This document discusses the use of surface activated chemical ionization (SACI) coupled with ion exchange chromatography for the analysis of polar low molecular weight drugs like morphine-3-glucoronide and morphine-6-glucoronide. Preliminary experiments showed that SACI produced better signals with less fragmentation compared to atmospheric pressure chemical ionization and electrospray ionization. SACI also showed less signal suppression during chromatographic analysis. The method was applied to urine samples without pre-treatment and showed good separation of analytes and low matrix effects. Future work will involve varying parameters and applying the LC-CEC-SACI-MS method to screen other drug metabolites in biological samples.

1. Ion exchange chromatography coupled to SACI for the analysis of high polar low molecular weight drugs Sara Crotti, ISB srl XIV Discussion group on Mass Spectrometry

2. Surface Activated Chemical Ionization

4. IEC coupled to Mass Spectrometry: SACI No discharge phenomena (low voltage source) Few application because of analyte degradation and ion suppression phenomena Never applied till now  ESI & APCI Discharge phenomena for the high voltages in API sources

5. Aim: IEC Analysis of M3G and M6G Optimal source: APCI, ESI or SACI? Morphine 3-glucoronide (M3G) Morphine 6-glucoronide (M6G)

6. Preliminary infusion of M3G 50ng/ml: APCI In source fragmentation MW 461 MW 285 0 2.0 3.0 4.0 5 x10 5 150 200 250 300 350 400 450 500 550 m/z 286 1.0 Morphine-3- Glucuronide in source fragment Intensity (counts/s) ‏

7. Preliminary infusion of M3G 50ng/ml: ESI Intensity (counts/s) ‏ 462 3.0 4.0 5.0 6 x10 5 2.0 1.0 0 150 200 250 350 400 450 550 m/z 300 500 [M+H] + Source noise

8. Preliminary infusion of M3G 50ng/ml: Intensity (counts/s) SACI 0.0 2.0 4.0 6.0 x10 5 150 200 250 300 350 400 450 500 550 m/z 462 286 8 Morphine-3- Glucuronide in source fragment [M+H] +

9. M3G and M6G separation with a linear gradient 5-100% B A : H 2 O B : H 2 O + 0.5% formic ac. + 10mM ammonium acetate SACI ESI Cation Exchange Chromatography (CEC) Injection of 500pg Chromatographic analysis:

10. Intensitiy (counts/s) Results : SACI ESI Signal suppression (in source current ~ 40000nA) Good signal (in source current ~ 2 4nA) ‏ 2 4 6 8 10 12 Time [min] 0 200 400 600 800 Intensitiy (counts/s) ‏ 2 4 6 8 10 12 Time [min] 0.0 0.5 1.0 1.5 2.0 5 x10 10.11 8.42 Morphine-6- Glucuronide Morphine-3- Glucuronide

11. SACI urine sample analysis: Urine sample Dil. 1:5 In H 2 0 10  l inj 2 4 6 8 10 12 Time [min] 0.0 0.5 1.0 1.5 2.0 x10 5 10.15 8.48 M3G M6G Intensitiy EIC m/z 462 m/z 286

12. Matrix effect: 20 negative urine spiked vs Standard solution % Var= (Au-As)/As*100 sample %Area variation urine vs Water samples 1 -6 2 -8 3 -6 4 -1 5 -5 6 -7 7 -4 8 -4 9 -5 10 -5 11 -5 12 -5 13 -0,5 14 -2 15 -1 16 -1 17 -4 18 -3 19 -3 20 -2

14. Future steps: Next experiments with potential and salt concentration variation Application of LC-CEC-SACI-MS for screening of other addictive drug metabolites in biological sample.

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