The following formulations of CLDs were used: Atorvastatin tablets 5 mg (Sawai, Osaka, Japan), Pravastatin sodium tablets 5 mg (Sawai, Osaka, Japan), Rosuvastatin tablets 2.5 mg (DSEP, Tokyo, Japan), Simvastatin tablets 5 mg (Teva Takeda Yakuhin, Aichi, Japan), Ezetimibe tablets 10 mg (DSEP, Tokyo, Japan). Magmitt tablets 330 mg (Nihon Shinyaku, Kyoto, Japan) were used as an MgO containing formulation. The tablets of atorvastatin, pravastatin, rosuvastatin, simvastatin, ezetimibe, and MgO are hereafter referred to as AS, PS, RS, SS, EZ, and MG, respectively. Atorvastatin calcium hydrate (99.8%), simvastatin (100%), and ezetimibe (96.3%) were purchased from Tokyo Chemical Industry (Tokyo, Japan). Pravastatin sodium (99.9%), rosuvastatin calcium (99.7%), distilled water, acetonitrile, methanol, trifluoroacetic acid, and sodium dihydrogen phosphate dihydrate were purchased from Fujifilm Wako Chemicals (Osaka, Japan). Distilled water, acetonitrile, and methanol were of high-performance liquid chromatography (HPLC) grade. An aqueous solution of phosphoric acid (0.5 M) and chloroform were purchased from Kanto Chemical (Tokyo, Japan). Weakly acidic cation exchange resin AMBERLITE® (IRC76) was purchased from ORGANO (Tokyo, Japan). A membrane filter DISMIC®—03 JP (0.50 μm) was purchased from Toyo Roshi Kaisha (Tokyo, Japan). Deuterated chloroform (CDCl3) containing 0.05% tetramethylsilane was purchased from Eurisotop (Massachusetts, USA). Deuterated dimethyl sulfoxide (DMSO-d6) containing 0.03% tetramethylsilane was purchased from Acros Organics (Massachusetts, USA).

One CLD tablet was placed with or without MG (one tablet) in a 50-mL plastic centrifuge tube containing 20 mL of warm distilled water (55°C). The centrifuge tube was allowed to stand at room temperature (ca. 25°C) for 10 min or 5 h. The mixture was then vortexed for 1 min to prepare a suspension. The subsequent treatments were immediately performed according to method I or II (see below). In separate experiments, the simple suspension prepared after 10 min soakage was centrifuged and the aqueous supernatant was directly subjected to HPLC analyses and pH measurements using InLab® Routine Pro (Mettler Toledo, Ohio, USA). The suspension preparation was performed three times for each CLD tablet in any of the experiments.

The simple suspensions were treated as follows, and the resulting solutions were analyzed by HPLC.

Method I: The simple suspension was completely transferred into a 100-mL volumetric flask, where the tube was washed by methanol (for AS, PS, RS, and EZ) or acetonitrile (for SS) and the washing solvent was put into the same flask. The mixture in the flask totaled 100 mL with the respective organic solvents, followed by sonication for 1 min unless otherwise noted.

Method II: For SS and EZ, the simple suspension was centrifuged, and the aqueous supernatant (18 mL) was neutralized by passing it through a column of the cation exchange resin. Eighteen milliliters of acetonitrile for SS or methanol for EZ were added to the residue of centrifugation, and the mixture was vortexed for 1 min and centrifuged again. The supernatant was also passed through the same cation exchange column. The same treatment was conducted again for the residue of the centrifugation. The effluents from the column were collected and totaled 250 mL by the respective organic solvents.

HPLC analyses were performed using a Shimadzu HPLC system (Kyoto, Japan) as described in our previous paper [29]. The reversed-phase HPLC analyses were performed modifying the conditions in the literature [30]. An Atlantis T3 column (4.6 × 250 mm, 5 μm; Waters, Milford, USA) fitted with a guard column (4.6 × 20 mm, 5 μm) was used. The mobile phases and detection wavelengths used are summarized in Table 1. The HPLC system was operated isocratically at a flow rate of 1.0 mL/min at 25°C. The samples were appropriately diluted and injected after being filtered using the membrane filter. The injection volume of the sample was 10 μL. Chromatograms were obtained within 30 min after the suspension preparation.

Standard stock solutions of atorvastatin calcium hydrate, pravastatin sodium, rosuvastatin calcium, simvastatin, and ezetimibe were prepared in methanol (ca. 100 μg/mL). The solutions for the validation were prepared from stock solutions. The calibration curves were constructed by triplicate analysis of the five concentrations of the samples, whose ranges were one fourth or one eighth to twice the expected drug concentrations in the diluted samples for the HPLC analyses. The expected drug concentrations for atorvastatin, pravastatin, rosuvastatin, simvastatin, and ezetimibe were 2.0, 2.0, 1.0, 2.0, and 4.0 μg/mL, respectively. Quality control samples were prepared at three concentrations (middle, high, low) within the ranges of the calibration curves. The three concentrations were half of the expected drug concentrations, the three-fold concentrations, and 40 to 70% less concentrations. The precision and repeatability of the HPLC methods were determined by intra- and inter-day variations through repeated analyses (N = 5) of quality control samples on three different days. Various diluted samples were analyzed five times, and the limit of detection (LOD) and limit of quantitation (LOQ) were determined based on the concentrations with signal to noise ratios of three and 10, respectively.

The degradation product from the SS was extracted with chloroform from the aqueous supernatants of the co-suspensions of SS and MG for structural analysis using nuclear magnetic resonance (NMR) spectroscopy. 1H and 1H-decoupled 13C NMR spectra and two-dimensional NMR spectra were measured with an AVANCE 500 NMR spectrometer (11.75 T, Bruker Japan, Kanagawa, Japan) using 5-mm sample tubes at 300 K. The aqueous supernatants of the co-suspensions of SS and MG were also injected into the HPLC apparatus in order to collect the fractions containing the degradation product. The HPLC conditions were the same as described above, except the mobile phase was a mixture of 0.1% trifluoroacetic acid and acetonitrile (15/85). The fractions were directly analyzed with a micro TOF-Q spectrometer (Bruker Daltonics, Kanagawa, Japan) in order to obtain the high-resolution electrospray ionization mass spectrum (ESI–MS).

The aqueous methanolic solution, which was obtained from the co-suspensions of EZ and MG, was injected into the HPLC apparatus to isolate the degradation product from EZ. The HPLC conditions were the same as described above, except the mobile phase was a mixture of 0.1% trifluoroacetic acid and acetonitrile (40/60). The NMR spectra and ESI–MS of the isolated degradation product were measured using the above spectrometers.

All data are expressed as the mean ± standard deviation of at least three experiments. All statistical analyses were performed using one-way ANOVA followed by Tukey’s test to determine significance. Statistical significance was set at p < 0.05.