Tedizolid phosphate standard samples were purchased from MedChemExpress (Monmouth Junction, NJ, USA), and TZD was purchased from Nacalai Tesque Corporation (Kyoto, Japan). Sivextro (tedizolid phosphate) and sodium acetate were purchased from MSD Corporation (Tokyo, Japan) and Nacalai Tesque Corporation, respectively. LZD, VCM, acetonitrile, and methanol were purchased from the FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Bovine serum albumin (BSA) was added to saline as pseudo-blood and was purchased from Iwai Chemical Corporation. A syringe filter (13 mm cellulose acetate 0.2 μm (AGC Techno Glass Company Limited, Tokyo, Japan) was used for sample solution pretreatment. A saline solution (Otsuka saline solution) was purchased from Otsuka Pharmaceutical Corporation (Tokyo, Japan).

The adsorption rates of tedizolid phosphate, TZD, LZD, and VCM onto two types of dialysis membranes were investigated (Table 1). The experiments were performed using two types of dialysis membranes: polysulfone (PS) (SNV-1.3, Toray Medical, Tokyo, Japan) and polymethylmethacrylate (PMMA) (CH-1.3, Toray Medical, Tokyo, Japan). First, tedizolid phosphate, TZD, LZD, and VCM were dissolved in 50 ml of saline containing 4% BSA. The drug concentrations used in the experiments were 3.5 μg/ml, 3.5 μg/ml, 20 μg/ml, and 50 μg/ml, referring to the maximum blood concentration (Cmax) of each drug at clinical administration.

The hollow fibers (1 cm wide, 0.1 g) of each dialysis membrane were soaked in solutions of tedizolid phosphate, TZD, LZD, and VCM, stirred for 1 h at room temperature, and collected. To 0.2 ml of this sample, an equal volume of methanol was added (0.2 ml), and the proteins were removed by centrifugation (10,000 g, 25 °C, 30 min). Protein-free samples were assayed by high- performance liquid chromatography (HPLC).

CHDF was performed using an in vitro model with a blood purification system (KM-8700EX, SANYO Electric Corporation, Japan) (Fig. 1). Saline solution containing 4% BSA was placed in a 1 L beaker and used as pseudo blood. Two types of dialysis membranes with the same composition (PS and PMMA) as those used for the affinity dialysis experiments were used. Drug concentrations were measured using HPLC. The drug concentration used in the experiment was the Cmax of each drug since the experiment will be comparing adsorption rates onto the dialysis membrane. The drug-containing pseudo-blood was circulated through the two types of dialysis membranes (PS and PMMA) and a hemoperfusion blood circuit (Kawasumi Chemical Industries, Tokyo, Japan) at a flow rate (Qb) of 100 ml/min. The flow rate of the dialysate (Qd) was set to 450 ml/hr, and the flow rates of the substitution solution (Qs) and ultrafiltrate (Qf) were set to 200 ml/hr. These flow rates were set based on the actual clinical flow rates. The pseudo-blood containing the drug solution was circulated in the circuit to equalize the concentration of the drug solution, and the experiment was then started. Samples were collected from the inlet (Cin), outlet (Cout), and filtrate (Cf) of the dialyzer at 0, 0.5, 1, and 2 h.

In vitro CHDF model and sampling points. Arrows indicate the flow of solutions, and dotted arrows indicate the sampling points

The elimination rates of tedizolid phosphate, TZD, LZD, and VCM were calculated as follows:

$$\text\ (\mathrm) = (\mathrm_0-\mathrm)\,/\mathrm_0 \times 100$$

C0: Initial drug concentration

Cn: Drug concentration at the time of sampling

The clearance of each drug in the in vitro CHDF (CLCHDF) was calculated using the following equation:

$$\mathrm_} =\mathrm_}\times (\mathrm_}-\mathrm_})/\mathrm_}$$

Analyses of tedizolid phosphate, TZD, LZD, and VCM were performed by HPLC using a Hibar Lichrosorb® RP-18 column (ODS, 5 μm, 4.0 × 120 mm, Kanto Chemical Corporation, Tokyo, Japan). The analyses of tedizolid phosphate and TZD were performed in accordance with previous reports [7,8,9]. The mobile phase for tedizolid phosphate was a mixture of 19.2 mM sodium acetate buffer (pH 7.4) and 15% acetonitrile. The mobile phase for TZD was a mixture of 19.2 mM sodium acetate buffer (pH 7.4) and 50% MeOH. The flow rate was 1.0 ml/min, and TZD was measured by UV absorbance at 251 nm. The LZD analysis was performed as previously described [10, 11]. The mobile phase was a mixture of 19.2 mM sodium acetate buffer (pH 7.4) and 30% acetonitrile at a flow rate of 1.0 ml/min. The LZD analysis was performed by measuring the UV absorbance at 253 nm. The VCM analysis was performed as previously described [12,13,14]. The mobile phase was a mixture of 50 mM sodium dihydrogen phosphate buffer (pH 2.5) and 10% acetonitrile at a flow rate of 1.0 ml/min.

Data are expressed as the mean ± SE. One-way analysis of variance (ANOVA) was applied to nonreplicated measurements. Repeated-measures ANOVA followed by the Tukey–Kramer test was applied for repeated or serial determinations. Statistical significance was set at P < 0.05.