Quantification of human plasma-busulfan concentration by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Busulfan, 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. METHODS: Busulfan 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. RESULTS: The 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. CONCLUSIONS: Our 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.

Physico-chemical stability of busulfan in injectable solutions in various administration packages.

BACKGROUND AND OBJECTIVES: Busulfan is used as part of a conditioning regimen prior to hematopoietic stem cell transplantation for the treatment of certain cancers and immune deficiency syndromes. Due to its instability in aqueous preparations, busulfan for infusion is prepared from a concentrate and has a relatively short shelf life once prepared. The purpose of this study was to identify the most suitable storage container and temperature to maximize the shelf life of busulfan therapeutic infusions prepared from Busilvex(®). METHODS: Busilvex(®) 6 mg/mL was diluted to 0.55 mg/mL with 0.9 % NaCl and aliquots dispensed into polypropylene syringes, polyvinyl chloride bags, and glass bottles. Three storage temperatures were evaluated: 2-8 °C, 13-15 °C (thermostatically controlled chamber), and room temperature (20 ± 5 °C). At set time points, samples were analysed for busulfan content, using a high-performance liquid chromatography (HPLC) system with ultraviolet detection. The change in pH and osmolarity on storage was also determined, and solutions were inspected visually for formation of a precipitate or colour change. To determine the contribution of precipitation to loss of busulfan content on storage, samples from one time series were treated with the solvent dimethylacetamide prior to HPLC separation and quantitation of busulfan. RESULTS: The results of the active substance content monitoring study over a 48-h period demonstrate that busulfan solution is stable at a 5 % threshold, at 2-8 °C for 16 h in syringes, 14 h in glass bottles, and 6 h in bags. In addition, the period of stability decreases as the temperature increases (4 h at 20 ± 5 °C). The solution is considered to be stable, subject to precipitation liable to be observed regardless of the temperature. CONCLUSION: The best stability was observed for busulfan solutions placed at 2-8 °C in syringes. This study demonstrated that precipitation, in addition to hydrolysis, has a significant influence on the busulfan content.

Early allogeneic blood stem cell transplantation after modified conditioning therapy during marrow aplasia: stable remission in high-risk acute myeloid leukemia.

Two patients with high-risk acute myeloid leukemia (AML) whose bone marrow aspirates showed more than 25% blasts between 2 and 4 weeks after the first induction chemotherapy immediately received modified conditioning therapy with intravenous busulfan at 50% of the usual dose and fludarabine, before hematologic recovery occurred. Unmanipulated G-CSF mobilized peripheral blood stem cells from an HLA-identical sibling donor were transfused and haematopoietic recovery was achieved in both recipients. Both of them are in continuing hematological remission with full donor chimerism 12 and 22 months after transplantation. Early treatment intensification with allogeneic cell therapy during marrow aplasia might cure high-risk AML patients who are unlikely to achieve remission with conventional chemotherapy protocols.

Prolonged disease-free survival after autografting for chemosensitive non-bulky low grade non-Hodgkin's Lymphoma.

The results of autografting in patients with relapsed low grade non-Hodgkin's Lymphoma (NHL) have generally been disappointing due to the failure to maintain remission and the late development of myelodysplasia. Most series have used regimens that include total body irradiation and purged stem cells. We evaluated the outcome in 32 patients with low grade NHL autografted using chemotherapy-only busulfan-based conditioning and unpurged stem cells. Seven of 10 patients with poor prognostic features at diagnosis remain alive in CR a median of 78 months (range 14-129) post-transplant. Twenty two patients with relapsed, chemosensitive, low bulk disease, most of whom did not have marrow involvement or an elevated LDH, were transplanted. Only five of the 22 have relapsed, with an 86 +/- 8% overall survival and 72 +/- 10% event free survival (EFS) after a median follow-up of 56.5 months. All but one patient has an EFS period longer, often substantially so, than their previous longest remission. No patient has developed myelodysplasia. These data suggest that in selected patients with poor prognosis or relapsed low grade NHL autografting has a favourable impact on the natural history of their disease and may result in long-term disease control.