Journal of Chromatography B, 969 (2014) 230–234
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Development and validation of a method for determination of plasma 25-hydroxyvitamin D3 3-sulfate using liquid chromatography/tandem mass spectrometry Tatsuya Higashi a,∗ , Ayaka Goto a , Misato Morohashi a , Shoujiro Ogawa a , Kenji Komatsu b , Takahiro Sugiura b , Tetsuya Fukuoka b , Kuniko Mitamura c a
Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan Shizuoka Saiseikai General Hospital, 1-1-1 Oshika, Suruga-ku, Shizuoka 422-8527, Japan c Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka-shi, Osaka 577-8502, Japan b
a r t i c l e
i n f o
Article history: Received 16 July 2014 Received in revised form 22 August 2014 Accepted 24 August 2014 Available online 28 August 2014 Keywords: 25-Hydroxyvitamin D3 3-sulfate LC/ESI-MS/MS Plasma Infant Vitamin D status
a b s t r a c t The quantification of plasma 25-hydroxyvitamin D3 3-sulfate [25(OH)D3 S] is expected to be helpful in the assessment of the vitamin D status, especially for infants. In this study, a simple and sensitive method for the quantification of 25(OH)D3 S in plasma using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. The plasma was deproteinized with acetonitrile, purified using an Oasis® HLB cartridge, and subjected to LC/ESIMS/MS operating in the negative-ion mode. Quantification was based on the selected reaction monitoring, and deuterated 25(OH)D3 S was used as the internal standard. This method enabled the reproducible (intra- and inter-assay relative standard deviations, 7.9% or lower) and accurate (analytical recovery, 95.8–105.3%) quantification of the plasma 25(OH)D3 S using a 20-L sample, and the limit of quantification was 2.5 ng/mL. The developed method was applied to the determination of plasma 25(OH)D3 S in infants; the result revealed that preterm infants have lower plasma 25(OH)D3 S concentrations. © 2014 Elsevier B.V. All rights reserved.
1. Introduction It is widely accepted that the measurement of 25hydroxyvitamin D3 [25(OH)D3 ], which is the major circulating metabolite of vitamin D3 , in plasma/serum is useful for the assessment of the vitamin D status and the diagnosis of several bone metabolic diseases, such as rickets and osteoporosis [1]. In addition to these diseases, vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes, such as type 1 diabetes [2], multiple sclerosis [3] and schizophrenia [4] in later life; screening for vitamin D deficiency in an infant is important for the early supplement of vitamin D to the diagnosed infant. Liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) is now the most commonly used method to determine vitamin D metabolites in various biological samples due to its high specificity and accuracy [5,6]. LC/ESI-MS/MS assays for 25(OH)D3 in dried blood spots have been
∗ Corresponding author. Tel./fax: +81 4 7121 3660. E-mail address: [email protected] (T. Higashi). http://dx.doi.org/10.1016/j.jchromb.2014.08.027 1570-0232/© 2014 Elsevier B.V. All rights reserved.
also developed for the assessment of the infant vitamin D status [7–10]. 25-Hydroxyvitamin D3 3-sulfate [25(OH)D3 S], the sulfated conjugate of 25(OH)D3 , is reported to be another major metabolite of vitamin D3 , and its circulating level is similar to or higher than that of 25(OH)D3 in adults or infants, respectively [5,11–14]. Although the biological role of 25(OH)D3 S is not still fully understood, it might be the storage form of vitamin D3 . Therefore, it is expected that the quantification of 25(OH)D3 S in plasma/serum is also helpful in the assessment of the vitamin D status, especially for infants. A method using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection has been reported for the quantification of plasma 25(OH)D3 S [13]; the method, however, employed a relatively-large volume of sample (0.5 mL) and complicated pretreatment procedures (2 steps of solid-phase extraction and ion-exchange chromatographic purification after deproteinization), and is not necessarily suited to evaluate the diagnostic value and biological role of 25(OH)D3 S. Based on this background information, the objective of this study was to develop and validate a stable isotope-dilution LC/ESI-MS/MS method for the determination of plasma 25(OH)D3 S requiring a small sample volume and simple pretreatment
T. Higashi et al. / J. Chromatogr. B 969 (2014) 230–234
procedure. The application of the method to the measurement of the plasma 25(OH)D3 S concentration of infants is also described. 2. Experimental 2.1. Chemicals and reagents
231
of the solvent, 25(OH)D3 S and IS were dissolved in the mobile phase A (60 L), 15 L of which was subjected to LC/ESI-MS/MS. 2.4. Linearity and calibration curves The plasma (20 L) was added to acetonitrile (50 L) containing IS (1.5 ng) and a graduated amount of standard 25(OH)D3 S (0.050, 0.10, 0.20, 0.50 or 1.0 ng; corresponding to 2.5, 5.0, 10, 25 or 50 ng/mL), and pretreated in the same way as described in Section 2.3. The peak area ratio [25(OH)D3 S/IS] (y) was plotted versus the added amount of 25(OH)D3 S (ng per tube) (x) with a weighting of 1/x to construct the regression lines. The same amounts of IS (1.5 ng) and 25(OH)D3 S (0.050, 0.10, 0.20, 0.50 or 1.0 ng) were pipetted into tubes. The mixture was dissolved in the mobile phase A (60 L), 15 L of which was subjected to LC/ESI-MS/MS. The peak area ratio [25(OH)D3 S/IS] (y) was plotted versus the amount of 25(OH)D3 S (ng per tube) (x), and the obtained regression line was used as the calibration curve.
25(OH)D3 S and 2 H3 -25(OH)D3 S [internal standard (IS)] were synthesized according to the method of Iida et al. [15] in our laboratories; 25(OH)D3 (Wako Pure Chemical Industries, Osaka, Japan) or 2 H -25(OH)D (IsoSciences, King of Prussia, PA, USA) was reacted 3 3 with the sulfur trioxide-trimethylamine complex in pyridine, and then the products were purified by silica-gel column chromatography with Merck silica-gel 60 (63–200 m; Darmstadt, Germany) and reversed-phase HPLC [column, J’sphere ODS-H80 (YMC, Kyoto, Japan) and mobile phase, methanol-10 mM ammonium formate (4:1, v/v)]. Structural confirmation of 25(OH)D3 S and IS was performed by negative ESI-MS/MS and UV spectroscopy (max 265 nm and min 228 nm). Their chemical purities were confirmed to be over 99.5% by HPLC. The isotopic purity of IS (the 2 H3 -form; 67%, 2 H -form; 33%, 2 H -form; 0% and 2 H -form; 0%) was determined 2 1 0 by LC/ESI-MS. A stock solution of 25(OH)D3 S was prepared as a 2.45 g/mL solution in ethanol; the concentration was determined by UV spectroscopy using the molar absorptivity (ε) of 18200 at 265 nm. Subsequent dilutions were carried out with ethanol to prepare 5.0, 10, 20, 50 and 100 ng/mL solutions. The ethanolic solution of IS (150 ng/mL) was also prepared. An Oasis® HLB cartridge (30 mg adsorbent; Waters, Milford, MA, USA) was successively washed with methanol (1 mL) and water (1 mL) prior to use. The method development and validation were carried out using a FFPLR Nisseki frozen plasma obtained from the Japan Red Cross Service (Tokyo). All other reagents and solvents were of analytical grade or LC/MS grade.
The intra- and inter-assay precisions were assessed by the repeated measurements (n = 5) of three plasma samples (A, B and C) on one day and over five days, respectively. The precision was determined as the relative standard deviation (RSD, %). The assay accuracy was examined using plasma samples A, B and C. The plasma (20 L) was added to acetonitrile (50 L) containing IS (1.5 ng) and 25(OH)D3 S (0.10 or 0.20 ng; corresponding to 5.0 or 10 ng/mL) (spiked sample) and pretreated in the same way as described in Section 2.3. The assay accuracy (analytical recovery) of 25(OH)D3 S was defined as F/(F0 + A) × 100 (%), where F is the concentration of 25(OH)D3 S in the spiked sample, F0 is the concentration of 25(OH)D3 S determined in the inter-assay precision test and A is the spiked concentration.
2.2. LC/ESI-MS/MS
2.6. Freeze/thaw stability
LC/ESI-MS/MS was performed using a Waters Quattro Premier XE triple quadrupole-mass spectrometer connected to an LC-2795 chromatograph. A J’sphere ODS H-80 (4 m, 150 × 2.0 mm i.d.) was used at the flow rate of 0.2 mL/min at 40 ◦ C. A gradient elution program with mobile phase A [methanol-10 mM ammonium formate (4:1, v/v)] and mobile phase B [methanol-10 mM ammonium formate (9:1, v/v)] was performed for the determination of 25(OH)D3 S; B = 0% maintained (0–3.5 min), 100% linearly increased (3.5–8.5 min) and maintained (8.5–15 min), and 0% maintained (15–20 min). 25(OH)D3 S and IS were analyzed in the negative-ion mode because they have a strong acidic group, a sulfate group, and the conditions were as follows: capillary voltage, 2.8 kV; cone voltage, 60 V; collision energy, 35 eV; source temperature, 120 ◦ C; desolvation temperature, 350 ◦ C; desolvation gas (N2 ) flow rate, 600 L/h; cone gas (N2 ) flow rate, 50 L/h; and collision gas (Ar) flow rate, 0.19 mL/min. The selected reaction monitoring (SRM) transitions for 25(OH)D3 S and IS were m/z 479.1 → 96.6 and m/z 482.1 → 96.6, respectively. Masslynx software (version 4.1, Waters) was used for the system control and data processing.
The freeze/thaw stability of 25(OH)D3 S in the plasma was examined by analyzing plasma samples A, B and C without/with 3 additional freeze/thaw cycles.
2.5. Precision and accuracy (analytical recovery)
2.7. Absolute recovery The plasma specimen not containing 25(OH)D3 S could not be prepared by the charcoal-treatment because 25(OH)D3 S is tightly bound to the plasma protein. Therefore, the absolute recovery from the plasma specimen was examined using IS [2 H3 -25(OH)D3 S, 1.5 ng/20 L plasma]. The recovery rate was calculated from the peak area ratio [25(OH)D3 S/IS] in samples I and II as described below; recovery = peak area ratio in sample II/peak area ratio in sample I. Sample I: The plasma (20 L) was added to acetonitrile (50 L) containing IS (1.5 ng) and pretreated in the same way as described in Section 2.3. Sample II: The plasma (20 L) was added to acetonitrile (50 L) and pretreated in the same way as described in Section 2.3. After the addition of IS (1.5 ng) to this pretreated plasma, the resulting sample was subjected to LC/ESI-MS/MS.
2.3. Pretreatment of plasma 2.8. Infant plasma sample The plasma (20 L) was added to acetonitrile (50 L) containing IS (1.5 ng), vortex-mixed for 30 s and centrifuged at 1000 × g (10 min). The supernatant was diluted with water (300 L), and the sample was passed through an Oasis® HLB cartridge. After washing with water (1 mL) and methanol–water (1:1, v/v) (1 mL), 25(OH)D3 S and IS were eluted with methanol (1 mL). After removal
The plasma samples from 28 Japanese infants (gestational age, 29.1–40.6 weeks and birth-weight, 1043–3524 g) including 18 preterm infants of both sexes were examined. Blood was collected from their dorsal hand vein within 60 days after birth at the Shizuoka Saiseikai General Hospital (Shizuoka, Japan). Written
T. Higashi et al. / J. Chromatogr. B 969 (2014) 230–234
informed consent was obtained from their parents. The experimental procedures were approved by the Ethics Committees of the Tokyo University of Science and Shizuoka Saiseikai General Hospital. 2.9. Determination of plasma 25(OH)D3 of infants The plasma 25(OH)D3 of the infants was determined using the LC/ESI-MS/MS method that had been developed and validated in our laboratories, the details of which will be reported elsewhere in the near future. Briefly, 25(OH)D3 was extracted from the plasma (10 L) with ethyl acetate, derivatized with 4-(4 -dimethylaminophenyl)-1,2,4-triazoline-3,5-dione [10] and subjected to LC/ESI-MS/MS. 2 H3 -25(OH)D3 was used as the IS and quantification was based on SRM. The LC/ESI-MS/MS conditions were the same as described in a previous study [10]. The measurable range was 2.5–50 ng/mL, with the inter- and intra-assay precisions of 0.998) and the slopes were reproducible [1.1612 ± 0.0557 (mean ± SD) and 4.8% (RSD)]. There was no significant difference between the slope of the line obtained from the standard solution and that obtained from the standard-added plasma. This result demonstrated that the plasma matrix had no impact on the determination of 25(OH)D3 S. Based on
T. Higashi et al. / J. Chromatogr. B 969 (2014) 230–234
60
50
50
0 5.5
6.0
6.5
7.0
7.5
Time (min) 100
Relative intensity (%)
Preterm infants
25(OH)D3S 100% = 1.75e4
IS 100% = 1.98e4
Plasma concentration (ng/mL)
Relative intensity (%)
100
233
40
30
20
10
50
0 25
30
35
40
45
Gestational age (week) Fig. 3. Correlations between gestational age and plasma concentrations of 25(OH)D3 S (filled circle) or 25(OH)D3 (open circle) in infants.
0 5.5
6.0
6.5
7.0
7.5
Time (min) Fig. 2. Chromatograms of 25(OH)D3 S (upper) in infant plasma spiked with IS (lower). The measured concentration of 25(OH)D3 S was 36.9 ng/mL. The LC/ESIMS/MS conditions are described in Section 2.2.
this result and the fact that the standard solution did not require the pretreatment steps, the calibration curve was constructed using the standard solution in the following studies. 3.3.2. Assay precision and accuracy The intra-assay (n = 5) RSDs did not exceed 7.9%, and a good inter-assay (n = 5) RSDs (not exceeding 2.8%) was also obtained, as shown in Table 1. A satisfactory assay accuracy (analytical recovery) ranging from 95.8 to 105.3% was obtained (Table 1). These data indicated that the present method is highly reproducible and accurate. 3.3.3. Stability 25(OH)D3 S in plasma was stable up to three additional freeze/thaw cycles; 98.3 ± 4.3 (mean ± SD, n = 3) of the initial measured value was obtained after three additional freeze/thaw cycles. Table 1 Precision and accuracy of 25(OH)D3 S assay.
Intact sample (intra-assay, n = 5) Measureda (ng/mL) Precision (RSD, %) Intact sample (inter-assay, n = 5) Measureda (ng/mL) Precision (RSD, %) Spiked sample (+ 5.0 ng/mL, n = 2) Measureda (ng/mL) Accuracy (%) Spiked sample (+ 10 ng/mL, n = 2) Measureda (ng/mL) Accuracy (%) a
Mean or mean ± SD.
Furthermore, it was possible to store the plasma at −20 ◦ C without loss of the 25(OH)D3 S for at least 1 month. 3.4. Determination of plasma 25(OH)D3 S of infants The developed method was applied to the determination of the plasma 25(OH)D3 S of the infants (Fig. 3). The plasma concentration of 25(OH)D3 S was 26.7 ± 12.7 ng/mL (mean ± SD, n = 28) with the range of 3.9–58.2 ng/mL, and significantly higher than that of 25(OH)D3 (8.0 ± 3.8 ng/mL); these agreed with previously reported results [12,14]. The plasma 25(OH)D3 concentrations of the infants were significantly lower than those of healthy adults (ca. 10–40 ng/mL [5]), whereas the plasma 25(OH)D3 S concentrations of full-term infants (gestational age, 37–40 weeks) were similar to or higher than those of healthy adults (ca. 10–30 ng/mL [14]). These results suggested that 25(OH)D3 S may play an important role in infants; 25(OH)D3 S may be utilized after deconjugation to 25(OH)D3 , in other words, it could be a storage form of vitamin D3 . This study revealed that the preterm infants have lower plasma 25(OH)D3 S concentrations (Pearson’s correlation coefficient test, p < 0.01), whereas the plasma 25(OH)D3 concentration was not related to the gestational age (p = 0.66). The low level of 25(OH)D3 S may be a possible cause of rickets that is more common in the preterm infants. 4. Conclusion
Plasma A
Plasma B
Plasma C
10.60 ± 0.84 7.9
25.45 ± 0.58 2.3
35.48 ± 1.05 3.0
10.45 ± 0.23 2.2
26.03 ± 0.73 2.8
34.40 ± 0.64 1.9
15.03 97.3
32.68 105.3
39.59 100.5
19.98 97.7
35.48 98.5
42.54 95.8
We have demonstrated the stable isotope-dilution LC/ESIMS/MS method for the determination of 25(OH)D3 S in human plasma. The method was simple, accurate and reproducible, and able to quantify 2.5–50 ng/mL of 25(OH)D3 S using a 20-L plasma. To the best of our knowledge, this is the first validated LC/MS/MS assay for the determination of plasma 25(OH)D3 S. Using the method, we found that preterm infants have lower plasma 25(OH)D3 S concentrations, which is suspected as a cause of prematurity rickets. Thus, this well-characterized method will prove helpful in the assessment of vitamin D status, especially for infants, and understanding the biological role of 25(OH)D3 S.
234
T. Higashi et al. / J. Chromatogr. B 969 (2014) 230–234
Acknowledgement This study was supported in part by a Grant-in-Aid for Scientific Research (C) for 2014–2016 (Grant Number 26460043) from the Japan Society for the Promotion of Science. References [1] B.W. Hollis, R.L. Horst, J. Steroid Biochem. Mol. Biol. 103 (2007) 473. [2] E. Hyppönen, E. Läärä, A. Reunanen, M.R. Järvelin, S.M. Virtanen, Lancet 358 (2001) 1500. [3] C.J. Willer, D.A. Dyment, A.D. Sadovnick, P.M. Rothwell, T.J. Murray, G.C. Ebers, Canadian Collaborative Study Group, BMJ 330 (2005) 120. [4] J. McGrath, D. Eyles, B. Mowry, R. Yolken, S. Buka, Schizophr. Res. 63 (2003) 73. [5] T. Higashi, K. Shimada, T. Toyo’oka, J. Chromatogr. B 878 (2010) 1654.
[6] J.M. El-Khoury, E.Z. Reineks, S. Wang, Clin. Biochem. 44 (2011) 66. [7] D. Eyles, C. Anderson, P. Ko, A. Jones, A. Thomas, T. Burne, P.B. Mortensen, B. Nørgaard-Pedersen, D.M. Hougaard, J. McGrath, Clin. Chim. Acta 403 (2009) 145. [8] T. Higashi, M. Suzuki, J. Hanai, S. Inagaki, J.Z. Min, K. Shimada, T. Toyo’oka, J. Sep. Sci. 34 (2011) 725. [9] D. Kvaskoff, P. Ko, H.A. Simila, D.W. Eyles, J. Chromatogr. B 901 (2012) 47. [10] S. Ogawa, S. Ooki, M. Morohashi, K. Yamagata, T. Higashi, Rapid Commun. Mass Spectrom. 27 (2013) 2453. [11] M. Axelson, FEBS Lett. 191 (1985) 171. [12] M. Axelson, N.J. Christensen, J. Steroid Biochem. 31 (1988) 35. [13] K. Shimada, K. Mitamura, N. Kitama, Biomed. Chromatogr. 9 (1995) 229. [14] T. Higashi, K. Mitamura, H. Ohmi, N. Yamada, K. Shimada, K. Tanaka, H. Honjo, Ann. Clin. Biochem. 36 (1999) 43. [15] T. Iida, G. Kakiyama, Y. Hibiya, S. Miyata, T. Inoue, K. Ohno, T. Goto, N. Mano, J. Goto, T. Nambara, A.F. Hofmann, Steroids 71 (2006) 18. [16] A.J. Brown, D.W. Coyne, Kidney Int. 82 (2012) 5.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Development and validation of a method for determination of plasma 25-hydroxyvitamin D3 3-sulfate using liquid chromatography/tandem mass spectrometry Tatsuya Higashi a,∗ , Ayaka Goto a , Misato Morohashi a , Shoujiro Ogawa a , Kenji Komatsu b , Takahiro Sugiura b , Tetsuya Fukuoka b , Kuniko Mitamura c a
Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan Shizuoka Saiseikai General Hospital, 1-1-1 Oshika, Suruga-ku, Shizuoka 422-8527, Japan c Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka-shi, Osaka 577-8502, Japan b
a r t i c l e
i n f o
Article history: Received 16 July 2014 Received in revised form 22 August 2014 Accepted 24 August 2014 Available online 28 August 2014 Keywords: 25-Hydroxyvitamin D3 3-sulfate LC/ESI-MS/MS Plasma Infant Vitamin D status
a b s t r a c t The quantification of plasma 25-hydroxyvitamin D3 3-sulfate [25(OH)D3 S] is expected to be helpful in the assessment of the vitamin D status, especially for infants. In this study, a simple and sensitive method for the quantification of 25(OH)D3 S in plasma using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. The plasma was deproteinized with acetonitrile, purified using an Oasis® HLB cartridge, and subjected to LC/ESIMS/MS operating in the negative-ion mode. Quantification was based on the selected reaction monitoring, and deuterated 25(OH)D3 S was used as the internal standard. This method enabled the reproducible (intra- and inter-assay relative standard deviations, 7.9% or lower) and accurate (analytical recovery, 95.8–105.3%) quantification of the plasma 25(OH)D3 S using a 20-L sample, and the limit of quantification was 2.5 ng/mL. The developed method was applied to the determination of plasma 25(OH)D3 S in infants; the result revealed that preterm infants have lower plasma 25(OH)D3 S concentrations. © 2014 Elsevier B.V. All rights reserved.
1. Introduction It is widely accepted that the measurement of 25hydroxyvitamin D3 [25(OH)D3 ], which is the major circulating metabolite of vitamin D3 , in plasma/serum is useful for the assessment of the vitamin D status and the diagnosis of several bone metabolic diseases, such as rickets and osteoporosis [1]. In addition to these diseases, vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes, such as type 1 diabetes [2], multiple sclerosis [3] and schizophrenia [4] in later life; screening for vitamin D deficiency in an infant is important for the early supplement of vitamin D to the diagnosed infant. Liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) is now the most commonly used method to determine vitamin D metabolites in various biological samples due to its high specificity and accuracy [5,6]. LC/ESI-MS/MS assays for 25(OH)D3 in dried blood spots have been
∗ Corresponding author. Tel./fax: +81 4 7121 3660. E-mail address: [email protected] (T. Higashi). http://dx.doi.org/10.1016/j.jchromb.2014.08.027 1570-0232/© 2014 Elsevier B.V. All rights reserved.
also developed for the assessment of the infant vitamin D status [7–10]. 25-Hydroxyvitamin D3 3-sulfate [25(OH)D3 S], the sulfated conjugate of 25(OH)D3 , is reported to be another major metabolite of vitamin D3 , and its circulating level is similar to or higher than that of 25(OH)D3 in adults or infants, respectively [5,11–14]. Although the biological role of 25(OH)D3 S is not still fully understood, it might be the storage form of vitamin D3 . Therefore, it is expected that the quantification of 25(OH)D3 S in plasma/serum is also helpful in the assessment of the vitamin D status, especially for infants. A method using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection has been reported for the quantification of plasma 25(OH)D3 S [13]; the method, however, employed a relatively-large volume of sample (0.5 mL) and complicated pretreatment procedures (2 steps of solid-phase extraction and ion-exchange chromatographic purification after deproteinization), and is not necessarily suited to evaluate the diagnostic value and biological role of 25(OH)D3 S. Based on this background information, the objective of this study was to develop and validate a stable isotope-dilution LC/ESI-MS/MS method for the determination of plasma 25(OH)D3 S requiring a small sample volume and simple pretreatment
T. Higashi et al. / J. Chromatogr. B 969 (2014) 230–234
procedure. The application of the method to the measurement of the plasma 25(OH)D3 S concentration of infants is also described. 2. Experimental 2.1. Chemicals and reagents
231
of the solvent, 25(OH)D3 S and IS were dissolved in the mobile phase A (60 L), 15 L of which was subjected to LC/ESI-MS/MS. 2.4. Linearity and calibration curves The plasma (20 L) was added to acetonitrile (50 L) containing IS (1.5 ng) and a graduated amount of standard 25(OH)D3 S (0.050, 0.10, 0.20, 0.50 or 1.0 ng; corresponding to 2.5, 5.0, 10, 25 or 50 ng/mL), and pretreated in the same way as described in Section 2.3. The peak area ratio [25(OH)D3 S/IS] (y) was plotted versus the added amount of 25(OH)D3 S (ng per tube) (x) with a weighting of 1/x to construct the regression lines. The same amounts of IS (1.5 ng) and 25(OH)D3 S (0.050, 0.10, 0.20, 0.50 or 1.0 ng) were pipetted into tubes. The mixture was dissolved in the mobile phase A (60 L), 15 L of which was subjected to LC/ESI-MS/MS. The peak area ratio [25(OH)D3 S/IS] (y) was plotted versus the amount of 25(OH)D3 S (ng per tube) (x), and the obtained regression line was used as the calibration curve.
25(OH)D3 S and 2 H3 -25(OH)D3 S [internal standard (IS)] were synthesized according to the method of Iida et al. [15] in our laboratories; 25(OH)D3 (Wako Pure Chemical Industries, Osaka, Japan) or 2 H -25(OH)D (IsoSciences, King of Prussia, PA, USA) was reacted 3 3 with the sulfur trioxide-trimethylamine complex in pyridine, and then the products were purified by silica-gel column chromatography with Merck silica-gel 60 (63–200 m; Darmstadt, Germany) and reversed-phase HPLC [column, J’sphere ODS-H80 (YMC, Kyoto, Japan) and mobile phase, methanol-10 mM ammonium formate (4:1, v/v)]. Structural confirmation of 25(OH)D3 S and IS was performed by negative ESI-MS/MS and UV spectroscopy (max 265 nm and min 228 nm). Their chemical purities were confirmed to be over 99.5% by HPLC. The isotopic purity of IS (the 2 H3 -form; 67%, 2 H -form; 33%, 2 H -form; 0% and 2 H -form; 0%) was determined 2 1 0 by LC/ESI-MS. A stock solution of 25(OH)D3 S was prepared as a 2.45 g/mL solution in ethanol; the concentration was determined by UV spectroscopy using the molar absorptivity (ε) of 18200 at 265 nm. Subsequent dilutions were carried out with ethanol to prepare 5.0, 10, 20, 50 and 100 ng/mL solutions. The ethanolic solution of IS (150 ng/mL) was also prepared. An Oasis® HLB cartridge (30 mg adsorbent; Waters, Milford, MA, USA) was successively washed with methanol (1 mL) and water (1 mL) prior to use. The method development and validation were carried out using a FFPLR Nisseki frozen plasma obtained from the Japan Red Cross Service (Tokyo). All other reagents and solvents were of analytical grade or LC/MS grade.
The intra- and inter-assay precisions were assessed by the repeated measurements (n = 5) of three plasma samples (A, B and C) on one day and over five days, respectively. The precision was determined as the relative standard deviation (RSD, %). The assay accuracy was examined using plasma samples A, B and C. The plasma (20 L) was added to acetonitrile (50 L) containing IS (1.5 ng) and 25(OH)D3 S (0.10 or 0.20 ng; corresponding to 5.0 or 10 ng/mL) (spiked sample) and pretreated in the same way as described in Section 2.3. The assay accuracy (analytical recovery) of 25(OH)D3 S was defined as F/(F0 + A) × 100 (%), where F is the concentration of 25(OH)D3 S in the spiked sample, F0 is the concentration of 25(OH)D3 S determined in the inter-assay precision test and A is the spiked concentration.
2.2. LC/ESI-MS/MS
2.6. Freeze/thaw stability
LC/ESI-MS/MS was performed using a Waters Quattro Premier XE triple quadrupole-mass spectrometer connected to an LC-2795 chromatograph. A J’sphere ODS H-80 (4 m, 150 × 2.0 mm i.d.) was used at the flow rate of 0.2 mL/min at 40 ◦ C. A gradient elution program with mobile phase A [methanol-10 mM ammonium formate (4:1, v/v)] and mobile phase B [methanol-10 mM ammonium formate (9:1, v/v)] was performed for the determination of 25(OH)D3 S; B = 0% maintained (0–3.5 min), 100% linearly increased (3.5–8.5 min) and maintained (8.5–15 min), and 0% maintained (15–20 min). 25(OH)D3 S and IS were analyzed in the negative-ion mode because they have a strong acidic group, a sulfate group, and the conditions were as follows: capillary voltage, 2.8 kV; cone voltage, 60 V; collision energy, 35 eV; source temperature, 120 ◦ C; desolvation temperature, 350 ◦ C; desolvation gas (N2 ) flow rate, 600 L/h; cone gas (N2 ) flow rate, 50 L/h; and collision gas (Ar) flow rate, 0.19 mL/min. The selected reaction monitoring (SRM) transitions for 25(OH)D3 S and IS were m/z 479.1 → 96.6 and m/z 482.1 → 96.6, respectively. Masslynx software (version 4.1, Waters) was used for the system control and data processing.
The freeze/thaw stability of 25(OH)D3 S in the plasma was examined by analyzing plasma samples A, B and C without/with 3 additional freeze/thaw cycles.
2.5. Precision and accuracy (analytical recovery)
2.7. Absolute recovery The plasma specimen not containing 25(OH)D3 S could not be prepared by the charcoal-treatment because 25(OH)D3 S is tightly bound to the plasma protein. Therefore, the absolute recovery from the plasma specimen was examined using IS [2 H3 -25(OH)D3 S, 1.5 ng/20 L plasma]. The recovery rate was calculated from the peak area ratio [25(OH)D3 S/IS] in samples I and II as described below; recovery = peak area ratio in sample II/peak area ratio in sample I. Sample I: The plasma (20 L) was added to acetonitrile (50 L) containing IS (1.5 ng) and pretreated in the same way as described in Section 2.3. Sample II: The plasma (20 L) was added to acetonitrile (50 L) and pretreated in the same way as described in Section 2.3. After the addition of IS (1.5 ng) to this pretreated plasma, the resulting sample was subjected to LC/ESI-MS/MS.
2.3. Pretreatment of plasma 2.8. Infant plasma sample The plasma (20 L) was added to acetonitrile (50 L) containing IS (1.5 ng), vortex-mixed for 30 s and centrifuged at 1000 × g (10 min). The supernatant was diluted with water (300 L), and the sample was passed through an Oasis® HLB cartridge. After washing with water (1 mL) and methanol–water (1:1, v/v) (1 mL), 25(OH)D3 S and IS were eluted with methanol (1 mL). After removal
The plasma samples from 28 Japanese infants (gestational age, 29.1–40.6 weeks and birth-weight, 1043–3524 g) including 18 preterm infants of both sexes were examined. Blood was collected from their dorsal hand vein within 60 days after birth at the Shizuoka Saiseikai General Hospital (Shizuoka, Japan). Written
T. Higashi et al. / J. Chromatogr. B 969 (2014) 230–234
informed consent was obtained from their parents. The experimental procedures were approved by the Ethics Committees of the Tokyo University of Science and Shizuoka Saiseikai General Hospital. 2.9. Determination of plasma 25(OH)D3 of infants The plasma 25(OH)D3 of the infants was determined using the LC/ESI-MS/MS method that had been developed and validated in our laboratories, the details of which will be reported elsewhere in the near future. Briefly, 25(OH)D3 was extracted from the plasma (10 L) with ethyl acetate, derivatized with 4-(4 -dimethylaminophenyl)-1,2,4-triazoline-3,5-dione [10] and subjected to LC/ESI-MS/MS. 2 H3 -25(OH)D3 was used as the IS and quantification was based on SRM. The LC/ESI-MS/MS conditions were the same as described in a previous study [10]. The measurable range was 2.5–50 ng/mL, with the inter- and intra-assay precisions of 0.998) and the slopes were reproducible [1.1612 ± 0.0557 (mean ± SD) and 4.8% (RSD)]. There was no significant difference between the slope of the line obtained from the standard solution and that obtained from the standard-added plasma. This result demonstrated that the plasma matrix had no impact on the determination of 25(OH)D3 S. Based on
T. Higashi et al. / J. Chromatogr. B 969 (2014) 230–234
60
50
50
0 5.5
6.0
6.5
7.0
7.5
Time (min) 100
Relative intensity (%)
Preterm infants
25(OH)D3S 100% = 1.75e4
IS 100% = 1.98e4
Plasma concentration (ng/mL)
Relative intensity (%)
100
233
40
30
20
10
50
0 25
30
35
40
45
Gestational age (week) Fig. 3. Correlations between gestational age and plasma concentrations of 25(OH)D3 S (filled circle) or 25(OH)D3 (open circle) in infants.
0 5.5
6.0
6.5
7.0
7.5
Time (min) Fig. 2. Chromatograms of 25(OH)D3 S (upper) in infant plasma spiked with IS (lower). The measured concentration of 25(OH)D3 S was 36.9 ng/mL. The LC/ESIMS/MS conditions are described in Section 2.2.
this result and the fact that the standard solution did not require the pretreatment steps, the calibration curve was constructed using the standard solution in the following studies. 3.3.2. Assay precision and accuracy The intra-assay (n = 5) RSDs did not exceed 7.9%, and a good inter-assay (n = 5) RSDs (not exceeding 2.8%) was also obtained, as shown in Table 1. A satisfactory assay accuracy (analytical recovery) ranging from 95.8 to 105.3% was obtained (Table 1). These data indicated that the present method is highly reproducible and accurate. 3.3.3. Stability 25(OH)D3 S in plasma was stable up to three additional freeze/thaw cycles; 98.3 ± 4.3 (mean ± SD, n = 3) of the initial measured value was obtained after three additional freeze/thaw cycles. Table 1 Precision and accuracy of 25(OH)D3 S assay.
Intact sample (intra-assay, n = 5) Measureda (ng/mL) Precision (RSD, %) Intact sample (inter-assay, n = 5) Measureda (ng/mL) Precision (RSD, %) Spiked sample (+ 5.0 ng/mL, n = 2) Measureda (ng/mL) Accuracy (%) Spiked sample (+ 10 ng/mL, n = 2) Measureda (ng/mL) Accuracy (%) a
Mean or mean ± SD.
Furthermore, it was possible to store the plasma at −20 ◦ C without loss of the 25(OH)D3 S for at least 1 month. 3.4. Determination of plasma 25(OH)D3 S of infants The developed method was applied to the determination of the plasma 25(OH)D3 S of the infants (Fig. 3). The plasma concentration of 25(OH)D3 S was 26.7 ± 12.7 ng/mL (mean ± SD, n = 28) with the range of 3.9–58.2 ng/mL, and significantly higher than that of 25(OH)D3 (8.0 ± 3.8 ng/mL); these agreed with previously reported results [12,14]. The plasma 25(OH)D3 concentrations of the infants were significantly lower than those of healthy adults (ca. 10–40 ng/mL [5]), whereas the plasma 25(OH)D3 S concentrations of full-term infants (gestational age, 37–40 weeks) were similar to or higher than those of healthy adults (ca. 10–30 ng/mL [14]). These results suggested that 25(OH)D3 S may play an important role in infants; 25(OH)D3 S may be utilized after deconjugation to 25(OH)D3 , in other words, it could be a storage form of vitamin D3 . This study revealed that the preterm infants have lower plasma 25(OH)D3 S concentrations (Pearson’s correlation coefficient test, p < 0.01), whereas the plasma 25(OH)D3 concentration was not related to the gestational age (p = 0.66). The low level of 25(OH)D3 S may be a possible cause of rickets that is more common in the preterm infants. 4. Conclusion
Plasma A
Plasma B
Plasma C
10.60 ± 0.84 7.9
25.45 ± 0.58 2.3
35.48 ± 1.05 3.0
10.45 ± 0.23 2.2
26.03 ± 0.73 2.8
34.40 ± 0.64 1.9
15.03 97.3
32.68 105.3
39.59 100.5
19.98 97.7
35.48 98.5
42.54 95.8
We have demonstrated the stable isotope-dilution LC/ESIMS/MS method for the determination of 25(OH)D3 S in human plasma. The method was simple, accurate and reproducible, and able to quantify 2.5–50 ng/mL of 25(OH)D3 S using a 20-L plasma. To the best of our knowledge, this is the first validated LC/MS/MS assay for the determination of plasma 25(OH)D3 S. Using the method, we found that preterm infants have lower plasma 25(OH)D3 S concentrations, which is suspected as a cause of prematurity rickets. Thus, this well-characterized method will prove helpful in the assessment of vitamin D status, especially for infants, and understanding the biological role of 25(OH)D3 S.
234
T. Higashi et al. / J. Chromatogr. B 969 (2014) 230–234
Acknowledgement This study was supported in part by a Grant-in-Aid for Scientific Research (C) for 2014–2016 (Grant Number 26460043) from the Japan Society for the Promotion of Science. References [1] B.W. Hollis, R.L. Horst, J. Steroid Biochem. Mol. Biol. 103 (2007) 473. [2] E. Hyppönen, E. Läärä, A. Reunanen, M.R. Järvelin, S.M. Virtanen, Lancet 358 (2001) 1500. [3] C.J. Willer, D.A. Dyment, A.D. Sadovnick, P.M. Rothwell, T.J. Murray, G.C. Ebers, Canadian Collaborative Study Group, BMJ 330 (2005) 120. [4] J. McGrath, D. Eyles, B. Mowry, R. Yolken, S. Buka, Schizophr. Res. 63 (2003) 73. [5] T. Higashi, K. Shimada, T. Toyo’oka, J. Chromatogr. B 878 (2010) 1654.
[6] J.M. El-Khoury, E.Z. Reineks, S. Wang, Clin. Biochem. 44 (2011) 66. [7] D. Eyles, C. Anderson, P. Ko, A. Jones, A. Thomas, T. Burne, P.B. Mortensen, B. Nørgaard-Pedersen, D.M. Hougaard, J. McGrath, Clin. Chim. Acta 403 (2009) 145. [8] T. Higashi, M. Suzuki, J. Hanai, S. Inagaki, J.Z. Min, K. Shimada, T. Toyo’oka, J. Sep. Sci. 34 (2011) 725. [9] D. Kvaskoff, P. Ko, H.A. Simila, D.W. Eyles, J. Chromatogr. B 901 (2012) 47. [10] S. Ogawa, S. Ooki, M. Morohashi, K. Yamagata, T. Higashi, Rapid Commun. Mass Spectrom. 27 (2013) 2453. [11] M. Axelson, FEBS Lett. 191 (1985) 171. [12] M. Axelson, N.J. Christensen, J. Steroid Biochem. 31 (1988) 35. [13] K. Shimada, K. Mitamura, N. Kitama, Biomed. Chromatogr. 9 (1995) 229. [14] T. Higashi, K. Mitamura, H. Ohmi, N. Yamada, K. Shimada, K. Tanaka, H. Honjo, Ann. Clin. Biochem. 36 (1999) 43. [15] T. Iida, G. Kakiyama, Y. Hibiya, S. Miyata, T. Inoue, K. Ohno, T. Goto, N. Mano, J. Goto, T. Nambara, A.F. Hofmann, Steroids 71 (2006) 18. [16] A.J. Brown, D.W. Coyne, Kidney Int. 82 (2012) 5.
