LCMS Vitamin D3 Method development
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- Erick Reynolds
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1 LCMS-85 Vitamin D3 Method development
2 Background Vitamin D3 Analysis is in demand in SAP region especially in Singapore and India Clinical Diagnostics and Clinical Research is demanding this application 25-OH Vit D3 Diagnotics 1,25 Di-OH Biomarker for many types of cancers hence more in research
3 Collaborative efforts The collaborator has an Agilent 646 and are disappointed with the frequency of maintenance The customer collaborated with Agilent (using the 649) for the last 9 months without any success in achieving their desired sensitivity of 5 ppt in plasma, therefore SAP offered an LCMS-85 trial Standards, IS and plasma blank were provided by the customer
4 Objectives and Experimental Conditions 5. Analytical Conditions: LC Conditions Column Flow Rate Mobile Phase Elution Mode Shim-pack FC-ODS (75mm x 2mm, 3 μm).5 ml/min A : Water with.1% Formic Acid B : Methanol with.1% Formic acid Gradient elution, LC program 8~1 minute Oven Temperature 45 ºC Injection Volume 5 & 5 ul MS and Interface Conditions Interface APCI MS Mode Positive MRM Block Temperature 2 ºC DL Temperature 2 ºC Nebulizing Gas Flow Nitrogen, 2.5 L/min Drying Gas Flow Nitrogen, 5 L/min 4
5 MRM Optimization MRM optimization result of 25-hydroxyl-vitamin D3 and its d6 internal standard Compound MRM Pause time Dwell Time Q1 Pre Bias CE Q3 Pre Bias 41.3 > OH-VD > > > OH-VD3-d > MRM optimization result of 1,25-dihydroxyl-vitamin D3 and its d3 internal standard Compound MRM Pause time Dwell Time Q1 Pre Bias CE Q3 Pre Bias > ,25-diOH-VD > > ,25-diOH > VD3-d >
6 25-hydroxyl vitamin D3 in Neat Solution Single std: VD3-1OH,.2 ppb (6) Single std: VD3-1OH, 1.56 ppb (13) 4 3 1:OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: :OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: min min 1:OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: Single std: VD3-1OH,.39 ppb (9) 3 2 Single std: VD3-1OH, 3.13 ppb (15) 1:OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: min min 75 5 Single std: VD3-1OH,.78 ppb (11) 1:OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: Single std: VD3-1OH, 6.25 ppb (17) 1:OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: min min 6
7 25-hydroxylvitamin D3 in Neat Solution Single std: VD3-1OH, 12.5 ppb (19) Single std: VD3-1OH, 1 ppb (25) :OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: :OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: min min Single std: VD3-1OH, 25 ppb (21) Single std: VD3-1OH, 2 ppb (27) 1:OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: :OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: min min Single std: VD3-1OH, 5 ppb (23) 1:OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: min 7
8 25-hydroxyl VD3 + IS (d6) in neat solution Batch: C:\LabSolutions\Data\11213_VitaminD3\APCI\13113 Zhan\1 Dec VD3-1OH\VD3 met-batch1dec-12 MeOH DP.lcb Method: C:\LabSolutions\Data\11213_VitaminD3\APCI\13113 Zhan\1 Dec VD3-1OH\VDs_FC-ODS75mm_14 DP.lcm Single std: VD3-1OH:.2,.39,.78, 1.56, 3.13, 6.25, 12.5, 25, 5, 1 and 2 ppb (11 point), two injection each Injection volume: 5 ul FC-ODS, 75 mm x 2 mm, 3 um MA: 7% B, MeOH with.1 FA Data file: from C:\LabSolutions\Data\11213_VitaminD3\APCI\13113 Zhan\1 Dec VD3-1OH\VD3-1OH_6.lcd to C:\LabSolutions\Data\11213_VitaminD3\APCI\13113 Zhan\1 Dec VD3-1OH\VD3-1OH_27.lcd Area Ratio Conc. Ratio Internal standard calibration curve of 25- dihydroxyl vitamin D3 in neat solution,.2 ppb ~ 2 ppb; IS 5 ppb 8
9 7. Results (3) blank serum + IS (d6) File 84 (repeat), ppb + IS 5 ppb in Serum :OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: File 21, ppb + IS ppb in serum 1:OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: min min :47.4>389.5(+) CE: :47.4>371.3(+) CE: D6 2:47.4>389.5(+) CE: :47.4>371.3(+) CE: min Quan: 7.81 ppb min Conclusion: The above results indicate that the serum blank contains 25-OH VD3 already!!! 9
10 25-hydroxyl VD3 + IS(d6) in Serum File 24,.2 ppb + IS 5 ppb in serum File 5,.2 ppb + IS 5 ppb in neat solu 5 1:OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: OHVD3 1:OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: OHVD min min 3 2:47.4>389.5(+) CE: :47.4>371.3(+) CE: D6 25-OHVD3-d6 (IS) 4 3 2:47.4>389.5(+) CE: :47.4>371.3(+) CE: D6 25-OHVD3-d6 (IS) min min.2 ppb 25-OHVD3 + 5 ppb d6 (IS) in Serum.2 ppb 25-OHVD3 + 5 ppb -d3 (IS) in solvent Note: Blank serum contains 25-OH VD3 1
11 25-hydroxyl VD3 + IS(d6) in Serum File 3, 1.56 ppb + IS 5 ppb in serum 1:OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: OHVD min File 56, 1.56 ppb + IS 5 ppb in neat solu 1:OHVD3 and epimer 41.3>383.3(+) CE: :OHVD3 and epimer 41.3>365.1(+) CE: :OHVD3 and epimer 41.3>17.(+) CE: OHVD min 4 3 2:47.4>389.5(+) CE: :47.4>371.3(+) CE: D6 25-OHVD3-d6 (IS) :47.4>389.5(+) CE: :47.4>371.3(+) CE: D6 25-OHVD3-d6 (IS) min min 1.56 ppb 25-OHVD3 + 5 ppb d6 (IS) in Serum 1.56 ppb 25-OHVD3 + 5 ppb -d3 (IS) in solvent Note: Blank serum contains 25-OH VD3 11
12 1,25 di-oh VitaminD3
13 7. Results (1) 1,25-dihydroxylvitamin D3 1:OH2D >381.3(+) CE: :OH2D >157.(+) CE: :OH2D >15.(+) CE: Single std: VD3-2OH, 12.5 ppt (4) min Single std: VD3-2OH, 25 ppt (43) 1:OH2D >381.3(+) CE: :OH2D >157.(+) CE: :OH2D >15.(+) CE: -44. VD3-di-OH VD3-di-OH min Single std: VD3-2OH, 5 ppt (45) 1:OH2D >381.3(+) CE: :OH2D >157.(+) CE: :OH2D >15.(+) CE: -44. VD3-di-OH Single std: VD3-2OH, 1 ppt (47) 1:OH2D >381.3(+) CE: :OH2D >157.(+) CE: :OH2D >15.(+) CE: min Single std: VD3-2OH, 2 ppt (49) 1:OH2D >381.3(+) CE: :OH2D >157.(+) CE: :OH2D >15.(+) CE: -44. VD3-di-OH min Single std: VD3-2OH, 4 ppt (51) 1:OH2D >381.3(+) CE: :OH2D >157.(+) CE: :OH2D >15.(+) CE: -44. VD3-di-OH VD3-di-OH min min 13
14 di-hydroxyl VD3 + IS(d3) in neat solution Area Ratio Conc. Ratio Internal standard calibration curve of 1,25- dihydroxyl vitamin D3 in neat solution, 22.7 ppt ~ ppt; IS ppt 14
15 1,25 di-hydroxyl VD3 + IS(d3) :OH2D >381.3(+) CE: :OH2D >157.(+) CE: :OH2D >15.(+) CE: -44. OH2-VD3 Di-OHVD :OH2D >381.3(+) CE: :OH2D >157.(+) CE: :OH2D >15.(+) CE: -44. OH2-VD3 Di-OHVD min min 2:OH2D3-D6 42.3>383.3(+) CE: :OH2D3-D6 42.3>366.3(+) CE: OH2-VD3-D3 Di-OHVD3-d3 (IS) :OH2D3-D6 42.3>383.3(+) CE: :OH2D3-D6 42.3>366.3(+) CE: -12. OH2-VD3-D3 Di-OHVD3-d3 (IS) min min ppt Di-OHVD ppt -d3 (IS) in Serum ppt Di-OHVD ppt -d3 (IS) in solvent 1 D LC/MS/MS method faced the problem of a serious interference
16 Advantage of 2-D LC Previous work revealed that with the APCI interface using MeOH-water-Formic acid mobile phase, the best sensitivity for standards in neat solution is 12.5 ppt (5 ul). The sensitivity in serum is far from the required concentration. The main problem iss peak interference in the MRM transition 399>281 at the low ppt level in serum. It is not possible to solve this peak interference problem by changing LC conditions.
17 2D-LC/MS/MS method 1:OH2D >381.3(+) CE: :OH2D >157.(+) CE: :OH2D >15.(+) CE: OH2-VD min 7 2:OH2D3-D6 42.3>383.3(+) CE: :OH2D3-D6 42.3>366.3(+) CE: OH2-VD3-D min Precise peak cut (<2 ul) in 1 st D separation Transfer to 2 nd separation and analysis
18 2D-LC/MS/MS system for precise peakcut in 1 st D and transfer to 2 nd D Pump A Mixer Auto sampler RP column Trapping loop 2 ul Switching Valve Detector Pump B Pump C Mixer Switching Valve nd Column Valve (2) = Valve (1) = Pump D Step 1: 1 st D column: sample loading & separation 2 nd D column: ready
19 2D-LC/MS/MS system for precise peakcut in 1 st D and transfer to 2 nd D Pump A Mixer Auto sampler RP column Trapping loop 2 ul Switching Valve Detector Pump B Pump C Mixer Switching Valve nd Column Valve (2) = Valve (1) = 1 Pump D Step 2: 1 st D column: peak-cut at RT (<2 ul) by time program 2 nd D column: ready
20 2D-LC/MS/MS system for precise peakcut in 1 st D and transfer to 2 nd D Pump A Mixer Trapping loop 2 ul Auto sampler 1st RP column 75 mm x 2 mm 5 Switching Valve Detector Pump B Pump C Mixer Switching Valve nd RP Column 5 mm x 2 mm Valve (1) = Valve (2) = Pump D Step 3: 1 st D column: washing & equilibration 2 nd D column: analysis
21 Advantage of 2-D LC
22 1,25-dihydroxylvitamin D3, 12.5 ppt :399.3>381.3(+) 1:399.3>157.(+) 12.5 ppt x 1 ul In neat solution :399.3>381.3(+) 1:399.3>157.(+) Area: :399.3>381.3(+) 4 1:399.3>157.(+) ppt x 1 ul In serum :399.3>381.3(+) 1:399.3>157.(+) Area: 3957
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