immunosuppressants in blood - trace organic · ralph hindle, vogon laboratory services, calgary, ab...

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



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”



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



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



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



MS2 Scan SirolimusMS2 Scan Sirolimus

Adduct m/z

H+ 914.6


Na+ 936.5

K+ 952.5



MS2 Scan TacrolimusMS2 Scan Tacrolimus

Adduct m/z

H+ 804.5


Na+ 826.5

K+ 842.4



Product Ion Scan Product Ion Scan –– Cyclosporine ACyclosporine ACollision Energy 75

Collision Energy 85

Collision Energy 80

Collision Energy 90



Effect of column temperatureEffect of column temperatureCyclosporine ACyclosporine A

80°C 60°C 40°C70°C



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



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



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)



10 µL Calibrant S10.5 pg Sirolimus & Tacrolimus7.0 pg Cyclosporine A

LowLow--level Calibrant in Blood Matrixlevel Calibrant in Blood Matrix



Calibration CurvesCalibration Curves

Correlation coefficients (R2)Tacrolimus 0.9997Sirolimus 0.9996Cyclosporine A 0.9999



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



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



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



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



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)



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



RuggednessRuggedness

Tacrolimus

Cyclosporine A



Alternative IonizationAlternative Ionization• APCI

• Multimode (ESI + APCI)

• Negative Ion



APCI APCI –– SirolimusSirolimus

Adduct m/z

H+ 914.6

NH4+ 931.6 Found it !!

Na+ 936.5

K+ 952.5



APCI APCI –– TacrolimusTacrolimus

Adduct m/z

H+ 804.5

NH4+ 821.5 Found it !!

Na+ 826.5

K+ 842.4



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



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

immunosuppressants in blood - trace organic · ralph hindle, vogon laboratory services, calgary, ab...

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