LC-MS/MS Screen for Penitrem A and Roquefortine C in Serum and Urine Samples Elizabeth R. Tor,* Birgit Puschner, Michael S. Filigenzi, Asheesh K. Tiwary, and Robert H. Poppenga California Animal Health and Food Safety Laboratory System, Toxicology Laboratory, University of California, Davis, California 95616 A rapid LC-MS/MS method, using a triple quadrupole/ linear ion trap mass spectrometer, was developed for determination of penitrem A and roquefortine C in serum and urine samples. Penitrem A and roquefortine C were extracted from samples with methylene chloride. The extracts were injected onto a liquid chromatograph coupled with a hybrid triple quadrupole/linear ion trap mass spectrometer. Seven replicate fortifications of serum at 0.001 μg/g (1 ppb) each of penitrem A and roquefortine C gave average recoveries of 90% with 10% CV (relative standard deviation) and 97% with 3% CV, respectively. Seven replicate fortifications of urine at 0.001 μg/g (1 ppb) each of penitrem A and roquefortine C gave average recoveries of 98% with 12% CV and 100% with 6% CV, respectively. This is the first report of a positive mass spectrometric identification and quantitation of both compounds in urine and serum samples from dog intoxi- cation cases. Penitrem A [12627-35-9], C 37 H 44 ClNO 6 , and roquefortine C [58735-64-1], C 22 H 23 N 5 O 2 , Figure 1, are tremorgenic mycotoxins produced most commonly by the fungi from the genera Penicil- lium, Aspergillus, and Claviceps. 1,2 Intoxication with these myc- otoxins has been documented in humans 3 and animals including dogs 4-10 and cattle. 11 Clinical signs of tremorgenic mycotoxin poisoning are well documented and include severe generalized muscle tremors and incoordination, 3-5 seizures 3-6 , muscle fas- ciculations, 6 and generalized convulsions, 9,10 followed by death in many cases. In severe intoxications increased muscle activity can ultimately lead to exhaustion, metabolic changes, rhabdomyolysis, and death. In most cases in dogs, vomiting occurs prior to the onset of neurological signs. Analytical methods for the diagnosis of tremorgenic mycotoxin poisoning are currently limited to the analysis of penitrem A and roquefortine C in source material (mold cultures, food) or GI contents. These include TLC, 8,12 GC/MS, 12,13 HPLC, 14 and LC/ MS techniques including triple quadrupole, 15 ion trap, 16-18 and time-of-flight mass spectrometry. 19 In clinical cases where penitrem A or roquefortine C poisoning is suspected a rapid, specific, and accurate method is needed to detect them at toxicologically significant, low ppb concentrations in urine and serum from live * Corresponding author. Phone: 530-752-6322. Fax: 530-752-3361. E-mail: ertor@ucdavis.edu. (1) Puschner, B. Vet. Clin. Small Anim. 2002, 32, 409-419. (2) Bu ¨gner, J.; Westphal, G.; Mo ¨nnich, A.; Hinnendahl, B.; Hallier, E.; Mu ¨ller, M. Toxicology 2005, 202, 199-211. (3) Lewis, P. R.; Donoghue, M. B.; Hocking, A. D.; Cook, L.; Granger, L. V. Med. J. Aust. 2005, 182, 582-584. (4) Boysen, S, R.; Rozanski, E. A.; Chan, D. L.; Grobe, T. L.; Fallon, M. J.; Rush, J. E. J. Vet. Med. Assoc. 2002, 221, 1441-1444. (5) Walter, S. L. Can. Vet. J. 2002, 43, 372-374. (6) Young, K. L.; Villar, D.; Carson, T. L.; Imerman, P. M.; Moore, R. A.; Bottoff, M. R. J. Vet. Med. Assoc. 2003, 222, 52-53. (7) Lowes, N. R.; Smith, R. A.; Beck, B. E. Can. Vet. J. 1992, 33, 535-537. (8) Hocking, A. D.; Holds, K.; Tobin, N. F. Aust. Vet. J. 1988, 65, 82-85. (9) Puls, R.; Ladyman, E. Can. Vet. J. 1988, 29, 569. (10) Richard, J. L.; Bacchetti, R. L.; Arp, L. H. Mycopathologia 1981, 76, 55-58. (11) Cysewski, S. J.; Baetz, A. L.; Pier, A. C. Am. J. Vet. Res. 1975, 36, 53-58. (12) Braselton, W. E.; Johnson, M. J. Vet. Diagn. Invest. 2003, 1, 42-45. (13) Braselton, W. E.; Rumler, P. C. J. Vet. Diagn. Invest. 1996, 8, 515-518. (14) Noroozian, E.; Lagerwerf, F.; Lingeman, H.; Brinkman, U. A.; Kerkhoff, M. A. J. Pharm. Biomed. Anal. 1999, 20, 611-619. (15) Sumarah, M. W.; Miller, J. D.; Blackwell, B. A. Mycopathologia 2005, 159, 571-577. (16) Rundberget, T.; Skaar, I.; O’Brien, O.; Flaoyen, A. Mycopathologia 2004, 157, 349-357. (17) Naude, T. W.; O’Brien, O. M.; Rundberget, T.; McGregor, A. D.; Roux, C.; Flaoyen, A. J. S. Afr. Vet. Assoc. 2002, 73, 211-215. (18) Rundberget, T.; Wilkins, A. L. J. Chromatogr., A 2002, 964, 189-197. (19) Nielsen, K. F.; Smedsgaard, J. J. Chromatogr., A 2003, 1002, 111-136. Figure 1. Penitrem A [12627-35-9] C37H44ClNO6 (top) and roque- fortine C [58735-64-1] C22H23N5O2 (bottom)stremorgenic mycotoxins produced most commonly by the fungus Penicillium crustosum. Anal. Chem. 2006, 78, 4624-4629 4624 Analytical Chemistry, Vol. 78, No. 13, July 1, 2006 10.1021/ac0601617 CCC: $33.50 © 2006 American Chemical Society Published on Web 05/09/2006