April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
ImmunosuppressantsImmunosuppressantsin Blood by LC/MS/MSin Blood by LC/MS/MS
Agilent 6410 QQQAgilent 6410 QQQ
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
Objectives using Agilent QQQObjectives using Agilent QQQ
MDL’s and required Linear Dynamic Range0.2 - 50 ug/L (Sirolimus and Tacrolimus)8 - 2,000 ug/L (Cyclosporine A)
Allow analysis of 100 samples per daySamples arriving in lab by 11:30am reported by 5:00pmMust work without changing sample preparation
Changes allowedChanges allowedUse of sodium adduct instead of ammoniumLC column and conditions
“See what you can can do with it”
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
Reference Instrumental MethodReference Instrumental Method
API 3000 with direct spray2.1 mm x 75 mm x 3.5 u Supelcosil C182 mM NH4OAc + 0.1% formic acid in water and MeOH90% B isocratic at 65ºCElectrospray Positive using NH4
+ adducts30 uL injections
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
CompoundsCompounds
NH
N CH3
CH3
CH3
O
NH
CH3
CH3
O
NCH3O
NHCH3
OCH3
O
N
CH3
CH3
CH3
CH3
CH3
O
O
CH3N
CH3
NH
CH3O
N
CH3CH3
N
CH3O
N
OCH3OH
OCH3CH3
CH3
CH3
CH3
O
O N
O
CH 3
O
O HHCH 3
O
CH 3
OCH 3
O H
OCH 3
O
OCH 3CH 3
O
C H 3CH 3
O HCH 3
Cyclosporine A, C62H111N11O12FW = 1201.8
Sirolimus, C51H79NO13FW = 913.6
O
O
C H 3 C H 2
O H
N
O
OCH 3 C H 3
O
O HH
OCH 3
CH 3
O
O
C H 3 C H 3
OCH 3
O H
Tacrolimus, C44H69NO12FW = 803.5
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
MS2 Scan Cyclosporine AMS2 Scan Cyclosporine A
Adduct m/z
H+ 1202.8
NH4+ 1219.9 Missing !!
Na+ 1224.8
K+ 1240.8
Adduct Mass m/z
2 H+ 1203.9 601.9
H+ + Na+ 1225.8 612.9
2 Na+ 1247.8 623.9
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
MS2 Scan SirolimusMS2 Scan Sirolimus
Adduct m/z
H+ 914.6
NH4+ 931.6 Missing !!
Na+ 936.5
K+ 952.5
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
MS2 Scan TacrolimusMS2 Scan Tacrolimus
Adduct m/z
H+ 804.5
NH4+ 821.5 Missing !!
Na+ 826.5
K+ 842.4
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
Product Ion Scan Product Ion Scan –– Cyclosporine ACyclosporine ACollision Energy 75
Collision Energy 85
Collision Energy 80
Collision Energy 90
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
Effect of column temperatureEffect of column temperatureCyclosporine ACyclosporine A
80°C 60°C 40°C70°C
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
RRLC ConditionsRRLC Conditions
Sodium adducts preferred, so sodium acetate used in mobile phaseSodium adducts preferred, so sodium acetate used in mobile phase
HPLC Agilent 1200 RRLC
Column Zorbax XDB-C8, RRHT, 2.1 mm x 50 mm x 1.8 µm
Column Temp. 80ºC Improved peak shape and resolution; quicker elution; reduced back pressure
Mobile Phase A: 1 mM sodium acetate + 0.02% glacial acetic acid in water Sodium adduct is favoured
B: 0.01 mM sodium acetate + 0.02% glacial acetic acid in methanol
Flow rate 0.4 mL/min
Injection volume 10 µL Injection volume is limited by sample’s solvent composition and mobile phase conditions
Time, min. %B Notes
0.00 15 Focus components
1.00 15 Elute salts with aqueous wash that may cause ion suppression
1.01 85 High organic to quickly elute drugs
5.30 85
Diverter Valve 2.3 min LC eluant to waste during initial part of run, to minimize contamination of QQQ source
Post-run time 1.5 min Allow 4-5 column volumes to equilibrate column before next run
Gradient
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
QQQ Acquisition ParametersQQQ Acquisition ParametersCompound R.T. MS1
ResolutionPrecursor Product
814.2 604.1
616.2
409.2
1112.2
1126.5
826.2
936.2
1224.8
1238.7
Wide
Wide
Wide
Wide
Wide
2.87
2.90
2.98
3.52
3.67
Dwell (ms)
Fragmentor(V)
CollisionEnergy (V)
Ascomycin - ISTD 200 260 45
Tacrolimus 200 260 45
Sirolimus 200 280 54
Cyclosporine A 200 300 85
Cyclosporine D - ISTD 200 300 80
Drying Gas 300°CNebulizer 30 psiGas Flow 12 L/minVcap 5500 V
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
Calibrant PreparationCalibrant PreparationCalibrants labeled S1 – S6 (µg/L - ppb)
Calibrant Sirolimus Tacrolimus Cyclosporine A
S1 0.5 0.5 8
S2 1.0 1.0 40
S3 5.0 5.0 200
S4 10.0 10.0 400
S5 30.0 30.0 1200
S6 50.0 50.0 2000
Sample preparation50 µl calibrant (at levels in table)+ 125 µL water+ 125 µL ZnSO4 (0.1M)+ 250 µL internal standard in MeOH= 550 µL total volume
Calibrant Sirolimus Tacrolimus Cyclosporine A
S1 0.05 0.05 0.7
S2 0.09 0.09 3.6
S3 0.46 0.46 18.2
S4 0.91 0.91 36.4
S5 2.73 2.73 109.1
S6 4.55 4.55 181.9
Actual concentration in A/S vial (pg/µL – ppb)
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
10 µL Calibrant S10.5 pg Sirolimus & Tacrolimus7.0 pg Cyclosporine A
LowLow--level Calibrant in Blood Matrixlevel Calibrant in Blood Matrix
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
Calibration CurvesCalibration Curves
Correlation coefficients (R2)Tacrolimus 0.9997Sirolimus 0.9996Cyclosporine A 0.9999
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
Result ComparisonResult Comparison
QC Sample Date Level I Level II Level IV
Reference Mar 25, 2007
Mar 30, 2007
Apr 6, 2007
92 237 1,530
Vogon 1 83 214 1,516
Vogon 2 83 194 1,527
QC Sample Date Level I Level II Level IV
Reference Mar 25, 2007
Mar 30, 2007
Apr 6, 2007
2.7 7.7 30.9
Vogon 1 2.6 9.1 32.4
Vogon 2 2.7 7.3 30.8
QC Sample Date Level I Level II Level IV
Reference Mar 25, 2007
Mar 30, 2007
Apr 6, 2007
2.0 4.6 24.0
Vogon 1 2.5 5.5 23.2
Vogon 2 2.2 5.5 23.8
Sirolimus
Tacrolimus
Cyclosporine A
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
Dynamic RangeDynamic RangeCyclosporine A standard in methanolCyclosporine A standard in methanol
Mobile phase contains0.01 mM sodium acetate+ 0.02% acetic acid
Sodium acetate added to aqueous phaseTo give 1 mM concentration
Broken Vial
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
Run time optimizationRun time optimization2.1 mm x 50 mm x 1.8 µ Zorbax XDB-C8 @ 0.4 mL/min gradient (~ 250 bar @ 80°C)
2.1 mm x 30 mm x 3.5 µ Zorbax SB-C18 @ 0.4 mL/min isocratic 85%B (~ 45 bar @ 80°C)
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
ProductivityProductivitySegment Reference Method
(3.1 min / run)Gradient Flush( 7.5 min / run)
Isocratic(2.2 min / run)
Initial Calibration- 3 blanks- 6 calibrants- 1 blank- 3 QC- 1 blank
43.4 min 105 min 30.8 min
Sample Batch- 10 samples- 1 blank
34.1 min 82.5 min 24.2 min
Ongoing QC- 1 QC- 1 blank
6.2 min 15.0 min 4.4 min
Total time for minimal batch
1h 24m 3h 23m 1h 0m
Minimum time per 100 samples*
7h 26m 18h 0min 5h 17m
Based on ideal run of 1 Calibration + 10 Sample batches + 10 On-going QC
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
RuggednessRuggedness
Tacrolimus
Cyclosporine A
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
Alternative IonizationAlternative Ionization• APCI
• Multimode (ESI + APCI)
• Negative Ion
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
APCI APCI –– SirolimusSirolimus
Adduct m/z
H+ 914.6
NH4+ 931.6 Found it !!
Na+ 936.5
K+ 952.5
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
APCI APCI –– TacrolimusTacrolimus
Adduct m/z
H+ 804.5
NH4+ 821.5 Found it !!
Na+ 826.5
K+ 842.4
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
APCI APCI –– Cyclosporine D Cyclosporine D istdistd
Adduct m/z
H+ 1216.8 Only significant adduct
NH4+ 1233.8 Still missing !!
Na+ 1238.8
K+ 1254.8
April 2007April 2007Ralph Hindle, Vogon Laboratory Services, Ralph Hindle, Vogon Laboratory Services,
Calgary, ABCalgary, AB
ConclusionsConclusions
MDL’s and required Linear Dynamic Range0.2 - 50 ug/L (Sacrolimus and Tacrolimus)8 - 2,000 ug/L (Cyclosporine A)
Allow analysis of 100 samples per day
Samples arriving in lab by 11:30am reported by 5:00pm
Must work without changing sample preparation
Perform analysis using NH4+ adducts
• Na+ adducts• Can use APCI
Bonus: only 10 uL injected, instead of 30 uL in Reference method
☺ 30% faster than Reference Method