Determination of Pharmaceuticals in Water by SPE and LC/MS/MS in Both Positive and Negative Ion Modes

Determination of Pharmaceuticals in Water by SPE and LC/MS/MS in Both Positive and Negative Ion Modes (50-6) Chin-Kai Meng Senior Applications Chemist Agilent Technologies March, 007 Page

Introduction Scientists suggested that man-made chemicals (for example, pesticides and pharmaceuticals) are disrupting the endocrine system. Compounds like antibiotics, over-the-counter medicines, and caffeine drain through the sewage system largely unaltered into rivers and streams, and even get into the drinking water supply. In order to monitor the trace pharmaceuticals in surface and ground water, an effective sample preparation and analysis method is required. Wastewater treatment facility near Atlanta, Georgia Page

39 Streams in 30 States were Monitored (during 999 and 000) USGS, June 00 Page 3

Most frequently detected chemicals (found in more than half of the streams): Coprostanol (fecal steroid) Cholesterol (plant and animal steroid) N-N-diethyltoluamide (insect repellent) Caffeine (stimulant) Triclosan (antimicrobial disinfectant) Detergent metabolites, steroids, and plasticizers generally were found at the highest concentrations USGS, June 00 Page

Sample Preparation Procedures - developed by the US Geological Survey National Water Quality Laboratory (NWQL) Filter water samples in the field or in the laboratory using 0.7-µm glass fiber filters. Pump -L of the filtered water sample, at a flow rate of 0 ml/min, through an Oasis HLB (SPE) cartridge containing g of sorbent. Elute the HLB column with 6 ml of methanol followed by ml of 0.% TFA in methanol. The resulting solvent extract is then concentrated to approximately 00 µl. Add internal standard (ISTD). The extract is reconstituted to ml. L final volume of ml Page 5

Multiple Reaction Monitoring (MRM) Quad Mass Filter (Q) Quad Mass Filter (Q3) Collision Cell ions from ESI 0 Q lets only target ion 0 pass through 0 Collision cell breaks ion 0 apart Q3 monitors only characteristic fragments 58 and 9 from ion 0 for quant and qual. 65 68 80 58 9 0 58 9 70 0 50 90 90 0 50 70 90 0 60 90 no chemical background Page 6

Method Parameters (Positive Ion Mode) MS: G60 QQQ ESI (+) Mass Range: 5-800 amu Scan Time: 300 ms Drying gas: 350 o C Gas Flow: 9 L/min Nebulizer: 0 psi Capillary: 3500 V LC: 00 Mobile A: 0.% Formic Acid in water add NH OH to ph 5.5 Mobile B: ACN Flow rate: 0.3 ml/min Column: ZORBAX Extend-C-8. x 00 mm,.8 µm, RRHT Column: 0 o C µl injection Refer to app note 5989-539, July 006 for MRM parameters. Time (min) B(%) 0 0 5 00 0 00.5 0 Page 7

Fragment Ion Has Higher m/z than the Precursor (product ion scan) + Product Ion (7.30 min) (375.5 -> **) pharm_pos_750frag_scan0.d x0 37.9 3.6 3. 3. 3.8.6...8.6.. 0.8 0.6 0. 0. 0.7 Doubly charged Azithromycin (precursor) 58.3 33.8 Singly charged fragment 00 0 0 60 80 00 0 0 60 80 300 30 30 360 380 00 0 0 60 80 500 50 50 560 580 600 60 Abundance vs. Mass-to-Charge (m/z) 590.8 Page 8

Multiple Reaction Monitoring (MRM) 5 pg on column Acetaminophen Salbutamol Cimetidine,7-dimethylxanthine x0 5.5 5.5 3.5 3.5.5 + MRM (30. -> 7.5) cal_.d Cotinine Codeine Caffeine Trimethoprim Thiabendazole Sulfamethoxazole Azithromycin Diltiazem Fluoxetine Dehydronifedipine Diphenhydramine Warfarin Cabamazepine Miconazole Nitrate salt * Metformin 0.5.5 3 3.5.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 0.5.5 3 Abundance vs. Acquisition Time (min) 9 compounds Page 9

Linearity,5,0,0,0,00,00,00,000 pg on column Compound Metformin HCl,7,-dimethylxanthine Acetaminophen Caffeine Cimetidine Codeine Cotinine Ranitidine HCL Salbutamol Trimethoprim Sulfamethoxazole Thiabendazole Carbamazepine Diltiazem HCl Diphenhydramine Dehydronifedipine Fluoxetine HCl Warfarin Miconazole Nitrate Salt R (linear fit) 0.9975 0.9998 0.985 0.999 0.9968 0.9989 0.997 0.8369 0.9850 0.9980 0.9998 0.9997 0.996 0.9997 0.9975 0.9985 0.998 0.9989 0.9989 R (quadratic fit) 0.9999 0.9998 0.9999 0.9997 0.9998 0.9997 0.9998 0.973 0.999 0.9999 0.9999 0.9998 0.9999 0.9997 0.9998 0.9993 0.9997 0.9997 0.9995 origin Included, no weighting Page 0

Area Repeatability, n = 6 5 pg of each analyte on column 9 8 8 7 # of compounds 6 5 3 5 3 3 0-5 5. - 0 0. - 5 > 5 %RSD Page

What s in Our Surface Water? (Actual sample extracts) x0 5 + TIC MRM (** -> **) 7008_pos.d 7008 x0 + TIC MRM (** -> **) 8008_pos.d 8008 x0 5 + TIC MRM (** -> **) 60083_pos.d 60083 x0 5 + TIC MRM (** -> **) 60093_pos.d 60093 x0 5 + TIC MRM (** -> **) 6009_pos.d 6009 x0 + TIC MRM (** -> **) 60096_pos.d 60096 x0 + TIC MRM (** -> **) 60097_pos.d 60097 x0 5 + TIC MRM (** -> **) 60098_pos.d 60098 5.5 6 6.5 7 7.5 8 8.5 9 9.5 0.5.5 3 3.5.5 5 5.5 6 6.5 Abundance vs. Acquisition Time (min) Page

Pharmaceuticals Screening (ESI+) x0 5 x0 x0 5 + TIC MRM (** -> **) 7008_pos.d Sulfamethoxazole (antibiotics) + TIC MRM (** -> **) 8008_pos.d + TIC MRM (** -> **) 60083_pos.d,7-dimethylxanthine x0 5 (metabolite of caffeine) + TIC MRM (** -> **) 60093_pos.d Diphenhydramine (Benadryl) 7008 8008 60083 60093 x0 5 + TIC MRM (** -> **) 6009_pos.d 6009 x0 + TIC MRM (** -> **) 60096_pos.d 60096 x0 x0 5 + TIC MRM (** -> **) 60097_pos.d Caffeine + TIC MRM (** -> **) 60098_pos.d Diltiazem HCl 60097 (lowering high blood pressure) 60098 5.5 6 6.5 7 7.5 8 8.5 9 9.5 0.5.5 3 3.5.5 5 5.5 6 6.5 Abundance vs. Acquisition Time (min) Page 3

What s in Our Surface Water? (Actual sample extracts) x0 + TIC MRM (** -> **) 69003_pos.d 69003 x0 6 + TIC MRM (** -> **) 69003_pos.d 69003 x0 3 + TIC MRM (** -> **) 69009_pos.d 69009 x0 5 + TIC MRM (** -> **) 69000_pos.d 69000 x0 3 + TIC MRM (** -> **) 6900_pos.d 6900 x0 3 + TIC MRM (** -> **) 6900_pos.d 6900 x0 5 x0 3 + TIC MRM (** -> **) 69003_pos.d + TIC MRM (** -> **) 6900_pos.d 69003 6900 5.5 6 6.5 7 7.5 8 8.5 9 9.5 0.5 Abundance vs. Acquisition Time (min) Page

Pharmaceuticals Screening (ESI+) x0 + TIC MRM (** -> **) 69003_pos.d Diphenhydramine (Benadryl) 69003 x0 6 x0 3 + TIC MRM (** -> **) 69003_pos.d + TIC MRM (** -> **) 69009_pos.d Trimethoprim and Sulfamethoxazole 69003 69009 x0 5 + TIC MRM (** -> **) 69000_pos.d Acetaminophen (Tylenol) + TIC MRM (** -> **) 6900_pos.d x0 3 (Antibiotics) 69000 6900 x0 3 + TIC MRM (** -> **) 6900_pos.d 6900 x0 5 x0 3 + TIC MRM (** -> **) 69003_pos.d + TIC MRM (** -> **) 6900_pos.d 69003 6900 5.5 6 6.5 7 7.5 8 8.5 9 9.5 0.5 Abundance vs. Acquisition Time (min) Page 5

Method Parameters (Negative Ion Mode) MS: G60 QQQ ESI (-) Mass Range: 9-800 amu Scan Time: 300 ms Drying gas: 00 o C Gas Flow: 9 L/min Nebulizer: 0 psi Capillary: 3500 V Refer to app note 5989-539, July 006 for MRM parameters. LC: 00 Mobile A: 0.0% Glacial Acetic Acid in water Mobile B: ACN Flow rate: 0.3 ml/min Column: ZORBAX Extend-C-8. x 00 mm,.8 µm, RRHT Column: 60 o C µl injection Time (min) B(%) 0 0 0 5 3 70 6 00 3 00 0 Page 6

Drying Gas Temp. Effect (for some compounds) -MS (5.7 min) neg_drug_frag60_3500v_00c.d (BP=0778) # Ketoprofen Precusor = 53 x0 5.5..3 3500V, 00 C.. 0.9 5.7 0.8 0.7 0.6 08.8.7 0. 0.3 0..6 8.7 0. 0 00.7 -MS (5.7 min) USGS_neg_drug_scan_frag60.d (BP=6089) # 36.5 0.6.5 8. 58.6 x0 5 08.7 3500V, 350 C.5..3.. 0.9 0.8 0.7 0.6 0. 0.3 0. 0. 0.7.6 00 05 0 5 0 5 30 35 0 5 50 55 60 Abundance vs. Mass-to-Charge (m/z) 5.6 8.6 38.6.5 3..6 8.5 58.5 Page 7

x0 5.5 0 x0 -BPC (69.0 m/z) MRM (96.0 m/z); from USGS_pharma_neg_0ppb.d Smooth () Hydrochlorothiazide -BPC (.0 m/z) MRM (79.0 m/z); from USGS_pharma_neg_0ppb.d Smooth () Aspirin Pharmaceuticals MRM, 0 pg on column, (ESI-) 0 -BPC (85.0 m/z) MRM (39.0 m/z); from USGS_pharma_neg_0ppb.d Smooth () 3 x0 Furosemide 0 -BPC (09.0 m/z) MRM (53.0 m/z); from USGS_pharma_neg_0ppb.d Smooth () 3 x0 Ketoprofen -BPC (7.0 m/z) MRM (3.0 m/z); from USGS_pharma_neg_0ppb.d Smooth () 3 x0 Clofibric Acid -BPC (70.0 m/z) MRM (9.0 m/z); from USGS_pharma_neg_0ppb.d Smooth () x0 0 -BPC (50.0 m/z) MRM (9.0 m/z); from USGS_pharma_neg_0ppb.d Smooth () Naproxen x0 Diclofenac Na salt 0 -BPC (6.0 m/z) MRM (05.0 m/z); from USGS_pharma_neg_0ppb.d Smooth () 3 x0 Ibuprofen 0 x0 3 -BPC (.0 m/z) MRM (9.0 m/z); from USGS_pharma_neg_0ppb.d Smooth () 3 min Gemfibrozil 7 min -BPC (60.0 m/z) MRM (33.0 m/z); from USGS_pharma_neg_0ppb.d Smooth () x0 Triclocarban 0 3 3. 3. 3.3 3. 3.5 3.6 3.7 3.8 3.9...3..5.6.7.8.9 5 5. 5. 5.3 5. 5.5 5.6 5.7 5.8 5.9 6 6. 6. 6.3 6. 6.5 6.6 6.7 6.8 6.9 7 7. 7. Abundance vs. Acquisition Time (min) Page 8

What s in Our Surface Water? (Actual sample extracts) x0 5 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 7008.d 7008 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 8008.d x0 8008 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 60083.d x0 5 60083 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 60093.d x0 60093 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 6009.d x0 3 6009 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 60096.d x0 60096 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 60097.d x0 60097 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 60098.d x0 5 60098 3. 3.5 3.6 3.7 3.8 3.9...3..5.6.7.8.9 5 5. 5. 5.3 5. 5.5 5.6 5.7 5.8 5.9 6 6. 6. 6.3 6. 6.5 6.6 6.7 6.8 6.9 7 7. 7. 7.3 7. 7.5 Abundance vs. Acquisition Time (min) Page 9

Pharmaceuticals Screening (ESI-) x0 5 x0 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 7008.d 7008 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 8008.d 8008 Hydrochlorothiazide (lowering high blood pressure) Gemfibrozil (lowering cholesterol) x0 5 x0 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 60083.d 60083 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 60093.d 60093 Ibuprofen x0 3 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 6009.d 6009 x0 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 60096.d 60096 x0 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 60097.d 60097 x0 5 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 60098.d 60098 3. 3.5 3.6 3.7 3.8 3.9...3..5.6.7.8.9 5 5. 5. 5.3 5. 5.5 5.6 5.7 5.8 5.9 6 6. 6. 6.3 6. 6.5 6.6 6.7 6.8 6.9 7 7. 7. 7.3 7. 7.5 Abundance vs. Acquisition Time (min) Page 0

What s in Our Surface Water? (Actual sample extracts) x0 3.5 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 69003.d 69003 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 69003.d x0 5 0.75 0.5 69003 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 69009.d x0 3 6 69009 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 69000.d x0 3 69000 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 6900.d x0 6900 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 6900.d x0 3 6900 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 69003.d x0 5 0.75 0.5 69003 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 6900.d x0 6900 3. 3.5 3.6 3.7 3.8 3.9...3..5.6.7.8.9 5 5. 5. 5.3 5. 5.5 5.6 5.7 5.8 5.9 6 6. 6. 6.3 6. 6.5 6.6 6.7 6.8 6.9 7 7. 7. 7.3 7. 7.5 7.6 Abundance vs. Acquisition Time (min) Page

Pharmaceuticals Screening (ESI-) x0 3.5 x0 5 0.75 0.5 x0 3 6 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 69003.d 69003 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 69003.d 69003 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 69009.d 69009 Hydrochlorothiazide (lowering high blood pressure) No Ibuprofen Gemfibrozil (lowering cholesterol) -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 69000.d x0 3 69000 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 6900.d x0 6900 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 6900.d x0 3 6900 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 69003.d x0 5 0.75 0.5 x0 69003 -TIC MRM (37.0, 39.0, 96.0, 79.0... m/z); from 6900.d 6900 Furosemide (lowering high blood P.) 3. 3.5 3.6 3.7 3.8 3.9...3..5.6.7.8.9 5 5. 5. 5.3 5. 5.5 5.6 5.7 5.8 5.9 6 6. 6. 6.3 6. 6.5 6.6 6.7 6.8 6.9 7 7. 7. 7.3 7. 7.5 7.6 Abundance vs. Acquisition Time (min) Page

Acknowledgement Standard solutions and water sample extracts from: Ed Furlong and Stephen L. Werner USGS National Water Quality Laboratory Denver, Colorado USA Page 3

Thank You for Your attention Questions? Page