Online Solid Phase Extraction and LC/MS/MS Detection of Thyroid Hormones in Biological Fluids Xiaoning Lu and David S. Bell Supelco, Div. of Sigma-Aldrich Bellefonte, PA 16823 USA T414067 sigma-aldrich.com/analytical
ntroduction Thyroid hormones play critical roles in the regulation of biological processes such as growth, metabolism, protein synthesis, and brain development. Specifically, thyroid hormones, 3,3,5,5 -tetraiodo-l-thyronine (thyroxine or T4) and 3,3,5-triiodo-L-thyronine (T3), are essential for development and maintenance of normal physiological functions. For a clinical laboratory, measurements of total T4 and total T3, along with estimates of free T4 (FT4) and free T3 (FT3), are important for the diagnosis and monitoring of thyroid diseases. Most clinical laboratories measure thyroid hormones using immunoassays. The immunoassay-based methods offer a relatively rapid, high patient sample throughput that lends itself to automation but are significantly compromised by problems with assay interference and are perturbed by changes in protein levels that alter the free hormone availability (1). 2
ntroduction (contd.) Liquid chromatography mass spectrometry (LC/MS) has been reported to offer superior specificity and speed over the immunoassays for determination of thyroid hormones in biological matrices such as serum and tissues. Nevertheless, the reported sample preparation procedures, typically by liquidliquid extraction followed by solid phase extraction (SPE), involve multiple time consuming steps, and are less compatible with automation (2-3). The present work exploited online SPE with LC/MS for rapid determination of T4, T3 and 3,3,5 -triiodo-l-thyronine (rt3) from biological matrices. 3
Experiment Figure 1. Chemical Structures of the Thyroids Analytes in this Study Thyroid compounds HO HO HO O O O N H 2 H COOH N H 2 H COOH N H 2 H COOH Thyroxine (T4), C 15 H 11 4 NO 4, Monoisotopic mass: 776.686 Da 3, 3', 5-Triiodothyronine (T3), C 15 H 12 3 NO 4, Monoisotopic mass: 650.790 Da 3, 3', 5'-Triiodothyronine (Reverse T3), C 15 H 12 3 NO 4, Monoisotopic mass: 650.790 Da 4
Sample Processing Procedure A 100 µl of rabbit plasma spiked with a desired amount of T4, T3 and rt3, were protein precipitated by addition of 25 µl zinc chloride and 200 µl methanol, agitated by vortex for 1 min, and then centrifuged at 9000 g x 3 min. The resulting supernatant was collected and directly injected for LC/MS analysis. Online SPE-LC/MS instrument: Shimadzu LCMS-8030 trapping column: Ascentis Express RP-Amide guard cartridge, 5 x 2.1 mm, (53514-U) or Ascentis Express C8 guard cartridge, 5 x 2.1 mm, (53509-U) HPLC column: Ascentis Express Phenyl Hexyl, 5 cm x 2.1 mm (53334-U) mobile phase: (A) water; (B) methanol, both with 0.1% acetic acid column pressure: 1800 psi (88 bar) det.: MS, ES(+), MRM mode (777.7/731.8 for T4; 651.8/605.5 for T3 or rt3). injection: 5 µl 5
Figure 2a. Configuration of the LC/MS System and Valve Positions for Online SPE-LC/MS 6
Figure 2b. A Representative Online SPE-LC/MS Program 7
Results and Discussion Column Screening for Separation of Thyroids Table 1. Ascentis Express Columns (5 cm x 2.1 mm.d., 2.7 µm) Screened in this Study Column Phase Sigma-Aldrich Part No. C18 C18 53822-U F5 pentafluorophenylpropyl 53567-U Phenyl Phenyl-Hexyl 53334-U LC-UV conditions: instrument: Agilent 1200 HPLC-DAD HPLC column: Ascentis Express Columns mobile phase: (A) water; (B) methanol, both with 0.1% acetic acid isocratic: 70% B flow rate: 0.2 ml/min temp.: 35 C det.: UV 210 nm with DAD scan column pressure: 1600-1750 psi injection: 1 µl 8
Figure 3a. Comparison of Separation of Thyroids by Different Phases All the tested phases have adequate selectivity to resolve all of the 3 thyroids. The C18 gives least retention. F5 and Phenyl offer better selectivity than C18. Phenyl-Hexyl phase tend to give better peak shapes than C18 and F5, and best selectivity. Therefore, it was selected for further method development. C18 F5 Phenyl 9
Figure 3b. Separation of Thyroids by Phenyl-Hexyl Phase under Optimized LC Conditions T3 rt3 T4 mobile phase: (A) water; (B) methanol, both with 0.1% acetic acid isocratic: 65% B 10
Online SPE-LC/MS Method As shown in Figure 2a, the online SPE-LC/MS system comprises a trapping column, a switching valve, and an LC/MS instrument. Figure 2b shows a typical online SPE-LC/MS program, which consists of three distinct steps. The valve switches between positions 1 for loading/washing (Step 1) and equilibration (Step 3), and position 2 for LC/MS analysis (Step 2), respectively. t should be noted the online SPE and LC/MS analysis use the same pumping system. n the Step 1, samples are loaded and washed with the mobile phase containing low organic (5% methanol in this case) and at higher flow rate where the salts or other interferences in the samples are washed off and directed to the waste. Subsequently (n Step 2), the samples are eluted, separated and detected by the LC/MS at optimum flow (0.25 ml/min) for both chromatographic separation and MS signals. Lastly, the system returns back to valve position 1 for re-equilibration under the sample loading/washing conditions. 11
Choice of Online SPE Trapps The choice of the right trapping column is critical for the successful online SPE-LC/MS method development as it affects both trapping efficiency (e.g. the recovery of the analytes) and the downstream LC separation. As a rule of thumb, the trapping column should be less retentive than the analytical column to avoid possible peak broadening. Two guard cartridges (traps), RP-Amide and C8, of the same dimensions, were evaluated and compared. As can be seen in Figure 3, both trapping columns led to comparable sharp peak shape (separation efficiency). However, the RP-Amide delivered slightly (15%) higher peak responses than the C8 trap under the trapping conditions with 5% methanol as the washing solvent. Further increasing methanol content to 20% in the washing solvent resulted in significant (about 50%) signal decrease with C8 traps, but almost no change in signals with RP-Amide traps (See Figures 4a and 4b). These results indicate better washing can be achieved with minimal sample loss with RP-Amide traps. 12
Figure 4. Comparison of the LC/MS Chromatograms with Online Trapping with C8 (4a) and RP-Amide (4b) Guard Cartridges (Under the Conditions with 5% Methanol as the Washing Solvent) ntensity / cps 0 100 200 300 (4a) C8 Trap T3 rt3 T4 Online SPE-LC/MS conditions as in Figure 2. 4.0 5.0 6.0 7.0 8.0 Time (min) T3 ntensity / cps 0 100 200 300 (4b) RP-Amide Trap rt3 Note the higher peaks with RP-Amide trap. T4 4.0 5.0 6.0 7.0 8.0 Time (min) 13
Figure 5. Comparison of the LC/MS Chromatograms with Online Trapping with C8 (5a) and RP-Amide (5b) Guard Cartridges (Under the Conditions with 20% Methanol as the Washing Solvent, 5c) ntensity / cps 0 100 200 300 (5a) C8 Trap Note the much lower response with C8 trap under 20% MeOH washing conditions. T3 rt3 T4 4.0 5.0 6.0 7.0 8.0 Time (min) T3 ntensity / cps 0 100 200 300 (5b) RP-Amide Trap rt3 T4 4.0 5.0 6.0 7.0 8.0 Time (min) 14
Figure 5. (contd.) (5c) 15
Conclusion An online SPE-LC/MS method was developed for the determination of thyroid hormones in biological fluids. The method exploits RP-Amide or C8 as the trapping column and Phenyl phase as the separation column, respectively. The preliminary experiments demonstrated that, under the optimized conditions, both RP-Amide and C8 effectively trapped the thyroid hormones extracted from spiked rabbit plasma sample which had been protein precipitated. ith both traps, sharp peak shapes were observed. However, the RP-Amide traps are advantageous over the commonly used C8 traps with higher signals and recoveries. Additionally, RP-Amide traps are more flexible with washing solvent as it is compatible with 100% aqueous mobile phases and can be used with up to 20% methanol with minimal sample loss. 16
References 1. Kahric-Janicic N, Soldin SJ, Soldin OP, West T, Gu J, Jonklaas J, Tandem mass spectrometry improves the accuracy of free thyroxine measurements during pregnancy. Thyroid. 2007;17(4): 303-11. 2. Susan S-C. Taia, Lorna T. Sniegoski and Michael J. Welch, Candidate Reference Method for Total Thyroxine in Human Serum Use of sotope- Dilution Liquid Chromatography-Mass Spectrometry with Electrospray onization. Clinical Chemistry, 2002; 48(4): 637-642. 3. Dongli Wang and Heather M. Stapleton, Analysis of thyroid hormones in serum by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem. 2010; 397(5): 1831 1839. Ascentis is a registered trademark of Sigma-Aldrich Co. LLC. Agilent is a registered trademark of Agilent Technologies, nc. Shimadzu is a registered trademark of Shimadzu Corporation. 17