Quantitative Analysis of Vit D Metabolites in Human Plasma using Exactive System
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1 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
2 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
3 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
4 Goal Results comparison between Exactive and QQQ method for quantitation of 25-OH-Vit D2 and 25-OH-Vit D3 in human plasma - 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
5 Exactive System Introduction 5 Clinical Research use only, Not for use in Diagnostics Procedure
6 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
7 Orbitrap Principle of Operation r z φ Hyper-logarithmic potential distribution: ideal Kingdon trap k 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, Clinical Research use only, Not for use in Diagnostics Procedure
8 Ethinyl-Estradiol at Different Mass Resolutions Resolution: 1k, 3k, 5k, k Butyl-Phthalate, (ubiquitous background ion) Relative Abundance Ethinyl-Estradiol, m/z Note:Ethinyl Estradiol used just to demonstrate the power of resolution 8 Clinical Research use only, Not for use in Diagnostics Procedure
9 ppb Hormone Sample (5 pg on-column) ppb Hormone sample different resolution settings Ethinyl-Estradiol Phthalate Estradiol Phthalate Estradiol Relative Abundance Relative Abundance RP =, Time (min) 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
10 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 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
11 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] for Vit-D2 and 2 for Vit-D3* 5 2 Required Precision [%] * 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
12 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
13 Exactive 3 Selected M/Z Analyte Monitored m/z 25-OH-D 3 25-OH-D (M+H) (M - H 2 O + H) (M - 2 x H 2 O) + H) (M+H) (M - H 2 O + H) (M - 2 x H 2 O) + H m/z with minimum interference [ 2 H 6 ] 25-OH-D 3 (Internal Standard) (M+H) (M - H 2 O + H) (M - 2 x H 2 O) + H 13 Clinical Research use only, Not for use in Diagnostics Procedure
14 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 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
15 Linearity and Accuracy 25OH-Vit D3 Vit D3 - Validation1 Vit D3 - Validation 2.65 Y = *X R^2 =.9918 W: 1/X^2.7 Y = *X R^2 =.9954 W: 1/X^ Area Ratio.35.3 Area Ratio ng/ml ng/ml.75.7 Vit D3 - Validation 3 Y = *X R^2 =.9946 W: 1/X^2 % Difference.65 Standard Validation_1 Validation_2 Validation_ ng/ml Precision just below set criteria ng/ml Area Ratio ng/ml ng/ml ng/ml ng/ml ng/ml Note: Calibration curve using neat standards 15 Clinical Research use only, Not for use in Diagnostics Procedure
16 Linearity and Accuracy 25OH-Vit D2 Vit D2 Validation 1 Vit D2 Validation Y = *X R^2 =.9919 W: 1/X^2 2.8 Y = *X R^2 =.9943 W: 1/X^ Area Ratio Area Ratio ng/ml ng/ml Vit D2 Validation Y = *X R^2 =.9996 W: 1/X^2 % Difference Standard Validation_1 Validation_2 Validation_ ng/ml ng/ml Area Ratio ng/ml ng/ml ng/ml ng/ml ng/ml Note: Calibration curve using neat standards 16 Clinical Research use only, Not for use in Diagnostics Procedure
17 Lower points in calibration curve Good ppecificity 1ng_std - m/z= SM: 5 RT: NL: 4.36E4 F: {,} + p APCI Full ms [ ] RT: D3-1ng/mL R e la tiv e In te n s ity %RSD>2% Time (min) 2ng_std - m/z= SM: 5 RT: NL: 5.39E4 F: {,} + p APCI Full ms [ ] R e la tiv e In te n s ity RT: 1.13 LOQ D3-2ng/mL Time (min) ng_std - m/z= SM: 5 RT: NL: 1.49E5 F: {,} + p APCI Full ms [ ] R e la tiv e In te n s ity RT: Time (min) D3-1ng/mL 1ng_std - m/z= SM: 5 RT: NL: 7.47E4 F: {,} + p APCI Full ms [ ] RT: R e la tiv e In te n s ity Time (min) LOQ D2-1ng/mL 2ng_std - m/z= SM: 5 RT: NL: 1.37E5 F: {,} + p APCI Full ms [ ] RT: D2-2ng/mL 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= SM: 5 RT: NL: 5.8E5 F: {,} + p APCI Full ms [ ] RT: D2-1ng/mL R e la tiv e In te n s ity Time (min) 17 Clinical Research use only, Not for use in Diagnostics Procedure
18 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 Mean Mean Mean %RSD %RSD %RSD %Rec ND ND ND %Rec %Rsc Clinical Research use only, Not for use in Diagnostics Procedure
19 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 Mean Mean Mean SD %RSD %RSD %Rec %Rec %Rec Clinical Research use only, Not for use in Diagnostics Procedure
20 Blinded Sample Results Comparison 2 Clinical Research use only, Not for use in Diagnostics Procedure
21 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
22 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 <1 <1.9 ND B <1 2.1 ND C D <1 <1.9 ND E <1 2.3 ND F <1 2. ND G <1 2.4 ND Note: External service lab used Access QQQ 22 Clinical Research use only, Not for use in Diagnostics Procedure
23 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: SM: 5B R e l a t i v e A b u n d a n c e RT:.9 AA: 1925 RT: 1.14 AA: RT:.37 AA: RT: 1.14 MA: RT: 1.15 MA: RT: 1.15 MA: RT: 1.14 MA: Time (min) NL: 5.21E5 m/z= F: FTMS {,} + p APCI Full ms [ ] MS ICIS Sample_A1 NL: 4.45E5 m/z= F: FTMS {,} + p APCI Full ms [ ] MS sample_a2 NL: 4.57E5 m/z= F: FTMS {,} + p APCI Full ms [ ] MS sample_a3 NL: 4.72E5 m/z= F: FTMS {,} + p APCI Full ms [ ] MS sample_a4 NL: 4.33E5 m/z= F: FTMS {,} + p APCI Full ms [ ] MS sample_a5 RT: SM: 5B R elative A bundance R elative A bundance R elative A bundance RT:.84 MA: RT:.84 MA: RT:.84 MA: Time (min) NL: 3.87E5 TIC F: + c APCI SRM ms [ , ] MS Sample_A_1 NL: 3.7E5 TIC F: + c APCI SRM ms [ , ] MS sample_a_2 NL: 2.84E5 TIC F: + c APCI SRM ms [ , ] 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
24 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: SM: 5B R e l a t i v e A b u n d a n c e Time (min) NL: 2.14E4 m/z= F: FTMS {,} + p APCI Full ms [ ] MS sample_a NL: 5.32E3 m/z= F: FTMS {,} + p APCI Full ms [ ] MS Sample_A1 NL: 3.29E4 m/z= F: FTMS {,} + p APCI Full ms [ ] MS sample_a3 NL: 3.53E4 m/z= F: FTMS {,} + p APCI Full ms [ ] MS sample_a4 NL: 1.74E4 m/z= F: FTMS {,} + p APCI Full ms [ ] MS sample_a5 RT: 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 Time (min) NL: 7.9E4 TIC F: + c APCI SRM ms [ , ].93 MS sample_a_ NL: 8.68E4 TIC F: + c APCI SRM ms [ , ] MS Sample_A_1 NL: 8.68E4 TIC F: + c APCI SRM ms [ , ] 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
25 Appendix 25 OH Vit D Analysis 25 Clinical Research use only, Not for use in Diagnostics Procedure
26 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
27 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
28 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) Clinical Research use only, Not for use in Diagnostics Procedure
29 25-OH-Vit D - Exactive Method Polarity: Positive Microscans: 1 Resolution: Ultra High ( ) Target: Balanced Max Inject time: 25 Scan range: Divert valve: switch events min to waste to detector to waste to detector 29 Clinical Research use only, Not for use in Diagnostics Procedure
30 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 Clinical Research use only, Not for use in Diagnostics Procedure
31 25-OH-Vit D - Autosampler method 31 Clinical Research use only, Not for use in Diagnostics Procedure
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