Monitoring carnitine and acetylcarnitine levels in biological fluids is a powerful tool for diagnostic studies. Research has recently shown that the analysis of carnitine and related compounds in clinical samples can be accomplished by different analytical approaches. Because of the polar and ionic nature of the analytes and matrix complexity, accurate quantitation is a highly challenging task. Thus, sample processing factors, preparation/cleanup procedures, and chromatographic/ionization/detection parameters were evaluated. On the basis of the results obtained, a rapid, selective, sensitive method based on hydrophilic interaction liquid chromatography-tandem mass spectrometry for the analysis of carnitine and acetylcarnitine in serum and urine samples is proposed. The matrix effect was assessed. The proposed approach was validated, the limits of detection were in the nanomolar range, and carnitine and acetylcarnitine levels were found within the micromolar range in both types of sample.
Inductively coupled plasma mass spectrometry instrumentation along with the dynamic reaction cell (DRC) technology has been increasingly used in the last decade for multielemental analysis of biological samples. This work reports the development of a method to assess concentrations of Li,
The high sensitivity that can be attained by enzymatic amplification via substrate cycling has been verified by on-line interfacing of a rotating bioreactor and continuous-flow/stopped-flow/continuous-flow processing [Raba, J.; Mottola, H. A. Anal. Biochem. 1994, 220, 297-302]. The determination of glucose levels was possible with a limit of detection of 0.2 fmol·L(-)(1) in the processing of as many as 30 samples per hour. Determination at such low levels is of interest in several situations encountered in fermentation biotechnology and clinical chemistry, and this determination in culture broths illustrates the capabilities of the proposed approach. The glucose oxidase/glucose dehydrogenase coupled system was used by immobilizing glucose oxidase (EC 1.1.3.4) on the top of a rotating disk while glucose dehydrogenase (EC 1.1.1.47) was immobilized on the top part of the flow-through cell. Substrate cycling was realized via NADH/NAD(+) that, in conjunction with glucose dehydrogenase, regenerates glucose, the substrate in the glucose oxidase-catalyzed reaction. This cycling permits generation of H(2)O(2) (detected at Pt ring electrode concentric to the rotating disk) beyond stoichiometric limitations. This permits a 100-fold increase in the sensitivity for glucose determination when compared with the determination involving no substrate cycling.
A rapid, selective and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to determine carnitine in human urine. Solid phase extraction approaches based on the use of polymeric and weak cationic exchange cartridges were evaluated and applied to the treatment of urine samples. After optimizing the various stages of SPE, a satisfactory set up for retaining substances interfering on carnitine's response was achieved for both type of cartridges. The UPLC separation was carried out on a reversed phase column. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. Residual matrix components were specific to urine samples and interfered on the carnitine signal (a response suppression of 50% was observed). It was then demonstrated that sample treatment by SPE could reduce the effect of the above mentioned interferents, without needing a preliminary derivatization step. The recovery percentage of carnitine obtained after the application of SPE was of approximately 83±7% and, consequently, the matrix effect was minimized. Thus, a sensitive, precise and reliable methodology was developed to determine traces of carnitine in biological fluids.
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