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This document discusses the development of assays to measure vitamin D and its metabolites over the past 40 years. It describes the key metabolites of clinical interest, including 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. Early assays like competitive protein binding assays had limitations but allowed the measurement of 25OHD levels. Later, immunoassays and liquid chromatography-mass spectrometry improved accuracy, specificity and automation. However, standardization between methods remains challenging. The optimal assay choice balances factors like turnaround time, cost and desired clinical information.
This document discusses the development of assays to measure vitamin D and its metabolites over the past 40 years. It describes the key metabolites of clinical interest, including 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. Early assays like competitive protein binding assays had limitations but allowed the measurement of 25OHD levels. Later, immunoassays and liquid chromatography-mass spectrometry improved accuracy, specificity and automation. However, standardization between methods remains challenging. The optimal assay choice balances factors like turnaround time, cost and desired clinical information.
This document discusses the development of assays to measure vitamin D and its metabolites over the past 40 years. It describes the key metabolites of clinical interest, including 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. Early assays like competitive protein binding assays had limitations but allowed the measurement of 25OHD levels. Later, immunoassays and liquid chromatography-mass spectrometry improved accuracy, specificity and automation. However, standardization between methods remains challenging. The optimal assay choice balances factors like turnaround time, cost and desired clinical information.
Vitamin D Assays: Past and Present Debates, Difculties,
and Developments William D. Fraser
Anna M. Milan Received: 13 April 2012 / Accepted: 30 November 2012 Springer Science+Business Media New York 2013 Abstract Clinical interest in Vitamin D and its purported roles not only in calcium and bone metabolism but in several other medical conditions (diabetes, cardiovascular disease, multiple sclerosis, cancer, psychiatric disorders, neuro-muscular disease) has led to a surge in laboratory requests for 25 hydroxy vitamin D and 1,25 dihydroxy vitamin D measurement. Circulating 25 hydroxy vitamin D concentration is routinely used as the best indicator of vitamin D status, but measurement of other metabolites, especially the physiologically active 1,25 dihyroxy vitamin D, are of clinical value. Over the last 40 years the devel- opment of assays for vitamin D and its metabolites from early competitive binding assays through to immunoassay and liquid chromatography aligned to mass spectrometry have demonstrated various analytical challenges, the advantages and disadvantages of each method are con- stantly changing with new technological developments. Immunoassay remains the predominant mode of measure- ment for 25-hydroxy vitamin D although problems with equimolar recovery of the D2 and D3 metabolites remain an issue. Standardisation of all assays has been improved but not resolved with the currently available reference materials as evidenced by the international vitamin D external quality assurance scheme, DEQAS. The choice of method for each laboratory remains a balance mainly between turn around time, convenience, cost and the specicity and accuracy of the information obtained. With increasing discussion and clinical interest surrounding other vitamin D metabolites the vitamin D assay debate is set to continue. Keywords Steroid hormones: vitamin D Assay Review Introduction The recent explosion of interest in vitamin D and its pos- sible roles in both classical processes (calcium and bone metabolism, neuromuscular function) and nonclassical diseases (arthritis, cardiovascular, cancer, diabetes, multi- ple sclerosis, psychiatric illness) has increased demand for measurement of vitamin D and its metabolites. In part this has been a chicken-and-egg situation, where the advances in technologies in the last 20 years have resulted in the development of assays that have improved sensitivity and specicity while lending themselves to automation; auto- mation has resulted in large numbers of vitamin D metabolite measurements being performed with ease on small sample volumes. The ability to generate a large number of vitamin D metabolite results has expanded the database, producing information that has resulted in the expansion of both clinical and research interest in vitamin D and signicant debate regarding its role in health and disease. There have been major advances in semiautomation and full automation of immunoassays utilizing nonradio- active tracers which have been incorporated into both W. F. has a consultant/advisory role and has received funding from IDS, Siemens, Nichols, Roche, and Abbott and has patents/ intellectual property with IDS. A. M. has stated no conict of interest. W. D. Fraser (&) Norwich Medical School, University of East Anglia, Norfolk, UK e-mail: w.fraser@uea.ac.uk A. M. Milan Department of Clinical Biochemistry and Metabolic Medicine, Royal Liverpool and Broadgreen University Hospital Trust, Liverpool, UK 1 3 Calcif Tissue Int DOI 10.1007/s00223-012-9693-3 specialist-dedicated immunoassay systems and the large analytical platforms of several major manufacturers. Increasing interest in the use of mass spectrometry (MS) detection allied to high-performance liquid chromatogra- phy (HPLC) separation has seen many laboratories invest- ing in tandem MS for measurement of vitamin D metab- olites. Each methodology has its advantages and disad- vantages, which will be discussed in this review. Vitamin D Metabolites of Current Interest There are well over 40 metabolites of vitamin D identied [1], and this could potentially result in difculties in establishing assays for specic molecules of interest. In practice, however, the vast majority of metabolites have a very short half-life in the circulation and thus, are currently of minimal interest and present little challenge in assay development. Although the parent sterol vitamin D has a half-life of close to 24 h [2], this is relatively short com- pared with 25-hydroxyvitamin D (25OHD), which has a half-life of 2130 days [3, 4]. Therefore, 25OHD mea- surement is a better indicator of vitamin D stores, whether obtained from sunlight (ultraviolet [UV] exposure) or dietary sources. The most potent physiologically active circulating metabolite produced by humans is 1,25-di- hdroxyvitamin D (1,25[OH] 2 D), which has a half-life of 415 h [58]; and while 25OHD circulates in nanomole per liter concentrations, 1,25(OH) 2 D is present in picomole per liter concentrations, which means that 1,25(OH) 2 D repre- sents the greater challenge in assay development. Vitamin D is derived from two major sources in humans, with approximately 8090 % produced by the action of sunlight (UVB) on the skin resulting in chole- calciferol (D 3 ). The other 20 % is derived from dietary sources, which can be animal cholecalciferol (D 3 ) or plant- derived ergocalciferol (D 2 ). Supplementation of foods and health products or physician-guided treatment with either D 2 or D 3 can increase the percentage derived from exog- enous sources, so assay technology needs to be able to measure both D 2 and D 3 metabolites. The production and metabolism of vitamin D is shown in Fig. 1, and it should be noted that a major rate-limiting step in this pathway is 25-hydroxylation, which takes place in the liver. This step is primarily dependent on the substrate concentration (vitamin D) [9, 10] and is the reason the well-recognized seasonal variability related to UVB exposure exists. 1-Hydroxylation mainly takes place in the kidney, but 1a-hydroxylase activity has also been demonstrated in cells of the placenta, bone, skin, and granuloma tissue (sarcoid, tuberculosis) [11] and requires 25OHD 2 /D 3 (total 25OHD) as the substrate. The rate of 1,25(OH) 2 D 2 /D 3 (total 1,25[OH] 2 D) production by the kidney can be inuenced by prevailing calcium (Ca) and parathyroid hormone (PTH) concentrations. For these reasons, as well as the short half-life, total 1,25(OH) 2 D is a poor indicator of overall vitamin D status as total 25OHD needs to decrease to around 10 nmol/L (4 ng/mL) for total 1,25(OH) 2 D to decrease signicantly [12]. Two other hydroxylases produce metabolites from 25OHD and 1,25(OH) 2 D. 24-Hydroxylase (renal and intes- tinal) produces 24,25-dihydroxyvitamin D (24,25[OH] 2 D), and 26-hydroxylase (renal and liver) produces 25,26- dihydroxyvitamin D (25,26[OH] 2 D). Their circulating con- centrations are not a good reection of vitamin D status, but their role in vitamin D physiology and pathology is being investigated. Measurement of 25OHD Development of 25OHD Assays Measurement of 25OHD has proven to be a major chal- lenge, with signicant variability in the results generated by many differing methods. Several factors contribute to the analytical problem, namely, 25OHD is hydrophobic and therefore unstable in water; its lipophilic nature means it strongly associates with vitamin D binding protein (VDBP); endogenous lipids coextract from plasma and serum, affecting binding and chromatographic separation; it exists in several molecular forms; direct natural sunlight degrades both 25OHD metabolites and internal standards employed in some assays, and a suitable reference standard material has not been available until very recently. Competitive Protein Binding Assay In the 1970s a pivotal method [13] was developed that employed preliminary solvent extraction of samples and chromatographic purication, resulting in elimination of VDBP. The extract (containing unknown, unlabeled 25OHD) was incubated with a known concentration of radioactively labeled 25OHD 3 , and they competed for an added limited amount of binding protein. Subsequent competitive protein binding (CPB) assays utilized either rachitic rat, chicken-, or human-derived binding protein and allowed correction for procedural losses during extraction and purication with an analytical recovery of 82 3.5 % (SD). In addition, they exhibited a shorter assay incubation time (1 h vs. 10 days) and measurement across a wide range of concentrations [1418]. However, these assays cross-reacted with several 25OHD metabo- lites, suffered from instability of the binding protein preparations, and could not be simplied by eliminating the chromatography step [19]; and although attempts were W. D. Fraser and A. M. Milan: Vitamin D Assays 1 3 made to automate a CPB-based assay, this proved prob- lematic, with inaccuracy, poor detection limit, low cross- reactivity with 25OHD 2 , poor precision, and overall unacceptable performance [2022]. Immunoassay Immunoassay methods were rst reported in the 1980s with a radioimmunoassay (RIA) described that initially utilized a 3 H-labeled 25OHD tracer [23], which was subsequently developed into a 125 I-labeled 25OHD assay with higher throughput and improved performance [24]. This assay formed the basis for a subsequent chemiluminescent detectionbased system, which has been automated [25]. A two-step extraction RIA [26] was produced by one com- mercial manufacturer and subsequently developed into an enzyme immunoassay (EIA) without the extraction step, which could also be automated [27]. Several manufacturers have produced 25OHD immunoassays where the solvent extraction and chromatographic separation have been replaced by various blocking agents that displace 25OHD from VDBP, with varying success. This approach facili- tates automation of these assays, but recent data suggest that some immunoassays employing these techniques may be affected by variations in VDBP concentrations, proba- bly due to variable displacement of 25OHD from VDBP and, in specic subjects, may be due to the increased afnity of 25OHD for certain variants of VDBP, resulting in marked variation in results [28]. Major differences in concentrations obtained with the same samples were reported, which serves to highlight some of the problems of standardization of current (and previous) assays that exist as well as VDBP effects. An area of concern in relation to immunoassays is the variability that exists in the detection of 25OHD 2 . Some assays claim to have 100 % cross-reactivity with exoge- nously added 25OHD 2 and 25OHD 3 and are therefore equipotent for the measurement of the two metabolites. Other assay manufacturers admit to lower cross-reactivity with exogenous 25OHD 2 (75 % [kit insert from IDS, Boldon, UK], 52 % [product insert from Abbott, North Chicago, IL]), while some assays were specically designed to measure only 25OHD 3 (product insert from Roche, Indianapolis, IN). Reports have conrmed the variability of commercial immunoassays to detect 25OHD 2 [21, 2931], and this is postulated to be due to differing binding afnities to VDBP, with 25OHD 3 exhibiting a stronger afnity than 25OHD 2 [32]. Of further concern is the fact that some assays have been shown to have prob- lems detecting 25OHD 2 . In a Vitamin D 2 (ergocalciferol) supplementation study, venous sampling following oral administration of Vitamin D 2 demonstrated a signicant underestimation of 25OHD 2 by three immunoassays [21]. There appears to be a change in vitamin D 2 following oral consumption and subsequent metabolism and circulation in the blood in some individuals, which leads to altered Fig. 1 Pathway of Vitamin D production W. D. Fraser and A. M. Milan: Vitamin D Assays 1 3 recognition of 25OHD 2 in vivo by the antibodies in several immunoassays. In some populations up to 30 % of samples can have 25OHD 2 present due to vitamin D 2 being in the food chain or to vitamin D 2 possibly being the only rec- ognized and approved form of supplementation. It is important to recognize this problem as serious misclassi- cation of patients may arise if the wrong type of immu- noassay is utilized in a population receiving vitamin D 2 . When interviewed, most experts agree that in an ideal situation an immunoassay reporting total 25OHD should be equipotent for 25OHD 2 and 25OHD 3 [33]. Currently, it would appear that only HPLC or tandem MS methods are able to meet this requirement. Almost all immunoassays show a high cross-reactivity with 24,25(OH) 2 D, which increases in concentration with increasing sun exposure; and as 25OHD increases and/or is metabolized to 1,25(OH) 2 D, this provides an increased supply of the two substrates for the 24-hydroxylase enzyme. Concentrations in the region of 1015 nmol/L have been recorded for 24,25(OH) 2 D in serum using gas chromatography-MS [34], with reported circulation levels of 1015 % that of 25OHD. HPLC and LC-MS/MS The rst direct UV detectionbased HPLC assays for 25OHD were published in 1977 [35, 36]. A cumbersome chloroform methanol extraction was followed by chroma- tography on Sephadex/silica gel columns, followed by HPLC with UV detection. Improvements in HPLC meth- ods centered around the introduction of reversed-phase HPLC mainly using C18 columns [37], improved internal standard material, improved sample extraction using chloroformmethanol [32] and methanolhexane [38], then extraction of samples using semiautomated technology employing acetonitrile post-sample precipitation [39]; and a combination of these approaches decreased HPLC run times to less than 10 min, therefore increasing sample throughput. The techniques of sample extraction allied to chroma- tography with MS detection methods offer increased specicity for the molecule of interest, so the combination of HPLC and MS in tandem, LC-MS/MS, has become a commonly used technique in routine clinical biochemistry laboratories. Early methods employed fast atom bom- bardment with Cookson-type reagents [40], resulting in derivatization of 25OHD, which improved detection of 25OHD. Isotope dilution-electrospray LC-MS/MS methods performed on bench top analyzers became popular in the mid-2000s with protein precipitation of the sample, liquid liquid extraction, short run times, and computer processing of chromatograms contributing to higher throughput and ease of use [4143]. Deuterated 25OHD 2 and D 3 internal standard material improves accuracy and veries recovery, with improved extraction processes aiding in the removal of phospholipids, thereby reducing the problem of ion suppression [44]. Recent technical developments have centered around reducing the manual component of sample preparation prior to chromatography and elimination of liquidliquid extraction using either separate automated solid-phase extraction (SPE) [45], online solid-phase extraction [46, 47], or online turbulent ow extraction [48]. All of these method changes contribute to increased throughput, improved reliability, and better cost-effec- tiveness of LC-MS/MS assays for 25OHD. A step change in technology has recently been published that has increased sample throughput to 300 samples/h, multiplex- ing samples by differential mass tagging in LC-MS/MS measurement with excellent assay performance (LLOQ 10 nmol/L [4 ng/mL] for 25OHD 2 , 5 nmol/L [2 ng/mL] for 25OHD 3 ) and CVs of 3.715.2 % across the reportable assay range [49]. Isotope dilution LC-MS/MS is currently considered the gold standard method for 25OHD measurement, being able to simultaneously quantitate 25OHD 2 and 25OHD 3 , with summation of the two values resulting in total 25OHD. One criticism made about LC-MS/MS, however, is that there is a multitude of home-brew or in-house methods available using different sample preparation and extraction methods, varying running conditions and buffers, different HPLC systems, and multiple MS detection systems which utilize different transitions for each molecule of interest. This is aside from the issues relating to standards and QC materials. A review of the International Vitamin D Exter- nal Quality Assurance Scheme (DEQAS) results for the LC-MS/MS group highlights the spread of results gener- ated by these methods. While the majority of the methods (7075 %) are positively biased against the all-laboratory trimmed mean (ALTM), some are close to the mean (1520 %) or negatively biased depending on the 25OHD concentration measured (510 %). There has also been concern raised regarding the presence of the 3-epi-25OHD epimer of 25OHD, which because of the achiral nature of LC-MS/MS cannot be separated from 25OHD by the majority of current methods. An epimer is a nonsuperim- posable/nonmirror image of a molecule that differs in conguration at one carbon atom and has the identical mass:charge ratio, and parent and product ion pairs fol- lowing ionization. Although mainly seen in younger chil- dren [50] and in specic disease states, a recent report has claimed that 3-epi-25OHD 3 is present in 99 % of samples tested, with concentrations ranging from 0.2 to 59.25 nmol/ L in samples from neonates to 80?-year-old adults [51]. The presence of an epimer may increase the total 25OHD concentration measured by LC-MS/MS methods compared to immunoassays as, apart from the Roche CPB assay, W. D. Fraser and A. M. Milan: Vitamin D Assays 1 3 immunoassays do not cross-react with 3-epi-25OHD 3 [52]. It is important therefore to utilize an assay which does not detect 3-epi-25OHD in neonates, and the presence of the epimer in the reference standards (as discussed later) should enable assay validation. The use of chiral chro- matographic columns, cyano columns, and longer run times can allow separation and quantication of 3-epi- 25OHD 3 [50, 5356], which should enable investigation of the importance of this metabolite in vitamin D physiology and pathophysiology. Isobars of 25OHD metabolites which can cause isobaric interference, have also been reported in a small number of subjects [54]; but their clinical impor- tance and contribution to 25OHD measurement need to be investigated further. In 2011 a reference method [56] was recognized by the Joint Committee for Traceability in Laboratory Medicine, and a further reference method accepted by European authorities has been published recently [55]. Comparison with these methods in the future should allow a signicant improvement in consistency and comparability of results between methods. Current 25OHD Methods A report from DEQAS (January 2012) lists 16 categories of methods and 1,119 users performing 25OHD measurement of quality-assurance samples (Table 1). Immunoassay methods dominate the scheme with 86 % of users, the next largest group being LC-MS/MS with 11 %. A high percentage of the immunoassay group used fully automated methods on either dedicated or multianalyte platforms (82 %). Evaluation of these automated assays has raised concerns about increased variability in performance with wider CVs [57], poor cross-reactivity with 25OHD 2 [58], inaccuracy of results [28], interference from heterophilic antibodies requiring a reformulation of the assay [59], and failure to achieve minimal performance goals [60]. Standardization of 25OHD Assays In the past the absence of a recognized international stan- dard material for 25OHD 2 and D 3 meant that most inves- tigators produced their own standard and calibrator materials for HPLC and LC-MS/MS methods. The approach adopted was to obtain the purest form of 25OHD 2 or D 3 available commercially and dissolve this in sufcient quantity in ethanol to enable spectrophotometric estimation of absorbance at both 265 nm (absorption maximum) and 228 nm (absorption minimum) to check for purity and then, using the appropriate molar absorption coefcient (MAC), calculate an accurate concentration of 25OH D 2 or D 3 in the sample. Several problems arise from this approach including the purity and source of the 25OHD, the need to dissolve 25OHD in an organic solvent, the labile nature of 25OHD in water, the diluent/matrix used to make sub- sequent standard dilutions resulting in the presence of VDBP of different species with varying afnities for 25OHD 2 and D 3 , and the marked variability in the MACs used. While the use of a commercially available common standard material can improve the consistency of perfor- mance of LC-MS/MS, the provenance of this material remains in doubt [61] and the subsequent benet of the approach questioned [62]. In 2009 the National Institute of Standards and Tech- nology (NIST) produced standard reference materials (SRM 972 and SRM 2972) and certied the reference values for 25OHD 2 , 25OHD 3 , and 3-epi-25OHD 3 in four different serum pools. Value assignment of SRM 972 was accom- plished using a combination of three isotope-dilution MS approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention [63]. Three of the four SRM 972 standards have exogenous metabolites or are diluted with equine serum and have proved unsuitable for standardizing commercial immunoassays. It has been possible to align most HPLCand LC-MS/MS methods using these standard materials, although the relatively low value for the highest available concentration, around 40 nmol/L (16 ng/mL), also limits the application of the material. Some commercial manufacturers have started to use NIST-aligned LC-MS/MS methods to calibrate their assay standards, and this may increase the harmonization of results obtained by different methods. Table 1 25-Hydroxyvitamin D participants in DEQAS January 2012 Method Number participating CV for samples analyzed (%) Diasorin Liaison total 401 9.613.7 IDS EIA 136 10.211.8 IDS-iSYS 131 8.112.9 LC-MS/MS 125 8.914.9 Automated IDS EIA 106 8.610.2 Roche total 25OHD 61 8.216.9 Siemens ADVIA Centaur 46 14.019.6 Abbott Architect 34 6.810.7 HPLC 31 15.435.6 Diasorin RIA 26 14.120.7 IDS RIA 11 5.215.3 Unknown 4 9.524.3 Roche 25OHD 3 3 7.139.7 DIAsource 2 7.636.7 Diazyme 25OHVitD 1 NA Chromatographic ligand 1 NA Binding assay NA not available W. D. Fraser and A. M. Milan: Vitamin D Assays 1 3 The DEQAS scheme was established in 1989 and has contributed signicantly to the improvement in assay per- formance and development. Over a 15-year period an improvement in mean interlaboratory imprecision (CV) from 32.0 % (1994) to 15.3 % (2009) was observed, and most major methods were giving results within plus or minus 7.4 % of the ALTM (2009) [64]. The expansion in LC-MS/MS technology and respondents to the scheme have transiently stopped this trend, with most LC-MS/MS methods positively biased against the ALTM, which is heavily based on immunoassay users. The introduction of SRMs for 25OHD will hopefully restore the improvement in interlaboratory imprecision. Measurement of 1,25(OH) 2 D 1,25(OH) 2 D is the biologically active form of vitamin D whose production and metabolism is tightly regulated; it is highly lipophilic, is relatively labile, and circulates at 1,000-fold lower concentration in blood than 25OHD. If 25OHD is considered a difcult analyte to measure [14], 1,25(OH) 2 D presents an even greater analytical challenge. It is essential to perform extraction of 1,25(OH) 2 D from serum/plasma to remove interfering substances and to remove 1,25(OH) 2 D from VDBP and albumin. A major problem that exists with solvent extraction methods is that the methods used in the past were the same as those used to extract 25OHD, so this major interferent was also present in the extract at concentrations 10 3 greater than that of 1,25(OH) 2 D. The specicity for 1,25(OH)D is therefore mainly dependent on the detection methods used. CPB The earliest published method used 20 mL of sample extracted using methanolchloroform and subsequent chromatographic purication [65]. Detection was performed using intestinal vitamin D receptor freshly isolated from chicken intestine, so this was a classical CPB assay utilizing [ 3 H]-1,25(OH) 2 D. The chromatin receptor was highly spe- cic for 1,25(OH) 2 D, and this enabled detection with a claimed assay sensitivity of 0.2 pmole of pure standard, although the lowest concentration detected in plasma was 26 pmol/L (10 pg/mL). Improvements in purication and incorporation of HPLC separation allowed simultaneous detection of 25OHD and a signicant reduction in sample requirement. Further developments in CPB assays came from the use of SPE sample using C-18 Sep-Pak and silica cartridge purication plus the move to utilizing vitamin D receptor isolated from calf thymus glands, which was much more stable than chick intestine preparations. This assay was able to detect low 1,25(OH) 2 D, 27.6 3.9 pmol/L (10.6 1.5 pg/mL), in chronic renal failure [66]. Com- mercial methods employing the calf thymus receptor assay became available, and a number of modications have been made to the method over several years; but the specicity of the thymus receptor for 1,25(OH) 2 D meant that the radio receptor assay (RRA) has remained a standard comparator/ reference method for over 35 years. Immunoassay In 1978 the rst report of an RIA for 1,25(OH) 2 D was published [67]. The assay required sample purication due to the relatively poor specicity of the rabbit antibody, and a tritiated tracer was employed but overall was relatively insensitive compared to the RRA, with a detection limit of 52 pmol/L (20 pg/mL). Improved antibody technology resulted in RIAs with better performance and detection limits, but antibody cross-reactivity with 25OHD and 24,25(OH) 2 D meant that extensive purication of 1,25(OH) 2 D from serum was still required. By manipula- tion of the extraction and chromatography or use of par- ticular antibodies, assays specic for 1,25(OH) 2 D 2 and 1,25(OH) 2 D 3 were produced [6870]. A major advance was the development and commercialization of an RIA for 1,25(OH) 2 D that required minimal sample prepurication, did not need internal standardization, utilized calibrators in an equivalent matrix to samples, and utilized a 125 I-labeled tracer that allowed gamma counting of the assay. This assay involved acetonitrile extraction, followed by treat- ment of the extract with sodium periodate, then a second extraction and purication of 1,25(OH) 2 D by solid-phase chromatography (C18OH and silica cartridges), followed by quantication by RIA. A detection limit of 6.2 pmol/L (2.4 pg/mL) was quoted, and recovery of 1,25(OH) 2 D 2 was 6471 % and that of 1,25(OH) 2 D 3 90101 %; and by converting the 24,25(OH) 2 D 3 and 25,26(OH) 2 D 3 to alde- hyde and ketone forms using sodium periodate, cross- reactivity with known metabolites was signicantly reduced [71]. Further analytical and clinical validation of the commercial assay (Diasorin, Stillwater, MN) was published in 2002 [72]. A highly novel approach to sample purication and extraction was adopted by IDS in a com- mercial assay utilizing immunoextraction separating 1,25(OH) 2 D from delipidated samples using an antibody bound to a mini-immunocapsule. Elution of the sample from the capsule, evaporation to dryness, then reconstitution prior to RIA results in a rapid, user-friendly assay [73]. The IDS RIA has been modied to a nonisotopic format utilizing avidin-linked horseradish peroxidase and tetramethylbenz- idine substrate to generate a signal at 450 nm. Both the RIA and EIA show excellent correlation with a RRA and a limit of detection of 9.4 pmol/L (3.6 pg/mL) [74]. Concerns have been raised about a possible contribution to 1,25(OH) 2 D W. D. Fraser and A. M. Milan: Vitamin D Assays 1 3 measurement from other 1a-hydroxylated metabolites [75], and cross-reactivity for 1,25(OH) 2 D 3 26,23-lactone, 1,24,25 (OH) 3 D 3 , and 1,25,26(OH) 3 D 3 has been demonstrated in both the Diasorin and IDS assays [71]. HPLC, LC-MS/MS, and GC-MS Measurement of 1,25(OH) 2 D by direct detection UV methods is not possible due to the low concentration in blood (picomoles per liter). 1,25(OH) 2 D has few ionizable polar groups, so techniques to increase the ionization efciency (namely, sample derivitization) have been incorporated in all of the published methods. An isotope dilution-mass fragmentography assay for 1,25(OH) 2 D was rst published in 1979 [76]. Extraction of 20 mL of serumwas performed using chloroformmethanol after addition of [26- 2 H 3 ]-1,25(OH) 2 D 3 and purication by liquid chromatography. The puried material was converted into the trimethylsilyl ether and analyzed by GC-MS. The lower limit of quantication (LLOQ) was 13 pmol/L (5 pg/ mL), with a CV of 5 %; but the large sample volume limited the general applicability of the assay. An LC/thermospray MS technique using positive and negative ion detection after online postcolumn Diels Alder derivitization obtained an LLOQ of 1 nmol/L [77]. 1,25(OH) 2 D 3 in rat and pig serum was measured by LC-MS/MS after extraction and purica- tion using both reverse phase and normal phase on a C18 cartridge, followed by generation of an ammonium adduct prior to positive electrospray ionization. The LLOQ was 20 pg/mL using 1 mL of sample [78]. Derivitization using 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), a Cookson- type reagent, of a SPE sample and measurement using ultraperformance liquid chromatography (UPLC) electro- spray tandem MS allowed simultaneous quantication of a prole of vitamin D metabolites (1,25[OH] 2 D 2 , 1,25[OH] 2 D 3 , 24,25[OH] 2 D 3 , 25OHD 2 , and 25OHD 3 ) with an LLOQ of 25 pg/mL and CV of 516 % for 1,25(OH) 2 D 3 [79]. PTAD was also used in a method that quantitated the same four metabolites with signicantly improved sensi- tivity, 5 ng/L (12 pmol/L) for 1,25(OH) 2 D 3 , that employed selective SPE and microow LC-MS/MS [80]. The SPE procedure enabled high sample loading on the UPLC col- umn, and on-column sample focusing prevented band broadening, allowing excellent separation and eliminating endogenous interference while minimizing ion suppression but with a run time of 27 min. Lithium acetate has been used to produce ionizable adducts in a method that uses a complex online sample processing procedure with the use of a per- fusion column followed by a chain of two monolithic col- umns to clean and enrich the sample prior to quantication on a highly sensitive LC-MS/MS [81]. Both 1,25(OH) 2 D 2 and 1,25(OH) 2 D 3 can be measured on 30 lL of sample with an LLOQ for 1,25(OH) 2 D 3 of 15 ng/L (36 pmol/L), a CV of 515 % across physiological concentrations, and a total run time per sample of 30 min. IDS immunoafnity column and reagents were incorporated into a sample preparation pro- cedure following protein precipitation and SPE. Lithium acetate was used to produce adducts prior to LC-MS/MS analysis. This method removed isobaric interferences and matrix effects, resulting in signicantly reduced ion suppression with the resultant LLOQs of 3.9 ng/L (9.1 pmol/L) for 1,25(OH) 2 D 2 and 3.4 ng/L (8.2 pmol/L) for 1,25(OH) 2 D 3 with interassay CVs of 2.57.0 % [82]. A very similar approach using the IDS columns for immu- noextraction but derivitizing using PTAD prior to UPLC MS/MS resulted in improved sensitivity with LLOQs of 0.65 ng/L (1.5 pmol/L) for 1,25(OH) 2 D 2 and 1.25 ng/L (3.0 pmol/L) for 1,25(OH) 2 D 3 and interassay CVs of 8.013.0 %. All of the LC-MS/MS methods described above are fairly labor-intensive, with manual workows and lim- ited throughput (Strathmann et al. [83] quote 7.5 h to process 21 samples). Future developments will likely incorporate more online processing, automated sample preparation, and mass tagging [49] to increase throughput. A comparison of LC-MS/MS measurement of 1,25(OH) 2 D and the IDS immunoassay has highlighted differences in recognition of 1,25(OH) 2 D 2 when high-dose vitamin D 2 (300,000 IU) is administered intramuscularly. The LC-MS/MS method detected a signicant increase in total 1,25(OH) 2 D, mainly due to an increase in 1,25(OH) 2 D 2 ; but the immunoassay failed to detect this increase in 1,25(OH)D [84]. This appears to be very similar to the situation observed with 25OHD immunoassays, discussed previously, where antibodies incorporated in the assay system are not able to recognize the 1,25(OH) 2 D 2 synthesized in vivo after exogenous administration of vitamin D 2 . Current 1,25(OH) 2 D Methods A recent report from DEQAS (January 2012) lists seven categories of methods and 123 users performing Table 2 1,25-Dihydroxyvitamin D participants in DEQAS January 2012 Method Number participating CV for samples analyzed (%) IDS RIA 63 15.119.2 DiaSorin RIA 28 22.324.1 IDS EIA 21 21.722.9 LC-MS/MS 5 14.723.9 Biosource CT (ELISA) 3 2.714.9 AMP RIA 2 2.721.7 In-house RIA 1 NA NA not available W. D. Fraser and A. M. Milan: Vitamin D Assays 1 3 1,25(OH) 2 D measurement of quality-assurance samples (Table 2). Immunoassay methods dominate the scheme with 96 % of users: 68 % are performing an IDS method, and 4 % report results by LC-MS/MS. The CVs reported are wide for all the methods at around 20 % for most samples circulated, and this reects the difculty of mea- surement and a lack of availability of an international standard material. Standardization There is no international standard material available, so most methods utilize in-house standards, produced as previously discussed for 25OHD using solvent dissolution, spectrophotometric analysis at 264 nm, and a MAC of 19,400 to calculate the concentration. Human serum stripped of 1,25(OH) 2 D 3 by activated charcoal can then be used to make calibrators of varying concentrations by adding dissolved 1,25(OH) 2 D 3 [71]. Some authors report using substrate addition technology to calculate concen- trations, and commercial standard material with a consen- sus mean concentration attached is also available. Conclusions Measurement of vitamin D and its metabolites is difcult. Many methods have been developed over the years, with immunoassay becoming the most popular method for both 25OHD and 1,25(OH) 2 D due to a combination of avail- ability, ease of use, relative cost, high throughput using a small sample volume, and rapid turnaround. Recently, questions have been asked regarding immunoassays, with problems of accuracy and specicity being demonstrated and the recognition of uctuations of assay performance noted in long-term surveys such as the National Health and Nutrition Examination Survey [85]. Many clinical labora- tories have moved to LC-MS/MS technology, with poten- tial greater specicity and accuracy of measurement. 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