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A Novel Simultaneous Determination of Sarpogrelate and its Active Metabolite (M-1) in Human Plasma, Using Liquid Chromatography-Tandem Mass Spectrometry: Clinical Application.

Yang JS, Kim JR, Cho E, Huh W, Ko JW, Lee SY - Ann Lab Med (2015)

Bottom Line: The calibration curves of sarpogrelate and M-1 were linear over the concentration ranges of 10-2,000 and 2-400 ng/mL, respectively (R(2)>0.99).The carry-over effect, precision, accuracy, and stability of the method met the criteria for acceptance.A simple, fast, robust, and reliable analytical method was successfully developed and applied to the high-throughput determination of sarpogrelate and its metabolite in real plasma samples in a pharmacokinetic study of healthy subjects.

View Article: PubMed Central - PubMed

Affiliation: Clinical Trial Center, Clinical Research Institute, Samsung Medical Center, Korea.

ABSTRACT

Background: This study describes a novel analytical method for simultaneously determining sarpogrelate and its metabolite (M-1) in human plasma, using liquid chromatography coupled with tandem mass spectrometry, with electrospray ionization in the positive ion mode.

Methods: Sarpogrelate, M-1, and labeled internal standard (d3-sarpogrelate) were extracted from 50 µL of human plasma by simple protein precipitation. Chromatographic separation was performed by using a linear gradient elution of a mobile phase involving water-formic acid (99.9:0.1, v/v) and acetonitrile-formic acid (99.9:0.1, v/v) over 4 min of run time on a column, with a core-shell-type stationary phase (Kinetex C18, 50 mm×2.1 mm i.d., 2.6-µm particle size, Phenomenex, USA). Detection of the column effluent was performed by using a triple-quadruple mass spectrometer in the multiple-reaction monitoring mode.

Results: The developed method was validated in human plasma, with lower limits of quantification of 10 ng/mL for sarpogrelate and 2 ng/mL for M-1. The calibration curves of sarpogrelate and M-1 were linear over the concentration ranges of 10-2,000 and 2-400 ng/mL, respectively (R(2)>0.99). The carry-over effect, precision, accuracy, and stability of the method met the criteria for acceptance.

Conclusions: A simple, fast, robust, and reliable analytical method was successfully developed and applied to the high-throughput determination of sarpogrelate and its metabolite in real plasma samples in a pharmacokinetic study of healthy subjects.

No MeSH data available.


Related in: MedlinePlus

Representative multiple reaction monitoring chromatograms of sarpogrelate (left panel), its metabolite (M-1; middle panel), and internal standard (IS; right panel) in (A) double-blank plasma, (B) blank plasma spiked with IS, (C) blank plasma spiked with sarpogrelate and M-1 at lower limit of quantification (LLOQ; 10.0 and 2.0 ng/mL, respectively) and IS, and (D) real plasma sample spiked with IS.
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Figure 1: Representative multiple reaction monitoring chromatograms of sarpogrelate (left panel), its metabolite (M-1; middle panel), and internal standard (IS; right panel) in (A) double-blank plasma, (B) blank plasma spiked with IS, (C) blank plasma spiked with sarpogrelate and M-1 at lower limit of quantification (LLOQ; 10.0 and 2.0 ng/mL, respectively) and IS, and (D) real plasma sample spiked with IS.

Mentions: Various mobile phase modifiers, such as formic acid and ammonium buffers, under gradient elution of acetonitrile in water were investigated, for optimization of mobile phase conditions. Addition of 0.1% formic acid, as an acidic modifier, to the mobile phase, rather than ammonium formate buffer, yielded an excellent response in positive ion electro-spray. A higher percentage (1.0%) of formic acid in the diluting solution, added to the extracted sample before LC-MS/MS analysis, improved separation of the analytes from unknown interfering substances, and minimized peak tailing (Fig. 1).


A Novel Simultaneous Determination of Sarpogrelate and its Active Metabolite (M-1) in Human Plasma, Using Liquid Chromatography-Tandem Mass Spectrometry: Clinical Application.

Yang JS, Kim JR, Cho E, Huh W, Ko JW, Lee SY - Ann Lab Med (2015)

Representative multiple reaction monitoring chromatograms of sarpogrelate (left panel), its metabolite (M-1; middle panel), and internal standard (IS; right panel) in (A) double-blank plasma, (B) blank plasma spiked with IS, (C) blank plasma spiked with sarpogrelate and M-1 at lower limit of quantification (LLOQ; 10.0 and 2.0 ng/mL, respectively) and IS, and (D) real plasma sample spiked with IS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446576&req=5

Figure 1: Representative multiple reaction monitoring chromatograms of sarpogrelate (left panel), its metabolite (M-1; middle panel), and internal standard (IS; right panel) in (A) double-blank plasma, (B) blank plasma spiked with IS, (C) blank plasma spiked with sarpogrelate and M-1 at lower limit of quantification (LLOQ; 10.0 and 2.0 ng/mL, respectively) and IS, and (D) real plasma sample spiked with IS.
Mentions: Various mobile phase modifiers, such as formic acid and ammonium buffers, under gradient elution of acetonitrile in water were investigated, for optimization of mobile phase conditions. Addition of 0.1% formic acid, as an acidic modifier, to the mobile phase, rather than ammonium formate buffer, yielded an excellent response in positive ion electro-spray. A higher percentage (1.0%) of formic acid in the diluting solution, added to the extracted sample before LC-MS/MS analysis, improved separation of the analytes from unknown interfering substances, and minimized peak tailing (Fig. 1).

Bottom Line: The calibration curves of sarpogrelate and M-1 were linear over the concentration ranges of 10-2,000 and 2-400 ng/mL, respectively (R(2)>0.99).The carry-over effect, precision, accuracy, and stability of the method met the criteria for acceptance.A simple, fast, robust, and reliable analytical method was successfully developed and applied to the high-throughput determination of sarpogrelate and its metabolite in real plasma samples in a pharmacokinetic study of healthy subjects.

View Article: PubMed Central - PubMed

Affiliation: Clinical Trial Center, Clinical Research Institute, Samsung Medical Center, Korea.

ABSTRACT

Background: This study describes a novel analytical method for simultaneously determining sarpogrelate and its metabolite (M-1) in human plasma, using liquid chromatography coupled with tandem mass spectrometry, with electrospray ionization in the positive ion mode.

Methods: Sarpogrelate, M-1, and labeled internal standard (d3-sarpogrelate) were extracted from 50 µL of human plasma by simple protein precipitation. Chromatographic separation was performed by using a linear gradient elution of a mobile phase involving water-formic acid (99.9:0.1, v/v) and acetonitrile-formic acid (99.9:0.1, v/v) over 4 min of run time on a column, with a core-shell-type stationary phase (Kinetex C18, 50 mm×2.1 mm i.d., 2.6-µm particle size, Phenomenex, USA). Detection of the column effluent was performed by using a triple-quadruple mass spectrometer in the multiple-reaction monitoring mode.

Results: The developed method was validated in human plasma, with lower limits of quantification of 10 ng/mL for sarpogrelate and 2 ng/mL for M-1. The calibration curves of sarpogrelate and M-1 were linear over the concentration ranges of 10-2,000 and 2-400 ng/mL, respectively (R(2)>0.99). The carry-over effect, precision, accuracy, and stability of the method met the criteria for acceptance.

Conclusions: A simple, fast, robust, and reliable analytical method was successfully developed and applied to the high-throughput determination of sarpogrelate and its metabolite in real plasma samples in a pharmacokinetic study of healthy subjects.

No MeSH data available.


Related in: MedlinePlus