concentration level of 113.6 nmol/L.

Creatinine standardization: a key consideration in evaluating whole blood creatinine monitoring systems for CKD screening

Early detection of CKD using point of care creatinine and eGFR testing improves patient management outcomes. We undertook a field study to evaluate the use of a whole blood creatinine/eGFR device to screen a rural Nicaraguan population to determine the variability between creatinine methods and specimen types. All specimens including capillary and venous dried blood spots (DBS) were tested with an isotope dilution liquid chromatography mass spectrometry (ID-LCMS) gold standard method. This is to our knowledge the first time a capillary whole blood (POC) method has been directly compared to the gold standard IDMS method, through the novel approach of using dried blood spots. Capillary and venous whole blood specimens were obtained and tested directly with the BCMS method, and then, DBS samples were prepared. Venous plasma specimens were tested using three laboratory analyzer creatinine methods. DBS were sent to the site performing ID-LCMS. Control samples were also prepared to assess the stability of shipment and storage of DBS. The ID-LCMS method was aligned using primary and secondary standards. Sixty-six (66) patients participated in the study, and the CKD prevalence rate was 7.8%. While all creatinine methods showed a good correlation to ID-LCMS, there was a positive bias (mean absolute bias range: 0.21–0.63 mg/dL). All methods used were 100% sensitive, but specificity varied from 62.7 to 94.9% with PPV ranging from 25 to 62.5%. A correction factor was used to align the values from each method to ID-LCMS which improved the specificity of each method. This study used a unique DBS approach to align capillary whole blood creatinine to ID-LCMS. To ensure reliability of BCMS for identifying screened patients with CKD, it is important to establish IDMS traceability and alignment prior to undertaking CKD studies.

Study of SI-traceable purity assessment of bovine lactoferrin using mass balance approach and amino acid-based isotope dilution liquid chromatography-mass spectrometry

The accurate measurement of bovine lactoferrin (bLF) attracts wide attention in food and nutraceutical applications as its important physiological and nutritional functions. We present SI traceable procedures for assessing bLF purity using mass balance method and amino acid (AA)-based isotope dilution mass spectrometry (IDMS). The mass balance method was revealed with a purity of 0.938 ± 0.011 g/g by deducting all aspects of impurities, including related structure impurities of 4.60%, ignition residue of 0.28%, Cl- of 1.10%, SO42- of 0.13%, and moisture of 0.17%. The AA-based IDMS quantitative result was 0.937 ± 0.027 g/g. Hydrolysis conditions were optimized and methodology validation including, accuracy, precision, were studied. Good consistency was achieved between the two independent strategies and bLF purity assigned via the weighted mean value of their results was 0.938 ± 0.015 g/g. These analyses are expected to be applicable to proteins quantification and development of LF certified reference materials.

Assessing the Validity of Normalizing Aflatoxin B1-Lysine Albumin Adduct Biomarker Measurements to Total Serum Albumin Concentration across Multiple Human Population Studies.

The assessment of aflatoxin B1 (AFB1) exposure using isotope-dilution liquid chromatography-mass spectrometry (LCMS) of AFB1-lysine adducts in human serum albumin (HSA) has proven to be a highly productive strategy for the biomonitoring of AFB1 exposure. To compare samples across different individuals and settings, the conventional practice has involved the normalization of raw AFB1-lysine adduct concentrations (e.g., pg/mL serum or plasma) to the total circulating HSA concentration (e.g., pg/mg HSA). It is hypothesized that this practice corrects for technical error, between-person variance in HSA synthesis or AFB1 metabolism, and other factors. However, the validity of this hypothesis has been largely unexamined by empirical analysis. The objective of this work was to test the concept that HSA normalization of AFB1-lysine adduct concentrations effectively adjusts for biological and technical variance and improves AFB1 internal dose estimates. Using data from AFB1-lysine and HSA measurements in 763 subjects, in combination with regression and Monte Carlo simulation techniques, we found that HSA accounts for essentially none of the between-person variance in HSA-normalized (R2 = 0.04) or raw AFB1-lysine measurements (R2 = 0.0001), and that HSA normalization of AFB1-lysine levels with empirical HSA values does not reduce measurement error any better than does the use of simulated data (n = 20,000). These findings were robust across diverse populations (Guatemala, China, Chile), AFB1 exposures (105 range), HSA assays (dye-binding and immunoassay), and disease states (healthy, gallstones, and gallbladder cancer). HSA normalization results in arithmetic transformation with the addition of technical error from the measurement of HSA. Combined with the added analysis time, cost, and sample consumption, these results suggest that it may be prudent to abandon the practice of normalizing adducts to HSA concentration when measuring any HSA adducts—not only AFB1-lys adducts—when using LCMS in serum/plasma.

Egy LC-MS/MS alapú élelmiszervizsgálati módszer nemzetközi szabványosítása: egységesen elfogadott vizsgálati eljárás kidolgozása Alternaria toxinokra

There are more than seventy varieties of Alternaria toxins, but researchers have so far identified only a few of them structurally. The objective of this paper is to present a nearly ten-year process, during which an international standard for the simultaneous analysis of five Alternaria toxins in food samples was developed. This long process includes the development of the need for the standard and, in addition to the preparation and evaluation of the standardization tender, the development of the method, its validation and documentation. The paper focuses mainly on the development and validation of the analytical method, which is the longest and most labor-intensive part of the process, but in order to understand the overall picture, it is also necessary to emphasize the first and final steps. The development of a standard is a task of great responsibility for both the preparers of the standard and those involved in the validation and documentation of the standard, as the use of standardized methods is expected by the customers of the laboratories. On the other hand, laboratories that choose unique, self-developed methods can ascertain the accuracy and precision of their procedure by comparing them with the standard method. In this process that went on for nearly ten years, the original analytical method underwent several changes; the goal of these improvements was to make the procedure as simple and reproducible as possible. This is how the use isotope dilution mass spectrometry was reached through derivatization. It is important to emphasize that one of the goals of standardization is to have an appropriate analytical method available to authority laboratories for the testing of legally prescribed food contaminants, which procedure is available to any laboratory, however, it is questionable, whether the cost of the test covers its application. Consequently, it is not necessarily the most cost-effective analysis which is recommended by the standard, which may be the cause of conflict between the professional and economic managers of a laboratory in the case of private laboratories. The final form of the liquid chromatography/isotope dilution mass spectrometry (LC-IDMS) standard method developed for Alternaria toxins is likely to be approved and published by the European Committee for Standardization (CEN) in the end of 2021 (the standard has been issued since the article was submitted: CEN EN 17521:2021 Foodstuffs - Determination of Alternaria toxins in tomato, wheat and sunflower seeds by SPE clean-up and HPLC-MS/MS. The Editor). The standard will contain the determination of tenuazonic acid (TEA), altenuane (ALT), alternariol (AOH), tentoxin (TEN) and alternariol monomethyl ether (AME).

Simultaneous toxicokinetics characterization of acrylamide and its primary metabolites using a novel microdialysis isotope-dilution liquid chromatography mass spectrometry method

Acrylamide (AA) is a toxicant in high-temperature processed foods and an animal carcinogen. Upon absorption, AA is metabolized to glycidamide (GA) or conjugates with glutathione (AA-GSH). Important advantages of microdialysis coupled with liquid chromatography-tandem mass spectrometry (MD-LC-MS/MS) include its minimization of potential losses during sample collection, storage and preparation, as well as an improvement in temporal resolution for toxicokinetics (TKs). We aimed to simultaneously study the TKs of AA and products of its primary metabolism using an isotope-dilution (ID) MD-LC-MS/MS method. MD probes implanted into the jugular vein/right atrium of anesthetized Sprague Dawley rats were connected to the ID-LC-MS/MS for continuous monitoring of AA, GA and AA-GSH in the blood every 15 min over 8 h following intraperitoneal AA administration (0.1 mg/kg or 5 mg/kg). AA, GA, and AA-GSH TKs followed linear kinetics: GA AUC/AA AUC = 0.11 and AA-GSH AUC/AA AUC = 0.011 at 5 mg/kg. Elimination half-life (Te1/2) values were 2.44 ± 0.70, 4.93 ± 2.37 and 3.47 ± 1.47 h for AA, GA and AA-GSH, respectively. GA TKs reached a plateau at 3–6 h, suggesting that metabolic saturation of AA and Te1/2 values of the analytes were prolonged with AA at 5 mg/kg. Our results demonstrate that oxidation of AA to GA overwhelmed the conjugation of AA with GSH. Our innovative MD-ID-LC-MS/MS method facilitates the simultaneous characterization of multiple TKs associated with toxicants and their active metabolites with excellent temporal resolution to capture metabolic saturation of AA to GA.

Quantitation of glycated albumin by isotope dilution mass spectrometry

BackgroundGlycated albumin is considered an alternative glycemic indicator in certain situations where HbA1c does not accurately reflect glycemic status. These patient cases are usually associated with decreased erythrocyte lifespan, gestational diabetes, or end-stage renal disease. The aim of our study was to develop an assay for absolute quantitation of glycated albumin based on isotope dilution liquid chromatography-mass spectrometry. MethodsThe plasma samples were reduced/alkylated, spiked with isotope-labeled standards RQIKKQTALV(D8)E and RQIKK(fructosyl)QTALV(D8)E and enzymatically digested by Glu-C. The samples were analyzed on an LC–MS system. Two MRM transitions (M3+ → (b9-3H2O)2+ and M3+ → (b10-3H2O)2+ or M3+ → b92+ and M3+ → b102+) were used for each peptide, then the percentage of glycation (MS GA%) was calculated. ResultsThe comparison study demonstrated a good linear correlation between our LC–MS/MS and Lucica method with r2 = 0.95. The intra-day CV for the low HbA1c sample was 2.2%, while CV for the high HbA1c sample was 0.64%. Inter-day CV for low HbA1c sample was 5.6%, while the CV for the high HbA1c sample was 5.7%. We found the LLOQ to be 0.12 nmol/ml for the non-glycated and glycated peptide. No interference from hemoglobin was observed up to 500 mg/dL concentration. ConclusionsThis is the first implementation of isotope dilution LC–MS assay for glycated albumin with simultaneously quantitation of glycated and non-glycated peptides. The method includes a simple sample preparation and has demonstrated a good analytical performance.

An isotope dilution liquid chromatography-mass spectrometry method for detection of melamine in milk powder

ABSTRACT Adulteration/unintentional contamination of milk with melamine could have negative health and economic implications especially in the developing countries due to insufficient laboratory support and surveillance. This paper presents an Isotope Dilution Liquid Chromatography-Mass Spectrometry (ID LC-MS) method developed for detection of melamine in powdered milk. The rapid sample preparation involved dissolution of 1g of milk powder in 2.5% formic acid, precipitation of protein with acetonitrile, spiking of samples with melamine (triamine-15N3) at 200 µg L–1 and detection of intrinsic 14N-melamine molecular ratio to the spike. The isotope dilution calibration procedure was free from matrix effects, unlike other methods where the detector sensitivity can fluctuate up to several orders of magnitude. Limit of detection of the method was 13 µg kg–1, and the recovery of melamine at 50, 100, and 250 µg kg–1 was 78.7–126.3%. The method was used to determine melamine levels in 22 milk powder products (local and imported) available in Sri Lanka. Melamine was detected in all the samples (range = 0.33–0.96 mg kg–1). Full cream milk powders (both local and imported) contained melamine in the range of 0.39–0.84 mg kg–1, and various health and pregnancy formulas contained <0.5 mg kg–1 of melamine. Two imported infant formula samples contained the highest levels of melamine (0.96 and 0.94 mg kg–1). Although these melamine levels are below the regulatory limit in Sri Lanka (1 mg kg–1), a monitoring programme would ensure consumer safety.

Quantitative Bioreactor Monitoring of Intracellular Bacterial Metabolites in Clostridium autoethanogenum Using Liquid Chromatography-Isotope Dilution Mass Spectrometry.

We report a liquid chromatography–isotope dilution mass spectrometry method for the simultaneous quantification of 131 intracellular bacterial metabolites of Clostridium autoethanogenum. A comprehensive mixture of uniformly 13C-labeled internal standards (U-13C IS) was biosynthesized from the closely related bacterium Clostridium pasteurianum using 4% 13C–glucose as a carbon source. The U-13C IS mixture combined with 12C authentic standards was used to validate the linearity, precision, accuracy, repeatability, limits of detection, and quantification for each metabolite. A robust-fitting algorithm was employed to reduce the weight of the outliers on the quantification data. The metabolite calibration curves were linear with R2 ≥ 0.99, limits of detection were ≤1.0 μM, limits of quantification were ≤10 μM, and precision/accuracy was within RSDs of 15% for all metabolites. The method was subsequently applied for the daily monitoring of the intracellular metabolites of C. autoethanogenum during a CO gas fermentation over 40 days as part of a study to optimize biofuel production. The concentrations of the metabolites were estimated at steady states of different pH levels using the robust-fitting mathematical approach, and we demonstrate improved accuracy of results compared to conventional regression. Metabolic pathway analysis showed that reactions of the incomplete (branched) tricarboxylic acid “cycle” were the most affected pathways associated with the pH shift in the bioreactor fermentation of C. autoethanogenum and the concomitant changes in ethanol production.

Determination of Serum Growth Hormone by Liquid Chromatography-Isotope Dilution Mass Spectrometry

Determination of Serum Growth Hormone by Liquid Chromatography-Isotope Dilution Mass Spectrometry

Determination of Total and A1-Type β-Casein in Milk and Milk-Derived Ingredients by Liquid Chromatography-Mass Spectrometry Using Characteristic Tryptic Peptides.

Gastrointestinal digestion of A1-type β-casein is conducive to β-casomorphin-7 with potential adverse digestive health effects. Monitoring of A1-type β-casein concentration in milk and milk-derived ingredients used in the formulation of A2-type nutritional products with associated health claims is important from a quality standpoint. New analytical methods were developed and validated for total and A1-type β-casein in milk and milk-derived ingredients. Data on total and A1-type β-casein concentrations in milk, nonfat dry milk, and whey protein concentrate was generated. The methods are based on a bottom-up proteomic approach using tryptic marker peptides and stable isotope dilution liquid chromatography-mass spectrometry. The measurement includes all protein sequences (intact, modified, and partial) which are potential sources of β-casomorphin-7. Total β-casein was quantified using a neat calibration curve. Recovery and between-day precision RSD were 98% and 5.8%, respectively. A1-type β-casein was quantified by the method of standard additions. Between-day precision RSD was 7.2% and limit of quantitation was 0.01% in nonfat dry milk. The mass fraction of A1-type β-casein in the β-casein standard was 0.444. Samples manufactured from A2-type milk contained 0.26-5.0% A1-type β-casein relative to total β-casein. The methods described enable the monitoring of the A1-type β-casein concentration in milk and milk-derived ingredients destined for the manufacture of A2-type products with associated health claims. New methods are presented for the analysis of total and A1-type β-casein in milk and milk-derived ingredients. The mass fraction of A1-type β-casein in a commercial β-casein standard was determined to enable its use as a calibrant.

An accuracy-based proficiency testing programme with metrological reference values for the mass fraction of acesulfame potassium and sucralose in cake mix flour

This paper describes a metrological approach to evaluate the measurement capability of laboratories participating in a then Asia Pacific Laboratory Accreditation Cooperation [APLAC, now Asia Pacific Accreditation Cooperation] proficiency testing (PT) programme on mass fraction of acesulfame potassium and sucralose in cake mix flour. The programme was jointly organised by the Chemical Metrology Laboratory of the Health Sciences Authority, Singapore, and the Singapore Accreditation Council. Through the extensive APLAC network of accreditation bodies, 41 nominated laboratories from 32 economies registered, of which 40 laboratories submitted their results. Reference values of artificial sweeteners in the programme for performance evaluation were obtained from a high-accuracy liquid chromatography isotope dilution mass spectrometry (LC-IDMS) method. One of the unique features of this PT programme was the provision of pure substance certified reference materials (CRMs) of acesulfame potassium and sucralose to the participating laboratories as calibration standards to ensure comparability and metrological traceability of results. The participating laboratories’ performance, based on z-scores, were evaluated using the assigned reference value determined from IDMS. Five and one of the laboratories’ results would be incorrectly evaluated using consensus value calculated from the laboratories’ results for performance evaluation and using commercial standard as calibrant, respectively. This PT programme demonstrates the importance of the use of metrologically traceable reference values as the assigned values for appropriate evaluation of accuracy and reliable assessment. The provision of CRMs also heightened the testing laboratories’ awareness on the importance of using CRMs as calibrants for accurate measurements.

Assessing a method and reference material for quantification of vitamin D binding protein during pregnancy

Vitamin D plays a vital role in successful pregnancy outcomes for both the mother and fetus. Vitamin D is bound to vitamin D binding protein (VDBP) in blood and is carried to the liver, kidneys and other target tissues. Accurate measurements of the clinically measured metabolite of vitamin D, 25-hydroxyvitamin D [25(OH)D], depend on complete removal from the binding protein. It has been found that VDBP concentrations increase in maternal serum during pregnancy, obfuscating the accuracy of 25(OH)D concentration measurements in pregnant women. Additionally, measurements of VDBP concentrations during pregnancy have been performed using immunoassays, which suffer from variations due to differences in antibody epitopes, making clinical comparisons difficult. Quantification of VDBP is also of interest because changes in VDBP expression levels may indicate negative outcomes during pregnancy, such as preterm delivery and restricted fetal growth. To address the need for accurate measurement of VDBP during pregnancy, a method using liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) was developed to quantify VDBP using isotopically labeled peptides as internal standards. This method was used to quantify VDBP in Standard Reference Material® (SRM) 1949 Frozen Human Prenatal Serum, which was prepared from separate serum pools of women who were not pregnant and women during each trimester of pregnancy. VDBP concentrations were found to be lowest in the serum pool from non-pregnant women and increased in each trimester. These data had good repeatability and were found to be suitable for reference value assignment of VDBP in SRM 1949.