CCQM-K107: Total elements and selenomethionine in human serum

Transcription

1 CCQM-K107: Total elements and selenomethionine in human serum Final Report Contact Point: Heidi Goenaga-Infante Tel: Date: 15 February 2016

2 CCQM-K107: Total elements and selenomethionine in human serum Final Report Heidi Goenaga Infante LGC, United Kingdom March 2016

3 CCQM-K107 final report Contents 1. Introduction 3 2. Rationale of this comparison 3 3. Participation in CCQM-K The K107 serum sample and Se species calibration standard 4 5. Instructions to the participants 6 6. Methods and instrumentation used 6 7. CCQM-K107 participants' results and discussion Results for total Fe Results for total K Results for total Mg Results for total Ca Results for SeMet SI traceability of results KCRV and associated uncertainty Degrees of equivalence Conclusions Acknowledgements References 25 Annex 1: Invitation to participate in the key comparison CCQM-K Annex 2: Protocol distributed to participants 30 Annex 3: Results report form 33 Annex 4: Methodology used for th determination of SeMet in the CCQM-K107 sample 38 Annex 5: Core competency summary table for HPLC-ID-ICP-MS 39 Annex 6: Core competency summary table for HPLC-ICP-MS 42 Annex 7: Core competency summary table for ETA-AAS 44 Annex 8: Core competency summary table for ID-TIMS 46 Annex 9: Core competency summary table for ID-ICP-MS 48 Annex 10: Core competency summary table for ICP-OES 49 Annex 11: Core competency summary table for ICP-MS 51 CCQM-K107 final report

4 1. Introduction The last IAWG key comparison for total elements in the clinical area (CCQM-K14: Ca in human serum) was organized in Therefore, participating in this newly proposed study will help NMIs to support relevant CMCs in this area. It will also help European NMI partners of the EMRP JRP 10 project (TraceBioactivity) to demonstrate acquired capability for the accurate quantification of Se species at low µg kg -1 levels in clinical matrices. Also, in terms of Se species (SeMet) 1, these are follow-up studies to the previous key comparison K60 (on wheat flour), in which the ability of NMIs and expert laboratories worldwide to deliver accurate results for SeMet in a complex sample (containing approximately 28 mg kg -1 SeMet) with 5 % expanded uncertainty was demonstrated. Therefore, the IAWG agreed that CCQM-K107 and parallel P146 should be carried out. The candidate human serum sample to be used in both CCQM-K107 and P146 is of high complexity but contains much lower concentrations of SeMet than those encountered in the CCQM-K60 wheat flour (approx fold lower). This significantly broadens the scope and degree of difficulty of earlier measurements in this field. Organisations which are a national metrological institute (NMI), or an appropriate designated institute (DI) in accordance with the CIPM MRA, are invited to participate in the key comparison or the pilot study. Other expert institutes, from countries that are members of the Metre Convention, may also participate in the pilot study provided that their contribution HSA added scientific value or where they may qualify later as a designated institute in the field under study. The process of nomination of expert laboratories for participation in a CCQM pilot study should preferably be nationally co-ordinated. Expert laboratories which respond to this invitation are requested to inform their national metrological institute of their participation in the pilot study and to advise the co-ordinating laboratory of the appropriate contact at their NMI. In accordance with the requirements of the CCQM President, the IAWG Chairman will be asked to formally notify each relevant NMI of the participation by an expert institute from their country. 2. Rationale for this comparison Routine tests that measure the concentration of electrolytes in serum are needed for diagnosis and management of renal, endocrine, acid-base, water balance and other conditions. For example, the reference range for K is mmol/l and values below 3.0 mmol/l or above 6.0 mmol/l are associated with heart problems. Increased levels of plasma K are also associated with renal failure. The typical Ca concentration range in healthy adults is mmol/l and routine clinical tests for Ca are used for screening D- and A-vitamin disorders, kidney insufficiency, bone diseases and leukaemia. Reference methods and measurements of electrolytes and also essential trace elements in clinical samples are increasingly needed for quality control of clinical tests. There is an increasing interest in the speciation of selenium (Se) compounds in clinical samples such as serum. Serum concentration of total Se but more importantly, individual Se- Species (e.g. GPx3, SelP, selenoalbumin) are bio-indicators of Se status 2,3. However, reference methods and materials are available only for total serum Se (e.g. NIST SRM909c; BCR-637; ERM DA120) and they are, therefore, urgently needed for species to provide reference values for nutrition and cancer clinical trials. CCQM K107 final report Page 3 of 57

5 Participants will be required to determine the analytes in the matrix material using their own calibration solutions. The proposed study will fully test the ability of laboratories to accurately quantify total elements (electrolytes at high mg kg -1 levels and essential elements at low mg kg-1 levels) and seleno-amino acids (at low µg kg -1 levels) of relevance to health, in limited amounts of human serum. 3. Participation in CCQM-K107 A total of eleventh NMIs participated in CCQM-K107 (11 participants for total elements and 7 for SeMet), as summarised in Table The CCQM K107 serum sample and Se species calibration standard The sample is frozen human serum prepared from blood obtained from the Royal Surrey County Hospital (Guildford, UK). It HSA been stored in 2 ml plastic screw-cap Nalgene cryovial, each containing 1.1 ml serum, at -70 C ± 10 C in the dark. The material HSA been certified by LGC (ERM - DA120a) for total Cu ( g kg -1 ), Zn ( g kg -1 ) and Se ( g kg -1 ) mass fractions. Confirmatory data was obtained from a PT scheme (TEQAS) with 50 participants. For information, the serum was obtained from a pooled sample of many patients and authorised for use as a test material in analytical chemistry. The serum sample HSA been tested for homogeneity (n=10 in duplicate) with 0.5g subsamples analysed by ID-ICP-MS. Cu u hom = 0.98%; Se u hom = 1.88% and Zn u hom = 1.84%. Short term stability data (storage at -20 C and 40 C for 2 weeks, 1 month) suggested that the sample should be transported frozen (e.g. using ice packs). Long term stability data (storage at -20 C for 1, 3 and 6 months) showed no significant change for the material occurred in the Cu, Zn and Se content. The material should be stored at (-20±5 C) in the original closed vial until it is first opened. Homogeneity testing for electrolytes was performed by PTB using microwave digestion and ion chromatography. LGC did perform homogeneity testing for Fe, Mg and SeMet using ICP-IDMS and HPLC-ICP-IDMS, respectively, before the sample was distributed. PTB analysed ten vials in duplicate (0.2 g subsample); the u hom for K and Ca were 0.10% and 0.11%, respectively. LGC analysed 10 vials in duplicate (0.3 g subsample); the u hom for Fe, Mg and SeMet were 1.70%, 1.15% and 2.20%, respectively. Participants were sent a number of vials according to their response to the questionnaire distributed by the coordinating laboratory. An average of approximately 5-10 vials was sent to each participant. All samples were shipped in dry ice. NIM China provided one ampoule of SeMet solution CRM (GBW10034) with mass fraction of 39.4 µg/g (as Se) and expanded uncertainty (k = 2) of 1.0 mg/g (as Se) to the participants which required this material for the determination of SeMet. The use of this material as calibrant was optional. Each ampoule of this material contains 2 ml of selenomethione solution. The certificate of analysis for this material with the instructions were sent to the participants. Core competency table templates for ICP-OES, ICP-MS, ICP-IDMS, ETA-AAS, HPLC-ICP- IDMS and HPLC-ICP-MS were provided by LGC. PTB provided a core competency table for ID- TIMS. CCQM K107 final report Page 4 of 57

6 Table 1. CCQM-K107 participants Lab No. Institute/ Organisation NMI (Y/N) Contact Person(s) Total Elements (Y/N) SeMet (Y/N) 1 2 National Metrology Institute of Japan (NMIJ) Government Laboratory, Hong Kong (GLHK) 3 PTB Y Tϋbìtak UME (UME) Health Sciences Authority Singapore (HSA) National Institute of Measurement Thailand (NIMT) EXHM/GCSL-EIM Chemical Metrology Laboratory, Hellenic Metrology Institute (EXHM) National Institute of Metrology, P.R. China (NIM China) Y Y Y N Y Y Y Tomohiro Narukawa (Tomohironarukawa@aist.go.jp) Zhu Yanbei (yb-zhu@aist.go.jp) Fung Wai-Hong (whfung@govtlab.gov.hk) Yau Ho-Pan (hpyau@govtlab.gov.hk) Claudia.Swart (claudia.swart@ptb.de) Olaf.Rienitz (olaf.rienitz@ptb.de) Alper Işleyen (alper.isleyen@tubitak.gov.tr) Murat Tunc (tunc.murat@tubitak.gov.tr) Süleyman Z. Can suleyman.can@tubitak.gov.tr Nilgϋn Tokman (nilgun.tokman@tubitak.gov.tr Richard Shin (richard_shin@hsa.gov.sg) Pranee Phukphatthanachai (pranee@nimt.or.th) Charun Yafa (charun_yafa@yahoo.com) Elias Kakoulides (metrology@gcsl.gr) Liuxing Feng (fenglx@nim.ac.cn) Y Y (no Fe) Y Y Y Y Y (only Fe) Y Y N Y Y N N N Y CCQM K107 final report Page 5 of 57

7 9 LGC Y 10 LNE Y 11 NIST Y Sarah Hill (sarah.hill@lgcgroup.com) Paola Fisicaro (paola.fisicaro@lne.fr) Maria Estela del Castillo (Maria-Estela.Del- Castillo@lne.fr) Steve Long (selong@nist.gov) Y Y (only Fe) Y Y Y Y 5. Instructions to the participants The CCQM-K107 sample was sent to all participants in January 2013 with an accompanying letter containing the protocol explaining the work to be conducted and a results report form for submission of data (see Annexes 2 and 3). The protocol and results report form included the following: scope of the study general instructions for handling and preservation of sample and calibration standard request for reporting results in mg kg -1 (for K, Ca and Mg) and in µg kg -1 (for Fe and SeMet), for at least three replicate analyses (SeMet) and five replicate analyses (total elements). request for a full description of the sample preparation and measurement procedures request for a full description of calibrants in terms of source, purity, uncertainty. request for uncertainty evaluation according to ISO principles (Guide to the Expression of Uncertainty in Measurement, ISO, Geneva, 1993, ISBN ) request for a full uncertainty budget No sample preparation and/or measurement techniques were prescribed by the coordinating laboratory. As a consequence, participants were free to develop and validate their own approaches. 6. Methods and instrumentation used For the determination of total Fe, seven (out of ten) of the key comparison participants used microwave acid digestion with isotope dilution ICP-MS (using 57 Fe spike except for NIMT which used 54 Fe spike). LGC, HSA and UME used sample dilution (with dilute nitric acid) with isotope dilution ICP-MS and ICP-MS with matrix matched external calibration, respectively. For the determination of total K, six (out of nine) of the key comparison participants used microwave acid digestion for sample preparation whilst LGC, HSA and UME used sample dilution with dilute nitric acid. Five of the participants used IDMS. PTB used ID TIMS, NMIJ used ICPMS with standard additions, UME used ICP-MS with matrix matched external calibration and GLHK used ICPOES with standard additions. CCQM K107 final report Page 6 of 57

8 For the determination of total Mg, five (out of eight) of the key comparison participants used microwave acid digestion for sample preparation whilst LGC, HSA and UME used sample dilution with dilute nitric acid. Most participants used IDMS calibration. The exceptions were UME, GLHK and NMIJ which used ICP-MS with matrix matched external calibration, ICP OES with gravimetric standard addition and ICP-MS with standard additions and internal standard correction, respectively. For the determination of total Ca, five (out of eight) of the key comparison participants used microwave acid digestion for sample preparation whilst LGC, HSA and UME used sample dilution with dilute nitric acid. Four (out of eight) of the participants used IDMS calibration. HSA and GLHK used ICP-OES with standard addition. UME used ICP-MS with matrix matched external calibration and NMIJ used ICP-MS with standard additions and internal standard correction. For the determination of SeMet, six (out of seven) participants used HPLC-ICP-MS with speciesspecific IDA ( 76 SeMet spike) after multi-step enzymatic hydrolysis or HCl hydrolysis, except for NIST, which used HPLC-ICP-MS with standard additions. A more detailed overview of the analytical methods and instrumental techniques used by each participant for the determination of SeMet is given in Annex CCQM-K107 participants results and discussion 7.1. Results for total Fe Results for total Fe are summarised in Table 2 and graphically displayed in Figure 1. All uncertainties reported are standard uncertainties (k=1). Table 2. Results for the determination of total Fe in CCQM-K107 serum sample Participant Mass fraction, µg kg -1 Standard uncertainty, µg kg -1 Expanded uncertainty, µg kg -1 UME LGC NIM China EXHM HSA NIST LNE NMIJ k = CCQM K107 final report Page 7 of 57

9 PTB NIMT Figure 1. Results for total Fe in K107 serum sample. Error bars depict standard uncertainties. The solid horizontal line is the median of all results. As can be seen in the graph, the NIMT mass fraction for Fe is significantly high biased from those reported by the rest of the participants. The first presentation of results to the working group was followed up by discussions with NIMT, which identified problems related to relatively high procedural blanks. Therefore, the working group decided to exclude the NIMT value from the calculations of the KCRV and its associated uncertainty. Figure 2 represents results obtained for Fe excluding the NIMT value as outlier. CCQM K107 final report Page 8 of 57

10 Figure 2. Results for total Fe in K107 serum sample. Error bars depict standard uncertainties. The solid horizontal line is the suggested KCRV of µg kg -1 ; the dashed lines show the standard uncertainty interval calculated with a coverage factor k= Results for total K Results for total K are summarised in Table 3 and graphically displayed in Figure 3. All uncertainties reported are standard uncertainties (k=1). Table 3. Results for the determination of total K in CCQM-K107 serum sample Participant Mass fraction, mg kg -1 Standard uncertainty, mg kg -1 Expanded uncertainty, mg kg -1 UME NIMT HSA k = CCQM K107 final report Page 9 of 57

11 PTB NMIJ NIST LGC GLHK NIM China Figure 3. Results for total K in K107 serum sample. Error bars depict standard uncertainties. The solid horizontal line is the suggested KCRV of mg kg -1 ; the dashed lines show the standard uncertainty interval calculated with a coverage factor k=1 CCQM K107 final report Page 10 of 57

12 7.3. Results for total Mg Results for total Mg are summarised in Table 4 and graphically displayed in Figure 4. All uncertainties reported are standard uncertainties (k=1). Table 4. Results for the determination of total Mg in CCQM-K107 serum sample Participant Mass fraction, mg kg -1 Standard uncertainty, mg kg -1 Expanded uncertainty, mg kg -1 UME HSA NIST LGC NIMT GLHK NIM China NMIJ k = CCQM K107 final report Page 11 of 57

13 Figure 4. Results for total Mg in K107 serum sample. Error bars depict standard uncertainties. The solid horizontal line is the suggested KCRV of mg kg -1 ; the dashed lines show the standard uncertainty interval calculated with a coverage factor k= Results for total Ca Results for total Ca are summarised in Table 5 and graphically displayed in Figure 5. All uncertainties reported are standard uncertainties (k=1). Table 5. Results for the determination of total Ca in CCQM-K107 serum sample Participant Mass fraction, mg kg -1 Standard uncertainty, mg kg -1 Expanded uncertainty, mg kg -1 NMIJ HSA LGC NIM China NIST UME k = CCQM K107 final report Page 12 of 57

14 GLHK NIMT Figure 5. Results for total Ca in K107 serum sample. Error bars depict standard uncertainties. The solid horizontal line is the suggested KCRV of mg kg -1 ; the dashed lines show the standard uncertainty interval calculated with a coverage factor k= Results for SeMet Results for SeMet are summarised in Table 6 and graphically displayed in Figure 6. All uncertainties reported are standard uncertainties for a coverage factor (k) of 1. Although LNE reported the uncertainty summarised in Table 6 and Figures 6 and 7, an error was found in the reported data. Whilst calculating the uncertainty for SeMet, there was no multiplication for the CCQM K107 final report Page 13 of 57

15 conversion factor of Se to SeMet (2.48). Therefore, LNE revised their uncertainty value which is now 1.2 (k=1) and 2.4 (k=2). Table 6. Results for the determination of SeMet in CCQM-K107 serum sample Participant Mass fraction, µg kg -1 Standard uncertainty, µg kg -1 Expanded uncertainty, µg kg -1 LGC LNE* * 1.3* 2 NMIJ UME NIM China PTB NIST k = *New LNE uncertainties: 1.2 (k=1) ad 2.4 (k=2). CCQM K107 final report Page 14 of 57

16 Figure 6. Results for SeMet in K107 serum sample. Error bars depict standard uncertainties. The solid horizontal line is the median of all results. As can be seen in the graph, the NIST mass fraction for SeMet is significantly high biased from those reported by the rest of the participants. The first presentation of results to the working group was followed up by discussions with NIST about possible errors that could be found with the procedures used for the determination of SeMet in the CCQM K107 serum sample. NIST identified problems that are well known to affect accuracy of SeMet determination when calibration techniques other than IDMS are used for quantification of this relatively labile species 1. Therefore, the working group decided to exclude the NIST value from the calculations of the KCRV and its associated uncertainty. Figure 7 represents results obtained for SeMet excluding the NIST value as outlier. Figure 7. Results for SeMet in K107 serum sample. Error bars depict standard uncertainties. The solid horizontal line is the suggested KCRV of µg kg -1 ; the dashed lines show the standard uncertainty interval calculated with a coverage factor k= SI Traceability of results Calibrant materials through which results (to be considered for the KCRV calculation) are traceable to the SI are summarised in Table 7. CCQM K107 final report Page 15 of 57

17 Table 7. Sources of SI traceability for the results provided for total elements and SeMet in the CQM K107 serum sample Participant Fe K Mg Ca SeMet UME NIST SRM 3126A NIST SRM 3141A NIST SRM 3131A NIST SRM 3109A NIM CRM GBW10034 LGC NIST SRM 3126A, lot NIST SRM 3141A, lot NIST SRM 3131A, lot NIST SRM 3109A, lot LGC in house characterised SeMet calibrant NIM China NIM CRM GBW NIM CRM GBW NIM CRM GBW NIM CRM GBW NIM CRM GBW10034 EXHM HSA NIST LNE NMIJ PTB GLHK NIMT NIST SRM 3126A n/a n/a n/a n/a NIST SRM 3126A NIST SRM 3126A, lot NIST SRM 3141A NIST SRM 3141A, lot NIST SRM 3131A NIST SRM 3131A, lot NIST SRM 3109A NIST SRM 3109A, lot High purity Iron primary standard BNM 001 n/a n/a n/a NIST SRM 3126A BAM-Aprimary-Fe-1 n/a NIST SRM 3126A NIST SRM 3141A BAM-Y-010 (KCl) NIST SRM 3141A NIST SRM 3141A NIST SRM 3131A n/a NIST SRM 3131A NIST SRM 3131A NIST SRM 3109A n/a NIST SRM 3109A NIST SRM 3109A n/a NIM CRM GBW10034 NIM CRM GBW10034 NIM CRM GBW10034 PTB in house characterised SeMet calibrant n/a n/a 8. KCRV and associated uncertainty Inspection showed no strong outliers among the valid data, and little evidence of over dispersion. Under these circumstances, robust estimators are unnecessary 4,5. Estimates and their associated uncertainties were accordingly calculated using the mean, median, the DerSimonian-Laird CCQM K107 final report Page 16 of 57

18 estimator (DSL) and, solely for comparison, the uncertainty-weighted mean (also known as the Graybill-Deal estimator). The results are shown in Table 8 and Figure 8. Please note that none of the uncertainties shown here incorporate explicit allowances for inhomogeneity of the test materials. The working group selected the median/made basis for all analytes, as represented in the figures in section 7. As discussed earlier, outlier values of total Fe (by NIMT) and SeMet (by NIST) were not included in these calculations. Table 8. Candidate estimates of KCRV and KCRV uncertainty Analyte Estimator KCRV u Df U Units Remark Ca Arithmetic mean mg/kg Ca Median/MADe mg/kg Chosen KCRV Ca DerSimonian-Laird mg/kg Between-lab SD: Ca Weighted mean mg/kg Fe Arithmetic mean µg/kg Fe Median/MADe µg/kg Chosen KCRV Fe DerSimonian-Laird µg/kg Between-lab SD: Fe Weighted mean µg/kg K Arithmetic mean mg/kg K Median/MADe mg/kg Chosen KCRV K DerSimonian-Laird mg/kg Between-lab SD: 0 K Weighted mean mg/kg Mg Arithmetic mean mg/kg Mg Median/MADe mg/kg Chosen KCRV Mg DerSimonian-Laird mg/kg Between-lab SD: 0.20 Mg Weighted mean mg/kg SeMet Arithmetic mean µg/kg SeMet Median/MADe µg/kg Chosen KCRV SeMet DerSimonian-Laird µg/kg Between-lab SD: 0 SeMet Weighted mean µg/kg CCQM K107 final report Page 17 of 57

19 Arithmetic mean Median/MADe DerSimonian-Laird Weighted mean Arithmetic mean Median/MADe DerSimonian-Laird Weighted mean Arithmetic mean Median/MADe DerSimonian-Laird Weighted mean mg/kg g kg mg/kg Ca (mg/kg) Fe g kg K (mg/kg) CCQM K107 final report Page 18 of 57

20 Arithmetic mean Median/MADe DerSimonian-Laird Weighted mean Arithmetic mean Median/MADe DerSimonian-Laird Weighted mean mg/kg g kg Mg (mg/kg) Selenomethionine g kg Figure 8: Candidate KCRV and KCRV uncertainties. Uncertainties shown are expanded using coverage factors based on Student s t for the appropriate degrees of freedom. 9. Degrees of equivalence Given the values xi (submitted by the participating NMIs) with standard uncertainties ui and a KCRV xk calculated as the median with standard uncertainty uk calculated as the MADe, the degree of equivalence di is (xi-xk) with standard uncertainty u(di) = (ui 2 +uk 2 ) 0.5 for results included in the estimate are summarised in Table 9. Figure 9 shows degrees of equivalence with error bars showing the uncertainty component of the degree of equivalence expressed as an expanded uncertainty with a coverage factor k=2. Empty circles (o) denote laboratories excluded from the KCRV calculation. Table 9. CCQM K107 - degrees of equivalence Analyte Laboratory x u U Units d U(d)* Ca NMIJ mg/kg Ca HSA mg/kg Ca LGC mg/kg Ca NIM mg/kg CCQM K107 final report Page 19 of 57

21 Ca NIST mg/kg Ca UME mg/kg Ca GLHK mg/kg Ca NIMT mg/kg Fe UME μg/kg Fe LGC μg/kg Fe NIM μg/kg Fe EXHM μg/kg Fe HSA μg/kg Fe NIST μg/kg Fe LNE μg/kg Fe NMIJ μg/kg Fe PTB μg/kg Fe NIMT μg/kg K UME mg/kg K NIMT mg/kg K HSA mg/kg K PTB mg/kg K NMIJ mg/kg K NIST mg/kg K LGC mg/kg K GLHK mg/kg K NIM mg/kg CCQM K107 final report Page 20 of 57

22 Table 9 (continued) Analyte Laboratory x u U Units d U(d)* Mg UME mg/kg Mg HSA mg/kg Mg NIST mg/kg Mg LGC mg/kg Mg NIMT mg/kg Mg GLHK mg/kg Mg NIM mg/kg Mg NMIJ mg/kg Selenomethionine LGC μg/kg Selenomethionine LNE (corre cted: 1.2) 1.3 (corr ected :2.4) μg/kg (corrected : 2.541) Selenomethionine NMIJ μg/kg Selenomethionine UME μg/kg Selenomethionine NIM μg/kg Selenomethionine PTB μg/kg Selenomethionine NIST μg/kg *Degree of equivalence uncertainties are shown to three decimal places and use a coverage factor k=2. The U(d) calculated for the LNE s d with the corrected uncertainty is CCQM K107 final report Page 21 of 57

23 CCQM K107 final report Page 22 of 57

24 CCQM K107 final report Page 23 of 57

25 Figure 9: Degrees of equivalence with error bars showing the uncertainty component of the degree of equivalence expressed as an expanded uncertainty with a coverage factor k=2. Empty circles (o) denote laboratories excluded from the KCRV calculation. The uncertainty associated with LNE s degree of equivalence for the revised value is larger than that represented in the graph (2.541). 10. Conclusions The performance of the majority of the K107 participants for all the measurands was very good, illustrating their ability to obtain accurate results for analytes such as electrolytes (at mg kg -1 level) and essential elements (at µg kg -1 level) and selenium species (at µg kg -1 level) in a complex biological fluid. Results agreed within the interval of ± 0.1% (for Ca) to ± 1.8% (for Fe). With regard to how far does the light shine, CMC claims based on SeMet measurements in this study may be applied to other biological matrices (e.g., tissues) provided that the concentration range is similar and due diligence is taken to ensure an appropriate extraction process is achieved and species specific spikes are available for quantitation by ID. Indeed, having accepted such conditions, application to quantitation of other organometallic species and other elements in similar matrices should be possible with the same level of performance. CMC claims based on total elements in this study may include other elements with similar core competencies (e.g. Se, Cu, Zn) in a wide range of biological materials (including liquids and CCQM K107 final report Page 24 of 57

26 solids) at a similar level of performance using the same measurement technique applied in CCQM-K107 provided that there are no additional factors (e.g. blank or dissolution issues). The core competency tables for all the measurands are summarised in Annexes They can be used to support a wide variety of CMCs using the core capability approach. 11. Acknowledgements The work described contains contributions of many scientists as described in Table 1. LGC particularly acknowledges the efforts of the Reference Materials team, especially, G. Holcombe and D. Curtis for help with organising the homogeneity and stability study. Also to Detlef Shiel from PTB for help with homogeneity measurements for the electrolytes and to S. Ellison for the statistical analysis of the K107 results. LGC also acknowledges the efforts of L. Evans, D. Kmiotek, Sarah Hill and Christian Deitrich for contributing to the homogeneity testing of the K107 sample. Also, special thanks to M. Sargent for very helpful discussions and for his assistance as chairman of the Inorganic Analysis WG of CCQM. The coordination of this study at LGC was funded, in part, by the UK National Measurement Office. 14. References [1] H. Goenaga Infante, R. Sturgeon, J. Turner, R. Hearn, M. Sargent, P. Maxwell, L. Yang, A. Barzev, Z. Pedrero, C. Cámara, V. Díaz Huerta, M. L. Fernández Sánchez, A. Sanz-Medel, K. Emese, P. Fodor, W. Wolf, R. Goldschmidt, V. Vacchina, J. Szpunar, L. Valiente, R. Huertas, G. Labarraque, C. Davis, R. Zeisler, G. Turk, E. Rizzio, L. G. Mackay, R. B. Myors, D. L. Saxby, S. Askew, W. Chao and W. Jun, Anal.Bioanal. Chem., 2008, 390, [2] P. Jitaru, H. Goenaga-Infante, S. Vaslin-Reimann, P. Fisicaro, Anal. Chim Acta, 2010, 657, [3] L. Hinojosa Reyes, j m. Marchante-Gaýon; J I García-Alsonso and A. Sanz-Medel,. J Anal At Spectrom., 2003, 18, [4] R Core Team (2014). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.URL [5] S L R Ellison. (2014). metrology: Support for metrological applications. R package version CCQM K107 final report Page 25 of 57

27 ANNEX 1 Call for Participants Key Comparison CCQM-K107 and Pilot Study CCQM-P146 Elements (K, Ca, Mg, Fe) and Selenomethionine in human serum From: The Co-ordinating Laboratory (LGC Limited) To: Members of the CCQM Inorganic Analysis Working Group (IAWG) Other expert institutes 15 November 2012 (Reply is requested even if the institute does not wish to participate) Dear Colleague, This letter is an invitation to participate in the key comparison CCQM-K107 and the parallel pilot study CCQM-P146, Elements and selenomethionine (SeMet) in human serum. The last IAWG key comparison for total elements in the clinical area (CCQM-K14: Ca in human serum) was organized in Therefore, participating in this newly proposed study will help NMIs to support relevant CMCs in this area. It will also help European NMI partners of the EMRP JRP 10 project (TraceBioactivity) to demonstrate acquired capability for the accurate quantification of Se species at low ng g -1 levels in clinical matrices. Also, in terms of Se species (SeMet), these are follow-up studies to the previous key comparison K60 (on wheat flour), in which the ability of NMIs and expert laboratories worldwide to deliver accurate results for SeMet in a complex sample (containing approximately 28 mg kg -1 SeMet) with 5 % expanded uncertainty was demonstrated. Therefore, the IAWG agreed that CCQM-K107 and parallel P146 should be carried out. The candidate human serum sample to be used in both CCQM-K107 and P146 is of high complexity but contains much lower concentrations of SeMet than those encountered in the CCQM-K60 wheat flour (approx fold lower). This significantly broadens the scope and degree of difficulty of earlier measurements in this field. Organisations which are a national metrological institute (NMI), or an appropriate designated institute (DI) in accordance with the CIPM MRA, are invited to participate in the key comparison or the pilot study. Other expert institutes, from countries that are members of the Metre Convention, may also participate in the pilot study provided that their contribution HSA added CCQM K107 final report Page 26 of 57

28 scientific value or where they may qualify later as a designated institute in the field under study. The process of nomination of expert laboratories for participation in a CCQM pilot study should preferably be nationally co-ordinated. Expert laboratories which respond to this invitation are requested to inform their national metrological institute of their participation in the pilot study and to advise the co-ordinating laboratory of the appropriate contact at their NMI. In accordance with the requirements of the CCQM President, the IAWG Chairman will be asked to formally notify each relevant NMI of the participation by an expert institute from their country. The results of the key comparison will be presented in the form of a report to the CCQM, available to participants and to members of the IAWG. The report will identify the results with the names of the participating institutes. Preliminary (A) and final (B) drafts of the report will be circulated to participants for comment and correction. The approved report will be submitted to the BIPM s Key Comparison Database (KCDB) and the results will be publicly available. A similar report will be prepared for the pilot study, for participants and members of the IAWG. A scientific paper describing the study may be published separately in an appropriate journal provided participants agree to this. A short description of the study is given below. A detailed study protocol will be sent to registered participants later. Rationale Routine tests that measure the concentration of electrolytes in serum are needed for diagnosis and management of renal, endocrine, acid-base, water balance and other conditions. For example, the reference range for K is mmol/l and values below 3.0 mmol/l or above 6.0 mmol/l are associated with heart problems. Increased levels of plasma K are also associated with renal failure. The typical Ca concentration range in healthy adults is mmol/l and routine clinical tests for Ca are used for screening D- and A-vitamin disorders, kidney insufficiency, bone diseases and leukaemia. Reference methods and measurements of electrolytes and also essential trace elements in clinical samples are increasingly needed for quality control of clinical tests. There is an increasing interest in the speciation of selenium (Se) compounds in clinical samples such as serum. Serum concentration of total Se but more importantly, individual Se- Species (e.g. GPx3, SelP, selenoalbumin) are bio-indicators of Se status. However, reference methods and materials are available only for total serum Se (e.g. NIST SRM909c; BCR-637; ERM DA120) and they are, therefore, urgently needed for species to provide reference values for nutrition and cancer clinical trials. Participants will be required to determine the analytes in the matrix material using their own calibration solutions. The proposed study will fully test the ability of laboratories to accurately quantify total elements (electrolytes at high mg kg -1 levels and essential elements at low mg kg -1 levels) and seleno-amino acids (at low g kg -1 levels) of relevance to health, in limited amounts of human serum. CCQM K107 final report Page 27 of 57

29 Sample The sample is frozen human serum prepared from blood obtained from the Royal Surrey County Hospital (Guildford, UK). It HSA been stored in 2 ml plastic screw-cap Nalgene cryovial, each containing 1.1 ml serum, at -70 C ± 10 C in the dark. The material HSA been certified by LGC (ERM - DA120a) for total Cu ( g/kg), Zn ( g/kg) and Se ( g/kg) mass fractions. Confirmatory data was obtained from a PT scheme (TEQAS) with 50 participants. For information, the serum was obtained from a pooled sample of many patients and authorised for use as a test material in analytical chemistry. The serum sample HSA been tested for homogeneity (n=10 in duplicate) with 0.5g subsamples analysed by ID-ICP-MS. Cu u hom = 0.98%; Se u hom = 1.88% and Zn u hom = 1.84%. Short term stability data (storage at -20 C and 40 C for 2 weeks, 1 month) suggested that the sample should be transported frozen (e.g. using ice packs). Long term stability data (storage at -20 C for 1, 3 and 6 months) showed no significant change for the material occurred in the Cu, Zn and Se content. The material should be stored at (-20±5 C) in the original closed vial until it is first opened. Homogeneity testing for electrolytes and SeMet content will be performed by PTB using ion chromatography and LGC using HPLC-ICP-IDMS, respectively, before the sample is distributed. Participants will be sent a number of vials according to their response to the questionnaire distributed by the coordinating laboratory. Measurands Total Ca ( mg kg -1 ), K ( mg kg -1 ), Fe (0.1-1 mg kg -1 ), Mg and selenomethionine ( mg kg -1 as Se) in a human serum sample. Method of analysis Participants are encouraged to use any method of their choice. It is recommended that preparation and dilution of solutions be carried out by weighing. Time schedule Deadline for registration of participation: 07 December 2012 Shipment of samples: 15 December 2012 Deadline for delivery of results: 28 February 2013 Presentation of results: April 2013 Draft A report: July Registration If you wish to participate in the key comparison CCQM-K107 and/or pilot study CCQM-P146 please use the registration sheet provided and specify which analyte(s) you wish to analyse and CCQM K107 final report Page 28 of 57

30 which method(s) will be used. Institutes not wishing to participate are also requested to sign and return the registration form. Please return the form by or fax no later than 07 December 2012 to: 2. Dr Heidi Goenaga-Infante LGC Limited Queens Road Teddington, Middlesex TW11 0LY United Kingdom Fax: I look forward to your participation in this study. Yours sincerely, Heidi Goenaga-Infante CCQM K107 final report Page 29 of 57

31 ANNEX 2 Key Comparison CCQM-K107 and Pilot Study CCQM-P146 Elements (K, Ca, Mg, Fe) and Selenomethionine in human serum Introduction Protocol Routine tests that measure the concentration of electrolytes in serum are needed for diagnosis and management of renal, endocrine, acid-base, water balance and other conditions. For example, the reference range for K is mmol/l and values below 3.0 mmol/l or above 6.0 mmol/l are associated with heart problems. Increased levels of plasma K are also associated with renal failure. The typical Ca concentration range in healthy adults is mmol/l and routine clinical tests for Ca are used for screening D- and A-vitamin disorders, kidney insufficiency, bone diseases and leukaemia. Reference methods and measurements of electrolytes and also essential trace elements in clinical samples are increasingly needed for quality control of clinical tests. There is an increasing interest in the speciation of selenium (Se) compounds in clinical samples such as serum. Serum concentration of total Se but more importantly, individual Se- Species (e.g. GPx3, SelP, selenoalbumin) are bio-indicators of Se status. However, reference methods and materials are available only for total serum Se (e.g. NIST SRM909c; BCR-637; ERM DA120) and they are, therefore, urgently needed for species to provide reference values for nutrition and cancer clinical trials. The IAWG agreed that CCQM-K107 and parallel P146 should be carried out because it conforms with the requirements of the IAWG rolling programme of key comparisons, supporting ongoing requirements to demonstrate core capabilities as well as specific groups of CMCs. The last IAWG key comparison for total elements in the clinical area (CCQM-K14: Ca in human serum) was organized in Therefore, participating in this newly proposed study will help NMIs to directly support relevant CMCs in this area. In terms of Se species (SeMet), these are follow-up studies to the previous key comparison K60 (on wheat flour), in which the ability of NMIs and expert laboratories worldwide to deliver accurate results for SeMet in a complex sample (containing approximately 28 mg kg -1 SeMet) with 5 % expanded uncertainty was demonstrated. The candidate human serum sample to be used in both CCQM-K107 and P146 is of high complexity but contains much lower concentrations of SeMet than those encountered in the CCQM-K60 wheat flour (approx fold lower). It will also help European NMI partners of the EMRP JRP 10 project (TraceBioactivity) to demonstrate acquired capability for the accurate quantification of Se species at low ng g -1 levels in clinical matrices. Participants will be required to determine the analytes in the matrix material using their own calibration solutions. The proposed study will fully test the ability of laboratories to accurately CCQM K107 final report Page 30 of 57

32 quantify total elements (electrolytes at high mg kg -1 levels and essential elements at low mg kg -1 levels) and seleno-amino acids (at low g kg -1 levels) of relevance to health, in limited amounts of human serum. This significantly broadens the scope and degree of difficulty of earlier measurements in this field and will provide a major extension of the current demonstration of core capabilities in the clinical field.. Sample The sample is frozen human serum prepared from blood obtained from the Royal Surrey County Hospital (Guildford, UK). It HSA been stored in 2 ml plastic screw-cap Nalgene cryovial, each containing 1.1 ml serum, at -70 C ± 10 C in the dark. The material HSA been certified by LGC (ERM - DA120a) for total Cu ( g/kg), Zn ( g/kg) and Se ( g/kg) mass fractions. Confirmatory data was obtained from a PT scheme (TEQAS) with 50 participants. The density of ERM-DA102a was measured as kg/l with a standard uncertainty of kg/l (0.99%). The material should be stored at (-20±5 C) in the original closed vial until it is first opened. The homogeneity of the candidate material was fully investigated for Ca and K by PTB (using ion chromatography), for Fe and Mg by LGC (using ID-ICP-MS) and for selenomethionine by LGC (using HPLC-ICP-IDMS) before it was distributed. The minimum recommended sample size is 0.3 g. Participants have been sent a number of vials according to their response to the questionnaire distributed by the coordinating laboratory. Other materials NIM China HSA kindly agreed to send 1 ampoule of the natural selenomethionine CRM (GBW10034) to each participant performing determination of selenomethionine in the CCQM serum. The use of this CRM (as calibrant) is optional. GBW10034 is a selenomethionine solution CRM (mass fraction of approx. 100 mg/kg as selenomethionine). Each ampoule of this material contains 2 ml of selenomethione solution. The certificate of analysis for this material with the instructions for use will also be sent to each participant. Analysis Results should be provided in mg/kg or g/kg, as appropriate. At least three independent results should be reported for selenomethionine and five for each total element concentration. Participants are free to use any suitable method but please include a full description of your method of analysis when reporting the results. Participants may, if they wish, obtain results by more than one method for each analyte. If the data are combined to report a single value, the demonstrated capability will be for the combined method. If results from more than one method are reported separately, the demonstrated capabilities will be for each method. However, please note that only one result from each institute (that with the smallest uncertainty) will be considered for calculation of the KCRV. A full uncertainty budget should also be included with your results, as indicated below. CCQM K107 final report Page 31 of 57

33 Uncertainty Evaluation Each laboratory should make an assessment of the experimental uncertainty according to ISO principles (Guide to the Expression of Uncertainty in Measurement, ISO, Geneva, 1993, ISBN ). Each variable contributing to the uncertainty of the results should be identified and quantified in order to be included in the combined standard uncertainty of the result. A full uncertainty budget must be reported, as part of the results. Contributions to the overall uncertainty will arise from the repeatability of the sample preparation, the repeatability of instrumental determination, determination of masses and volumes, concentration of primary and internal standards, and any other parameter specific to each method of analysis chosen by the participant. Results should be submitted using the results report form provided and sent to Rita Harte at LGC, by post, (Rita.Harte@lgc.co.uk) or fax, ( ) no later than 31 March Dr. Heidi Goenaga-Infante Study Co-ordinator LGC Limited Queens Road Teddington Middlesex TW11 0LY United Kingdom Fax: hgi@lgc.co.uk CCQM K107 final report Page 32 of 57

34 ANNEX 3 Key Comparison CCQM-K107 Elements (K, Ca, Mg, Fe) and Selenomethionine (SeMet) in human serum NAME : INSTITUTE : DEPARTMENT : ADDRESS : COUNTRY : TEL : FAX : Results Report Please return this results report by 31 March, Report your results and uncertainties in mass fraction using the units in the table below. Details concerning the analysis of replicates, details of the method, calculation of results, and associated uncertainties should be given in the following pages of your report. Analyte Matrix Unit Mass fraction Standard uncertainty Expanded uncertainty k Ca Serum mg kg -1 K Serum mg kg -1 Mg Serum mg kg -1 Fe Serum mg kg -1 SeMet Serum g kg -1 Method used for each analyte (if more than one method per analyte is used, please provide results/details on a separate sheet): CCQM K107 final report Page 33 of 57

35 Sample preparation: Analyte determination: DATE: SIGNATURE: CCQM K107 final report Page 34 of 57

36 Results for the analysis of replicate samples Date of Analysis: Mid April 25 th May, Additional information Determina tion Ca (mg kg -1 ) K (mg kg -1 ) Mg (mg kg -1 ) Fe ( g kg -1 ) SeMet ( g kg -1 ) Sample no. CCQM K107 final report Page 35 of 57

37 Report at least three replicates for SeMet and five for total Ca, K, Fe and Mg. If more than 5 determinations are carried out, please insert more lines. Method(s) used for total element and SeMet analysis Further information and details can be added in pages below, or in a separate report if preferred. If you use a separate report, please provide a complete description of the method(s) used for the determination, including the following information as appropriate: 1. Details of sample handling, including sample digestion/extraction and weight taken. 2. Measurement technique. 3. Calibration procedure. Principle (e.g. ICP-MS): Conditions used: Instrument used: 4. Details of the source of your calibration standard(s) together with the purity and associated uncertainty. 5. Any other relevant information. An uncertainty calculation should be prepared as described in the study protocol. Description Brief outline, or a reference to a published procedure, any special precautions to minimise loss or transformation of analyte, interferences etc. CCQM K107-Draft B report Page 1

38 Sample digestion or extraction: - Weight taken: - Extraction conditions including extractant/acids used, digestion/extraction time: - Procedure for extract/digest handling and preservation: - Time between extraction and measurement: Measurement procedure: Principle (e.g. ICP-MS): Conditions used: Instrument used: Calibration standard(s) used : Source, purity and uncertainty of standard(s): Typical standard solution concentration : Typical uncertainty of concentration : Type of calibration : IDMS [ x ]; External calibration [ ]; Standard addition [ ] - one-point [ ] - two-point [ ] - multi-point [ ] 2. If another method was used, please provide full details of the method below or in a separate report. CCQM K107-Draft B report Page 2

39 ANNEX 4 Methodology used for the determination of SeMet in the CCQM-K107 serum sample CCQM K107-Draft B report Page 3

40 ANNEX 5 Inorganic Core Capabilities Summary Table CCQM Study: CCQM-K107 Institute(s): PTB, LNE, UME, LGC, NIM China, NMIJ Method: HPLC-ID-ICP-MS Analyte(s): Selenomethionine (SeMet) Instructions: List in the appropriate column (as NIST, PTB, LGC, etc.) the institutes which did or did not demonstrate each capability. Where the table includes multiple analytes add the element symbols or All in parenthesis after each institute - e.g. LGC (As, Ca). Provide a brief summary of the challenges encountered in the final column, highlighting any aspects where this measurement presented an unusually high degree of difficulty. This should be a consensus agreed with all participants except where there is a valid reason for it to be different at a specific institute. This also requires explanation. Please add rows for any other capabilities which were used but which have not been included in this table. Capabilities/Challenges Not tested Tested Specific challenges encountered Extraction of species from the sample matrix Single- or multi-steps procedures used to efficiently extract the analyte(s) of interest from the sample matrix with preservation of the analyte (s) integrity Species pre-concentration Techniques and procedures used to increase the concentration of the species to be analysed by HPLC- ICP-IDMS. Includes solvent evaporation, freeze-drying, etc. Achieving compatibility of LC conditions with ICP-MS Selected chromatographic conditions (e.g mobile phsae composition and flow rate) selected to be compatible with conventional nebulisation ICP-MS Selectivity of LC separation Development of separation systems to separate the target species from other species of the same element and/or to minimise matrix interferences from other NIM, PTB, LNE, LGC NMIJ, NIM, UME, PTB, LNE, LGC NMIJ, UME, PTB NMIJ, NIM, UME, PTB, LNE, LGC NIM, PTB, LNE, LGC, Some participants such as NMIJ, UME and PTB did need to perform species preconcentration due to the very low concentration of SeMet and the complexity of the serum sample. CCQM K107-Draft B report Page 4