Metabolome analysis via comprehensive two-dimensional liquid chromatography: identification of modified nucleosides from RNA metabolism.

Modified nucleosides derived from the RNA metabolism constitute an important chemical class, which are discussed as potential biomarkers in the detection of mammalian breast cancer. Not only the variability of modifications, but also the complexity of biological matrices such as urinary samples poses challenges in the analysis of modified nucleosides. In the present work, a comprehensive two-dimensional liquid chromatography mass spectrometry (2D-LC-MS) approach for the analysis of modified nucleosides in biological samples was established. For prepurification of urinary samples and cell culture supernatants, we performed a cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. In order to establish a 2D-LC method, we tested numerous column combinations and chromatographic conditions. In order to determine the target compounds, we coupled the 2D-LC setup to a triple quadrupole mass spectrometer performing full scans, neutral loss scans, and multiple reaction monitoring (MRM). The combination of a Zorbax Eclipse Plus C18 column with a Zorbax Bonus-RP column was found to deliver a high degree of orthogonality and adequate separation. By application of 2D-LC-MS approaches, we were able to detect 28 target compounds from RNA metabolism and crosslinked pathways in urinary samples and 26 target compounds in cell culture supernatants, respectively. This is the first demonstration of the applicability and benefit of 2D-LC-MS for the targeted metabolome analysis of modified nucleosides and compounds from crosslinked pathways in different biological matrices.

Determination of opioid analgesics in hair samples using liquid chromatography/tandem mass spectrometry and application to patients under palliative care.

Hair testing procedures allow a cumulative reflection of long-term drug abuse and are useful as a test for compliance in clinical toxicology. In the present study, liquid chromatography coupled with tandem mass spectrometry was used to determine analgesic opioid drugs in hair samples. The procedure used a simple methanolic extraction, and the evaporated extract was analyzed directly. A selective and sensitive procedure for the simultaneous determination of bisnortilidine, nortilidine, tilidine, buprenorphine, codeine, oxycodone, fentanyl, norfentanyl, hydromorphone, morphine, normorphine, oxymorphone, methadone, piritramide, and tramadol was developed and fully validated. The method fulfilled validation criteria and was shown to be sensitive, with limits of detection ranging from 0.008 to 0.017 ng/mg hair matrix, and precision ranging between 3.1% and 14.9 %. The applicability of the method was shown by analysis of authentic hair samples from patients receiving opioids for the treatment of cancer pain (eg, fentanyl was detected in concentrations up to 0.292 ng/mg, tramadol in concentrations up to 0.612 ng/mg of hair of 1 patient). Hair analysis was shown to be a complementary and useful tool in monitoring the drug-taking behavior of patients consuming opioid analgesics for the treatment of pain. In self-reports and medical records especially, the ingestion of tramadol and methadone was found to be dramatically underreported. In summary, hair analyses gave important additional information for the medical treatment of patients, the results often coming as a surprise to even the attending physicians.

The significance of putative urinary markers of illicit heroin use after consumption of poppy seed products.

After consumption of poppy seeds various substances were detected in urine or blood samples using an immunoassay and a sophisticated liquid chromatographic-tandem mass spectrometric procedure. These compounds are widely considered to be putative markers of heroin (HER) abuse whereas acetylcodeine was regarded as a marker for illicit preparations ("street HER"). Besides positive urinary opiate immunoassay results during a 48 hours monitoring period, peak concentrations of morphine (MOR), codeine and their glucuronides appeared 4 to 8 hours after ingestion of poppy seeds, and concentrations of total MOR higher than 10 microg/mL were observed. Also, in serum samples taken up to 6 hours after consumption, MOR glucuronides were found. Free MOR was only detected in traces (1 to 3 ng/mL) within 2 hours of consumption. In addition, 3 of 6 onsite opiate sweat tests revealed positive results 6.5 hours after ingestion. Furthermore, it was demonstrated that neither noscapine (NOS) nor papaverine (PAP) was detectable in urine or blood samples after the consumption of poppy seeds containing up to 94 microg NOS and up to 3.3 mug PAP. NOS and PAP were rapidly metabolized, whereas desmethylpapaverine and, especially, its glucuronide were found in urine samples of poppy seed consumers even 48 hours after consumption. According to these results PAP metabolites should not be regarded as markers of illicit HER abuse. In conclusion, only acetylcodeine can be regarded as a specific marker but has the problem of a short half-life. Therefore, we suggest that NOS and PAP, but not their metabolites, might be used cautiously as additional markers of illicit HER abuse as they have not been detected after oral intake of poppy seeds in normal doses. But it must be kept in mind that in some cases poppy seeds with an unusually high content of these alkaloids could be available, and that these substances are also agents in some pharmaceuticals.