Agilent Quantitative Analysis of Opiates in Urine Using Accurate Mass LC/MSD TOF Application
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1. ng mL and 300 ng mL obtained by LC MSD TOF direct injection of urine Urine direct injection Spike 1000 ng mL Urine direct injection Spike 300 ng mL Morphine Codeine Acetylmorphine Morphine Codeine Acetylmorphine 241 446 715 66 7 93 8 176 222 402 653 78 5 94 8 203 238 426 683 73 7 93 199 195 338 687 73 7 96 5 201 200 351 588 76 6 94 3 185 Mean 219 2 392 6 665 2 Mean 73 8 94 5 192 8 sD 21 2 46 8 48 4 SD 45 1 3 11 8 RSD 97 11 9 7 3 RSD 6 1 1 4 6 1 These are typical concentration and cut off range of immunoassay Note that difference between spiked value and measured concentration represents degree of ion suppression at source Table 3 preparation Accubond Evidex 5 mL Urine Spike 6 ng mL Expected Conc 60 pg pL Morphine Codeine Acetylmorphine 6 97 8 62 3 74 8 56 9 57 4 21 10 8 41 4 03 9 24 8 5 3 81 7 07 8 15 3 48 9 46 8 99 3 5 7 66 8 91 3 79 Mean 8 4 8 7 3 8 SD 1 2 0 5 0 3 RSD 14 4 5 3 6 9 www agilent com chem Quantitative results of spikes at 6 ng mL and 60 ng mL obtained by LC MSD TOF with Accubond Evidex SPE sample Accubond Evidex 5 mL Urine Spike 60 ng mL Expected Conc 600 pg L Morphine Codeine Acetylmorphine 508 499 182 567 543 193 525 504 183 521 502 191 595 532 193 591 532 192 582 540 196 Mean 555 6 521 7 190 0 SD 36 6 19 2 5 4 RSD 6 6 3 7 2 8 Difference in spiked value and measured concentration represents both recovery of SPE method and ion suppression if any C2. 5 mL 10 N NaOH and then adjusted to pH 6 5 7 5 with 2 5 mL 0 5 M phosphoric acid The heating step was not included because acid hydrol ysis of glucuronides were not expected After con ditioning this solution was loaded onto the cartridge rinsed and then eluted with the pre scribed solution of methylene chloride isopropanol ammonium hydroxide The eluant was taken to dryness with nitrogen no heat and then reconstituted in 0 5 mL 40 60 water acetonitrile a Agilent Technologies Instrument Agilent 1100 Series LC MSD TOF with Agilent 1100 binary pump and well plate autosampler Table 1 Experimental Conditions LC Conditions Column ZORBAX XDB C18 2 1 mm x 50 mm 3 5 pm P N 971700 902 Mobile Phases A Acetonitrile with 0 1 formic acid B Water with 0 1 formic acid Gradient 35 to 95 A in 5 min then to 100 in 6 min Flow rate 0 35 mL min MS Conditions Standard autotune conditions with calibrant delivery system pro viding constant low flow of 2 uM purine and HP 921 calibrant to dual ESI for continuous auto calibration Results Shown in Figure 1 upper panel is the total ion chromatogram TIC and in the lower panel over laid extracted ion chromatograms EICs for mor phine codeine and acetylmorphine in a direct injection of urine at 300 ng mL The EIC has a mass window of 20 ppm 0 002 u Accurate mass spectra for these opiates is given in Figure 2 Table 1 shows the quantitative results obtained wi
3. Quantitative Analysis of Opiates in Urine Using Accurate Mass LC MSD TOF e Application e J e o o c e ee a e e o Forensics J e e Author Jerry Zweigenbaum Agilent Technologies Inc 2850 Centerville Road Wilmington DE 19808 USA Abstract Urine samples were quantitatively analyzed at the 6 ng mL level using liquid chromatography mass selec tive detector time of flight The advantage of accurate mass measurement to enhance selectivity is presented The instrumental detection limit is 2 pg on column with a signal noise ratio of 5 1 Introduction Until now quantitative time of flight TOF has not been broadly applicable This application demon strates that the Agilent liquid chromatography mass selective detector time of flight LC MSD TOF can routinely quantify compounds at low levels in matrices important to the forensic scientist Both direct injection of urine and solid phase extraction SPE are performed to demon strate the robustness sensitivity and selectivity of the LC MSD TOF Experimental Sample preparation Direct injection samples were spiked at the speci fied concentrations with no further handling Accubond II Evidex SPE Cartridges part number 188 2946 were used as per extraction protocol for opiates see step by step instructions that comes with cartridges Five milliliters of either blank or spiked urine was treated with 0 5 mL concentrated HCl 0 7
4. onclusions The data shown demonstrates the ability of LC MSD TOF to confirm with accurate mass mea surement and quantify with selective narrow mass window e Direct injection of urine shows the robustness of the LC MSD TOF e Typical clean up SPE shows excellent sensitivity e High mass resolution and accuracy of every spectrum provides the selectivity for reduction of chemical noise for quantitation and confirmation For More Information For more information on our products and services visit our Web site at 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 material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2005 Printed in the USA January 14 2005 5989 2206EN ea Agilent Technologies
5. th direct injection Table 2 shows the results obtained with the solid phase extraction SPE 1 71 2 41 1 41 y 14 67 EE A e E e E e E E A E a e 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 10 0 11 0 120 130 14 0 Time min Codeine Acetylmorphine EIC of opiates Morphine 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 10 0 110 120 13 0 14 0 Time min Figure 1 The upper panel shows the TIC of a direct urine injection spiked with 300 ng mL of each opiate The lower panel shows the EIC of each compound 286 1443 800 2 Morphine amp 600 Z Exact mass 286 1437 error 1 85 ppm Zo 400 4 g a z 3 200a T 0 T T T T ig T 1 286 0 287 0 288 0 m z amu Max 859 8 counts a 80 300 1580 z 600 gt 4 Codeine 7 E S 400 E Exact Mass 300 1594 error 4 7 ppm i r 200 4 N 0 4 T T I T i T T T i T ji 300 0 300 2 300 4 300 6 300 8 301 0 301 2 301 4 301 6 m z amu Max 3718 5 counts 2 eo 328 1545 Cc m 3 E Acetylmorphine 400 4 D Exact mass 328 1543 error 0 5 ppm D J 200 4 cS iy T i T T if i T T i if T T T T if i T T g T T T ij T ii i T T i T T T T T 327 0 327 5 328 0 328 5 329 0 329 5 330 0 330 5 331 0 m z amu Figure 2 Mass spectra of M H ions for opiates showing both mass resolution and mass accu racy at 2 pg on column Table 2 Quantitative results in ng mL of spikes at 1000
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Agilent Quantitative Analysis of Opiates in Urine Using Accurate Mass LC/MSD TOF Application