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

1. size column idis Triclocarban x 5 Clofibric acid zl Naproxen 4 4 4 Ibuprofen EE UN Ibuprofen d3 Gemfibrozil 3 Diclofenac 34 sodium salt 2 4 Furosemide i Hydrochlorothiazide Aspirin Enalaprilat U J MJ T T T T T T T T T T T T T T T T T T T T 32 34 36 38 4 42 44 46 48 5 52 54 56 58 6 62 64 66 6 8 7 Abundance vs acquisition time min Figure 1 Negative ion mode TIC of 11 pharmaceuticals A few compounds for example ketoprofen Figure 2 are sensitive to heat from the drying gas Higher drying gas temperature 350 C lowers the intensity of the precursor ions Therefore in the negative ion mode the drying gas temperature was set to 200 C Ketoprofen 405 4 Precursor 253 114 252 7 3500V 200 C 0 84 06 208 8 pais 02 4 214 6 228 7 208 7 212 7 3500V 350 C 0 8 252 6 214 6 0 l A T T T T T T j T j j T T T 200 205 210 215 220 225 230 235 240 245 250 255 260 Abundance vs mass to charge m z Figure 2 Higher drying gas temperature lowers precursor intensity for certain compounds In Figure 3 it was interesting to see that the fragment ion actually had a higher m z value than the precursor ion For azithromycin the doubly charged ion showed higher inten sity than the singly charged ion and was chosen as the pre cursor Therefore depending
2. MW 129 151 239 252 180 176 299 194 290 201 253 749 255 414 236 309 344 308 416 MW 297 180 348 330 254 214 230 294 206 209 250 314 Precursor 130 4 152 3 240 4 253 4 181 3 177 3 300 4 195 3 291 4 202 3 254 3 375 5 256 5 415 4 237 4 310 4 345 4 309 4 417 2 Precursor 296 179 347 329 253 213 229 294 205 208 249 313 Quant ion 71 5 110 3 148 4 94 9 123 9 118 3 164 9 137 9 122 8 131 3 156 0 157 9 167 1 177 6 194 0 148 5 283 9 163 3 159 3 Quant ion 269 122 114 285 209 127 170 250 161 164 121 160 Collision V 15 18 15 17 20 29 30 22 2b 3b 15 16 5 18 20 0 2 15 30 Frag V 140 120 120 140 80 80 80 100 80 80 120 140 Dwell 300 30 30 30 30 30 30 30 30 100 100 100 100 100 100 100 100 100 300 Collision V 20 15 10 15 5 10 10 10 Segment 1 Cc O1 01 O1 5 4 4 C9 CO CO F2 h2 h2 h2 h23 n2 DNDN LY Dwell 70 70 70 70 70 70 70 100 100 100 150 150 Segment 1 4 4 C0 c0 C0 P2 l2 l2 aca Results and Discussion The total ion chromatogram TIC in negative ion mode is shown in Figure 1 The analysis time in negative ion mode is less than 7 minutes for the 11 analytes Their peak widths are about 0 1 minute using a 1 8 um particle size column The narrower peak width gives a higher signal to noise s n ratio compared to a 3 5 um or larger particle
3. ACN 3 CITUR ACN Flow rate 0 3 mL min Flowrate DEA ient 0 Gradient Time B Gradient Time a 0 0 15 100 A 2 52 20 100 21 5 0 3 10 6 100 Injection volume 1 0 uL 13 100 MS G6410A QQQ 14 0 lonization ESI Injection volume 1 0 pL Mass range 125 to 800 amu MS G6410A Q00 Scan time 300 ms eer AC lonization ESI Capillary 3500 V Mass range 120 800 amu Nebulizer P 40 psi Divina dae 9 L min Scan time 300 ms ying gas Capillary 3500 V Gas temperature 350 C i Skimmer 35V Nebulizer P 40 psi Drying gas 9 L min Gas temperature 200 C Skimmer 35 V The MRM parameters for positive ion mode and negative ion mode are listed in Tables 1 and 2 respectively Table 1 Positive lon Mode MRM Method Parameters Name RT Metformin HCI 0 856 Acetaminophen 4 591 Salbutamol 4 717 Cimetidine 4 815 1 7 Dimethylxanthine 4 89 Cotinine 5 24 Codeine 5 321 Caffeine 5 493 Trimethoprim 5 935 Thiabendazole 7 194 Sulfamethoxazole 7 309 Azithromycin 7 326 Diphenhydramine 8 446 Diltiazem HCI 8 693 Carbamazepine 8 912 Fluoxetine HCI 9 71 Dehydronifedipine 10 635 Warfarin 11 152 Miconazole nitrate salt 12 865 Table2 Negative lon Mode MRM Method Parameters Name RT Hydrochlorothiazide 3 42 Aspirin 3 49 Enalaprilat 3 71 Furosemide 4 51 Ketoprofen 5 17 Clofibric acid 5 20 Napoxen 5 20 Diclofenac sodium salt 5 84 Ibuprofen 6 03 Ibuprofen d3 6 03 Gemfibrozil 6 49 Triclocarban 6 66
4. quadratic fit Sapata a Metformin HCI 0 9975 0 9999 Trimethoprim ae 1 7 Dimethylxanthi 0 9998 0 9998 iN Y Ne MR 3 Sulfamethoxazole 10 7 Acetaminophen 0 9852 0 9999 Thiabendazole 53 Caffeine 0 9992 0 9997 Carbamazepine 28 Cimetidine 0 9968 0 9998 Diltiazem HCI 4 7 Codeine 0 9989 0 9997 Diphenhydramine 3 7 Cotinine 0 9971 0 9998 Dehydronifedipine 94 Salbutamol 0 9850 0 9994 puema D n Trimethopri 0 9980 0 9999 eee li rimetioprim i Miconazole nitrate salt 2 9 Azithromycin 0 9633 0 9998 Sulfamethoxazole 0 9998 0 9999 Table 5 shows the linearity results of all 11 pharmaceuticals Thiabendazole 0 9997 0 9998 ESI over the range of 10 20 40 80 400 and 800 pg on Carbamazepine 0 9926 0 9999 column All the R values were above 0 99 except triclocar M ban which was about 0 97 Diltiazem HCI 0 9997 0 9997 Diphenhydramine 0 9975 0 9998 Dehydronifedipine 0 9985 0 9993 Table 5 Linearity 10 20 40 80 400 and 800 pg on Column ESI Fluoxetine HCI 0 9984 0 9997 Origin Included No Weighting Warfarin 0 9989 0 9997 Compound R linear fit Miconazole nitrate salt 0 9989 0 9995 Hydrochlorothiazide 0 9999 Aspirin 0 9977 Table 4 shows the repeatability results from six injections Enalaprilat 0 9981 of 5 pg of each analyte on column In general the RSDs are Furosemide 0 9997 below 15 except for fluoxetine which was at 23 Ketoprofen 0 9988 Clofibric acid 0 9997 Naproxen 0 9994 Diclofenac Na salt 0 9993 Ibuprofen 0 9997 Ibuprofen d3 0 9
5. 0 ss0t00 a d 103 10 690102 2 mes mre A abends 105 0 75 4 690103 0 25 4 s 690104 B 34 3 6 5 52 54 5 6 5 8 6 6 2 6 4 6 6 6 8 7 7 2 7 4 7 6 Abundance vs acquisition time min Figure 7 Pharmaceuticals screening in negative ion mode 10 sea Gemfibrozil 4 470018 for cholesterol 14 1024 Hydrochlorothiazide 4 480081 5 NT high blood pressure 1 fib lA S NAA eds lev dt tu MENU 108 820093 Ibuprofen 104 3 a 920083 k 10 3 620094 h 1 4 104 6 J 4 4 620096 2 4 the 104 6 4 4 620097 2 4 10 1 620098 3 4 3 6 5 5 4 5 8 6 6 6 8 7 6 Abundance vs acquisition time min Figure 8 Pharmaceuticals screening in negative ion mode Figure 6 shows several of the pharmaceuticals for example diphenhydramine and acetaminophen that were common to several of the water samples Some of the antibiotics were also found in the samples Interestingly enough in Figures 7 and 8 the most common pharmaceuticals in the water samples were related to high blood pressure and cholesterol medications Conclusions Using SPE and LC MS MS 19 pharmaceuticals in positive ion mode and 11 pharmaceuticals in negative ion mode were analyzed at low picogram level on column without any derivatization Good linearity was observed for analytes from 1 pg to 1 ng on column Repeatability study from
6. six injections of target analytes at b pg on column showed RSDs below 1596 except for fluoxetine at 2396 This method was applied to water sample extracts finding that several target pharmaceutical drugs were commonly found among the analyzed samples Reference 1 USGS SOP Instrumental Analysis for Determination of Human Health Phar maceuticals in Water by Chemically Modified Styrene Divinylbenzene Resin Based Solid Phase Extraction and High Performance Liguid Chromatography Mass Spectrometry by Steve Werner 2006 Acknowledgments The author gratefully acknowledges the assistance of Stephen Werner and Ed Furlong of the National Water Quality Lab United States Geological Survey Lakewood CO for providing the sample preparation procedures and extracts used in this work For More Information For more information on our products and services visit our Web site at www agilent com chem www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this mater ial Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2008 Published in the USA September 17 2008 5989 5319EN Agilent Technologies
7. 998 Gemfibrozil 0 9993 Triclocarban 0 9655 Once the method is established one can screen and quan titate target analytes in water Figures 6 7 and 8 are MRM analyses of actual water sample extracts in positive and negative ion modes Diphenhydramine 104 E 690031 ATATIA ATEA EA NETIA APEA APA PER TR OO PETA PA PA TTPA T PEA PEEN PA PE TRENT PA PATTA OOA PA PITIT PATEAT PEA PAEA TPE TT TUN PHTPERI PROPRE RRERTPERTTTER lans bosssss listaus TPE RIT saaa IR 196 M 690032 lias Il m 690091 7 ub Trimethoprim and sulfamethoxazole 105 E Acetaminophen 690100 alu joa n Ju m alu 103 l 690101 buanwa baboso latda oaoot Y 103 J 690102 idu oup dtt idl ld 405 i 690103 TNT T ji lh Wh l ih 40 10 690104 A j T T T T T T T T T T T T T T 5 5 6 6 5 7 7 5 8 8 5 9 9 5 VI 10 5 11 11 5 12 Abundance vs acquisition time min Figure 6 Pharmaceuticals screening in positive ion mode 103 10 Hvdrochlorothiazid Gemfibrazil 15 4 690031 yore Morounerive for cholesterol 05 for high blood pressure inmi ui wui u snou u MuR JA po anor 105 0 75 4 690032 103 5 g 690091 Furosemide acl for high blood pressure 104 3 690101 14 pp gt 104
8. Author Chin Kai Meng Agilent Technologies Inc 2850 Centerville Road Wilmington DE 19808 USA Determination of Pharmaceuticals in Water by SPE and LC MS MS in Both Positive and Negative lon Modes Application Note Environmental Abstract Using solid phase extraction SPE and liquid chromatography tandem mass spectrometry LC MS MS 19 pharmaceuticals in positive ion mode and 11 pharmaceuticals in negative ion mode were analyzed at low picogram level on column without any derivatization Good linearity was observed for analytes from 1 pg to 1 ng on column Repeatability from six injections of analytes at b pg on column showed RSDs below 15 for all target compounds except for fluoxetine at 23 RE Agilent Technologies Using the Multiple Reaction Monitoring MRM technique any interference and matrix signal from organic matters in the water can be minimized from the target compound sig nals for better confirmation and quantitation In this appli cation note SPE and LC MS MS methods are described to analyze 19 pharmaceuticals in positive ion mode and 11 pharmaceuticals in negative ion mode Introduction Many articles in leading medical journals and newspapers reported sexual development and reproductive problems in animals and humans for example low sperm counts geni tal deformities male fish making eggs and others Scien tists suggested that man made chemicals for example pesticides and pharmaceuticals a
9. e high 3 447 x10 10 pg on column 51 Hydrochlorothiazide 0 3 525 x10 1 A Aspirin 0 4 544 x10 054 Furosemide 0 5 216 x10 Ketoprofen 0 54 5 254 x10 ae 1 Clofibric acid 0 54 xit 5 233 H Naproxen 04 x10 5 876 24 Diclofenac Na salt 0 zii 6 051 05 Ibuprofen 04 x10 6 490 2 Gemfibrozil A x10 6 653 0 55 Triclocarban 0 T ij T T T T ij T Hi T T T if T T Hi T ij T T T T T T ij T ij T n T T i T i T T T T T 3 32 34 36 38 4 42 44 46 48 5 52 54 56 58 6 62 64 66 68 7 2 Abundance vs acquisition time min Figure 5 Overlay of MRM results from the 10 pharmaceuticals in negative ion mode Table 3 shows the linearity results of all 19 pharmaceuticals Table4 Repeatability from Six Injections at 5 pg pL 5 pg on column ESI over the range of 1 5 10 20 40 100 200 400 and ESI 1 000 pg on column Two calibration models were used a Compound RSD linear model and a quadratic model that both included Metformin HCI 12 4 origin with no weighting Some of the compounds showed 1 7 Dimethylxanthine 8 6 significant fitting improvement from the linear model to the Acetaminophen 6 1 quadratic model This is the nature of these compounds Caffeine 57 Cimetidine 4 1 Table 3 Linearity 1 5 10 20 40 100 200 400 and 1 000 pg on Codei 162 Column ESI Origin Included No Weighting ocene i F F i Cotinine 10 5 ompoun linear fit
10. on the precursor chosen it is sometimes necessary to set the upper mass of the product ion scan to be higher than the precursor ion Figure 4 shows the overlaid chromatograms of 19 pharma ceuticals each at 5 pg on column from the positive ion mode MRM analysis A04 4 n 374 9 3 Doubly charged Singly azithromycin charged precursor fragment 24 E 590 8 1 47 158 3 0 I I I I I I I I I I I I I I I I I I I I I 100 140 180 220 260 300 340 380 420 460 500 540 580 620 Abundance vs mass to charge m z Figure 3 Doubly charged precursor results in a fragment at higher m z 10 64 Salbutamol Diphenhydramine Cabamazepine 55 1 5 5pg on column Cimetidine 1 7 dimethylxanthine 45 1 43 Sulfamethoxazole Acetaminophen 3 55 Thiabendazole Azithromycin Dehydronifedipine 3 Diltiazem 25 Cotinine Warfarin 24 Toe Fluoxetine Miconazole affeine i 154 Trimethoprim nitrate salt 1 4 0 5 Metformin 0 T T T T T T T T T T f T f ry ek T T T T T 1 15 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 395 10 105 11 115 12 125 13 Abundance vs acquisition time min Figure 4 Overlaid MRM chromatograms of the 19 pharmaceuticals in positive ion mode Figure 5 shows the overlaid chromatograms of 10 pharma ceuticals each at 10 pg on column from the negative ion mode MRM analysis In both Figures 4 and 5 the analysis times were relatively short and s n ratios wer
11. re disrupting the endocrine system s Experimental Compounds like antibiotics over the counter medicines and caffeine drain through the sewage system largely unal tered into rivers and streams and even get into the drinking water supply in very small amounts In order to monitor the trace pharmaceuticals in surface and ground water an effective sample preparation and analysis method is required 2 Sample Preparation Procedure See Reference 1 for more information 1 Filter water samples in the field or laboratory using 0 7 um glass fiber filters Pump 1 L of the filtered water sample at a flow rate of 10 mL min through an Oasis HLB SPE cartridge con In 1999 the U S Geological Survey National Water Quality d taining 0 5 g of sorbent Laboratory NWOQL developed and implemented an Oasis HLB solid phase extraction SPE and a high performance 3 Elute the HLB column with 6 mL of methanol followed liquid chromatography HPLC mass spectrometry MS by 4 mL of 0 196 trifluoroacetic acid TFA in methanol method to analyze pharmaceuticals Instrumentation Positive lon Mode Negative lon Mode LC 1200 LC LC 1200 LC Column ZORBAX Extend C 18 RRHT lum ZORBAX Extend G 18 RRHT 2 1 mmx 100 mm 1 8 um 2 1 mm x 100 mm 1 8 um Column temperature 40 C Col mi temperature E Mobile phases A 0 196 formic acid in water Mouile phases ees Glacial acetic acidiin add NH OH buffer to pH 5 5 cel B Acetonitrile

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

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