Cryogenic cooling of the NMR radio frequency coils and electronics to give greatly enhanced sensitivity is arguably the most significant recent advance in NMR spectroscopy. Here we report the first cryogenic probe built in flow configuration and demonstrate the application to LC-NMR-MS studies. This probe provides superior sensitivity over conventional noncryogenic flow NMR probes, allowing the use of 100 microL of untreated urine (40% less material than previous studies that required preconcentration) and yet revealing drug metabolites hitherto undetected by LC-NMR-MS at 500 MHz. Besides the known sulfate and glucuronide metabolites, previously undetected metabolites of acetaminophen were directly observable in a 15-min on-flow experiment. Simultaneous MS data also provided knowledge on the NMR-silent functional moieties. Further, stop-flow LC-NMR-MS experiments were conducted for greater signal-to-noise ratios on minor metabolites. The cryoflow probe enables the NMR analysis of lower concentrations of metabolites than was previously possible for untreated biofluids. This strategy is generally applicable for samples containing mass-limited analytes, such as those from drug metabolism studies, biomarker and toxicity profiling, impurity analysis, and natural product analysis.

ABSTRACT:In discovery and development, having a qualified metabolite standard is advantageous. Chemical synthesis of metabolite standards is often difficult and expensive. As an alternative, biological generation and isolation of metabolites in the nanomole range are readily feasible. However, without an accurately defined concentration, these isolates have limited utility as standards. There is a significant history of NMR as both a qualitative and a quantitative technique, and these concepts have been merged recently to provide both structural and quantitative information on biologically generated isolates from drug metabolism studies. Previous methodologies relied on either specialized equipment or the use of an internal standard to the isolate. We have developed a technique in which a mathematically generated signal can be inserted into a spectrum postacquisition and used as a quantitative reference: artificial signal insertion for calculation of concentration observed (aSICCO). This technique has several advantages over previous methodologies. Any region in the analyte spectra, free from interference, can be chosen for the reference signal. In addition, the magnitude of the inserted signal can be modified to appropriately match the intensity of the sample resonances. Because this is postacquisition quantification, no special equipment or pulse sequence is needed. Compared with quantitation via the addition of an internal standard (10 mM maleic acid), the signal insertion method produced similar results. For each method, precision and accuracy were within ؎5%, stability of signal response over 8 days was ؎5%, and the dynamic range was more than 3 orders of magnitude: 10 to 0.01 mM.

The sex pheromone of the longtailed mealybug, identified as 2-(1,5,5-trimethylcyclopent-2-en-1-yl)ethyl acetate, represents the first example of a new monoterpenoid skeleton. A [2,3]-sigmatropic rearrangement was used in a key step during construction of the sterically congested tetraalkylcylopentene framework.