Improved tandem mass spectrometry (MS/MS) derivatized method for the detection of tyrosinemia type I, amino acids and acylcarnitine disorders using a single extraction process.

BACKGROUND: Succinylacetone (SUAC), a specific marker for tyrosinemia type I (Tyr I) cannot be detected by the routine LC-MS/MS screening of amino acids (AA) and acylcarnitines (AC) in newborns. The current derivatized methods require double extraction of newborn dried blood spots (DBS); one for AA and AC and the second for SUAC from the blood spot left after the first extraction. We have developed a method in which AA, AC and SUAC are extracted in a single extraction resulting in significant reduction in labor and assay time. METHODS: The 3.2 mm DBS were extracted by incubating at 45 °C for 45 min with 100 µl of acetonitrile (ACN)-water-formic acid mixture containing hydrazine and stable-isotope labeled internal standards of AA, AC and SUAC. The extract was derivatized with n-butanolic-HCl and analyzed by LC-MS/MS. RESULTS: The average inter-assay CVs for, AA, AC and SUAC were 10.1, 10.8 and 7.1% respectively. The extraction of analytes with ACN-water mixture showed no significant difference in their recovery compared to commonly used solvent MeOH. The concentration of hydrazine had considerable impact on SUAC extraction. CONCLUSION: We developed a new MS/MS derivatized method to detect AA/AC/SUAC in a single extraction process for screening Tyr I along with disorders of AA and AC.

Structural determination of aspericins A-C, new furan and pyran derivates from the marine-derived fungus Rhizopus sp. 2-PDA-61, by 1D and 2D NMR spectroscopy.

Three new furan and pyran derivatives named aspericins A-C (1-3), as well as a known asperic acid (4), have been isolated from the marine-derived fungus Rhizopus sp. 2-PDA-61. The complete (1)H and (13)C NMR assignments for the new compounds were carried out using (1)H, (13)C, DEPT, COSY, HMQC, HMBC, and NOESY NMR experiments. Compounds 1-3 were evaluated for their cytotoxic activities on P388, A549, HL-60, and BEL-7420 cell lines by the MTT and SRB methods.

Improved MS/MS analysis of succinylacetone extracted from dried blood spots when combined with amino acids and acylcarnitine butyl esters.

BACKGROUND: The utilization of succinylacetone (SUAC) as the primary metabolic marker for tyrosinemia Type I is now well known, thus new methods have been developed to analyze SUAC as a first tier test in newborn screening. One approach is to prepare a SUAC hydrazine derivative from the dried blood spots (DBS) previously utilized in the extraction of acylcarnitine (AC) and amino acids (AA). The final derivatized products of SUAC, AA and AC are combined in a single tandem mass spectrometric (MS/MS) analysis. However, butyl esterification techniques may result in contamination of underivatized acylcarnitines by as much as 20%. We have developed a simple wash step to improve the combined analysis of SUAC, AA and AC in DBS by MS/MS. METHODS: AA and AC were extracted with methanol containing labeled internal standard from 3.2mm punches taken from the DBS specimen. The previously extracted blood spot that remains after removal of the methanol extraction solvent was used in the preparation of SUAC with and without additional washing of the blood spot. The butyl ester eluates of AA and AC, and SUAC hydrazine derivatives were recombined and measured by MS/MS. RESULTS: Three additional methanol wash steps of the remaining DBS punches prior to SUAC derivatization reduced the presence of underivatized acylcarnitines, resulting in a 4-fold reduction of underivatized palmitoylcarnitine. Palmitoylcarnitine butyl ester is detected at m/z 456 while the underivatized species is detected at m/z 400, which is also the mass of dodecanoylcarnitine butyl ester. The linearity of the SUAC assay was unchanged by the additional wash steps. For butyl esterification methods, the preferred analytic procedure, the presence of AC can compromise the results of a newborn screen for the actual concentrations of acylcarnitines. It is essential to remove any underivatized acylcarnitines prior to SUAC analysis. CONCLUSION: The additional methanol wash steps did not alter SUAC assay results but did remove underivatized acylcarnitines which could result in the incorrect quantification of acylcarnitines.

Monitoring seasonal variation in apple fruit volatile emissions in situ using solid-phase microextraction.

Emissions of volatiles from apple fruits (Malus domestica Borkh.) were monitored in situ over the course of a growing season (from early June to mid September) for two apple varieties, Golden Delicious and Maigold. Results indicate a characteristic time-course of volatile emissions as the sampling date was a statistically significant factor for nine of the 13 compounds considered. The amounts of volatiles collected were greatest early and late in the season. The temporal effect on emissions was generally much larger than the effect of variety, which was significant for only four of the 13 compounds considered. The possible sources of variation which are not explained by the statistical models are discussed, and it is considered that they are most likely related to differences in the emissions from individual fruits.