Simultaneous determination of tryptophan and 8 metabolites in human plasma by liquid chromatography/tandem mass spectrometry.

Tryptophan (Trp) is an essential amino-acid and the precursor of many biologically active substances such as kynurenine (KYN) and serotonin (5HT). Its metabolism is involved in different physiopathological states, such as cardiovascular diseases, cancer, immunomodulation or depression. Hence, the quantification of Trp catabolites, from both KYN and 5HT pathways, might be usefulfor the discovery of novel diagnostic and follow-up biomarkers. We have developed a simple method for quantification of Trp and 8 of its metabolites,involved in both KYN and 5HT pathways, using liquid chromatography coupled to tandem mass spectrometry. We also validated the methodin human plasma samples, according to NF EN ISO 15189 criteria. Our method shows acceptable intra- and inter-day coefficients of variation (CV) (<12% and <16% respectively). The linearity entirelycovers the human plasma range. Stabilities of whole blood and of residues weredetermined, as well as the use of 2 different types of collectiontube, enabling us to adapt our process. Matrix effects and reference values showed good agreement compared to the literature. We propose here a method allowing the simultaneous quantification of a panel of Trp catabolites, never used before to our knowledge. This method, witha quickchromatographic runtime (15min) and simple sample preparation, has beenvalidated according to NF EN ISO 15189 criteria. The method enables the detailed analysis of these metabolic pathways, which are thought to be involved in a number of pathological conditions.

Four years of LC-MS/MS method for quantification of 25-hydroxyvitamin D (D2+D3) for clinical practice.

Many methods for routine total plasma/serum 25-hydroxyvitamin D (25OHD) measurements are available from automated immunoassays to the most specific LC-MS/MS techniques. These last ones are nowadays numerous, still perfectible but more powerful than immunoassays in specific illnesses. We presented a robust method for simple quantification of 25-hydroxyvitamin D(2) (25OHD(2)) and 25-hydroxyvitamin D(3) (25OHD(3)) in human plasma by LC-APCI-MS/MS. This method is reliable and easy to perform for clinical measurements as we report a 4-year of clinical laboratory use. A brief off-line sample pretreatment (protein precipitation with addition of the internal standard) was realized then the supernatant was loaded into 96-well plates and analyzed by an online SPE-LC/MS/MS method on an APCI mode. 25OHD(2) and 25OHD(3) were both measured. The chromatographic system was thought and optimized for providing a dedicated line for this measurement on a shared instrument. The linearity was tested up to 380 nmol/L for both 25OHD(2) and 25OHD(3). The limit of quantification (LOQ) was 7 and 8 nmol/L for 25OHD3 and 25OHD(2), respectively. Routine imprecision and bias were found in agreement with recommended limits for routine testing, CV≤10% and bias≤5%. Since July 2010, our participation to DEQAS was successfully validated. This simple robust online SPE-LC/MS/MS method is suitable for routine measurement of 25OHD(2) and 25OHD(3) in human adult plasma. The assay operates for 4 years and has performed more than 40,000 patient samples on a shared instrument.