Quantification of imatinib in human serum: validation of a high-performance liquid chromatography-mass spectrometry method for therapeutic drug monitoring and pharmacokinetic assays

Figures & data

Figure 1 Chemical structures of imatinib (A) and tamsulosin (B) used as internal standard.

Table 1 Chromatographic linear gradient scheme. Both methanol and water contain 10 mM of ammonium acetate and 0.1% of formic acid

Time (minutes)	Flow rate (mL per minute)	Methanol %	Water %
Initial condition	0.4	40	60
1.60	0.4	100	0
1.80	0.8	100	0
2.50	0.8	100	0
3.00	0.8	40	60
3.45	0.8	40	60
3.60	0.4	40	60
4.00 (stop)	0.4	40	60

Figure 2 Chromatograms of (A) sample at lower limit of quantification level plus internal standard prepared in solvent. Extracted blank serum samples, (B) normal, (C) hemolyzed, and (D) hyperlipemic. Responses (vertical axes) are normalized to highest peak.

Figure 3 Chromatograms of sample at lower limit of quantification level plus internal standard extracted as per the protocol.

Table 2 Recovery and matrix effect results for analyte and internal standard

	Analyte			Internal standard
	Level
	QCL	QCM	QCH	5 μg/mL
Absolute recovery
Normal serum	108.8	81.5	107.3	76.0
Hemolyzed serum	84.6	92.4	86.8	92.4
Hyperlipemic serum	104.8	87.1	105.7	80.1
Mean	99.4	87.0	99.9	82.8
CV	11.59			10.32
Mean recovery	95.4			82.8
Relative recovery
Normal serum	102.2	97.2	102.4	84.6
Hemolyzed serum	98.0	103.8	101.5	95.4
Hyperlipemic serum	98.8	89.8	93.3	92.7
Mean	99.7	96.9	99.1	90.9
CV	4.69			6.18
Mean recovery	98.5			90.9
Matrix effect
Normal serum	6.4	−16.2	4.8	−10.3
Hemolyzed serum	−13.7	−11.0	−14.5	−3.2
Hyperlipemic serum	6.1	−3.0	13.3	−13.6
Mean	−0.4	−10.1	1.2	−9.0
Mean recovery	−3.1			−9.0

Note: All values are expressed as a percentage (%).

Abbreviations: CV, precision; QCL, low level quality control; QCM, medium level quality control; QCH, high level quality control.

Figure 4 Calibration curves of first, second, and third batches of linearity test from top to bottom, with respective equations and coefficients of correlation (R^2).

Table 3 Results of precision and accuracy experiments, intrabatch and interbatch

Control name	LLOQ			QCL			QCM			QCH
Nominal concentration (μg/mL)	0.500			1.50			4.50			8.10
Intrabatch
Batch	1st	2nd	3rd	1st	2nd	3rd	1st	2nd	3rd	1st	2nd	3rd
Replicates (n)	6	6	6	6	6	6	6	6	6	6	6	6
Mean (ng/mL)	0.445	0.442	0.491	1.502	1.602	1.603	4.254	4.265	4.645	7.631	7.129	7.511
Precision (CV%)	4.3	3.5	1.5	2.8	2.1	3.4	3.5	2.5	3.0	3.3	1.7	1.0
Accuracy (%)	89.0	88.5	98.2	100.2	106.8	106.9	94.5	94.8	103.2	94.2	88.0	92.7
Interbatch
Replicates (n)	18			18			18			18
Mean (ng/mL)	0.459			1.569			4.388			7.424
Precision (CV%)	5.8			4.1			5.1			3.7
Accuracy (%)	91.9			104.6			97.5			91.7

Abbreviations: CV, precision; QCL, low level quality control; QCM, medium level quality control; QCH, high level quality control; LLOQ, lower limit of quantification.

Table 4 Limits of stability approved for imatinib

Test	Time	Conditions*
Short-term	18 hours	Bench top, laboratory light, ambient temperature
Freeze and thaw cycles	3 cycles	Frozen at −20°C, thawed at ambient temperature
Post processing	88 hours	Inside autosampler, 8°C
Long-term	92 days	Frozen at −20°C

Note:

* Ambient temperature, 22°C ± 3°C.

Table 5 Stability test results for imatinib

Test type	Range	Lower QC, 1.5 μg/mL		Higher QC, 8.1 μg/mL
		Fresh	Test	Fresh	Test
Short-term	18 hours
	Average (μg/mL)	1.585	1.589	6.947	6.832
	CV (%)	0.9	3.8	0.8	4.6
	Variation (%)	0.2		−1.7
Freeze and thaw	3 cycles
	Average (μg/mL)	1.712	1.698	8.789	8.182
	CV (%)	3.1	2.4	2.8	1.1
	Variation (%)	−0.8		−6.9
Post processing	88 hours
	Average (μg/mL)	1.489	1.436	7.735	6.874
	CV (%)	2.5	2.4	4.2	2.5
	Variation (%)	−3.6		−11.1
Long-term	92 days
	Average (μg/mL)	1.447	1.640	7.828	8.263
	CV (%)	5.1	3.4	3.4	6.2
	Variation (%)	13.3		5.6

Abbreviations: CV, precision; QC, quality control.

Table 6 Method enhancements and performance compared with a previous validated method

Parameter	Previous method	Current method	Enhancement/performance
Analytical run time	13 minutes	4 minutes	3.2 times less
Solvent spent per sample (approximately)	5.4 mL	1.6 mL	3.4 times less
Costs of internal standard (approximately)	US$12,000.00*	US$2,080.00**	5.8 times less
Mean intra-assay method accuracy	102.2%	96.4%	Equivalent
Mean intra-assay method precision	2.0%	2.7%	Equivalent
Mean inter-assay method accuracy	101.1%	96.4%	Equivalent
Mean inter-assay method precision	5.5%	4.6%	Equivalent

Notes:

* CDN isotopes, Canada supply, deuterium-labeled imatinib, 200 mg, May 2013

** United States Pharmacopeia, USA supply, tamsulosin, 200 mg, May 2013.