Quantification of Busulfan in Plasma using Luxon Ion Source

Authors: Serge Auger, Pierre Picard and Jean Lacoursière
Themes: High-Throughput, Busulfan, LDTD-MS/MS
From: Phytronix Technologies, Québec, Canada
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Introduction

Busulfan (Bu) is an alkylating agent commonly used in patients being prepared for hematopoietic stem cell transplantation (HSCT) for various types of hematologic malignancies such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) in addition to non-malignancies (thalassemia)1,2. In the clinical doses, Bu is considered as a potent cytotoxic drug which causes severe and prolonged myelosuppression.

Our goal for this application note is to develop a diagnostic tool to rapidly go through a quantification of Busulfan in plasmas, which will allow a rapid diagnosis.

LDTD®-MS/MS offers specificity combined with an ultra-fast analysis for an unrivaled quantification method. To develop this application, we focused on performing an efficient sample preparation, which will increase the speed of the analysis process and confirm diagnoses more quickly.

Luxon Ionization Source

The Luxon Ion Source® (Figure 1) is the second-generation sample introduction and ionization source based on the LDTD® technology for mass spectrometry. Luxon Ion Source® uses Fiber-Coupled Laser Diode (Figure 2) to obtain unmatchable thermal uniformity providing more precision, accuracy, and speed. The process begins with dry samples which are rapidly evaporated using indirect heat. The thermally desorbed neutral molecules are carried into a corona discharge region. High efficiency protonation and strong resistance to ionic saturation characterize this type of ionization and is the result of the absence of solvent and mobile phase.

Figure 1 – Luxon Ion Source®
Figure 2 – Schematic of the Luxon Ionization Source

Sample Preparation Method

Stock solutions of Busulfan was prepared in acetonitrile at 1 mg/mL. Then, negative EDTA-K2 plasmas were spiked to generate a calibration curve and QCs.

Automated Sample Extraction

An automated system (Figure 3) is used to extract the samples using the following conditions:

Figure 3 – Automated extraction system – Azeo Liquid Handler

 

LDTD®-MS/MS Parameters

LDTD

Model: Luxon S-960, Phytronix

Carrier gas: 9.0 L/min (air)

Laser pattern: 3-second ramp to 45% power

MS/MS

MS model: QTrap® System 5500, Sciex

IonSpray Voltage: 5500

Temperature: GS1 and GS2 set to zero.

CUR: 30

Scan Time: 20 msec

Analysis Method: Positive MRM mode

 

Transition CE (V)
Busulfan-Quan 247.0 → 55.0 22
Busulfan-d8-Quan 255.0 → 62.0 22
Busulfan-Qual 247.0 → 151.0 11
Busulfan-d8-Qual 273.0 → 147.0 11

 

Results and Discussion

Validation Test

Calibration curves ranging from 25 to 2000 ng/mL were prepared in negative EDTA-K2 plasmas. A set of QCs were prepared in the same matrix. Replicate extractions were deposited onto a LazWell™ plate and dried before analysis. The peak area against the internal standard (IS) ratio was used to normalize the signal.

Linearity

The calibration curves were plotted using the peak area ratio and the nominal concentration of standards. For the linearity test, the following acceptance criteria was used:

Table 2 shows the inter-day correlation coefficients for Busulfan (Quan and Qual transition). Values greater than 0.995 are obtained. Figure 4 shows a typical calibration curve result for Busulfan-Quan.

 

Figure 4 – Busulfan-Quan calibration curve

 

Table 2 – Inter-day calibration curve correlation coefficients
Busulfan-Quan Busulfan-Qual
Curve 1 0.99909 0.99936
Curve 2 0.99980 0.99979
Curve 3 0.99957 0.99982
Curve 4 0.99928 0.99979
Curve 5 0.99985 0.99982
Curve 6 0.99968 0.99976

Precision and Accuracy

For the accuracy and precision evaluation, the following acceptance criteria were used:

For the intra and inter-run precision and accuracy experiment, each QC was analyzed in sextuplicate, on six different runs. Table 3 and 4 show the intra and inter-run precision and accuracy results for Busulfan-Quan. The obtained %CV was below 15% and the accuracy was within 15% of the nominal value. Similar results were obtained with the Busulfan-Qual transition.

Table 3 – Intra-Run Precision and Accuracy of Busulfan-Quan
Busulfan-Quan LLOQ QC-L QC-M QC-H ULOQ
Conc (ng/mL) 25 75 1000 1500 2000
N 6 6 6 6 6
Mean (ng/mL) 28.1 76.2 987.4 1513.2 2060.8
%CV 4.7 1.4 2.4 0.8 1.9
%Nom 112.3 101.7 98.7 100.9 103.0

 

Table 4 – Inter-Run Precision and Accuracy of Busulfan-Quan
Busulfan-Quan QC-L QC-M QC-H
Conc (ng/mL) 75 1000 1500
N 36 36 36
Mean (ng/mL) 76.8 980.6 1505.1
%CV 4.4 2.3 2.4
%Nom 102.4 98.1 100.3

Wet Stability of Sample Extracts

Following the extraction, sample extracts are kept at 4°C in closed containers. After 1 day, sample extracts are spotted on a LazWell™ plate, dried and analyzed. The precision and accuracy results of QCs are reported in Table 5. All the results are within the acceptable criteria range for 1 day at 4°C.

Dry Stability of Samples Spotted on LazWell™

Extracted samples are spotted onto a LazWell™ plate, dried and kept at room temperature for 1 hour before analysis. The precision and accuracy results of QCs are reported in Table 5. All the results are within the acceptable criteria range for 1 hour at room temperature.

Table 5 – Wet and Dry Stability of Busulfan-Quan
Parameters Dry Stability
(1 hour / RT)
Wet Stability
(1 day / 4°C)
QC QC-L QC-M QC-H QC-L QC-M QC-H
Conc (ng/mL) 75 1000 1500 75 1000 1500
N 4 4 4 4 4 4
Mean (ng/mL) 77.0 1003.9 1510.7 77.7 985.1 1483.2
%CV 2.4 1.2 1.9 1.4 1.6 2.3
%Nom 102.6 100.4 100.7 103.6 98.5 98.9

Matrix Effect Study

Eight different plasmas were spiked at QC-M level, extracted, and analysed. The precision and accuracy results of the different matrix spiked are reported in Table 6. All the results are within the acceptable criteria range.

Table 6 – Matrix effect results
M1 M2 M3 M4 M5 M6 M7 M8
Conc (ng/mL) 1000 1000 1000 1000 1000 1000 1000 1000
N 3 3 3 3 3 3 3 3
Mean (ng/mL) 978.2 1007.9 1020.0 986.5 988.8 1044.0 1036.0 1031.0
%CV 0.9 1.7 1.4 0.9 0.8 1.0 1.8 3.4
%Nom 97.8 100.8 102.0 98.7 98.9 104.4 103.6 103.1

Conclusion

The Luxon Ion Source® combined with the Sciex QTrap® 5500 mass spectrometer system enables the rapid quantification of Busulfan in plasmas. This method of analysis can thus be applied at the clinical level in order to evaluate the concentration of Busulfan for a quick dosage adjustment.