A sensitive LC/MS/MS assay of 25OH vitamin D3 and 25OH vitamin D2 in dried blood spots.
ABSTRACT Low levels of 25 hydroxyvitamin D (25OHD) during early development is associated with a range of adverse health outcomes. While a number of methods exist to measure 25OHD in sera, none have been specifically developed to examine dried blood spots (DBS).
We describe an assay where 25 hydroxyvitamin D(3) (25OHD3) and 25 hydroxyvitamin D(2) (25OHD2) are extracted from 3.2 mm DBS punches, derivatised with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) prior to analysis with LC/MS/MS. We assessed assay precision, relative accuracy and examined the impact of storage conditions in samples stored for up to 22 years.
The new assay had good accuracy and precision, and was highly sensitive, being capable of detecting <1 nmol/l 25OHD3 and 2 nmol/l 25OHD2. CDER sensitivity criteria were slightly higher at 7.7 nmol/l for 25OHD3 and 10.7 nmol/l for 25OHD2. The mean 25OHD3 concentration in 118 archived DBS was 20.8+/-11.4, (4.8 to 67.8 nmol/l). 25OHD2 was detected in only two of these samples. 25OHD3 concentrations were significantly higher in DBS collected in summer compared to winter (p<0.0001).
Both 25OHD3 and 25OHD2 can be reliably quantified in archived 3.2 mm dried blood spots. We can not be certain that the levels we measure in archived samples are exactly the same as when they were collected. However, the fact that the DBS levels reflect the well-known seasonal variation in this vitamin and when corrected for sera, values fall within the normal range for 25OHD3, means that DBS are a useful tissue repository for testing a range of hypotheses linking developmental hypovitaminosis D and adverse health outcomes.
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ABSTRACT: Dried blood spots (DBS) on filter paper have been used in human medicine since the 1960s, predominantly for screening in-borne metabolic disorders and more recently, for toxicology. Despite its 50-year existence, this technology has not been adopted by veterinarians for routine diagnoses and research. We have validated a novel DBS analytical procedure for the routine measurement of toxic heavy metals using 50 µL of whole blood on a single DBS by inductively coupled plasma mass spectrometry (ICP-MS). Targeted heavy metals are arsenic, cadmium, mercury, lead, selenium and thallium. The limits of quantitation (LOQ) on DBS are: arsenic 1.7 µg/L, cadmium 4.0 µg/L, mercury 13.7 µg/L, lead 13.3 µg/L, selenium 6.3 µg/L and thallium 1.5 µg/L. These LOQs suffice for routine diagnoses of heavy metal intoxication in domesticated and wildlife species as well as for basic, applied and epidemiological studies. The technique is ideal for population studies involving investigations of wildlife exposure to heavy metals and other environmental pollutants. The small blood volume involved (50 µL) makes it feasible to study small animals (birds, reptiles, amphibians and small mammals) that were previously excluded, or difficult to study due to the relatively large sample volumes required by current gold standard blood collection techniques.Journal of analytical toxicology 07/2013; · 2.11 Impact Factor
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ABSTRACT: Over the past several years, dried blood spot (DBS) sampling technique has emerged as a pertinent method in both qualitative and quantitative bioanalysis context. In the DBS method, the blood sample is directly soaked on to a paper (with or without treatment). After drying it can be analyzed by modern analytical, immunological, or genomic detection systems. Several advantages of the DBS technique such as low blood volume requirement, transportation and storage without special treatment, better analytes stability, enhanced clinical cooperation in clinical trials, and reduced unforeseeable exposure of analysts to biohazards, make it the most appropriate blood sampling technique. This review illustrates the information available on the DBS method which may serve as a single window for investigators in the field of bioanalysis. Also, it explores the proficiency and appliance of the DBS method in pharmacokinetic (PK), therapeutic drug monitoring (TDM), toxicokinetic (TK), metabolomic, and disease diagnosis. Copyright © 2014 John Wiley & Sons, Ltd.Drug Testing and Analysis 04/2014; · 3.17 Impact Factor
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ABSTRACT: 25-Hydroxyvitamin D3 [25(OH)D3 ] is the best-established indicator of vitamin D status. 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD), a representative Cookson-type reagent, has often been employed for enhancing the sensitivity in the trace determination of 25(OH)D3 in a neonatal dried blood spot (DBS), which contains only 2.65 μL of whole blood, using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). The objective of this study was the development of a novel Cookson-type reagent surpassing PTAD in terms of sensitivity and specificity in the LC/ESI-MS/MS assay of 25(OH)D3 . A novel Cookson-type reagent, 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), was synthesized from 4-dimethylaminobenzoyl chloride. The DAPTAD-derivative of 25(OH)D3 was prepared and its LC/ESI-MS/MS behavior was examined. The applicability of the DAPTAD-derivatization in the determination of 25(OH)D3 in neonatal DBSs was also examined. The derivatization was completed at room temperature within 1 h. The DAPTAD-derivative of 25(OH)D3 provided a characteristic product ion derived from the cleavage of the vitamin D skeleton during MS/MS. The limit of detection of the DAPTAD-derivative during selected reaction monitoring was 0.25 fmol on the column, which was 30 and 2 times lower than those of the intact 25(OH)D3 and the PTAD-derivative, respectively. The DAPTAD-derivatization followed by LC/ESI-MS/MS enabled the detection of a trace amount (in the low-ng/mL range) of 25(OH)D3 in DBSs with a simple pretreatment (only methanol extraction) and short chromatographic run time (10 min). The DAPTAD-derivatization was also useful for the separation of 25(OH)D3 from a potent interfering metabolite, 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3 ]. On the contrary, the assay using the PTAD-derivatization might lead to overestimation of the true 25(OH)D3 levels due to the co-elution of 25(OH)D3 and 3-epi-25(OH)D3 . We developed DAPTAD for enhancing the sensitivity and specificity of the LC/ESI-MS/MS assay of 25(OH)D3 . Our new method using DAPTAD can reduce the overestimation of the 25(OH)D3 levels, and will prove helpful in the diagnosis of vitamin D deficiency in infants. Copyright © 2013 John Wiley & Sons, Ltd.Rapid Communications in Mass Spectrometry 11/2013; 27(21):2453-2460. · 2.51 Impact Factor