Ann Lab Med.  2014 Jan;34(1):7-14. 10.3343/alm.2014.34.1.7.

Quantification of Human Plasma-Busulfan Concentration by Liquid Chromatography-Tandem Mass Spectrometry

Affiliations
  • 1Department of Laboratory Medicine, Seoul National University Hospital, Seoul, Korea. cloak21@snu.ac.kr
  • 2Department of Pharmacology and Clinical Pharmacology, Seoul National University Hospital, Seoul, Korea.
  • 3Department of Pediatrics, Seoul National University Hospital, Seoul, Korea.
  • 4Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seongnam, Korea.

Abstract

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 -20degrees C and -80degrees C, but decreased by 25% when the plasma was stored for 24 hr at room temperature, and by <5% in 24 hr at 4degrees C. 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.

Keyword

Busulfan; Drug monitoring; Tandem mass spectrometry; Liquid chromatography; Hematopoietic stem cell transplantation

MeSH Terms

Busulfan/*blood/standards
Child
Child, Preschool
*Chromatography, High Pressure Liquid/standards
Hematopoietic Stem Cell Transplantation
Humans
Infant
Quality Control
Reference Standards
*Tandem Mass Spectrometry/standards
Busulfan

Figure

  • Fig. 1 Chemical structure of busulfan (butane-1,4-diyl dimethanesulfonate).

  • Fig. 2 Mass spectra and chromatograms of busulfan and internal standard obtained from the analysis of the lowest calibration solution (25 ng/mL). The MRM chromatograms (left panels) show well-separated busulfan and glipizide as indicated by different retention times. The right panels show the MRM transitions of busulfan and glipizide.Abbreviations: IS, internal standard; MRM, multiple reaction monitoring; ESI, electrospray ionization.

  • Fig. 3 Linearity of plasma busulfan analysis. At each concentration, 4 repeated test results are plotted and the mean values for each concentration are used for drawing a trend line. The R2 value of the trend line is presented.

  • Fig. 4 Chromatogram at the limit of detection, 1.56 ng/mL. The signal to noise ratio (SNR) at the concentration is calculated as 4.0.

  • Fig. 5 Stability of plasma busulfan on storing at room temperature, 4℃, -20℃, and -80℃.Abbreviation: RT, room temperature.

  • Fig. 6 Distribution and change of plasma busulfan concentrations in clinical samples. The central box represents the values from the lower to upper quartile (25-75 percentiles). The middle line represents the median value. A line extends from the minimum to the maximum value, excluding "outside" and "far out" values displayed as separate points (marked as ⋄).


Cited by  1 articles

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Kyunghoon Lee, Soo Young Moon, Serim Kim, Hyun-Jung Choi, Sang-Guk Lee, Hyung-Doo Park, Soo-Youn Lee, Sang Hoon Song,
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