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Technical Briefs |
1
Department of Hematology, Christian Medical College Hospital, Vellore, India
a author for correspondence: fax 91-416-232035, e-mail
bala{at}hemato.cmc.ernet.in
Busulfan is widely used as a component of myeloablative conditioning therapy for bone marrow transplantation (1)(2)(3)(4)(5). Busulfan is most often used at a fixed total dose of 16 mg/kg. Wide interindividual variation in the bioavailability of busulfan has been recognized. Pharmacokinetic analyses to achieve target plasma concentrations and dose adjustments are increasingly being used to improve the outcome of bone marrow transplantation (6)(7)(8). Studies on busulfan kinetics have demonstrated that busulfan concentrations in stored plasma samples are stable for up to 3 months at -20 °C and that blood samples for busulfan analysis should be centrifuged within 3 h of collection and plasma frozen if not analyzed immediately (9)(10)(11). The stability of busulfan in blood and plasma samples stored for longer time periods and at lower temperatures has not been reported. Because pharmacokinetic analysis of busulfan requires collection of multiple samples at frequent intervals, such data will be useful in planning transport and analysis of blood and plasma samples.
This study was undertaken to determine the stability of busulfan in whole blood samples stored up to 24 h at 4 °C and in plasma samples stored up to 2 years at -80 °C.
Busulfan analysis was performed on plasma samples by HPLC as described previously (12). Briefly, busulfan in plasma was extracted with toluene and derivatized with 1 mol/L tetrafluorothiophenol under alkaline conditions for 2 h at 70 °C. The tetrafluorothiophenol derivative of busulfan thus formed was reextracted with 1 mol/L sodium hydroxide and toluene. The toluene layer was then removed and evaporated to dryness under nitrogen gas at 60 °C. The residue was dissolved with 200 µL of 800 mL/L methanol, vortex-mixed, and filtered through 0.45 µm microspin filters (Millipore India Ltd). A 20-µL aliquot of the sample was injected into an isocratic HPLC system (Bio-Rad) by an automatic sampler. Detection was performed using an ultraviolet detector (Bio-Rad) at 275 nm. A calibration curve was created with each set of samples using known concentrations of busulfan calibrator in acetone. This method did not involve the use of an internal standard. The minimum quantifiable concentration of busulfan was 50 µg/L, with linearity up to 2000 µg/L. The detection limit of the assay, corresponding to a signal-to noise-ratio of 3, was 25 µg/L. The inter- and intraday CVs were <3% for the calibrators used for the calibration curve (12). Samples analyzed both by this HPLC method and the gas chromatography–mass spectrometry method showed good correlation (r2 = 0.95) (13). The area under the busulfan concentration–time curve (AUC) was calculated using TOPFIT by noncompartmental analysis (14).
For the blood collection and stability studies, the protocol was approved by the Institutional Review Board. Whole blood samples (8–9 mL) were collected 0, 0.5, 1, 1.5, 2, 4, and 6 h after the 13th dose of busulfan from three patient with ß-thalassemia before bone marrow transplantation. Each sample was divided into six aliquots. Plasma was separated from one aliquot by centrifugation at 1900g for 10 min at 4 °C immediately after collection and stored at -80 °C until analysis [immediate sample (IS)]. The other five aliquots were processed similarly at 2, 4, 6, 12, and 24 h. Busulfan concentrations in all plasma samples were analyzed within 48 h of sample collection.
Linear regression analysis comparing the busulfan concentrations in the
IS with those obtained by centrifuging whole blood 2, 4, and 6 h
after collection showed a variation <5%
(R2 = 0.999, 0.997, and 0.991,
respectively; Fig. 1
). However, a 7.7–9.8% decrease occurred in busulfan
concentrations (r2= 0.91 and 0.88,
respectively) in plasma samples separated after 12 and 24 h
compared with the IS.
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The AUC calculated from plasma samples centrifuged and separated within
2–6 h of collection showed good agreement with that calculated from
the IS with a maximum CV of 4.7%; samples collected after 12 and
24 h showed CVs of 5.4% and 7.2%, respectively. No data
regarding the stability of busulfan in whole blood are currently
available. Henner et al. (15) determined the stability of
busulfan in buffer (50 mmol/L phosphate–0.15 mol/L sodium chloride, pH
7.4) and plasma incubated at 0, 22, and 37 °C for periods up to
22 h. They suggested that samples in buffer were stable for >22 h
with <15% decomposition at all three temperatures, but plasma samples
were stable at 0 and 22 °C only; plasma samples incubated at
37 °C decomposed with a half-time of 
14 h. It was concluded that
busulfan is relatively stable at physiologic temperatures (half-life,
14 h), unlike other alkylating drugs such as carmustine
(half-life, 20–25 min). Our data show that busulfan is stable in whole
blood samples with a <5% reduction in concentration and AUC when
stored at 4 °C and a <10% reduction in concentration and AUC up to
24 h.
To examine the stability of busulfan in plasma at -80 °C, blood
samples were collected as described above, centrifuged immediately
after collection, and stored at -80 °C in five different aliquots.
The busulfan concentration in one aliquot was measured within 48 h
of storage (IS). The other four aliquots were analyzed after 3 months,
6 months, 1 year, and 2 years of storage at -80 °C, respectively.
The AUC was calculated as mentioned earlier. Good correlation was found
between busulfan concentrations obtained for the IS and for the other
aliquots after storage with a maximum decrease of 7.8% at 2 years
(Table 1B
). The decrease in AUC values was 4.7%, 6.9%, and 7.3% at 6,
12, and 24 months, respectively. These data suggest that plasma samples
collected for busulfan assay can be stored at -80 °C for up to 6
months with a <5% reduction in concentration and AUC and a <10%
reduction in these values for up to 2 years. Henner et al
(13) reported that plasma samples supplemented with
0.5–20 µmol/L busulfan and stored at -20 °C for 0, 16, and 57
days showed values identical to that of the plasma sample analyzed
immediately after the addition of busulfan (± 5%). Our data show that
busulfan concentrations in plasma samples stored at -80 °C are
stable for up to 2 years.
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In conclusion, we have shown that busulfan concentrations are stable in whole blood for 24 h at 4 °C and in plasma for 2 years at -80 °C. There is a <5% variation in concentration and AUC if plasma is isolated within 6 h of blood collection and analyzed within 6 months of storage. These data will be very useful for evaluation of busulfan kinetics in situations where sample analysis cannot be undertaken immediately.
Acknowledgments
This study was supported by an Indian Council of Medical Research Grant for the project "Advanced Center for Bone Marrow Transplantation for Thalassaemia in India" (56/2/93-BMS II).
References
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, after 6 h; 
, after
12 h; 
, after 24 h.