IMMUNOSUPPRESSANT DRUGS BY LC/MS/MS Page 1 of 22

Transcription

TITLE:
IMMUNOSUPPRESSANT DRUGS BY
LC/MS/MS
DEPT OF LAB MEDICINE
CLINICAL CHEMISTRY
DOCUMENT NO:
CC-80023
REVIEW BY PREP/SUPVR:
EFFECTIVE DATE:
August 1, 2004
REVISION:
H-1
Page 1 of 22
LAB MANAGER:
Timothy Shizume, MT (ASCP)
DIRECTOR:
Richard Donabedian, M.D.
DIRECTOR:
Michael Hodsdon, M.D.
DIRECTOR:
Herbert Malkus, PhD.
I.
PRINCIPLE:
The Waters Quattro micro API tandem mass spectrophometer coupled to a Waters 2795 Alliance
HPLC, run in the multiple reaction monitoring (MRM) mode, offers a rapid sensitive assay for
the simultaneous measurement of Cyclosporin A, Tacrolimus (fk 506), and Rapamycin
(sirolimus). All aspects of system operation and data acquisition are controlled by using Mass
Lynx nt 4.0 software with automated data processing by the Quan Lynx software.
The samples, controls, and standards are pretreated with zinc sulfate to lyse the cells and then
methanol containing the internal standard (ascomycin) which solublizes the drugs and precipitates
the blood proteins. The samples are then centrifuged and the supernatant is transferred to the
HPLC autosampler. The samples are the injected into a C18 guard column. The drugs are eluted
off the column with a step gradient of 50% methanol to 100% methanol both containing 2mM
ammonium acetate and 0.1% formic acid. The drugs are detected by monitoring the
fragmentation of selected ammoniated precursor ions to specific product ions by using positive
electrospray ionization. The MRM transitions used are:
1.
2.
3.
4.
II.
Cyclosporin A : 1220.3 > 1203.5
Rapamycin:
931.7 > 864.5
Tacrolimus:
821.6 > 768.5
Ascomycin:
809.7 > 756.6
Specimen preparation: Sample collection, Handling, and Storage
Only whole blood specimens (EDTA) are to be used. If samples are to be tested within 8 hours of
collection, they may be stored at room temperature (18 - 25°C ). Samples can be stored
refrigerated (2-8°C) for up to one week or frozen (-20°C) for up to 3 months. Avoid repeated
thawing and freezing. Mix all samples thoroughly before assaying
III.
Materials:
A.
Equipment:
1.
2.
3.
4.
5.
6.
7.
8.
Waters/Micro Mass LC/MS-MS
Centrifuge- ABBOTT microfuge
Fisher vortex genie 2
Sample rocker
Eppendorf repeater plus pippetor -( cardinal ca t# 5063-20)
Finnpipette 40 - 200 uL -(cardinal cat # p5055-905)
Corning pipette tips (0-200) -(VWR cat # 29442682)
Eppendorf combitip plus (5.0 mL) - (cardinal cat. # p5063-207)
TITLE:
LC/MS/MS
EFFECTIVE DATE:
August 1, 2004
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
B.
REVISION:
H-1
DEPT. OF LAB MEDICINE
CLINICAL CHEMISTRY
DOCUMENT NO:
CC-80023
Page 2 of 22
Eppendorf combitip plus (10.0mL) - (cardinal cat # p5063-208)
Globe conical microcentrifuge tubes - (globe cat # 111558)
24 position microcentrifuge tube plate - (waters #405000560)
Security guard kit - (phenomenex # k50-4282)
C18 cartridge (4.0 x 3.00 mm) - (phenomenex # AJO-4287)
Hamilton 500 ul gastight syringe
Replacement needles for syringe
Peek tubing 1/16th OD x 0.005 ID
Peek tubing 1/16th OD x 0.02 ID
One piece fingertight fitting
Standard polymer tubing cutter
Replacement blades for cutter
15 ml blue max conical tube- (cardinal # c3917-16)
50 ml blue max conical tube- (cardinal # c3978 - 50)
0.5 ml Sarstedt screw capped polyethylene vials - (Sarstedt # 72.730)
Reagents:
1.
2.
3.
4.
5.
6.
7.
HPLC grade Methanol - (cardinal # 230-4)
ACS grade Formic acid- (sigma # f0507)
ACS grade Zinc Sulfate- (sigma # 22,137-6)
ACS grade Ammonium Acetate- (sigma # 73594)
Cyclosporin A - (LC laboratories # c-6000)
Tacrolimus - (LC laboratories # f-4900)
Rapamycin- (LC laboratories # r-5000)
8.
Ascomycin - (sigma # a3835)
9.
1M Ammonium acetate
a.
b.
c.
10.
Add 38.5 g of ammonium acetate to a 500 mL volumetric flask.
Fill to mark with type I water. Mix well to dissolve.
Store in a 500 mL polypropylene bottle. Stable for one year at room
temperature.
2mM ammonium acetate/0.1% formic acid in water - Mobile phase A and purge
solvent
a.
b.
c.
d.
e.
To a 3.5 liter carboy add 3 liters of type I water
With a volumetric pipet add 6 mL of 1 M ammonium acetate
With a volumetric pipet add 3 mL of ACS grade formic acid
Swirl to mix
Store at room temperature. Stable for 1 month
TITLE:
LC/MS/MS
EFFECTIVE DATE:
August 1, 2004
11.
Page 3 of 22
To a 3.5 liter carboy add 3 liters of methanol
With a volumetric pipet add 6 mL of 1 M ammonium acetate
With a volumetric pipet add 3 mL of ACS grade formic acid
Swirl to mix
Store at room temperature. Stable for 1 month
50% Methanol in water - mobile phase C and D
a.
b.
c.
13.
DOCUMENT NO:
CC-80023
2mM ammonium acetate/0.1% formic acid in methanol - Mobile phase B and
wash solvent
a.
b.
c.
d.
e.
12.
REVISION:
H-1
CLINICAL CHEMISTRY
To a 2 liter flask add 1 liter of type I water
Add 1 liter of methanol, swirl to mix.
Store at room temperature, stable for one year.
10% aqueous methanol - seal wash
a.
Using a graduated cylinder add 100 mL hplc grade methanol to a 1 liter
erlenmeyer flask
b.
Add 900 mL type I water, swirl to mix.
c.
Store in a polypropylene bottle at room temperature. Stable for 1 year
14.
0.1M Zinc sulfate - lyse solution
a.
b.
c.
15.
Add 13.1 g of zinc sulfate to a 500 mL volumetric flask.
Fill to mark with type I water, mix well to dissolve.
Store in a polypropylene bottle at room temperature. Stable for one year
5 ng/mL ascomycin in methanol - precipitating solution
a.
Add 50 uL of ascomycin intermediate stock solution to a 50 mL
volumetric flask.
b.
Fill to mark with methanol, swirl to mix.
c.
Store in a polypropylene bottle at room temperature. Make fresh daily.
C.
STANDARD AND QC PREPARATION:
1. Stock Standards
a.
Cyclosporin A stock standard - 1mg/mL
(1)
Weigh out 5 mg of Cyclosporin A and place in a 15 mL blue
TITLE:
LC/MS/MS
EFFECTIVE DATE:
August 1, 2004
(2)
(3)
b.
(2)
(3)
(2)
(3)
max conical tube.
Using a 5 mL volumetric pipet add 5 mL of hplc grade methanol.
Vortex well to dissolve.
Store at - 20°C. Stable for 2 years.
Weigh out 5 mg of Tacroliumus and place in a 15 mL blue max
conical tube.
Weigh out 5 mg of Rapamycin and place in a 15 mL blue max
conical tube.
Ascomycin stock standard - 1 mg/mL
(1)
(2)
(3)
2.
Page 4 of 22
Rapamycin stock standard - 1 mg/mL
(1)
d.
DOCUMENT NO:
CC-80023
Tacrolimus stock standard - 1 mg/ml
(1)
c.
REVISION:
H-1
CLINICAL CHEMISTRY
Weigh out 1 mg of Ascomycin and place in a 15 mL blue max
conical tube.
Intermediate stock standards
a.
Cyclosporin A intermediate stock standard - 20 ug/mL
(1)
(2)
(3)
(4)
b.
Using a volumetric pipet add 0.5 mL of cyclosporin A stock
standard to a 25 mL volumetric flask
Fill to mark with whole blood
Mix on rocker for 2 hours for complete mixing
Store in 15 mL blue max conical tube. Stable at -20°C for 4
months.
Tacrolimus and Rapamycin intermediate stock standard - 2 ug/mL
(1)
add 50 uL each of Tacrolimus and rapamycin stock standards to
TITLE:
LC/MS/MS
EFFECTIVE DATE:
August 1, 2004
(2)
(3)
(4)
c.
DOCUMENT NO:
CC-80023
Page 5 of 22
a 25 mL volumetric flask
Fill to mark with whole blood
Mix on rocker for 2 hours for complete mixing
Store in 15 mL blue max conical tube. Stable at -20°C for 4
months.
Ascomycin intermediate stock standard – 5ug/mL
(1)
(2)
(3)
3.
REVISION:
H-1
CLINICAL CHEMISTRY
To a 10 mL volumetric flask add 8 mL of methanol
Add 50 uL of ascomycin stock standard, fill to mark with
methanol.
Store in a 15 mL blue falcon tube at - 20°C. Stable for 2 years.
Working standards
a.
Thaw standards at room temperature. Mix well on tube rocker for 1 hour
before using.
b.
Add the indicated volume of intermediate stock standards to 10 mL
volumetric flasks using the following list:
Working Standard
#1
#2
#3
#4
#5
#6
c.
d.
4.
Cyclosporin
Int. stock std.
Conc.
ng/mL
5 uL
25 uL
50 uL
250 uL
500 uL
1000 uL
10
50
100
500
1000
2000
Rapamycin
& Tacrolimus
Int. stock std
10 uL
25 uL
50 uL
100 uL
250 uL
400 uL
Conc
ng/mL
2
5
10
20
50
80
Fill to mark with whole blood. Mix well.
Aliquot into 2 mL screw cap vials. Store at 2 – 8 degrees C for 7 days.
Negative Control
a.
b.
c.
Obtain a fresh, EDTA blood sample from the carboxyhemoglobin area.
Extract sample to test for the presence of any immunosuppressive drugs.
If negative, re-label as negative control. Store at 2 – 8 degrees C. Stable
for one week.
TITLE:
LC/MS/MS
EFFECTIVE DATE:
August 1, 2004
5.
d.
b.
c.
Remove vial from freezer and allow to warm to room temperature.
Thoroughly mix by gentle swirling and inversion.
Aliquot 0.5 mL into screw cap vials. Store at 2 – 8 degrees C., stable for
45 days after thawing. Do not refreeze.
Frozen vials are stable at –20 degrees C. until expiration date.
Add 0.5 mL cyclosporine stock standard to a 25 mL volumetric flask
containing 20 mL of methanol.
Add 50 uL each of rapamycin and tacrolimus stock standard; fill to mark
with methanol.
Store at –20 degrees C. in a 15 mL blue falcon tube. Stable for 2 years.
Test mixture:
a.
b.
c.
d.
e.
D.
Page 6 of 22
Test mix intermediate stock standard:
a.
7.
DOCUMENT NO:
CC-80023
Rap/Tac/CsA whole blood controls (More Diagnostics):
a.
b.
c.
6.
REVISION:
H-1
CLINICAL CHEMISTRY
Add 13 mL of deionized water to a 50 mL Erlenmeyer flask.
Add 13 mL of 0.1 M zinc sulfate.
Add 25 mL of methanol.
Add 25 uL of ascomycin intemediate stock standard.
Add 100 uL of test mix intermediate stock standard.
TUNING SOLUTIONS:
1.
Cyclosporine A tuning solution:
a.
b.
c.
d.
2.
Add 16 mL of mobile phase B to a 25 mL Erlenmeyer flask.
Add 4 mL of mobile phase A.
Add 200 uL of Cyclosporine A stock solution. Mix well.
Store in a 15 mL blue max conical tube at –20 degrees C.. Stable for 1
year.
Tacrolimus tuning solution:
a.
b.
c.
d.
Add 200 uL of tacrolimus stock solution. Mix well.
Store in a 15 mL blue max conical tube at –20 degrees C.. Stable for one
year.
TITLE:
LC/MS/MS
EFFECTIVE DATE:
August 1, 2004
3.
Page 7 of 22
Add 200 uL of rapamycin stock solution. Mix well.
Store in a 15 mL blue max conical tube at –20 degrees C. Stable for one
year.
Ascomycin tuning solution:
a.
b.
c.
d.
IV.
DOCUMENT NO:
CC-80023
Rapamycin tuning solution:
a.
b.
c.
d.
4.
REVISION:
H-1
CLINICAL CHEMISTRY
Add 200 uL of ascomycin stock solution. Mix well.
Store in a 15 mL blue max conical tube at –20 degrees C. Stable for 1
year.
METHOD:
A.
STARTUP:
The instrument is always left in the OPERATE mode with the API (nitrogen) gas
flowing.
1.
2.
3.
4.
5.
6.
Check that the nitrogen generator pressure is < 90 psi.
Check that the argon outlet pressure is approx. 10 psi.
Record the level of argon in the tank.
Check the mobile phase and seal wash levels and fill as needed.
Empty the nitrogen waste water and HPLC waste containers.
Click on instrument side bar, click on “inlet method”
a.
b.
c.
d.
e.
7.
Click on “load method” (third icon from right on toolbar)
Click on “waters 2795” and drag down to “wet prime”
Set flow to mL/min. to 5.0
Set solvent B% to 50
Click on “prime”
While still on the inlet method page:
a.
b.
c.
Click on “waters 2795” and drag down to “refresh syringe”
Check that the settings are 6 strokes and replacement volume of 600uL.
Click on ok.
TITLE:
LC/MS/MS
EFFECTIVE DATE:
August 1, 2004
REVISION:
H-1
CLINICAL CHEMISTRY
DOCUMENT NO:
CC-80023
Page 8 of 22
8. On the instrument side bar click on “startup”, this will turn on the collision (argon)
gas and the HPLC system.
9. On the instrument page, click on MS tune, check that the collision cell piriani reads
approx. 3.00e-3; if not adjust by using the knob on the front upper left side of the
quattro micro (just under the tubing labeled NEB).
B.
SAMPLE PRETREATMENT:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
C.
Gently mix all standards, controls, and samples on the tube rocker. If it is the first
run of the day - the negative control, all standards and 4 levels of QC will be run
with the patients. In each of the following runs 2 QC and a negative control need
to be extracted along with the patients.
Label a microcentrifuge tube for each standard, control or patient, with no more
than 24 samples per run.
Using 20-200 uL Finnpipette add 50 uL of each standard, control or patient to it’s
corresponding tube.
Using the eppendorf repeater plus, set to 2.5, with a 5 mL syringe tip, dispense
250 uL of type I water to each tube.
250 uL of lyse solution(zinc sulfate) to each tube.
500 uL of precipitating solution(methanol containing 5 ng/mL ascomycin) to
each tube, capping each tube immediately after adding the
precipitating solution.
Place all tubes on the vortex genie. Vortex for 30 seconds at a setting of 10.
Let tubes sit for 5 minutes.
Centrifuge samples for 5 minutes at 13.4 k in the Abbott microcentrifuge.
Transfer supernatant to a clean labeled centrifuge tube, place each tube in a 24
position microcentrifuge rack, starting with sample one in upper left hand corner.
Load rack into autosampler.
PROGRAMMING A SAMPLE LIST:
1.
2.
3.
4.
5.
On the Mass Lynx page - click on the icon for new sample list, second icon from
the list on the toolbar. A one line sample list will appear.
In the file name box - enter the file name as month, day, year, then A denoting
first run(B as second etc.) then 1 as first sample.
Example: on January 1st 2004, first run, first sample, should read 010104a1.
Leave file text box blank for now.
In MS file box- double click on box, the select file box will appear; double click
on “immun.exp.”
In Inlet file box- double click on box, the inlet methods box will appear, double
click on “immun”.
TITLE:
LC/MS/MS
EFFECTIVE DATE:
August 1, 2004
6.
7.
8.
9.
10.
11.
12.
13.
14.
V.
REVISION:
H-1
CLINICAL CHEMISTRY
DOCUMENT NO:
CC-80023
Page 9 of 22
In the Bottle box- type in position of rack in autosampler followed by a
semicolon, then the bottle position on the rack. For example, a rack in position 1
with a bottle in the first position would be 1:1.
In the Injection box type in 30 (this is an injection volume of 30 uL).
In the Sample type box - double click on box, click on analyte.
Anywhere on the first line click with right mouse button, drag down to “add” and
click on it, the samples box will appear , type in the number of samples to be
added to complete the sample list. Click on ok, the computer will add the samples
and sequentially fill in the test file names and bottle numbers.
In the Text box fill in the QC , calibrator, or patient accession number, pressing
enter after typing in the name ( this moves the cursor down one sample).
In the Sample type box change any calibrator from analyte to standard. Then
enter the value of that standard in the Conc boxes. Conc A = Cyclosporin, Conc
B = rapamycin, Conc C = Tacroliumus.
On the tool bar click on “file”, drag down to “save as”. Enter sample list name as
month, day, year, a for first run, b for second etc. Example the first run on
January 1st 2004 would be saved as: 010104a
Click on the run button - this is the sixth icon on the tool bar, a start sample list
box will appear. Check that the acquire sample data, auto process samples, and
auto quantify samples are all selected. Also check that the correct samples are
being run. Click on ok.
A create quanlynx data set box appears - If it is the first run of the day, the
“integrate samples”, “calibrate standards”, and “quantify samples” boxes should
all be checked. On following runs the calibrate standards box should not be
checked and in the quantify area the curve saved for that day should be selected.
Click on ok.
DATA ANALYSIS:
A.
To review results when the sample list is complete:
1.
2.
3.
4.
5.
6.
On the Masslynx main page, click on “Quanlynx” in the left side bar.
Click on “view results”.
In the Quanlynx page click on “open data set” icon- this is the 1st icon on the
toolbar.
Either type in or scroll to find the sample list needed, click on the sample list,
then click on open.
A window will open containing the sample summary list, a calibration window,
and a chromatogram window.
If it is the first run of the day, check the calibration curve and QC for each
compound. To change between compounds, click on the next compound icon in
the tool bar (10th from the left) or previous compound icon (9th from the left).
TITLE:
LC/MS/MS
EFFECTIVE DATE:
August 1, 2004
7.
d.
10.
11.
B.
Page 10 of 22
Move the cursor over the point and click with the right mouse button.
Click on “exclude”, a box will appear “do you wish to add a comment ?”
Click on no. the computer will now recalculate all the results using the
new curve.
If you want to undo changes to the curve, follow steps a-c.
To save the calibration curves for the daya.
b.
c.
9.
DOCUMENT NO:
CC-80023
If a point needs to be excluded from the calibration curve, do the following:
a.
b.
c.
8.
REVISION:
H-1
CLINICAL CHEMISTRY
Click on file, drag down to export.
Click on calibration, enter the file name as the month, day, and year.
Click on save
Review all samples for correct retention time, peak shape and whether there is
baseline tailing. To scroll down the sample list click on the next sample icon on
the toolbar ( 8th from the left). This will cause the chromatogram window to
change to the corresponding chromatogram.
If everything is ok then click on the printer button on the tool bar, This will cause
the reports to be printed.
If any changes were made remember to save results before closing the page.
To review results as the sample list is running:
1.
2.
3.
4.
5.
6.
7.
On the masslynx main page open the sample list to be reviewed by clicking on
the open sample list icon on the toll bar (3rd from left).
Either type in or scroll to find the sample list, click on the sample list, then click
on open.
Highlight samples to be reviewed by clicking on first sample number and
dragging down to the last number. Only samples that have been completed can be
reviewed.
Click on process samples
A create quanlynx data set box appears - If it is the first run of the day, integrate
samples, calibrate standards, and quantify samples boxes should all be checked.
On following runs the calibrate standards box should not be checked and in the
quantify area the curve saved for that day should be selected. Click on ok.
The quanlynx page will open showing the quantification of the highlighted
samples.
When finished close out the page but don’t save any results.
TITLE:
LC/MS/MS
EFFECTIVE DATE:
August 1, 2004
VI.
DOCUMENT NO:
CC-80023
Page 11 of 22
CALCULATIONS AND METHOD OF REPORTING RESULTS:
A.
Verify results:
1.
2.
3.
B.
Identify peaks by retention time. Make sure correct peaks are being picked
Check integration of each sample.
Take concentration readings off printouts.
Reporting Results:
1.
Analytical measurement range:
a.
b.
c.
Cyclosporine: 10 - 2000 ng/mL
Rapamycin : 2 - 80 ng/mL
Tacroliumus: 2 - 80 ng/mL
(1)
(2)
2.
results less than 10 ng/mL for cyclosporin are reported as “less
than 10 ng/mL” (#16). Results less than 2 ng/mL for tacroliumus
or rapamycin are reported as “less than 2 ng/mL”(#82).
results greater than 2000 ng/mL for cyclosporin or 80 ng/mL for
rapamycin or tacroliumus are to be repeated on dilution: 100 uL
of sample plus 100 uL of negative control. Mix well. Follow
sample pretreatment steps 3-9 using 50 uL of the above dilution.
Multiply answer by 2.
Clinically reportable range:
a.
b.
c.
VII.
REVISION:
H-1
CLINICAL CHEMISTRY
Cyclosporine: 10 – 4000 ng/mL
Rapamycin: 2 – 160 ng/mL
Tacrolimus: 2 – 160 ng/mL
QUALITY CONTROL:
Values for the More Diagnostics whole blood controls, levels 1 - 4, should be within + 2 SD
limits. All four levels must be run on the first run of the day. At least two controls are to be run on each
subsequent run. All controls must fall within acceptable range. If controls are not within acceptable range,
repeat controls, troubleshoot, or recalibrate assay as needed. Notify section coordinator if still
unacceptable.
TITLE:
LC/MS/MS
EFFECTIVE DATE:
August 1, 2004
VIII.
REVISION:
H-1
CLINICAL CHEMISTRY
DOCUMENT NO:
CC-80023
Page 12 of 22
REFERENCE RANGE:
A.
Cyclosporin
1.
2.
Therapeutic range: 100 - 500 ng/mL (trough)
Critical values:Trough = > 750 ng/mL
Peak = > 1500 ng/mL
Call trough values > 500 mg/mL to the physician or nurse. Call peak values >1500
ng/mL to physician or nurse. All values greater than > 750 ng/mL must be written in the
overdose book for follow up by the laboratory resident.
B.
Tacrolimus and Rapamycin:
1.
2.
Therapeutic range: 5 - 20 ng/mL (trough)
Critical value >25 ng/mL.
Call results >20 ng/mL to the physician or nurse. All values >25 ng/mL must be written
in the overdose book for follow up by the laboratory resident.
IX.
MAINTENANCE:
A.
Daily
1.
2.
3.
4.
5.
6.
Check waste bottles empty as needed
Check argon and nitrogen tank pressure
Record argon tank volume - change tank when below 500
Preform wet prime and refresh syringe
Check collision cell piriani
Make working internal standard solution
B. Weekly
1.
Gas ballast rotary pump
a.
b.
c.
d.
e.
Shut off vacuum system isolation valve by moving handle to the right.
Turn gas ballast control on top of rotary pump completely
counterclockwise.
Leave open for 20 minutes to drain oil.
Close gas ballast knob by turning clockwise.
Open vacuum system isolation valve by moving handle to the left.
TITLE:
LC/MS/MS
EFFECTIVE DATE:
August 1, 2004
2.
3.
DOCUMENT NO:
CC-80023
Page 13 of 22
Clean centrifuge.
Clean cone gas cone and sample cone
a.
b.
c.
d.
e.
f.
g.
h.
i.
j.
k.
l.
m.
n.
X.
REVISION:
H-1
CLINICAL CHEMISTRY
In tune page, set source temperature to 20° C; when cool, turn off API
gas.
Open source enclosure cover by pulling clips forward, releasing the door.
Turn isolation valve fully to the right.
Remove PTFE tube attached to the cone gas cone.
Remove the two screws that secure the cone retainer clip using the 2.5
mm hex wrench. Remove the retainer clip.
Remove the cone gas cone and sample cone from the ion block.
Carefully separate the gas cone from the sample cone, being careful not
to drop the sample cone. Remove the O ring.
Place sample cone and gas cone with tips facing upwards into a beaker
containing 50% methanol/water. If parts are very dirty, use a solution of
45% methanol, 45% water, and 10% formic acid.
Place beaker in sonicator for 30 minutes. If formic acid was used during
cleaning, then sonicate parts again in a beaker of distilled water for 20
minutes, and then in a beaker of methanol for 10 minutes.
Carefully remove one part at a time from the beaker to a kimwipe and
then blow the part completely dry with nitrogen.
Reassemble cone gas cone, sample cone, and O ring.
Secure back on instrument with retainer clip and two screws- do not over
tighten the screws!
Replace PTFE tubing- move isolation valve to the open position (to the
left).
Turn on API gas and return source temperature to 120° C.
SPECIAL NOTES:
A. Clinical Significance:
Cyclosporine, tacrolimus and sirolimus are immunosuppressant with widespread
application in the suppression of immune-mediation rejection of transplanted organs.
Although differences exist (detailed below), they all function primarily by inhibition of T
cell-mediated immunity.
Cyclosporine is a cyclic polypeptide immunosuppressant consisting of 11 amino acids. It is
produced as a metabolite of the fungus species Tolypocladium inflatum Gams. The
mechanism of action of cyclosporine is not fully known; however, its actions appear to be
dependent upon binding to intracellular sites of action. Because of its high lipophilicity,
cyclosporine enters cells easily to gain access to the site of action. The intracellular protein
TITLE:
LC/MS/MS
EFFECTIVE DATE:
August 1, 2004
REVISION:
H-1
CLINICAL CHEMISTRY
DOCUMENT NO:
CC-80023
Page 14 of 22
most closely linked to the immunosuppressive activity of cyclosporine is cyclophilin. By
binding to cyclophilin, the antigenic response of helper T lymphocytes is inhibited; the
production of interleukin-2 and interferon-gamma is suppressed. In addition, production of
the receptor site for interleukin-2 on T lymphocytes is inhibited by cyclosporine. The action
of macrophages does not appear to be affected by cyclosporine. Cyclosporine has also been
shown to bind with low affinity to calmodulin. Binding to calmodulin, at high
concentrations may interfere with phosphorylation of proteins. The significance of the
effect of cyclosporine on calmodulin is controversial, but may be related to toxicity.
Tacrolimus is a macrolide antibiotic produced by the soil fungus Streptomyces
tsukubaensis. It suppresses both humoral (antibody) and cell-mediated immune responses.
The compound is chemically distinct from cyclosporine but both agents elicit similar
immunosuppressant effects. The immunosuppressive activity of tacrolimus is, however,
more marked than that of cyclosporine. In vitro, tacrolimus prevents activation of T
lymphocytes in response to mitogenic or antigenic stimulation. Studies on cultured CD4+
(T-helper) lymphocytes have demonstrated that tacrolimus is at least 100 times more potent
than cyclosporine (weight basis) in selectively inhibiting secretion of various cytokines
(i.e., interleukin-2, interleukin-3, interferon-gamma). The exact mechanism of action for
tacrolimus is unknown; however, tacrolimus inhibits T-lymphocyte activation. The
proposed mechanism for this effect is binding to an intracellular protein, FKBP-12. A
complex is formed which inhibits the phosphatase activity of calcineurin. This may prevent
dephosphorylation and translocation of nuclear factor of activated T-cells which is believed
to initiate gene transcription for lymphokines. This results in inhibition of T-lymphocyte
activation.
Sirolimus is a macrocyclic triene antibiotic (structurally related to tacrolimus) with
immunosuppressive, antitumor, and antifungal properties; it is produced by fermentation of
Streptomyces hygroscopicus (actinomycete). Sirolimus has been demonstrated to block the
response of T- and B-cell activation by cytokines, which prevents cell-cycle progression
and proliferation; in contrast, tacrolimus (FK-506) and cyclosporine inhibit the production
of cytokines. Intracellularly, sirolimus forms a complex with cytosolic FK-binding proteins
(FKBPs; immunophilins), primarily FKBP-12, considered essential for functionality; the
sirolimus-FKBP complex binds to and modulates the activity of a sirolimus effector protein
(SEP), with resultant cell-cycle arrest in the G1 to S phase of the cell cycle. In contrast,
tacrolimus (and cyclosporine) block T-cell cycle progression at the G0 to G1 stage. Like
sirolimus, tacrolimus also forms a complex with FKBP12, whereas cyclosporine complexes
with a cyclophilin (mainly cyclophilin A); both complexes subsequently inhibit the
phosphatase calcineurin, with resultant inhibition of cytokine gene transcription. Sirolimus
appears to be less nephrotoxic than cyclosporine and tacrolimus; this may be related to a
decreased effect on calcineurin. In preclinical studies (in vitro, in vivo), additive or
synergistic immunosuppressive effects were observed when sirolimus was combined with
tacrolimus, cyclosporine, mycophenolate mofetil, and brequinar. However, antagonistic
effects of sirolimus and tacrolimus have reported in some in vitro immunologic models. In
addition to transplantation applications, preclinical data suggest a potential role for
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sirolimus in corticosteroid-resistant chronic asthma and rheumatoid arthritis.
B.
Reference Ranges:
1. Cyclosporine
Trough (C0) Values: 100 – 500 ng/mL
Peak (C2) Values:
Target Cyclosporine Peak(C2) Levels for Renal Transplant (+/- 20%)
1 month posttransplant: 1500 - 2000 ng/mL
2 months posttransplant:
1500 ng/mL
1300 ng/mL
4 – 6 months posttransplant:
1100 ng/mL
900 ng/mL
> 12 months posttransplant:
800 ng/mL
Target Cyclosporine Peak(C2) Levels for Liver Transplant (+/- 20%)
1000 ng/mL
800 ng/mL
600 ng/mL
Critical Values: call all trough values > 500 ng/mL or peak values > 1500 ng/mL to
physician or nurse.
2. Tacrolimus
Critical Values: call all values > 25 ng/mL to physician or nurse.
3. Sirolimus
Critical Values: call all values > 25 ng/mlLto physician or nurse.
C. Pharmacokinetics:
1.
Cyclosporine
a. Dosage and Administration
Cyclosporine is available as a traditional suspension known as Sandimmune® (Novartis
Pharma, Basle, Switzerland) and a newer “microemulsion” with improved
bioavailability known as Neoral® (Novartis Pharma, Basle, Switzerland). The two
formulations are non-equivalent and cannot be used interchangeably.
Dosage, Oral: Dependent upon type of transplant and formulation:
Neoral®
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Renal: 9 ± 3 mg/Kg/day, divided twice daily
Liver: 8 ± 4 mg/Kg/day, divided twice daily
Heart: 7 ± 3 mg/Kg/day, divided twice daily
Sandimmune®
Initial dose of 14 – 18 mg/kg/day for non-renal and 10 – 14 mg/kg/day for
renal transplants. Continue initial dose for 1 – 2 weeks and then taper by 5%
per week to a maintenance dose of 5 – 10 mg/kg/day or as low as 3
mg/kg/day for renal transplants.
Dosage, IV: Only the Sandimmune formulation may be given intravenously at an initial
dose of 5 – 6 mg/kg/day, infused over 2 – 6 hours. However, given the risk of
anaphylaxis during IV administration of cyclosporine, use should be limited to those
patients unable to tolerate oral dosing and patients should be switched to oral
administration as quickly as possible.
b. Absorption
Absorption of the Sandimmune formulation is erratic, incomplete and strongly
dependent on the patient population, ranging from less than 10% in liver transplant
patients to as great as 89% in renal transplant patients. Absorption also depends on the
presence of food and bile acids and GI motility. Neoral is better absorbed and less
dependent on food, bile acids and GI motility compared to Sandimmune, but still erratic
and incomplete. Overall cyclosporine exposure as determined by mean relative AUC
for Neoral compared to Sandimmune ranged from 1.24 ± 0.34 to 1.51 ± 0.59. The dose
normalized AUC in de novo renal transplant patients dosed with Neoral was 23%
greater than in those patients dosed with Sandimmune. The dose normalized AUC in de
novo liver transplant patients dosed with Neoral 28 days after transplantation was 50%
greater than in those patients administered with Sandimmune. The increase in AUC is
accompanied by an increase in peak blood Cyclosporin concentration (Cmax) in the
range of 40% to 106% in renal transplant patients and approximately 90% in liver
transplant patients. AUC and Cmax are also increased (Neoral relative to Sandimmune)
in heart transplant patients, but data are very limited. Although the AUC and Cmax
values are higher on Neoral relative to Sandimmune, the pre-dose trough concentrations
(dose-normalized) are similar for the two formulations. This resulted in the
implementation of peak (C2 – two hours after the dose) levels for monitoring of
cyclosporine, which better correlate with total drug exposure (AUC) and, consequently,
both efficacy and toxicity.
c. Distribution
Cyclosporine is distributed largely outside the blood volume. The steady state volume
of distribution during intravenous dosing has been reported as 3-5 L/Kg in solid organ
transplant patients. In blood, the distribution is concentration dependent. Approximately
33-47% is in plasma, 4-9% in lymphocytes, 5-12% in granulocytes, and 41-58% in
erythrocytes. At high concentration, the binding capacity of leukocytes and erythrocytes
becomes saturated. In plasma, approximately 90% is bound to proteins, primarily
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lipoproteins. Cyclosporine is secreted in human milk.
d. Metabolism and Elimination
Cyclosporin is extensively metabolized by the cytochrome P-450 III-A enzyme system
in the liver, and to a lesser degree in the gastrointestinal tract, and the kidney. At least
25 metabolites have been identified from human bile, feces, blood and urine. The
biological activity of the metabolites and their contributions to toxicity are considerably
less than those of the parent compound. Only 0.1% of the dose is excreted in the urine
as unchanged drug. Elimination is primarily biliary with only 6% of the dose (parent
drug and metabolites) excreted in the urine. Neither dialysis nor renal failure alters
cyclosporine clearance significantly. The terminal half-life in plasma or blood is 19
hours (range 10-27 hours) for Sandimmune and approximately 8.4 hours (range 5 to 18
hours) for Neoral.
e. Potential Toxicity and Adverse Reactions
The principal adverse reactions of cyclosporine therapy are renal dysfunction, tremor,
hirsutism, hypertension, and gum hyperplasia. Rare reactions are allergy, anemia,
anorexia, confusion, conjunctivitis, edema, fever, brittle fingernails, gastritis, hearing
loss, hiccups, hyperglycemia, muscle pain, peptic ulcer, thrombocytopenia, tinnitus,
anxiety, chest pain, constipation, depression, hair breaking, hematuria, joint pain,
lethargy, mouth sores, myocardial infarction, night sweats, pancreatitis, pruritus,
swallowing difficulty, tingling, upper GI bleeding, visual disturbance, weakness or
weight loss. Cyclosporine can cause nephrotoxicity and hepatoxicity when used in high
doses. It is not unusual for serum creatinine and BUN levels to be elevated during
cyclosporine therapy. These elevations in renal transplant patients do not necessarily
indicate rejection and each patient must be fully evaluated before dosage adjustment is
initiated. Hypertension is a common side effect of cyclosporine therapy. Mild or
moderate hypertension is encountered more frequently than severe hypertension and the
incidence decreases over time. Antihypertensive therapy may be required. Control of
blood pressure can be accomplished with any of the common antihypertensive agents.
However, since cyclosporine may cause hyperkalemia, potassium-sparing diuretics
should not be used. Calcium antagonists can be effective agents in treating cyclosporine
associated hypertension. However, care should be taken since interference with
cyclosporine metabolism may require dosage adjustment.
f. Blood Level Monitoring
Blood level monitoring of cyclosporine is useful in patient management. The 24 h
trough values of 100-500 ng/mL (whole blood) are suggested to minimize side effects
and rejection events. This range does not apply for patients who are being dosed twice
daily. Values from 150-350 ng/mL have been proposed as targets for 12 hour troughs.
More recently, peak blood levels drawn two hours after an oral dose have been
demonstrated to better correlate with total drug exposure and both the efficacy and
toxicity of cyclosporine. Optimal peak values continue to be defined; however, a
consensus statement from the Consensus on Neoral C2: ExpertReview in
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Transplantation (CONCERT) Conference in Paris, France recommended the following
target values.
Target Cyclosporine Peak(C2) Levels for Renal Transplant (+/- 20%)
1 month posttransplant: 1500 - 2000 ng/mL
1500 ng/mL
1300 ng/mL
1100 ng/mL
900 ng/mL
800 ng/mL
Target Cyclosporine Peak(C2) Levels for Liver Transplant (+/- 20%)
1000 ng/mL
800 ng/mL
600 ng/mL
Renal and liver functions should be assessed repeatedly by measurement of BUN,
serum creatinine, serum bilirubin, and liver enzymes.
2.
Tacrolimus
Tacrolimus is a potent macrolide immunosuppressant derived from Streptomyces
tsukubaensis, and has actions similar to those of cyclosporine. It is used to prevent or
reverse rejection in patients receiving organ transplants and has been tried in a few
patients with refractory auto-immune or immune-mediated disorders. Tacrolimus is
also applied topically in the management of moderate to severe atopic eczema.
For transplantation, the initial dose recommended in the UK for liver graft recipients is
100 to 200 micrograms/Kg daily by mouth, in 2 divided doses. The initial oral dose in
kidney transplant patients is 150 to 300 micrograms/Kg daily in 2 divided doses.
Administration should begin about 6 hours after completion of liver grafting and within
24 hours of a kidney transplant. If the patient's condition does not permit oral
administration, therapy may be commenced intravenously, by continuous 24-hour
infusion: suggested initial doses are 10 to 50 micrograms/Kg daily for liver transplants
and 50 to 100 micrograms/Kg daily for kidney transplants. In the USA, initial oral
doses in patients with liver grafts are 100 to 150 micrograms/Kg daily, in 2 divided
doses, and in kidney grafts 200 micrograms/Kg daily, in 2 divided doses. The
recommended starting dose of intravenous tacrolimus is 30 to 50 micrograms/Kg daily.
The manufacturer recommends that oral tacrolimus should be taken consistently with
respect to the timing of ingestion of food. Children generally require doses 1.5 to 2
times greater than those recommended in adults to achieve the same blood
concentrations.
b. Absorption
Absorption of tacrolimus following oral administration is reported to be erratic. Peak
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serum levels are seen 1 to 3 hours after an oral dose. Oral bioavailability varies widely,
although a bioavailability of about 15 to 20% seems common. Bioavailability of
tacrolimus appears to be influenced by the type and timing of meals. Food, particularly
high-fat meals, significantly reduced bioavailability, compared with the fasting state.
Ingestion of tacrolimus up to 1.5 hours after a meal also considerably reduced
absorption. The manufacturer therefore recommends that tacrolimus should be taken
consistently with respect to the timing of ingestion of food. Gastrointestinal metabolism
of tacrolimus is thought to be extensive, significantly affecting its bioavailability, and
differences in this metabolism may account for apparent differences in availability
according to ethnic origin.
c. Distribution
Following intravenous administration it is widely distributed to the tissues; in the blood,
about 80% is bound to erythrocytes, and variations in red cell binding account for much
of the variability in pharmacokinetics. The portion in plasma is approximately 99%
bound to plasma proteins.
Tacrolimus is extensively metabolised in the liver, principally by cytochrome P450
isoenzyme CYP3A4, and excreted, primarily in bile, almost entirely as metabolites.
Some metabolism may occur in the gastrointestinal tract. Less than 2% of the
administered dose is recovered in the urine. Whole-blood elimination half-life has been
reported to range from about 12 to 16 hours in transplant patients. The half-life of
tacrolimus can be increased by hepatic dysfunction.
Systemic exposure to tacrolimus may produce nephrotoxicity and neurotoxicity. The
most common adverse effects after systemic administration include tremor, headache,
paraesthesias, nausea and diarrhea, hypertension, leucocytosis, and impaired renal
function. Anemia, leucopenia, and thrombocytopenia also occur commonly.
Disturbances of serum electrolytes, notably hyperkalaemia occur frequently. Other
adverse effects include mood changes, sleep disturbances, confusion, dizziness, tinnitus,
visual disturbances, and convulsions; disturbances of carbohydrate metabolism or frank
diabetes mellitus; ECG changes and tachycardia, as well as hypertrophic
cardiomyopathy (particularly in children); constipation, dyspepsia, and gastrointestinal
hemorrhage; dyspnea, asthma, pleural effusions; alopecia, hirsutism, skin rash and
pruritus; and arthralgia or myalgia, spasm, leg cramps, peripheral edema, liver
dysfunction, and coagulation disorders.
Tacrolimus injection is formulated with polyethoxylated castor oil: anaphylactoid
reactions have occurred, and appropriate means for their management should be
available in patients given the injection. Use of tacrolimus should be avoided in patients
hypersensitive to macrolides. Dosage reduction may be necessary in patients with
hepatic impairment. Care is also required in patients with pre-existing renal impairment,
and dosage reduction may prove advisable in such patients. Monitoring of blood
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concentrations of tacrolimus is recommended in all patients. Renal and hepatic
function, blood pressure, serum glucose and electrolytes, and hematological and cardiac
function, as well as visual function should be monitored regularly. As with other
immunosuppressants, patients receiving tacrolimus are at increased risk of infection and
malignancy. Intra-uterine devices should be used with caution during
immunosuppressive therapy as there is an increased risk of infection. Concomitant use
of live vaccines should be avoided for the same reason. Tacrolimus may affect visual or
neurological function, and patients so affected should not drive or operate dangerous
machinery.
Twelve hour tacrolimus trough whole blood concentrations should be measured in all
patients. The therapeutic trough whole blood concentration is in the range of 5 – 20
ng/mL for adult and pediatric liver transplant patients during months 1 to 12. For adult
kidney transplant patients, the recommended trough whole blood concentrations are 7 –
20 ng/mL and 5 – 15 ng/mL at months 1 to 3 and months 4 to 12, respectively. Renal
and hepatic function tests and serum electrolytes are also indicated.
3.
Sirolimus
Sirolimus oral solution and tablets are recommended as a component of a regimen that
includes cyclosporine and corticosteroids. A 1-time loading dose of 6 milligrams (mg)
is administered as soon as possible after kidney transplantation. The maintenance
dosage is 2 mg administered once daily. For patients 13 years or older who weigh less
than 40 Kg, the recommended loading dose is 3 mg/square meter (m(2)). The
maintenance dosage is 1 mg/m(2)/day. Sirolimus should be taken consistently with or
without food. Sirolimus should be taken 4 hours after cyclosporine capsules or oral
solution.
In patients with low to moderate immunological risk of organ rejection, withdrawal of
cyclosporine is recommended 2 to 4 months after renal transplantation. At 2 to 4
months posttransplant, cyclosporine should be discontinued over a 4 to 8 week period.
Sirolimus dose should be adjusted to achieve whole blood trough concentrations
between 12 and 24 ng/mL. Sirolimus concentrations will decrease when cyclosporine is
discontinued unless sirolimus dose is increased. The sirolimus dose will need to be
approximately 4 fold higher in the absence of cyclosporine.
Due to the long half-life of sirolimus, frequent dose adjustments can lead to overdosing
or underdosing. Once a maintenance sirolimus dose is adjusted, no additional
adjustments should be made for at least 7 to 14 days. Maximum sirolimus dose is 40
milligrams per day (mg/day). If addition of loading dose causes estimated daily dose to
exceed 40 mg/day, the loading dose should be administered over 2 days. Monitor
sirolimus trough concentrations for at least 3 to 4 days after loading dose(s). Dosage
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adjustment of sirolimus is NOT required with renal impairment. Similar to tacrolimus
(which is also metabolized in the liver), dose reductions should be considered for
sirolimus in patients with hepatic dysfunction, in which case close monitoring of wholeblood concentrations is indicated.
b. Absorption
Peak concentrations are reached in 1 to 3 hours after an oral dose of sirolimus with 15 –
30% biovailability. After administration of the oral solution and tablets with a high fat
meal, the maximum concentration was reduced, and the time to maximum
concentration was increased. The total exposure to drug (AUC) was also increased. To
reduce variability of exposure to drug, sirolimus should be taken consistently with or
without food.
c. Distribution
At steady state, the total body volume of distribution is 12 L/Kg. There is extensive
uptake by formed blood elements. The highest concentration is found in red blood cells
(95%), followed by plasma (3%), lymphocytes (1%), and granulocytes (1%); usual
blood/plasma ratios are about 30. Sequestration in erythrocytes is presumably related to
their high content of the intracellular protein FKBP-12, and is higher with sirolimus
than with cyclosporine or tacrolimus.
Similar to tacrolimus and cyclosporine, sirolimus is extensively metabolized in the liver
and is substrates for CYP450 IIIA4 and p- glycoprotein; it is also extensively
metabolized by O-demethylation and/or hydroxylation. Sirolimus has an extremely long
half-life of 57 – 63 hours, in renal transplant patients on a steady dosing regimen.
The most frequent adverse effects of sirolimus include peripheral edema, lymphocele,
hyperlipidaemia, hypercholesterolaemia, hypertension, asthenia, gastrointestinal
disturbances, acne, pain, and headache. Anemia, thrombocytopenia, and leucopenia
have occurred, especially at higher doses. Arthralgia, hypokalaemia, and tremor are
frequent. Infections, including cytomegalovirus and Pneumocystis carinii pneumonia,
are also common, and antimicrobial prophylaxis for pneumonia is recommended for the
first year following transplantation. Renal impairment may occur, and there are reports
of hepatotoxicity, and rarely, fatal hepatic necrosis. Excess mortality, graft loss, and
hepatic artery thrombosis has been associated with the use of sirolimus in
immunosuppressive regimens in liver transplant recipients and therefore use in such
patients is not recommended. Interstitial lung disease has been reported, including some
fatalities, although other cases resolved upon discontinuation or dose reduction of
sirolimus. Abnormal wound healing following transplant surgery has been reported
with the use of sirolimus; bronchial anastomotic dehiscence, including some fatal cases,
has occurred in lung transplant recipients and such use is not recommended.
Immunosuppressants may reduce the response to vaccines, and the use of live vaccines
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should be avoided. Intra-uterine devices should be used with caution during
immunosuppressive therapy, as there is an increased risk of infection.
An excellent correlation exists between trough whole-blood levels and the area under
the time-concentration curve (AUC) for sirolimus. Therefore, trough whole-blood
levels, drawn within 1 hour prior to the next oral dose, are recommended for monitoring
therapy. Effective prevention of rejection have been observed with trough levels in the
range of 4 – 20 ng/mL. Because of the extremely long half-life of sirolimus, new
steady-state concentrations occur 5 to 7 days after initiation of therapy or a change in
dosage. Whole blood trough concentrations of sirolimus should be monitored if
cyclosporine dosage is markedly changed or discontinued (as in cyclosporine- sparing
regimen). Additional recommended laboratory monitoring parameters include
lipid/lipoprotein profile, complete blood counts with differential, renal and liver
function tests, and blood glucose periodically during therapy. Closely monitor renal
function with long-term use of sirolimus in combination with cyclosporine. In patients
with elevated or increasing serum creatinine levels, consider adjustment of
immunosuppressive regimen, including discontinuation of sirolimus and/or
cyclosporine.
XI.
REFERENCES:
1. Levy, G. et al. (2004) “Patient Management by Neoral C2 Monitoring: An International
Consensus Statement.” Transplantation 73, S12 – S19.
2. Cyclosporine, tacrolimus and sirolimus entries in the MICROMEDEX® Healthcare Series,
Thomson MICROMEDEX, Greenwood Village, Colorado (Series Vol. 121 expires 9/2004).
XII. HISTORY:
H-1 Written by Katherine Rogers, 08/04.

IMMUNOSUPPRESSANT DRUGS BY LC/MS/MS Page 1 of 22

Transcription

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