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Development of an LC-ESI-MS/MS analysis for nine basic
pharmaceuticals in environmental waters
Jet C. Van De Steene and Willy E. Lambert
Laboratory of Toxicology, Ghent University, Harelbekestraat 72,
9000 Gent, Belgium
[email protected]
OVERVIEW
•9 pharmaceuticals: flubendazole, rabeprazole,
cinnarizine, domperidone,
ketoconazole, miconazole,
itraconazole, pipamperone and
propiconazole (=pesticide).
•LC-ESI-MS/MS analysis in surface waters
•Development and optimisation of:
* solid-phase extraction
* chromatography
RESULTS and DISCUSSION
1. Optimisation sample preparation [3]
• Several SPE tested in view of recovery and
matrix effect: Oasis HLB (Waters), phenyl, C8
(Varian), polymeric and SCX sorbents
(Phenomenex)
Oasis HLB best for recoveries but major
matrix effects
•Evaluation of matrix effect
Phenyl second best
INTRODUCTION
Pharmaceutical pollution in the environment
(surface water, effluents, sediments…) is of
growing concern. After excretion by humans and
animals, and improper disposal of unused drugs,
pharmaceuticals enter the waste water
treatment plant, but are not fully degraded and
are disposed in the aquatic environment.
Research towards this pollution is necessary
because of possible effects on fauna and flora,
acute or chronic [1]. The aim of this work was to
develop an LC-ESI-MS/MS method for
identifiying and quantifiying these
pharmaceuticals in effluent and surface water
samples.
BUT: still prominent matrix suppression!!
• Sample clean-up necessary because of high
matrix effect: NH2 column.
• No retention of the analytes if extract is
applied in chloroform/MeOH (80:20).
•SPE:*Speedisk phenyl for extraction (JTBaker):
conditioning: 3 ml MeOH and H2O; 100 ml
sample; wash: 3 ml 40%MeOH in H2O; dry 2 min;
elute: 2 x 0.5 ml MeOH.
*NH2 for sample clean-up (Varian):
conditioning: 5 ml chloroform/MeOH (80:20);
extract (MeOH) diluting with 4 ml chloroform,
applying to column and collect directly into
centrifugation tube
ME% (CV%)
area
1
Flubendazole (d) 78.2(9)
Pipamperone (a) 40.0(5)
Cinnarizine (b)
2.6(15)
Ketoconazole ( c) 37.5(9)
Rabeprazole (d)
37.8(5)
Itraconazole (b)
12.3(5)
Domperidone (a) 57.8(10)
Propiconazole ( c) 60.9(7)
Miconazole (b)
5.5(16)
• Post-column split
To diminish matrix effects, a split was installed
after the column, so less
flow/sample/interferences entered source.
major reduction in matrix effects!
• 2 columns tested:
*A Chromolith C18 (4.6 mm i.d.x 100 mm)
*A pentafluorophenyl column (4.6 mm i.d.x 100
mm x 5 µm particle size).
with same buffers and split.
PFP-column showed same results for matrix
effects, but chromatography was better for
the basic compounds, so the PFP-column was
further used.
•Detector: triple quadrupole MS: API 4000
(Applied Biosystems) with electrospray
ionisation
•Experiments to evaluate matrix effect were in
correspondence to the strategy applied by
Matuszewski et al. [2]:
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
Recoveries (RE) were calculated by spiking the
samples, before extraction, at a concentration
of 10 ng/l(C).
The MS/MS responses were compared with B:
RE= C/B x 100
• Method of quantification:
First, structural analogues were used as
internal standards (domperidone analogue (a),
itraconazole analogue (b), hexaconazole (c),
cambendazole (d)).
Finally, the standard addition method was used.
• Limit of detection: S/N=3
Limit of quantification: S/N=10.
3
137.5(8)
73.8(15)
55.7(18)
69.0(4)
76.1(8)
122.6(4)
97.1(4)
92.4(3)
66.8(13)
4. Results
• For quantification the standard addition
method must be used.
• Different matrices (surface waters, effluent,
influent) have to be examined. To be sure that
there is linearity in every matrix, a 10-times
diluted extract is also analysed. If the result is
not more than a tenth of the original extract,
linearity is showed for that matrix.
•
Recoveries and limits of detection and
quantification of the whole SPE-LC-ESI-MS/MS
method are summarized in Table 2.
Table 2: Recoveries (surface water spiked at 200
ng/l; n=5) and limits of detection and
quantification
Intensity, cps
1.3e5
•Gradient elution with (A) H2O/ ACN (95:5) and
(B) H2O/ACN (5:95), both containing 2 mM
ammoniumacetate and 2 mM acetic acid
2
132.7(1)
78.4(3)
55.8(17)
59.1(6)
67.3(5)
140.8(14)
113.1(3)
99.0(4)
77.4(15)
2. Optimisation chromatography
•HPLC: Agilent 1100 Series
•Column: In the first experiments a Chromolith
C18 column (4.6 mm i.d.x 100 mm)(Merck) was
used.Then a comparison was made with a
pentafluorophenyl column (4.6 mm i.d x 100
mm; 5-µm particle size) (Varian).
2
82.5(4)
42.8(5)
4.8(20)
39.3(9)
41.7(6)
11.7(1)
62.0(6)
65.7(5)
4.8(22)
area ratio
3
1
96.6(2) 130.0(1)
51.4(1) 85.4(7)
22.4(20) 26.5(15)
62.6(10) 58.7(8)
53.5(15) 63.5(13)
57.2(10) 148.7(10)
69.1(14) 122.4(3)
83.7(1) 95.2(3)
30.9(8) 49.2(13)
less matrix effect!
EXPERIMENTAL
•Filtration was done onto a combination of a
1.5-µm glass fibre filter and a 0.45-µm
membrane filter. Before extraction, the pH of the
water samples was adjusted to 7 using a 5 %
ammonia-solution or a 20% acetic acid-solution.
Table 1: Matrix effect in different surface water
samples (area ratio: with structural
analogues: see experimental) with CV% (n=5)
7.05
1.2e5
1.1e5
1.0e5
9.0e4
8.0e4
7.0e4
6.0e4
5.0e4
4.0e4
3.0e4
Flubendazole
Propiconazole
Pipamperone
Cinnarizine
Ketoconazole
Miconazole
Rabeprazole
Itraconazole
Domperidone
LOD (ng/l) LOQ (ng/l) Recovery % (CV%)
0.05
0.5
87.0(3)
0.05
5
93.7(2)
0.5
5
87.6(2)
0.05
0.05
83.5(4)
5
5
80.4(4)
0.1
10
77.7(9)
0.5
1
84.8(4)
5
10
72.9(4)
0.05
0.5
88.8(8)
CONCLUSION
2.0e4
1.0e4
0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Time, min
Figure 1: chromatogram with the
pentafluorophenylcolumn.
From left to right: rabeprazole (tr= 5.67 min),
domperidone (tr=6.89 min), flubendazole (tr=7.05
min), pipamperone (tr=7.22 min), ketoconazole
(tr=8.67 min), propiconazole (tr=8.88 min),
itraconazole (tr=9.70 min), miconazole (tr=11.01 min),
cinnarizine (tr=11.61 min)
3. Matrix effects and quantification
• Matrix effect for 3 different surface water
samples: with and without structural
analogues as internal standards: see Table 1
different results!!
• Accurate quantification?
No labeled internal standards available, so
the standard addition method must be applied.
• An LC-ESI-MS/MS method for the quantitative
analysis of nine basic pharmaceuticals in
surface water was developed: sample
preparation and chromatography were
optimised in view of matrix effects.
• Matrix effects could not be ruled out so for
correct quantification the standard addition
method is used.
• LODs in the range of 0.05 and 5 ng/l.
LOQs in the range of 0.05 and 10 ng/l.
References
[1] C.G. Daughton and Ternes T.A., Environmenal
health perspectives 107: 907-938 Suppl. 6
1999.
[2] B.K. Matuszewski, M.L. Constanzer, C.M.
Chavez- Eng. Analytical Chemistry 75:30193030 2003.
[3] J.C. Van De Steene, K.A. Mortier and W.E.
Lambert, Journal of Chromatography A
1123:71-81 2006.