Anal Bioanal Chem (2014) 406:6647–6654 DOI 10.1007/s00216-014-8095-y
RESEARCH PAPER
Analysis of urinary vitamin D3 metabolites by liquid chromatography/tandem mass spectrometry with ESI-enhancing and stable isotope-coded derivatization Shoujiro Ogawa & Satoshi Ooki & Kenta Shinoda & Tatsuya Higashi
Received: 13 June 2014 / Revised: 28 July 2014 / Accepted: 6 August 2014 / Published online: 29 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The determination of the urinary vitamin D3 metabolites might prove helpful in the assessment of the vitamin D status. We developed a method for the determination of trace vitamin D3 metabolites, 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3], in urine using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using an ESI-enhancing reagent, 4-(4′dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), and its isotope-coded analogue, 2H4-DAPTAD (d-DAPTAD). The urine samples were treated with β-glucuronidase, purified with an Oasis® hydrophilic–lipophilic balanced (HLB) cartridge, and then subjected to the derivatization. The DAPTAD derivatization enabled the highly sensitive detection (detection limit, 0.25 fmol on the column), and the use of d-DAPTAD significantly improved the assay precision [the intra- (n=5) and inter-assay (n=3) relative standard deviations did not exceed 9.5 %]. The method was successfully applied to urine sample analyses and detected the increases of the urinary 25(OH)D3 and 24,25(OH)2D3 levels due to vitamin D3 administration. Keywords Vitamin D3 metabolite . Human urine . LC/ESIMS/MS . Isotope-coded derivatization . Vitamin D status
Introduction It is widely accepted that the measurement of 25hydroxyvitamin D3 [25(OH)D3], which is the major circulating S. Ogawa : S. Ooki : K. Shinoda : T. Higashi (*) Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan e-mail: [email protected]
metabolite of vitamin D3, in plasma/serum is useful for the assessment of the vitamin D status and for the diagnosis of several bone metabolic diseases, such as rickets and osteoporosis [1]. 25(OH)D3 is metabolized to the hormonally active form, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] in the kidney, whereas excess 25(OH)D3 is excreted into the urine after conversion to catabolic metabolites. We have reported that some side chain-oxidized metabolites including 24R,25dihydroxyvitamin D3 [24,25(OH)2D3] are present in urine as the glucuronidated conjugates [2]. However, quantitative analysis of the urinary vitamin D3 metabolites has not been carried out, though it might also prove helpful in the assessment of the vitamin D status. A highly sensitive technique is required for the quantitative analysis of the urinary vitamin D3 metabolites because their concentrations are predicted to be extremely low (possibly in the pg/mL range). Recently, liquid chromatography (LC) coupled with electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) has been used for the determination of serum/plasma 25(OH)D3 (normal range, 10–40 ng/mL) due to its specificity and versatility [3–5]. However, the ionization efficiency of vitamin D3 metabolites is not high in ESI, and therefore, this insufficient sensitivity might become a major problem in the analysis of the picogram-abundant urinary metabolites. The use of 4-(4′-dimethylaminophenyl)1,2,4-triazoline-3,5-dione (DAPTAD), a derivatization regent that we recently developed, will probably be able to solve this problem; the DAPTAD derivatization improved the detection limit of 25(OH)D3 to 0.25 fmol (equivalent to 0.1 pg) [6]. Matrix effects caused by endogenous components often occur during the measurement of low abundant metabolites using LC/ESI-MS/MS. The ionization efficiency is also sometimes different between two single runs even for the same
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analyte. A stable isotope-labeled analogue of the analyte is widely used as an internal standard (IS) to overcome the matrix effects and run-to-run ionization differences for the accurate quantification. However, only a limited number of stable isotope-labeled analogues are commercially available for vitamin D3 metabolites and their syntheses are not always easy. The stable isotope-coded derivatization is an alternative way to introduce a stable isotope-coded moiety to the analyte, and the resulting derivative can be used as a substitute for a stable isotope-labeled IS [7, 8]. For example, the vitamin D3 metabolites in a urine sample are derivatized with the H-coded reagent (DAPTAD), while standard vitamin D3 metabolites are separately derivatized with the isotope ( 2H)-coded reagent (d-DAPTAD) and spiked into the sample solution prior to the LC/MS/MS analysis (Fig. 1). Since the isotopic pairs of the derivatives elute at almost the same time in a single run, the matrix effects and ionization process for the DAPTAD-derivatized vitamin D3 metabolites are expected to be identical with the d-DAPTAD-derivatized vitamin D 3 metabolites. Thus, stable isotope-coded derivatization can improve the analytical performance and precision without a stable isotope-labeled IS. DAPTAD contains the dimethylamino group, and therefore, its 2H-coded analogue can be easily prepared. In this study, we developed a method for the quantitative analysis of 25(OH)D 3 and 24,25(OH) 2 D 3 in the βglucuronidase-treated urine by LC/ESI-MS/MS combined with ESI-enhancing and stable isotope-coded derivatization. The application of the method to the analysis of urine samples obtained from healthy subjects who had received a vitamin D3 supplement was also described.
25(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3, and 3-epi-25hydroxyvitamin D3 [3-epi-25(OH)D3] were purchased from Wako Pure Chemical Industries (Osaka, Japan), the Duphar B. V. Co. (Amsterdam, The Netherlands), the Funakoshi Co. (Tokyo, Japan), and the Cayman Chemical Company (Ann Arbor, MI, USA), respectively. 3-Epi-24,25dihydroxyvitamin D3 [3-epi-24,25(OH)2D3] was the same as used in a previous study [9]. The stock solutions of the vitamin D3 metabolites were prepared as 100-μg/mL solutions in ethanol, and their concentrations were confirmed by UV spectroscopy using the molar absorptivity (ε) of 18,200 at 265 nm. Subsequent dilutions were carried out with ethanol to prepare 0.40, 1.0, 2.0, 4.0, 10, and 20 ng/ mL solutions. DAPTAD was the same as used in a previous study [6]. d-DAPTAD (2H4-DAPTAD) was synthesized from 2H4-4-dimethylaminobenzoic acid [10]; this starting material was treated with diphenylphosphoryl azide to prepare the carbonyl azide, which was then converted to d-DAPTAD as previously reported [6]. The DAPTAD and d-DAPTAD solutions (in ethyl acetate) could be used for the derivatization for at least 2 months when stored at −18 °C [6]. The isotopic purity of d-DAPTAD was determined by LC/ESI-MS (selected ion monitoring) after it was reacted with 25(OH)D3. By monitoring the respective protonated molecules of the 2H4-, 2H3-, 2H2-, 2H1-, and 2 H0-forms (m/z 623.6, 622.6, 621.6, 620.6, and 619.6, respectively), the isotopic purity of d-DAPTAD was found to be greater than 99.9 % (the 2H3-, 2H2-, 2H1-, and 2H0-forms
Derivatization with DAPTAD Mix
Standard vitamin D3 metabolites
LC/ESIMS/MS
Derivatization with d-DAPTAD
Peak area ratio
Urine samples
Materials and reagents
Intensity
Fig. 1 Scheme of quantification procedure of vitamin D3 metabolites in urine based on electrospray ionization (ESI)enhancing and isotope-coded derivatization
Experimental
Time
Concentration
O
R OH
N N
N
R CHX2 N CHX2
OH
O
O
DAPTAD: X = H d-DAPTAD: X= 2H
N N
N O
HO
25(OH)D3: R=H 24,25(OH)2D3: R= OH
HO
CHX2 N CHX2
Urinary vitamin D3 metabolite analysis by LC/ESI-MS/MS
were not detected at all). β-Glucuronidase originating from E. coli (type IX-A) was obtained from Sigma-Aldrich, Japan (Tokyo); this enzyme was reported to efficiently hydrolyze the glucuronidated vitamin D3 metabolites [11]. The Oasis® HLB cartridges (60 mg; Waters Assoc., Milford, MA, USA) were successively washed with ethyl acetate (2 mL), methanol (2 mL), and water (2 mL) prior to their use. All other reagents and solvents were of analytical grade or LC/MS grade. LC/ESI-MS/MS LC/ESI-MS/MS was performed using a Waters Quattro Premier XE triple quadrupole-mass spectrometer connected to an LC-2795 chromatograph. A YMC-Pack Pro C18 RS (5 μm, 150×2.0 mm i.d.; YMC, Kyoto) 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 (8:3, v/v)] and mobile phase B [methanol–10 mM ammonium formate (9:1, v/v)] was performed; B=0 % maintained (0–5 min), 40 % linearly increased (5–10 min) and maintained (10–13 min), 100 % linearly increased (13–15 min) and maintained (15– 19 min), and 0 % maintained (19–24 min). The derivatized vitamin D3 metabolites were analyzed in the positive ion mode, and the conditions were as follows: capillary voltage, 3.3 kV; cone voltage, 35 V [25(OH)D3-DAPTAD and 25(OH)D3-d-DAPTAD] or 40 V [24,25(OH)2D3-DAPTAD and 24,25(OH)2D3-d-DAPTAD]; collision energy, 25 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 the respective compounds are as follows: 25(OH)D 3 -DAPTAD m/z 619.6 → 341.1, 25(OH)D 3 -dDAPTAD m/z 623.6→345.1, 24,25(OH)2D3-DAPTAD m/z 635.1→341.0, and 24,25(OH)2D3-d-DAPTAD m/z 639.1→ 345.0. MassLynx software (version 4.1, Waters) was used for the system control and data processing.
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The urine samples were analyzed within 7 days after collection. A urine sample (1.0 mL) was incubated with βglucuronidase (1,000 Fishman units) in 0.1 mol/L sodium acetate–acetic acid buffer (pH 5.0, 1 mL) at 37 °C for 2 h. To the reaction mixture, acetonitrile (1 mL) was added and then subjected to centrifugation (1,500×g, 10 min). The supernatant was passed through an Oasis® HLB cartridge. After washing with water (2 mL), methanol–water (7:3, v/v, 2 mL), and hexane (1 mL), the metabolites were eluted with ethyl acetate (1 mL), from which the solvents were evaporated under a N2 gas stream. The residue was subjected to derivatization with DAPTAD. The standards of 25(OH)D3 (200 pg) and 24,25(OH) 2 D 3 (1.0 ng) were derivatized with dDAPTAD, and one tenth of the derivatives were added to the DAPTAD-derivatized urine sample. The solvent was evaporated from the mixture, and the residue was dissolved in the mobile phase (60 μL), 15 μL of which was injected into the LC/ESI-MS/MS (the concentrations of d-DAPTAD derivatives in the sample were 0.83 and 4.0 pmol/mL for 25(OH)D3 and 24,25(OH)2D3, respectively). Calibration curves for 25(OH)D3 and 24,25(OH)2D3
The standard vitamin D3 metabolites and pretreated urine samples were dried and then dissolved in ethyl acetate (50 μL) containing DAPTAD or d-DAPTAD (10 μg). The mixture was kept at room temperature for 1 h, and then, ethanol (20 μL) was added to the mixture to terminate the reaction [6].
The vitamin D metabolite-free urine sample was prepared by the absorptive removal of the metabolites with charcoal; the urine (15 mL) was stirred with the charcoal (1.5 g, Norit®, Nacalai Tesque, Kyoto) for 15 h and then the charcoal was removed by filtering. Neither 25(OH)D3 nor 24,25(OH)2D3 was detected in the charcoal-treated urine by the proposed method. Although some endogenous components, such as steroids [12], were removed together with the vitamin D3 metabolites by the charcoal treatment, the charcoal-treated urine was used to construct the calibration curves. The standards of 25(OH)D3 (4.0–100 pg) and 24,25(OH)2D3 (4.0– 200 pg) were spiked in the vitamin D metabolite-free urine, and then, the resulting samples were pretreated as previously described. The pretreated urine samples were derivatized with DAPTAD. The standards of 25(OH)D 3 (200 pg) and 24,25(OH)2D3 (1.0 ng) were derivatized with d-DAPTAD, and one tenth of the derivatives were added to the DAPTAD-derivatized urine samples. The samples were subjected to LC/ESI-MS/MS. The peak area ratio [DAPTAD derivative/d-DAPTAD derivative] (y) was plotted versus the concentration of 25(OH)D3 or 24,25(OH)2D3 (pg/mL) (x) with a weighting of 1/x to construct the calibration curve.
Collection and pretreatment of the urine sample
Assay precision
Urine samples were collected from healthy male subjects between 9:00 am and 11:00 am and stored at 4 °C. The subjects understood the purpose and significance of this experiment and donated their urine after signing an agreement.
The assay precision was examined using a pooled urine sample collected from healthy male subjects. The intra- (n=5) and inter-assay (n=3) precisions were assessed by the repeated measurements of two urine samples (A and B) on 1 day and
Derivatization
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over 3 days, respectively. The precision was determined as the relative standard deviation (RSD, %). Recovery rates of vitamin D3 metabolites during solid phase extraction The recovery rates of 25(OH)D3 and 24,25(OH)2D3 during the solid phase extraction (SPE) were calculated from the peak area ratio [DAPTAD derivative/d-DAPTAD derivative] in samples I and II as described below: recovery rate=peak area ratio in sample II/peak area ratio in sample I. Sample I The standards of 25(OH)D 3 (20 pg) and 24,25(OH)2D3 (100 pg) were spiked in the vitamin D metabolite-free urine sample (1 mL), and then, the resulting sample was pretreated as previously described. The pretreated urine sample was derivatized with DAPTAD. The standards of 25(OH)D3 (200 pg) and 24,25(OH)2D3 (1.0 ng) were derivatized with d-DAPTAD, and one tenth of the derivatives were added to the DAPTAD-derivatized urine sample. Sample II The vitamin D metabolite-free urine sample (1 mL) was pretreated as previously described. After the addition of the standards of 25(OH)D3 (20 pg) and 24,25(OH)2D3 (100 pg) to this pretreated urine, the resulting sample was derivatized with DAPTAD. The standards of 25(OH)D3 (200 pg) and 24,25(OH)2D3 (1.0 ng) were derivatized with d-DAPTAD, and one tenth of the derivatives were added to the DAPTAD-derivatized urine sample. Stability of vitamin D3 metabolites in urine at 4 °C Five different urine samples were analyzed immediately after their collection and after 7 days of storage at 4 °C, and the measured values were compared. Quantitative analyses of 25(OH)D3 and 24,25(OH)2D3 in β-glucuronidase-treated urine Urine samples were obtained from healthy male subjects (n= 20, age range: 22–35 years) known not to have received vitamin D supplements. The samples were prepared as described in the “Experimental” section. The 25(OH)D3 and 24,25(OH)2D3 concentrations were corrected by the urinary creatinine, which was measured by an enzymatic method at SRL, Inc. (Tokyo). Vitamin D3 administration study The vitamin D3 supplement [1,000 IU (25 μg)/body] (DHC Corporation, Tokyo) was orally administered to eight healthy male (22–35 years old) subjects at 10:00 am once daily for 7 days (from day 1 to day 7). Their urine was collected just
S. Ogawa et al.
before the first administration on day 1 and at 10:00 am on 1 day after the last administration (day 8). The statistical analysis was performed using the Wilcoxon signed-rank test. A p value of 10. Without the use of the d-DAPTAD derivatives, the precision of the calibration curves significantly decreased. When the peak area (absolute value, y) was plotted versus the 25(OH)D3 or 24,25(OH)2D3 concentration (pg/mL) (x), the slopes varied considerably [185.3±15.7 (mean±SD, RSD 8.5 %) for 25(OH)D3 and 265.2±25.2 (RSD 9.5 %) for 24,25(OH)2D3]. Furthermore, large RSDs of the back-calculated concentrations were found at each point, especially for 25(OH)D3 (14.4–45.4 %). Thus, the use of d-DAPTAD worked well in reducing the matrix effects and run-to-run ionization differences. Table 1 Precision and accuracy of calibration curve Nominal concentration Back-calculated RSD (%) REa (%) (pg/mL) concentration (pg/mL, mean±SD, n=5) 25(OH)D3 4.0 10 20 40 100 24,25(OH)2D3 4.0 10 20 40 100 a
4.44±0.54 8.50±0.93 20.5±0.45 41.2±2.52 99.7±3.76
12.2 10.9 2.2 6.2 3.8
11.1 −15.0 2.5 0.3 −0.3
3.64±0.34 9.86±0.74 21.8±2.26 40.5±2.77 99.5±3.81
9.3 7.5 10.4 6.8 3.8
−9.0 −1.4 9.0 1.3 −0.5
(Back-calculated concentration− nominal concentration)/nominal concentration×100 (%)
Urinary vitamin D3 metabolite analysis by LC/ESI-MS/MS
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Table 2 Assay precision 24,25(OH)2D3
25(OH)D3
Urine A Intra-assay Inter-assay Urine B Intra-assay Inter-assay a
Measureda (pg/mL, mean±SD)
Precision (RSD, %)
Measureda (pg/mL, mean±SD)
Precision (RSD, %)
6.87±0.20 7.12±0.39
2.9 5.5
24.0±2.28 25.1±0.97
9.5 3.9
30.5±1.03 28.8±2.14
3.4 7.4
114±5.27 117±3.09
4.6 2.6
n=5 for intra-assay precision and n=3 for inter-assay precision
The intra-assay (n=5) RSDs did not exceed 9.5 %, and good inter-assay (n=3) RSDs (not exceeding 7.4 %) were also obtained, as shown in Table 2. These data indicated that the present method has a satisfactory reproducibility. It was possible to store the urine at 4 °C without any significant loss of the 25(OH)D3 and 24,25(OH)2D3 for at least 7 days; the average measured values after storing at 4 °C for 7 days were 93.7 and 94.3 % (n=5) of those
(a) 24,25(OH)2D3 concentration (ng/g creatinine)
25(OH)D3 concentration (ng/g creatinine)
25 20 15 10
5
100 80 60 40 20 0
0 Before
Before
After
After
(b) p < 0.05
24,25(OH)2D3 concentration (ng/g creatinine)
25(OH)D3 concentration (ng/g creatinine)
25 20 15 10 5 0
100
p < 0.05
80
After
Vitamin D3 administration study The changes in the 25(OH)D3 and 24,25(OH)2D3 levels in a urine sample after the oral administration of vitamin D3 (1,000 IU/body/day) were examined using the developed method. Vitamin D3 was administered for 7 days, and the urine was collected just before the administration on day 1 and at 1 day after the last administration (day 8). The 25(OH)D3 and 24,25(OH)2D3 concentrations of the respective subjects are shown in Fig. 4a; the concentrations of both metabolites were elevated for all the subjects by the administration. Box and whisker plots also showed that the 25(OH)D3 and 24,25(OH)2D3 levels significantly increased after the vitamin D3 administration (p
RESEARCH PAPER
Analysis of urinary vitamin D3 metabolites by liquid chromatography/tandem mass spectrometry with ESI-enhancing and stable isotope-coded derivatization Shoujiro Ogawa & Satoshi Ooki & Kenta Shinoda & Tatsuya Higashi
Received: 13 June 2014 / Revised: 28 July 2014 / Accepted: 6 August 2014 / Published online: 29 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The determination of the urinary vitamin D3 metabolites might prove helpful in the assessment of the vitamin D status. We developed a method for the determination of trace vitamin D3 metabolites, 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3], in urine using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using an ESI-enhancing reagent, 4-(4′dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), and its isotope-coded analogue, 2H4-DAPTAD (d-DAPTAD). The urine samples were treated with β-glucuronidase, purified with an Oasis® hydrophilic–lipophilic balanced (HLB) cartridge, and then subjected to the derivatization. The DAPTAD derivatization enabled the highly sensitive detection (detection limit, 0.25 fmol on the column), and the use of d-DAPTAD significantly improved the assay precision [the intra- (n=5) and inter-assay (n=3) relative standard deviations did not exceed 9.5 %]. The method was successfully applied to urine sample analyses and detected the increases of the urinary 25(OH)D3 and 24,25(OH)2D3 levels due to vitamin D3 administration. Keywords Vitamin D3 metabolite . Human urine . LC/ESIMS/MS . Isotope-coded derivatization . Vitamin D status
Introduction It is widely accepted that the measurement of 25hydroxyvitamin D3 [25(OH)D3], which is the major circulating S. Ogawa : S. Ooki : K. Shinoda : T. Higashi (*) Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan e-mail: [email protected]
metabolite of vitamin D3, in plasma/serum is useful for the assessment of the vitamin D status and for the diagnosis of several bone metabolic diseases, such as rickets and osteoporosis [1]. 25(OH)D3 is metabolized to the hormonally active form, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] in the kidney, whereas excess 25(OH)D3 is excreted into the urine after conversion to catabolic metabolites. We have reported that some side chain-oxidized metabolites including 24R,25dihydroxyvitamin D3 [24,25(OH)2D3] are present in urine as the glucuronidated conjugates [2]. However, quantitative analysis of the urinary vitamin D3 metabolites has not been carried out, though it might also prove helpful in the assessment of the vitamin D status. A highly sensitive technique is required for the quantitative analysis of the urinary vitamin D3 metabolites because their concentrations are predicted to be extremely low (possibly in the pg/mL range). Recently, liquid chromatography (LC) coupled with electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) has been used for the determination of serum/plasma 25(OH)D3 (normal range, 10–40 ng/mL) due to its specificity and versatility [3–5]. However, the ionization efficiency of vitamin D3 metabolites is not high in ESI, and therefore, this insufficient sensitivity might become a major problem in the analysis of the picogram-abundant urinary metabolites. The use of 4-(4′-dimethylaminophenyl)1,2,4-triazoline-3,5-dione (DAPTAD), a derivatization regent that we recently developed, will probably be able to solve this problem; the DAPTAD derivatization improved the detection limit of 25(OH)D3 to 0.25 fmol (equivalent to 0.1 pg) [6]. Matrix effects caused by endogenous components often occur during the measurement of low abundant metabolites using LC/ESI-MS/MS. The ionization efficiency is also sometimes different between two single runs even for the same
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analyte. A stable isotope-labeled analogue of the analyte is widely used as an internal standard (IS) to overcome the matrix effects and run-to-run ionization differences for the accurate quantification. However, only a limited number of stable isotope-labeled analogues are commercially available for vitamin D3 metabolites and their syntheses are not always easy. The stable isotope-coded derivatization is an alternative way to introduce a stable isotope-coded moiety to the analyte, and the resulting derivative can be used as a substitute for a stable isotope-labeled IS [7, 8]. For example, the vitamin D3 metabolites in a urine sample are derivatized with the H-coded reagent (DAPTAD), while standard vitamin D3 metabolites are separately derivatized with the isotope ( 2H)-coded reagent (d-DAPTAD) and spiked into the sample solution prior to the LC/MS/MS analysis (Fig. 1). Since the isotopic pairs of the derivatives elute at almost the same time in a single run, the matrix effects and ionization process for the DAPTAD-derivatized vitamin D3 metabolites are expected to be identical with the d-DAPTAD-derivatized vitamin D 3 metabolites. Thus, stable isotope-coded derivatization can improve the analytical performance and precision without a stable isotope-labeled IS. DAPTAD contains the dimethylamino group, and therefore, its 2H-coded analogue can be easily prepared. In this study, we developed a method for the quantitative analysis of 25(OH)D 3 and 24,25(OH) 2 D 3 in the βglucuronidase-treated urine by LC/ESI-MS/MS combined with ESI-enhancing and stable isotope-coded derivatization. The application of the method to the analysis of urine samples obtained from healthy subjects who had received a vitamin D3 supplement was also described.
25(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3, and 3-epi-25hydroxyvitamin D3 [3-epi-25(OH)D3] were purchased from Wako Pure Chemical Industries (Osaka, Japan), the Duphar B. V. Co. (Amsterdam, The Netherlands), the Funakoshi Co. (Tokyo, Japan), and the Cayman Chemical Company (Ann Arbor, MI, USA), respectively. 3-Epi-24,25dihydroxyvitamin D3 [3-epi-24,25(OH)2D3] was the same as used in a previous study [9]. The stock solutions of the vitamin D3 metabolites were prepared as 100-μg/mL solutions in ethanol, and their concentrations were confirmed by UV spectroscopy using the molar absorptivity (ε) of 18,200 at 265 nm. Subsequent dilutions were carried out with ethanol to prepare 0.40, 1.0, 2.0, 4.0, 10, and 20 ng/ mL solutions. DAPTAD was the same as used in a previous study [6]. d-DAPTAD (2H4-DAPTAD) was synthesized from 2H4-4-dimethylaminobenzoic acid [10]; this starting material was treated with diphenylphosphoryl azide to prepare the carbonyl azide, which was then converted to d-DAPTAD as previously reported [6]. The DAPTAD and d-DAPTAD solutions (in ethyl acetate) could be used for the derivatization for at least 2 months when stored at −18 °C [6]. The isotopic purity of d-DAPTAD was determined by LC/ESI-MS (selected ion monitoring) after it was reacted with 25(OH)D3. By monitoring the respective protonated molecules of the 2H4-, 2H3-, 2H2-, 2H1-, and 2 H0-forms (m/z 623.6, 622.6, 621.6, 620.6, and 619.6, respectively), the isotopic purity of d-DAPTAD was found to be greater than 99.9 % (the 2H3-, 2H2-, 2H1-, and 2H0-forms
Derivatization with DAPTAD Mix
Standard vitamin D3 metabolites
LC/ESIMS/MS
Derivatization with d-DAPTAD
Peak area ratio
Urine samples
Materials and reagents
Intensity
Fig. 1 Scheme of quantification procedure of vitamin D3 metabolites in urine based on electrospray ionization (ESI)enhancing and isotope-coded derivatization
Experimental
Time
Concentration
O
R OH
N N
N
R CHX2 N CHX2
OH
O
O
DAPTAD: X = H d-DAPTAD: X= 2H
N N
N O
HO
25(OH)D3: R=H 24,25(OH)2D3: R= OH
HO
CHX2 N CHX2
Urinary vitamin D3 metabolite analysis by LC/ESI-MS/MS
were not detected at all). β-Glucuronidase originating from E. coli (type IX-A) was obtained from Sigma-Aldrich, Japan (Tokyo); this enzyme was reported to efficiently hydrolyze the glucuronidated vitamin D3 metabolites [11]. The Oasis® HLB cartridges (60 mg; Waters Assoc., Milford, MA, USA) were successively washed with ethyl acetate (2 mL), methanol (2 mL), and water (2 mL) prior to their use. All other reagents and solvents were of analytical grade or LC/MS grade. LC/ESI-MS/MS LC/ESI-MS/MS was performed using a Waters Quattro Premier XE triple quadrupole-mass spectrometer connected to an LC-2795 chromatograph. A YMC-Pack Pro C18 RS (5 μm, 150×2.0 mm i.d.; YMC, Kyoto) 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 (8:3, v/v)] and mobile phase B [methanol–10 mM ammonium formate (9:1, v/v)] was performed; B=0 % maintained (0–5 min), 40 % linearly increased (5–10 min) and maintained (10–13 min), 100 % linearly increased (13–15 min) and maintained (15– 19 min), and 0 % maintained (19–24 min). The derivatized vitamin D3 metabolites were analyzed in the positive ion mode, and the conditions were as follows: capillary voltage, 3.3 kV; cone voltage, 35 V [25(OH)D3-DAPTAD and 25(OH)D3-d-DAPTAD] or 40 V [24,25(OH)2D3-DAPTAD and 24,25(OH)2D3-d-DAPTAD]; collision energy, 25 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 the respective compounds are as follows: 25(OH)D 3 -DAPTAD m/z 619.6 → 341.1, 25(OH)D 3 -dDAPTAD m/z 623.6→345.1, 24,25(OH)2D3-DAPTAD m/z 635.1→341.0, and 24,25(OH)2D3-d-DAPTAD m/z 639.1→ 345.0. MassLynx software (version 4.1, Waters) was used for the system control and data processing.
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The urine samples were analyzed within 7 days after collection. A urine sample (1.0 mL) was incubated with βglucuronidase (1,000 Fishman units) in 0.1 mol/L sodium acetate–acetic acid buffer (pH 5.0, 1 mL) at 37 °C for 2 h. To the reaction mixture, acetonitrile (1 mL) was added and then subjected to centrifugation (1,500×g, 10 min). The supernatant was passed through an Oasis® HLB cartridge. After washing with water (2 mL), methanol–water (7:3, v/v, 2 mL), and hexane (1 mL), the metabolites were eluted with ethyl acetate (1 mL), from which the solvents were evaporated under a N2 gas stream. The residue was subjected to derivatization with DAPTAD. The standards of 25(OH)D3 (200 pg) and 24,25(OH) 2 D 3 (1.0 ng) were derivatized with dDAPTAD, and one tenth of the derivatives were added to the DAPTAD-derivatized urine sample. The solvent was evaporated from the mixture, and the residue was dissolved in the mobile phase (60 μL), 15 μL of which was injected into the LC/ESI-MS/MS (the concentrations of d-DAPTAD derivatives in the sample were 0.83 and 4.0 pmol/mL for 25(OH)D3 and 24,25(OH)2D3, respectively). Calibration curves for 25(OH)D3 and 24,25(OH)2D3
The standard vitamin D3 metabolites and pretreated urine samples were dried and then dissolved in ethyl acetate (50 μL) containing DAPTAD or d-DAPTAD (10 μg). The mixture was kept at room temperature for 1 h, and then, ethanol (20 μL) was added to the mixture to terminate the reaction [6].
The vitamin D metabolite-free urine sample was prepared by the absorptive removal of the metabolites with charcoal; the urine (15 mL) was stirred with the charcoal (1.5 g, Norit®, Nacalai Tesque, Kyoto) for 15 h and then the charcoal was removed by filtering. Neither 25(OH)D3 nor 24,25(OH)2D3 was detected in the charcoal-treated urine by the proposed method. Although some endogenous components, such as steroids [12], were removed together with the vitamin D3 metabolites by the charcoal treatment, the charcoal-treated urine was used to construct the calibration curves. The standards of 25(OH)D3 (4.0–100 pg) and 24,25(OH)2D3 (4.0– 200 pg) were spiked in the vitamin D metabolite-free urine, and then, the resulting samples were pretreated as previously described. The pretreated urine samples were derivatized with DAPTAD. The standards of 25(OH)D 3 (200 pg) and 24,25(OH)2D3 (1.0 ng) were derivatized with d-DAPTAD, and one tenth of the derivatives were added to the DAPTAD-derivatized urine samples. The samples were subjected to LC/ESI-MS/MS. The peak area ratio [DAPTAD derivative/d-DAPTAD derivative] (y) was plotted versus the concentration of 25(OH)D3 or 24,25(OH)2D3 (pg/mL) (x) with a weighting of 1/x to construct the calibration curve.
Collection and pretreatment of the urine sample
Assay precision
Urine samples were collected from healthy male subjects between 9:00 am and 11:00 am and stored at 4 °C. The subjects understood the purpose and significance of this experiment and donated their urine after signing an agreement.
The assay precision was examined using a pooled urine sample collected from healthy male subjects. The intra- (n=5) and inter-assay (n=3) precisions were assessed by the repeated measurements of two urine samples (A and B) on 1 day and
Derivatization
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over 3 days, respectively. The precision was determined as the relative standard deviation (RSD, %). Recovery rates of vitamin D3 metabolites during solid phase extraction The recovery rates of 25(OH)D3 and 24,25(OH)2D3 during the solid phase extraction (SPE) were calculated from the peak area ratio [DAPTAD derivative/d-DAPTAD derivative] in samples I and II as described below: recovery rate=peak area ratio in sample II/peak area ratio in sample I. Sample I The standards of 25(OH)D 3 (20 pg) and 24,25(OH)2D3 (100 pg) were spiked in the vitamin D metabolite-free urine sample (1 mL), and then, the resulting sample was pretreated as previously described. The pretreated urine sample was derivatized with DAPTAD. The standards of 25(OH)D3 (200 pg) and 24,25(OH)2D3 (1.0 ng) were derivatized with d-DAPTAD, and one tenth of the derivatives were added to the DAPTAD-derivatized urine sample. Sample II The vitamin D metabolite-free urine sample (1 mL) was pretreated as previously described. After the addition of the standards of 25(OH)D3 (20 pg) and 24,25(OH)2D3 (100 pg) to this pretreated urine, the resulting sample was derivatized with DAPTAD. The standards of 25(OH)D3 (200 pg) and 24,25(OH)2D3 (1.0 ng) were derivatized with d-DAPTAD, and one tenth of the derivatives were added to the DAPTAD-derivatized urine sample. Stability of vitamin D3 metabolites in urine at 4 °C Five different urine samples were analyzed immediately after their collection and after 7 days of storage at 4 °C, and the measured values were compared. Quantitative analyses of 25(OH)D3 and 24,25(OH)2D3 in β-glucuronidase-treated urine Urine samples were obtained from healthy male subjects (n= 20, age range: 22–35 years) known not to have received vitamin D supplements. The samples were prepared as described in the “Experimental” section. The 25(OH)D3 and 24,25(OH)2D3 concentrations were corrected by the urinary creatinine, which was measured by an enzymatic method at SRL, Inc. (Tokyo). Vitamin D3 administration study The vitamin D3 supplement [1,000 IU (25 μg)/body] (DHC Corporation, Tokyo) was orally administered to eight healthy male (22–35 years old) subjects at 10:00 am once daily for 7 days (from day 1 to day 7). Their urine was collected just
S. Ogawa et al.
before the first administration on day 1 and at 10:00 am on 1 day after the last administration (day 8). The statistical analysis was performed using the Wilcoxon signed-rank test. A p value of 10. Without the use of the d-DAPTAD derivatives, the precision of the calibration curves significantly decreased. When the peak area (absolute value, y) was plotted versus the 25(OH)D3 or 24,25(OH)2D3 concentration (pg/mL) (x), the slopes varied considerably [185.3±15.7 (mean±SD, RSD 8.5 %) for 25(OH)D3 and 265.2±25.2 (RSD 9.5 %) for 24,25(OH)2D3]. Furthermore, large RSDs of the back-calculated concentrations were found at each point, especially for 25(OH)D3 (14.4–45.4 %). Thus, the use of d-DAPTAD worked well in reducing the matrix effects and run-to-run ionization differences. Table 1 Precision and accuracy of calibration curve Nominal concentration Back-calculated RSD (%) REa (%) (pg/mL) concentration (pg/mL, mean±SD, n=5) 25(OH)D3 4.0 10 20 40 100 24,25(OH)2D3 4.0 10 20 40 100 a
4.44±0.54 8.50±0.93 20.5±0.45 41.2±2.52 99.7±3.76
12.2 10.9 2.2 6.2 3.8
11.1 −15.0 2.5 0.3 −0.3
3.64±0.34 9.86±0.74 21.8±2.26 40.5±2.77 99.5±3.81
9.3 7.5 10.4 6.8 3.8
−9.0 −1.4 9.0 1.3 −0.5
(Back-calculated concentration− nominal concentration)/nominal concentration×100 (%)
Urinary vitamin D3 metabolite analysis by LC/ESI-MS/MS
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Table 2 Assay precision 24,25(OH)2D3
25(OH)D3
Urine A Intra-assay Inter-assay Urine B Intra-assay Inter-assay a
Measureda (pg/mL, mean±SD)
Precision (RSD, %)
Measureda (pg/mL, mean±SD)
Precision (RSD, %)
6.87±0.20 7.12±0.39
2.9 5.5
24.0±2.28 25.1±0.97
9.5 3.9
30.5±1.03 28.8±2.14
3.4 7.4
114±5.27 117±3.09
4.6 2.6
n=5 for intra-assay precision and n=3 for inter-assay precision
The intra-assay (n=5) RSDs did not exceed 9.5 %, and good inter-assay (n=3) RSDs (not exceeding 7.4 %) were also obtained, as shown in Table 2. These data indicated that the present method has a satisfactory reproducibility. It was possible to store the urine at 4 °C without any significant loss of the 25(OH)D3 and 24,25(OH)2D3 for at least 7 days; the average measured values after storing at 4 °C for 7 days were 93.7 and 94.3 % (n=5) of those
(a) 24,25(OH)2D3 concentration (ng/g creatinine)
25(OH)D3 concentration (ng/g creatinine)
25 20 15 10
5
100 80 60 40 20 0
0 Before
Before
After
After
(b) p < 0.05
24,25(OH)2D3 concentration (ng/g creatinine)
25(OH)D3 concentration (ng/g creatinine)
25 20 15 10 5 0
100
p < 0.05
80
After
Vitamin D3 administration study The changes in the 25(OH)D3 and 24,25(OH)2D3 levels in a urine sample after the oral administration of vitamin D3 (1,000 IU/body/day) were examined using the developed method. Vitamin D3 was administered for 7 days, and the urine was collected just before the administration on day 1 and at 1 day after the last administration (day 8). The 25(OH)D3 and 24,25(OH)2D3 concentrations of the respective subjects are shown in Fig. 4a; the concentrations of both metabolites were elevated for all the subjects by the administration. Box and whisker plots also showed that the 25(OH)D3 and 24,25(OH)2D3 levels significantly increased after the vitamin D3 administration (p
