Hyun-Jung Park; Ae-Kyung Hwang; A-Reum Kim; Soo-Hyeon Kim; Eun-Hwa Kim; Sang-Heon Cho; Jong-Lyul Ghim; Sangmin Choe; Jin-Ah Jung; Seok-Joon Jin; Kyun-Seop Bae; Hyeong-Seok Lim

doi:10.12793/tcp.2016.24.1.22

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Park, Hwang, Kim, Kim, Kim, Cho, Ghim, Choe, Jung, Jin, Bae, and Lim: Development of a validated liquid chromato-graphy–tandem mass spectrometry assay for the quantification of simvastatin acid, the active metabolite of simvastatin, in human plasma

Original Article

Translational and Clinical Pharmacology 2016;24(1):22-29.

Published online: 15 March 2016

DOI: https://doi.org/10.12793/tcp.2016.24.1.22

Development of a validated liquid chromato-graphy–tandem mass spectrometry assay for the quantification of simvastatin acid, the active metabolite of simvastatin, in human plasma

Hyun-Jung Park^1,, Ae-Kyung Hwang^1,, A-Reum Kim¹, Soo-Hyeon Kim¹, Eun-Hwa Kim¹, Sang-Heon Cho², Jong-Lyul Ghim³, Sangmin Choe⁴, Jin-Ah Jung³, Seok-Joon Jin⁵, Kyun-Seop Bae⁶, Hyeong-Seok Lim^6,^∗

¹Pharmacokinetic and Pharmacogenetic Laboratory, Clinical Research Center, Asan Medical Center, Pungnap-2-dong, Seoul, Republic of Korea

²Department of Clinical Pharmacology, Inha University Hospital, Inha University School of Medicine, Incheon, Republic of Korea

³Department of Pharmacology, Inje University College of Medicine, Busan, Republic of Korea

⁴Division of Clinical Pharmacology, Clinical Trials Center, Pusan National University Hospital, Busan, Republic of Korea

⁵Department of Anesthesiology, University of Ulsan College of Medicine, Asan Medical Center, Pungnap-2-dong, Seoul, Republic of Korea

⁶Department of Clinical Pharmacology and Therapeutics, Asan Medical Center, University of Ulsan College of Medicine, Republic of Korea, Seoul, Republic of Korea

^∗Correspondence: H. S. Lim; Tel: +82-2-3010-4613, Fax: +82-2-3010-4622, E-mail: mdhslim@gmail.com

H.-J.Park and A.-K.Hwang contributed equally to this study.

Received 7 September 20153 November 2015 Accepted 13 November 2015

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

Simvastatin is a lipid-lowering drug that is metabolized to its active metabolite simvastatin acid (SA). We developed and validated a sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method to quantitate SA in human plasma using a liquid–liquid extraction method with methanol. The protonated analytes generated in negative ion mode were monitored by multiple reaction monitoring. Using 500-mL plasma aliquots, SA was quantified in the range of 0.1–100 ng/mL. Calibration was performed by internal standardization with lovastatin acid, and regression curves were generated using a weighting factor of 1/x². The linearity, precision, and accuracy of this assay for each compound were validated using quality control samples consisting of mixtures of SA (0.1, 0.5, 5, and 50 ng/mL) and plasma. The intra-batch accuracy was 95.3–107.8%, precision was –2.2% to –3.7%, and linearity (r²) was over 0.998 in the standard calibration range. The chromatographic running time was 3.0 min. This method sensitively and reliably measured SA concentrations in human plasma and was successfully used in clinical pharmacokinetic studies of simvastatin in healthy Korean adult male volunteers.

Keywords: simvastatin acid, plasma, LC/MS/MS

References

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Figure 1.

Chemical structure of simvastatin acid (Source: http://pub-chem.ncbi.nlm.nih.gov/compound/64718#section=Top).

Figure 2.

Full-scan product ion spectra of [M−H]+ for simvastatin acid and the lovastatin acid internal standard.

Figure 3.

A typical calibration curve for simvastatin acid concentrations in human plasma.

Figure 4.

Representative chromatograms of simvastatin acid in human plasma.

Table 1.

Tandem mass spectrometry parameters

Analyte	MRM transition (m/z)	Time (msec)	DP∗ (volts)	EP∗ (volts)	CE∗ (volts)	CXP∗ (volts)
Simvastatin acid	435.12 > 115.0	150	–90	–8	–36	–7
Lovastatin acid	435.12 > 115.0	150	–70	–8	–36	–5

^∗ DP, declustering potential; EP, entrance potential; CE, collision energy; CXP, collision cell exit potential.

Table 2.

Inter-batch precision and accuracy for calibration of standard data

Analyte	Nominal concentration (ng/mL)	Mean calculated concentration^‡ (ng/mL)	Accuracy∗ (%)	Precision^† (%)
Simvastatin	0.10	0.10	100.8	0.9
acid	0.50	0.47	94.9	3.5
	2.00	2.06	103.1	2.2
	10.00	10.27	102.7	2.4
	50.00	49.56	99.1	2.2
	100.00	99.32	99.3	3.3

^∗ [(mean calculated concentration)/(nominal concentration)] × 100.

^† Relative standard deviation.

^‡ Mean concentrations calculated from weighted (1/x²) linear regression curves using all five replicates at each concentration.

Table 3.

Intra- and inter-batch precision and accuracy of quality control samples in human plasma

	Intra-day (n=5)				Inter-day (n=5)
Analyte	Nominal Conc. (ng/mL)	Mean calculated Conc.^‡ (ng/mL)	Accuracy∗ (%)	Precision^† (%)	Mean calculated Conc.^‡ (ng/mL)	Accuracy∗ (%)	Precision^† (%)
Simvastatin	0.10	0.10	104.6	3.7	0.10	103.2	5.7
acid	0.50	0.48	95.3	2.9	0.47	93.5	3.5
	5.00	5.38	107.6	2.2	4.95	99.1	2.23
	50.00	53.86	107.8	3.7	50.22	100.4	4.7

^∗ [(mean calculated concentration)/(nominal concentration)] × 100.

^† Relative standard deviation.

Table 4.

Stability of simvastatin acid in human plasma samples

Additional freeze/thaw cycles	QC 0.1 ng/mL		QC 0.5 ng/mL		QC 5 ng/mL		QC 50 ng/mL
Additional freeze/thaw cycles	Observed Conc. (ng/mL)	CV (%)	Observed Conc. (ng/mL)	CV (%)	Observed Conc. (ng/mL)	CV (%)	Observed Conc. (ng/mL)	CV (%)
0	0.11	3.7	0.46	2.9	5.31	2.12	52.0	3.7
	0.10		0.48		5.30		52.5
	0.10		0.48		5.30		56.0
	0.10		0.50		5.56		56.0
	0.11		0.48		5.44		52.8
3	0.11	5.6	0.48	2.4	5.24	2.0	51.5	3.9
	0.11		0.50		5.29		53.1
	0.10		0.48		5.44		55.6
Percent difference		3.3		2.3		–1.1		–0.9

(a) Freeze/thaw stability of simvastatin acid in human plasma

Storage period (h)	QC 0.1 ng/mL		QC 0.5 ng/mL		QC 5 ng/mL		QC 50 ng/mL
Storage period (h)	Observed Conc. (ng/mL)	CV (%)	Observed Conc. (ng/mL)	CV (%)	Observed Conc. (ng/mL)	CV (%)	Observed Conc. (ng/mL)	CV (%)
0	0.08	4.34	0.44	3.1	4.99	3.7	51.4	4.96
	0.09		0.46		5.36		55.4
	0.08		0.47		5.45		55.1
	0.08		0.47		5.09		56.8
	0.08		0.45		5.15		50.7
24	0.08	4.5	0.44	2.2	4.93	3.3	50.9	3.63
	0.08		0.45		5.15		53.3
	0.08
Percent difference		–3.9		–3.7		–1.8		–1.7

(b) Autosampler stability of simvastatin acid in extracted sample

Storage period (h)	QC 0.1 ng/mL		QC 0.5 ng/mL		QC 5 ng/mL		QC 50 ng/mL
Storage period (h)	Observed Conc. (ng/mL)	CV (%)	Observed Conc. (ng/mL)	CV (%)	Observed Conc. (ng/mL)	CV (%)	Observed Conc. (ng/mL)	CV (%)
0	0.09	5.7	0.49	3.5	4.94	2.3	47.5	4.7
	0.11		0.47		5.08		48.1
	0.10		0.46		5.01		51.8
	0.11		0.45		4.95		53
	0.10		0.46		4.78		50.7
24	0.11	6.4	0.45	6.5	4.91	2.5	49.2	6.2
	0.11		0.46		5.16		47.1
	0.099		0.51		5.07		53.2
Percent difference		3.0		1.3		1.9		–0.8

Storage period (h)	QC 0.1 ng/mL		QC 0.5 ng/mL		QC 5 ng/mL		QC 50 ng/mL
Storage period (h)	Observed Conc. (ng/mL)	CV (%)	Observed Conc (ng/mL)	c. CV (%)	Observed Conc. (ng/mL)	. CV (%)	Observed Conc. (ng/mL)	CV (%)
0	0.08	4.4	0.44	3.1	4.99	3.7	51.4	5.0
	0.09		0.46		5.36		55.4
	0.08		0.47		5.45		55.1
	0.08		0.47		5.09		56.8
	0.08		0.45		5.15		50.7
6	0.10	3.7	0.46	3.0	5.14	3.7	52.8	5.0
	0.10		0.49		5.52		57.0
	0.10		0.50		5.62		56.6
	0.09		0.50		5.25		58.4
	0.10		0.48		5.30		52.1
Percent difference		16.6		5.3		3.0		2.8

(d) Stability of simvastatin acid extracts after storage for 6 h at room temperature

^∗ [(mean calculated concentration of stability QC samples–mean calculated concentration of QC samples) × 100]/mean calculated concentration of QC samples.

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