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Tackling matrix effects during development of a liquid chromatographicelectrospray ionisation tandem mass spectrometric analysis of
pharmaceuticals in aqueous environmental samples
Jet Van De Steene, Kjell Mortier and Willy Lambert
Laboratory of Toxicology, Ghent University, Harelbekstraat 72, B-9000 Gent, Belgium
[email protected], www.toxicologie.ugent.be
OVERVIEW
•LC-MS analysis of 9 pharmaceuticals in
environmental waters
RESULTS and DISCUSSION
Internal standards
Several SPE tested: see experimental
•Comparison of matrix effects related to
2 SPE-columns: Oasis HLB and Phenyl
•Best way to tackle matrix effects
Oasis HLB gave the best results and this procedure
was optimised.
Matrix effect was tested: very high: see Table 1
•Strategies to diminish matrix effects
during method development
•Search for appropriate internal standards in environmental
analysis is complicated: many other compounds could be
present in water (e.g. other drugs from the same therapeutic
classes as analyte)
Phenyl (2nd best) procedure optimisation
Comparison ME of Oasis and phenyl: see Table 1
INTRODUCTION
•A thorough search and the application of 4 internal standards
(Figure 1 + 2)(structural analogues) was beneficial and again
reduced matrix effect: see Table 3
Conclusion: phenyl gave less ME then Oasis HLB
•Matrix effects are a major issue when dealing with
LC-MS development. Co-eluting compounds
arising from the matrix can result in signal
suppression or enhancement[1]. During method
development attention should be paid to
overcome matrix effects as much as
possible[2,3].
Optimisation of sample preparation
•Good recoveries are obtained after the thorough sample
clean-up (Table 3)
Addition of a ferric nitrate solution before extraction
(coagulation with humic acids and DOM)
Application of an alkaline wash step (4.5% NH3 in
water/methanol (60:40,v/v))
The use of a second SPE-column:
NH2- or florisil- column
(trapping humic acids and organic material)
•Solutions to diminish matrix effects during the
development of a LC-ESI-MS-MS analysis of 9
pharmaceuticals were examined:
optimisation of sample preparation
decrease of flow rate entering the source
use of appropriate internal standards
Table 3: Matrix effect (ME) and recovery (RE) of the final
procedure (phenyl+NH2+split+internal standards)
n=5
level= 10 ng/L
Conclusion: a more efficient sample clean-up and less ME
is achieved with use of a NH2-column:
see Table 1
Table 1: Comparison of matrix effect (ME):
Oasis HLB – Phenyl - Phenyl+NH2
EXPERIMENTAL
ME (CV%)
n=5 level= 10ng/L
•Internal standards: see Figure 1
•Standards: see Figure 2
•HPLC: Agilent HP1100
•Column: Chromolith Performance RP-18e column
(100 x 4.6 mm i.d)(Merck, Darmstadt,Germany)
•Gradient elution
•Eluents: A:water/acetonitrile (95:5, by vol.)
B:water/acetonitrile (5:95, by vol.)
both containing 2mM ammoniumacetate
and 2 mM acetic acid
ME with IS(CV%)
RE with IS(CV%)
Flubendazole
104.7(4)
84.2(7)
Pipamperone
59.0(7)
107.1(2)
Cinnarizine
52.6(8)
116.0(4)
Ketoconazole
92.3(6)
91.3(11)
Rabeprazole
55.9(7)
64.4(7)
Itraconazole
122.4(7)
96.4(10)
Domperidone
112.6(7)
102.0(3)
Propiconazole
75.0(13)
113.3(10)
Miconazole
70.6(14)
124.8(5)
Oasis HLB
Phenyl
Phenyl + NH2
Flubendazole
51.1(1)
51.6(3)
70.6(12)
Pipamperone
30.6(9)
67.9(5)
32.3(8)
Cinnarizine
5.3(16)
7.3(7)
22.8(15)
Ketoconazole
20.4(18)
22.1(11)
62.5(12)
Rabeprazole
44.7(1)
63.0(5)
23.7(11)
Itraconazole
82.3(12)
85.2(7)
78.9(15)
Domperidone
44.9(6)
80.9(12)
79.8(13)
Propiconazole
34.4(16)
35.9(4)
72.4(13)
The application of a 2nd SPE-phase: an NH2column
Miconazole
10.1(5)
8.2(13)
35.1(15)
Applying a post-column split (1:5)
CONCLUSION
•Matrix effect is diminished by:
The use of a more selective phenyl SPE-phase
The use of 4 internal standards (structural
analogues)
Reduction of flow rate entering the source
•Detector: API 4000 system from Applied
Biosystems (Ontario, Canada) used in positive
ionisation and MRM-mode.
A post-column split (1:5) was installed. Less
compounds enter the source, so matrix effect should be
less pronounced[5]. Table 2 displays the results.
•A SPE-LC-ESI-MS-MS method is developed, eliminating almost
matrix effects without compromising the recovery of the individual
compounds
Conclusion: ME is decreased with post-column split
•SPE columns tested: Oasis HLB (Waters),
C8,phenyl(both Varian), Strata X-polymer
RP sorbent and Strata-X polymeric SCX/RP
sorbent (both Phenomenex)
ACKNOWLEDGEMENTS
Table 2: Application of a post-column split (1:5): comparison of
matrix effect (ME)
•Procedure Oasis HLB:
conditioning: 5 ml acetonitrile/isopropanol (10:90,
by vol.), 5 ml methanol, 5 ml water;
sample application (100 ml; 10ng/L);
wash: 3 ml 4.5% NH3 in water/methanol(50:50, by vol.);
elution: 2 x 2 ml acetonitrile/isopropanol (10:90, by vol.)
This work was partly supported by Janssen Pharmaceutica, Beerse, Belgium
ME (CV%)
n= 5 level= 10 ng/L
REFERENCES
Phenyl + NH2
•Procedure Phenyl:
conditioning: 5 ml methanol, 5 ml water; sample application
(100 ml; 10 ng/L);wash: 5 ml water/methanol (60:40, by vol.);
elution: 2 x 0.5 ml methanol
•Procedure Phenyl+NH2:
After elution of the phenyl-column with 2 x 0.5 ml
methanol: dilution with 4 ml chloroform.
Conditioning of NH2-column with 5 ml chloroform/
methanol (80:20, by vol.); application of extract,
and directly collecting into a centrifuge tube.
1. Taylor P.J. Matrix effects: The Achilles heel of quantitative high-performance liquid
chromatography-electrospray-tandem mass spectrometry. Clinical Biochemistry
2005;38:328-334.
2. Petrovic M., Hernando M.D., Diaz-Cruz M.S., Barcelo D. Liquid chromatographytandem mass spectrometry for the analysis of pharmaceutical residues in
environmental samples: a review. Journal of Chromatography A 2005;1067:1-14.
3. Benijts T., Dams R., Lambert W., De Leenheer A. Countering matrix effects in
environmental liquid chromatography-electrospray ionization tandem mass
spectrometry water analysis for endocrine disrupting chemicals. Journal of
Chromatography A 2004;1029:153-159.
4. Matuszewski B.K., Constanzer M.L., Chavez-Eng C.M. Strategies for the
assessment of matrix effect in quantitative bioanalytical methods based on HPLCMS/MS. Analytical Chemistry 2003;75:3019-3030.
5. Kloepfer A., Quintana J.B., Reemtsma T. Operational options to reduce matrix
effects in liquid chromatography-electrospray ionization-mass spectrometry analysis
of aqueous environmental samples. Journal of Chromatography A 2005;1067:153-160.
Phenyl + NH2;split
Flubendazole
70.6(12)
84.0(4)
Pipamperone
32.3(8)
46.8(2)
Cinnarizine
22.8(15)
36.3(6)
Ketoconazole
62.5(12)
89.8(9)
Rabeprazole
23.7(11)
44.9(4)
Itraconazole
78.9(15)
77.3(8)
Domperidone
79.8(13)
89.2(2)
Propiconazole
72.4(13)
75.5(5)
Miconazole
35.1(15)
39.5(15)
Figure 2: Pharmaceuticals of interest (internal standard used)
•Experiments to evaluate matrix effect were in
correspondence to the strategy applied by
Matuszewski et al. [4]:
MS/MS responses of known amounts of standards (A)
were compared with those measured in a blank water
extract spiked, after extraction, with the same analyte
amount (B).
Absolute matrix effect(ME%): B/A x 100
ME%>100%: signal enhancement
ME%<100%: signal suppression
Cl
O
O
N
N
H
F
N
Cl
H3C
N
O
O CH3
N
H
N
O
Flubendazole (2)
O
O
Cl
N
N
CH3
O
N
N
H3C
N
Propiconazole (3)
N
N
O
O
Cl
O
Figure 1: Internal standards (IS)
HN
O
N
N
N
HN
O
O
NH
H
N
N
S
N
H3C
N
H3C
S
N
O
Cl
O
O
Cl
O
N
O
Itraconazole (4)
Recoveries (RE) were calculated by spiking the
samples at a concentration of 10 ng/l(C).
The MS/MS responses were compared with B:
RE= C/B x 100
N
N
N
Cl
O
CH3
N
N
Cl
Domperidone analogue(IS1)
F
H3 C
O
Ketoconazole (3)
NH
O
Domperidone (1)
N
H
Cl
Fenbendazole (IS2)
F
Cl
N
N
N
N
OH
N
N N
Hexaconazole (IS3)
O
N
N
N
N
NH2
O
O
Rabeprazole (2)
N
N
N
Cl
N
Cl
N
O
O
O
N
Cl
O
Cl
Cl
Cl
Pipamperone (1)
Itraconazole analogue (IS4)
Cinnarizine (4)
Miconazole (4)