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II.11. A NALYSIS OF VITAMIN D METABOLITES
II.11.2. C HROMATOGRAPHIC ANALYSES OF VITAMIN D METABOLITES
SPE eluent: 2 % (v/v) formic acid
in methanol
II.11.2. C
HROMATOGRAPHIC ANALYSES OF VITAMIND
METABOLITESIn the following, the finally optimized and established methods for the separation and detection of vitamin D metabolites in prepared samples via HPLC coupled to UV detection (HPLC‐UV), HPLC coupled to scintillation detection (radioactive HPLC), and HPLC coupled to tandem mass spectrometry (LC‐MS/MS) are described.
AN A L Y S IS O F V I T A M IN D3 ME T A B O L IT E S V I A UV DE TE C T I O N
In order to develop a basic HPLC method for the separation of different vitamin D3 metabolites (vitamin D3, 25‐(OH)‐D3, 1α‐(OH)‐D3, 24,25‐(OH)2‐D3, 1α,25‐(OH)2‐D3) a Perkin Elmer Series 200 HPLC system comprising of a Series 200 Autosampler with 50 μl sample loop, two Series 200 Micro Pumps, a Series 200 Mixer, a Series 200 Vacuum Degasser, and a Series UV/Vis Detector was used. 30‐50 μl of sample, which was reconstituted with methanol and diluted with MilliQ‐H2O (1:1), were injected. The metabolites were separated on a Kinetex 2.6 μm C18 100 Å 50 mm x 4.6 mm (Phenomenex) reversed phase column with SecurityGuard Standard C18 4 mm x 2 mm (Phenomenex) cartridge using a gradient of mobile phase A (5 mM ammonium acetate, 0.1 % formic acid, and 80 % methanol in MilliQ‐H2O) and mobile phase B (5 mM ammonium acetate and 0.1 % formic acid in methanol) at a constant flow of 1 ml/min. The total HPLC run time was 23 min and covered a column equilibration step (step 0; 5 min) prior to the sample injection as well as the gradient steps necessary for the separation of vitamin D3 metabolites (step 1‐3; 18 min). Details of the HPLC program are listed in Table II‐27. The elution of the vitamin D3 metabolites was monitored at 265 nm with a sampling rate of 50 pts/s. The HPLC runs were controlled and analyzed by the TotalChrom Workstation Version 6.3.2 (Perkin Elmer) software.
Table II‐27: HPLC‐UV method for the separation of vitamin D3 metabolites.
Vitamin D3 metabolites were separated on a Kinetex 2.6 μm C18 100 Å 50 mm x 4.6 mm column with SecurityGuard Standard C18 4 mm x 2 mm cartridge using a Perkin Elmer Series
autosampler wash solution: 100 % methanol
AN A L Y S IS O F 3H‐L A B E L E D V IT A M I N D3 ME T A B OL I T E S
Since UV‐detection of vitamin D3 metabolites possesses only limited sensitivity, a method analyzing commercially available 3H‐labeled vitamin D3 metabolites (1α,25‐(OH)2‐D3 and 25‐(OH)‐D3,) or possible cellular derivatives (e.g., 24,25‐(OH)2‐D3) was also generated.
As with 3H‐labeled steroids, 3H‐labeled vitamin D3 metabolites were analyzed on a Beckman Coulter HPLC system, comprising of a LC‐502E autosampler with 20 μl sample loop and a System Gold 125 Solvent Module as binary pump, but coupled to an in‐line connected System Gold 166 UV detector (Beckman Coulter) and Berthold LB 506 D Radioactivity Monitor. This system was controlled by the 32 Karat software Version 3.0 (Beckman Coulter).
20 μl of sample in 2 % formic acid in methanol were injected and the metabolites were separated on a Kinetex 2.6 μm C18 100 Å 50 mm x 4.6 mm (Phenomenex) reversed phase column with SecurityGuard Standard C18 4 mm x 2 mm (Phenomenex) cartridge using a gradient of mobile phase A (0.05 % formic acid in MilliQ‐H2O) and mobile phase B (5 mM ammonium acetate and 0.1 % formic acid in methanol) at a constant flow of 1 ml/min. The total run time was 22 min. Details of the HPLC program are listed in Table II‐28. For the detection of 3H‐labeled metabolites via the scintillation detector, the mobile phase was mixed with the Ready Flow III (Beckman Coulter) scintillation cocktail, supplied by a MCP‐Z Standard gear pump (Ismatec) with a constant flow of 1 ml/min before entering the detection chamber.
For the determination of the retention times of vitamin D3 metabolites not available as
3H‐labeled compounds, 3H‐labeled metabolites were spiked with unlabeled metabolites and also analyzed via UV absorption at 265 nm using the in‐line System Gold 166 UV detector (Beckman Coulter).
Table II‐28: “Radioactive” HPLC method for the separation of vitamin D3 metabolites.
total time
[min] module function value duration
0.0 Pump % B 70
0.0 Pump Relay on 1 0.0
0.0 Pump Relay on 2 0.0
0.0 Det166‐2 Autozero
1.0 Pump % B 80 0.1
7.0 Pump % B 90 0.1
14.0 Pump % B 98 0.1
18.0 Pump % B 70 0.1
22.0 Pump Relay on 1 0.0
22.0 Pump Relay on 2 0.0
22.0 Det166‐2 Stop Data
Vitamin D3 metabolites were separated on a Kinetex 2.6 μm C18 100 Å 50 mm x 4.6 mm column with SecurityGuard Standard C18 4 mm x 2 mm cartridge using a Beckman Coulter Gold HPLC system with in‐line 166 UV detector and Berthold scintillation detector.
mobile phase A: 0.05 % (v/v) formic acid
in MilliQ‐H2O
autosampler wash solution: 100 % methanol
Analysis of vitamin D metabolites and itraconazole
via LC‐MS/MS
Within this doctoral thesis, a liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS) method was finally developed and applied as a potent tool for the analysis of vitamin D metabolites (vitamin D3, 25‐(OH)‐D3, 1α‐(OH)‐D3, 24,25‐(OH)2‐D3, 1α,25‐(OH)2‐D3, vitamin D2, 25‐(OH)‐D2, and 1α,25‐(OH)2‐D2) as well as itraconazole in cell culture, mouse tumor, or human plasma samples with high sensitivity and specificity.
Using this technique, compounds are first separated on a HPLC column and then directly passed into a mass spectrometer that allows for the detection of specific mother‐daughter‐ion pairs (multiple reaction monitorings = MRMs). For MRM detection, molecules entering the mass spectrometer are initially ionized in the source and then directed to a quadrupole (Q1) that filters the molecules according to their mass‐to‐charge ratio (m/z). Selected mother ions become fragmented in the collision cell (Q2) and the resulting daughter ions are once more filtered according to their m/z in another quadrupole (Q3). Finally, the daughter ions are conducted to a pulse‐counting channel electron multiplier (CEM) detector and the detected signals are processed by the software.
The LC‐MS/MS method used a HPLC system consisting of a HTC‐xt autosampler (CTC‐PAL) with a 20 μl sample loop, a 1260 Infinity degasser (Agilent), a 1260 Infinity binary pump model G1312B (Agilent), and a 1260 TCC column oven model G1316A (Agilent) for the LC separation of vitamin D metabolites on a Kinetex 2.6 μm C18 column, whereas a QTrap 5500 mass spectrometer (AB Sciex) was used for MS/MS analysis. The complete LC‐MS/MS set‐up [Figure II‐6] was controlled by the Analyst 1.6 software (AB Sciex).
Figure II‐6: Schematic illustration of the LC‐MS/MS set‐up.
The LC‐MS/MS set‐up used for the analysis of vitamin D metabolites included a HTC‐xt autosampler with a 20 μl sample loop, a 1260 Infinity degasser, a 1260 Infinity binary pump, and a 1260 TCC column oven as well as a QTrap 5500 mass spectrometer.
For the analysis of vitamin D metabolites via LC‐MS/MS, 25 μl of sample, which was reconstituted in methanol and diluted with MilliQ‐H2O (2:1) [II.11.1.4] were injected and separated on a Kinetex 2.6 μm C18 100 Å 50 mm x 2.1 mm (Phenomenex) reversed phase column with SecurityGuard Standard C18 4 mm x 2 mm (Phenomenex) cartridge using a gradient of mobile phase A (0.05 % formic acid and 0.15 mM lithium acetate in MilliQ‐H2O) and mobile phase B (0.05 % formic acid in methanol) at a constant flow of 0.3 ml/min. The total HPLC run time for the separation of vitamin D metabolites was 13 min. The HPLC method (described in detail in Table II‐29) included a column equilibration step (auto‐equilibration step: 5 min) which was followed by the sample injection and the gradient steps for the chromatographic separation of metabolites (step 1‐4: 8 min). The column oven temperature was constantly set to 30 °C.
To reduce the carryover of hydrophobic vitamin D metabolites, after each sample a 5 min washing step was performed. For this, 25 μl of 100 % methanol were injected and the LC gradient method listed in Table II‐29 was run.
MS/MS analysis was performed via ESI in positive‐ion mode and MRM detection using the optimized parameter settings listed in Table II‐30. These parameters resulted from infusion (flow: 7 μl/min) and FIA optimization analyses with single compounds in appropriate dilutions (< 1 μg/ml) in either 100 % methanol or 0.5‐1 mM lithium acetate in methanol using the “Automatic Optimization tool” of the Analyst 1.6 software (see III.3.3.1). To increase the number of reading points per peak, at least when more than ten vitamin D MRMs were simultaneously monitored, scheduled MRM detection was applied [Table II‐30].
The results from LC‐MS/MS runs were visualized and analyzed by the Analyst 1.6 software (AB Sciex). Thereby, the three times 13C isotope labeled 25‐(OH)‐D2 [25,26,27‐13C3] was used as internal standard (IS) for the normalization of signals and quantification (either relative or quantitative via calibration curves using a linear fit without weighting) of vitamin D metabolites in cell culture samples (see III.3.5.1).
equilibration 5.0 300 20.0 80.0
0 0.0 300 20.0 80.0
equilibration 0 0 0 0
0 0.0 300 0.0 100.0
1 4.0 300 0.0 100.0
2 4.5 300 20.0 80.0
3 5.0 300 20.0 80.0
Vitamin D metabolites were separated using a Kinetex 2.6 μm C18 100 Å 50 mm x 2.1 mm column with SecurityGuard Standard C18 4 mm x 2 mm cartridge and an Agilent Infinity 1260 HPLC system coupled to a QTrap 5500 mass spectrometer [Figure II‐6].
mobile phase B: 0.1 % (v/v) formic acid
in methanol
autosampler wash solution ‐II: 50 % (v/v) methanol
in MilliQ‐H2O
Please note that, beside the final mobile phase A (0.15 mM lithium acetate and 0.05 % formic acid in MilliQ‐H2O) and the MRMs listed in Table II‐30, different other MRMs as well as an initial mobile phase A without lithium ions (0.05 % formic acid in MilliQ‐H2O) were used within the method development process. The ionization parameter settings of MRMs not used for the final method but mentioned in the results are listed in the appendix [VI.8].
Table II‐30: MRMs and ionization parameter settings for the analysis of vitamin D metabolites (and itraconazole) on a QTrap 5500 via LC‐MS/MS.
compound adduct Q1 [m/z] itraconazole H+ 705.203 392.200 ‐ 41 10 47 16 H+ 705.203 256.000 ‐ 41 10 51 32 source = TurboSpray CUR = 40 CAD = Medium IS = 5500 TEM = 600 GS1 = 40 GS2 = 50 The MRM detection window for scheduled MRMs was set to 120 sec. If continuous MRM monitoring was performed, the time was set to 150 msec.
CAD, collision gas; CE, collision energy; CUR, curtain gas; CXP, collision cell exit potential; DP, declustering potential; EP, entrance potential; GS1, ion source gas 1; GS2, ion source gas 2; IS, ion spray voltage; source, ion source; TEM, temperature; tscheduled, scheduled time.