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ApplicationNOTE CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRIC AND TANDEM MASS SPECTROMETRIC ANALYSIS AND IDENTIFICATION OF NON-STEROIDAL ANTI-INFLAMMATORY DRUGS AND THEIR METABOLITES IN HUMAN URINE * Alison E. Ashcroft, Hilary J. Major, Waters Corporation, Tudor Road, Altrincham, Cheshire, WA14 5RZ, UK. Ian D. Wilson, Zeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK. Abstract Capillary electrophoresis (CE) was coupled via a triaxial sheath flow interface directly to the negative ionization electrospray (ES) atmospheric pressure source of a commercially available tandem quadrupole mass spectrometer (MS). The CE eluent was supplemented by a solvent make-up flow and this, together with a low flow nebulizer gas, produced a reliable and robust CE-ES-MS interface. Human urine extracts, after independent administration of three non-steroidal antiinflammatory drugs, were analyzed and the major metabolites identified using a combination of molecular weight (i.e. (M-H)-) and fragmentation data generated from both in-source collision induced dissociation and low energy tandem MS (MS-MS). The in-source collision induced dissociation and MS-MS product ion scanning spectra provided similar information. * These data have appeared in part in Anal. Proc., 32, 459, 1995. MS-MS precursor ion scanning was used for the ibuprofen-containing urine extract to screen specifically for glucuronide metabolites, all of which produced a common fragment ion at m/z 175 in negative ionization electrospray. In conclusion, on-line CE-ES-MS (-MS) proved to be a practical, viable technique for metabolite monitoring and identification of the drugs described, while minimizing sample clean-up. ApplicationNOTE Introduction Capillary electrophoresis (CE) and related techniques such as micellar electrophoresis have found increasing use in the area of drug and metabolite analysis in biological fluids1 due to the low sample and solvent consumption and high separation efficiencies. In parallel, CE coupled with electrospray (ES) - mass spectrometry (MS) is emerging as a viable on-line technique for a variety of routine analyses2,3, and has been applied to drug analysis4,5. In this paper, the high resolution of CE combined with the sensitivity and specificity of ES-MS and tandem ES-MS has been used to produce a practical method for the detection and identification of three non-steroidal antiinflammatory drugs and their metabolites in solid phase extracts of human urine. The drugs chosen were ibuprofen, flurbiprofen and aspirin, (Schematic 1), all widely administered and all of which produce highly polar metabolites amenable to negative ionization ES. The aim of the analyses was to produce both molecular mass information and to generate structurally useful fragment ions to aid the identification of the metabolites. By studying known drugs the validity of this approach can be estimated, and if successful the methodology then applied to more novel drug metabolism projects. Experimental All solvents were of HPLC grade purity, and all reagents and chemicals of analytical grade purity. All were used without further purification. Drug Administration and Sample Preparation Urine was obtained from a healthy male volunteer for the period 0 to 2 hr post-administration of a standard therapeutic dose of one of the chosen drugs: ibuprofen (400 mg), flurbiprofen (100 mg), or aspirin (600 mg), and then stored at -20°C until required for extraction. Solid phase extraction (SPE) of the urine was performed using C18 bonded SPE cartridges containing 500 mg of sorbent (Bond Elut, Varian, supplied by Jones Chromatography Ltd., Hengoed, UK). The cartridges were activated prior to extraction by washing with methanol (5 mL) followed by 0.1 M HCl (5 mL). Samples of urine (2 mL) were acidified to pH 2 with 0.1M HCl and then applied to the activated cartridges. The cartridges were then washed with 0.1M HCl (1 mL) followed by elution with methanol (5 mL). The collected methanolic eluates were reduced to dryness using a stream of nitrogen, and stored at 20°C until required for analysis. For CE-MS the dry residues were reconstituted in methanol (200 (µL). Capillary Electrophoresis CE was performed with an uncoated fused silica capillary (75 µm i.d. x 375 µm o.d. x 100 cm length) using a Beckman P/ACE System 2100 (Beckman Instruments Inc., Palo Alto, USA) with inline UV detection (200 nm) 19 cm from the injection end of the capillary. New CE capillaries were flushed with 1M NaOH, then water and finally the buffer in use for the analyses. In-between subsequent analyses the CE capillary was flushed with buffer only. A pressure injection (5 or 10 seconds) representing approximately 30 nL was used to introduce the sample into the capillary and then a voltage (20 kV) applied to effect separation. The CE buffer used was 20 mM aqueous ammonium acetate taken to pH 9 with aqueous ammonia solution. Mass Spectrometry The CE capillary was taken directly into CO2H CH 3 CH the atmospheric pressure ES source via the CE-ES triaxial sheath flow probe/interface of a Micromass® quadrupole mass spectrometer. The low up flow of water: propan-2-ol (20:80, v:v, 8 (µL/minute) which was introduced coaxially through the probe thus ensuring that mixing of the two mobile phases CO2H F CH 2 Altrincham, Cheshire, UK) tandem capillary necessitated the use of a make- OCOCH 3 CH Quattro II™ (Waters Corporation, flow of liquid emerging from the CE CO2H CH 3 CH CH 3 CH 3 C 15 H 13 FO2 244 C 9H 8 O 4 180 Flurbiprofen Aspirin (acetyl salicylic acid) C 13 H 18 O2 206 Ibuprofen Schematic 1. Chemical structures and formulae of ibuprofen, flurbiprofen and aspirin. took place at the probe tip inside the source and hence electrophoretic Ibuprofen disc 3, 10sec inj., 20mM amm. ac. pH 9, 20kV, CV=30. resolution was undisturbed. ZEN CE57 100 Scan ES- B 20.65 (a) The probe tip was held at -4 kV and C nitrogen was used as both the drying gas 22.97 and nebulizing gas, the latter being A introduced through the triaxial probe. 20.23 % The source temperature was maintained D at 70°C. Negative ionization was used 24.11 for all the analyses and mass accuracy 7.01 E 7.47 was ensured by calibration on a 31.10 24.42 14.24 separate introduction of a sugar mixture 0 using negative ionization ES analysis. All analyses were acquired at unit resolution ZEN CE57 100 200nm AN AL OG 6.90 (b) (50% valley definition). 3.54 3.95 Molecular mass information (i.e. (M-H)2.51 ions) was obtained using a low sampling % cone voltage (30 V). In order to generate fragment ions in the source, this value 4.52 was increased to 50 V. For tandem mass spectrometry (MS-MS) analyses, argon gas was introduced into the collision cell, which is situated between the first and second quadrupole analyzers, and a collision energy of 15 - 20 eV set. 0 T ime 10.00 20.00 30.00 40.00 Figure 1. (a) CE-MS total ion electropherogram for the separation of ibuprofen metabolites isolated from human urine by solid phase extraction. The order of elution of the peaks was: A. hydroxy ibuprofen glucuronide, 20.23 min.; B. ibuprofen glucuronide, 20.65 min.; C. hydroxy ibuprofen, 22.97 min.; D. ibuprofen, 24.11 min.; E. carboxylated ibuprofen glucuronide, 31.10 min. (b) in-line CE-UV ( 200 nm) electropherogram. ApplicationNOTE Results and Discussion Ibuprofen Following oral administration to humans, ibuprofen is rapidly excreted6 as a mixture including the drug and its hydroxy and carboxylic acid metabolites, together with the glucuronide conjugates of these three species. Ibuprofen and its metabolites are efficiently extracted by SPE onto C18 bonded silica which renders this a rapid, simple and convenient clean-up procedure from urine. The CE-ES-MS analysis of the ibuprofen urine extract produced the UV and total ion mass spectrometric electropherograms shown in Figure 1. The difference in retention times between the two traces results from the fact that the UV detection is taking place approximately one-fifth of the way along the separation column. A low sampling cone voltage (30V) had been set in the ionization source for this analysis to optimize the production of the (M-H)- ions, and from these ions the molecular masses of the components contained in this extract were established. The major components detected had molecular masses consistent with those expected for ibuprofen (MW 206), its side-chain hydroxylated metabolite (MW 222), the glucuronides of both these species (MWs 382 and 398 respectively), and the glucuronide of the side-chain oxidized carboxylic acid metabolite (MW 412), (Schematic 2). CH 3 CO2H CH 3 CO2G CH CH CH 2 CH 2 COH CH CH 3 CH 3 CH 3 CH 3 222 382 hydroxylated metabolite of ibuprofen glucuronide metabolite of ibuprofen CH 3 CO2G CH 3 CO2H(G) CH CH CH 2 CH 2 COH CH CH 3 CH 3 CH 3 CO2G(H) 398 412 hydroxylated glucuronide metabolite of ibuprofen carboxylated glucuronide metabolite of ibuprofen Schematic 2. Chemical structures of the major ibuprofen metabolites. Figure 2 shows the electropherograms of the (M-H)- ions of these components, in comparison with the total ion Ibuprofen disc 3, 10sec inj., 20mM amm. ac. pH 9, 20kV, CV=30. a) ZEN CE57 100 electropherogram. The CE-ES-MS peak electropherogram is typically 12 seconds for these compounds. E % width measured from the Scan ES411 31.10 7.63 21.89 0 b) ZEN CE57 100 Scan ES205 24.11 D % 1.63 4.57 7.63 8.50 12.43 16.00 24.99 20.65 0 ZEN CE57 c) 100 30.63 35.44 39.05 41.53 44.68 46.80 Scan ES221 22.97 C % 23.33 24.47 0 d) ZEN CE57 100 Scan ES381 20.65 B % 0 e) ZEN CE57 100 Scan ES397 20.29 A % 2.82 0 f) ZEN CE57 100 Scan ES- 20.65 22.97 20.23 % 24.11 7.01 24.42 14.24 31.10 0 T ime 10.00 20.00 30.00 40.00 Figure 2. CE-MS total ion (f) and selected ion electropherograms, (a) - (e), for the separation of ibuprofen metabolites isolated from human urine by solid phase extraction, showing the (M-H)- ions of ibuprofen, m/z 205; hydroxy ibuprofen, m/z 221; ibuprofen glucuronide, m/z 381; hydroxy ibuprofen glucuronide, m/z 397; carboxylated ibuprofen glucuronide, m/z 411. ApplicationNOTE The analysis was repeated using a higher sampling cone voltage (50V) in order to achieve in-source collision induced dissociation and generate structurally informative fragment ions for all of the components in the urine extract. The low and high cone voltage spectra obtained for the glucuronide conjugates of ibuprofen, hydroxylated ibuprofen, and carboxylated ibuprofen are compared in Figure 3. As these spectra show, in-source collision induced dissociation generates structurally useful information by producing diagnostic fragment ions. For example, the three glucuronide conjugates all exhibit a fragment ion at m/z 175 resulting from cleavage of the glucuronic acid portion of the molecules, and all three show a complementary loss of 176 amu from their (M-H)- ions to form the (M-H)- ions of the corresponding aglycones (i.e. m/z 205, 221, and 235) and/or decarboxylated aglycones (i.e. m/z 161, 177, and 191) of ibuprofen, hydroxylated ibuprofen and carboxylated ibuprofen respectively. It can also be observed that the intensity of the sodium adduct ions (M+Na-H)-, compared to the (M-H)- ions, is greater when a higher sampling cone voltage is used, implying that the sodiated adduct is the more stable of the two. These results indicate the potential of in-source collision induced dissociation on a single quadrupole mass spectrometer to produce structurally informative fragment ions. Ibuprofen disc 3, 10sec inj., 20mM amm. ac. pH 9, 20kV, CV=30. a) ZEN CE57 100 Scan ES- 411 % 433 220 0 b) ZEN CE58 100 Scan ES- 113 175 433 % 73 65 191 193 257 411 0 c) ZEN CE57 100 Scan ES- 397 % 398 0 d) ZEN CE58 100 Scan ES- 113 175 397 193 % 0 e) ZEN CE57 100 Scan ES- 381 % 382 0 f) ZEN CE58 100 Scan ES- 113 175 381 193 % 205 0 m/z 50 100 150 200 250 300 350 400 450 500 Figure 3. The low and high sampling cone voltage spectra of ibuprofen glucuronide (e and f), hydroxy ibuprofen glucuronide (c and d), and carboxylated ibuprofen glucuronide (a and b). For more specific fragmentation, and hence structural information, MS-MS can Ibuprofen disc 3, 20sec inj., 20mM amm. ac. pH 9, 20kV, D aughters, CE=15eV, Ar=3x10-3mbar. a) ZEN CE55 100 3: D aughters of 411ES- 175 be used with on-line CE-ES. Figure 4 shows the MS-MS product ion spectra 113 obtained from selectively and independently transmitting the (M-H)- ions % of the same three glucuronide conjugates through the first quadrupole analyzer into 193 the collision cell, and then monitoring the fragment ions arising directly from these using the second quadrupole analyzer. 411 235 157 0 b) ZEN CE55 100 1: D aughters of 397ES- 175 193 113 Many similarities between the product ion MS-MS spectra and the in-source collision induced dissociation spectra are apparent, in particular the characteristic % 177 glucuronide ions described for Figure 3. MS-MS data have the advantage of not 221 exhibiting solvent-related ions and 85 103 contain only those fragment ions which arise directly from the selected precursor. 397 131 0 c) ZEN CE55 100 2: D aughters of 381ES- 113 175 193 % 161 205 8589103 133 157 381 0 m/z 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 Figure 4. CE-MS-MS product ion spectra of the ibuprofen glucuronide conjugates: (a) carboxylated ibuprofen glucuronide; (b) hydroxy ibuprofen glucuronide; (c) ibuprofen glucuronide. ApplicationNOTE a) 1, 10sec inj., 20mM amm. ac. pH 9, 20kV, Parents of m/z 175. b) Ibuprofen disc(28.306) Ibuprofen disc 1, 10sec inj., 20mM amm. ac. pH 9, 20kV, Parents of m/z 175. ZEN CE47 100 Parents of 175ES- 18.16 Parents of 175ES411 ZEN CE47 808 100 T IC A C % 18.54 413 209 333 341 B c) 0 ZEN CE47 519 (18.190) 100 397 416 423 444 447 Parents of 175ES- A % % 409 0 d) ZEN CE47 528 (18.540) 100 Parents of 175ES- 381 C 28.31 B % 449 384 439 0 T ime 5.00 10.00 15.00 20.00 25.00 30.00 451 0 m/z 200 220 240 260 280 300 320 340 360 380 400 420 440 Figure 5. CE-MS-MS ibuprofen metabolite precursor ion data from m/z 175: (a) total ion electropherogram monitoring the precursor ions from m/z 175 in order to detect the glucuronide conjugates, showing A. hydroxy ibuprofen glucuronide; B. ibuprofen glucuronide; C. carboxylated ibuprofen glucuronide; (b) The MS-MS precursor ion spectrum for carboxylated ibuprofen glucuronide; (c) The MS-MS precursor ion spectrum for hydroxy ibuprofen glucuronide; (d) The MS-MS precursor ion spectrum for ibuprofen glucuronide. Ester glucuronide conjugates are very common metabolites of carboxylate-containing drugs and the ready fragmentation of these metabolites to generate a common ion at m/z 175 can be exploited using MS-MS to detect and identify selectively these conjugates in complex media such as urine extracts. This approach is illustrated in Figure 5 which shows the electropherogram from the precursor ion scan of m/z 175. In this case the second quadrupole analyzer was set to transmit only the m/z 175 ion while the first quadrupole analyzer was scanned routinely, with the result that only those components which produce a fragment at m/z 175 are monitored. The electropherogram shows three distinct components, whose precursor ion spectra show specifically those ions from which the m/z 175 fragment arises directly. The data indicate that the three components selectively monitored are indeed the three glucuronide conjugates discussed, and this screening procedure provides a rapid and specific means of identifying a particular set or type of compound in a complex extract. Flurbiprofen Flurbiprofen is another non-steroidal anti- Flurbiprofen, disc 2, 20kV, 20mM amm ac pH 9, 5sec inj. ZEN CE32 100 Scan ES- 17.56 inflammatory with some structural 17.34 similarities to ibuprofen. In humans, after A oral administration, it is also subject to B both “phase 1” metabolic hydroxylation reactions and also “phase 2” reactions to form glucuronide conjugates. After solid phase extraction from human urine, online CE-ES-MS was used to separate and identify the components in the extract. Figure 6 shows the total ion mass spectrometric electropherogram from the CE-ES-MS analysis of the flurbiprofen urine extract using a low sampling cone voltage to optimize the (M-H)- ions. The % peaks corresponding to flurbiprofen (MW 244), flurbiprofen glucuronide (MW 420) and the glucuronide of the hydroxylated metabolite of flurbiprofen (MW 436) have been identified on the electropherogram, with retention times of 20.60, 17.56 and 17.34 minutes respectively. C 20.60 0 T ime 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 Figure 6. CE-MS total ion electropherogram for the separation of flurbiprofen metabolites isolated from human urine by solid phase extraction. The order of elution of the peaks was: A. hydroxy flurbiprofen glucuronide, 17.34 min.; B. flurbiprofen glucuronide, 17.56 min.; C. flurbiprofen, 20.60 min. ApplicationNOTE The analysis was also performed with a high sampling cone voltage to induce in-source fragmentation, and complementary pairs of spectra for the two glucuronide conjugates are presented in Figure 7. As the spectra indicate, the (M-H)- ions are easily distinguished together with diagnostic fragment ions at m/z 175 for the glucuronic acid moieties, and their corresponding decarboxylated aglycone ions. Although the two glucuronides were barely resolved on the CE-MS electropherogram, clean spectra without interfering ions from other components have been obtained for both components. Flurbiprofen, disc 2, 20kV, 20mM amm ac pH 9, 5sec inj., CVF. CE32 335 (17.345) a) ZEN 100 Scan ES- 435 % 436 0 ZEN CE35 309 (16.000) b) 100 Scan ES- 175 435 215 113 % 89 457 103 193 133 0 c) ZEN CE32 338 (17.500) 100 Scan ES- 419 % 420 0 d) ZEN CE35 307 (15.894) 113 100 Scan ES- 175 % 477 199 419 193 133 0 50 100 150 200 250 300 350 400 450 m/z 500 Figure 7. The low and high sampling cone voltage spectra of hydroxy flurbiprofen glucuronide (a and b), and flurbiprofen glucuronide (c and d). Aspirin Aspirin (acetylsalicylic acid)7 is readily Aspirin, cartridge 5, 20kV, 20mM amm ac pH 9, 5sec inj. ZEN CE34 a) 100 metabolized in humans to salicylic acid 27.68 % (2-hydroxybenzoic acid), which is then mostly conjugated to glycine and excreted in urine as salicylhippuric acid. Scan ES- 28.45 29.90 22.20 0 ZEN CE34 b) 100 The glucuronide metabolites (both ester Scan ES137 29.90 % and ether) of salicylic acid are also produced to a lesser extent, and in addition minor metabolites include the 0 ZEN CE34 c) 100 hydroxylated metabolite gentisic acid, its % sulphate and glucuronic acid conjugate, and gentisuric acid (the glycine Scan ES194 28.40 28.19 0 ZEN CE34 d) 100 Scan ES187 27.68 conjugate). % The CE-ES-MS total ion electropherogram from the analysis with in-line UV detection 0 ZEN CE34 e) 100 using a low sampling cone voltage to Scan ES178 22.20 % optimize the (M-H)- ions is illustrated in Figure 8. The peaks were assigned as 0 ZEN CE34 f) 100 21.53 minutes), salicylhippuric acid (MW 195; 138; 29.90 minutes). 28.19 27.88 % 28.40 minutes), and salicylic acid (MW Scan ES195 28.45 follows: hippuric acid (MW 179; 22.20 0 g) ZEN CE34 100 Scan ES355 17.75 % 0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 T ime 40.00 Figure 8. (a) CE-MS total ion and (b) - (g) selected ion electropherograms for the separation of aspirin metabolites isolated from human urine by solid phase extraction. ApplicationNOTE The analysis was repeated at a high sampling cone voltage and also using on-line MS-MS experiments in order to generate structural information. For example, Figure 9 compares the low sampling cone voltage spectrum for the component of retention time 28.40 minutes (Figure 8) with the MS-MS product ion spectrum monitoring fragments of m/z 194. The assignment of salicylhippuric acid was made by observation of the (M-H)- ion at m/z 194 in the former spectrum and the diagnostic fragment ions at m/z 150 and 93 in the latter spectrum. Aspirin, cartridge 5, 20kV, 20mM amm ac pH 9, 5sec inj. a) ZEN CE34 549 (28.399) 100 194 Scan ES- % 195 0 b) ZEN CE62 570 (29.476) 100 D aughters of 194ES- 150 93 % 194 0 m/z 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 Figure 9. (a) The low sampling cone voltage spectrum of salicylhippuric acid compared with (b) its MS-MS product ion spectrum monitoring fragment ions from m/z 194. A Constant Neutral Loss (CNL) CE-ES-MSMS experiment monitoring those ions Aspirin cartridge 5, 10sec inj., 20mM amm. ac. pH 9, 20kV, N eutral loss of 44. a) ZEN CE61 514 (26.583) 100 178 N eutral L oss 44ES- which lose 44 amu was performed on this urine extract to highlight specifically those components containing carboxylate functional groups by scanning both quadrupole analyzers with an offset of 44 amu. Figure 10 shows two components of % the mixture monitored by this method: salicylhippuric acid (MW 195) and 113 hippuric acid (MW 179), both glycine conjugates. The method could constitute a rapid screening technique for complex extracts. 0 b) ZEN CE61 670 (34.643) 100 N eutral L oss 44ES194 % 0 m/z 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 Figure 10. On-line CE-ES-MS-MS Constant Neutral Loss analysis monitoring ions which lose 44 amu: (a) hippuric acid (MW 179), and (b) salicylhippuric acid (MW 195). ApplicationNOTE Summary These studies show that CE coupled to negative ionization ES-MS can be applied to the detection and identification of a range of non-steroidal anti-inflammatory drugs and their metabolites in urine, with minimal sample clean-up necessary. Molecular ion species (i.e. (M-H)-) ions were generated to confirm molecular weights, and in addition the use of in-source collision induced dissociation and low energy tandem mass spectrometry facilitated identification of the metabolites produced. Selective screening of the urine extracts for the presence of ibuprofen glucuronide metabolites was achieved by on-line CE-ES-MS-MS monitoring the precursor ions of the characteristic dehydrated glucuronic acid ion. The system used was operated for several days without any maintenance, due in part to the fact that CE requires only minimum amounts of dirty matrices to be injected. An environmental advantage of this technique over LC-MS methods is that solvent consumption is minimized. The results for each analysis were generated by injecting only 30 nL of the available, reconstituted urine samples, which were collected following standard therapeutic doses of the drugs. The ability to detect metabolites in this way could have a significant impact in the determination of such substances in biological matrices for both drug metabolism studies and in areas such as forensic toxicology and the detection of drugs of abuse. References 1. D.K. Lloyd in “Biofluid and Tissue Analysis for Drugs, including Hypolipodaemics”, eds. E. Reid, H.M. Hill, and I.D. Wilson, Royal Society of Chemistry, Cambridge, UK, p. 41 (1994). 2. J. Cai, J. Henion, J. Chromatogr., 703, 667 (1995). 3. M.W.F. Nielen, J. Chromatogr., 712, 269 (1995). 4. I.M. Johansson, R. Powelka, J.D. Henion, J. Chromatogr., 559, 515 (1991). 5. A.J. Tomlinson, L.M. Benson, K.L. Johnson, S. Naylor, J. Chromatogr., 621, 239 (1993). 6. S.S. Adams, R.G. Bough, E.E. Cliffe, B. Lessel, R.F.N. Mills, Toxicology and Applied Pharmacology, 15, 310 (1969). 7. K. Florey ed. in “Analytical Profiles of Drug Substances”, Academic Press, New York, p. 1 (1979). ApplicationNOTE WATERS CORPORATION 34 Maple St. Milford, MA 01757 U.S.A. T: 508 478 2000 F: 508 872 1990 www.waters.com/micromass Made in the United Kingdom Waters and Micromass are registered trademarks of Waters Corporation. All other trademarks are the property of their respective owners. ©2002 Waters Corporation October 2002 (WAN216) 720000514EN For research use only. Not for use in diagnostic procedures. Certificate No: 951387