Journal List > Transl Clin Pharmacol > v.24(1) > 1082637

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

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.

References

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Figure 1.
Chemical structure of simvastatin acid (Source: http://pub-chem.ncbi.nlm.nih.gov/compound/64718#section=Top).
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Figure 2.
Full-scan product ion spectra of [M−H]+ for simvastatin acid and the lovastatin acid internal standard.
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Figure 3.
A typical calibration curve for simvastatin acid concentrations in human plasma.
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Figure 4.
Representative chromatograms of simvastatin acid in human plasma.
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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
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
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
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

(c) Stability of simvastatin acid extracts after storage for 24 h at room temperature

Storage period (h) QC 0.1 ng/mL QC 0.5 ng/mL QC 5 ng/mL QC 50 ng/mL
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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