( PDF ) Rev Osteoporos Metab Miner. 2010; 2 (2): 55-60

Mata-Granados JM1,2,3, Ferreiro-Verab C2, Luque de Castro MD2, Quesada Gómez JM1,3
1 Departamento de I+D+i – Grupo Sanyres – Córdoba
2 Departamento de Química Analítica – Campus de Rabanales – Universidad de Córdoba – RETICEF – Córdoba
3 Unidad de Metabolismo Mineral – Hospital Reina Sofía – RETICEF – Córdoba


The determination of metabolites of vitamin D is very important in bone metabolism, in coronary disease, cancer, innate immunology, etc. Unfortunately, variation in methods for determining the metabolites of vitamin D limits the ability of clinicians to monitor the status, supplementation and toxicity of vitamin D.
In this work, an automatic method of determining the most important metabolites of vitamin D is presented. 0.2 ml of serum is injected into an XLC-MS/MS (eXtraction Liquid Chromatography-tandem Mass Spectrometry) platform to be cleaned and preconcentrated through extraction in the solid phase (SPE). The analytes retained in the SPE cartridge are eluated directly by the mobile chromatographic phase containing 10% water in methanol, with 5 mM of ammonium formate as ionizing agent, at a flow of 0.3 ml/min for the separation of the analytes, and their later detection through triple quadrupole mass spectrometry (MS/MS).
The limits of detection varied between 3.5 and 8.2 pg/ml. The coefficients of variation within the trial varied between 1.5 and 2.3% during the same day, and between 2.5-3.9% over a week. The recuperation varied between 97 and 99.7% for all analytes. The total time taken for the analysis was 20 minutes.
Thus, the proposed method is robust, cheap and appropriate for use in clinical and research laboratories.
Keywords: Metabolites of vitamin D, Healthy population, Deficiency in vitamin D.

Lack of vitamin D constitutes one of the most prevalent deficiencies in the world. It affects more than half of the population: babies, young people, adults, postmenopausal women and older people, in whom, if they have osteoporotic fractures, the prevalence of low levels of vitamin D reaches 100%1. In Spain, in spite of its geographical position and climate which facilitate adequate sunshine, this situation is faithfully reproduced.
Deficiency in vitamin D, in addition to its role in the etiopathology and treatment of rickets or osteomalacia, contributes to multiple extra-skeletal pathologies1,2. In fact vitamin D deficiency is associated with an increased risk of suffering diabetes mellitus4, arterial hypertension5, cardiac insufficiency6, cardiovascular disease7, peripheral arterial disease, acute myocardial infarction8, cancer9, as well as risk of suffering infections10, autoimmune and inflammatory diseases11, and mortality12,13. In addition, the taking of the usual doses of treatment of osteoporosis is associated with a reduction in rates of mortality14. All of which has increased interest in the metabolism of the endocrine system of vitamin D, and the quantification of its key metabolites.
The state of vitamin D is determined by the blood concentration of 25-hydroxyvitamin D [25(OH)D]1,2, which includes the concentrations of 25(OH)D3 and [25(OH)D2, although it is not clear if both have the same activity, or the same weight as vitamin D2 or D3 in the maintenance of the state of 25-(OH)D in humans15,16. For its quantification, methods based on liquid chromatography (LC)17,18, on protein competition tests by chemiluminescence19, on high and low frequency radioimmunoassay by sampling20,21, on automatic methods of chemiluminscence19, and on liquid chromatography tandem mass spectrometry (LC-MS/MS)22,23, are normally used. These new methods have generated much controversy due to the fact that the interlaboratory studies carried out have not shown concordant results between these different methods , nor among those based on immunoassay24-26. The clinical application of LC-MS/MS has improved the selectivity in the determination of 25(OH)D, although the coefficients of variation continue to be high (20%) among the different laboratories which use it, due to the nonexistence of standardised procedures for the analysis of vitamin D, since they are methods developed by and, generally very dependent on, the operator. The implementation of international measures of standardisation which certify the quality of the methodology, such as those conducted by DEQAS (vitamin D External Quality Assessment Scheme)27, have shown the differences in the determination vitamin D and have developed measures for their standardisation, such as the use of the same standards for the calibration of the method28.
The determination of 1,25(OH)2 dihydroxyvitamin D3 is necessary in cases of renal insufficiency, hypoparathyroidism, pseudohypoparathyroidism, screening for hypercalcemia, etc. This determination is more complicated than with 25(OH)D because the concentration is much lower and its stability less. Most of the methods use I125 as a marker in radioimmunoassay after a process of extraction. The structural similarity between the metabolites of vitamin D means that the specificity of the method is always in question, since there is no robust study of interference, due to the difficulty of finding a reference method. LC-MS/MS is used for the determination of calcitriol, using precipitation of proteins and previous extraction in solid phase29 and, recently, applying a derivation of Diels-Alder to improve the ionisation efficiency30. In spite of the fact that current methods based on LC-MS/MS provide high sensibility and sensitivity, a consistent platform, totally automated, with high frequency of sampling, precision and exactitude, which makes unnecessary the presence of expert operators, is demanded. From the experience of the group it is thought that the most appropriate platform is XLC-MS/MS (eXtraction Liquid Chromatography-tandem Mass Spectrometry), since it is a closed system which avoids the loss of analytes, and is totally automatic, for which reason it has low coefficients of variation.
This research seeks to fill this gap through the development of an automatic method based on the on-line coupling of solid phase extraction with liquid chromatography and tandem mass spectrometry to determine vitamins D3 and D2, the metabolites 25-hydroxyvitamin D3 and D2, 24,25(OH)2 dihydroxyvitamin D3, and 1,25(OH)2 dihydroxyvitamin D3, and their application in serum from blood donors.

Material and method

Solvents and standards
Ammonium formate, 25-hydroxyvitamin D3 (25(OH) D3) and 25-hydroxyvitamin D2 (25(OH)D2), vitamin D2 and vitamin D3, were obtained from Sigma (Sigma–Aldrich, St. Louis, MO, USA). 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), were provided by Roche (Basle, Switzerland). Methanol, acetonitrile and formic acid were obtained from Scharlau (Barcelona, Spain).
The reserve solutions were prepared by dissolving a known quantity of analytes (25(OH)D2, 25(OH)D3, vitamin D3, vitamin D2, 1,25(OH)2D3 and 24,25(OH)2D3) in methanol. From the reserve solutions the working solutions were prepared by dilution of an appropriate volume in methanol, calculating the exact concentration by photometry.

The separation chromatography was carried out in reverse phase mode with an Agilent 1200 Series (Palo Alto, CA, USA) chromatograph, followed by electrospray ionisation (ESI) in positive mode, and detection by means of mass spectrometry in tandem (Agilent 6410 Triple Quadrupole). The analyses were processed through the MassHunter Workstation Software (Agilent) programme for qualitative and quantitative analysis. The automatic solid phase extractor used was a Prospekt2 (Spark Holland, Emmen, Netherlands) system, and (Midas) autosampler with a sample loop of 0.2ml. The solid phase extraction cartridge was a Hysphere C18 (Spark Holland) of 10 x 2 mm. The analytic column used was Synergi Hydro-RP (Phenomenex, Torrance, CA, USA) of 2.5 µm particle size, 100 x 2,0 mm.
The extraction of blood was carried out using the standard process. Once obtained the blood was centrifuged at 4° for 10 minutes, aliquoted and frozen at -80°C until used.

The autosampler fills the sample loop (0.2ml) and initiates the sequence of operations described in Table 1. Basically, the process of extraction starts with the preparation of the cartridge by means of an activation of the stationary phase with methanol, conditioner and equilibration with an aqueous solution of 0.2% formic acid in 30% acetonitrile. With this same solution, the sample is pulled towards the cartridge. In these conditions the analytes are retained in the absorbent contained in the cartridge, and then 30% acetonitrile is used as a washing solution for interferences. Subsequently, the stages of chromatographic elution and separation commence, putting the mobile phase in contact with the extraction cartridge by the twisting of a valve. The elution time is 7 minutes. The initial mobile phase was 5 mM of ammonium formate contained in 90% methanol at a flow rate of 0.3 ml/min. In minute 2 a lineal gradient of 5 minutes was programmed to obtain 5 mM of ammonium formate in 100% methanol. The temperature of the column was 15° C. The total time for the analysis was 20 minutes.
The eluite of the chromatographic column was ionised using ESI in positive mode and monitored by MS/MS in multiple reaction monitoring (MRM) mode. The flow and temperature of the drying gas (nitrogen) in the ESI was 13 l/min and 350° C, respectively. While the pressure of the nebulizer was 35 psi and the capillary voltage 4,000 V. The scanning time for each transition of MS/MS was 50 minutes.


The limit of detection (LOD) according to the definition of the IUPAC (International Union of Pure and Applied Chemistry) is the minimum quantity of analyte detectable, and is calculated experimentally as the concentration which corresponds to 3 times the standard deviation of the noise to signal ratio calculated in 10 samples. The limit of quantification (LOQ) according to IUPAC is the minimum quantifiable quantity of analyte, generally corresponding to the smallest concentration of the calibration line and is calculated as the concentration which corresponds to a signal 10 times the standard deviation of noise to signal ratio in 10 samples. The individual values of these limits and the regression coefficients are found in Table 2.

Evaluation of the precision of the method
The coefficients of variation, intra- (repeatability) and inter-trial (reproducibility) were calculated over seven days, carrying out daily measurements with replicas in a serum of known concentration31. The results are shown in Table 2.

Evaluation of the exactitude of the method
The exactitude of the method and the matrix effect was studied using samples with or without being fortified with standard solutions. The recuperation was calculated with two configurations of the Prospekt2, a double cartridge for non-fortified samples and a single cartridge for fortified samples32. This was done because the recuperation may not be adequate for two reasons: first, due to a poor retention in the cartridge of the compound under study, which can be seen in the double cartridge, because what is not retained in the first one is retained in the second; the second reason is a low elution, which may be probed by means of a fortified sample whose concentration is known.
The recuperation in the double cartridge system is calculated as the quantity in the first cartridge/[quantity in cartridge 1+quantity in cartridge 2]. The configuration of a single cartridge is calculated as [final concentration – initial concentration/concentration added evaluated in a single sample in five repetitions on the same day under identical conditions. It is understood that the initial concentration is the concentration of analyte present in the sample before adding a known quantity of it, that is to say, the quantity of a blank sample. The results are found in Table 3.

Application of the method
Samples were analysed from 92 blood donors. A representative chromatogram of a sample fortified with standard solutions appears in Figure 1. Levels of vitamin D3 were detected: 10.4 ± 4.8 ng/ml; but levels of vitamin D2 were not. Blood levels of 25(OH)D (21.3 ± 5.7 ng/ml) correspond to the sum of 25(OH)D2 and 25(OH)D3. The share of 25(OH)D2 out of a total 25(OH)D is 1.9 %. 5% of the population studied had blood levels of 25(OH)D < 10 ng/mL, 42% < 20 ng/ml, 40% between 20 and 30 ng/ml, and only 18% were > 30 ng/ml. Blood levels of 24,25(OH)2D3 were 4.1 ± 1.6 ng/ml and of 1,25(OH)2D3, 48.2 ± 11.4 pg/ml.


The analysis of vitamin D and its metabolites represent a great challenge, due to the high lipophilic nature of these compounds, which means that they are found strongly bonded to their transporter proteins; bonds which need to be broken for their analysis by liquid chromatography. The cleaning of the extracts is essential, since other endogenous lipids will be co-extracted along with the vitamin D metabolites, which results in unclean extracts which may distort the shape of the chromographic peaks and curtail the life of the column. This makes necessary the use of selective and highly sensitive systems such as mass spectrometry for an exact quantification. It also means that the use of a closed system is critical, which avoids the breakdown of the metabolites of vitamin D by light. The advance in methodologies to ensure the determination of vitamin D has not improved the variations previously shown in the measurement of 25(OH)D24,25.
The disparity in the results affects all laboratories using the same, or different, methodologies. The use of methods with a low level of automisation makes the measurement of vitamin D highly dependent on the user, and requires rigorous quality control to assure the results. Methods based on RIA are not all equal, given different specificity for 25(OH)D3 and 25(OH)D2; therefore, there are some which overestimate levels of 25(OH)D and others which give lower values for some of the metabolites26,33.
HPLC is commonly recognised as the gold standard for the determination of vitamin D metabolites3,17,18,34, but it has high equipment costs and possesses a low frequency of samples, given the obligatory precipitation of proteins and/or liquid-liquid extraction using the methods described above, which has made its establishment in laboratories as a routine technique difficult. The methods which exist for the quantification of 1,25(OH)2D3 are laborious and take much time; therefore, more rapid, cheaper and simpler methods are required, which also reduce the risk to health associated with the use of radioisotopes. In recent studies an EIA kit had a poor correlation with a typical analysis by RIA35. Kissmeyer et al.29, published in 2001 an method using LC-MS/MS for determining 1,25(OH)2D3, but this required 1-mL of serum, in addition to an earlier precipitation of proteins and drying stage in a flow of nitrogen, and later reconstitution, which leads to low frequency of samples and relatively high coefficients of variation, because all the stages of treatment of the sample before the chromatographic analysis were manual.
In conclusion, the proposed method is an improvement on existing methods since it allows the rapid and automated determination of the concentrations of vitamins D3 and D2, and the metabolites 24,25(OH)2D3, 1,25(OH)2D3, 25(OH)D3 and 25(OH)D2 using a small quantity of serum, permitting both research into the physiology and physiopathology of the endocrine system, and clinical association studies, or their use in normal practice.
The use of an on-line system for the extraction means that there is no loss of analytes by degradation, and the total automation of the process of analysis means that the precision and accuracy is improved and the need for an expert user is avoided.
Therefore, the proposed method is rapid, with a high sensitivity, exactitude and precision. The main inconvenience it presents is the high cost of the equipment used, although the application of the method is cheap.


The authors thank Sanyres for funding the research and the regional centre for blood donation for their collaboration in the collection of samples. And to the Roche laboratories for kindly and disinterestedly providing the metabolites 1,25 dihydroxyvitamin D3 and 24,25 dihydroxyvitamin D3.

1. Holick MF. Vitamin D deficiency. N Engl J Med 2007;357:266-81.
2. Quesada JM. Insuficiencia de calcifediol. Implicaciones para la salud. Drugs of Today 2009;(supl A):1-31.
3. Mata-Granados JM, Luque de Castro MD, Quesada Gomez JM. Inappropriate serum levels of retinol, alpha-tocopherol, 25 hydroxyvitamin D3 and 24,25 dihydroxyvitamin D3 levels in healthy Spanish adults: simultaneous assessment by HPLC. Clin Biochem 2008;41:676-80.
4. Danescu LG, Levy S, Levy J. Vitamin D and diabetes mellitus. Endocrine 2009;35:11-7.
5. Forman JP, Giovannucci E, Holmes MD, Bischoff-Ferrari HA, Tworoger SS, Willett WC, et al. Plasma 25-hydroxyvitamin D levels and risk of incident hypertension. Hypertension 2007;49:1063-9.
6. Pilz S, März W, Wellnitz B, Seelhorst U, Fahrleitner-Pammer A, Dimai HP, et al. Association of vitamin D deficiency with heart failure and sudden cardiac death in a large cross-sectional study of patients referred for coronary angiography. J Clin Endocrinol Metab 2008;93:3927-35.
7. Kendrick J, Targher G, Smits G, Chonchol M. 25-hydroxyvitamin D deficiency is independently associated with cardiovascular disease in the Third National Health and Nutrition Examination Survey. Atherosclerosis 2009;205:255-60.
8. Giovannucci E, Liu Y, Hollis BW, et al. 25-hydroxyvitamin D and risk of myocardial infarction in men: A prospective study. Arch Intern Med 2008;168:1174-80.
9. Garland CF, Gorham ED, Mohr SB, Garland FC. Vitamin D for cancer prevention: global perspective. Ann Epidemiol 2009;19:468-83.
10. Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR, et al. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 2006;311:1770-3.
11. Arnson Y, Amital H, Shoenfeld Y. Vitamin D and autoimmunity: new aetiological and therapeutic considerations. Ann Rheum Dis 2007;66:1137-42.
12. Dobnig H, Pilz S, Scharnagl H, Renner W, Seelhorst U, Wellnitz B, et al. Independent association of low serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin d levels with all-cause and cardiovascular mortality. Arch Intern Med 2008;168:1340-9.
13. Ginde AA, Scragg R, Schwartz RS, Camargo CA Jr. Prospective study of serum 25-hydroxyvitamin d level, cardiovascular disease mortality, and all-cause mortality in older U.S. Adults. Am Geriatr Soc 2009;57:1595-603.
14. Autier P, Gandini S. Vitamin D supplementation and total mortality: A metaanalysis of randomized controlled trials. Arch Intern Med 2007;167:1730-7.
15. Armas LAG, Hollis B, Heaney RP. Vitamin D2 is much less effective than vitamin D3 in humans. J Clin Endocrinol Metab 2004;89:5387-91.
16. Holick MF, Biancuzzo RM, Chen TC, Klein EK, Young A, Bibuld D, et al. Vitamin D2 is as effective as vitamin D3 in maintaining circulating concentrations of 25-hydroxyvitamin D. J Clin Endocrinol Metab 2008;93:677-81.
17. Álvarez JC, De Mazancourt PJ. Rapid and sensitive high-performance liquid chromatographic method for simultaneous determination of retinol, alpha-tocopherol, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human plasma with photodiode-array ultraviolet detection. J Chromatogr B 2001;755:129-35.
18. Lensmeyer GL, Wiebe DA, Binkley N, Drezner MK. HPLC method for 25-hydroxyvitamin D measurement: comparison with contemporary assays. Clin Chem 2006;52:1120-6.
19. Roth HJ, Zahn I, Alkier R, Schmidt H. Validation of the first automated chemiluminescence protein-binding assay for the detection of 25-hydroxycalciferol. Clin Lab 2001;47:357-65.
20. Hollis BW, Kamerud JQ, Selvaag SR, Lorenz JD, Napoli JL. Determination of vitamin D status by radioimmunoassay with an 125I-labeled tracer. Clin Chem 1993;39:529-33.
21. Ersfeld DL, Rao DS, Body JJ, Sackrison Jr. JL, Miller AB, Parikh N, et al. Analytical and clinical validation of the 25 OH vitamin D assay for the LIAISON automated analyzer. Clin Biochem 2004;37:867-74.
22. Maunsell Z, Wright DJ, Rainbow SJ. Routine isotope-dilution liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of the 25-hydroxy metabolites of vitamins D2 and D3. Clin Chem 2005;51:1683-90.
23. Priego Capote F, Ruiz Jiménez J, Mata-Granados JM, Luque de Castro MD. Identification and determination of fat-soluble vitamins and metabolites in human serum by liquid chromatography/triple quadrupole mass spectrometry with multiple reaction monitoring. Rapid Commun Mass Spectrom 2007;21:1-10.
24. Carter GD, Carter R, Jones J, Berry J. How accurate are assays for 25-hydroxyvitamin D? Data from the international vitamin D external quality assessment scheme. Clin Chem 2004;50:2195-7.
25. Binkley N, Krueger D, Cowgill CS, Plum L, Lake E, Hansen KE, et al. Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization. J Clin Endocrinol Metab 2004;89:3152-7.
26. Souberbielle JC, Fayol V, Sault C, Lawson-Body E, Kahan A, Cormier C. Assay-specific decision limits for two new automated parathyroid hormone and 25-hydroxyvitamin D assays. Clin Chem 2005;51:395-400.
27. Binkley N, Krueger D, Gemar D, Drezner MK. Correlation among 25-hydroxy vitamin D assays. J Clin Endocrinol Metab 2008;93:1804-8.
28. Carter GD, Jones JC. Use of a common standard improves the performance of iquid chromatography-tandem mass spectrometry methods for serum 25-hydroxyvitamin-D. Ann Clin Biochem 2009;46:79-81.
29. Kissmeyer AM, Sonne K. Sensitive analysis of 1·,25-dihydroxyvitamin D3 in biological fluids by liquid chromatography-tandem mass spectrometry. J Chromatogr A 2001;935:93-103.
30. Aronov PA, Hall LM, Dettmer K, Stephensen CB, Hammock BD. Metabolic profiling of major vitamin D metabolites using Diels-Alder derivatization and ultra-performance liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 2008;391:1917-30.
31. Massart DL, Vanderginste BGM, Buydens LMC, De Jong S, Lewi PJ, Smeyers-verbeke J. Handbook of Chemometrics and Qualimetrics, Part A. Amsterdam: Elsevier, 1997.
32. Bert Ooms JA, Mark Van Gils GJ, Duinkerken AR, Halmingh O. Development and validation of protocols for solid-phase extraction coupled to LC and LC–MS. Am lab 2000;32:52-7.
33. Leventis P, Garrison L, Sibley M, Peterson P, Egerton M, Levin G et al. Underestimation of serum 25-hydroxyvitamin D by the Nichols Advantage Assay in patients receiving vitamin D replacement therapy. Clin Chem 2005;51:1072-4.
34. Turpeinen U, Hohenthal U, Stenman UH. Determination of 25-hydroxyvitamin D in serum by HPLC and immunoassay. Clin Chem 2003;49:1521-4.
35. Kimball SM, Reinhold V. A comparison of automated methods for quantitation of serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. Clin Biochem 2007;40:1305-10.