Validation of LC-MS/MS Method for Determination of Bivalirudin in Human Plasma: Application to a Pharmacokinetic Study

Yo Han Kim; Hyun Jeong Park; Hee Youn Choi; Hyeong-Seok Lim; Kyun-Seop Bae

doi:10.12793/jkscpt.2013.21.2.166

Journal List > J Korean Soc Clin Pharmacol Ther > v.21(2) > 1055126

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Kim, Park, Choi, Lim, and Bae: Validation of LC-MS/MS Method for Determination of Bivalirudin in Human Plasma: Application to a Pharmacokinetic Study

Original Article

J Korean Soc Clin Pharmacol Ther 2013;21(2):166-173.

Published online: 11 January 2013

DOI: https://doi.org/10.12793/jkscpt.2013.21.2.166

Validation of LC-MS/MS Method for Determination of Bivalirudin in Human Plasma: Application to a Pharmacokinetic Study

Yo Han Kim, MD^1,², Hyun Jeong Park³, Hee Youn Choi, MD^1,², Hyeong-Seok Lim, MD, PhD^1,², Kyun-Seop Bae, MD, PhD^1,²

¹Department of Clinical Pharmacology and Therapeutics, Asan Medical Center, Seoul, Korea

²University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea

³Clinical Trial Center, Asan Medical Center, Seoul, Korea

E-mail: ksbae@amc.seoul.kr

Received 14 November 201317 December 2013 Accepted 20 December 2013

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background:

Bivalirudin is a direct thrombin inhibitor for patients with unstable angina undergoing percutaneous coronary intervention.

Methods:

A sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of bivalirudin, in human plasma using nafarelin as internal standard (IS). Chromatographic separation was performed using a Shiseido MG3 mm column (2.0 × 50 mm) with a gradient mobile phase consisting of water and acetonitrile containing 0.1 % formic acid at a flow rate of 0.4 mL/min, and total run time was within 5 min. Detection and quantification was performed by the mass spectrometer using a multiple reaction-monitoring mode at m/z 1091.0 → 650.3 for bivalirudin, and m/z 662.1 → 249.3 for IS.

Results:

The assay was linear over a concentration range of 10 - 10000 ng/mL with a lower limit of quantification of 10 ng/mL in human plasma.

Conclusion:

This method was successfully applied for pharmacokinetics study after intravenous administration of bivalirudin to healthy Korean male volunteers.

Keywords: Bivalirudin, Liquid chromatography, Pharmacokinetics

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Figure 1.

Chromatograms of (A) a human blank plasma sample; (B) a human plasma sample spiked with 10 ng/mL of bivalirudin and IS (nafarelin); (C) a subject’s plasma sample at 5 min after intravenous bolus administration of bivalirudin 0.375 mg/kg.

Figure 2.

Mean plasma concentration time curves of bivalirudin after single intravenous bolus injection. Data are expressed as mean ± SD (each group, n=6).

Table 1.

Intra- and inter-day precision and accuracy data for this assay of bivalirudin in human plasma (n=5)

	QC 10 ng/mL	QC 20 ng/mL	QC 5000 ng/mL	QC 8000 ng/mL
Intra-day
Mean (ng/mL)	11.00	19.90	5201.08	8182.33
Precision (%)	7.03	10.16	5.03	4.76
Accuracy (RE*, %)	10.02	-0.48	4.02	2.28
Inter-day
Mean (ng/mL)	10.95	21.35	5295.03	8375.37
Precision (%)	8.02	5.78	5.13	6.34
Accuracy (RE*, %)	9.46	6.73	5.90	4.69

RE = 100% × (spiked concentration - mean calculated concentration) / spiked concentration.

Table 2.

Stability of bivalirudin in human plasma

Storage conditions	QC 20 ng/mL	QC 8000 ng/mL
Short-term (room temperature for 24 h)	-12.37%	-13.01%
Long-term (-70 °C for 40 days)	-0.89%	-7.68%
Three Freeze/thaw cycles	3.66%	-9.07%
After extraction (stored in HPLC autosampler at 4 °C for 24 h)	-7.66%	0.37%

Data are presented as percent difference from baseline (%).

Table 3.

Suitability of bivalirudin in human plasma

Analyte concentration (ng/mL)	Calculated concentration (ng/mL)	Accuracy (%)
20	18.82	94.08
	18.39	91.96
5000	4983.39	99.67
	4873.87	97.48
50000(Dilution 1/10)*	51346.64	102.69
	53721.03	107.44
8000	8205.98	102.57
	8663.37	108.29

QC samples of 50000 ng/mL were diluted 1:10 to prepare QC sample of 5000 ng/mL. Those diluted QC samples were used to analyze diluted samples of bivalirudin in case of the unexpected high concentration over 10000 ng/mL.

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