Analysis of 25-Hydroxyvitamin D In Serum Using Automated On Line Sample Preparation Technique With High Resolution Orbitrap Mass Spectrometer Matt Berube, Joe Dibussolo, Catherine Lafontaine, Yang Shi, and Francois Espourteille Thermo Fisher Scientific, Franklin, MA
Overview Purpose: A high throughput method for the quantitation of vitamin D using online sample preparation and high resolution, accurate mass (HRAM) quantitation with an Exactive Plus orbitrap mass spectrometer for clinical research use. Methods: Solvent precipitated serum is injected onto an extraction column under turbulent flow conditions. A 2 microliter loop containing organic solvent is used to transfer the Vitamin D to a solid core HPLC column for separation and analysis using an Exactive Plus Orbitrap MS. Results: The method provided reliable quantitation of 25-hydroxyvitamin D 2 and 25- hydroxyvitamin D 3 levels in serum with a lower limit of quantitation of 2 ng/ml. Introduction Blood levels of 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 are commonly tested by clinical researchers. In the last decade, liquid chromatography coupled with triple quadrupole mass spectrometry (LC-MS/MS) has become a popular technique for vitamin D testing. Due to their higher resolving power over triple stage quadrupole mass spectrometers, high-resolution benchtop orbitrap mass spectrometers are better able to resolve analytes from sample matrix. In addition, the ease of initial method set up and daily use provide an advantage over triple quadrupole mass spectrometers for clinical research. A method has been created that allows the precipitated serum to be injected onto an HPLC system with minimal sample preparation and analyzed by an Exactive Plus benchtop Orbitrap mass spectrometer. Total method time is 7.75 minutes on a Transcend TLX-1 system. Throughput can be increased to a sample every 3.7 minutes by using a Transcend TLX-2 multiplexed UHPLC system or 1.9 minutes for a Transcend TLX-4 system. FIGURE 1. Liquid Chromatography M FIGURE 2. Plumbing Diagram for onli Methods Standards Standard solutions of 25-hydroxyvitamin D 2, 25-hydroxyvitamin D 3, and deuterated 25- hydroxyvitamin D 3 internal standard were obtained from Cerilliant, Inc. Six calibrators at 2, 5 1, 25, 5 and1ng/ml and three QCs at 5ng/mL, 4 ng/ml and 8 ng/ml were prepared by fortifying bovine serum albumin diluent with 2 ng/ml 25- hydryoxyvitamin D 2 and D 3 standard mix. Precipitating reagent was prepared by adding deuterated D 6-25-hydroxyvitamin D 3 to acetonitrile for a final concentration of 75 ng/ml. In addition, pooled patient serum samples were crashed 2 to 1 with acetonitrile and spiked with analytes for a final concentration of 2 ng/ml for 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3, and 5ng/mL of D 6 25-hydroxyvitamin D 3 internal standard. Sample Preparation 1. Aliquot 1 µl of calibrators and controls into centrifuge tubes. 2. Add 2 µl of precipitating reagent containing internal standard to each tube 3. Vortex each tube for 3 seconds. 3. Centrifuge tubes at 5, RCF for 1 minutes. 4. Aliquot supernatants into autosampler vials for analysis. The supernatant should contain internal standard at a concentration of 5 ng/ml. Liquid Chromatography Transcend TLX-2 system in focus mode configuration (Figure 2). Extraction column: Thermo Scientific TurboFlow TM XL C-18P.5x 5 mm Separation column: Thermo Scientific Accucore TM C18 3 x 5 mm 2.6uM Injection Volume: 5µL Mobile Phases: Loading A, Eluting A: Water.1% Formic Acid Loading B, Eluting B: Acetonitrile.1% Formic Acid Loading C: 4% Acetonitrile, 4% Isopropanol, 2% Acetone Total method time: 7.75 minutes Cycle time when multiplexed: 3.7 minutes Mass Spectrometry The Thermo Scientific Exactive Plus be with an APCI source in positive ionizati 35 to 425 m/z, using a lock mass of 3 was added in order to maintain mass a Mass Spectrometer Parameters: Scan Mode: Full Scan Range: m/z 35 425 Fragmentation: None Resolution: 7, Polarity: Positive Microscans: 1 AGC Target: 3e6 Maximum Inject Time: 2 Data Analysis Calibration curves and QCs were run i addition, 8 pooled serum sample rep hydroxyvitamin D 2 and 25-hydroxyvitam D 3 internal standard were injected in or Scientific Xcalibur software was use 2 Analysis of 25-Hydroxyvitamin D In Serum Using Automated On Line Sample Preparation Technique With High Resolution Orbitrap Mass Spectrometer
D using online uantitation with an. column under solvent is used to and analysis using itamin D 2 and 25- of 2 ng/ml. e commonly tested coupled with triple r technique for age quadrupole trometers are better of initial method set s spectrometers for pitated serum to be d analyzed by an d time is 7.75 sed to a sample system or 1.9 FIGURE 1. Liquid Chromatography Method FIGURE 2. Plumbing Diagram for online TurboFlow Extraction Results Summary The lower limit of quantitation (LLOQ) w in BSA as indicated in Figure 3. Limits o triplicate injections of the standard solut and the coefficient of variation (CV) valu ng/ml for both 25-hydroxyvitamin D 2 an improvement over the LLOQ of 1 ng/m Orbitrap MS. The correlation coefficien regression analysis of the standard curv (Figure 6). A relative standard deviation serum fortified with analytes at 2 ng/m for a total internal standard concentratio injections were less than 1 percent for performed using a neat standard of 2n hydroxyvitamin D 3 with 5 ng/ml D 6-25- ten times on the TurboFlow column and analytical column only, and area counts 97 percent and 99 percent for 25-hydro respectively. Consistent results were ac of 25-Hydroxyvitamin D 2 and 25-hydrox hydroxyvitamin D 3 up to about 6 injec FIGURE 3. Chromatograms at LLOQ of RT:. - 3.6 SM: 5G 1 RT: 1 9 8 7 6 5 4 3, and deuterated 25- Inc. ng/ml, 4 ng/ml and t with 2 ng/ml 25- s prepared by adding entration of 75 to 1 with acetonitrile 5-hydroxyvitamin D 2 3 internal standard. RT: 1.6 2 1..2.4.6.8 1. 1.2 1.4 1.6 RT:. - 3.6 SM: 5G 1 9 8 7 6 5 4 3 RT: 1.63 2 1..2.4.6.8 1. 1.2 1.4 1.6 RT:. - 3.6 SM: 5G 1 bes. dard to each tube e supernatant L. ). 5 mm 2.6uM Mass Spectrometry The Thermo Scientific Exactive Plus benchtop orbitrap mass spectrometer was used with an APCI source in positive ionization mode. Full scan data was collected from 35 to 425 m/z, using a lock mass of 391.28429 (Di iso-octyl Phthalate). Lock mass was added in order to maintain mass accuracy during the batch runs. Mass Spectrometer Parameters: Scan Mode: Full Ion Source: APCI Scan Range: m/z 35 425 Sheath Gas Flow Rate: 3 units Fragmentation: None Aux Gas Flow Rate: 5 units Resolution: 7, Sweep Gas Flow Rate: 1 unit Polarity: Positive Discharge Current: 3.5 µa Microscans: 1 Capillary Temperature: 25 C AGC Target: 3e6 S-Lens RF Level: 6 Maximum Inject Time: 2 Vaporizer Temp: 5 C Data Analysis Calibration curves and QCs were run in triplicate each day across four days. In addition, 8 pooled serum sample replicates containing 2 ng/ml 25- hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 and 5ng/mL of D 6-25-hydroxyvitamin D 3 internal standard were injected in order to test robustness of the method. Thermo Scientific Xcalibur software was used to collect data and analyze the results. 9 8 7 6 5 4 3 2 1..2.4.6.8 1. 1.2 1.4 1.6 FIGURE 4. Chromatograms of 4 ng/m RT: 1.22-2.38 1 9 8 7 6 5 4 3 2 1 SM: 5G RT: 1.67 25-Hyd 1.25 1.3 1.35 1.4 1.45 1.5 1.55 1.6 1.65 1.7 1.75 RT: 1.64 1 25-Hydroxyvita 9 8 7 6 5 4 3 2 1 1.73 1.24 1.27 1.31 1.38 1.41 1.45 1.49 1.51 1.53 1.57 1.25 1.3 1.35 1.4 1.45 1.5 1.55 1.6 1.65 1.7 1.7 RT: 1.63 1 d6 25-Hydroxyvita 9 8 7 6 5 4 3 2 1 1.25 1.3 1.35 1.4 1.45 1.5 1.55 1.6 1.65 1.7 1.75 Thermo Scientific Poster Note PN6368_E 6/12S 3
Results Summary The lower limit of quantitation (LLOQ) was determined to be 2 ng/ml for both analytes in BSA as indicated in Figure 3. Limits of quantitation (LOQs) were estimated from the triplicate injections of the standard solutions. The signal to noise was greater than 1 and the coefficient of variation (CV) values were less than 5 percent at the LLOQ of 2 ng/ml for both 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3. This is an improvement over the LLOQ of 1 ng/ml that was previously achieved on the Exactive Orbitrap MS. The correlation coefficients obtained using 1/X weighted linear regression analysis of the standard curves were greater than.99 for both analytes (Figure 6). A relative standard deviation (%RSD) test was performed in pooled human serum fortified with analytes at 2 ng/ml and crashed with internal standard solution for a total internal standard concentration of 5 ng/ml. The RSDs of ten replicate injections were less than 1 percent for both analytes. A recovery study was also performed using a neat standard of 2ng/mL 25-hydroxyvitamin D 2 and 25- hydroxyvitamin D 3 with 5 ng/ml D 6-25-hydroxyvitamin D 3. The standard was injected ten times on the TurboFlow column and analytical column, and ten times on the analytical column only, and area counts were compared. The relative recoveries were 97 percent and 99 percent for 25-hydroxyvitamin D 2 and 25-Hydroxyvitamin D 3, respectively. Consistent results were achieved with pooled serum containing 2 ng/ml of 25-Hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 and 5 ng/ml D 6 25- hydroxyvitamin D 3 up to about 6 injections as indicated in Figure 7. FIGURE 3. Chromatograms at LLOQ of 2 ng/ml with 5 ng/ml internal standard RT:. - 3.6 SM: 5G 1 9 8 7 6 5 4 3 2 1 RT: 1.67 NL: 1.1E5 25-Hydroxyvitamin D2 395.32965-395.3323 [35.-425.] MS FIGURE 5. QC Replicate Injections. each day over four days and results c 1. 9. 8. 7. 6. 5. 4. 3. 2. 1...5 1 1.5 1. 9. 8. 7. 6. 5. 4. 3. 2. 1...5 1 1.5 FIGURE 6. Calibration Curves Vit Vit..2.4.6.8 1. 1.2 1.4 1.6 1.8 2. 2.2 2.4 2.6 2.8 3. 3.2 3.4 3.6 RT:. - 3.6 SM: 5G 1 9 8 7 6 5 4 3 RT: 1.64 NL: 1.73E5 25-Hydroxyvitamin D3 383.32969-383.33199 [35.-425.] MS.4 25-OH Y = -.81351+.3 ctrometer was used as collected from thalate). Lock mass uns. 2 1..2.4.6.8 1. 1.2 1.4 1.6 1.8 2. 2.2 2.4 2.6 2.8 3. 3.2 3.4 3.6 RT:. - 3.6 SM: 5G RT: 1.63 NL: 8.39E6 1 d6 25-Hydroxyvitamin D3 9 389.36733-389.36967 8 [35.-425.] MS 7 6 5 4 3 2 1..2.4.6.8 1. 1.2 1.4 1.6 1.8 2. 2.2 2.4 2.6 2.8 3. 3.2 3.4 3.6 FIGURE 4. Chromatograms of 4 ng/ml QC injection.3.2.1. 2 4 : 3 units units : 1 unit µa 25 C s four days. In L 25-25-hydroxyvitamin he method. Thermo yze the results. RT: 1.67 NL: 1.43E6 1 25-Hydroxyvitamin D2 9 395.32886-395.33282 8 7 [35.-425.] MS 6 5 4 3 2 1 1.25 1.3 1.35 1.4 1.45 1.5 1.55 1.6 1.65 1.7 1.75 1.8 1.85 1.9 1.95 2. 2.5 2.1 2.15 2.2 2.25 2.3 2.35 RT: 1.64 NL: 2.13E6 1 25-Hydroxyvitamin D3 9 383.32892-383.33276 F: FTMS 8 + p APCI corona Full 7 l ock ms [35.-425.] MS 6 QC2 5 4 3 2 1 1.73 1.8 1.24 1.27 1.31 1.38 1.41 1.45 1.49 1.51 1.53 1.57 1.87 1.91 1.97 1.99 2.4 2.8 2.13 2.15 2.19 2.25 2.28 2.3 1.25 1.3 1.35 1.4 1.45 1.5 1.55 1.6 1.65 1.7 1.75 1.8 1.85 1.9 1.95 2. 2.5 2.1 2.15 2.2 2.25 2.3 2.35 RT: 1.63 NL: 7.96E6 1 d6 25-Hydroxyvitamin D3 9 389.36655-389.3745 8 7 [35.-425.] MS 6 5 4 3 2 1 1.25 1.3 1.35 1.4 1.45 1.5 1.55 1.6 1.65 1.7 1.75 1.8 1.85 1.9 1.95 2. 2.5 2.1 2.15 2.2 2.25 2.3 2.35.6.5.4.3.2.1 25-OH Y =.183722+.59. 2 4 4 Analysis of 25-Hydroxyvitamin D In Serum Using Automated On Line Sample Preparation Technique With High Resolution Orbitrap Mass Spectrometer
FIGURE 5. QC Replicate Injections. Three replicates of each QC level were run each day over four days and results compared. FIGURE 7. Serum injection replicat variability increased. Vitamin D 2 ml for both analytes estimated from the as greater than 1 nt at the LLOQ of 2 his is an eved on the Exactive hted linear for both analytes ed in pooled human l standard solution of ten replicate study was also 2 and 25- andard was injected times on the ive recoveries were xyvitamin D 3, containing 2 ng/ml D 6 25- e 7. nternal standard NL: 1.1E5 395.32965-395.3323 1. 9. 8. 7. 6. 5. 4. 3. 2. 1...5 1 1.5 2 2.5 3 3.5 4 4.5 Day Vitamin D 3 1. 9. 8. 7. 6. 5. 4. 3. 2. 1...5 1 1.5 2 2.5 3 3.5 4 4.5 Day Area 2 18 16 14 12 1 8 6 4 2 2 [35.-425.] MS 3. 3.2 3.4 3.6 NL: 1.73E5 383.32969-383.33199 [35.-425.] MS FIGURE 6. Calibration Curves.4 25-OH_Vitamin_D2 Y = -.81351+.392862*X R^2 =.994 W: 1/X Variability increased during replicate injections. The system ran serum inj blanks or washing in between injectio mass spectrometer source becoming cleaning. Future experiments will se variability occurs, possibly by increas occurs during the HPLC method, as w during the sample run. 3. 3.2 3.4 3.6 NL: 8.39E6 389.36733-389.36967 [35.-425.] MS.3.2.1 Conclusions Reliable detection of 25-hydrox by clinical researchers. Higher resolving power with Ex 3. 3.2 3.4 3.6 NL: 1.43E6 395.32886-395.33282. 2 4 6 8 1 ng/ml Lower LLOQ with Exactive Plu Cycle time of 1.9 minutes when Method robustness of approxim changes. [35.-425.] MS 25-OH_Vitamin_D3 Y =.183722+.59339*X R^2 =.9989 W: 1/X Ease of method set up compar 2.15 2.2 2.25 2.3 2.35.6 References NL: 2.13E6 383.32892-383.33276 F: FTMS + p APCI corona Full l ock ms [35.-425.] MS QC2 3 2.15 2.19 2.25 2.28 2.3 2.15 2.2 2.25 2.3 2.35 NL: 7.96E6 389.36655-389.3745.5.4.3.2.1 1. Shah I, James R,Barker J, Pet in Vitamin D analysis: A nove isobars. Nutrition Journal 211 Acknowledgement We would like to thank Keeley Murph 2.15 2.2 2.25 2.3 2.35 [35.-425.] MS. 2 4 6 8 1 ng/ml All trademarks are the property of Thermo Fisher Sc This information is not intended to encourage use o intellectual property rights of others. For Research U Thermo Scientific Poster Note PN6368_E 6/12S 5
level were run FIGURE 7. Serum injection replicates. Approximately 6 injections before variability increased. 4 4.5 Area 2 18 16 14 12 1 8 Vitamin D3 Vitamin D2 4 4.5 6 4 2 2 4 6 8 1 Injection Number W: 1/X Variability increased during replicate serum injections after approximately 6 injections. The system ran serum injections constantly during this time period with no blanks or washing in between injections. Increased variability could be due to the mass spectrometer source becoming dirty after so many serum injections with no cleaning. Future experiments will seek to extend the number of injections before variability occurs, possibly by increasing the amount of washing of the columns that occurs during the HPLC method, as well as steps to keep the MS source cleaner during the sample run. Conclusions Reliable detection of 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3 in serum by clinical researchers. Higher resolving power with Exactive+ orbitrap mass spectrometer 1 Lower LLOQ with Exactive Plus Cycle time of 1.9 minutes when using a Transcend TLX-4 multiplexing system. Method robustness of approximately 6 serum injections between column changes. Ease of method set up compared to triple quadrupole MS methods. W: 1/X References 1. Shah I, James R,Barker J, Petroczi A, and Naughton D: Misleading measures in Vitamin D analysis: A novel LC-MS/MS assay to account for epimers and isobars. Nutrition Journal 211, 1:46. Acknowledgements We would like to thank Keeley Murphy for his advice and assistance. 1 All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. For Research Use Only. Not for use in diagnostic procedures. 6 Analysis of 25-Hydroxyvitamin D In Serum Using Automated On Line Sample Preparation Technique With High Resolution Orbitrap Mass Spectrometer
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