Christian Lutz Deitrich

There is an urgent need for reference methodologies to assign SI traceable values to quality control (QC) materials for protein biomarkers to validate biochemical tests currently used in disease diagnosis and patient-specific therapeutics. Here we describe a novel methodology specifically developed for the SI traceable quantification of human serum ferritin light chain (FTL), an established biomarker for the diagnosis of iron-related disorders such as hemochromatosis. Natural and ³⁴S isotopically enriched human FTL standards were recombinantly expressed using the HEK-293 mammalian cell system. Both standards were fully characterized for their total sulfur content, isotopic abundance, species distribution and primary sequence before using them for isotope dilution analysis (IDA). Treatment of serum spiked with FTL was optimized to selectively remove high abundant serum proteins without affecting significantly the integrity of the FTL. This was achieved by using methanol protein precipitation in combination with heat treatment and ultrafiltration. Complementary asymmetrical field-flow-fractionation (AF4) and anion-exchange high performance liquid chromatography (AE-HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) were used to monitor the impact of sample treatment on protein removal and FTL recovery. Both standards, natural and enriched, were used with the optimal sample treatment in a double species-specific isotope dilution analysis (SS-IDA) workflow for the determination of FTL in the WHO "4th International Standard for Ferritin" (NIBSC 19/118) resulting in a protein mass fraction of 11.0 with an expanded uncertainty (U, k = 2) of 1.0 μg mL^-1 (RSU of 9.4%). The method was validated by standard addition experiments, and a full uncertainty budget was established. For the first time sulfur SS-IDA has been used to assign an SI traceable value to a relatively low abundance serum protein biomarker at supraphysiological concentrations in a WHO standard. This methodology will be invaluable for adding SI traceability to existing QC materials and for the certification of new biomarker reference materials.

Main text: KRISS and NMIJ jointly coordinated the CCQM-K158 comparison on elements and inorganic As in polished rice flour. The study comprised two parts, Part A and Part B. Successful participation in formal international comparisons is essential for documenting calibration and measurement capability claims (CMCs) by national metrology institutes (NMIs) and designated institutes (DIs). CCQM-K158 specifically supports CMCs within the food category, corresponding to the sample matrix labeled "High organic content" in the new CC table for broad claims. Simultaneously, the CCQM-P200 comparison ran in parallel with the key comparison CCQM-K158. In Part A, sixteen NMIs/DIs participated. Participants were requested to measure the mass fractions of Cu, Hg, K, Na, Pb, and Sb in rice flour, expressed in mg·kg-1, on a dry mass basis. Most NMIs/DIs adopted microwave-assisted acid digestion in a closed vessel for sample pretreatment, except for NIS, which used open vessel acid digestion with heating. Majority of the participants used isotope dilution (ID) ICP-MS, except for K and Na. ICP-MS or ICP-OES with standard addition calibration and k0 INAA were also applied specially for elements of which ID is impossible or difficult to access. ICP-MS, ICP-OES, AAS methods were also used with external calibration. In Part B, fifteen NMIs/DIs participated. Participants were requested to evaluate the mass fractions, expressed in mg·kg-1, of total arsenic and inorganic arsenic (the sum of As (III) and As (V)) in polished rice flour. For total As, thirteen NMIs/DIs reported their analytical results, where all the NMIs/DIs, except for JSI, adopted ICP-MS with a microwave acid digestion. JSI adopted k0-INAA after pelletizing the sample. For inorganic As, seven NMIs/DIs reported their analytical results, where all the NMIs/DIs adopted liquid chromatography-ICP-MS with a thermostatically controlled extraction using diluted acids. The results of all the participating NMIs/DIs were evaluated against the key comparison reference value (KCRV). The KCRV and associated uncertainty were estimated from reported results, excluding outliers, using NIST decision tree (NDT) as an estimator of the KCRVs. Successful participation in CCQM-K158 Part A demonstrates measurement capabilities for determining mass fractions of alkali and alkaline earth (K, Na), transition (Cu, Hg, Pb), and metalloid/semi-metal (Sb) elements in mass fraction range above 0.05 mg kg-1, in high organic content matrices such as grains, beans, and related samples. Similarly, successful participation in CCQM-K158 Part B demonstrates measurement capabilities to determine mass fractions of total arsenic and water-soluble arsenic species such as inorganic arsenic (the sum of As (III) and As (V)), mono-, di-, tri-, and tetra-methyl arsenic compounds (MAA, DMAA, TMAO, TeMA), arsenocholine (AsC), and arsenobetaine (AsB), in mass fraction range above 0.05 mg·kg-1 in grains, beans, and related samples. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database https://www.bipm.org/kcdb/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

The hyphenation of HPLC with its high separation ability and ICP-MS with its excellent sensitivity, allows the analysis of Pt drugs in biological samples at the low nanomolar concentration levels. On the other hand, LC-MS provides molecular structural confirmation for each species. Using a combination of these methods, we have investigated the speciation of the photoactive anticancer complex diazido Pt(IV) complex trans, trans, trans-[Pt(N₃)₂(OH)₂(py)₂] (FM-190) in aqueous solution and biofluids at single-digit nanomolar concentrations before and after irradiation. FM-190 displays high stability in human blood plasma in the dark at 37 °C. Interestingly, the polyhydroxido species [{Pt^IV(py)₂(OH)₄} + Na]⁺ and [{Pt^IV(py)₂(N₃)(OH)₃} + Na]⁺ resulting from the replacement of azido ligands, as determined by LC-MS, were the major products after photoirradiation of FM-190 with blue light (463 nm). This finding suggests that such photosubstituted Pt(IV) tri- and tetra-hydroxido species could play important roles in the biological activity of this anticancer complex. Density Functional Theory (DFT) and Time-Dependent DFT (TDDFT) calculations show that these Pt(IV) species arising from FM-190 in aqueous media can be formed directly from a singlet excited state. The results highlight how speciation analysis (metallomics) can shed light on photoactivation pathways for FM-190 and formation of potential excited-state pharmacophores. The ability to detect and identify photoproducts at physiologically-relevant concentrations in cells and tissues will be important for preclinical development studies of this class of photoactivatable platinum drugs.

Main text: The platinum group elements (PGEs) play an important role in reducing emissions from automotive vehicles through their use in catalytic convertors but also for catalysis in the pharmaceutical industry. The immense economic value of platinum (Pt), palladium (Pd) and rhodium (Rh) highlights the importance of highly accurate measurements. Therefore, there is a need for National Metrology Institutes (NMIs) and Designated Institutes (DIs) to demonstrate measurement capability in this space. A pilot comparison (CCQM-P63) for precious metals in automotive catalyst took place in 2006, but with a limited number of institutes participating. Furthermore, this study was performed over 17 years ago. Therefore, there was a need to maintain existing capability and demonstrate new capability in a key comparison, in order to claim calibration and measurement capability claims (CMCs). With the core capability matrix, this study falls into the "Difficult to dissolve metals/metal oxides" which will support CMC categories 8 (Metal and metal alloys), 9 (Advanced materials) and 14 (Other materials). Eleven NMIs and DIs participated in the Key Comparison CCQM-K160 Platinum Group Elements in Automotive Catalyst. Participants were requested to evaluate the mass fractions of Pt, Pd and Rh in mg/kg in an unused autocatalyst material (cordierite ceramic base). The Key Comparison Reference Values (KCRVs) and Degrees of Equivalence (DoEs) were calculated utilising the NIST Decision Tree for the measurands. The participants utilised a number of sample preparation and analytical methods including hot plate digestion, microwave digestion and sodium fusion, followed by either atomic absorption spectroscopy (AAS), inductively coupled plasma optical emission spectroscopy (ICP-OES) or inductively coupled plasma mass spectrometry (ICP-MS) detection. Several calibration techniques were used, namely external calibration, standard addition, isotope dilution mass spectrometry (IDMS) and an exact matching procedure. Additionally, one participant employed instrumental neutron activation analysis (INAA) with k0 standardisation which is a direct solid analysis method. The majority of participants claimed traceability to NIST primary calibrants or their own CRMs. Furthermore, several matrix CRMs were included or spiked samples for quality control. All institutes were required to determine the dry mass fraction using the stipulated protocol. The NIST decision tree was implemented for the calculation of the KCRVs and DoEs. The participant results overall showed good agreement with the KCRV, despite the variety of dissolution procedures and measurement techniques for this highly complex matrix and challenging measurands. Successful participation in CCQM-K160 demonstrated measurement capabilities for the determination of mass fraction of Pt, Pd and Rh in the mg/kg range and will support broad scope CMC claims for a wide range of challenging matrices. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database https://www.bipm.org/kcdb/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

This work demonstrates for the first time the potential of monolithic chromatography coupled to double species-specific isotope dilution analysis (IDA) inductively coupled plasma mass spectrometry (ICPMS) for the detection and accurate quantification of adducts of plasma/serum proteins with carboplatin, an anti-cancer drug. The results of Pt fractionation in blood incubated with Pt drugs, to mimic cancer scenarios in real life, suggested that approximately 83% of the Pt in drug-incubated blood is found to be associated with plasma/serum proteins. Such studies were performed with cisplatin (instead of carboplatin), as cisplatin shows faster kinetics compared to carboplatin and Pt–DNA adducts have been demonstrated to be the same using both drugs. These results provide for the first time solid evidence to support that adducts of Pt drugs with plasma proteins are key competitors for the drugs binding to DNA in cancer models. The distribution of Pt associated with albumin (HSA), transferrin (Tf) and immunoglobulin (IgG) in human serum was investigated in reconstituted serum samples incubated with clinical concentrations of carboplatin for ten days at 37 °C. Two approaches were examined for the separation and detection of the three Pt–protein adducts. Asymmetrical flow field-flow fractionation (AF4) could not achieve baseline separation of the three proteins in a real matrix; however, its coupling with ICPMS was found to be particularly useful to provide detailed information on the formation of different adduct isoforms during the optimisation of the conditions for calibrant and spike production (e.g., by incubation of the individual proteins with either natural or ¹⁹⁴Pt-enriched carboplatin). Conjoint Liquid Chromatography (CLC) based on one affinity (Protein G) and two weak anion exchange (DEAE) disks coupled to ultraviolet-visible spectroscopy (UV-Vis) and ICPMS revealed that Pt–HSA adducts represented more than 96% of the protein-bound Pt in the serum, whereas Pt–Tf and Pt–IgG represented only about 2% each. Therefore, a reference methodology based on the use of double species-specific IDA ICPMS has been developed and validated for the determination of carboplatin–HSA adducts using a simplified separation method with two DEAE monolithic disks. Limits of detection achieved for carboplatin–HSA adducts with this method were 0.005 and 0.003 ng g⁻¹ for ¹⁹⁴Pt and ¹⁹⁵Pt, respectively. These values are about two orders of magnitude lower than those previously reported in the literature. An expanded uncertainty (k = 2) of 25 ng g⁻¹ (RSU of 4.7%) was achieved for a mass fraction of carboplatin–HSA adducts of about 528 ng g⁻¹. The reference methodology developed here will be invaluable for evaluation of dose-dependent response to treatment for individual patients in cancer trials, aimed at finding more effective therapies.

Recent evidence has highlighted the prevalence of mild-to-moderate iodine deficiency in women of childbearing age and pregnant women, with important public health ramifications due to the role of iodine, which is required for thyroid hormone production, in neurodevelopment. Cow milk contributes the greatest amount to iodine intakes in several countries. The objective of this study was to investigate the effect of increased cow milk consumption on iodine status, thyroid hormone concentrations, and selenium status. A 12-wk randomized controlled trial was conducted in 78 low–moderate milk-consuming (P = 0.014) and week 12 (51.6 compared with –3.8 µg/L; P = 0.045) were significantly greater in the intervention group compared with the control group. However, despite being higher within the intervention group at weeks 6 and 12, the change in the iodine:creatinine ratio from baseline was not significantly different between groups at either week 6 (P = 0.637) or week 12 (P = 0.178). There were no significant differences in thyroid hormone concentrations or selenium status between groups at any time point. The present study shows that the consumption of additional cow milk can significantly increase UIC in women of childbearing age. These results suggest that cow milk is a potentially important dietary source of iodine in this population group. This trial was registered at www.clinicaltrials.gov as NCT02767167.

Cow’s milk is the most important dietary source of iodine in the UK and Ireland, and also contributes to dietary selenium intakes. The aim of this study was to investigate the effect of season, milk fat class (whole; semi-skimmed; skimmed) and pasteurisation on iodine and selenium concentrations in Northern Ireland (NI) milk, and to estimate the contribution of this milk to consumer iodine and selenium intakes. Milk samples (unpasteurised, whole, semi-skimmed and skimmed) were collected weekly from two large NI creameries between May 2013 and April 2014 and were analysed by inductively coupled plasma-mass spectrometry (ICP-MS). Using milk consumption data from the National Diet and Nutrition Survey (NDNS) Rolling Programme, the contribution of milk (at iodine and selenium concentrations measured in the present study) to UK dietary intakes was estimated. The mean ± standard deviation (SD) iodine concentration of milk was 475.9 ± 63.5 µg/kg and the mean selenium concentration of milk was 17.8 ± 2.7 µg/kg. Season had an important determining effect on the iodine, but not the selenium, content of cow’s milk, where iodine concentrations were highest in milk produced in spring compared to autumn months (534.3 ± 53.7 vs. 433.6 ± 57.8 µg/kg, respectively; p = 0.001). The measured iodine and selenium concentrations of NI milk were higher than those listed in current UK Food Composition Databases (Food Standards Agency (FSA) (2002); FSA (2015)). The dietary modelling analysis confirmed that milk makes an important contribution to iodine and selenium intakes. This contribution may be higher than previously estimated if iodine and selenium (+25.0 and +1.1 µg/day respectively) concentrations measured in the present study were replicable across the UK at the current level of milk consumption. Iodine intakes were theoretically shown to vary by season concurrent with the seasonal variation in NI milk iodine concentrations. Routine monitoring of milk iodine concentrations is required and efforts should be made to understand reasons for fluctuations in milk iodine concentrations, in order to realise the nutritional impact to consumers.

Platinum–DNA adducts, and in particular Pt–GG, have been identified as the major cytotoxic species during chemotherapy treatment with platinum containing drugs. This paper reports for the first time a strategy based on the use of double species-specific isotope dilution analysis (IDA) for the quantification of carboplatin–GG adducts formed by exposing human lung carcinoma cells to carboplatin. The main challenge posed by the use of carboplatin in this pre-clinical application, in comparison with most previously reported studies using cisplatin, includes the relatively low reactivity of this drug, thus demanding for improved limits of detection to be achieved in order to perform accurate quantification of the adducts at low ng Pt per mg DNA levels with relatively small uncertainty in micro-volumes of the biological sample. This was alleviated by developing micro-flow HPLC reversed phased methodology coupled to sector field ICP-MS (R = 300), showing a limit of detection of 0.2 ng Pt per mg DNA. To perform IDA, carboplatin–GG calibrants and spikes (¹⁹⁴Pt-enriched GG adducts) were synthesized in house and characterised for Pt mass fraction, Pt distribution and structural composition. In order to assess the accuracy of the developed procedure, in the absence of certified reference materials, a reference sample prepared by incubation of calf thymus DNA with carboplatin and characterised in house (e.g. for its total P and Pt contents and Pt–GG concentration) was analysed in parallel. The use of this sample as a quality control of the cleavage of DNA and recovery of Pt adducts from real samples makes the described strategy particularly novel. Moreover, spike recovery experiments on the cell samples with the reference carboplatin–DNA sample were undertaken. The validated methodology was applied to cultured human lung carcinoma cells exposed to carboplatin; Pt–GG adducts were found to be at a level of 5.54 ng Pt per mg DNA with a relatively expanded combined uncertainty (k = 2) of approximately 20%. The major contributing factors to the overall measurement uncertainty were the mass fraction of Pt in the natural carboplatin–GG standard, the measured isotope ratio precision of sample and calibration blends and the blend to blend variation. The SI traceable methodology presented here will be invaluable for the provision of reference values to clinical measurements and cancer clinical trials.

Cow's milk is the most important dietary source of iodine and it also contributes to dietary selenium intakes in many European countries, including the UK and Ireland (1-4). Iodine and selenium are necessary for the maintenance of thyroid hormone homeostasis and have important roles in growth and development (5). While the UK and Ireland were historically thought to be iodine sufficient, emerging evidence shows that many of the population are now iodine deficient (6,7). The aim of the present study was to investigate the effect of season, milk fat class and pasteurisation on iodine and selenium concentrations in Northern Ireland (NI) milk. Milk samples (unpasteurised, whole, semi-skimmed and skimmed) were collected from two large NI creameries between May 2013 and April 2014. Monthly samples of each milk type were analysed by inductively coupled plasma-mass spectrometry (ICP-MS) for iodine and selenium content.

A species-specific isotope dilution analysis (SS IDA) method was developed for the first time for the determination of selenoprotein P (SEPP1) in human plasma/serum at the protein level by double affinity HPLC-ICP-MS. In this regard, a standard and a spike of SEPP1 were produced by cell-free E. coli protein synthesis, where Se (ICP tag) was introduced in the form of selenomethionine (SeMet) allowing for the absolute SEPP1 quantification by ICP-MS. A complete characterization of the standard and the spike was carried out in terms of isotopic composition and Se mass fraction by collision-cell ICP-MS to ensure SI-traceability. Method development and validation were conducted using the reference materials BCR-637, extensively analysed for its Se species, and the SRM 1950, which provides reference values for Se species (including SEPP1). Stability of the isotope ratio R_77/76 in the sample blends was tested for one month with negligible change. Relative expanded uncertainties of 5.7% and 7.7% were achieved for BCR-637 and SRM 1950 for mass fractions of 55.5 and 63.9 ng g⁻¹ Se for SEPP1, respectively. The developed SEPP1 SS IDA methodology could be a valuable tool to establish references values for selenoproteins in clinical chemistry and showed the potential of cell-free protein synthesis for the preparation of future stable isotope-labeled intact selenoproteins.