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Clinical Chemistry 44: 2506-2510, 1998;
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(Clinical Chemistry. 1998;44:2506-2510.)
© 1998 American Association for Clinical Chemistry, Inc.

Drug Monitoring and Toxicology

Routine analysis of plasma busulfan by gas chromatography–mass fragmentography

Wai-Kai Lai1, Chi-Pui Pang1,a, Lap-Kay Law1, Raymond Wong2, Chi-Kong Li2 and Patrick Man-Pan Yuen2

Departments of
1 Chemical Pathology and
2 Paediatrics, the Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong.
a Address correspondence to this author at: Department of Opthalmology and Visual Sciences, the Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong. Fax: 852-26482943; e-mail cppang{at}cuhk.edu.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Busulfan (BU) is a widely used alkylating agent for antineoplastic therapy and marrow ablation in preparation for bone marrow transplantation (BMT). High-dose BU often leads to successful preparation and low relapse but is associated with veno-occlusive disease of liver. We established a protocol to determine postdosage plasma BU concentrations by gas chromatography–mass fragmentography in an attempt to relate clinical outcome to plasma BU concentrations. We used nonisotopic pusulfan as the internal standard. After extraction into ethyl acetate, BU and pusulfan were iodinated into 1,4-diiodobutane and 1,5-diiodopentane, respectively. Gas chromatography–mass spectrometry (GC–MS) analysis was carried out on an Hewlett–Packard (HP) 5890II gas chromatograph with a 30-m 100% methyl silicon narrow bore, fused-silica capillary column interfaced with an HP 5970A mass spectrometer. Helium was the carrier gas. The sample molecules were identified by total ion monitoring and quantified by selective ion monitoring of m/z 183 and 197. The calibration curve was linear to 4 mg/L. The limit of quantification was 0.04 mg/L, and the analytical recovery was ~97%. The within-day and between-day imprecision (CV) was <6% and 9%, respectively. In a preliminary study of 12 children, the BU areas under the BU-time curve were 616–949 µmol · min/L after the first dose and 793-1143 µmol · min/L after the fifth dose. We conclude that the GC–MS procedure is suitable for routine analysis of plasma BU.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Busulfan (BU;1 1,4-butanediol dimethanesulfonate; C6H14O6S2) is a widely used alkylating agent for antineoplastic therapy and marrow ablation in preparation for bone marrow transplantation (BMT) of patients with hematological malignancies, non-Hodgkin lymphomas, immune deficiencies, and thalassemias (1)(2)(3)(4). High-dose BU, usually used in combination with other cytotoxic agents such as cyclophosphamide or with total body irradiation, often leads to successful preparation and low relapse for both children and adults. However, high-dose BU is also associated with serious organ toxicity such as central nervous system toxicity or veno-occlusive disease (VOD) of the liver (5). VOD is one of the major complications of high-dose BU and occurs in almost 20% of patients after BMT; up to 50% of such cases are fatal (6)(7).

BU is available only in oral form, and the plasma concentration is affected by absorption rate. It is also extensively metabolized. There is strong interindividual variation in drug disposition and the metabolic clearance rate, which is higher in younger children than in older children (8)(9). In a fixed milligram per kilogram of body weight dosage, children have substantially lower plasma BU concentrations than adults (10).

Pharmacokinetic adjustments in dose on the basis of plasma determinations reduce toxicity in some patients, especially the occurrence of VOD, (11). Plasma BU has been shown to be an important determinant of graft rejection and regimen-related toxicity (12). Area-under-the-curve (AUC) measurements of BU kinetics are necessary for adjustment of individual dosing, especially for children undergoing BMT (8)(13). Results of a study on BU-AUC determination in 66 BMT patients suggested an association between BU exposure and occurrence of VOD (14). BU concentrations are highly variable in children. Recently, however, one report found no association between BU concentration and posttransplant mortality or graft reject in 64 children with homozygous ß-thalassemia receiving BU and high-dose cyclophosphamide along with bone marrow from HLA-identical sibling donors (15). Whether BU pharmacokinetics are predictive in those patients receiving bone marrow from HLA-partially matched, -related, or -unrelated donors remains to be investigated.

At the Prince of Wales Hospital in Hong Kong, >100 BMTs have been performed during the last 4 years; approximately one-half have received BU. There had been occurrences of severe or fatal VOD, apparently associated with high BU. Therefore, establishing plasma BU concentrations in BMT is necessary. Existing methods of plasma BU determination invariably involve chromatography. HPLC is not sufficiently specific or sensitive, mainly because of high imprecision (16)(17)(18). A recently published HPLC methodology provided good limits of quantification and good imprecision for high, but not for low, concetrations of BU (19). Gas chromatography (GC) with electron capture detection provides good sensitivity but lacks specificity (20)(21)(22). GC–mass fragmentography offers excellent specificity and reliability (23)(24). To preserve the specificity and sensitivity of a GC–mass spectrometry (GC-MS) technique while operating at a lower cost, we optimized a GC–MS procedure suitable for routine analysis of BU in a clinical laboratory.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
materials
BU was from Sigma Chemical Co. The pusulfan (PU; 1,5-pentanediol dimethanesulfonate) was kindly provided by R. Younker (Fred Hutchinson Cancer Research Center, Seattle, WA). The helium gas (99.9995% purity) was supplied by Hong Kong Oxygen & Acetylene Co. Stock solutions of 200 mg/L BU and PU were prepared in ethyl acetate and stored in the dark under refrigeration.

study subjects
We obtained venous blood samples (1 mL) after the first and fifth doses (24 h later) from 12 thalassemia patients, ages 6 and 16 years, undergoing BMT and receiving 1 mg/kg BU orally every 6 h for 16 doses as a standard practice for conditioning. Verbal consent was obtained from the patients and their parents. The study was approved by the Ethics Committee of the Chinese University of Hong Kong.

sample treatment
A previously described procedure (25) was modified. To 0.25 mL of plasma in a 10-mL tube, 40 µL of internal standard (5.5 mg/L PU in ethyl acetate) and 5 mL of ethyl acetate were added. After the mixture was vortex-mixed vigorously for 15 min, the organic phase was collected by centrifugation and reacted with 0.5 mL of 4 mol/L sodium iodide in a shaker at 200 rpm for 1 h at 60 °C. After iodination, the extract was washed with water to remove excess sodium iodide and evaporated to dryness under a stream of nitrogen. The residue was reconstituted in 60 µL of ethyl acetate, and 1 µL was injected into the GC–MS system.

gc–ms analysis
A Hewlett–Packard (HP) 5890 II gas chromatograph equipped with an HP 7636A autosampler was interfaced with an HP 5970A mass spectrometer. Samples were injected onto an HP-1 100% methyl silicon capillary fused-silica column (30 m x 0.2 mm i.d.; 0.33 µm film thickness). Helium was the carrier gas, at a column pressure of 160 kPa and a column flow rate of 1.13 mL/min. The injector temperature was 250 °C, and the split ratio of inlet to column gas flow was 15:1. The iodinated alkane residues were separated by a 1-ramp GC temperature program: initial temperature at 80 °C, increased at 25 °C/min to 200 °C, with a solvent delay of 1 min. The GC analysis was completed in 6 min. MS was carried out in electron ionization mode, and both total and selected ions were monitored.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
gc–ms
The iodine derivatives of BU and PU (1,4-diiodobutane and 1,5-diiodopentane, respectively) were different by one methylene group but were well separated by the GC method (Fig. 1 ). They were identified by total ion monitoring and quantified by selected ions, 1,4-diiodobutane (from BU) at m/z 183 and 1,5-diiodopentane (from PU) at m/z 197 (Fig. 2 ).



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  		Figure 1. GC–MS analysis of BU and PU by total ion monitoring of their iodinated alkane derivatives, 1,4-diiodobutane (from BU) and 1,5-diiodopentane (from PU).

Presented as an overlay of a blank plasma and a plasma sample of a child given BU. Retention times were in minutes. The amount of PU as internal standard was 0.22 µg and that of BU in the plasma sample was 0.4 µg.



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  		Figure 2. The mass spectra and major fragment ions of 1,4-diiodobutane (A) and 1,5-diiodopentane (B).

analytical performance
The mean regression equation for 17 calibration curves was: y = 1.5386 (±0.1273)x - 0.0068 (±0.0587); r = 0.9979 (±0.00275). The calibration curves were obtained by plotting the peak intensity ion ratio m/z 183/197 of 1,4-diiodobutane and 1,5-diiodopentane against the BU calibrator at seven concentrations (0.04, 0.2, 0.4, 1.0, 2.0, 3.0, and 4.0 mg/L). The r values for these 17 calibration curves ranged from 0.9897 to 1.0000. By least-squares linear regression analysis, a typical linearity equation from 0.04 to 4 mg/L was: y = 1.0727x + 0.0976 (r = 0.9987), where y is the target BU concentration (mg/L), and x is the measured BU concentration (mg/L).

The imprecision (CV) of the instrument (GC–MS) was estimated by analysis of two calibration samples, at BU concentrations of 0.4 and 4 mg/L, 14 times within 1 day. The corresponding CVs were 5.4% and 4.6%.

Two quality-control BU samples (BU concentrations, 0.2 and 2 mg/L) were used for both imprecision of the entire procedure, including sample preparation and GC–MS analysis, and accuracy studies. The calculated value for each sample was read off a calibration curve obtained on the same day. The imprecision of the whole procedure was as follows: within-day CV, 5.9% and 5.6% (n = 8); and between-day CV, 7.1% and 8.4% (n = 11), for the BU calibrator at 0.2 and 2 mg/L, respectively. Accuracy was reported as percentage of bias [(measured concentration - true concentration)/true concentration] (26). The bias was less than ±5% with no significant differences between the intra- and interday accuracy at both concentrations (P >0.1, Student t-test). The recoveries of the BU calibrator at concentrations of 0.4, 2.0, and 4.0 mg/L, as determined over 7 consecutive days, were 98.8% ± 7.4%, 95.5% ± 7.9%, and 95.6% ± 6.4%, respectively.

The lowest limit of detection, defined as the minimum concentration of BU corresponding to twice the signal-to-noise ratio, was 0.002 mg/L BU in plasma. The lowest limit of quantification (LLQ) was defined as the lowest measured concentration of BU with a CV <15% after repeated analysis (26)(27). The LLQ of this method was 0.04 mg/L, with a bias of +1.91% (n = 17).

The iodine derivatives of the BU calibrator added to plasma at three concentrations (0.04, 0.4, and 2.0 mg/L) were left in the automated sample processor at room temperature for GC–MS determination after 5 days. The measured concentrations varied slightly from the baseline (time zero), with average differences of -2.6%, +4.2%, and -5.2%, respectively.

In our preliminary study of plasma BU in 12 children who suffered from transfusion-dependent thalassemia and who received BU as part of the preparative regimen for hematopoietic stem cell transplantation, we determined the BU AUC after the first and fifth doses 24 h later. BU was administered every 6 h after the first dose, and blood samples were taken every 30 min for BU analysis to obtain AUC values. The BU AUC ranged from 616 to 949 (µmol · min/L) after the first dose and from 793 to 1143 µmol · min/L after the fifth dose. Seven children developed severe VOD, and their BU AUC values were all >750 and 900 µmol · min/L after the first and fifth doses, respectively.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have established a robust and reliable method of determination of plasma BU by GC–MS, which is regarded as the definitive methodology for BU analysis in clinical laboratories (24). We succeeded in using a small plasma volume of 0.25 mL; therefore, ~0.5 mL of whole blood was sufficient for each analysis. This is important for the pharmacological investigation of BU for dosage adjustment by serial blood collection from young children undergoing BMT.

Although PU resembles BU in chemical structure, the retention time of 1,5-diiodopentane is distinct from that of 1,4-diiodobutane. This GC–MS method had advantages over other methods in that there were no interfering peaks in plasma. Furthermore, the peaks of the selected ions were sharp, with good baseline resolution and minimum tailing.

The calibration curve was highly linear, with r >0.99, which was comparable to that of the method described by Chen et al. (21). At <6%, the instrument CV was less than the CV for the GC–MS method developed by Vassal et al. (24). The analytical precision and accuracy were within allowable limits.

The limit of detection and LLQ of this method were determined by adjustment of the solvent volume for extraction, the sample injection volume, and the dwell time on the mass spectrometer. The LLQ was in the microgram per liter range and was thus applicable to the measurement of BU concentrations in human plasma. If the peak ion ratio m/z 183/197 was either too high or too low, the plasma volume could be adjusted correspondingly to fit within the calibration curve. If there is a constraint on plasma volume, such as from young children, we can prepare a more concentrated extract by reconstitution in a smaller volume of ethyl acetate before injection into the GC–MS system.

We found the iodine derivatives, 1,4-diiodobutane and 1,5-diiodopentane, chemically stable in room temperature for at least 5 days. When we used PU as an internal standard, good reproducibility, sensitivity, accuracy, and specificity were obtained. No expensive isotopic reagents are required. The iodinated alkane derivatives are stable, and the GC–MS procedure is automated. Moreover, each GC–MS analysis required <10 min. The rapid turnaround time in our protocol allowed the analysis of up to 40 plasma samples in 24 h, with ~3 h of actual manual labor, including reporting. Our new GC–MS method is sufficiently rapid for same-day analysis and reporting of plasma BU results before the second dose after 6 h. This method is applicable to the investigation of plasma BU concentration-time profiles for clinical purposes. We have carried out AUC analyses of 12 Chinese children undergoing BMT who developed transfusion-dependent thalassemia. Our preliminary results indicated a possible association between BU toxicity with high BU AUC values in the fifth dose. We are in the process of determining BU AUC values in all our patients undergoing BMT and receiving BU to determine the need to adjust the BU dose and to assess the clinical value of BU monitoring by AUC measurement in predicting severe treatment-related complications.


   Footnotes
 
1 Nonstandard abbreviations: BU, busulfan; BMT, bone marrow transplantation; VOD, veno-occlusive disease; AUC, area under the curve; GC, gas chromatography; MS, mass spectrometry; PU, pusulfan; HP, Hewlett–Packard; and LLQ, lowest limit of quantification.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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