Separation and Quantitation of Oxysterol Compounds Using LC-MS/MS
2 likes368 views
A method for the separation and quantitation of 16 oxysterols and cholesterol-related compounds using a Shimadzu LCMS-8060 system was developed, achieving low detection and quantitation limits via APCI, ESI, and DUIS sources. The optimally configured DUIS source enables analysis of all compounds in a single run with improved sensitivity for challenging analytes like zymosterol and desmosterol. Overall, the method demonstrates efficiency in detecting low picogram levels of oxysterol-related compounds, aiding research related to neurodegenerative diseases.
Separation and Quantitation of Oxysterol Compounds Using LC-MS/MS
- 1. Separation and Quantitation of Oxysterol Compounds Using LC-MS/MS Shimadzu Scientific Instruments, Columbia, MD, USA
- 2. Summary A mixture of 16 oxysterols and cholesterol related compounds were separated using a Shimadzu Nexera UHPLC and analyzed by a Shimadzu LCMS-8060 system. Detection and quantitation limits were obtained using APCI, ESI, and Dual Ion (DUIS) sources.
- 3. Background There is high demand for oxysterol quantitation due to their correlation with neurodegenerative diseases. The ratios of various oxysterols in biological fluids are used by researchers to study disease states. An LCMS oxysterol quantitation method was developed using a Shimadzu LCMS-8060. Detection and quantitation limits were determined using multiple reaction monitoring (MRM) mode for each analyte.
- 4. Method Oxysterol standards were obtained in methanol and diluted with 10:90 H2O:MeOH. Standards included the compounds indicated in Table 1. An MRM method was determined and optimized using LabSolutions 5.82 on a Shimadzu triple quadrupole mass spectrometer (LCMS-8060). A Shim-pack XR-ODS III column was used for the separation. Injection volume was 2 µL with autosampler temperature being set at 10°C. Flow rate was initially at 0.4 mL/min and adjusted to 0.7 mL/min toward the end of the run to speed the elution of highly retained compounds. Compound name Abbreviation 24 (S)-hydroxycholesterol 24HC (D7)22-hydroscycholesterol 22HC(d7) 25-hydroxycholesterol 25HC 27-hydroxycholesterol 27HC (D7)7α-hydroxycholesterol 7αHC(d7) 7α-hydroxycholesterol 7αHC 7β-hydroxycholesterol 7βHC 7-Ketocholesterol 7KC (D7) 7-Ketocholesterol 7KC(d7) 7α-hydroxycholestenone 7αHCn (D3) Vitamin D3 VitD3(d3) Zymosterol Zymo Desmosterol Desmo 7α,27 dihydroxycholestenone 7α,27diHC,3one Cholesterol CH 7dehydrocholesterol 7DHC Table 1. Oxysterol Related Compounds
- 5. Experimental MRM transitions were developed and optimized on the triple quadrupole mass spectrometer using DUIS, APCI, and ESI sources. In cases where two precursor ions showed a strong signal, MRM development and optimization were done on both precursor ions. This may provide an option for better sensitivity in different matrices for future applications. The DUIS method established nebulizing, heating, and drying gas flows at 3, 17, and 3 L/min. Interface, desolvation line, and heat block temperatures were set at 300°C, 250°C, and 400°C, respectively. With the APCI source, temperatures for the interface, desolvation line, and heat block were set at 350°C, 200°C, and 200°C, respectively. Drying gas flow rate was 5 L/min. For ESI, nebulizing, heating, and drying gas flows were set at 3, 10, and 10 L/min. Interface, desolvation line, and heat block temperatures were 300°C, 250°C, and 400°C.
- 6. Experimental Compound Name Retention time (min) DUIS APCI ESI Target MRM Reference MRM Target MRM Reference MRM Target MRM Reference MRM 24HC 4.0 385.45>367.30 385.45>324.30 367.35>91.05 367.35>104.85 385.10>367.25 385.10>109.00 24HC 4.0 367.30>281.10 367.30>104.90 385.40>367.30 385.40>104.90 367.45>95.15 367.45>105.25 22HC(d7) 4.2 374.40>91.10 374.40>255.25 374.40>104.80 374.40>133.15 374.20>104.90 374.20>132.95 25HC 4.1 385.30>367.40 385.30>324.25 367.35>95.00 367.35>135.10 367.35>105.15 367.35>147.15 25HC 4.1 367.35>81.15 367.35>105.10 385.35>367.30 385.35>133.10 385.20>367.30 385.20>324.45 27HC 4.5 385.05>67.15 385.05>93.10 385.10>135.10 385.10>149.10 385.15>95.05 385.15>324.35 7αHC(d7) 7.6 374.35>159.15 374.35>91.15 374.40>145.30 374.40>159.25 374.20>158.95 374.20>144.80 7αHC 7.7 367.35>117.10 367.35>66.90 367.45>144.95 367.45>95.00 367.15>145.40 367.15>159.35 7αHC 7.7 385.30>367.40 385.30>367.40 385.10>367.25 385.10>159.10 385.45>367.35 385.45>159.05 7βHC 7.9 385.30>367.30 385.30>324.35 367.15>159.10 367.15>145.30 367.10>95.15 367.10>158.95 7βHC 7.9 367.35>81.10 367.35>104.85 385.10>367.30 385.10>159.00 385.10>367.15 385.10>158.95 7KC 8.3 401.15>95.10 401.15>383.30 401.10>80.95 401.10>383.35 401.45>95.30 401.45>383.30 7KC(d7) 8.1 408.30>390.15 408.30>95.15 408.40>81.15 408.40>95.25 408.40>390.25 408.40>95.05 7αHCn 6.5 401.25>383.30 401.25>97.10 401.35>383.45 401.35>97.10 401.10>382.95 401.10>96.90 VitD3(d3) 3.0 386.25>232.20 386.25>368.45 386.40>368.35 386.40>92.90 386.40>368.30 386.40>95.20 Zymo 12.8 367.30>95.00 367.30>80.90 367.15>95.20 367.15>109.20 367.15>95.10 367.15>80.95 Desmo 13.3 367.25>95.05 367.25>95.05 367.35>135.25 367.35>104.90 367.15>95.00 367.15>104.95 7α,27diHC,3one 2.2 417.25>399.10 417.25>381.30 417.30>399.25 417.30>381.45 417.35>399.30 417.35>97.25 CH 17.4 369.30>161.10 369.30>94.90 369.40>95.20 369.40>146.95 369.40>147.25 369.40>135.25 7DHC 14.5 367.25>95.05 367.25>158.90 367.30>145.25 367.30>159.25 367.15>145.10 367.15>159.20 Table 2. MRM transitions of oxysterol related compounds Multiple MRM transitions were determined from each precursor ion, the two most intense are shown.
- 7. Results Chromatographic separation was accomplished using a Shimadzu Nexera UHPLC system with a Shim-pack XR-ODS III column. Challenging separations of 24HC and 25HC as well as 7αHC and 7βHC were achieved to allow individual quantitation of these analytes. Maximum operating pressure for a typical run was about 11,000 psi. Figure 1. Chromatogram of the 16 oxysterol related compounds. 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min 0 2500000 5000000 7a,27diHC,3one VitD3(d3) 7aHCn 7βHC 7aHC 7aHC(d7) 27HC24HC CH Intensity
- 8. Results 4.0 5.0 6.0 7.0 8.0 min 24HC 27HC 7βHC 7aHC Figure 2. Chromatogram showing separation of key components.
- 9. Detection limits determined using ESI, APCI, and DUIS sources are shown below. Limits of detection (LOD) and limits of quantitation (LOQ) were determined by a signal to noise ratio of 3:1 and 10:1, respectively. Results LOD LOQ LOD LOQ LOD LOQ 24 (S)-hydroxycholesterol 20 30 30 50 20 50 (D7) 22-hydroscycholesterol 10 20 30 50 10 20 25-hydroxycholesterol 20 30 50 100 20 30 27-hydroxycholesterol 20 50 50 50 20 100 (D7) 7a-hydroxycholesterol 20 20 10 10 10 10 7a-hydroxycholesterol 4 10 10 10 10 20 7b-hydroxycholesterol 10 10 10 30 10 20 7-Ketocholesterol 1 4 4 10 1 4 (D7) 7-Ketocholesterol 0.5 2 2 4 0.5 2 7a-hydroxycholestenone 0.5 4 4 4 1 4 (D3) Vitamin D3 30 300 50 50 20 30 Zymosterol 100 >2000 30 300 300 300 Desmosterol 300 >2000 30 50 100 300 7a,27 dihydroxycholestenone 0.25 1 2 4 0.5 2 Cholesterol 100 1000 100 500 100 500 7dehydrocholesterol 100 300 100 300 100 500 ESI APCI DUIS Table 3. LODs and LOQs determined by LCMS-8060 with ESI, APCI, and DUIS sources. Values shown are picograms on-column. Green highlighting indicates lower or lowest limits. Yellow highlighting indicates those limits which are somewhat higher than the lowest limits. Red highlighting indicates the highest limits.
- 10. Results Although ESI was able to obtain low detection limits for most oxysterol related compounds, it had difficulty reaching practical quantitation limits for zymosterol and desmosterol, even in neat standards. APCI allowed better quantitation for these two compounds; however, the LODs and LOQs for many other compounds were sacrificed. DUIS offers the benefit of both APCI and ESI by allowing quantitation of zymosterol and desmosterol without significantly sacrificing the LODs of other compounds. Therefore, DUIS is the optimal source for analyzing this oxysterol related mixture. The dual ion DUIS source is able to quantify all sixteen analytes in a single run instead of two runs using ESI and APCI sources separately. In the case of (D3) Vitamin D3, detection and quantitation was even lower using DUIS vs. APCI and ESI. This is likely the result of probe position optimization, which is possible with DUIS (and ESI) but not with APCI.
- 11. Results MRM transitions for sixteen oxysterol related compounds were identified and optimized using the LCMS-8060. A UHPLC column was used to obtain separation. 24HC, 25HC, 7αHC, and 7βHC were chromatographically separated in a manner sufficient for individual quantitation. The total run time for this method was 21 minutes. Limits of detection and limits of quantitation for oxysterol related compounds were determined using APCI, ESI, and DUIS sources. Although LOD and LOQ vary per compound, the LCMS-8060 was able to reach detection limits in the low picogram (mass on-column) range for oxysterol related compounds. DUIS was shown to be the optimal source for this oxysterol related compound mixture analysis. It demonstrates the advantages of both ESI and APCI and allows all sixteen compounds to be analyzed in a single run.
- 12. Conclusion A fast and sensitive method using a triple quadrupole mass spectrometer was developed to assist future applications for oxysterol related compound quantitation.
- 13. Need More Info? Thank you for viewing this presentation. Should you have any questions or require additional information about our research, products, or services, please visit our Web site at: www.ssi.shimadzu.com Follow us on Twitter @shimadzussi