SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

F7
Direct Analysis of Opiates in Urine by Liquid ChromatographylMass Spectrometry!
Mass Spectrometry
Leslie E. Edinboro, MS, PhD, *,1,2 Ronald C. Backer, PhD. 3 & Alphonse Poklis, PhD.l
1. Department of Pathology, School of Medicine, 2. Department of Pharmaceutics, School of Pharmacy,
Virginia Commonwealth University, Richmond, Virginia, 3. AmeriTox Laboratory, Midland, Texas.
A method for the direct analysis of ten opiate compounds in urine was developed using Liquid
ChromatographylMass SpectrometrylMass Spectrometry (LCIMSIMS). Opiates included were: morphine-
3-f3-glucuronide, morphine-6~~-glucuronide, morphine, oxymorphone, hydromorphone, norcodeine,
codeine, oxycodone, 6-monoacetylmorphine (6MAM) and hydrocodone. Deuterated internal standards
used were morphine-d 3 and 6MAM-~. Urine samples were prepared by addition of the internal standard
solution, centrifugation to remove large particles and direct injection into the LCIMSIMS. Gradient reverse
phase separation was accomplished on a phenyl column using acetonitrile and water modified with formic
acid and ammonium formate. An electrospray ionization (ESI) interface was used to introduce the LC
eluent into the MS. Multiple Reaction Monitoring (MRM) was utilized for detection in the MSIMS mode
based upon pre-established precursor: product mJz transitions. Separation and detection of all compounds
was accomplished within six minutes. Selectivity and possible ion suppression were evaluated using 10
different blank urines. Linearity was established for all opiates except 6MAM from 50 nglmL to 10,000
nglmL; 6MAM from 0.25 ng/mL to 50 ngimL. Correlation coefficients (r) for all opiates was > 0.99. Interrun
precision (%CV) ranged from 1.1 % to 16.7%. Intra-run precision ranged from 1.3% to 16.3%.
Accuracy (% Bias) ranged from -7.3% to 13.6% and -8.5% to 11.8 for inter and intra-run respectively. 89
urine samples previously analyzed by GCIMS were re-analyzed by the LCIMSIMS method. The qualitative
results found an 88% agreement for negative samples between the two methods and 94% for positive
samples. The LCIMSIMS method identified 19 samples with additional opiates in the positive samples.
Ten samples were oxymorphone positive that the GCIMS method was not setup to detect. Of these, 90%
were associated with GCIMS positiYe oxycodone samples, which metabolically are justified. Additionally
three samples were· hydromorphone positive for GCIMS hydrocodone positive samples, which also is
likely. The remaining positive samples were oxymorphone, oxycodone, hydrocodone and hydromorphone.
Quantitatively it was difficult to compare the two methods as the GCIMS method utilized a glucuronidase
step that was not included in the LCIMSIMS method. Therefore only the morphine results could be
compared if the LCIMSLMS results for the glucuronide metabolites were summed on a molar basis with the
morphine. The R2 was 0.78. The difference between the methods is probably due to the efficiency of the
GCIMS glucuronidase procedure. Overall the direct injection LCIMSIMS method performed well and
permitted the rapid analysis « 6,0 minutes) of urine samples without extensive sample preparation for
several opiates simultaneously. Additionally the method provided a lower limit of quantitation for 6MAM
than previously reported utilizing direct injection techniques. The method could be further improved with
the addition of compound specific deuterated internal standards and other glucuronide metabolites, i:e.,
hydromorphone.
Keywords: Opiates, LCIMS/MS, Urine
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