SIMULTANEOUS QUANTIFICATION OF 10 CYTOTOXIC DRUGS BY …
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Pharmacie des Hôpitaux Universitaires de Genève SIMULTANEOUS QUANTIFICATION OF 10 CYTOTOXIC DRUGS BY A VALIDATED LC-ESI-MS/MS METHOD Susanne Nussbaumer, Sandrine Fleury-Souverain, Paola Antinori, Farshid Sadeghipour, Pascal Bonnabry, Jean-Luc Veuthey, Laurent Geiser Drug Analysis 2010 21-24 September 2010, Antwerp, Belgium
Transcript of SIMULTANEOUS QUANTIFICATION OF 10 CYTOTOXIC DRUGS BY …
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SIMULTANEOUS QUANTIFICATION OF 10 CYTOTOXIC
DRUGS BY A VALIDATED LC-ESI-MS/MS METHOD
Susanne Nussbaumer, Sandrine Fleury-Souverain, Paola Antinori, Farshid Sadeghipour,
Pascal Bonnabry, Jean-Luc Veuthey, Laurent Geiser
Drug Analysis 2010
21-24 September 2010, Antwerp, Belgium
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HUG – Geneva University Hospitals - Pharmacy
2000 beds
50’000 hospitalizations/year
Geneva University Hospitals
Pharmacy
50 cytotoxic preparations per day Patient administrationPrescription
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High risk formulations
Environmental
Contamination (waste water, air, surfaces,…)
Patient: - medication error
- therapeutic drug monitoring
Health care professionalsContact with cytotoxic agents during preparation steps
Need of simple, validated methods for the
determination of different cytotoxic agents
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Aim of the work
- Quality control
- Environmental samples
Overview of published work
Our goal
Environmental:
- specific method for one single drug
- multi-compounds method
Biological sample analysis:
one compound/family with metabolites
Quality control: one compound or
HPLC-UV/Vis – FIA assays
1 method – 1 application area
validation ?
Generic multi-compounds
method
Complete validation
Different applications
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Cytotoxic preparations at HUG pharmacy in 2009
0 200 400 600 800 1000 1200 1400
fluorouracil
cisplatine
cyclophosphamide
etoposide phosphate
gemcitabine
carboplatine
irinotecan
doxorubicin
cytarabine
epirubicin
paclitaxel
methotrexate
oxaliplatine
docetaxel
mitomycine
ifosfamide
azacitidine
vincristine
bortezomib
fludarabine
analyzed (> 50%)
20 most prepared drugs
not analyzed
Nb of preparation per year
Compromise:
- prescription frequency
- toxicity
- analytical considerations
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Analytical strategy = LC-ESI-MS/MS
HPLC
Flow rate: 0.2 ml/min
Time[min]
Water(%)
Aceto-nitrile (%)
Formicacid 1%
0 88 2 10
2.0 88 2 10
2.5 69 21 10
10 69 21 10
13 60 30 10
13.5 40 50 10
15.5 40 50 10
16 88 2 10
21 88 2 10
Column Zorbax C18 SB (Agilent)
2.1 x 100 mm 3.5 μm
1.97
2.62
00
50100 6.00
6.02
7.49
11.3512.18
13.66
15.33
Time (min)
15.26
00
50100
00
50100
00
50100
00
50100
00
50100
0 4 8 12 16 200
50100
00
50100
00
50100
14.81
Irinotecan
Ifosfamide
Etoposide phosphate
Gemcitabine
Cyclophosphamide
Methotrexate
Cytarabine
Vincristine
Doxorubicin
Epirubicin
[13C, 2H3]
Methotrexate
Rela
tive
Ab
un
da
nce
Injection: 25 μL
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Analytical strategy = LC-ESI-MS/MS
Tandem mass spectrometry
Susanne Nussbaumer, Drug Analysis 2010, Antwerp, Belgium
ESI parameters
Spray voltage 4000 V
Sheath gaz pressure 45 psi
Aux gaz pressure 2 psi
Capillary
temperature
325°C
Drug Parent
(m/z)
Product
(m/z)
CE
(eV)
cytarabine 244.0 112.3 15
gemcitabine 264.7 112.3 20
methotrexate 455.2 308.0 20
[13C, 2H3]
methotrexate
459.2 312.2 20
Etoposide
phosphate
691.0 691.0 15
ifosfamide
cyclophosphamide
261.1 92.3 , 140.2
154.1, 232.9
20
irinotecan 587.9 587.3 20
doxorubicin
epirubicin
544.6 379.2,397.1 15
vincristine 413.3 353.2 30
MS/MS conditions
7 time segments - 9 scan events
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Validation
Calibration model
200
100
50
25
20
10
5
2.5
2
1
0.5
0.25
200
100
50
25
20
10
5
2.5
2
1
0.5
0.25
CS
[ng·mL-1]
VS
[ng·mL-1]
Serie 1
Injection in triplicate
CS
[ng·mL-1]
VS
[ng·mL-1]
Serie 2
Injection in triplicate
CS
[ng·mL-1]
VS
[ng·mL-1]
Serie 3
Injection in triplicate
200
100
50
25
20
10
5
2.5
2
1
0.5
0.25
200
100
50
25
20
10
5
2.5
2
1
0.5
0.25
200
100
50
25
20
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5
2.5
2
1
0.5
0.25
200
100
50
25
20
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2
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0.5
0.25
Validation protocol with simple sample preparation:
3 series at 12 concentration levels, calibration standard (CS), validation standard (VS)
Internal standard:[13C, 2H3] methotrexate
(50 ng·mL-1)
Quality control
Environmental
monitoring
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Validation results
Cytarabin Gemcitabin Methotrexat Etoposide
phosphate
Ifosfamid Cyclophos-
phamid
Irinotecan Doxo
rubicin
Epirubicin Vincristin
Trueness 99-102% 96-109% 97-104% 99-106% 98-101% 99-104% 90-106% 86-103% 89-103% 91-100%
Repeatability 1-3% 1-5% 1-2% 1-3% 1-8% 1-4% 1-4% 2-10% 2-9% 1-7%
Intermediate
precision
1-4% 2-9% 1-2% 1-7% 2-8% 2-5% 3-10% 3-10% 2-13% 1-10%
LOD [ng·mL-1] 0.025 0.25 0.01 0.5 0.25 0.25 0.025 0.5 0.5 0.25
LOQ [ng mL-1] 0.25 0.5 0.25 1 1 0.5 1 2 2 1
Linearity
[ng·mL-1]
1-200 1-200 1-200 1-200 1-200 1-200 1-200 2-200 2-200 1-200
r2 0.9996 0.9994 0.9997 0.9994 0.9993 0.9990 0.9992 0.9962 0.9976 0.9988
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Validation results: cyclophosphamide
Susanne Nussbaumer, Drug Analysis 2010, Antwerp, Belgium
concentration [ng/mL]
0 50 100 150 200
30%
0%
30%
Re
lati
ve
bia
is
Environmental monitoring
1-200 ng/mL: 30%
Calibration model: weighted linear regression (1/x)
Cyclophosphamide
Trueness 99-104%
Repeatability 1-4%
Intermediate precision 2-5%
LOD [ng·mL-1] 0.25
LOQ [ng mL-1] 0.5
Linearity range [ng·mL-1] 1-200
Determination coefficient (r2) 0.9990
LOQ: 0.5 ng/mL
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Environmental application: spiked surfaces
0%
20%
40%
60%
80%
100%
cyta
rab
ine
ge
mc
ita
bin
e
me
tho
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xa
te
eto
po
sid
e p
ho
sp
ha
te
ifo
sfa
mid
e
cyc
lop
ho
sp
ha
mid
e
irin
ote
ca
n
do
xo
rub
icin
e
vin
cri
sti
ne
ep
iru
bic
in
polypropylene infusion bag
stainless steel
Reco
very
Wiping (with blotting paper)
Extraction in formic acid 0.1%
by ultrasonication
Contamination of 2 surfaces:
- Stainless steel
- Polypropylene infusion bag
LC-MS/MS analysis
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Validation results: cyclophosphamide
Susanne Nussbaumer, Drug Analysis 2010, Antwerp, Belgium
concentration [ng/mL]
0 50 100 150 200
30%
0%
30%
Re
lati
ve
bia
is
Environmental monitoring
1-200 ng/mL: 30%
Quality control at
200 ng/mL: 5%
Calibration model: weighted linear regression (1/x)
Cyclophosphamide
Trueness 99-104%
Repeatability 1-4%
Intermediate precision 2-5%
LOD [ng·mL-1] 0.25
LOQ [ng mL-1] 0.5
Linearity range [ng·mL-1] 1-200
Determination coefficient (r2) 0.9990
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Quality control of drug formulations by LC-MS/MS
CYT/10-115058
Gemcitabine 1800 mg in 340 mL NaCl 0.9%
97% ± 2%
CYT/10-114026
Methotrexate 30 mg in 62 mL glucose 5%
97% ± 2%
CYT/10-113521
Ifosfamide 2316 mg in 1080 mL glucose 5%
97% ± 2%
CYT/10-116360
Cyclophosphamide 860 mg in 293 mL glucose 5%
100% ± 2%
CYT/10-116778
Doxorubicin 44 mg in 72 mL in NaCl 0.9%
106% ± 4%
CYT/10-115322
Epirubicin 190 mg in 145 mL NaCl 0.9%
98% ± 4%
CYT/10-116578
Vincristine 1 mg in 51 mL glucose 5%
93% ± 2%
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Conclusion
Generic LC-MS/MS method for the simultaneous quantification of 10 cytotoxic drugs
LC-ESI-MS/MS method has exhibited
good quantitative performance
Application for quality control of
pharmaceutical formulations and
environmental samples.
Safety for patient and operator
handling cytotoxic compounds
Susanne Nussbaumer, Drug Analysis 2010, Antwerp, Belgium
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Acknowledgement
Susanne Nussbaumer, Drug Analysis 2010, Antwerp, Belgium
Pharmacy, Geneva University Hospitals (HUG),
Switzerland
Swiss Centre for Applied Human Toxicology, Geneva,
Switzerland
School of Pharmaceutical Sciences, University of Geneva,
University of Lausanne, Switzerland
