1. Jet C. Van De Steene and Willy E. Lambert
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
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
Evaluation of matrix effect
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.
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.
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
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).
Gradient elution with (A) H2O/ ACN (95:5) and (B) H2O/ACN (5:95), both containing 2 mM ammoniumacetate and 2 mM acetic acid
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.
RESULTS and DISCUSSION
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
Phenyl second best
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).
less matrix effect!
2. Optimisation chromatography
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.
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.
Table 1: Matrix effect in different surface water samples (area ratio: with structural analogues: see experimental) with CV% (n=5)
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
CONCLUSION
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: Suppl
[2] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez- Eng. Analytical Chemistry 75:
[3] J.C. Van De Steene, K.A. Mortier and W.E. Lambert, Journal of Chromatography A 1123: