Etimicin sulfate (90.7% purity), acting as the standard of etimicin, was purchased from National Institutes for Food and Drug Control (Beijing, China). The standard for carbamazepine-D₁₀ (99% purity), acting as the internal standard (IS), was provided by Shanghai Zzbio (Shanghai, China).

HPLC-grade methanol and acetonitrile were obtained from Honeywell Burdick & Jackson (Morris Plains, NJ, USA). HPLC-grade acetic acid was purchased from Shanghai Aladdin Biochemical Technology (Shanghai, China). AR-grade ammonia solution and sodium hydroxide were sourced from Xilong Scientific (Shantou, China). Water was purified by the Milli-Q™ system from Millipore (Bedford, MA, USA). Blank human serum and blank human urine were collected from Chinese healthy volunteers (HVs) in Peking Union Medical College Hospital (Beijing, China) after written informed consent had been provided.

LC-MS/MS was done on an Acquity ultra-high-performance liquid chromatography system (Waters, Milford, MA, USA) coupled with a QTRAP 5500 mass spectrometer (Sciex, Toronto, ON, Canada) equipped with an electrospray ionization source operating in positive mode. Data were acquired and processed by Sciex Analyst™ 1.7.1.

An XTerra MS C18 column (2.1 × 150 mm, 3.5 μm; Waters) was used with water–ammonia solution–acetic acid (96:3.6:0.2, v/v/v) as mobile phase A and methanol as mobile phase B. Optimal peak shapes and retentions of analytes were achieved by isocratic elution (50% mobile phase B) at a flow rate of 0.35 ml/min with a column temperature of 40°C. The injection volume of each sample was 10 μL and the autosampler temperature was set to 10°C. The analytical time for each sample was 5 min.

MS parameters were optimized: gas 1, 55 psi; gas 2, 55 psi; curtain gas, nitrogen, 35 psi; collision gas, medium; temperature, 550°C. Compound-specific MS parameters for etimicin were optimized: declustering potential (DP), 180 V; entrance potential (EP), 8 V; collision energy (CE), 36 V; collision cell exit potential (CXP), 14 V. Compound-specific MS parameters for the IS were: DP, 100 V; EP, 8 V; CE, 29 V; CXP, 14 V. The following parent → product ion transitions were used for multiple-reaction monitoring: etimicin, m/z 478.280 → 191.110; IS, m/z 247.100 → 204.197; both had a dwell time of 150 ms.

Two sets of etimicin stock solution, both prepared with methanol–water (50/50, v/v) at 1.00 mg/ml, were used for the preparation of calibration standards and QC samples, respectively. A stock solution of the IS (1.00 mg/ml) was also prepared with methanol–water (50/50, v/v). These stock solutions were stored at −80°C.

For the preparation of calibration standards and QC samples, two series of working solutions of etimicin of suitable concentrations were prepared with methanol–water (50/50, v/v) by diluting the two sets of etimicin stock solution. Calibration standards in serum were prepared at 50.0, 100, 500, 1,000, 2500, 5000, 10,000, and 20,000 ng/ml by spiking the stock solution or working solutions of calibration standards in blank human serum. Similarly, calibration standards in urine were prepared by blank human urine at 50.0, 100, 250, 500, 1,000, 2500, 5000, and 10,000 ng/ml. Meanwhile, the lower limit of quantitation (LLOQ), low quality control (LQC), medium quality control (MQC), and high quality control (HQC) samples in human serum and human urine were prepared with stock solutions or working solutions of QC samples at 50.0, 150, 1,500, 15,000 ng/ml and 50.0, 150, 750, 7,500 ng/ml, respectively. Working solutions of the IS were prepared with methanol–water (50/50, v/v) at 40.0 ng/ml and 100 ng/ml for the pretreatment of urine samples and serum samples, respectively. The calibration standards and QC samples stated above were stored at −80°C, as were the two IS working solutions for urine (uISWS) preparation and serum (sISWS) preparation.

Serum samples were prepared by protein precipitation. A serum sample (50 μL) was first mixed with sISWS (50 μL), and mixed with methanol (400 μL) subsequently to precipitate the proteins in the matrix. After vortex-mixing for 1 min, the mixture was centrifuged (17,000 × g, 10 min, room temperature). Then, the supernatant (200 μL) was mixed fully with mobile phase A (200 μL). This solution was diluted five-fold by mobile phase A–mobile phase B (50:50, v/v) before LC-MS/MS.

A urine sample (25 μL) was first mixed with uISWS (25 μL) and then with sodium hydroxide (50 μL) subsequently. After vortex-mixing, 900 μL of mobile phase A–mobile phase B (50:50, v/v) was added to the mixture, followed by vortex-mixing for 1 min. Then, the mixtures were centrifuged (17,000 × g, 10 min, room temperature). The supernatant was used for LC-MS/MS.

We developed two LC-MS/MS bioanalytical methods for the determination of etimicin in human samples of serum and urine. Following the guidelines for the validation of bioanalytical methods set by China National Medical Products Administration (Chinese Pharmacopoeia Commission, 2020), US Food and Drug Administration (US Food and Drug Administration, 2018), and European Medicines Agency (European Medicines Agency, 2011), these two methods were validated comprehensively including accuracy, precision, linearity, selectivity, matrix effect, recovery, dilution integrity, stability, and carry-over. For each analytical batch of serum samples or urine samples, dual eight-point calibration curves, ranging from 50.0 ng/ml to 20,000 ng/ml for serum samples and 50.0 ng/ml to 10,000 ng/ml for urine samples, were analyzed to quantify the analyte.

A phase I clinical study of etimicin was conducted in Peking Union Medical College Hospital. The study protocol was approved (EC# I-22PJ127) by the ethics committee of Peking Union Medical College Hospital and conducted in compliance with the Declaration of Helsinki 1964 and its later amendments. The study cohort was Chinese HVs, aiming to investigate the comprehensive pharmacokinetics and breakpoint of etimicin.

This was an open-label, dose-escalation, phase I study, in which subjects received single and multiple doses of etimicin by intravenous infusion for 1.0 h. Eligible individuals were enrolled and assigned to four groups (A–D). HVs in groups A, B, and C were administered a single dose of etimicin (120, 200, and 300 mg, respectively) on day-1. Group D was used to explore the pharmacokinetic characteristics of etimicin after administration of multiple doses. Individuals in group D received etimicin (150 mg) once a day on day-1, received etimicin (150 mg) twice a day at a time interval of 12 h from day-2 to day-5, and received etimicin (150 mg) once a day on day-6.

For single-dose groups, blood samples were collected from a peripheral vein pre-dose as well as 0.5 (except group A), 1, 1.25, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, and 24 h after dosing. For the multiple-dose group, blood samples were collected at certain time points on day-1 and day-6 (pre-dose as well as 1, 1.25, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, and 24 h after dosing) as well as pre-dose on day-4 and day-5. Urine samples of single-dose groups were collected pre-dose as well as 0–2, 2–4, 4–8, 8–12, and 12–24 h after dosing.

Blood samples were stored at room temperature for 60 min initially, and then centrifuged (2000 × g, 10 min, 4°C) to obtain serum samples. The acquired serum samples were frozen at −80°C before LC-MS/MS analysis. Urine samples were also stored at −80°C until LC-MS/MS analysis.

The pharmacokinetic parameters of etimicin were calculated through non-compartment analysis by Phoenix WinNonlin 8.3.1 (Certara, Princeton, NJ, USA).

In addition, incurred sample reanalysis (ISR) was conducted during the analysis of clinical serum samples and urine samples. For the ISR assessment, more than 10% of the clinical samples should be chosen and they should be reanalyzed according to fresh calibration curves. The time point of the ISR samples should be close to the time point of peak concentration or near the end of the terminal phase of the pharmacokinetic profile. The ISR concentration should be within ±20% of the mean of original concentration and ISR concentration. This requirement should be met by at least three-thirds of the ISR samples.

The two methods were developed for the quantitative determination of etimicin in human samples of serum or urine by LC-MS/MS. First, MS parameters were optimized with solutions of etimicin and the IS at 500 ng/ml in water–methanol (50:50, v/v), respectively. To achieve optimal sensitivity, a series of MS parameters were tuned in positive mode: desolvation temperature, gas settings, DP, EP, CE, and CXP. Typical product ion mass spectra for etimicin and IS are shown in Figure 1.

Different types of columns from Waters were explored: Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm), Acquity UPLC CSH C18 (2.1 × 50 mm, 1.7 μm), Acquity UPLC BEH HILIC (2.1 × 50 mm, 1.7 μm), and XTerra MS C18 (2.1 × 150 mm, 3.5 μm). We also investigated modifiers of the mobile phase: formic acid, acetic acid, and ammonia solution. Finally, optimal peak separations and peak shapes were achieved on the Waters XTerra MS C18 column (2.1 × 150 mm, 3.5 μm) with water–ammonia solution–acetic acid (96:3.6:0.2, v/v/v) as mobile phase A and methanol as mobile phase B. Ion-pairing agents were not used in the mobile phases, which ensured that the LLOQ could be sufficiently low. Under isocratic elution of 50% mobile phase B, the typical retention time of etimicin and the IS was 1.5 min and 3.7 min, respectively (Figure 2).

Different sample-preparation methods were explored to reduce interference from the matrix: protein precipitation, liquid–liquid extraction, and solid-phase extraction. In consideration of succinctness and rapidity, protein precipitation was finally adopted for serum samples. Methanol was the optimal solvent for protein precipitation, and had a good recovery and only a minimal matrix effect. Besides, considering that the methods were developed for a human pharmacokinetic study, a deuterated reagent was adopted as the IS to remove endogenous interference introduced by the sample-preparation process. A deuterated version of etimicin is not available commercially, so we chose carbamazepine-D₁₀ as the IS. The retention time and ionization of the IS were not similar to those of etimicin, but the matrix effect and recovery of the IS and etimicin were comparable.

The two validated LC-MS/MS methods were applied to a phase I pharmacokinetic study of etimicin in Chinese HVs. Currently, six HVs have been enrolled in each group of this trial.

So far, 444 serum samples and 108 urine samples have been analyzed. The average serum concentration–time curves of etimicin in Chinese HVs after a single dose and multiple doses are shown in Figure 5. The pharmacokinetic parameters of etimicin for the same dose groups are shown in Table 4. The maximum serum concentrations of etimicin after a single dose were 6,910–20200 ng/ml at a dose range of 100–300 mg, and were observed at the end of the 1-h intravenous infusion. The half-life and clearance of etimicin in serum were 2.56–2.89 h and 5.80–7.50 L/h after a single dose, respectively, and were approximately consistent in a dose range of 100–300 mg. Obvious accumulation was not observed after multiple doses of etimicin at 150 mg. The half-life and clearance of etimicin were 5.16 h and 5.90 L/h, respectively, after administration of multiple doses. The analysis of etimicin excretion in urine is shown in Figure 6. According to the urinary-excretion profile, 79.4–84.9% of the etimicin dose was eliminated through urine 24 h after a single dose of etimicin.

Additionally, 12.6% and 29.6% of the clinical serum samples and clinical urine samples were selected for the ISR assessment, with respective passing rates of 82.1% and 80.6%. The ISR assessments for these two bioanalysis methods both met the acceptance criterion.

Several pharmacokinetic studies using a single dose of etimicin have been reported (Zhang and Yin, 1999; Yin et al., 2000; Wang et al., 2002; Ouyang, 2007). In those studies, pharmacokinetics was evaluated in only one single-dose group (150 mg or 200 mg) and drug concentrations were measured by micro-bioassays. The single-dose or multiple-dose pharmacokinetics of etimicin in a certain dose-escalation range have not been clarified before. Based on the two reliable and rapid LC-MS/MS methods developed in this paper, we captured the detailed and comprehensive human pharmacokinetic profile of etimicin for the first time, including the single-dose pharmacokinetics in 120 mg-300 mg and the multiple-dose pharmacokinetics. These pharmacokinetic parameters will be further used in the exploration of the breakpoint in Chinese by Monte Carlo simulation, which will advance the rational use of antibiotics in clinical practice.