BackgroundBusulfan, an alkylating agent administered prior to hematopoietic stem cell transplantation, has a narrow therapeutic range and wide variability in metabolism. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for rapid and accurate quantification of plasma busulfan.MethodsBusulfan was separated and detected using an LC system containing a C18 column equipped with MS/MS. The sample was eluted with a mobile phase gradient for a total run time of 10 min. Plasma busulfan concentration was quantified against a 6-point standard curve in a multiple reaction monitoring mode at mass-to-charge (m/z) 264.1 > 151.1. Precision, recovery, matrix effect, linearity, detection capability, carryover, and stability were evaluated. The range of plasma busulfan concentration was obtained by analyzing samples from 9 children receiving busulfan.ResultsThe coefficients of variation of within-run and within-laboratory precision were all below 5%. Recoveries were all within the range of 100-105%. Linearity was verified from 0 to 5,000 ng/mL. Limit of detection and limit of quantification were 1.56 and 25 ng/mL, respectively. Carryover rate was within allowable limits. Plasma busulfan concentration was stable for 2 weeks at -20℃ and -80℃, but decreased by 25% when the plasma was stored for 24 hr at room temperature, and by <5% in 24 hr at 4℃. The plasma busulfan concentrations were between 347 ng/mL and 5,076 ng/mL.ConclusionsOur method using LC-MS/MS enables highly accurate, reproducible, and rapid busulfan monitoring with minimal sample preparation. The method may also enable safe and proper dosage.

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BackgroundGenetic abnormalities in adult AML are caused most frequently by somatic mutations in exon 12 of the NPM1 gene, which is observed in approximately 35% of AML patients and up to 60% of patients with cytogenetically normal AML (CN-AML).MethodsWe performed mutational analysis, including fragment analysis and direct sequencing of exon 12 of the NPM1 gene, on 83 AML patients to characterize the NPM1 mutations completely.ResultsIn this study, NPM1 mutations were identified in 19 (22.9%) of the 83 AML patients and in 12 (42.9%) of the 28 CN-AML patients. Among the 19 patients with NPM1 mutations, type A NPM1 mutations were identified in 16 (84.2%) patients, whereas non-A type NPM1 mutations were observed in 3 (15.8%) patients. Two of the 3 non-A type NPM1 mutations were novel: c.867_868insAAAC and c.869_873indelCTTTAGCCC. These 2 novel mutant proteins display a nuclear export signal motif (L-xxx-L-xx-V-x-L) less frequently and exhibit a mutation at tryptophan 290 that disrupts the nucleolar localization signal.ConclusionsThis study suggests that novel NPM1 mutations may be non-rare and that supplementary sequence analysis is needed along with conventional targeted mutational analysis to detect non-A types of NPM1 mutations.

Robinsoniella peoriensis has recently been identified as a Gram-positive, spore-forming, anaerobic rod originally recovered from swine manure storage pits. To date, 6 cases of R. peoriensis infection have been reported, including 2 cases of bacteremia, 1 of abdominal fluid collection, and 3 of wound infection. In the present study, we report a 76-yr-old man with R. peoriensis bacteremia who developed aspiration pneumonia. Gram staining of a purified colony revealed Gram-positive, rod-shaped bacteria. Biochemical identification using API 20 A (bioMérieux, France) indicated presence of Clostridium spp. We performed both 500-bp and full-gene sequencing of 16S rRNA of the isolate. The sequence was analyzed with MicroSeq ID 16S rRNA Library v2.0 (Applied Biosystems, USA), GenBank Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/genbank), and EzTaxon database v2.1 (http://www.eztaxon.org). The 500-bp 16S rRNA sequence of the blood culture isolate showed 99.16-99.79% similarity with R. peoriensis and the full-gene 16S rRNA sequence showed 98.87-99.50% similarity with R. peoriensis. The organism was confirmed as R. peoriensis by using all of the mentioned databases except for MicroSeq, which did not include the RNA sequence of this bacterium. This case suggests that identification of R. peoriensis might be challenging in clinical laboratories with no access to molecular methods, as certain commercial identification systems may not identify, or may misidentify, this organism. To the best of our knowledge, this is the first report of the isolation of R. peoriensis in Korea.