A collaborative study on the use of single radial immunodiffusion for the assay of rabies virus glycoprotein

The single radial immunodiffusion (SRD) technique has been applied to the assay of the glycoprotein content of rabies vaccines produced in cell cultures. Fourteen laboratories in seven countries participated in a collaborative study to evaluate the reproducibility of the SRD technique; some laboratories also examined vaccines in the mouse protection (NIH) test and by enzyme immunoassay. Good agreement was found between potency estimates using the SRD technique: the geometric coefficients of variation for combined potency estimates of all laboratories were about 10%. SRD assays appear to have a role for the in vitro assay of antigen content of vaccine and could complement results obtained in in vivo assays which are subject to wide variability.     

Modified use of a commercial ELISA kit for deoxynivalenol determination in rice and corn silage

The analysis of deoxynivalenol (DON) in silage samples using enzyme-linked immunosorbent assay (ELISA) often leads to an overestimation. To better analyze DON in rice and corn silages using a commercially available ELISA kit, a cleanup method using a MultiSep #226 column was developed. As a result, overestimation of DON by the influence of specific cross-reaction with acetyldeoxynivalenol (AcDON) was confirmed. In samples where AcDON was not detected by liquid chromatography with mass spectrometry (LC-MS), no samples showed a significant difference (P?<?0.05) in DON amounts between ELISA with cleanup and LC-MS analysis. For the recovery study, blank silage was spiked with 0.5 or 1.0 mg/kg DON. The mean recoveries of DON determined by ELISA with cleanup and LC-MS analysis were 112 and 96 %, respectively, and the relative standard deviation for the repeatability (RSD_r) were 8.2 and 9.8 %, respectively. No samples showed a significant difference (P?<?0.05) in DON concentration determined by either ELISA or LC-MS analysis. A collaborative study to validate this rapid method was carried out using four samples, two rice and two corn silage, by 10 participating laboratories. Each sample was analyzed using blind duplicates. The mean values of DON detected were 1.5–2.3 mg/kg, RSD_r and the relative standard deviation for the reproducibility (RSD_R) were 4.1–12.7 and 7.6–23.4 %, respectively, and the HorRat values were 0.5–1.6. Therefore, the overestimation of DON by the influence of nonspecific cross-reaction with sample matrix was reduced by the cleanup method using a MultiSep #226 column, and analysis of DON in silage was improved. This use of this method for estimation of DON contamination in silage allows rapid detection at the place of use that is likely to result in improved animal health.     

Bestimmung von Deoxynivalenol in Brot und Bier

Analytical procedures for the determination of deoxynivalenol (DON) in bread and beer, using enzyme immunoassay (EIA) and HPLC methods, were developed. For determination of DON by EIA, aqueous raw extracts of bread or degassed beer were extracted by liquid-liquid partitioning with ethyl acetate, the organic solvent evaporated, and the residue redissolved in phosphate buffered saline (PBS) for analysis. For determination by HPLC (UV detection at 218 nm), DON in bread extracts or beer was purified on immunoaffinity chromatographic columns. In bread, detection limits for DON of 15 µg/kg (EIA) and 7 µg/kg (HPLC) were achieved, with mean recoveries of 81%. In beer, the detection limit for DON was 2 µg/l both in EIA and HPLC, with recoveries of 91–93%. Both methods showed good agreement of the results for naturally contaminated sample materials, with r²=0.993 for bread and r²=0.823 for beer, respectively.     

Towards the development of innovative multi-mycotoxin reference materials as promising metrological tool for emerging and regulated mycotoxin analyses

The interest in LC-MS/MS multi-mycotoxin methods unveiled an urgent need for multi-mycotoxin reference material. A multi-fusariotoxin, including deoxynivalenol (DON); zearalenone (ZEN); T-2 toxin (T-2); HT-2 toxin (HT-2); enniatin A, A1, B, and B1 (ENNs); and beauvericin (BEA), contaminated wheat flour was obtained by inoculation Fusarium spp. strains. The candidate material has successfully passed the homogeneity test and submitted to an international interlaboratory study achieved by 19 laboratories from 11 countries using their routine analytical method. The dispersion of the results for ZEN and BEA did not allow the derivation of reliable consensus values, while the assignment was only possible for DON, HT-2, T-2, and ENN A. No link was found between the methods used by the participants and the results. Significant changes in dry matter contents (≥±1.4 % of the initial dry matter) and significant changes in ergosterol contents (≥±10 %) did not occur. Using the mycotoxin contents in wheat flour stored at ?80 °C as reference values, statistically significant decreases were observed only for T-2 contents at +24 °C, in contrast to the storage at ?20 and +4 °C. For the other involved toxins, the candidate material was found to be stable at ?20, +4, or +24 °C. Based on the T-2 decreases, a shelf life of 6 years was derived from isochronous study when the material is kept at ?20 °C. At room temperature (e.g., +24 °C) or higher, this time validity drastically decreases down to 6 months. The development of this metrological tool is an important step towards food and feed quality control using multi-mycotoxin analyses. In vivo animal experiments using multi-mycotoxin-contaminated feeds dealing with the carryover or mitigation could further benefit from the methodology of this work.     

Determination of deoxynivalenol-sulfonate (DONS) in cereals by hydrophilic interaction chromatography coupled to tandem mass spectrometry

Deoxynivalenol (DON) is one of most widespread mycotoxins in cereal commodities, and animal feed is prevalently contaminated at high concentrations. This poses a problem in animal nutrition as especially pigs are very sensitive to DON. An effective process for the reduction of the DON concentration is the treatment of contaminated feed with sodium bisulfite (SBS) whereby DON is transformed into DON-sulfonate (DONS). Although the success of this treatment has been confirmed in several feeding studies, it is unexplained if the decrease of DON is accompanied with a coincident increase of DONS. For this reason, we developed a method for the analysis of DONS using hydrophilic interaction chromatography coupled to tandem mass spectrometry. In order to investigate the correlation between DON and DONS concentrations during SBS-treatment, DON-contaminated wheat was treated with SBS and stored for up to 36 days. At defined timepoints of this treatment, samples were analyzed for DON and DONS using stable isotope labeled standards. The preparation, purification, and structure elucidation of DONS, and the HILIC-HPLC-MS/MS method for the analysis of DONS as well as the results of two storage experiments are presented in this paper.     

Immunoassay and antibody microarray analysis of the HUPO Plasma Proteome Project reference specimens: systematic variation between sample types and calibration of mass spectrometry data

Four different immunoassay and antibody microarray methods performed at four different sites were used to measure the levels of a broad range of proteins (N = 323 assays; 39, 88, 168, and 28 assays at the respective sites; 237 unique analytes) in the human serum and plasma reference specimens distributed by the Plasma Proteome Project (PPP) of the HUPO. The methods provided a means to (1) assess the level of systematic variation in protein abundances associated with blood preparation methods (serum, citrate-anticoagulated-plasma, EDTA-anticoagulated-plasma, or heparin-anticoagulated-plasma) and (2) evaluate the dependence on concentration of MS-based protein identifications from data sets using the HUPO specimens. Some proteins, particularly cytokines, had highly variable concentrations between the different sample preparations, suggesting specific effects of certain anticoagulants on the stability or availability of these proteins. The linkage of antibody-based measurements from 66 different analytes with the combined MS/MS data from 18 different laboratories showed that protein detection and the quality of MS data increased with analyte concentration. The conclusions from these initial analyses are that the optimal blood preparation method is variable between analytes and that the discovery of blood proteins by MS can be extended to concentrations below the ng/mL range under certain circumstances. Continued developments in antibody-based methods will further advance the scientific goals of the PPP.     

International reference standards in coagulation

Measurement of coagulation factor activity using absolute physico-chemical techniques is not possible and estimation therefore relies on comparative bioassay relative to a reference standard with a known or assigned potency. However the inherent variability of locally prepared and calibrated reference standards can give rise to poor agreement between laboratories and methods. Harmonisation of measurement between laboratories at the international level relies on the availability of a common source of calibration for local reference standards and this is provided by the World Health Organization (WHO) International Standards which define the International Unit for the analyte. This article describes the principles, practices and problems of biological standardisation and the development and use of reference standards for assays of coagulation factors, with particular emphasis on WHO International Standards for both concentrates and plasma.     

Sample clean-up methods,immunoaffinity chromatography and solid phase extraction,for determination of deoxynivalenol and deepoxy deoxynivalenol in swine serum

Concentrations of deoxynivalenol (DON) and deepoxy deoxynivalenol (DOM-1) in animal blood are important parameters for studies in toxicology and biological detoxification of DON. Clean-up methods, using either immunoaffinity chromatography (IAC) or solid phase extraction (SPE), were compared in order to determine the free form of DON or DOM-1 and the sum amount (free form plus glucuronide conjugated form of DON or DOM-1), respectively, in swine serum. Detection was achieved by high performance liquid chromatography with ultraviolet detection (HPLC-UV). Compared with the SPE-HPLC method, the IAC-HPLC method provided lower quantitation limit (DON: 18 vs 42 ng/ml; DOM-1: 21 vs 30 ng/ml) and higher recoveries (DON: 93.4–102.7% vs 63.7–85.3%; DOM-1: 85.5–91.1% vs 68.0–82.6%). Compared with previously published methods, the developed IAC-HPLC method removed analytical interferences from swine serum in one quick and easy step, and eliminated steps of extraction with organic solvent and/or pre-purification using SPE cartridges. This IAC-HPLC method was used to analyze swine serum samples collected from pigs that were evaluated in a feeding trial of a microbiological detoxification of DON. No DON or DOM-1 were detected in serum samples from pigs given a toxin-free diet or a microbial control diet. In serum samples from pigs given a DON diet (5 mg/kg of DON), free form DON and sum free DON + conjugated DON were 38.8 ± 13.7 and 49.8 ± 14.1 ng/ml, respectively. In serum samples from those given a detoxified-DON diet (DON was transformed to DOM-1), free form DOM-1 was detected but not quantified, and the sum DOM-1 was found as 47.5 ± 6.3 ng/ml.     

Effects of Feed Contaminant Deoxynivalenol on Plasma Cytokines and mRNA Expression of Immune Genes in the Intestine of Broiler Chickens

An experiment was conducted to investigate the individual and combined effects of dietary deoxynivalenol (DON) and a microbial feed additive on plasma cytokine level and on the expression of immune relevant genes in jejunal tissues of broilers. A total of 40 broiler chicks were obtained from a commercial hatchery and divided randomly into four groups (10 birds per group). Birds were reared in battery cages from one day old for 5 weeks. The dietary groups were 1) control birds fed basal diet; 2) DON group fed basal diet contaminated with 10 mg DON/ kg feed; 3) DON + Mycofix group fed basal diet contaminated with 10 mg DON/ kg feed and supplemented with a commercial feed additive, Mycofix® Select (MS) (2.5 kg/ton of feed); 4) Mycofix group fed basal diet supplemented with MS (2.5 kg/ton of feed). At 35 days, the plasma levels of tumor necrosis factor alpha (TNF-α) and interleukin 8 (IL-8) were quantified by ELISA test kits. Furthermore, the mRNA expression of TNF-α, IL-8, IL-1β, interferon gamma (IFNγ), transforming growth factor beta receptor I (TGFBR1) and nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NF-κβ1) in jejunum were quantified by qRT-PCR. The results showed that the plasma TNF-α decreased in response to DON, while in combination with MS, the effect of DON was reduced. DON down-regulated the relative gene expression of IL-1β, TGFBR1 and IFN-γ, and addition of MS to the DON contaminated diet compensates these effects on IL-1β, TGFBR1 but not for IFN-γ. Furthermore, supplementation of MS to either DON contaminated or control diet up-regulated the mRNA expression of NF-κβ1. In conclusion, DON has the potential to provoke and modulate immunological reactions of broilers and subsequently could increase their susceptibility to disease. The additive seemed to have almost as much of an effect as DON, albeit on different genes.     

Broth disk elution method for anaerobic bacteria: a collaborative study to assess its reliability for clinical purposes

A collaborative study involving seven laboratories was undertaken to evaluate the reproducibility and the reliability of the broth disk elution test against anaerobic bacteria by comparing with the reference agar dilution method. A two breakpoint broth test was also evaluated. Assays were performed using the same testing conditions (i.e. medium, temperature, atmosphere and incubation time). One hundred Gram-negative and Gram-positive clinical isolates were initially studied. Overall agreement of 98.5% and 97.5%, were found for disk elution and the two breakpoint tests, respectively. In order to assess the reliability of the disk elution test, two different lots (LOT1 and LOT2) of disks of piperacillin and clindamycin were selected, to obtain two final concentrations after dilution (10 and 60 mg/mL; 1 and 4 mg/mL, respectively). Two hundred and eighty assays were performed against one strain of both Bacteroides fragilis(piperacillin MIC, 8.0 mg/mL; clindamycin MIC, <0.5 mg/mL) and Bacteroides thetaiotaomicron(piperacillin MIC, 16.0 mg/mL; clindamycin MIC, <0.5 mg/mL). With LOT 1, considering both species and both antibiotics, the agreement among six laboratories ranged from 85% to 100% (P > 0.05) with the higher concentration. Overall agreement among all laboratories was 91%. No optimal agreement (>90%) for clindamycin-Bacteroides thetaiotaomicron using the LOT1 (77%) was found. Since this finding was not observed with LOT2 (100% agreement), discrepancies were attributed to variation between lots. Overall agreement with LOT2 was 100% for all centres. The present study indicates that the broth disk elution method proved to be a reliable and suitable alternative for routine susceptibility testing for anaerobic bacteria, as a resistance screening method for clinical purposes.     

Measurement of 25-hydroxyvitamin D in the clinical laboratory: Current procedures, performance characteristics and limitations

In this review we describe procedures, performance characteristics and limitations of methods available for the measurement of 25-hydroxyvitamin (25OHD) since the year 2000. The two main types of methods are competitive immunoassay and those based on chromatographic separation followed by non-immunological direct detection (HPLC, LC-MS/MS). Lack of a reference standard for 25OHD has, until recently, been a major issue resulting in poor between-method comparability. Fortunately this should soon improve due to the recent introduction of a standard reference material in human serum (SRM 972) from the National Institute of Standards and Technology (NIST). For immunoassay, specificity can be an issue especially in relation to the proportion of 25OHD2 that is quantified whereas HPLC and LC-MS/MS methods are able to measure the two major vitamin D metabolites 25OHD2 and 25OHD3 independently. HPLC and LC-MS/MS require more expensive equipment and expert staff but this can be offset against lower reagent costs. Increasingly procedures are being developed to semi-automate or automate HPLC and LC-MS/MS but run times remain considerably longer than for immunoassays especially if performed on automated platforms. For most HPLC and LC-MS/MS methods extraction and procedural losses are corrected for by the inclusion of an internal standard which, in part, may account for higher results compared to immunoassay. In general precision of immunoassay, HPLC and LC-MS/MS are comparable and all have the required sensitivity to identify severe vitamin D deficiency. Looking to the future it is hoped that the imminent introduction of a standard reference method (or methods) for 25OHD will further accelerate improvements in between method comparability.     

Minimal inhibitory concentrations of Candida species to five antifungals by using the reference dilution micromethod and the E-test.

It is accepted that the frequency of candidosis has increased during the last decade, specially in hospitalized patients. The more frequent use of azole antifungals and the recognition of isolates of Candida sp resistant to these and other drugs such as 5-fluorocytosine constitute a great need for a reproducible and useful C. albicans in vitro susceptibility testing method for monitoring antifungal therapy in clinical mycological laboratories. The E-test is a novel agar diffussion technique for testing the susceptibility of yeasts against a defined continous gradient of drug and could be used by most clinical laboratories. In this study the E-test and the NCCLS reference microbroth method (M27-P guidelines) were used to determine the MICs of amphotericin B, 5-flucytosine, itraconazole, fluconazole and ketoconazole for 50 clinical isolates of Candida albicans, Torulopsis glabrata, C. tropicalis and Hansenula anomala and five reference ATCC strains. The main purpose of the study was to compare the results obtained by the two methods. In general good agreement (+/- 1 dilution) was otained between both methods, despite differences observed for some species-antifungal combinations in which the MICs were lower by the E-test than by the microbroth method. MICs for C. albicans and T. glabrata to amphotericin B were < 0.50 microg/mL. Two isolates of C. albicans and two others of H. anomala, showed MIC < 8 microg/mL for 5- flucytosine. All isolates of T. glabrata and 40% of C. albicans showed MICs > 16 microg/mL for fluconazole. The results of this study indicate that E-test is an alternative for susceptibility testing to the NCCLS reference method. Because its simplicity it seems to be an easier test for routine clinical laboratories.     

First Evaluation after Implementation of a Quality Control System for the Second Line Drug Susceptibility Testing of Mycobacterium tuberculosis Joint Efforts in Low and High Incidence Countries

Three networks/projects involving 27 European countries were established to investigate the quality of second-line drug (SLD) susceptibility testing with conventional and molecular methods. 1. The “Baltic-Nordic TB-Laboratory Network” comprised 11 reference laboratories in the Baltic-Nordic States. They performed SLD testing in the first phase with a panel of 20 Mycobacterium tuberculosis strains. After several laboratories made technical changes a second panel of 10 strains with a higher proportion of resistant strains were tested. Although the concordance for Ofloxacin, Kanamycin, and Capreomycin was consistently high, the largest improvements in performance were achieved for the analysis of Ofloxacin resistant (from 88.9 to 95.0%), and Capreomycin resistant (from 71.0 to 88.9%) strains. 2. Within the FP7 TB PAN-NET project (EU Grant agreement 223681) a quality control panel to standardize the EQA (External Quality Assurance) for first-line drugs (FLD) and SLD testing for phenotypic and molecular methods was established. The strains were characterized by their robustness, unambiguous results when tested, and low proportion of secondary drug resistances. 3. The (European Reference Laboratory Network-TB) ERLN-TB network analyzed four different panels for drug resistance testing using phenotypic and molecular methods; in two rounds in 2010 the 31 participating laboratories began with 5 strains, followed by 10 strains and 6 additional crude DNA extracts in 2011 and 2012 were examined by conventional DST and molecular methods. Overall, we demonstrated the importance of developing inter-laboratory networks to establish quality assurance and improvement of SLD testing of M. tuberculosis.     

Bestimmung von deoxynivalenol mittels LC - MS/MS (Tandem Massenspektrometrie)

A sensitive and selective method using high-performance liquid chromatography in combination with atmospheric pressure chemical ionization tandem mass spetrometry (LC-APCI-MS/MS) has been developed for the determination of Deoxynivalenol (DON) in trace levels. The extract was purified with a MultiSep? column followed by the Vicam? DON immunoaffinity column. Quantification is based on an external standard method using positive Multiple Reaction Monitoring (MRM). The limit of detection was 5 μg/kg with a signal to noise ratio of 3:1.     

Use of enzyme-linked immunosorbent assay to screen for aflatoxins,ochratoxin A,and deoxynivalenol in dry pet foods

The objective of this study was to perform a market survey on dry pet foods using enzyme-linked immunosorbent assay (ELISA) to detect total aflatoxins (AFs), ochratoxin A (OTA), and deoxynivalenol (DON). Pet food products (n?=?58) marketed for dogs, cats, birds, and rabbits were tested in duplicate with ELISA, and results above the limit of quantitation were confirmed using liquid chromatography tandem mass spectrometry (LC-MS/MS). OTA was detected in one product (rabbit food) and AFs were detected in two products (one dog treat and one bird treat). In contrast, DON was detected in the majority (74%) of products tested. Bird and rabbit products were the most affected by DON, with levels above 0.5 μg/g in 50 and 80% of samples, respectively. One rabbit sample tested positive for both OTA and DON. Overall, the findings of this study revealed a low incidence of AFs and OTA in commercial pet food. Although DON was detected in numerous products, the levels were well below those associated with acute toxic effects.     

Determination of aldosterone in human serum by isotope dilution gas chromatography/mass spectrometry using a new heptafluorobutyryl derivative.

The formation of a new derivative of aldosterone with heptafluorobutyric anhydride for gas chromatography/mass spectrometry (GC/MS) is presented. The highest and also the most prominent ion of this derivative is observed at m/z 734. The new derivative is, under the described conditions, reproducibly formed, is stable and has good gas chromatographic properties. These characteristics make the new derivative extremely suitable for selective, sensitive, precise and accurate analysis of aldosterone in serum by GC/MS in association with isotope dilution. This is proven by the agreement between the measurement results obtained by two laboratories for samples of three batches of lyophilized control serum. The sample pretreatment procedures used in each laboratory are described.     

Determination of serum uric acid using high-performance liquid chromatography (HPLC)/isotope dilution mass spectrometry (ID-MS) as a candidate reference method

Uric acid is an important diagnostic marker of catabolism of the purine nucleosides, and accurate measurements of serum uric acid are necessary for proper diagnosis of gout or renal disease appearance. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) has been described. An isotopically labeled internal standard, [1,3-(15)N(2)] uric acid, was added to serum, followed by equilibration and protein removal clean up to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) analyses. (M-H)(-) ions at m/z 167 and 169 for uric acid and its labeled internal standard were monitored for LC/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for uric acid (Standard Reference Materials SRM909b) with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added uric acid. The method performed well against the established reference method of ion-exchange followed by derivatization isotope dilution (ID) gas chromatography mass spectrometry (ID-GC/MS). The results of this method for uric acid agreed well with the certified values and were within 0.10%. The amounts of uric acid recovered and added were in good agreement for the three concentrations. This method was applied to determine uric acid in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.08-0.18% and between-set CVs of 0.02-0.07% for LC/MS analyses. Liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analysis was also performed. The LC/MS and LC/MS/MS results were in very good agreement (within 0.14%). This LC/MS method, which demonstrates good accuracy and precision, and is in the speed of analysis without the need for a derivatization stage, qualifies as a candidate reference method. This method can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.     

Application of deglycosylation to SDS PAGE analysis improves calibration of influenza antigen standards

Each year the production of seasonal influenza vaccines requires antigen standards to be available for the potency assessment of vaccine batches. These are calibrated and assigned a value for haemagglutinin (HA) content. The calibration of an antigen standard is carried out in a collaborative study amongst a small number of national regulatory laboratories which are designated by WHO as Essential Regulatory Laboratories (ERLs) for the purposes of influenza vaccine standardisation. The calibration involves two steps; first the determination of HA protein in a primary liquid standard by measurement of total protein in a purified influenza virus preparation followed by determination of the proportion of HA as determined by PAGE analysis of the sample; and second, the calibration of the freeze-dried reference antigen against the primary standard by single radial immunodiffusion (SRD) assay. Here we describe a collaborative study to assess the effect of adding a deglycosylation step prior to the SDS-PAGE analysis for the assessment of relative HA content. We found that while the final agreed HA value of the samples tested was not significantly different with or without deglycosylation, the deglycosylation step greatly improved between-laboratory agreement.     

Analysis of Fusarium toxins in maize and wheat using thin layer chromatography

Thin layer chromatography (TLC) methods for identifying and quantifying deoxynivalenol (DON), fumonisin B1 (FB1) and zearalenone in grain samples were compared to immunoassay (ELISA) and high performance liquid chromatography (HPLC) methods to determine the reliability of the less expensive TLC. There was a very good agreement between levels of DON measured by TLC and competitive-direct ELISA, and between levels of fumonisin B1 measured by TLC and HPLC, over a wide range of concentrations. Correlation coefficients (Pearson's) were 0.978, 0.914 and 0.953 for DON in maize, DON in wheat and FB1 in maize respectively. A lower correlation coefficient (r = 0.672) was obtained when zearalenone was quantified by TLC and HPLC. Possible reasons for this are discussed. A cost comparison of the various methods revealed that TLC was the least expensive for sample analysis. It is recommended that researchers choose which analytical method to use based upon individual considerations of cost and precision. This revised version was published online in June 2006 with corrections to the Cover Date.     

Analytical Bias in the Measurement of Serum 25-Hydroxyvitamin D Concentrations Impairs Assessment of Vitamin D Status in Clinical and Research Settings

Measured serum 25-hydroxyvitamin D concentrations vary depending on the type of assay used and the specific laboratory undertaking the analysis, impairing the accurate assessment of vitamin D status. We investigated differences in serum 25-hydroxyvitamin D concentrations measured at three laboratories (laboratories A and B using an assay based on liquid chromatography-tandem mass spectrometry and laboratory C using a DiaSorin Liaison assay), against a laboratory using an assay based on liquid chromatography-tandem mass spectrometry that is certified to the standard reference method developed by the National Institute of Standards and Technology and Ghent University (referred to as the ‘certified laboratory’). Separate aliquots from the same original serum sample for a subset of 50 participants from the Ausimmune Study were analysed at the four laboratories. Bland-Altman plots were used to visually check agreement between each laboratory against the certified laboratory. Compared with the certified laboratory, serum 25-hydroxyvitamin D concentrations were on average 12.4 nmol/L higher at laboratory A (95% limits of agreement: -17.8,42.6); 12.8 nmol/L higher at laboratory B (95% limits of agreement: 0.8,24.8); and 10.6 nmol/L lower at laboratory C (95% limits of agreement: -48.4,27.1). The prevalence of vitamin D deficiency (defined here as 25-hydroxyvitamin D <50 nmol/L) was 24%, 16%, 12% and 41% at the certified laboratory, and laboratories A, B, and C, respectively. Our results demonstrate considerable differences in the measurement of 25-hydroxyvitamin D concentrations compared with a certified laboratory, even between laboratories using assays based on liquid chromatography-tandem mass spectrometry, which is often considered the gold-standard assay. To ensure accurate and reliable measurement of serum 25-hydroxyvitamin D concentrations, all laboratories should use an accuracy-based quality assurance system and, ideally, comply with international standardisation efforts.