Quantitative Analysis of Vit D Metabolites in Human Plasma using Exactive System Marta Kozak Clinical Research Applications Group Thermo Fisher Scientific San Jose CA Clinical Research use only, Not for use in Diagnostics Procedure
Calibrators and Controls Six calibrators at 2, 5 1, 25, 5 andng/ml in BSA Three QCs at 5ng/mL, 4 ng/ml and 8 ng/ml in BSA Cals and QC s prepared by fortifying bovine serum albumin diluent with 2 ng/ml 25-hydryoxyvitamin D2 and D3 standard mix. Precipitating reagent was prepared by adding deuterated D6-25- hydroxyvitamin D3 to acetonitrile for a final concentration of 75ng/mL. Pooled donor serum samples were crashed 2 to 1 with Acetonitrile and spiked with analytes for a final concentration of 2 ng/ml for 25- hydroxyvitamin D2 2 Clinical Research use only, Not for use in Diagnostics Procedure
Presentation Overview Goals Introduction to Exactive Sample preparation and LC/MS method for 25 OH-Vit D Calibration curves, LOQ and results on QCs Blinded sample results comparison Appendix QQQ: Triple Quadrupole MS/MS system 3 Clinical Research use only, Not for use in Diagnostics Procedure
Goal Results comparison between Exactive and QQQ method for quantitation of 25-OH-Vit D2 and 25-OH-Vit D3 in human plasma Exactive@Thermo - Off line sample prep and 2 min gradient QQQ@Service lab - Protein precipitation followed by Turboflow and 6min gradient Evaluate Exactive system for analysis of 1, 25 di hydroxy Vit D3 4 Clinical Research use only, Not for use in Diagnostics Procedure
Exactive System Introduction 5 Clinical Research use only, Not for use in Diagnostics Procedure
Exactive Benchtop LC-MS Resolution, at 1 scan per second 1, at 1 scans per second Mass accuracy Sub ppm Dynamic range >1, within a spectrum Scan speed Up to 1 scans per second Mass range m/z 5-4 Polarity switching One positive and one negative scan < 1 second (25K Resolution) 6 Clinical Research use only, Not for use in Diagnostics Procedure
Orbitrap Principle of Operation r z φ Hyper-logarithmic potential distribution: ideal Kingdon trap k 2 2 2 U ( r, z) = { z r / 2 + Rm ln( r / R m )} 2 Characteristic frequencies: Frequency of rotation ω φ Frequency of radial oscillations ω r Frequency of axial oscillations ω z ω z = k m / q ω z R m 2 R m ω ϕ = 1 ω r = ω z 2 2 R R 2 Makarov A. Anal. Chem. 2, 72, 1156-1162. 7 Clinical Research use only, Not for use in Diagnostics Procedure
Ethinyl-Estradiol at Different Mass Resolutions Resolution: 1k, 3k, 5k, k Butyl-Phthalate, 279.1599 (ubiquitous background ion) 9 8 7 Relative Abundance 6 5 4 3 Ethinyl-Estradiol, 279.17434 2 1 279.12 279.14 279.16 279.18 279.2 m/z Note:Ethinyl Estradiol used just to demonstrate the power of resolution 8 Clinical Research use only, Not for use in Diagnostics Procedure
ppb Hormone Sample (5 pg on-column) ppb Hormone sample measured @ different resolution settings Ethinyl-Estradiol Phthalate Estradiol Phthalate Estradiol Relative Abundance Relative Abundance 5 5 5 5 18.3 19.32 17.22 18.17 16.4 18.76 17.73 RP =, 17 18 19 Time (min) 16.68 17.59 17.81 18.7 18.94 17 18 19 Time (min) 19.4 RP = 1, Note:Ethnyl Estradiol used just to demonstrate the power of resolution The isobaric phthalate background ion interferes with the Ethinyl-Estradiol ion. At resolution of 1, the steroid mass is off because the isobaric ions are not resolved. 9 Clinical Research use only, Not for use in Diagnostics Procedure
Exactive method Sample preparation Standards preparation µg/ml 25-OH-D3 and 25-OH-D2 mix in Ethanol Sample preparation µl plasma + 2 µl IS 5 ng/ml [2H6]-25OH-D3 Serial dilutions down to ng/ml 1 µl std + 99 ul of 5% Ethanol Calibration standards 1 to 2 ng/ml Vortex Centrifuge 1 min @ 132 RPM Supernatant 5 µl inject Note: Stock IS in ACN; Standards, QC and samples were processed using identical procedure 1 Clinical Research use only, Not for use in Diagnostics Procedure
Data acceptance criteria Current generally acceptable LC/MS precision Samples Cal Standard 1 Cal Standard 2 Cal Standard 3 Cal Standard 4 Cal Standard 5 Cal Standard 6 Cal Standard 7 QC 1* QC 2 QC 3 Concentration [ng/ml] 1 2 1 2 5 2 5 for Vit-D2 and 2 for Vit-D3* 5 2 Required Precision [%] 2 15 15 15 15 15 15 2 15 15 * QC1 spiked with 5 ng/ml of Vit-D3 and Vit-D2. Only total concentration was analyzed for QC1 sample. 11 Clinical Research use only, Not for use in Diagnostics Procedure
25OH-Vit D2 and D3 Exactive mass spectrum D2 - H2O D3 - H2O D3 (2 x H2O) D2 - (2 x H2O) D2 12 Clinical Research use only, Not for use in Diagnostics Procedure
Exactive 3 Selected M/Z Analyte Monitored m/z 25-OH-D 3 25-OH-D 2 41.3411 (M+H) 383.336 (M - H 2 O + H) 365.32 (M - 2 x H 2 O) + H) 413.3411 (M+H) 395.335 (M - H 2 O + H) 377.3199 (M - 2 x H 2 O) + H m/z with minimum interference [ 2 H 6 ] 25-OH-D 3 (Internal Standard) 47.3787 (M+H) 389.3679 (M - H 2 O + H) 371.3576 (M - 2 x H 2 O) + H 13 Clinical Research use only, Not for use in Diagnostics Procedure
Exactive LC/MS Method LC method Thermo Hypersil aqgold 5 x 2.1 mm, 5um (1, 25 Vit D3) Thermo Hypersil aqgold 5 x 2.1 mm, 5µm (25 OH Vit D) Mobile phase A: water containing.1% formic acid Mobile phase B: methanol containing.1% formic acid Column Temp: 5 Deg C Exactive MS method HESI source Full scan MS acquisition @ 5, Resolution External mass calibration Extract Ion Chromatogram for molecule of interest (m/z)@ better than 5ppm mass accuracy Confirm the identity using mass accuracy and RT Quantitate using peak area in the chromatogram Note: Details in appendix 14 Clinical Research use only, Not for use in Diagnostics Procedure
Linearity and Accuracy 25OH-Vit D3 Vit D3 - Validation1 Vit D3 - Validation 2.65 Y =.576964+.27313*X R^2 =.9918 W: 1/X^2.7 Y =.56991+.298291*X R^2 =.9954 W: 1/X^2.65.6.6.55.55.5.5.45.45.4.4 Area Ratio.35.3 Area Ratio.35.3.25.25.2.2.15.15.1.1.5.5. 2 4 6 8 12 14 16 18 2 22 ng/ml. 2 4 6 8 12 14 16 18 2 22 ng/ml.75.7 Vit D3 - Validation 3 Y =.571591+.317597*X R^2 =.9946 W: 1/X^2 % Difference.65 Standard Validation_1 Validation_2 Validation_3.6.55 1 ng/ml Precision just below set criteria.5.45 2 ng/ml 2.2 1.5.8 Area Ratio.4.35 1 ng/ml -1.5-9.6-6.5.3.25 2 ng/ml -.2 4.9 7.8.2.15 5 ng/ml -7.5-2.4-7.9.1.5 ng/ml 2.9-2.3-1.7. 2 4 6 8 12 14 16 18 2 22 2 ng/ml 13. 7.9 7.6 Note: Calibration curve using neat standards 15 Clinical Research use only, Not for use in Diagnostics Procedure
Linearity and Accuracy 25OH-Vit D2 Vit D2 Validation 1 Vit D2 Validation 2 2.8 Y =.43257+.119399*X R^2 =.9919 W: 1/X^2 2.8 Y =.89+.121633*X R^2 =.9943 W: 1/X^2 2.6 2.6 2.4 2.4 2.2 2.2 2. 2. 1.8 1.8 1.6 1.6 Area Ratio 1.4 1.2 Area Ratio 1.4 1.2 1. 1..8.8.6.6.4.4.2.2.. 2 4 6 8 12 14 16 18 2 22 2 4 6 8 12 14 16 18 2 22 ng/ml ng/ml Vit D2 Validation 3 2.6 Y = -.622327+.121577*X R^2 =.9996 W: 1/X^2 % Difference 2.4 2.2 Standard Validation_1 Validation_2 Validation_3 2. 1 ng/ml 5.4-4.4.6 1.8 1.6 2 ng/ml -1.6 9.7 -.7 Area Ratio 1.4 1.2 1 ng/ml -4.3-4.4-2.9 1. 2 ng/ml 7.5 2.1 2..8.6 5 ng/ml -8.6-5.9-1.4.4 ng/ml.8-5.3.3.2. 2 4 6 8 12 14 16 18 2 22 2 ng/ml 9.7 8.2 2.1 Note: Calibration curve using neat standards 16 Clinical Research use only, Not for use in Diagnostics Procedure
Lower points in calibration curve Good ppecificity 1ng_std - m/z= 365.32-365.32 SM: 5 RT:.75-1.5 NL: 4.36E4 F: {,} + p APCI Full ms [ 35.-42.] RT: 1.12 95 9 D3-1ng/mL 85 8 75 7 65 R e la tiv e In te n s ity 6 55 5 %RSD>2% 45 4 35 3 25 2 1.2 15 1.47 1.77.87.97 5.8.9 1. 1.1 1.2 1.3 1.4 Time (min) 2ng_std - m/z= 365.32-365.32 SM: 5 RT:.76-1.51 NL: 5.39E4 F: {,} + p APCI Full ms [ 35.-42.] R e la tiv e In te n s ity 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5.78.83.88.96 RT: 1.13 LOQ D3-2ng/mL.8.9 1. 1.1 1.2 1.3 1.4 1.5 Time (min) 1.25 1.32 1.44 1ng_std - m/z= 365.32-365.32 SM: 5 RT:.76-1.51 NL: 1.49E5 F: {,} + p APCI Full ms [ 35.-42.] R e la tiv e In te n s ity 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 RT: 1.13 1.25 1.31 1.41.81.89.98 1.3 1.48.8.9 1. 1.1 1.2 1.3 1.4 1.5 Time (min) D3-1ng/mL 1ng_std - m/z= 395.33-395.33 SM: 5 RT:.79-1.54 NL: 7.47E4 F: {,} + p APCI Full ms [ 35.-42.] RT: 1.16 95 9 85 8 75 7 65 R e la tiv e In te n s ity 6 55 5 45 4 35 3 25 2 15 1 1.29 5 1..86.92.8.9 1. 1.1 1.2 1.3 1.4 1.5 Time (min) LOQ D2-1ng/mL 2ng_std - m/z= 395.33-395.33 SM: 5 RT:.78-1.53 NL: 1.37E5 F: {,} + p APCI Full ms [ 35.-42.] RT: 1.16 95 9 85 D2-2ng/mL 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 1.31 1.41 1.35.83.91.99 1.5.8.9 1. 1.1 1.2 1.3 1.4 1.5 Time (min) Note:Extracted Ion Chromatogram (XIC)@accuracy of 5 ppm R e la tiv e In te n s ity 1ng_std - m/z= 395.33-395.33 SM: 5 RT:.8-1.55 NL: 5.8E5 F: {,} + p APCI Full ms [ 35.-42.] RT: 1.17 95 D2-1ng/mL 9 85 8 75 7 65 6 55 5 45 R e la tiv e In te n s ity 4 35 3 25 2 15 1 5.86.99 1.37.8.9 1. 1.1 1.2 1.3 1.4 1.5 Time (min) 17 Clinical Research use only, Not for use in Diagnostics Procedure
QC Results - 25OH-Vit D3 inter and intra assay Excellent accuracy, precision and recovery QC1 (22 ng/ml) QC2 (5 ng/ml) QC3 (2 ng/ml) Replicate Val 1 Val 2 Val 3 Replicate Val 1 Val 2 Val 3 Replicate Val 1 Val 2 Val 3 1 19.6 21.5 2.3 1 47.4 5.2 48.2 1 29 215 24 2 21.3 19.9 2.1 2 53.1 49.7 54.9 2 215 224 29 3 19. 2.9 19.9 3 55.8 54.9 47.7 3 218 212 21 4 22.1 22.6 2.4 4 53.5 53.9 49.1 4 2 212 24 5 21.5 23.1 19.3 5 49.6 54.7 47.3 5 22 213 27 Mean 2.7 21.6 2. Mean 51.9 52.7 49.4 Mean 212 215 25 %RSD 6.5 5.8 2.2 %RSD 6.4 4.7 6.3 %RSD 3.8 2.3 1.5 %Rec ND ND ND %Rec 14 15 98.9 %Rsc 16 18 12 18 Clinical Research use only, Not for use in Diagnostics Procedure
QC Results - 25OH-Vit D2 inter and intra assay Excellent accuracy, precision and recovery QC1 (5 ng/ml) QC2 (5 ng/ml) QC3 (2 ng/ml) Replicate Val1 Val 2 Val 3 Replicate Val 1 Val 2 Val 3 Replicate Val 1 Val 2 Val 3 1 5. 4.4 4.3 1 47.3 45.6 49.7 1 189 197 23 2 4.8 4.1 4.7 2 51.1 48.5 48.1 2 191 29 29 3 4.2 5.1 3.9 3 47.3 48.9 51.6 3 191 186 184 4 5.3 4.8 4.4 4 54.2 45. 49.1 4 177 188 199 5 4.9 4.9 4.4 5 52.2 54. 48. 5 191 187 19 Mean 4.8 4.6 4.4 Mean 5.4 48.4 49.3 Mean 188 193 22 SD.37.4.28 %RSD 6. 7.3 3. %RSD 3.4 5. 8.9 %Rec 96.9 93. 87.4 %Rec 11 96.8 98.6 %Rec 93.9 96.7 11 19 Clinical Research use only, Not for use in Diagnostics Procedure
Blinded Sample Results Comparison 2 Clinical Research use only, Not for use in Diagnostics Procedure
Blinded sample - Results comparison Seven blinded samples from service lab analyzed Comparison with service labs 6 min TurboFlow TM QQQ method 5 replicate analysis on Exactive to asses precision 21 Clinical Research use only, Not for use in Diagnostics Procedure
Blinded samples Results summary Good agreement between Exactive and service lab QQQ results Vitamin D3 Vitamin D2 Sample # Exactive (ng/ml) Clinical Service lab Lab (ng/ml) % Diff Exactive (ng/ml) Clinical Service Lab lab (ng/ml) % Diff A 39.6 33.5-15.4 <1 <1.9 ND B 34. 34.8 2.3 <1 2.1 ND C 14.6 16.6 13.7 4.3 7.2 67.4 D 19.7 2.4 3.5 <1 <1.9 ND E 32.3 36.3-12.4 <1 2.3 ND F 25.5 25.8-1.2 <1 2. ND G 34.2 34..6 <1 2.4 ND Note: External service lab used Access QQQ 22 Clinical Research use only, Not for use in Diagnostics Procedure
Chromatograms - Blinded sample A Exactive 39.6 ng/ml Vitamin D 3 Access 33.5 ng/ml c:\xcalibur\...\rawfiles\sample_a5 7/13/29 4:47:5 PM c:\xcalibur\...\rawfiles\sample_a_3 7/14/29 3:31:6 PM RT:. - 2.2 SM: 5B R e l a t i v e A b u n d a n c e 5 5 5 5 5 RT:.9 AA: 1925 RT: 1.14 AA: 25893.21 RT:.37 AA: 21678.49.61.65.72.78.93 1.1 1.38 1.44 1.49 1.57 1.68 1.83 1.87 1.99 RT: 1.14 MA: 2488.8.14.23.36.5.64.72.9.93 1.5 1.3 1.35 1.45 1.54 1.72 1.78 1.91 RT: 1.15 MA: 2178738 1.33.3.14.22.35.41.45.58.63.69.82.89.94 1.37 1.48 1.59 1.67 1.75 1.79 1.95 RT: 1.15 MA: 225493.5.9.2.27.33.43.49.59.68.76.91 1.3 1.29 1.36 1.51 1.63 1.68 1.73 1.91 1.95 RT: 1.14 MA: 1964455.8.14.19.35.48.55.59.77.86.9 1.5 1.33 1.49 1.6 1.65 1.69 1.82 1.9..1.2.3.4.5.6.7.8.9 1. 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2. Time (min) NL: 5.21E5 m/z= 365.3182-365.3218 F: FTMS {,} + p APCI Full ms [35.-42.] MS ICIS Sample_A1 NL: 4.45E5 m/z= 365.3182-365.3218 F: FTMS {,} + p APCI Full ms [35.-42.] MS sample_a2 NL: 4.57E5 m/z= 365.3182-365.3218 F: FTMS {,} + p APCI Full ms [35.-42.] MS sample_a3 NL: 4.72E5 m/z= 365.3182-365.3218 F: FTMS {,} + p APCI Full ms [35.-42.] MS sample_a4 NL: 4.33E5 m/z= 365.3182-365.3218 F: FTMS {,} + p APCI Full ms [35.-42.] MS sample_a5 RT:. - 2. SM: 5B R elative A bundance R elative A bundance R elative A bundance 8 6 4 2 8 6 4 2 8 6 4.9.24.47.54 RT:.84 MA: 176719.96 1.5.74 1.19 1.28 1.46 1.74.37 RT:.84 MA: 193612 1.67 1.86.75.97 1.7.11.17.26.38.54.63 1.13 1.18 1.45 1.64 1.75 1.8 1.93 RT:.84 MA: 844399 2.74.97 1.7 1.14 1.19.11.21.39.47.55 1.46 1.57 1.77 1.84 1.96..2.4.6.8 1. 1.2 1.4 1.6 1.8 2. Time (min) NL: 3.87E5 TIC F: + c APCI SRM ms2 383.28 [257.13-257.153, 365.75-365.125] MS Sample_A_1 NL: 3.7E5 TIC F: + c APCI SRM ms2 383.28 [257.13-257.153, 365.75-365.125] MS sample_a_2 NL: 2.84E5 TIC F: + c APCI SRM ms2 383.28 [257.13-257.153, 365.75-365.125] MS sample_a_3 Note: 5 injections on Exactive and 3 injections on Access 23 Clinical Research use only, Not for use in Diagnostics Procedure
Chromatograms - Blinded sample A Exactive <LOQ Vitamin D 2 Access <LOQ See appendix for other 6 samples c:\xcalibur\...\rawfiles\sample_a5 7/13/29 4:47:5 PM C:\Xcalibur\...\Rawfiles\Sample_A_1 7/14/29 3:25:28 PM RT:. - 2.2 SM: 5B R e l a t i v e A b u n d a n c e 5 5 5 5 5.3.15.23.34.67.12.26.33.43.56.68.9.9.87.8.28.32.49.55.64.71.76.13.19.3.43.49.59.75.13.18.24.28.52.59.71.74 1.19 1.15..1.2.3.4.5.6.7.8.9 1. 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2. Time (min) 1.17.88.93 1.16.87 1.13 1.25 1.29 1.46 1.52 1.67 1.42 1.82 1.89 1.46 1.63 1.76 1.84 1.42 1.48 1.52 1.67 1.76 1.89 NL: 2.14E4 m/z= 395.3285-395.3325 1.98 F: FTMS {,} + p APCI Full ms [35.-42.] MS sample_a2 1.94 1.94 1.55 1.52 1.63 1.89 1.76 1.63 1.83 1.57 1.92 1.98 1.97 NL: 5.32E3 m/z= 395.3285-395.3325 F: FTMS {,} + p APCI Full ms [35.-42.] MS Sample_A1 NL: 3.29E4 m/z= 395.3285-395.3325 F: FTMS {,} + p APCI Full ms [35.-42.] MS sample_a3 NL: 3.53E4 m/z= 395.3285-395.3325 F: FTMS {,} + p APCI Full ms [35.-42.] MS sample_a4 NL: 1.74E4 m/z= 395.3285-395.3325 F: FTMS {,} + p APCI Full ms [35.-42.] MS sample_a5 RT:. - 2. SM: 5B R e l a ti v e A b u n d a n c e R e l a ti v e A b u n d a n c e R e l a ti v e A b u n d a n c e 8 6 4 2 8 6 4 2 8 6 4 2.79.84.95 1..3.25.3.46.62 1.24 1.45 1.62 1.67 1.84 1.94.76.81.85.94.7.15.26.42.56.66 1.25 1.46 1.59 1.66 1.89..2.4.6.8 1. 1.2 1.4 1.6 1.8 2. Time (min) 1.18 1.1 1.6 1.15.2.2.39.57 1.26 1.46 1.65 1.8 1.96.79 NL: 7.9E4 TIC F: + c APCI SRM ms2 395.29.84 [269.93-269.143, 377.175-377.225].93 MS sample_a_3 1.1 1.3 1.15 NL: 8.68E4 TIC F: + c APCI SRM ms2 395.29 [269.93-269.143, 377.175-377.225] MS Sample_A_1 NL: 8.68E4 TIC F: + c APCI SRM ms2 395.29 [269.93-269.143, 377.175-377.225] MS sample_a_2 Note: 5 injections on Exactive and 3 injections on Access 24 Clinical Research use only, Not for use in Diagnostics Procedure
Appendix 25 OH Vit D Analysis 25 Clinical Research use only, Not for use in Diagnostics Procedure
Reagents 25-OH-D 3 was purchased from Cerilliant, 25-OH-D 2 was purchased from Sigma. Both compounds were stored in -2 deg.c. [ 2 H 6 ]-25-OH-D 3 (Internal Standard) was purchased from Medical Isotopes and it was stored in -7 deg.c. 26 Clinical Research use only, Not for use in Diagnostics Procedure
Method validation Confirm method accuracy Total quantity in QC sample Endogenous quantity in minus blank plasma measured using IS alone = 25-OH-D3 = 16.5 ng/ml 25-OH-D2 =.8 ng/ml Actual spiked amount in QC sample Intra assay variability - 5 replicates of each QC sample Inter assay variability - 5 replicates of each QC sample in 3 different batches 27 Clinical Research use only, Not for use in Diagnostics Procedure
25-OH-Vit D - Ionization source parameters Exactive Mass Spectrometer Ionization APCI, positive, Ion Max source, Discharge current 5. Vaporizer temperature (deg C) 39 Sheath gas 15 Ion Sweep Gas Pressure:. Aux gas 5 Capillary temperature (deg C) 275 28 Clinical Research use only, Not for use in Diagnostics Procedure
25-OH-Vit D - Exactive Method Polarity: Positive Microscans: 1 Resolution: Ultra High ( ) Target: Balanced Max Inject time: 25 Scan range: 35-42 Divert valve: switch events 4 1-.95 min to waste.95-1.4 to detector 1.4-1.9 to waste 1.9-2. to detector 29 Clinical Research use only, Not for use in Diagnostics Procedure
25-OH-Vit D - LC method LC Conditions Surveyor autosampler and pump Mobile Phase A: water containing.1% formic acid B: methanol containing.1% formic acid Column 5 x 2.1 mm id packed with 3 µm, C18-aQ stationary phase Injection volume: 5 µl Flow rate: 7 µl/min Gradient Time (min) % A %B 2 8.1 2 8.2 5 95 1.4 5 95 1.5 2 8 2. 2 8 3 Clinical Research use only, Not for use in Diagnostics Procedure
25-OH-Vit D - Autosampler method 31 Clinical Research use only, Not for use in Diagnostics Procedure