Published online Mar 12, 2016.
https://doi.org/10.12793/tcp.2016.24.1.22
Development of a validated liquid chromatography-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/χ2. 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.
Introduction
Simvastatin is widely used for the treatment of hypercholesterolemia. It acts by inhibiting 3-hydroxy-3-methylglutaryl coenzyme (HMG-CoA) reductase, the rate-limiting enzyme of the HMG-CoA reductase pathway, which is the metabolic pathway responsible for the endogenous production of cholesterol.[1, 2, 3, 4] Simvastatin is a pro-drug that undergoes rapid hydrolysis to several metabolites. The active carboxylate form of the drug is β-hydroxyacid simvastatin (simvastatin acid [SA]), which competitively inhibits HMG-CoA reductase at 436.58 g/mol and is principally biotransformed from simvastatin by cytochrome P450 3A4/5 (CYP3A4/5) (Fig. 1).[5, 6] Furthermore, manipulation of this HMG-CoA pathway is suggested to be useful in treating certain forms of cancer, as well as heart disease, increasing the interest in simvastatin.[7]
Figure 1
Chemical structure of simvastatin acid (Source: http://pubchem.ncbi.nlm.nih.gov/compound/64718#section=Top).
Less than 5% of orally administered simvastatin reaches the systemic circulation due to its extensive first-pass hepatic extraction.[8] The metabolism of simvastatin into SA is reversible, so there is also inter-conversion between simvastatin and SA.[9, 10]
Previously, we conducted clinical pharmacokinetic studies[11] of simvastatin and SA in healthy Korean volunteers and, based on the pharmacokinetic data from these studies, we performed population pharmacokinetic analysis to more precisely explore the complex pharmacokinetics of simvastatin and SA. Therefore, sensitive and validated methods for the quantification of SA in plasma were required. The use of liquid chromatography-tandem mass spectrometry (LC/MS/MS) to simultaneously quantify simvastatin and SA was previously reported, with a lower limit of quantification (LLOQ) of 0.5 ng/mL.
The objective of our present study was to develop and validate a more sensitive LC/MS/MS method to quantitate SA in human plasma, which could be applied in pharmacokinetic studies of simvastatin and SA.[12]
Methods
Chemicals and reagents
SA was purchased from Sigma-Aldrich. The acetonitrile, methanol, water, and ethyl ether used were of HPLC grade, whereas the ammonium acetate and acetic acid were of analytical grade. Deionized water was prepared using a Milli-Q Plus Ultra-Pure water system.
Preparation of calibration standards and quality control samples
Stock solutions (1 mg/mL) of SA and lovastatin acid (internal standard) were prepared by dissolving accurately weighed standard compounds in methanol. Dilution factors were calculated by weighing the amounts of standard solution and dilution solvent added. Aliquots of each stock standard solution were diluted with the respective solvent to provide working standard solutions. Calibration standards of SA (0.1 to 100 ng/mL) were freshly prepared and vortexed for approximately 30 s before processing. We also prepared validation quality control (QC) samples of SA (0.1, 0.5, 5, and 50 ng/mL) in human plasma; these QC samples were stored at -70℃.
Plasma sample extraction
Plasma samples were stored in polypropylene tubes at -70℃ until analysis. Sample pretreatment was performed in an ice bath. SA and the internal standard lovastatin acid were obtained by liquid-liquid extraction. To 500 µL of a plasma sample (or blank plasma) in a 10-mL tube, we added 10 µL of the internal standard solution in methanol (100 ng/mL). After mixing, 2 mL of ethyl ether was added, and the samples were vortexed for 2 min and centrifuged at 2,600 × g for 15 min (4℃).
The organic layer from each tube was transferred to a second tube and solvent was evaporated at 45℃ for 90 min in a Speed Vac at room temperature. The residue was dissolved in 50 µL reconstitution solvent [1 mM ammonium acetate (acetic acid [pH 4.5]):acetonitrile, 20:80, v/v] by vortex mixing for 2 min. After centrifugation for 5 min at 15,000 × rpm (4℃), each clean supernatant was transferred to a glass autosampler vial with insert and 10-µL samples were injected into the LC/MS/MS.
Flow injection analysis, a sample introduction technique in which a sample is introduced into the LC/MS/MS without first passing through a column for separation, was used to optimize the LC/MS/MS parameters and for rapid ion selection. To minimize the run time of the assay, several columns were evaluated, with CAPCELL PAK (Shiseido) found to give the best chromatography results for SA and lovastatin acid, as determined by the signal-to-noise ratio and peak width using a mobile phase consisting of 1 mM ammonium acetate (acetic acid [pH 4.5]):acetonitrile (20:80, v/v). We prepared a mobile phase volume sufficient for the anticipated number of samples to be analyzed.
LC/MS/MS conditions
The HPLC system (Symbiosis Pharma) consisted of a pump, degasser, and temperature-controlled autosampler. Isocratic chromatography was performed using a CAPCELL PAK column (3 µm, 50 mm × 2.0 mm). Samples were evaporated using a Speed Vac. The mobile phase was prepared by mixing 1 mM ammonium acetate (acetic acid, pH 4.5) and acetonitrile (20:80, v/v) and was pumped at a flow rate of 0.2 mL/min. The column oven was maintained at 30℃. The samples were placed in the autosampler at a temperature of 4℃. Using an electrospray ionization source, the LC elute was injected directly into an API 4000 triple quadrupole mass spectrometer (Applied Biosystems Sciex, Ontario, Canada), with detection by electrospray mass spectrometry in negative ion mode. The resulting multiple reaction monitoring (MRM) chromatograms were quantified using Analyst software version 1.4.2 (Applied Biosystems Sciex).
The mass spectrometric signals of all analytes were optimized by continuous infusion, using the automatic quantitative optimization function provided by the manufacturer, and further optimized by flow injection optimization, also controlled automatically by the software. Highest signal intensities were obtained with turbo-ionspray or assisted electrospray in the negative ion mode.
Method validation
The method was validated according to the guidelines on bioanalytical method validation of the US Food and Drug Administration (FDA). In terms of selectivity, accuracy, intra-batch and inter-batch precision, stability, and linearity, the quantification process of SA in human plasma was fully validated.
Each analytical run included a blank plasma sample, a zero-level standard (blank plasma plus internal standard), a set of calibration standards, and QC samples. The intra-day precision and accuracy of the method were assessed by analyzing five replicates of plasma standards at all concentrations used to construct the calibration curve. The initial inter-batch precision and accuracy were determined by analyzing five replicates of the QC samples. Calibration curves were analyzed by weighted linear regression (1/x2) of drug:internal standard peak area ratios.
For validation, we prepared calibration standards (seven standards of the analytes) in control human plasma. Linear regression of the ratio of the areas of the analyte and internal standard peaks versus the concentration was weighted by 1x2. Concentrations were back-calculated from the constructed calibration curves, with deviations from the concentrations allowed within ±20% for the LLOQ and within ±15% for other concentrations, with coefficients of variation (CVs) less than 20% and 15%, respectively. Five replicates of each human plasma sample were analyzed in three analytical runs, together with a calibration curve independently prepared from the QC samples containing 0.1, 0.5, 5, and 50 ng/mL of SA. Intra-batch precision was assessed by analyzing five samples, prepared by spiking blank samples, in the same batch, whereas inter-batch precision was assessed by analyzing samples prepared on five different days.
Accuracies were determined as the percentage difference in the measured concentration from the nominal concentration, and CVs were used to report the precision. Intra- and inter-assay accuracies within ±20% at the LLOQ and within ±15% at the other concentrations were allowed.
To investigate whether endogenous matrix constituents interfered with the assay, six individual batches of control, drug-free, human plasma were processed and analyzed according to the described procedures. The responses of the three compounds at the LLOQ concentration were compared with the response of the blank samples. All validation experiments were performed with the blank matrix from different individuals, instead of using a blank matrix pool.
All stability testing in plasma was performed in triplicate at the LLOQ and the HOQ (highest of quantification) concentrations; the results were compared with those of freshly prepared samples. The stability of the SA stock solutions after incubation at room temperature for 6 h was determined in triplicates. The effects of three freeze/thaw cycles on SA concentrations in plasma were evaluated by assaying samples after they had been frozen (-70℃ for 24 h) and thawed (room temperature for 3 h) on three separate days. The stability of SA in plasma during sample preparation was evaluated by assaying samples before and after 24-h storage at room temperature. Autosampler storage stability was determined by storing the reconstituted QC samples at two concentrations for 24 h at 4℃ before analysis.
Results and Discussion
Method development
We developed an assay for the quantification of SA in human plasma using LC/MS/MS. The procedure consisted of a simple liquid-liquid extraction with ethyl ether followed by LC/MS/MS. We also reduced the sample preparation time by conducting liquid-liquid extraction in a single step, versus the two steps used in a previous study.[11] MS detection was adopted in this analysis because it has high sensitivity and selectivity, which is particularly important in the analysis of structurally analogous compounds. SA and the internal standard lovastatin acid responded best to negative ionization and, under electrospray ionization conditions, protonated molecular ions ([M-H]-) were present as major peaks. The protonated analytes generated in negative ion mode were monitored by MRM.
Mass transitions of mass to charge ratios (m/z) 435.12 > 115.0 and 421.22 > 101.2 were optimized for SA and lovastatin acid, respectively, which are different from the m/z 437 > 303 for SA and m/z 423 > 285 for lovastatin acid described in a previous study.[11] CAD and curtain gas were operated at 8 and 20 L/min. Finally, the ion spray voltage was kept at -4,500 V, with a source temperature of 600℃. A complete overview of the MS/MS transitions, declustering potential, entrance potential, collision energy, and collision cell exit potential has been compiled.
The full-scan mass spectrum of each compound was acquired in negative ion mode via spectra infusion of 10 µL/min of each compound. Full-scan product ion spectra and fragmentation pathways of SA and lovastatin acid are shown in Figure 2. The [M-H]- ions of SA and lovastatin acid analyzed in unit resolution were observed at m/z 435.12 and 421.22, respectively. After fragmentation in the collision cell, the [M-H]- ions of SA and lovastatin acid yielded a product ion at m/z 115.0 and 101.2, respectively. The MRM transitions chosen for the quantitative experiments are summarized in Table 1.
Figure 2
Full-scan product ion spectra of [M-H]+ for simvastatin acid and the lovastatin acid internal standard.
Table 1
Tandem mass spectrometry parameters
Assay validation
The assay was found to be linear within the concentration ranges using linear regression with 1/x2 weighting (Fig. 3). The peak area ratios, relative to the internal standard, were determined in the range of 0.1-100 ng/mL for SA in plasma, and the linearity of the method was confirmed, as can be seen in the correlation coefficient (r2) of 0.9986-0.9997 for the standard calibration curve. The accuracy of the standard curve was within 94.9-103.1%, the precision was within 0.9-3.5% CV, and the LLOQ was 0.1 ng/mL (Table 2).
Figure 3
A typical calibration curve for simvastatin acid concentrations in human plasma.
Table 2
Inter-batch precision and accuracy for calibration of standard data
The accuracies and precision of the assays were determined by calculating the intra- and inter-batch variations of five replicates each of the LLOQ and low, mid, and high concentrations of SA (0.1, 0.5, 5, and 50 ng/mL). The mean assay accuracies were between 93.5% and 107.8%, and the intra- and inter-batch precisions were ≤ 5.7% CV (Table 3). The specificity of the method was assessed by analyzing both human blank plasma and plasma containing SA and internal standard from six individual subjects. No interference peaks were observed at the retention times of either analytes or the internal standard (Fig. 4).
Figure 4
Representative chromatograms of simvastatin acid in human plasma.
Table 3
Intra- and inter-batch precision and accuracy of quality control samples in human plasma
Analyte stability was determined under various processes and storage conditions. The stabilities of the SA stock solutions at room temperature for 6 h were 16.6%, 5.3%, 3.0%, and 2.8%, respectively, whereas their stabilities in plasma following freeze-thaw cycling (-70℃ for 24 h, followed by room temperature for 3 h) were 3.3%, 2.3%, -1.1%, and -0.9%, respectively (Table 4). The stabilities of SA QC samples at room temperature for 24 h were 3.0%, 1.3%, 1.9%, and -0.8% (Table 4). The stabilities of plasma extracts of SA at their QC concentrations, when reconstituted and kept in autosamplers for 24 h, were -3.9%, -3.7%, -1.8%, and -1.7% (Table 4). All stability data were within a 20% deviation range at the LLOQ and within a 15% deviation range after freeze/thaw cycling, incubation in plasma at room temperature for 24 h, stock solution at room temperature for 6 h, and storage in an autosampler in plasma for 24 h, all of which showed stability based on the predefined criteria.
Table 4
Stability of simvastatin acid in human plasma samples
CONCLUSION
We have developed and validated a sensitive LC/MS/MS method for the measurement of SA in plasma. All of the stability data were within ±20% at the LLOQ and within ±15% at the other concentrations. The LLOQ for SA was 0.1 ng/mL, which is lower than the 0.5 ng/mL obtained using the previously reported method.[11] The findings indicate that all frozen samples should be assayed immediately after thawing and not refrozen. The linearity, precision, and accuracy of this method were validated in the studied concentration ranges for SA. This method has been successfully applied to clinical pharmacokinetic studies.[12]
Conflict of interest:There is no conflict of interests.
Acknowledgements
None.
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