BSMS 7th Annual Meeting High throughput LC High throughput LC - - MS/MS of MS/MS of Pyrimethamine Pyrimethamine and and Sulfadoxine Sulfadoxine in limited volume plasma samples: in limited volume plasma samples: combining selectivity from chromatography and fast ion combining selectivity from chromatography and fast ion separation by MS separation by MS M.P. M.P. Bouche Bouche 1 1 P. P. Risha Risha ² ² , T. , T. Decaestecker Decaestecker 1 1 , C. , C. Vervaet Vervaet ³ ³ , C. Van , C. Van Peteghem Peteghem 4 4 , and J. Van Bocxlaer , and J. Van Bocxlaer 1 1 1 1 Laboratory of Medical Biochemistry and Clinical Analysis, Ghent Laboratory of Medical Biochemistry and Clinical Analysis, Ghent , Belgium , Belgium 2 2 Muhimbili Muhimbili University College of Health Sciences, Dar University College of Health Sciences, Dar es es Salaam, Tanzania Salaam, Tanzania 3 3 Laboratory of Pharmaceutical Technology, Ghent, Belgium Laboratory of Pharmaceutical Technology, Ghent, Belgium 4 4 Laboratory of Food Analysis, Ghent, Belgium Laboratory of Food Analysis, Ghent, Belgium
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BSMS 7th Annual Meeting
High throughput LCHigh throughput LC--MS/MS of MS/MS of PyrimethaminePyrimethamine and and SulfadoxineSulfadoxinein limited volume plasma samples:in limited volume plasma samples:
combining selectivity from chromatography and fast ion combining selectivity from chromatography and fast ion separation by MSseparation by MS
M.P. M.P. BoucheBouche 11
P. P. RishaRisha ²², T. , T. DecaesteckerDecaestecker 11, C. , C. VervaetVervaet ³³, C. Van , C. Van PeteghemPeteghem 44, and J. Van Bocxlaer , and J. Van Bocxlaer 11
11 Laboratory of Medical Biochemistry and Clinical Analysis, GhentLaboratory of Medical Biochemistry and Clinical Analysis, Ghent, Belgium, Belgium22 MuhimbiliMuhimbili University College of Health Sciences, Dar University College of Health Sciences, Dar eses Salaam, TanzaniaSalaam, Tanzania
33 Laboratory of Pharmaceutical Technology, Ghent, BelgiumLaboratory of Pharmaceutical Technology, Ghent, Belgium44 Laboratory of Food Analysis, Ghent, BelgiumLaboratory of Food Analysis, Ghent, Belgium
BSMS 7th Annual Meeting
IntroductionIntroduction
PyrimethaminePyrimethamine + Sulfadoxine+ Sulfadoxine::
Prevention and treatment of Malaria P. falciparumMalaria P. falciparum
Cloroquine in Eastern Africa
Antifolate-sulfonamide combination: new firstnew first--line drugline drug
N
N NH2
NH2Cl
H2N SO2NH N
N
OCH3H3CO
PYRPYRSULSUL
BSMS 7th Annual Meeting
Drug resistanceDrug resistance::
Due toDue to exposure to sublethal concentrationssublethal concentrations
Suboptimal dosageUse of substandard drug formulations
Therefore:
Tablet formulations on African marketTablet formulations on African market::
Acceptable quality ?Acceptable quality ?Constant quality (shelf time under tropical conditions) ?Constant quality (shelf time under tropical conditions) ?
In vitroIn vitro availabilityavailability::
- Dissolution tests- ≠ between formulations
In vivoIn vivo biobio--availabilityavailability::
-- Clinical trial-- Same ≠ ?
BSMS 7th Annual Meeting
AimAim
ChallengeChallenge:
HugeHuge sample load
Repetitive samples / volunteer
Largely different concentrationsLargely different concentrations
From an From an analytical viewpointanalytical viewpoint::
Sound simultaneoussimultaneous quantitationquantitation of both drugs in human plasma samples
FansidarFansidar®®Tablet: 25 mg PYR + 500 mg SUL
BSMS 7th Annual Meeting
LCLC--MS/MS method: developmentMS/MS method: development
Limited volume samples Fast analysis
Rapid sample clean-upFast chromatography
↓
«« StripStrip and and shootshoot »»Short columnShort column
Selectivity
Sensitivity
↓
Tandem Tandem MSMS
FocusFocus
BSMS 7th Annual Meeting
ChromatographyChromatography::
10 µL10 µL supernatant injected
XTerra MS C18 column (3.5 µm p. s., 50*1 mm)
Elution gradient:
ISIS: Sulfamerazine
Sample pretreatmentSample pretreatment::
250 µL crude plasma
Deproteinization: 100 µL100 µL ZnSOZnSO4 4 0.1 M0.1 M+ 100 µL AcCN+ 100 µL AcCN
Removal of lipids: 300 µL300 µL CHClCHCl33
H2O/ AcCN + 0.5% formic acid ammoniumformate (20 mM)
«« Single step Single step -- one tubeone tube »»
BSMS 7th Annual Meeting
MS/MS conditionsMS/MS conditions::
Triple quadrupole (Q. Ultima, Micromass)
ESI +ESI +
MRMMRM
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z0
100
%
176.89
128.07
110.81
162.98
232.97
198.14
216.05248.90
176.89
248.90
198.14
232.97
Product ion spectrum Product ion spectrum PYRPYR
[[M+HM+H]]++
249.1
233.1
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 4000
100
%
156.03
107.98
92 .09
140.02
311.11245 .03
156.0
311.1
Product ion spectrum Product ion spectrum SULSUL
[[M+HM+H]]++
m/z
BSMS 7th Annual Meeting
MS/MS conditionsMS/MS conditions::
Compound Precursor ion Product ion Cone voltage (V)
Collision energy (eV)
Ion m/z m/z
PYR [M+H]+ 249.1 233.1 30 30
SUL [M+H]+ 311.1 156.0 70* 15
IS [M+H]+ 265.2 110.0 35 14
Detuned to extend linear dynamic range
BSMS 7th Annual Meeting
LCLC--MS/MS method: results & discussionMS/MS method: results & discussion
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50Time0
100
%
0
100
%
0
100
%
2.71
1.92
1.06
PYR PYR
SULSUL
ISIS
Fortified Fortified plasma plasma samplesample
40 ng/mL PYR40 ng/mL PYR
4 µg/mL SUL4 µg/mL SUL
BSMS 7th Annual Meeting
Sample cleanSample clean--upup
«« Single step Single step -- one tubeone tube »»
Fast, efficient, simple
No unduly dilution
ChromatographyChromatography
Fast
Partial separationPartial separation1 mm-column → sensitivity ↑↑
MS/MSMS/MS
Selectivity → MRM
Sensitivity
Clear supernatantClear supernatant
Sensitivity Sensitivity in bioin bio--analysis ?analysis ?
BSMS 7th Annual Meeting
LowLow [PYR] ↔↔ HighHigh [ SUL]
[[ SULSUL]]: 0.1 - 50 µg/mL[[PYRPYR]]: 5 - 1000 ng/mL
Plasma concentration
Peak
are
a ra
tioProblemsProblems:: carry-over
saturation (ionisation and detector level)
⇒⇒ Reconciliation in oneReconciliation in one--acquisition method ?acquisition method ?
1. Quantitative interval
2. Detuning for SUL: conecone voltage
BSMS 7th Annual Meeting
LCLC--MS/MS method: validationMS/MS method: validation
LinearityLinearity::
Range: [[SULSUL]] 0.1 - 50 µg/mL [PYR] 5 - 1000 ng/mL
Weighed linear regression: 1/x
SULSUL R= 0.9978 R= 0.9978 PYR R= 0.9984R= 0.9984
LODLOD: : SUL 0.01 0.01 µµg/mLg/mLPYR 1 ng/mL1 ng/mL
SelectivitySelectivity: : common antimalarial drugs + sulfonamidesMRMMRM
LOQLOQ: : Lowest calibration point
BSMS 7th Annual Meeting
Other parametersOther parameters::
Compound SUL PYR
Conc. level
1
µg/mL
10
50
10
ng/mL
100
1000
Total reproducibility (CV, %) (n=5)
9.8 9.7 6.5 14.9 8.5 8.2
Within-day reproducibility (CV, %) (n=7)
7.0 3.8 3.6 9.9 5.1 4.8
Accuracy
(Recovery ± SD, %) (n=5)
103.7 ± 8.7
103.9 ± 6.9
100.2 ± 4.4
100.9 ± 6.2
100.7 ± 9.7
101.8 ± 4.7
BSMS 7th Annual Meeting
ConclusionConclusion
SensitivitySensitivity::
«« No longer an issueNo longer an issue » » for majority of applications
ShiftShift towards reproducibility and carry-over
Trend: «« fast analysisfast analysis »»Tempting respons to ever increasing demands
HoweverHowever:
Rudimentary sample cleanRudimentary sample clean--up (+ fast chromatography)up (+ fast chromatography)
≠≠«« Quick & dirtyQuick & dirty »»
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