LC-ESI-MS/MS analysis of nine basic pharmaceuticals in LC-ESI-MS/MS analysis of nine basic pharmaceuticals in influent, effluent and surface waterinfluent, effluent and surface water

Jet C. VanJet C. Van De Steene De Steene and Willy E. Lambert and Willy E. LambertLaboratory of Toxicology, University Ghent, Harelbekestraat 72, B-9000 Gent, Belgium Laboratory of Toxicology, University Ghent, Harelbekestraat 72, B-9000 Gent, Belgium Jet.Vandesteene@ugent.beJet.Vandesteene@ugent.be

n=5; level=20 ng/l Accuracy (%) Precision (CV%) LOD (ng/l) LOQ (ng/l) Recovery (%) (CV%;n=5) at 20 ng/l at 20µg/lFlubendazole 103.1 6 0.05 0.5 90.0 (6) 104.8(15)Propiconazole 92.1 6 0.05 5 108.3(7) 90.8(3)Pipamperone 93.1 9 0.5 5 82.3(6) 87.5(6)Cinnarizine 96.5 6 0.05 0.05 91.7(6) 92.4(7)Ketoconazole 105.9 5 5 5 88.1(8) 82.1(5)Miconazole 94.5 3 0.1 10 55.8(9) 79.1(9)Rabeprazole 102.1 15 0.5 1 106.4(4) 93.8(4)Itraconazole 124.5 18 5 10 64.1(17) 69.2(6)Domperidone 105.0 8 0.05 0.5 64.9(17) 61.0(8)

Overview• Analysis of nine basic pharmaceuticals: flubendazole, rabeprazole, cinnarizine, domperidone, ketoconazole, miconazole, itraconazole, pipamperone and propiconazole (=pesticide).•Qualitative and quantitative LC-ESI-MS/MS analysis in influent, effluent and surface waters•Quantification with the standard addition method•Method validation on influent, effluent and surface water

Introduction

Pharmaceutical pollution in the environment is of growing concern. Pharmaceuticals are excreted by humans, or disposed improperly, and enter the waste cyclus. They are not fully degraded in waste water treatment plants and end up in surface waters. Other pharmaceuticals enter the environment by application of manure on fields. These pharmaceuticals are designed to interact with living beings, so effects on fauna and flora – acute or chronic- are certainly possible, and some are already proven [1]. The aim of this work was to develop an LC-ESI-MS/MS method to identify and quantify these pharmaceuticals in influent, effluent and surface water samples.

References

[1] C.G. Daughton and Ternes T.A., Environmenal Health Perspectives 107: 907-938 Suppl. 6 1999. [2] J.C. Van De Steene, K.A. Mortier and W.E. Lambert, Journal of Chromatography A 1123:71-81 2006.

Experimental*Filtration: combination of a 1.5-µm glass fibre filter and a 0.45-µm membrane filter. Before extraction, the pH of the samples was adjusted to 7.

*SPE:*Speedisk phenyl for extraction (JTBaker): conditioning: 3 ml MeOH and 3 ml H2O; 100 ml surface water, 10 ml or 1 ml effluent, 1 ml influent 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) diluted with 4 ml chloroform, apply to

column and collect directly into centrifugation tube; dry residue; reconstitution in 100 µl H2O/ACN (80:20); inject 20 µl.*HPLC: *Agilent 1100 Series *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 in MRM-mode: 2 MRM-transitions + ratio’s were monitored.Results and discussionA. Quantification with the standard addition method

Matrix effects were tested on different blank surface water samples. Results showed that matrix effects were, in spite of intensive sample clean-up, not eliminated and different for the different samples [2]. Because no labelled internal standards are commercially available, the only way to perform accurate quantification, is to use the standard addition method. A prerequisite to perform standard addition is linearity. Dilution of the extracts is done to proof

and/or obtain linearity. Validation of influent and effluent is done on samples from the pharmaceutical company where these pharmaceuticals are manufactured or formulated. There is no blank influent or effluent available, so only precision data could be obtained. Because concentrations were very high (influent: range of 1-3000 µg/l; effluent: range from 1-300000 ng/l), extracts were diluted. Depending on the concentration, the original, or 10-, 50- or 100-fold diluted extracts were used. The added amount of standard depends on concentration found/suspected.

B. Chromatogram C. Stability data

Table: MRM-transitions and capacity factors of the analytes

Figure 1: Influent with standard addition of 1 mg/L, extract: 50-fold diluted.Total ion chromatogram. From left to right: rabeprazole (tr=5.61 min), domperidone (tr=6.80 min), flubendazole (tr=7.07 min), pipamperone (tr=7.21 min), ketoconazole

(tr=8.41 min), propiconazole (tr=8.72 min), itraconazole (tr=9.38 min), miconazole (tr=10.59 min), cinnarizine (tr=11.71 min).

D. Validation on surface water E. Validation on effluent and influent

ConclusionA qualitative and quantitative SPE- LC-ESI-MS/MS method is presented.Quantification with the standard addition method.Validation on surface water: linearity, accuracy, precision, LOD, LOQ, recovery and stability.Precision data on influent and effluent.Application on influent and effluent samples.

MRM-transitions Capacity factor amu k'

314.20281.90314.20122.80342.16159.30342.16122.80376.40165.40376.40122.90369.30167.10369.30115.20531.16148.30531.16120.10416.90161.00416.90123.00360.20242.20360.20195.10705.21392.00705.21159.30426.20175.10426.20147.10

Domperidone 5.89

Rabeprazole4.88

Itraconazole 5.60

Ketoconazole 4.14

Miconazole 4.41

Pipamperone 3.26

Cinnarizine 3.34

Compound

Flubendazole 2.48

Propiconazole 3.10

TIC: from Sample 12 (I4-SA50) of 070405stalen.wiff (Turbo Spray) Max. 2,0e6 cps.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24Time, min

0,0

1,0e5

2,0e5

3,0e5

4,0e5

5,0e5

6,0e5

7,0e5

8,0e5

9,0e5

1,0e6

1,1e6

1,2e6

1,3e6

1,4e6

1,5e6

1,6e6

1,7e6

1,8e6

1,9e6

2,0e6

Inte

ns

ity, c

ps

7,216,80

5,61

11,71

8,72 10,59

9,38

Conc. effluent (ng/l) CV% (n=5) Conc. influent (µg/l) CV% (n=5)flubendazole 1094 8 155 8pipamperone 294233 10 213 15rabeprazole <LOD - <LOD -domperidone 23566 2 2400 4propiconazole 82 4 0.16 5ketoconazole 1115 9 143 9itraconazole 15 13 6 8cinnarizine 5 11 4 13miconazole 81 11 15 4

7,07

8,41

20 ng/l; n=3 stability sample at 4°C autosampler stability, 5hflubendazole 112.0 96.9pipamperone 94.3 100.3rabeprazole 21.4 93.0domperidone 107.2 94.7propiconazole 94.1 98.6ketoconazole 102.2 91.7itraconazole 95.9 108.0cinnarizine 121.0 92.7miconazole 87.5 92.2

extract -18°C 1 week extract freeze-thaw (3cycles)flubendazole 94.7 98.1pipamperone 93.0 92.6rabeprazole 83.0 81.4domperidone 92.9 97.3propiconazole 121.1 100.5ketoconazole 90.0 84.5itraconazole 122.7 104.0cinnarizine 101.3 96.7miconazole 93.1 89.2