Candidate reference method for determination of vitamin D from dried blood spot samples.

Background The current millennium has seen an explosion in vitamin D testing with the overarching aim of requests to clinically stratify patients as replete or deficient in vitamin D. At a population level, dried blood spot (DBS) sampling offers a less invasive and more practical application for assessment of vitamin D status. We have therefore aimed to develop a sensitive and robust DBS vitamin D method that is traceable to serum for use in population-based studies. Methods Blood spots, calibrators and controls were prepared by punching a 3.2 mm DBS from filter paper and placed into a 96-well micro-plate. The DBS disk was eluted with a combination of water-methanol and internal standard (ISTD) solution followed by supported-liquid extraction and derivatisation. The extract was analysed by liquid-chromatography tandem-mass spectrometry in positive electrospray-ionisation mode with 732.5 > 673.4 and 738.4 > 679.4 m/z ion-transitions for derivatised vitamin D and the ISTD, respectively. Vitamin D results were made traceable to the National Institute of Standards and Technology reference material through the inclusion of Chromsystems vitamin D calibrators. Results 25-Hydroxy-vitamin D3 and its related ISTD were detected at a retention time of 7 min. The seven-point calibration-curve consistently demonstrated a coefficient of determination of 0.99 with an experimentally determined reportable range of 0.5-376 nmol/L. Method validation studies using DBS samples demonstrated 12.9% between-assay imprecision at 45 nmol/L, 84% average recovery and high correlation with plasma vitamin D (correlation coefficient = 0.86). Conclusions We have successfully developed an analytical method for vitamin D quantitation from DBSs which will be applied to our population-based vitamin D research study. This approach improves traceability of DBS results and potentially could be used broadly for other DBS measurands that require comparison to serum/plasma for their interpretation.

Determination of haemoglobin derivatives in aged dried blood spot to estimate haematocrit.

Introduction Dried blood spot (DBS) sample applications now encompass analytes related to clinical diagnosis, epidemiological studies, therapeutic drug monitoring, pharmacokinetic and toxicokinetic studies. Haematocrit (Hct) and haemoglobin (Hb) at very high or low concentrations may influence the accuracy of measurement quantification of the DBS sample. In this study, we aimed to predict the Hct of the punched DBS through primary spectrophotometric estimation of its haemoglobin-derivative (Hb-drv) content. Methods Formic acid solution was used to elute Hb-drv content of 3.2 mm spotted blood from its dry matrix. Direct spectrometry measurement was utilised to scan the extracted Hb-drv in the visible spectrum range of 520-600 nm. The linear relationship between an individual's Hct percentage and Hb-drv concentration was applied to estimate the Hct level of the blood spot. De-identified whole blood samples were used for the method development and evaluation studies. Results The Hb-drv estimation is valid in samples >2 months old. Method validation experiments DBS demonstrate linearity between 82.5 and 207.5 g/L, average coefficient of variation of 3.6% (intra-assay) and 7.7% (inter-assay), analytical recovery of 84%, and a high positive correlation (r=0.88) between Hb-drv and the original whole blood Hct. The Bland-Altman difference plot demonstrates a mean difference of 2.4% between the calculated DBS Hct and the directly measured Hct from fresh whole bloods. Conclusions We have successfully developed a simple Hb-drv method to estimate Hct in aged DBS samples. This method can be incorporated into DBS analytical work-flow for the in-situ estimation of Hct and subsequent correction of the analyte of interest as required.

Analyte stability during the total testing process: studies of vitamins A, D and E by LC-MS/MS.

BACKGROUND: There are limited evidence based studies demonstrating the stability of fat-soluble vitamins (FSV) measured in blood. This study aimed to examine the effects of light, temperature and time on vitamins A, D and E throughout the total testing process. METHODS: Four experiments were conducted. Three investigated the sample matrix, of whole blood, serum and the extracted sample, against the variables of temperature and light; and the fourth experiment investigated the sample during the extraction process against the variable of light. All samples were analysed via our simultaneous FSV method using liquid chromatography-tandem mass spectrometry technology. The allowable clinical percentage change was calculated based on biological variation and desirable method imprecision for each analyte. The total change limit was ±7.3% for 25-OH-vitamin D3, ±11.8% for retinol and ±10.8% for α-tocopherol. RESULTS: Vitamins D and E were stable in the investigated conditions (concentration changes <4%) in the pre-analytical and analytical stages. Vitamin A showed photosensitivity in times >48 h with concentration changes of -6.8% (blood) and -6.5% (serum), both are within the allowable clinical percentage change. By contrast, the extracted retinol sample demonstrated a concentration change of -18.4% after 48 h of light exposure. However, vitamin A in the serum and extracted solution was stable for one month when stored at -20°C. CONCLUSIONS: Blood samples for vitamins D and E analyses can be processed in normal laboratory conditions of lighting and temperature. The required conditions for vitamin A analysis are similar when performed within 48 h. For longer-term storage, serum and vitamin A extracts should be stored at -20°C.

A simultaneous quantitative method for vitamins A, D and E in human serum using liquid chromatography-tandem mass spectrometry.

Non-classical roles of fat-soluble vitamins (FSVs) in many pathologies including cancer have been identified. There is also evidence of hormonal interactions between two of these vitamins, A and D. As a result of this enhanced clinical association with disease, translational clinical research and laboratory requests for FSV measurement has significantly increased. However there are still gaps in the analytical methods available for the measurement of these vitamins. This study aimed to develop a method for simultaneous quantification of 25-hydroxyvitamin-D2 (25-OHD2), 25-hydroxyvitamin-D3 (25-OHD3) and its 3-epimer (epi-25-OHD3), retinol and α-tocopherol in human serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The procedure was developed and validated across two LC-MS/MS platforms, using commercial calibrators referenced to certified reference materials, controls, and deuterated internal standards. The samples were prepared by liquid-liquid extraction prior to injection and LC separation (using a Pursuit-PFP column) on two Agilent MS/MS systems (6410 and 6490) in electrospray ionisation positive mode with multiple reaction monitoring. Identification and quantification of 25-OHD3 from its 3-epimer as well as 25-OHD2, retinol and α-tocopherol were achieved. The dynamic ranges were 4-160 nmol/L for 25-OHD2 and epi-25-OHD3, 4-200 nmol/L for 25-OHD3, 0.1-4.0µmol/L for retinol and 4-70µmol/L for α-tocopherol with correlation (r(2)) of 0.997-0.998. Based on participation in an external quality assurance program, the overall performance of the simultaneous methods were: imprecision (CV%) and inaccuracy (average bias) 3.0% and 3.2 nmol/L, respectively, for 25-OHD3; 5.0% and 0.04µmol/L, respectively, for retinol; and 4.7% and 0.2µmol/L, respectively, for α-tocopherol. In summary, two simple LC-MS/MS methods were successfully developed and validated for the simultaneous quantification of the three vitamin D metabolites (25-OHD2, 25-OHD3 and 3-epimer of 25-OHD3), vitamin A (retinol) and vitamin E (α-tocopherol) in serum.

Advantages and Challenges of Dried Blood Spot Analysis by Mass Spectrometry Across the Total Testing Process.

INTRODUCTION: Through the introduction of advanced analytical techniques and improved throughput, the scope of dried blood spot testing utilising mass spectrometric methods, has broadly expanded. Clinicians and researchers have become very enthusiastic about the potential applications of dried blood spot based mass spectrometric applications. Analysts on the other hand face challenges of sensitivity, reproducibility and overall accuracy of dried blood spot quantification. In this review, we aim to bring together these two facets to discuss the advantages and current challenges of non-newborn screening applications of dried blood spot quantification by mass spectrometry. METHODS: To address these aims we performed a key word search of the PubMed and MEDLINE online databases in conjunction with individual manual searches to gather information. Keywords for the initial search included; "blood spot" and "mass spectrometry"; while excluding "newborn"; and "neonate". In addition, databases were restricted to English language and human specific. There was no time period limit applied. RESULTS: As a result of these selection criteria, 194 references were identified for review. For presentation, this information is divided into: 1) clinical applications; and 2) analytical considerations across the total testing process; being pre-analytical, analytical and post-analytical considerations. CONCLUSIONS: DBS analysis using MS applications is now broadly applied, with drug monitoring for both therapeutic and toxicological analysis being the most extensively reported. Several parameters can affect the accuracy of DBS measurement and further bridge experiments are required to develop adjustment rules for comparability between dried blood spot measures and the equivalent serum/plasma values. Likewise, the establishment of independent reference intervals for dried blood spot sample matrix is required.

Vitamins D and A can be successfully measured by LC-MS/MS in cord blood diluted plasma.

OBJECTIVES: In widely used protocols for the collection and isolation of cord blood mononuclear cells, investigators are left with substantial volumes of diluted plasma which could be used for other measurements. The aim of this study was to ascertain the validity of umbilical cord blood (UCB) diluted plasma samples for vitamin D, A and E analysis compared to UCB serum samples. DESIGN & METHODS: Twenty UCB matched samples of diluted plasma and serum were collected. The samples were analysed by two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods on two separate occasions. RESULTS: The results of 25(OH)D3 obtained by the two laboratories demonstrated close agreement with a mean difference of 0.14nmol/L [95% confidence interval (95% CI), -6.8 to 7.1]. Both methods demonstrate close agreement for 25(OH)D3 in UCB serum versus diluted UCB plasma; mean difference 2.2nmol/L [95% CI, -9.5 to 13.9] and 4.1nmol/L [95% CI, -14.5 to 6.1] for the results from Lab A and Lab B, respectively. Vitamin A was quantified by Lab A in UCB serum and diluted UCB plasma; mean difference 0.07µmol/L [95% CI, -0.41 to 0.28]. Results of 25(OH)D3 epimer and vitamin E in the diluted UCB plasma were below the limit of quantification, and could not be compared with UCB serum. CONCLUSIONS: Diluted UCB plasma can be used for the quantification of retinol and 25(OH)D3 by LC-MS/MS. By contrast, quantification of 25(OH)D3 epimer and vitamin E in diluted UCB plasma is not supported by this study due to limitations in analytical sensitivity.

Comparison of three commercial calibrators for alpha-tocopherol using liquid chromatography-tandem mass spectrometry.

OBJECTIVES: Alpha-tocopherol is the predominant form of vitamin E in plasma and is routinely measured to assess vitamin E status. Agreement between vitamin E assays is essential to provide consistent result interpretation. Lack of agreement among calibrators is potentially a significant obstacle to method harmonization. The aim of this study was to ascertain the agreement between commercial secondary calibrators for analysis of serum/plasma alpha-tocopherol using two methods of isotope dilution liquid chromatography-tandem mass spectrometry (LC-MSMS). DESIGN AND METHODS: Three commercial single level calibrators (Bio-Rad Laboratories, Chromsystems Diagnostics and RECIPE) were prepared in quintuplicate in conjunction with an in-house calibrator set for alpha-tocopherol. Samples were analyzed by two methods using an Agilent-6490 LC-MSMS. RESULTS: The linearity of both methods ranged from at least 4 to 70µmol/L. The expanded within run imprecision of both LC-MSMS methods was ±6% at 95.4% confidence interval across the assay range. The percentage observed difference for the commercial calibrators was calculated from the observed mean against the given value of the calibrator: Bio-Rad (bias +1.4% in both methods); Chromsystems (bias +5.4% [first method] and +5.0% [second method]); and RECIPE (bias -8.9% [first method] and -9.8% [second method]). CONCLUSIONS: Results demonstrate an overall discordance between the commercial calibrators that is greater than the assay uncertainty. It is observed that the greatest deviation of the three calibrators is traceable to a different standard reference material. This lack of harmonization means that results from different laboratories may not be comparable.

Preparation of template DNA and labeling techniques.

A variety of probes can be used for in situ hybridization, depending on the application and the labeling strategy. In general, RNA probes (riboprobes) are now more commonly used. However, some laboratories may still prefer to use DNA probes for in situ hybridization, and a number of techniques for this type of labeling have been described in this chapter. The preparation of plasmid DNA and the subsequent use of polymerase chain reaction products as labeling templates are discussed in detail. Both radioactive and nonradioactive labeling procedures are described and the latest nonradioactive detection methods are outlined.

Signaling pathways of the LGR7 and LGR8 receptors determined by reporter genes.

Although much is known about the pleiotropic effects mediated by relaxin, the exact signaling pathways involved remain relatively elusive. This study examines LGR7 and LGR8 signaling using reporter gene technology. The greatest response was observed at the CRE reporter (indicates activation of cAMP-PKA and p38/JNK pathways), although INSL3 treatment of LGR8 produced a lower response than H2 relaxin treatment of LGR7. AP1 (which indicates activation of JNK pathways) was stimulated to a lesser degree. Three other reporters produced no response. The reporter gene studies suggest that ligand stimulation of LGR7 and LGR8 involves cAMP-PKA and p38/JNK signaling.